Split (1)

train · ~9.11M rows (showing the first 93.5k)

identifier stringlengths 11 32	pdf_url stringlengths 17 4.62k ⌀	lang stringclasses 120 values	error stringclasses 1 value	title stringlengths 2 500 ⌀	source_name stringlengths 1 435 ⌀	publication_year float64 1.9k 2.02k	license stringclasses 3 values	word_count int64 0 1.64M	text stringlengths 1 9.75M
https://openalex.org/W4387572654	https://drpress.org/ojs/index.php/fbem/article/download/12157/11840	English	null	Exploration on the Development Path of Agricultural, Cultural and Tourism Industry Integration Under the Background of Rural Revitalization	Frontiers in business, economics and management	2,023	cc-by	5,499	1. Introduction g In recent years, Chaxi Village, located in hongjiang city, Hunan Province, relies on its characteristic industries, local culture and agricultural landscape, implements the rural revitalization strategy, and constantly promotes the deep integration of agriculture, culture and tourism. The village's tourism and economic development have achieved phased results, and it has won more than ten provincial and Huaihua municipal honorary titles, such as "beautiful countryside" and "Boutique Village Demonstration Village", which has certain representativeness. This study takes Chaxi Village in hongjiang city City, Hunan Province as a case, and based on field research, theoretically summarizes the path and mode of rural revitalization in Chaxi Village, with a view to providing reference for other traditional agricultural villages in Hunan Province to realize rural revitalization. The issue of agriculture, countryside and farmers has always been a key concern of the party and the country. The rural revitalization strategy is an important starting point for the work of agriculture, countryside and farmers in the new period, aiming at achieving strong agriculture, rich farmers and beautiful countryside[1]. Some rural areas have continued to promote the process of rural revitalization, and the results have gradually become prominent. But generally speaking, the implementation of rural revitalization strategy still faces many challenges. Backward infrastructure, serious hollowing-out phenomenon, lack of support from characteristic industries and lack of vitality in economic development are still important factors restricting rural development at present. Although some achievements have been made in promoting rural development, the key is that the countryside relies on its internal driving force to form a virtuous circle to promote the sustainable development of rural economy[2]. How to fully tap the unique resource advantages of rural areas and promote rural revitalization according to local conditions is an important proposition at present. The integration of primary, secondary and tertiary industries in rural areas has opened up new paths and methods for rural revitalization. The integration of agriculture, culture and tourism can enhance the hematopoietic function in poor areas and is an important means to realize rural revitalization[3]. Under the guidance of the government, many villages in China have made full use of local resources to plan and develop scientifically, and promoted industrial prosperity through the deep integration of agriculture, culture and tourism, so that the villages can regain their vitality[4][5]. 1. Introduction The deep integration of agriculture, literature and tourism is of great significance for boosting rural revitalization, accelerating rural agricultural modernization and promoting rural prosperity and development[6]. At present, all kinds of rural revitalization models are blooming all over the country, and it is urgent to summarize them at the academic level in Frontiers in Business, Economics and Management ISSN: 2766-824X \| Vol. 11, No. 2, 2023 Frontiers in Business, Economics and Management ISSN: 2766-824X \| Vol. 11, No. 2, 2023 Shaohua Bin School of Business, Hunan University of Science and Technology, Xiangtan, Hunan 411201, China Abstract: The issue of agriculture, countryside and farmers has always been a key concern of the party and the country. The rural revitalization strategy is an important starting point for the work of agriculture, countryside and farmers in the new period. The deep integration of agriculture, culture and tourism is of great significance for boosting rural revitalization, accelerating rural agricultural modernization and promoting rural prosperity and development. Taking Chaxi Village in hongjiang city City, Hunan Province as a case, this study summarizes the Chaxi model of "taking agriculture as the carrier, culture as the core and tourism as the engine" to promote rural revitalization, and provides reference for the rural revitalization strategies of other traditional agricultural villages in Hunan Province from four aspects: the deep integration of agriculture, literature and tourism to promote industrial revitalization, beautiful countryside to help ecological revitalization, rural civilization to empower culture revitalization, and external education to help talent revitalization. order to better promote them to other traditional agricultural villages. -- A Case Study of Chaxi Village, Hongjiang City, Hunan Province -- A Case Study of Chaxi Village, Hongjiang City, Hunan Province Shaohua Bin 4.1. The dimension of industrial revitalization The long-term development of rural areas can not be separated from industrial revitalization. Chaxi Village is a traditional agricultural village, and the villagers have been planting for generations. However, in the early stage, due to many problems such as limited output, limited funds, irregular management, lack of effective sales channels, and meager sales profits of primary agricultural products, industrial development also faces many difficulties. p y According to local conditions, Chaxi Village reclaimed idle hillsides and low-yield dry fields, made full use of local unique climatic conditions and soil rich in trace elements to cultivate sugar orange, and constantly conducted research on purification, selection and cultivation techniques, so as to improve the quality of qianyang sugar orange. The village regularly invites experts for technical training, and adopts a series of efficient cultivation and management measures such as thinning flowers and fruits, pruning, fertilizing, and preventing diseases and pests, thus realizing standardized and specialized production of citrus bases. At present, Sugar Orange has become a pillar industry in Chaxi Village. The planting area of Sugar Orange in the village is more than 10,000 mu, with an annual output of 15 million kilograms. Sugar Orange in the producing area has been selected as one of the top 100 agricultural products in China and a geographical indication product in China, and won the honorary title of "Famous Fruit in China". Chaxi Village has promoted the village collective industry by establishing the production and marketing mode of "Party branch+cooperative+farmer". At present, three citrus production and sales companies and seven professional cooperatives have been established, which effectively help farmers solve the problems of financial difficulties, irregular management and no channels for sales by integrating resources through cooperatives. 2. Rural Cultural Tourism and Rural Revitalization Combing the above research, it is found that the traditional common development modes are mainly the integration of literature and tourism, and the integration of agriculture, literature and tourism has gradually emerged in recent years, and achieved certain results. The integration of agriculture, literature and tourism has fully utilized the advantages of rural agricultural resources and regional cultural characteristics into rural tourism development, effectively stimulated rural vitality, promoted the spread of rural culture and maximized the utilization of rural resources. It is of great theoretical significance and practical value to explore the specific path of the integration of agriculture, literature and tourism to promote rural revitalization. 3.1. Brief introduction of Chaxi Village Chaxi Village is located in the north of Qiancheng Town, hongjiang city, 4 kilometers away from Qiancheng Town, with a village area of 12.5 square kilometers. It governs 18 villagers' groups, with a total population of 2,608. There are 101 people in party member, and the leading industries are sugar orange and rice. At present, there are more than 10,000 mu of sugar orange bases in the village, with an annual output of more than 15 million kilograms. Sugar orange is the supporting industry of Chaxi Village, and colorful rice is planted. While making full use of agricultural resources, Chaxi Village deeply digs local cultural resources, builds tourist attractions such as educated youth huts, effectively practices the rural development model integrating leisure sightseeing, cultural experience and rural tourism, and effectively promotes the rural revitalization strategy. Chaxi Village has successively won more than ten provincial and Huaihua municipal honors, such as Hunan Advanced Party Organization, Hunan Tongxin beautiful countryside, "beautiful countryside" and "Boutique Village". The development of a single industry is difficult to achieve real industrial revitalization, and industrial prosperity needs the joint promotion of primary, secondary and tertiary industries. Based on the agricultural background of large- scale and specialized cultivation of sugar oranges in Chaxi Village, the local government took the lead in building a citrus Expo Park covering an area of more than 120 mu, which integrates picking, sightseeing and leisure. Large-scale festivals such as hongjiang city Citrus Flower Festival and Qianyang Sugar Orange Picking Festival are held in specific seasons such as March and December every year, attracting more than 7,000 tourists every year. During the sugar orange 2. Rural Cultural Tourism and Rural Revitalization Through the extension of industrial chain, the maximum utilization of resources can be truly realized, so as to increase farmers' income as much as possible and accelerate the development of rural economy [10]. In his research, Liao Yonglun emphasized that "the community supports the rural cultural tourism", and the development of rural tourism needs the support of characteristic agriculture and cultural experience, and at the same time, it effectively uses the Internet platform to accelerate the integration of resources [11]. Combing the above research, it is found that the traditional common development modes are mainly the integration of literature and tourism, and the integration of agriculture, literature and tourism has gradually emerged in recent years, and achieved certain results. The integration of agriculture, literature and tourism has fully utilized the advantages of rural agricultural resources and regional cultural characteristics into rural tourism development, effectively stimulated rural vitality, promoted the spread of rural culture and maximized the utilization of rural resources. It is of great theoretical significance and practical value to explore the specific path of the integration of agriculture, literature and tourism to promote rural revitalization. "agriculture+culture+tourism", and affirmed the remarkable effect of the integration of agriculture, culture and tourism in promoting rural revitalization [8]. Luo Xianju pointed out in his research that rural areas should fully tap their resource characteristics and conform to the development trend of industrial integration, and emphasized the importance of industrial chain in industrial revitalization, emphasizing the effective extension, replenishment, strengthening and expansion of the chain based on rural reality, and establishing a multifunctional rural industrial integration system to realize the industry bigger and stronger [9]. Liu Lanlan believes that the integration of agriculture, culture and tourism can be realized by fully tapping the agricultural and cultural resources in rural areas and integrating tourism elements. Through the extension of industrial chain, the maximum utilization of resources can be truly realized, so as to increase farmers' income as much as possible and accelerate the development of rural economy [10]. In his research, Liao Yonglun emphasized that "the community supports the rural cultural tourism", and the development of rural tourism needs the support of characteristic agriculture and cultural experience, and at the same time, it effectively uses the Internet platform to accelerate the integration of resources [11]. 2. Rural Cultural Tourism and Rural Revitalization The integration of rural primary, secondary and tertiary industries is an important means to improve the quality and efficiency of rural development, and rural industrial integration has always been a key research field in academic circles. The research on the integration mode of agriculture, culture and tourism has increased in recent years, and farmhouse music is the earliest form of combination of agriculture and tourism in China. Cao Lili made clear the characteristics of five types of development models in analyzing the integration model of Zhejiang agriculture, culture and tourism, and pointed out that different rural areas should combine their resource characteristics and advantages to choose the appropriate integration model according to local conditions [7]. Yang Yun and others took the ancient village of Anyi in Jiangxi Province as a case, deeply analyzed the current situation of local resources, emphasized the development model and specific measures of 22 interviews, online search and other forms. The researchers went to Chaxi Village for field research three times from July 5 to July 31, 2023.And set up an interview outline around the "integration of agriculture, culture and tourism" and "rural revitalization" in Chaxi Village according to the theme of this study. Interviews were conducted with 9 people, including the town government staff, Chaxi village committee cadres, homestay operators, cooperative contractors and ordinary villagers. The average interview duration was 1.5 hours/person, and about 50,000 words of recorded text were sorted out. After understanding the development of Chaxi village in depth, the interview text was coded step by step to form the main point of this paper. "agriculture+culture+tourism", and affirmed the remarkable effect of the integration of agriculture, culture and tourism in promoting rural revitalization [8]. Luo Xianju pointed out in his research that rural areas should fully tap their resource characteristics and conform to the development trend of industrial integration, and emphasized the importance of industrial chain in industrial revitalization, emphasizing the effective extension, replenishment, strengthening and expansion of the chain based on rural reality, and establishing a multifunctional rural industrial integration system to realize the industry bigger and stronger [9]. Liu Lanlan believes that the integration of agriculture, culture and tourism can be realized by fully tapping the agricultural and cultural resources in rural areas and integrating tourism elements. 3.2. Research methods and processes In this study, the relevant materials of rural revitalization in Chaxi Village were collected through field research, in-depth 23 cultural walls, and the overall image of the village has been greatly improved. In 2022, Chaxi Village completed the renovation of 148 household toilets, demolished 12 abandoned miscellaneous houses and broken walls, rectified 5 black and odorous water bodies, and implemented 46 village micro-renovations, and solidly promoted the work of "purification" of rural positions, rural toilet revolution, courtyard construction, garbage classification and reduction, and "hollow house" demolition. In order to solve the problems of domestic garbage and sewage treatment in rural areas, Chaxi Village has set up a special sewage treatment system and composting buckets, and encouraged villagers to actively participate in the construction of maintaining the village appearance through strengthening methods such as "red and black list" and "love supermarket". Environmental remediation is actually implemented in practice, making Chaxi truly livable and suitable for traveling. picking festival, fruit farmers can sell their own products such as sugar oranges and citrus nectar to tourists. At the same time, Chaxi Village has built a "concentric orchard" of more than 300 mu, and launched a "fruit tree adoption" activity. Individuals or groups adopt fruit trees at a certain price, and the output belongs to the adopters, and they can participate in the cultivation and picking of fruit trees all the time. This form of fruit tree adoption connects tourists with Chaxi Village, which increases the rate of tourists revisiting and broadens the income channels of fruit farmers. In addition to sugar orange, rice is also the main crop in Chaxi village, and colorful rice has been planted in recent years. As an important carrier of farming culture, the unique and beautiful paddy field landscape formed by colorful rice is an indispensable tourism element in rural tourism. Chaxi Village launched the rice fish project, and the rice fish cultured in rice fields tastes delicious. Visitors can not only taste the original ecological farmhouse food, but also harvest the rural tourism experience of catching fish in the fields. 4.3. The dimension of cultural revitalization The civilization of rural customs is embodied in the good atmosphere formed by the villagers' good words and deeds, which includes four cultural cornerstones: clan culture, family style and family training culture, rural sage culture and socialist core values culture. Chaxi Village pays attention to cultural construction and builds the first rural revitalization hall in Hunan Province. Based on the village history and cultural accumulation, it has created cultural landscapes such as educated youth huts and Shijia Courtyard. The family instructions of Shijia Courtyard vividly interpret the core values of socialism, which is not only the binding criterion of villagers' words and deeds, but also the carrier of the inheritance of simple rural customs in Chaxi. Educated youth culture is not only a nostalgic memory, but also a admiration for the pioneering spirit. The endless colorful rice fields in front of the educated youth hut are also the imprint of farming culture. Educated youth huts and Shijia Courtyard have become important windows for tourists to learn about Chaxi culture. In the past two years, the educated youth cabin has carried out 782 person-times of party spirit education and training, and received 185 person-times of research students. It is a gathering place to carry forward the educated youth culture and promote the development of cultural tourism. Under the guidance and support of the Party Committee of Chaxi Village, hongjiang city Chaxi Village has cultivated and developed characteristic industries such as characteristic homestay, educated youth cultural experience, health and leisure around the characteristic themes of green mountains and green waters, folk customs, ecological leisure and educated youth culture, and explored a new path to help rural revitalization through the deep integration of agriculture, culture and tourism. Chaxi Village has established the Rural Civilization Council, built a civilized practice station in the new era, formulated village rules and regulations, adhered to the principle of "judging morality by virtue, cultivating morality by culture and promoting morality by regulations", formulated the Measures for the Management of Villagers' Rule of Virtue Points System and the Measures for the Management of Love Supermarket, and implemented the points exchange of rule of virtue evaluation and love supermarket points. Volunteers of civilized practice in the new era, such as "red vest", "literary team" and "party 3.2. Research methods and processes Chaxi Village has established a garbage collection and transfer system of "household classification, village collection" and a sanitation civilization evaluation system around garbage control, water control, toilet control, house control and wind control, vigorously implemented projects such as upgrading roads, improving water and toilets in rural areas, and successively created three beautiful roads, and declared to create 560 beautiful courtyard demonstration households, integrating farm house features, courtyard landscape, local culture and other elements to create beautiful countryside. In the 1980s and 1990s, a large number of educated youth went to the countryside. Chaxi Village was the point where educated youth went to the countryside at that time, and the educated youth culture has also become a nostalgic memory. The spirit of "hard work and hard struggle" of educated youth is still of great significance to the times. Chaxi Village has restored and renovated the educated youth hut, which displays relevant exhibits during the period of educated youth going to the countryside, and also creates an educated youth canteen with the theme of food. The educated youth cabin is not only the experience point of the educated youth culture network, but also a gathering place to promote the development of cultural tourism. Shijia Courtyard with architectural characteristics of Ming and Qing Dynasties and its excellent family style and family instruction are not only the catalyst of simple and civilized rural style in Chaxi Village, but also an important carrier for tourists to feel the rural culture. Chaxi Village has fully tapped the existing local cultural heritage, built tourist attractions such as educated youth huts and Shijia Courtyard, and skillfully integrated local cultural elements into rural tourism. Chaxi Village has planned the first batch of farmhouse homestays, parent-child picking bases and starry sky campsites, so that tourists can truly feel the fun of living in farmhouse houses, eating farmhouse meals and doing farm work. 4.4. The dimension of talent revitalization Brain drain is a common problem encountered in rural areas at present. Only when talents flourish can rural areas prosper. Many talents are reluctant to come to the countryside because of the imperfect rural infrastructure, poor salary and treatment, and greater intensity of grassroots work. There is no fresh blood injection in the countryside for a long time, and it is even more difficult to innovate. On the introduction of this channel from outside, Selected Graduates, directly under the provincial government, is the assistant to the village secretary, and the resident working group composed of capable people at the town level has added fresh blood to Chaxi Village, which is an important guarantee for the revitalization of talents in Chaxi Village. In view of talent training, Chaxi Village, combined with the actual situation, launched the activity of "attracting fellow villagers, returning to their hometown and building their hometown", set up a love hometown association in Chaxi Village, and called on truly capable talents to return to and build Chaxi. Under the extremely difficult situation of attracting talents to the countryside, the Party Committee of Chaxi Village pays attention to selecting the capable talents in the left-behind villages, implements the system of "one examination and one evaluation" for village-level reserve cadres, and brings the outstanding talents who really want to be officers, can be officers and accomplish things into the team of village-level reserve cadres. At the same time, we are committed to developing the rich expert into party member, cultivating party member into a rich expert, and selecting the rich expert from party member into the village "two committees" cadres. The Party Committee of Chaxi Village gives full play to the role of party member as a leader in getting rich, guides farmers' professional and technical knowledge in planting sugar oranges, and conducts professional training to guide more farmers to join the trend of getting rich in the sugar orange industry. The problem of talent shortage in rural industrial management and tourism services in Chaxi Village is still quite obvious, and the lack of high-quality talents is a major challenge to the road of rural revitalization in Chaxi Village. 5. The Main Challenges Faced by The Integration of Agriculture, Literature and Tourism in Chaxi Village member micro-propaganda group", are active in country lanes, cultural squares and under big trees, and take cultural infiltration to awaken the villagers' consciousness of actively participating in the cultural construction of Chaxi Village in a subtle way. Chaxi Village integrates educated youth culture research, farming culture and natural landscape, which not only enriches the tourism format of Chaxi Village and revives its characteristic culture, but also enhances the villagers' cultural self-confidence and sense of ownership, makes the villagers participate in the construction and inheritance of Chaxi culture independently and consciously, and cultivates a new civilized atmosphere to revitalize the culture of Chaxi Village. 4.2. The dimension of ecological revitalization Chaxi Village has successively won many honors, such as Tongxin beautiful countryside in Hunan Province, Hunan Rural Governance Demonstration Village, beautiful countryside and so on. Chaxi Village has made remarkable achievements in village environmental improvement. The roads in Chaxi Village are clean and tidy, and the vegetation on both sides of the road is clean and beautiful under the embellishment of Gesang flowers and hydrangeas. The dirty walls on the roadside have been transformed into artistic 24 5.1. The lack of high-quality professionals restricts the development of agricultural tourism Talent is the first resource, and high-quality talent is an important support to realize industrial prosperity. Due to the slow development of rural economy and relatively backward infrastructure, young people in rural areas are reluctant to stay in their hometowns and choose to go out to work, and outsiders are even more reluctant to come in. The phenomenon of "hollowing out" in rural areas is still widespread, and the shortage of high-quality talents in rural areas hinders the efficient promotion of rural revitalization strategy. At present, in addition to grassroots cadres, Chaxi Village is short of all-round high-quality talents to serve the in-depth construction of Chaxi Village. Chaxi village is rich in cultural tourism resources, but it lacks high-quality tourism talents to interpret the cultural heritage of Chaxi village to tourists in a more vivid way. In addition, Chaxi Village has started to operate homestays, but all of them are individually operated by farmers, lacking in management, with few rooms and outstanding homestay characteristics. The management form of homestay mainly shows that farmers vacate several rooms in their own houses for tourists to use, only considering the accommodation requirements of guests, but not fully considering the living experience of guests. Whether it is the development of rural tourism products, the operation of homestays, the systematic training and guidance of farmers, and a series of activities need high-quality talents to check. Only limited to the superficial practice of form can not make the rural revitalization achieve good results. 5.2. The lack of breadth and depth of industrial integration restricts the development of agricultural tourism The supporting industry of Chaxi Village is sugar orange, and agriculture and tourism can be integrated and connected to some extent by holding sugar orange picking festival and "concentric orchard". However, at present, the industrial chain of sugar orange in Chaxi Village is not complete, and its industrial value has not been fully highlighted. It is only limited to the tourism activities of enjoying flowers and picking fruits. The products sold are still mainly primary agricultural products such as fresh fruits, and no fruit processing base has been established. In addition, under the fierce competition of Mayang Sugar Orange, Gannan Navel Orange and other similar fruits, the brand promotion of Qianyang Sugar Orange still needs to be further strengthened. With the rise of rural tourism in Chaxi, the demand for tourism professionals has also increased. The Party Committee of Chaxi Village trained the remaining labor force in Chaxi Village in tourism service skills, encouraged the villagers to set up homestays and catering services, made full use of the idle resources in the village, tapped the potential ability of the villagers, fully mobilized the enthusiasm of the villagers, and opened up a practical road to rejuvenating talents. 5.3. The connotation of tourism products is not deeply explored, which restricts the development of agricultural cultural tourism 5.3. The connotation of tourism products is not deeply explored, which restricts the development of agricultural cultural tourism At present, Chaxi Village has developed cultural At present, Chaxi Village has developed cultural 25 landscapes with cultural connotations, such as educated youth huts, Shijia Courtyard, educated youth canteen, and citrus Expo Park. At the same time, some rural tourism products have made full use of the characteristics of farming culture. As the carrier of educated youth culture and simple rural customs, the exhibits in educated youth huts and Shijia Courtyard are limited, mainly photos and some ancient objects, which are less attractive, lack of tour guides to explain, and their presentation methods are not vivid enough. Chaxi Village has not deeply explored the cultural tourism products, and there are still shortcomings in the development of cultural tourism products, and the tourism attraction is obviously insufficient. circular agriculture, develop pollution-free rural eco-tourism industry with the help of continuous innovation of agricultural science and technology, effectively use rural ecological resources, and promote the protection and development of rural culture and the development of rural economy. Give full play to the main role of villagers' ecological protection, create an ecological space for rural life, strengthen villagers' awareness of ecological environmental protection through various educational methods and publicity channels, strengthen the construction of rural ecological human settlements and comprehensive environmental management, develop rural ecological environmental protection services, and enhance the supervision ability of government departments on rural ecological environment. 6.4. External introduction and internal education to help revitalize talents Talent, as the first resource, is an important support for rural revitalization. In order to make the countryside full of vitality, it is inevitable that high-quality talents will actively participate in rural construction. Rural areas need to pay attention to the "capable person effect", activate the resources of capable people, mobilize the rich leaders in rural areas to join the team to help rural areas, share the experience of getting rich, and be able to lead everyone, and at the same time enhance confidence for other villagers to get rid of poverty. In addition, we focus on optimizing the ranks of grassroots village cadres. At present, the comprehensive quality of grassroots cadres in many rural areas needs to be improved, and their age is too large, and their information literacy is far from enough. It is urgent to cultivate leaders of grassroots party organizations with a strong sense of foresight, responsibility, service and local feelings. While attracting foreign talents through various talent introduction policies, we should strengthen the training of villagers, cultivate new professional farmers, such as breeding experts, e-commerce experts and planting experts, promote the construction of rural professionals, and give full play to the talents of the left- behind villagers, so that farmers can give full play to their strengths and serve the development of rural industries. 6.3. Rural civilization empowers cultural revitalization Culture is the soul of tourism. At present, most rural areas still stay in shallow rural tourism and leisure activities such as farmhouse music, and a single tourism experience can't leave a deep memory for tourists. China's rural areas should fully tap the existing local cultural heritage and integrate local cultural elements into rural tourism according to local conditions. Build a farmhouse, a farmhouse garden and an orchard picking base, give full play to the advantages of farming culture, and let tourists really feel the fun of living in a farmhouse, eating farmhouse meals and doing farm work. Effectively tap the characteristics of rural culture to carry out research activities, and build a rural tourism complex that integrates cultural research, farming culture and natural landscape. At the same time, it is necessary to stimulate the villagers' cultural self-confidence and sense of ownership, turn them into customs with cultural people, and make them participate in the construction and inheritance of rural culture independently and consciously, so as to cultivate a new civilization and revitalize the culture. 6.1. The deep integration of agriculture, literature and tourism to promote industrial revitalization The achievements made by Chaxi Village in the practice of rural revitalization largely depend on the supporting industry of sugar orange, giving full play to its industrial advantages. At the same time, various measures of Chaxi Village have achieved the integration of agriculture, farming culture and tourism to a certain extent. Industrial integration is an important basis for multi-dimensional increase of product added value and promotion of product value chain. Vigorously promoting the strategy of rural industrial integration and realizing the development of rural primary, secondary and tertiary industries can be started from the following aspects: first, we should build industrial integration bases and platforms, support major grain producing areas to focus on the intensive processing of agricultural products, build a close connection mechanism between pre-production, mid-production and post-production, and form an industrial integration pattern with multi-agent participation, multi- factor aggregation, multi-format development and multi- mode promotion. Secondly, it is necessary to extend the industrial chain, build a supply chain, and realize the deep integration of rural industries from products to business, from business to market. Finally, it is necessary to build a number of characteristic product bases around industrial integration, avoid homogenization, create differentiated products, and enhance the formation of rural industries to improve quality and efficiency and core competitiveness. 6.2. beautiful countryside helps rural ecological revitalization. Rural capacity and rural appearance play a decisive role in the development of rural tourism. In rural areas, the garbage collection and transfer system of "household classification and village collection" and the evaluation system of sanitation civilization can be established around garbage control, water control, toilet control, house control and wind control, and projects such as upgrading roads and improving water and toilets in rural areas can be vigorously implemented, so as to integrate elements such as farm house style, courtyard landscape and local culture, and promote the ecological revitalization of tea creek while building beautiful countryside. Rural areas should establish the concept of 26 [6] Yan Yan, Hao Xin, Li Bo. The path of integrated development of rural "agricultural tourism" [J]. Journal of Nantong Vocational University, 2023,37(02):9-12. References [1] Zhu Lijiao, Qi Jianli, Sun Ye. Deepening the integration of agriculture, literature and tourism to help rural revitalization in an all-round way [J]. Hebei Agriculture, 2023(05):10-11. [7] Cao Lili. Research on the integration and development of rural tourism industry in the new era [M]. Northwest A&F University Press:, 201910.117. [2] Tao Jun-mei. The integration of agriculture, literature and tourism empowers the path of rural revitalization in Guizhou [J]. Contemporary County Economy, 2023(09):78-80. [8] Yang Yun, Jia Gui. Research on the path of rural revitalization of ancient villages under the background of the integration of agriculture, literature and tourism [J]. Journal of Yunnan Agricultural University (Social Science), 2021,15(05):19-24. [3] wujun. The integration of agricultural, cultural and tourism development paints a new picture of rural revitalization [J]. Jiangsu Rural Economy, 2023(06):37. [9] Luo Xianju. Promoting rural revitalization in ethnic areas with the deep integration of agriculture, culture and tourism: mechanism and promotion strategy [J]. Price Theory and Practice, 2022(02):188-191+203. [4] Min Zhongxiu, Wang Yongqiang, Ding Yumei. Study on the development countermeasures of "agricultural tourism" industry integration from the perspective of rural revitalization- taking three key rural tourism villages in Gannan as an example [J]. Journal of Gansu Normal University, 2023,28(03):28-34. [10] Liu Lanlan. Promoting rural revitalization through the integration of agriculture, literature and tourism [J]. Henan Daily, 2019-12-04(006) [5] Li Yuqiong. On the path of rural revitalization promoted by the integration of agriculture, culture and tourism in Kunming [J]. Yunnan Agriculture, 2023(06):42-44. [11] Liao Yonglun. The integration and development of agriculture, literature and tourism opens up a new path for rural revitalization [J] . Guizhou Daily, 2020-04-15(009) 27 27
W4389048820.txt	https://riviste.unimi.it/index.php/RIPS/article/download/13305/12450	en		PALEONTOLOGICAL DESCRIPTIONS OF SOME RUDISTS FROM THE UPPER CRETACEOUS OF OSTUNI (BR - ITALY)	Rivista italiana di paleontologia e stratigrafia	2,020	cc-by-sa	0
https://openalex.org/W2085225969	https://escholarship.org/content/qt3j69q4hj/qt3j69q4hj.pdf?t=plscfk	English	null	High-resolution frequency domain second harmonic optical coherence tomography	Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE	2,007	cc-by	4,879	Copyright Information py g This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Irvine UC Irvine Previously Published Works Title High-resolution frequency-domain second-harmonic optical coherence tomography. Permalink https://escholarship.org/uc/item/3j69q4hj Journal Applied Optics, 46(10) ISSN 1559-128X Authors Su, Jianping Tomov, Ivan V Jiang, Yi et al. Publication Date 2007-04-01 DOI 10.1364/ao.46.001770 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title High-resolution frequency-domain second-harmonic optical coherence tomography. Permalink https://escholarship.org/uc/item/3j69q4hj Journal Applied Optics, 46(10) ISSN 1559-128X Authors Su, Jianping Tomov, Ivan V Jiang, Yi et al. Publication Date 2007-04-01 DOI 10.1364/ao.46.001770 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title High-resolution frequency-domain second-harmonic op Permalink https://escholarship.org/uc/item/3j69q4hj Journal Applied Optics, 46(10) ISSN 1559-128X Authors Su, Jianping Tomov, Ivan V Jiang, Yi et al. Publication Date 2007-04-01 DOI 10.1364/ao.46.001770 Copyright Information This work is made available under the terms of a Crea License, available at https://creativecommons.org/licen Peer reviewed UC Irvine UC Irvine Previously Published Works High-resolution frequency-domain second-harmonic optical coherence tomography. 1. Introduction tion improvement. Recently, second-harmonic OCT (SH-OCT), which combines the sample structural sensitivity of second-harmonic generation (SHG) with the coherence gating of OCT to obtain molecular contrast, has been reported.1–6 The SH is generated only by molecules that are noncentrocymmetric, and hence contrast is a function of the molecular structure of the specimen and its orientation relative to the laser beam. In biological materials, collagen is the most abundant protein and possesses noncentro- cymmetric structure.7,8 Because collagen is the pre- dominant structural component of most biological tissues, as well as a strong SHG source, it is predicted that structural changes in collagen will result in the change of the second harmonic generation signal. In- deed, the experimental studies have found that the SH responded to the structural modiﬁcations of col- lagen, such as thermal denaturation, nonenzymatic glycation, and partial enzymatic cleavage.8 These modiﬁcations simulate the possible changes that may take place in several pathophysiologic conditions, including thermal injury, diabetes, aging, abnormal wound healing, and malignant transformation of dys- plastic nevi. Optical coherence tomography (OCT) is a recently developed imaging modality based on coherence- domain optical technology. OCT takes advantage of the short coherence length of broadband light sources to perform micrometer-scale, cross-sectional imaging of biological tissues. OCT can provide imaging reso- lutions that approach those of conventional histopa- thology and can be performed in situ and in vivo. Despite its advantages, a serious limitation of OCT is the relatively low imaging contrast compared to his- topathology and optical microscopy. The imaging con- trast in OCT originates from the inhomogeneities of sample scattering properties. However, the linear scattering properties of pathological tissue are often optically similar to the scattering properties of nor- mal tissue. For example, many cancers originate in the epithelium that has a thickness suitable for OCT imaging, but in their early stages when these cancers are developing through cell dysplasia, changes in the tissue morphology and the refractive index between normal and diseased tissues are very small and dif- ﬁcult to detect. Therefore to meet the challenges found in OCT clinical applications, imaging contrast enhancement is as important as the imaging resolu- SHG is a powerful contrast mechanism in nonlin- ear optical microscopy.9–12 SHG microscopy is known for its capability for high-resolution optical 3D sec- tioning of samples because signals only arise from the focal point of the objective where sufﬁcient peak power can occur. The authors are with the Department of Biomedical Engineer- ing, Beckman Laser Institute, University of California, Irvine, Irvine, California 92697. Z. Chen’s e-mail address is z2chen@uci.edu. Received 23 June 2006; accepted 15 September 2006; posted 27 September 2006 (Doc. ID 72297); published 13 March 2007. 0003-6935/07/101770-06$15.00/0 © 2007 Optical Society of America High-resolution frequency-domain second-harmonic optical coherence tomography Jianping Su, Ivan V. Tomov, Yi Jiang, and Zhongping Chen We used continuum generated in an 8.5 cm long ﬁber by a femtosecond Yb ﬁber laser to improve threefold the axial resolution of frequency domain second-harmonic optical coherence tomography (SH-OCT) to 12 m. The acquisition time was shortened by more than 2 orders of magnitude compared to the time-domain SH-OCT. The system was applied to image biological tissue of ﬁsh scales, pig leg tendon, and rabbit eye sclera. Highly organized collagen ﬁbrils can be visualized in the recorded images. Polarization dependence on the SH has been used to obtain polarization resolved images. © 2007 Optical Society of America OCIS codes: 170.4500, 170.3880, 190.4160. OCIS codes: 170.4500, 170.3880, 190.4160. Powered by the California Digital Library University of California eScholarship.org 2. Methods h compressed in external grating pairs to 170 fs with an average power of 260 mW at 50 MHz repetition rate. The output spectra have an approximate Gauss- ian shape with a linewidth of 13.5 nm (FWHM), which allowed us to record FD SH-OCT images with 30 m axial resolution.6 The experimental setup is shown in Fig. 1. A self- starting, stretched-pulse Yb ﬁber laser generating femtosecond pulses centered at 1.03 m wavelength with a 50 MHz repetition rate is used as the light source.15 The mode-locked pulses from the ﬁber os- cillator are temporally stretched in a 10 m single mode ﬁber and ampliﬁed in yttrium-doped ﬁbers pumped by two diode lasers. The output pulses are We need a light source with a broader spectrum to increase the axial resolution. For our study, we used a continuum generation in the ﬁber to broaden the spectra. Spectral broadening in a ﬁber is also associ- ated with pulse broadening. However, pulse broad- ening is not desirable because the SH efﬁciency is quadratic dependent on the pump intensity. We stud- ied the temporal and spectral broadening of our laser pulse in several ﬁbers. In Fig. 2, we summarize the results for a Corning HI 780 ﬁber. For a 1 m long ﬁber, spectral broadening up to 80 nm was recorded, but at the same time the pulse duration increased to 4 ps. This leads to about 2 orders of magnitude de- crease of SH efﬁciency. We also noted that the output spectra from the ﬁber was not smooth, but exhibited modulation. In the experiment, we selected an 8.5 cm long ﬁber, and the output spectra width was about 40 nm at 10% peak intensity level. The pulse dura- tion of the continuum spectra was less then 400 fs. The 100 mW continuum output power from the ﬁber was incident on a 2:1 beam splitter (BS1). The more powerful beam was used in the sample arm where 20 microscope objectives were used to focus the beam on the sample. Fig. 1. (Color online) Schematic of the FD SH-OCT experimental system. FSL, femtosecond ﬁber laser; HWP, half-wave plate; DM, dichroic mirror; NLC, nonlinear crystal; OBJ, objective; BS, beam splitter; CF, color ﬁlter. The average excitation power at the exit of the objective was about 50 mW, and the typical pulse energy density at the focus was about 3 mJcm2. 1. Introduction Because SHG is a coherent process, the scattering beam may be highly forward directed. The directionality of SHG depends on the distribu- tion and orientation of the induced dipoles within the focal volume and has been investigated.13,14 However, 1770 APPLIED OPTICS Vol. 46, No. 10 1 April 2007 Fig. 2. (Color online) Relationship between ﬁber length (Corning HI 780) and output spectra bandwidth. Without ﬁber continuum generation, the corresponding SH resolution is 30 m. A 1 m long ﬁber generates continuum with corresponding SH resolution of 8 m; however, the continuum pulse duration broadens to 4 ps. Experimentally, the pulse duration was measured by an auto- correlator. because transmission images are difﬁcult to acquire in thick samples or in vivo studies, SHG microscopy that uses backscattered light has been demonstrated.9 In comparison to SHG microscopy, SH-OCT has the advantage of decoupling the axial and transverse scans and is able to obtain two-dimensional tomo- graphic images with a one-dimensional scan. because transmission images are difﬁcult to acquire in thick samples or in vivo studies, SHG microscopy that uses backscattered light has been demonstrated.9 In comparison to SHG microscopy, SH-OCT has the advantage of decoupling the axial and transverse scans and is able to obtain two-dimensional tomo- graphic images with a one-dimensional scan. Recently a time-domain (TD) SH-OCT system that utilizes the nonlinear optical effect of SHG to produce highly contrasting images of biological tissues was reported.1–3 This technique combines the sample struc- tural sensitivity of SHG with the coherence gating of OCT. The images of SH-OCT can show collagen ﬁbers that are not obvious in conventional OCT images. However, the TD SH-OCT has limited sensitivity and imaging speed. Frequency-domain (FD) OCT has been attracting much attention because of its poten- tial higher speed and sensitivity. Recently, FD SH- OCT with an axial resolution of the order of 30 m has been reported.4,6 Here we report the development of a FD SH-OCT system with an axial resolution of 12 m. Fig. 2. (Color online) Relationship between ﬁber length (Corning HI 780) and output spectra bandwidth. Without ﬁber continuum generation, the corresponding SH resolution is 30 m. A 1 m long ﬁber generates continuum with corresponding SH resolution of 8 m; however, the continuum pulse duration broadens to 4 ps. Experimentally, the pulse duration was measured by an auto- correlator. 1 April 2007 Vol. 46, No. 10 APPLIED OPTICS 2. Methods h This energy density was much less than the tissue damage threshold estimated in the range 0.5–1.0 Jcm2.8,16 The sample was situated on a motorized stage for lateral movement with submicrometer resolution. In this setup the interferometers for the fundamental and Fig. 1. (Color online) Schematic of the FD SH-OCT experimental system. FSL, femtosecond ﬁber laser; HWP, half-wave plate; DM, dichroic mirror; NLC, nonlinear crystal; OBJ, objective; BS, beam splitter; CF, color ﬁlter. 1 April 2007 Vol. 46, No. 10 APPLIED OPTICS 1771 second harmonic only partially overlapped. Both in- terferometers used commercially available spectrom- eters (Avantes), and the length of the reference arms was independently adjustable (Fig. 1). The recom- bined fundamental beams in BS1 were directed to a spectrometer with a resolution of 1 0.15 nm at 1030 nm range (AvaSpec-3648). The spectrometer was equipped with a CCD camera with 3648 pixels 8 200 m pixel. The integration time was adjust- able from 10 s to 600 s. NA but at the expense of the depth range. Because the SH efﬁciency has a square dependence on the fundamental intensity, it beneﬁts from focusing. For a given Gaussian fundamental pump intensity, both axial and lateral resolution of SH-OCT are improved compared to fundamental radiation OCT, and they scale as 12 of the fundamental radiation resolution. For FD OCT, the measuring range, z 2 4n, is determined by the resolution of the spectrom- eter, , where is the wavelength of the radiation, and n is the refractive index of the sample.18 In prac- tice, it is desirable to match the DOF and measuring range. However, the efﬁciency of SHG has square dependence on the intensity of the fundamental ra- diation; therefore, strong focusing of the laser beam is used. Because of this, the measuring range of a SH- OCT system is usually longer than the sample focus- ing optics DOF. The scanning of both focusing optics and reference delay may be used to effectively cover the whole measuring range but at the expense of the speed of image recording. The backscattered SH signal from the sample was reﬂected by a dichroic mirror and directed to beam splitter BS2. 3. Results and Discussion Using continuum radiation generated in the ﬁber, SH-OCT images of ﬁsh scales from salmon and pig leg tendon were recorded. Figure 3 shows the image of the ﬁsh scales obtained simultaneously with both fundamental and second harmonic interferometers. For these images, 50 mW of fundamental power was incident on the sample. The SH spectrometer CCD integration time was set at 20 ms, and the averaging of 50 A-scans was used. The CCD integration time of the fundamental spectrometer was set at 0.8 ms and the averaging of 50 A-scans. In Fig. 3, the left-hand side shows an OCT image of ﬁsh scales obtained by the fundamental beam. Boundaries between differ- ent layers can be clearly identiﬁed. For clarity, the SH image is displayed on the right-hand side. The polarization of the SH is parallel to the fundamental beam polarization. The SH-OCT image on the right- hand side shows layerlike distribution of collagen Compared with other tomographic modalities, one of the advantages of OCT techniques is their high resolution. The axial resolution l 0.442 is de- termined by the bandwidth of the light source, . The lateral resolution is determined by the size of the focal spot used in the experiment. In the case of a Gaussian beam, the lateral resolution is deﬁned as w0 2 NA, where NA is the numerical aper- ture of the illumination optics. To achieve high lat- eral resolution, tight focusing is used in an OCT system. However, focusing has some effects on the OCT measuring range. Complementary depth of focus, DOF 2NA2, deﬁnes a depth over which lateral resolution is considered constant. Thus higher lateral resolution is achieved with optics with high Fig. 3. (Color online) Fish scale OCT and SH-OCT images. On the left-hand side is the ﬁsh scale OCT image; on the right-hand side, the SH-OCT image is shown. The length of both images is 3 mm, and the depth of the fundamental image is 0.46 mm, and the depth of the SH image is 0.27 mm. The acquisition time for the SH-OCT image was 10 min. Fig. 3. (Color online) Fish scale OCT and SH-OCT images. On the left-hand side is the ﬁsh scale OCT image; on the right-hand side, the SH-OCT image is shown. 2. Methods h In the reference arm, a type I -barium borate (BBO) crystal 0.1 mm thick was used to gen- erate a reference second harmonic signal without spectral narrowing.17 A dichroic mirror and a delay line were used before the reference SH signal was combined with the sample SH signal in BS2. A vari- able neutral density ﬁlter was included in the refer- ence arm to vary the reference signal. In both arms of the SH interferometer, half-wave plates (HWP2) were used to control the SH polarization. The BS2 transmitted 80% of the sample signal and reﬂected 20% of the reference signal. The output from BS2 after passing a color ﬁlter that blocks fundamental radiation was coupled to a ﬁber connected to the input of a spec- trometer. The spectrometer was equipped with a 1200 linemm grating and a 2048 pixels (14 56 m pixel) thermoelectric cooled CCD camera (AvaSpec- 2048TEC). The integration time varied from 2 ms to 60 s. The resolution of the spectrometer for the 515 nm light was 2 0.15 nm. In order to decrease the mechanical vibrations and thus use longer inte- gration time, the experimental setup was built on an optical table isolated from ﬂoor motion using a pneu- matic suspension system. For our experimental setup, the measuring ranges in the air determined by the spectrometer resolution were 0.44 mm for the SH and 1.77 mm for the fun- damental radiation, respectively. However, the DOF of the sample focusing optics was about 0.27 mm with an estimated transverse resolution of about 10 m. The relative position of both working ranges in the sample was adjusted by tuning the reference arm length in the respective interferometers. 3. Results and Discussion The length of both images is 3 mm, and the depth of the fundamental image is 0.46 mm, and the depth of the SH image is 0.27 mm. The acquisition time for the SH-OCT image was 10 min. 1772 APPLIED OPTICS Vol. 46, No. 10 1 April 2007 Fig. 4. (Color online) Pig leg tendon OCT and SH-OCT images. On the left-hand side is the pig leg tendon OCT image; on the right-hand side, the SH-OCT image is shown. The length of both images is 1.5 mm, and the depth of the fundamental image is 0.47 mm, and the depth of the SH image is 0.27 mm. The acquisition time for the SH-OCT image was 5 min. Fig. 4. (Color online) Pig leg tendon OCT and SH-OCT images. On the left-hand side is the pig leg tendon OCT image; on the right-hand side, the SH-OCT image is shown. The length of both images is 1.5 mm, and the depth of the fundamental image is 0.47 mm, and the depth of the SH image is 0.27 mm. The acquisition time for the SH-OCT image was 5 min. ﬁbrils. Highly ordered sections of the collagen bun- dles in the ﬁsh scales are clearly visible. The mea- sured axial resolution of the SH image was 12 m, and the measured axial resolution of the fundamen- tal image was 17 m. signal corresponds to an averaged effect over the il- luminated area. The regional variation in collagen ﬁbril orientation is not well understood, and it will require a submicrometer resolution study for better understanding.19 In the measurements, the input beam on BS1 was with vertical polarization, and the SH generated by the BBO crystal in the reference arm was, corre- spondingly, with horizontal polarization. A half-wave plate, HWP1, in the sample arm allowed rotation of the polarization of the fundamental radiation inci- dent on the sample. SH polarization in both arms of the SH interferometer may be controlled indepen- dently by two half-wave plates, HWP2. We obtained images from the same area of the sample for both vertical and horizontal polarization of the fundamen- tal beam. For each fundamental beam polarization, two SH images were recorded: one with SH polariza- tion parallel to fundamental polarization and the other with SH polarization perpendicular to it. 3. Results and Discussion We used samples of ﬁsh scales and rabbit eye sclera; in both samples, polarization anisotropy of SH signal was observed. The SH signal with polarization par- allel to the fundamental polarization was stronger for both vertical and horizontal fundamental radiation polarization. Figure 5 depicts images acquired from the same area of the ﬁsh scale and rabbit eye sclera with output SH polarization oriented parallel and perpendicular to the fundamental polarization. The information regarding the ordered nature of the col- lagen ﬁbrils was obtained by measuring the anisot- ropy parameter11 Images of pig leg tendon are shown in Fig. 4. The experimental setup was the same as for the ﬁsh scales sample. Both SH and fundamental radiation had parallel polarizations. Figure 4 is organized sim- ilarly to Fig. 3; the left-hand side is the OCT image of pig leg tendon. The upper layer is the tendon sheath while the layer underneath has tendon ﬁber bundles, which are made up of highly aligned collagen ﬁbers that generate strong SH signals. The right-hand side is the SH-OCT image, which shows the distribution of collagen ﬁbers. As a tension-bearing element in the tendon, collagen appears in parallel, cablelike bun- dles oriented in the same direction. SHG depends on the orientation, anisotropy, and local symmetry properties of the tissue. SH efﬁciency in collagen depends on the orientation of the collagen ﬁbrils relative to the incident electrical ﬁeld polariza- tions.8 It was shown that SH intensity reaches a max- imum when the collagen ﬁbers are parallel to the beam polarization, and conversely, SH intensity is at a minimum for beam polarization perpendicular to the ﬁbers. This can be used to obtain information about the degree of ﬁbrillar alignment of collagen along a given axis. We used the polarization depen- dence of SHG to obtain information about the order- liness of ﬁbrillar structure in some tissue samples. Because the diameter of the laser beam used in the experiment was considerably larger than the individ- ual ﬁbril diameter, the detected second harmonic Ipar IperpIpar 2Iperp, Fig. 5. FD SH-OCT images of ﬁsh scales and rabbit eye sclera showing polarization anisotropy. Polarization of the fundamental and second harmonic radiation are (a) ﬁsh scales, perpendicular; (b) ﬁsh scales, parallel; (c) ﬁsh scales, overlay of both SH polarizations; (d) rabbit eye sclera, perpendicular; (e) rabbit eye sclera, parallel; (f) rabbit eye sclera, overlay of both SH polarizations. 3. Results and Discussion Fig. 5. FD SH-OCT images of ﬁsh scales and rabbit eye sclera showing polarization anisotropy. Polarization of the fundamental and second harmonic radiation are (a) ﬁsh scales, perpendicular; (b) ﬁsh scales, parallel; (c) ﬁsh scales, overlay of both SH polarizations; (d) rabbit eye sclera, perpendicular; (e) rabbit eye sclera, parallel; (f) rabbit eye sclera, overlay of both SH polarizations. 1 April 2007 Vol 46 No 10 APPLIED OPTICS 1773 Fig. 5. FD SH-OCT images of ﬁsh scales and rabbit eye sclera showing polarization anisotropy. Polarization of the fundamental and second harmonic radiation are (a) ﬁsh scales, perpendicular; (b) ﬁsh scales, parallel; (c) ﬁsh scales, overlay of both SH polarizations; (d) rabbit eye sclera, perpendicular; (e) rabbit eye sclera, parallel; (f) rabbit eye sclera, overlay of both SH polarizations. 1 April 2007 Vol. 46, No. 10 APPLIED OPTICS 1773 1773 dence on the input intensity, a decrease of the pulse duration to 40 fs would allow the same SH intensity to be generated by input radiation of about 5 mW average power. Thus femtosecond pulses with dura- tions in the 10–20 fs range (available by commercial lasers) are highly desirable for FD SH-OCT, because a substantial gain in SH efﬁciency will be achieved, and at the same time the axial resolution will be improved to the 1–2 m range. FD SH-OCT may offer several distinct advantages for imaging ordered, or partially ordered, biological tissues. First, the nonlin- ear (SHG) signal from tissue tends to be a more sen- sitive indicator of molecular structure and symmetry changes than linear behavior. Second, coherence gat- ing extends the capability of high-resolution detec- tion of SHG signals at locations deep inside the sample. Third, SHG signals are produced intrinsi- cally so imaging does not require staining the sample with dyes or ﬂuorophores. Fourth, decoupled axial and transverse scans enable two-dimensional tomo- graphic imaging of a sample with only one dimension moving of the probing beam, which is essential for in vivo endoscopic applications. where Ipar and Iperp are the intensities of the SH sig- nals parallel and perpendicular to the fundamental radiation. Using the integrated data presented in Fig. 5, we obtained a value for of 0.65 for the ﬁsh scales sample. This value indicates a highly ordered nature of the collagen ﬁbrils in ﬁsh scales. References 1. Y. Jiang, I. Tomov, Y. Wang, and Z. Chen, “Second-harmonic optical coherence tomography,” Opt. Lett. 29, 1090–1092 (2004). 2. Y. Jiang, I. Tomov, Y. Wang, and Z. Chen, “High-resolution second-harmonic optical coherence tomography of collagen in rat-tail tendon,” Appl. Phys. Lett. 86, 133901–3 (2005). 3. B. E. Applegate, C. Yang, A. M. Rollins, and J. A. Izatt, “Polarization-resolved second-harmonic-generation optical co- herence tomography in collagen,” Opt. Lett. 29, 2252–2254 (2004). When the SH generating tissue thickness is larger than the wavelength, the SH is generated mainly in a forward direction, and a detected signal in the backward direction is a result of backscattering from underlying structures. In these cases, care must be taken when interpreting the recorded images. We assume that our samples have similar collagen struc- tures as the ones studied in Refs. 12 and 20; there- fore, detected SH signals are due mainly to backward generated SH. A qualitative support for this assump- tion was that the strongest backward detected SH is not correlated to the strongest SH observed in a for- ward direction in the samples used in this study. 4. M. V. Sarunic, B. E. Applegate, and J. A. Izatt, “Spectral do- main second-harmonic optical coherence tomography,” Opt. Lett. 30, 2391–2393 (2005). 5. C. Vinegoni, J. S. Bredfeldt, D. L. Marks, and S. A. Boppart, “Nonlinear optical contrast enhancement for optical coherence tomography,” Opt. Express 12, 331–341 (2004). 6. J. Su, I. V. Tomov, Y. Jiang, and Z. Chen, “Frequency domain second harmonic optical coherence tomography,” Proc. SPIE 6079, 607901–6 (2006). 7. I. Freund and M. Deutsch, “Second-harmonic microscopy of biological tissue,” Opt. Lett. 11, 94–96 (1986). In conclusion, we demonstrated FD SH-OCT using continuum generated in an optical ﬁber to increase threefold the axial image resolution reported.4,6 The acquisition time for recording an A line was by more than 2 orders of magnitude faster compared to TD SH-OCT with the same setup.1,2 In our experiment the average input power on the sample was about 50 mW, well below the estimated damage threshold, but still high according to the American National Standards Institute guidelines. In the measure- ments, the continuum pulse duration was about 400 fs. Because SH efﬁciency had a quadratic depen- 8. B. M. Kim, J. Eichler, K. M. Reiser, A. M. Rubenchik, and L. B. 3. Results and Discussion For rabbit sclera, we measured around 0.1, which shows a more ran- dom distribution of collagen ﬁbrils. Figures 5(c) and 5(f) show the images from both samples when images of both SH polarizations overlap. These polarization- resolved images display the difference in collagen ﬁ- ber orientation relative to incident laser polarization. Polarization-resolved images will be critical in appli- cations of SHG for differentiating between normal and abnormal tissue.20 One important feature of the SHG process is that the geometry of the emitted SH radiation depends on the size and shape of the collagen ﬁbrils. Theo- retical studies13,14 have shown that SH emission is highly asymmetric due to phase-matching condi- tions. For objects with an axial size of the order of or larger than the SH wavelength, the SH emission is predominantly forward directed. However, for objects with an axial size of less than 10 (approximately 50 nm in our experiment), forward emission and backward emission are nearly equal. Recently, high- resolution SH microscopy experiments12,21 have con- ﬁrmed these theoretical predictions. The measured tendon SH forwardbackward ratio was close to 1. Be- cause the tendon ﬁbrils were in size comparable to or larger than the SH wavelength, the results were ex- plained by the ﬁbril model that assumes a thin hollow tube ﬁbril shape.12 Studies of collagen in sclera have conﬁrmed the tubelike structures of sclera collagen ﬁbrils.21 The SHG in such ﬁbrils is generated in the shell, which has a thickness of less than 40 nm. This work was supported by research grants from the National Science Foundation (BES-86924), National Institutes of Health (EB-00293, NCI-91717, RR-01192), Air Force Ofﬁce of Scientiﬁc Research (FA9550-04-1-0101), and the Beckman Laser Insti- tute Endowment. 1 April 2007 Vol. 46, No. 10 APPLIED OPTICS 1775 References Da Silva, “Collagen structure and nonlinear susceptibility: effects of heat, glycation and enzymatic cleavage on second harmonic signal intensity,” Laser Surg. Med. 27, 329–335 (2000). 9. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nature Biotechnol. 21, 1369–1377 (2003). 10. S. Yazdanfar, L. H. Laiho, and P. T. C. So, “Interferometric second harmonic generation microscopy,” Opt. Express 12, 2739–2745 (2004). 11. P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J. Malone, and W. A. Mohler, “Three-dimensional high- 1774 APPLIED OPTICS Vol. 46, No. 10 1 April 2007 B. C. Stuart, “Plasma mediated ablation of biological tissues with nanosecond-to-femtosecond laser pulses: relative role of linear and nonlinear absorption,” IEEE J. Sel. Top. Quantum Electron. 2, 801–809 (1996). resolution second harmonic generation imaging of endogenous structural proteins in biological tissues,” Biophys. J. 81, 493– 508 (2002). 12. R. M. Williams, W. R. Zipfel, and W. W. Webb, “Interpreting second harmonic generation images of collagen I ﬁbrils,” Bio- phys. J. 88, 1377–1386 (2005). 17. Y. R. Shen, The Principles of Nonlinear Optics (Wiley, 1984). 18. R. Leitgeb, C. K. Hitzenberger, and A. Fercher, “Performance of fourier domain vs. time domain optical coherence tomogra- phy,” Opt. Express 11, 889–894 (2003). 13. J. Mertz and L. Moreausx, “Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers,” Opt. Commun. 196, 325–330 (2001). 19. P. Stoller, P. M. Celliers, K. M. Reiser, and A. M. Rubenchik, “Quantitative second harmonic generation microscopy in col- lagen,” Appl. Opt. 42, 5209–5219 (2003). 14. L. Moreaux, O. Sandre, and L. Mertz, “Membrane imaging by second-harmonic generation microscopy,” J. Opt. Soc. Am. B 17, 1685–1694 (2000). 20. P. J. Campagnola, H. A. Clark, W. A. Mohler, A. Lewis, and L. M. Loew, “Second harmonic imaging microscopy for visu- alizing biomolecular arrays in cells, tissues and organisms,” Nature Biotechnol. 21, 1356–1360 (2003). 15. H. Lim, Y. Jiang, Y. Wang, Y. C. Huang, Z. Chen, and F. W. Wise, “Ultrahigh-resolution optical coherence tomography with a ﬁber laser source at 1 m,” Opt. Lett. 30, 1171–1173 (2005). 21. M. Han, G. Giese, and J. F. Bille, “Second harmonic generation imaging of collagen ﬁbrils in cornea and sclera,” Opt. Express 13, 5791–5797 (2005). 16. A. A. Oraevsky, L. B. Da Silva, A. M. Rubenchik, M. D. Feit, M. E. Glinsky, M. D. Perry, B. M. Mammini, W. Small, and
https://openalex.org/W1994609873	https://europepmc.org/articles/pmc4062065?pdf=render	English	null	Mutation signatures of carcinogen exposure: genome-wide detection and new opportunities for cancer prevention	Genome medicine	2,014	cc-by	13,696	Abstract Exposure to environmental mutagens is an important cause of human cancer, and measures to reduce mutagenic and carcinogenic exposures have been highly successful at controlling cancer. Until recently, it has been possible to connect the chemical characteristics of mutagens to actual mutations observed in human tumors only indirectly. Now, next-generation sequencing technology enables us to observe in detail the DNA-sequence-level effects of well-known mutagens, such as ultraviolet radiation and tobacco smoke, as well as endogenous mutagenic processes, such as those involving activated DNA cytidine deaminases (APOBECs). We can also observe the effects of less well-known but potent mutagens, including those recently found to be present in some herbal remedies. Crucially, we can now tease apart the superimposed effects of several mutational exposures and processes and determine which ones occurred during the development of individual tumors. Here, we review advances in detecting these mutation signatures and discuss the implications for surveillance and prevention of cancer. The number of sequenced tumors from diverse cancer types and multiple geographic regions is growing explosively, and the genomes of these tumors will bear the signatures of even more diverse mutagenic exposures. Thus, we envision development of wide-ranging compendia of mutation signatures from tumors and a concerted effort to experimentally elucidate the signatures of a large number of mutagens. This information will be used to link signatures observed in tumors to the exposures responsible for them, which will offer unprecedented opportunities for prevention. * Correspondence: steve.rozen@duke-nus.edu.sg 2Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore 3Duke-NUS Centre for Computational Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore Full list of author information is available at the end of the article © 2014 Poon et al.; licensee BioMed Central Ltd. The licensee has exclusive rights to distribute this article, in any medium, for 12 months following its publication. After this time, the article is available under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mutation signatures of carcinogen exposure: genome-wide detection and new opportunities for cancer prevention ong Ling Poon1,2, John R McPherson2,3, Patrick Tan2,4,5,6, Bin Tean Teh1,2 and Steven G Rozen2,3* Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 New opportunities for detecting mutagen exposures in human tumors Thus, while not obviating the need for mechanistic studies of the bio- chemical mechanisms of mutagenicity, cataloging mutations by next-generation sequencing provides information about a critical endpoint: the actual mutations that occur in cell lines or in human cancers in response to mutagenic exposures. The long-term promise is that the epidemiological connec- tion of specific mutagens to signatures actually observed in tumors will indicate which mutagenic exposures are true sub- stantial contributors to the burden of human cancer (Figure 1). (a) Signatures of single mutagens (c) Experimental exposure (e) (f) Sequence exome or genome of exposed cells (b) (i) Mixtures of mutation signatures Somatic mutation catalogs from many thousands of tumors NMF Match extracted signatures to experimentally determined signatures Correlate signature strength to exposures or endogenous factors Signatures due to known factors Signatures due to unknown factors (g) (d) Observation of signature in tumors with known predominant exposure Contribution of each signature to each tumor + (j) (k) (l) (m) (n) One-nucleotide signature (h) Extended signature Reporter constructs Set of extended signatures Figure 1 Linking mutation signatures to exposures or endogenous mutational processes. One can either (a) focus on signatures of one mutagen at a time or (b) study mixtures of signatures. One can study signatures of single mutagens either (c) via experimental approaches, or (d) via observation of mutation signatures in the exome or genome sequence of tumors with a known predominant mutational exposure. Some tumor exomes harbor only a handful of somatic point mutations, and presumably these tumors arise from causes other than mutagenesis. For many cancers, typical numbers of somatic point mutations in exomes are 60 to 300 [9-12]. Highly mutated cancers sometimes have >3,500 mutations per exome [13]. Typical numbers for genomes of cancers such as those of the lung or stomach are >15,000 [14,15], and a few highly mutated genomes harbor >400,000 somatic point mutations [16]. Among experimental approaches, one can use (e) reporter constructs and observe mutations in short sequences. This allows inference of relatively simple signatures, for example (g) signatures involving only single nucleotide mutations. (f) By sequencing the exome, or, ideally, the genome of a mutagen-exposed, clonal cell line, one can (h) infer a more informative, extended signature, for example one that includes the trinucleotide contexts of single-nucleotide mutations. One can also infer extended signatures by sequencing the exomes or genomes of tumors with known, predominant exposures (d). New opportunities for detecting mutagen exposures in human tumors assays for mutagenicity became proxies for tests of carcino- genicity, with the Ames test, performed in a bacterial system, as a well-known example [7]. However, tests of mutagenicity in artificial systems do not fully connect mutagenic expo- sures to the patterns of mutation observed in cancers. Mutagenic environmental exposures are important causes of human cancer. This was first understood from Percival Pott's 18th century epidemiological observation of scrotal cancer in chimney sweeps [1]. Causality was eventually confirmed experimentally by using coal tar to induce can- cer in rabbits [2]. Soon thereafter, polycyclic aromatic hy- drocarbons were identified as carcinogens in coal tar [3]. Much later, once the role of DNA as an information mol- ecule was understood, the biochemical mechanisms for polycyclic aromatic hydrocarbon mutagenesis were eluci- dated [4]. This led to a broader appreciation of the roles of DNA damaging agents in mutagenesis and to extensive study of numerous other mutagens [5,6]. Subsequently, More recently, it has become clear that specific mutagens produce characteristic patterns of somatic mutations in the DNA of malignant cells. We describe these patterns, called 'mutation signatures', in detail below. Briefly, mutation signatures usually include the relative frequencies of the various nucleotide mutations (such as A > C, A > G, A > T, C > A) plus, ideally, their trinucleotide contexts, that is, the identities of the bases on both sides of the mutated nucleo- tides. Previously, our knowledge of these signatures was based on short lengths (such as a few kilobases) of DNA sequence. With the advent of next-generation sequencing, it is now possible to infer these signatures from the se- quences of all the exons in the genome ('whole exome') or from the sequence of the entire genome ('whole genome'). Page 2 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Characterization of mutagenicity based directly on observed mutations across whole exomes or genomes offers several advantages over previous approaches, including that many more mutations can be detected, which provides far greater statistical power and allows the parsing of the superimposed mutation signatures stemming from several exposures. Ac- tual mutation signatures are the end result of a series of bio- chemical and biological processes, including the metabolism of pro-mutagens to active forms, biochemical damage to DNA, the efforts of the cell to repair the damage, and, rarely, selection for or against the resulting mutations. New opportunities for detecting mutagen exposures in human tumors One can also infer extended signatures by sequencing the exomes or genomes of tumors with known, predominant exposures (d). Extraction of mutation signatures from mixtures of signatures (b) requires somatic mutation catalogs from the exomes or genomes of large numbers of tumors. The most recent studies have looked at thousands of catalogs. (i) Procedures based on NMF (non-negative matrix factorization) allow (j) simultaneous inference of a set of extended mutation signatures and the contributions of each inferred signature to each tumor's mutations. (k) Extended signatures derived from mixtures of signatures (j) can be matched to extended signatures that were experimentally determined or inferred from known predominant exposures (h), thereby providing information on (l) exposures that contributed to tumors with mixtures of mutation signatures. (m) Alternatively, extended signatures extracted from mixtures can be correlated with information on mutagenic exposures or on endogenous mutagenic factors, allowing inference of the causes of mutation signatures (l). (n) The causes of some signatures will remain unknown and require further research. g mutation signatures to exposures or endogenous mutational processes. One can either (a) focus on signatures of on Figure 1 Linking mutation signatures to exposures or endogenous mutational processes. One can either (a) focus on signatures of one mutagen at a time or (b) study mixtures of signatures. One can study signatures of single mutagens either (c) via experimental approaches, or (d) via observation of mutation signatures in the exome or genome sequence of tumors with a known predominant mutational exposure. Some tumor exomes harbor only a handful of somatic point mutations, and presumably these tumors arise from causes other than mutagenesis. For many cancers, typical numbers of somatic point mutations in exomes are 60 to 300 [9-12]. Highly mutated cancers sometimes have >3,500 mutations per exome [13]. Typical numbers for genomes of cancers such as those of the lung or stomach are >15,000 [14,15], and a few highly mutated genomes harbor >400,000 somatic point mutations [16]. Among experimental approaches, one can use (e) reporter constructs and observe mutations in short sequences. This allows inference of relatively simple signatures, for example (g) signatures involving only single nucleotide mutations. (f) By sequencing the exome, or, ideally, the genome of a mutagen-exposed, clonal cell line, one can (h) infer a more informative, extended signature, for example one that includes the trinucleotide contexts of single-nucleotide mutations. New opportunities for detecting mutagen exposures in human tumors Extraction of mutation signatures from mixtures of signatures (b) requires somatic mutation catalogs from the exomes or genomes of large numbers of tumors. The most recent studies have looked at thousands of catalogs. (i) Procedures based on NMF (non-negative matrix factorization) allow (j) simultaneous inference of a set of extended mutation signatures and the contributions of each inferred signature to each tumor's mutations. (k) Extended signatures derived from mixtures of signatures (j) can be matched to extended signatures that were experimentally determined or inferred from known predominant exposures (h), thereby providing information on (l) exposures that contributed to tumors with mixtures of mutation signatures. (m) Alternatively, extended signatures extracted from mixtures can be correlated with information on mutagenic exposures or on endogenous mutagenic factors, allowing inference of the causes of mutation signatures (l). (n) The causes of some signatures will remain unknown and require further research. Signatures of single mutagens (e) (i) (j) (g) Extended signature Correlate signature strength to exposures or endogenous factors (k) Match extracted signatures to experimentally determined signatures (l) Signatures due to unknown factors Signatures due to known factors Figure 1 Linking mutation signatures to exposures or endogenous mutational processes. One can either (a) focus on signatures of one mutagen at a time or (b) study mixtures of signatures. One can study signatures of single mutagens either (c) via experimental approaches, or (d) via observation of mutation signatures in the exome or genome sequence of tumors with a known predominant mutational exposure. Some tumor exomes harbor only a handful of somatic point mutations, and presumably these tumors arise from causes other than mutagenesis. For many cancers, typical numbers of somatic point mutations in exomes are 60 to 300 [9-12]. Highly mutated cancers sometimes have >3,500 mutations per exome [13]. Typical numbers for genomes of cancers such as those of the lung or stomach are >15,000 [14,15], and a few highly mutated genomes harbor >400,000 somatic point mutations [16]. Among experimental approaches, one can use (e) reporter constructs and observe mutations in short sequences. This allows inference of relatively simple signatures, for example (g) signatures involving only single nucleotide mutations. (f) By sequencing the exome, or, ideally, the genome of a mutagen-exposed, clonal cell line, one can (h) infer a more informative, extended signature, for example one that includes the trinucleotide contexts of single-nucleotide mutations. Signatures of single mutagens To date, the signatures of carcinogenic mutagens have been established either in vitro or in human cancers that are primarily caused by one exposure (Table 1). We elaborate first on the mutation signature of aristolochic acid (AA), which has been established both in vitro and in human cancers [16-19]. AA is a powerful mutagen that is found in some herbal remedies and that causes upper urinary-tract urothelial cancer (UTUC) [16-19]. It also probably contributes to liver cancer (hepatocellular carcinoma, HCC) [16]. Thus, in addition to providing an example of the signature of a single mutagen, AA also il- lustrates the use of signatures to detect likely carcino- genic exposures that were previously unsuspected. We describe below the state of the art for determining mutation signatures by next-generation sequencing, the implications of this approach for detecting the carcino- genic impacts of mutagenic exposures, and its promise for prevention. We start by describing signatures of sin- gle mutagens. New opportunities for detecting mutagen exposures in human tumors One can also infer extended signatures by sequencing the exomes or genomes of tumors with known, predominant exposures (d). Extraction of mutation signatures from mixtures of signatures (b) requires somatic mutation catalogs from the exomes or genomes of large numbers of tumors. The most recent studies have looked at thousands of catalogs. (i) Procedures based on NMF (non-negative matrix factorization) allow (j) simultaneous inference of a set of extended mutation signatures and the contributions of each inferred signature to each tumor's mutations. (k) Extended signatures derived from mixtures of signatures (j) can be matched to extended signatures that were experimentally determined or inferred from known predominant exposures (h), thereby providing information on (l) exposures that contributed to tumors with mixtures of mutation signatures. (m) Alternatively, extended signatures extracted from mixtures can be correlated with information on mutagenic exposures or on endogenous mutagenic factors, allowing inference of the causes of mutation signatures (l). (n) The causes of some signatures will remain unknown and require further research. Page 3 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 apart superimposed signatures from multiple mutagenic processes, and conclude with a vision of how this could improve prevention. The observation of known signatures in tumors might also implicate previously unsuspected exposures in par- ticular cancers. This information on causal exposures then could provide foci for prevention efforts. Neverthe- less, despite the low cost and ubiquity of next-generation sequencing, detailed mutation signatures of only a few known carcinogens have been elucidated experimentally so far. Indeed, in a recent groundbreaking survey of mu- tation signatures across many types of human cancers, most mutation signatures are ascribed to particular expo- sures by statistical association rather than recapitulation of the signatures in experimental systems [8]. Mutation signature of aristolochic acid Molecule schematics in (b-e) reproduced DNA n ion Ultraviolet light (h) Cyclobutane pyrimidine dimers (i) Deamination 5′ 3′ Py C (j) Error free TLS 5′ 3′ Py U 5′ 3′ Py U 5′ 3′ C C 5′ 3′ U U 5′ 3′ U U 5′ Py A 5′ A A (k) Replication 5′ 3′ Py T 3′ 5′ Py A 5′ 3′ A A 3′ 5′ T T C>T mutation CC>TT mutation (g) Pyrimidine N + (e) Adenosine adduct in DNA (a) Aristolochia plant (b) Aristolochic acid (c) Metabolic activation (d) Aristolactam nitrenium ion (f) A>T mutation N + (e) Adenosine adduct in DNA (a) Aristolochia plant (b) Aristolochic acid (c) Metabolic activation (d) Aristolactam nitrenium ion Ultraviolet light (h) Cyclobutane pyrimidine dimers (i) Deamination 5′ 3′ Py C (j) Error free TLS 5′ 3′ Py U 5′ 3′ Py U 5′ 3′ C C 5′ 3′ U U 5′ 3′ U U 5′ Py A 5′ A A (k) Replication 5′ 3′ Py T 3′ 5′ Py A 5′ 3′ A A 3′ 5′ T T C>T mutation CC>TT mutation (g) Pyrimidine Ultraviolet light (h) (g) Pyrimidine (g) Pyrimidine Ultraviolet light (h) Cyclobutane pyrimidine dimers (h) Cyclobutane pyrimidine dimers (c) Metabolic activation (d) Aristolactam nitrenium ion N + (e) Adenosine adduct in DNA ) Replication (e) Adenosine adduct in DNA (f) A>T mutation Figure 2 Mechanisms of mutagenesis of aristolochic acid and UV light. Preparations of (a) plants from the genus Aristolochia contain (b) aristolochic acid. Aristolochic acid I is shown; in aristolochic acid II, OCH3 is replaced by H. Aristolochic acid is (c) metabolically activated to (d) aristolactam nitrenium ions by one or more of several enzymes, including NQO1 (NAD(P)H dehydrogenase, quinone 1), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), and NADPH-hemoprotein reductase [45]. (e) The aristolactam nitrenium ions form covalent adducts with adenosine bases, and (f) these adducts lead to A > T mutations. (g) Pyrimidines exposed to ultraviolet (UV) radiation form (h) cyclobutane pyrimidine dimers (CPD). (i) Either the cytosine (C) (left) or the CC dipyrimidines in CPD (right) undergo deamination, resulting in uracil (U). Py denotes pyrimidine. (j) Error-free trans-lesion DNA synthesis (TLS) induces C > T and CC > TT mutations at the sites of U-containing CPDs through DNA replication of the U-containing DNA strand (k). Photograph of Aristolochia plant (a) by ST Pang. Signatures of single mutagens We then describe approaches for teasing Table 1 Examples of exogenous mutagens and endogenous mutagenic processes Mutagen Dominant mutations* Extended context* Studies reporting mutation signatures Prevalence Challenges Exogenous Aristolochic acid A > T (C\|T)AG > (C\|T)TG [16-19] Widely used in traditional medicines [20]; exposure to AA is widespread in Taiwan [17] No unusual challenges UV radiation C > T;strand bias; CC > TT TC > TT (C\|T)C > (C\|T)T [6,8,9,21-23] Prevalence of signature: 87% of melanoma [8] No unusual challenges Tobacco smoke Primarily C > A, some C > G and C > T CG > AGCG > TG;CG > GG [8,14,24-26] Extensive epidemiological evidence of the role of tobacco smoke in cancer [27,28] Contains multiple carcinogens with individually unknown signatures Aflatoxin B1 Primary G > T; some G > A NA [29-34] [29,35] Signature in extended context not known Temozolomide C > T CC > TC; CT > TT [8,36,37] Present in 10% of glioblastomas; 9% of melanoma [8] No unusual challenges Benzene C > T;C > A NA [38] Exposure associated with risk of leukemia [39,40] Several mutagenic metabolites and signature in extended context not known Endogenous Activated APOBEC C > T TCA > TTA [8,41,42] Present in 16 tumor types [8] Signatures 2 and 13 are similar [8], except 2 has C > T and 13 has higher C > G Mutated DNA polymerase epsilon C > T TCG > TTG; TCT > TAT [8,13] Present in 13.7% of uterus cancer and 36.7% of colorectal cancer [8] No unusual challenges Mismatch repair deficiency (MSI) C > T;C > A CG > TG; CT > AT; homopolymer and microsatellite length changes [8] Present in 9 tumor types [8] No unusual challenges Correlated with patient age C > T CG > TG [8] A majority of tumors of most types have this signature [8] Interpretation of two similar signatures in [8] not clear *The ' > ' symbol indicates a change from one nucleotide to another; a vertical line indicates alternative nucleotides. NA, not applicable. Table 1 Examples of exogenous mutagens and endogenous mutagenic processes Page 4 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Mutation signature of aristolochic acid species in the genus are known to be used medicinally, and although the AA content of most species is unknown, 23 of the medicinally used species contain AA [46]. AA is metabolized to aristolactam nitrenium ions, which form covalent adducts with adenosines in DNA (Figure 2b-e) [45,47]. These adducts then lead to A > T mutations (mutations from adenine to thymine; Figure 2f). These were initially observed as somatic mutations in the AA is a natural compound found in plants in the genus Aristolochia (Figure 2a). These plants are used in trad- itional herbal remedies for weight loss and a plethora of health problems, including menstrual symptoms, snakebites, rheumatism, arthritis, and gout [43,44]. Although challenging to document, the use of these plants probably remains widespread [20,45]. Indeed, 99 ut [43,44]. se of these Indeed, 99 [45,47]. These adducts then lead to A > T mutations (mutations from adenine to thymine; Figure 2f). These were initially observed as somatic mutations in the Ultraviolet light (h) Cyclobutane pyrimidine dimers (i) Deamination 5′ 3′ Py C (j) Error free TLS 5′ 3′ Py U 5′ 3′ Py U 5′ 3′ C C 5′ 3′ U U 5′ 3′ U U 5′ Py A 5′ A A (k) Replication 5′ 3′ Py T 3′ 5′ Py A 5′ 3′ A A 3′ 5′ T T C>T mutation CC>TT mutation (g) Pyrimidine acid and UV light. Preparations of (a) plants from the genus Aristolochia contain (b) hic acid II, OCH3 is replaced by H. Aristolochic acid is (c) metabolically activated to (d) nzymes, including NQO1 (NAD(P)H dehydrogenase, quinone 1), CYP1A2 (cytochrome hemoprotein reductase [45]. (e) The aristolactam nitrenium ions form covalent adducts T mutations. (g) Pyrimidines exposed to ultraviolet (UV) radiation form (h) cyclobutane or the CC dipyrimidines in CPD (right) undergo deamination, resulting in uracil (U). Py esis (TLS) induces C > T and CC > TT mutations at the sites of U-containing CPDs through otograph of Aristolochia plant (a) by ST Pang. An AA-like signature in liver cancer Unexpectedly, recent examination of mutation signa- tures in hepatitis B virus-exposed human HCCs revealed some with obvious AA-like signatures (Figure 3g,h) [16], although this cancer type apparently was not previously linked to AA exposure [48]. The signature shows a large proportion of A > T somatic mutations with strand bias (as seen in AA-exposed UTUCs; Figure 3e,g) and a tri- nucleotide context that strongly resembles that in AA- associated UTUC (compare Figure 3c and Figure 3h). It is possible that exposure to AA in conjunction with hepatitis B virus infection may contribute synergistically to HCC formation, much as hepatitis and aflatoxin do (see below). As AA had not been previously implicated as a risk factor for HCC, this finding may represent a new paradigm, in which environmental exposures contrib- uting to specific cancers are deduced from observations of mutation signatures. It is likely that Aristolochia-containing herbal remedies are the source of AA exposure in these cancers. If so, appropriate measures to minimize exposure should be taken - for example, through education and more aggressive enforcement of bans on Aristolochia- containing remedies. Recently, high-throughput next-generation sequencing has provided the means to catalog and analyze somatic mutations far more completely, whether by whole- exome or whole-genome sequencing. Recent work has shown a remarkable preponderance of A > T mutations in AA-associated UTUCs from Taiwan (Figure 3a) [16-19]. For comparison, in gastric cancer or other non- AA-associated cancers, A > T somatic mutations are rare (Figure 3b) [8,14,24]. By way of technical explanation, if we consider a single DNA strand as a point of reference, there are 12 possible single-nucleotide mutations: four nucleotides times three possible mutations for each nucleotide. In some parts of the genome, it makes sense to use a particular strand as the reference sequence. In particular, in regions of the genome that are transcribed, we can use the transcribed strand, that is, the strand that serves as a template for the RNA polymerase, as the point of reference. However, in the non-transcribed regions, neither strand in particu- lar is the obvious choice for the reference sequence. Therefore, the usual practice in the study of mutation signatures has been to not distinguish complementary mutations, but rather to group them together. An AA-like signature in liver cancer For ex- ample, A > C mutations are grouped with the comple- mentary T > G mutations, A > G mutations are grouped with T > C mutations, and so on. Mutation signature of aristolochic acid Molecule schematics in (b-e) reproduced from [14], with permission from Oxford University Press. Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Page 5 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 tumor suppressor gene TP53 in UTUCs in the UK, Taiwan, and the Balkans [17,47]. This was highly unusual, as A > T mutations are rare in other types of human cancer, including UTUCs unrelated to AA exposure [18]. Furthermore, the AA-associated mutations in TP53 tended to occur in the context of CAG > CTG (C followed by the mutated A followed by G, in 5' to 3' order) [19]. In addition, the A > T mutations in AA-associated UTUCs are less common on the transcribed strands of genes than on the non-transcribed strands (Figure 3e). This strand bias suggests that AA adducts occurring on the transcribed strand were often corrected by transcription-coupled nucleotide excision repair. Similar strand bias is not seen for the relatively infrequent A > T mutations seen in other cancers, such as gastric cancer (Figure 3f). However, analysis of approximately 1 kb of sequence in a single gene (TP53) [19] offers limited statistical power to determine the sequence contexts in which the A > T mutations occur. In addition, the approach of assessing physical mutation signatures in TP53, a key tumor suppressor gene, runs the risk of bias caused by conflation of physical mutation signatures with the ef- fects of intense selection during tumor evolution. Ultraviolet radiation l l ( ) d Ultraviolet (UV) radiation induces several kinds of muta- tions, primarily C > T (Figure 2g-k, Table 1) [6,9]. It also induces double mutations CC > TT, in which adjacent cytosines mutate to thymines as a result of cytosine di- mers generated by UV light. Earlier studies indicated that UV-induced C > T mutations often occur after a pyrimidine (C or T) [9,21,22]. Analysis of mutation cata- logs from melanomas indicates that the trinucleotide context is often TCC [8]. As with AA-induced A > T mutations, there is strand bias: UV-induced mutations are less likely to occur on the transcribed strand [8]. With the availability of catalogs of somatic mutations from sequencing data, it has become possible to investigate the nucleotides that neighbor AA-induced A > T mutations. The trinucleotide sequence contexts of AA-associated mu- tations show a dramatic overrepresentation of cytosines and thymines immediately 5′ of mutated adenines (that is, [C\|T]A; mutated adenine in bold) and overrepresentation of guanines 3′ of mutated adenines (that is, AG) (Figure 3c) [16-19]. This preference of A > T mutations for the (C\|T)AG context has not been observed in non- AA-associated cancers (such as gastric cancer; Figure 3d), suggesting that this sequence context is a particular characteristic of AA mutagenesis. Tobacco smoke b k Tobacco smoking causes the vast majority of lung can- cers and contributes strongly to many other cancers, in- cluding liver, colorectal, breast, prostate, and bladder cancers [49]. Tobacco smoke contains many mutagenic carcinogens, including polycyclic aromatic hydrocarbons and N-nitrosamines [25,50,51]. The mutation signature Poon et al. Tobacco smoke b k Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 (See figure on previous page.) l h d ( g p p g ) Figure 3 Aristolochic acid signatures in upper urinary-tract urothelial cancer and hepatocellular carcinoma. (a,b) Mean counts of each of six different somatic single-nucleotide mutations in exome data from (a) AA-associated UTUCs (AA-UTUC, n = 9) and (b) gastric cancers (n = 15). (c,d) Trinucleotide contexts for somatic mutations in (c) AA-UTUCs (n = 9) and (d) gastric cancers (n = 15). The height of each bar (the y axis) represents the proportion of all observed mutations that fall into a particular trinucleotide mutational class, for example CAG > CTG and TAG > CTG (indicated). Along the x axis the mutations are organized first by the nucleotide mutation itself: C > T (blue bars), C > G (black bars), C > A (red bars), A > T (gray bars), A > G (green bars), A > C (pink bars). For each single-nucleotide mutation (such as A > T) there are 16 possible trinucle- otide contexts (AAA > ATA, AAC > ATC, and so on) The heights of the bars indicate the observed proportions of mutations aggregated over all exomes studied. (e,f) Mean counts of somatic single-nucleotide mutations in (e) AA-associated UTUCs (n = 9) and (f) gastric cancer (n = 15), shown separately for non-transcribed (N) and transcribed (T) strands. The lower mutation counts on the transcribed strand suggest transcription- coupled repair (see main text). (f) Analogous data for gastric cancer do not show strand bias (n = 15). (g) Probable AA-exposed HCCs show a preponderance of A > T mutations with strand bias similar to that observed in AA-associated UTUCs (n = 11). (h) Trinucleotide context for mutations in probable AA-exposed HCC is highly similar to that for AA-associated UTUCs (c). Plotted using data from [16]. exposure and hepatitis infection is far greater than each individual risk [29,35]. of tobacco smoke was studied primarily in the context of the TP53 gene, in which exposure to tobacco-smoke mutagens often results in G > T mutations [25]. Only a few studies extended the mutation signature to a trinu- cleotide context, and the preference for particular nucle- otides 5' or 3' of the mutated nucleotides is weak (Table 1) [8,24], possibly reflecting the complex mix of mutagens present in tobacco smoke. Aflatoxin B1 fl Aflatoxins are byproducts of mold growing on food [52], and among the aflatoxins, aflatoxin B1 (AFB1) is thought to be the most carcinogenic and is the most studied [53]. The International Agency for Research on Cancer (IARC) classifies AFB1 as a Group I carcinogen (an agent that is definitely carcinogenic to humans) [54]. AFB1 is metabolized to an epoxide compound that can form a covalent bond with the N7 atom of guanine, thereby leading to G > T mutations (Table 1) [35]. In addition, AFB1 can induce 8-hydroxy-2'-deoxyguanosine, which also produces predominantly G > T mutations in in vitro experimental models [52]. The mutation signature of AFB1 has been primarily studied in the TP53 gene, and indeed particular somatic mutations in TP53 are used as biomarkers for aflatoxin exposure in tumors [55,56]. How- ever, the extended mutation signature of AFB1 has not been studied (Table 1). Exposure to AFB1 is through food, but unfortunately, its contamination in food is difficult to detect. Consequently, convincing evidence that AFB1 is carcinogenic relied on studies showing that people with AFB1-derived adducts were more likely to develop cancer [29,35]. The predominant cancer associated with AFB1 is HCC, and the risk associated with combined AFB1 Tobacco smoke b k There are chal- lenges in dissecting the tobacco-smoke mutation signa- ture, because the signatures from different constituent mutagens are likely to differ, and their effects on differ- ent organs and tissues are also likely to differ [51]. Thus, it would be highly informative to examine experimen- tally the signatures of individual mutagenic components of tobacco smoke in the genomes of exposed cell lines from different tissues (Figure 1f). Temozolomide Temozolomide is an alkylating agent commonly used for chemotherapeutic treatment of melanoma and cen- tral nervous system tumors [57,58]. Temozolomide is quickly absorbed and undergoes spontaneous break- down to form an active compound (methyltriazen-1-yl imidazole-4-carboxamide), which forms several DNA adducts: N7-methylguanine (70%), N3-methyladenine (9%), and O6-methylguanine (5%) [59]. Both the N7- methylguanine and N3-methyladenine lesions are rapidly repaired by base excision repair [60]. However, the O6-methylguanine adducts sometimes are not repaired, leading to point mutations [61,62]. Although the mecha- nisms of temozolomide genotoxicity have been intensively studied in a therapeutic context, to our knowledge, the mutation signature of temozolomide has not been studied in experimental systems. However, Alexandrov et al. [8] detected a clear association between a CC > TC signature and temozolomide treatment in glioblastoma and mela- noma patients (Table 1). Tobacco smoke b k Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Page 6 of 14 (c) (d) (e) (f) (g) (h) AA-UTUC exomes Gastric cancer exomes Exomes from probable AA-exposed HCC tumors CAG TAG (a) (b) AA-UTUC exomes Gastric cancer exomes AA-UTUC exomes by strand Gastric cancer exomes by strand Exomes from probable AA- exposed HCC tumors, by strand AAG TCG GCG CCG CG CAG TAG Somatic mutation count Somatic mutation count Somatic mutation count Somatic mutation count Somatic mutation count Proportion of observed mutations Proportion of observed mutations Proportion of observed mutations Preceding nt Following nt 0.00 0.05 0.10 0.15 C>T C>G C>A A>T A>G A>C C>T C>G C>A A>T A>G A>C C>T C>G C>A A>T A>G A>C 700 600 500 400 300 200 100 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C 800 600 400 200 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 12 10 8 6 4 2 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C 60 50 40 30 20 10 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C 25 15 20 10 5 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 0.00 0.05 0.15 0.20 0.10 0.00 0.02 0.04 0.06 A re 3 (See legend on next page.) (c) (d) (e) (f) (g) AA-UTUC exomes Gastric cancer exomes CAG TAG (a) (b) AA-UTUC exomes Gastric cancer exomes AA-UTUC exomes by strand Gastric cancer exomes by strand Exomes from probable AA- exposed HCC tumors by strand AAG TCG GCG CCG CG Somatic mutation count Somatic mutation count Proportion of observed mutations Proportion of observed mutations Preceding nt Following nt C>T C>G C>A A>T A>G A>C C>T C>G C>A A>T A>G A>C 800 600 400 200 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 25 15 20 10 5 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 0.00 0.05 0.15 0.20 0.10 0.00 0.02 0.04 0.06 A (a) AA-UTUC exomes Somatic mutation count 800 600 400 200 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) (a) (b) AA-UTUC exomes Gastric cancer exomes Somatic mutation count Somatic mutation count 800 600 400 200 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 25 15 20 10 5 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) (c) AA-UTUC exomes CAG TAG (a) (b) AA-UTUC exomes Gastric cancer exomes Somatic mutation count Somatic mutation count Proportion of observed mutations Preceding nt Following nt C>T C>G C>A A>T A>G A>C 800 600 400 200 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 25 15 20 10 5 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) 0.00 0.05 0.15 0.20 0.10 (b) Gastric cancer exomes Somatic mutation count 25 15 20 10 5 0 C>T C>G C>A A>T A>G A>C (G>A) (G>C) (G>T) (T>A) (T>C) (T>G) (c) AA-UTUC exomes CAG TAG Proportion of observed mutations Preceding nt Following nt C>T C>G C>A A>T A>G A>C 0.00 0.05 0.15 0.20 0.10 (d) Gastric cancer exomes AAG TCG GCG CCG CG Proportion of observed mutations C>T C>G C>A A>T A>G A>C 0.00 0.02 0.04 0.06 A (e) (f) (g) AA-UTUC exomes by strand Gastric cancer exomes by strand Exomes from probable AA- exposed HCC tumors, by strand Somatic mutation count Somatic mutation count Somatic mutation count 700 600 500 400 300 200 100 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C 12 10 8 6 4 2 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C 60 50 40 30 20 10 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C (e) AA-UTUC exomes by strand Somatic mutation count 700 600 500 400 300 200 100 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C (f) Gastric cancer exomes by strand Somatic mutation count 12 10 8 6 4 2 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C (g) Exomes from probable AA- exposed HCC tumors, by strand Somatic mutation count 60 50 40 30 20 10 0 N T C>T N T C>G N T C>A N T A>T N T A>G N T A>C (e) (f) (g) Somatic mutation count (h) Exomes from probable AA-exposed HCC tumors CAG TAG Proportion of observed mutations 0.00 0.05 0.10 0.15 C>T C>G C>A A>T A>G A>C Figure 3 (See legend on next page.) (h) Exomes from probable AA-exposed HCC tumors Page 7 of 14 Poon et al. Endogenous mutagenic processes There are also endogenous mutagenic processes, which are sometimes unleashed during cancer development. For example, the APOBEC genes encode DNA cytidine deaminases that, when upregulated, promote C > G and (c) (a) (b) (d) Age 1B Tobacco smoke “Signature 5” TCA TCA TCT TCT ACG CCG GCG TCG CCC TCC CCT CCA CCC (e) Tumors with somatic mutations per Mb < 15 and > 7.5 Somatic mutations prevalence (Number mutations per megabase) C>A APOBEC C>G C>T T>A T>C T>G 0% 5% 10% 15% 20% 25% 0% 5% 10% 0% 5% 10% 0% 15 12 9 6 3 0 5% Age 1B APOBEC Tobacco smoke Signature 5 Signature R2 Figure 4 Mutation signatures in lung adenocarcinoma. (a-d) Four signatures that are prominent in lung adenocarcinoma [8]. The x axes are organized as in Figure 3c. However, unlike in Figure 3c, the y axes in these plots represent proportions of mutations in inferred rather than observed signatures. (a) Signature of APOBEC-induced mutagenesis. (b) 'Age 1B', one of two signatures that correlated with age. (c) Tobacco smoke. (d) 'Signature 5' from [8], due to an unknown exposure or mutational process. (e) Almost all lung adenocarcinomas have mutations that are overlays of several of the signatures above. Signature R2 is an additional signature that may partly represent sequencing errors [8]. All panels adapted from [8] with permission from Macmillan Publishers Ltd. (a) (b) (c) (e) Tumors with somatic mutations per Mb < 15 and > 7.5 Figure 4 Mutation signatures in lung adenocarcinoma. (a-d) Four signatures that are prominent in lung adenocarcinoma [8]. The x axes are organized as in Figure 3c. However, unlike in Figure 3c, the y axes in these plots represent proportions of mutations in inferred rather than observed signatures. (a) Signature of APOBEC-induced mutagenesis. (b) 'Age 1B', one of two signatures that correlated with age. (c) Tobacco smoke. (d) 'Signature 5' from [8], due to an unknown exposure or mutational process. (e) Almost all lung adenocarcinomas have mutations that are overlays of several of the signatures above. Signature R2 is an additional signature that may partly represent sequencing errors [8]. All panels adapted from [8] with permission from Macmillan Publishers Ltd. Figure 4 Mutation signatures in lung adenocarcinoma. (a-d) Four signatures that are prominent in lung adenocarcinoma [8]. The x axes are organized as in Figure 3c. Benzene O Occupational exposure to benzene is of particular con- cern, as it is widely used in a variety of industries, in- cluding manufacture of petrochemicals and other chemicals, as well as in manufacture of shoes, lubricants, dyes, detergents, drugs, and pesticides [63]. Non- occupational exposures occur from automobile exhaust and gasoline fumes, industrial emissions, and especially cigarette smoking and second hand smoke [63]. Benzene is classified as a Group 1 carcinogen by IARC [64]. It is benzene's metabolites, such as phenol, hydroquinone, and related hydroxyl metabolites, that have been linked to leukemia in experimental models in vitro and in vivo [65,66]. Benzene metabolites can exert their genotoxic effect through the formation of DNA adducts, oxidative stress, damage to the mitotic apparatus, and inhibition of topoisomerase II function [65]. Although the Page 8 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 C > T mutations especially in the TC(A\|T) context (mutated base in bold; Figure 4a, Table 1) [41,42,67]. Endogenous mutagenic processes arising in cancer devel- opment can also consist of inactivation of DNA repair or proofreading mechanisms. A well-known example is microsatellite instability, caused by defects in the DNA mismatch repair mechanism [68]. As another example, it was recently shown that, in some cancers, inactivation of the proofreading domain of DNA polymerase delta 1 or epsilon (POLD1 or POLE) leads to very high mutation rates [13]. POLE mutations were associated with very high rates of TCT > TAT and TCG > TTG mutations [8] (Table 1). genotoxic mechanisms of benzene have been studied, its mutation signature is poorly understood. Thus far, re- search using a reporter gene has found a preponderance of C > T and C > A mutations [38] (Table 1). However, there has been no genome-wide analysis of benzene's mutation signature in cell line models or in benzene- associated leukemias. Endogenous mutagenic processes However, unlike in Figure 3c, the y axes in these plots represent proportions of mutations in inferred rather than observed signatures. (a) Signature of APOBEC-induced mutagenesis. (b) 'Age 1B', one of two signatures that correlated with age. (c) Tobacco smoke. (d) 'Signature 5' from [8], due to an unknown exposure or mutational process. (e) Almost all lung adenocarcinomas have mutations that are overlays of several of the signatures above. Signature R2 is an additional signature that may partly represent sequencing errors [8]. All panels adapted from [8] with permission from Macmillan Publishers Ltd. Page 9 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Aging by itself is a major risk factor for cancer devel- opment, and the majority of tumors are diagnosed in older patients [69-71]. DNA damage and mutations ac- cumulate with age [72]. Interestingly, there are different age-related mutation patterns in different tissues due to differences in functional characteristics such as mitotic rate, transcriptional activity, metabolism, and specific DNA repair mechanisms [73]. Two distinct yet similar age-related mutation signatures have been detected in cancers (Table 1), and at least one of the two is present in the overwhelming majority of tumors [8]. negative, because one cannot have a negative contribution of a mutation signature to a tumor, and because a muta- tion signature cannot have a negative proportion of muta- tions of a given class; this is the origin of the term non-negative matrix factorization. We emphasize that NMF simultaneously detects the signatures present in the somatic mutation catalogs of multiple tumors and deter- mines the contribution of each signature to the somatic mutations in each tumor. There are, of course, numerous fine points, salient among which is the question of how to find the right number, N, of signatures. This depends on the num- ber of mutation catalogs (and the number of muta- tions) available for analysis, as well as on the actual diversity of mutational processes represented in the sampled tumors. A large international effort recently generated somatic mutation catalogs from 7,042 tu- mors encompassing 30 cancer types, and these cata- logs allowed discernment of 21 mutation signatures [8]. Mixtures of signatures In most tumors, somatic mutation catalogs comprise the superimposed results of several mutational exposures and processes. For example, lung adenocarcinomas usually show the signature of tobacco smoke [8,14,24] (Figure 4c). In addition, these tumors often simultaneously show mu- tation signatures due to exposure to endogenous activated DNA cytidine deaminases (APOBECs; Figure 4a), signa- tures of mutations that accumulate with age (Figure 4b), and other signatures of unknown origin [8] (Figure 4d). Given that the catalog of somatic mutations in a tumor often represents an overlay of several mutational processes, a key challenge is to dissect out and assess the contribution of each process. Building on initial work [74], recent strides have been made in computa- tional techniques for meeting this challenge. Specifically, it is now possible to simultaneously discover the existence of multiple signatures and assess the relative contribution of each signature to each tumor's catalog of somatic mu- tations [8,75,76]. Figure 5 explains the process of com- bining three mutation signatures to reconstruct a close approximation to the observed somatic mutation cata- log of a tumor. Based on association with clinically documented expo- sures or correspondence to previously known mutational profiles, the origins of 11 of the 21 signatures in [8] were identifiable. Three signatures were attributed to exogen- ous exposures: tobacco smoke, UV radiation, and temozo- lomide. Other signatures were attributed to endogenous processes, including activation of APOBEC genes, mis- match repair deficiency, mutations in the POLE gene, and mutations in the BRCA1 or BRCA2 breast cancer genes. Finally, there were two signatures for which the level of the contribution to mutations in tumors was strongly correlated with the patient's age. Discovering the signatures relies on a computational analysis called non-negative matrix factorization (NMF). The input to NMF consists of the observed catalogs of somatic mutations from tens [75] to several thousands [8] of tumors. For each of the observed catalogs (one for each tumor), NMF sets up an equation such as the one shown in Figure 5. Then, for a pre-specified number, N, of undefined component signatures, NMF finds the N specific signatures and the contributions of each specific signature (the 'pie chart' circle, Figure 5b) that, for all the tumors simultaneously, provide the closest recon- structions of the observed catalogs. Endogenous mutagenic processes Across all the tumors analyzed, every cancer type had at least two mutation signatures; the cancers with the most signatures were those of the liver (seven sig- natures) and stomach and uterus (six signatures each). Figure 4e shows the example of lung adenocar- cinomas, which usually show mixtures of several mu- tational processes. Mixtures of signatures For example, in Signature 1, C > T mutations make up almost half of the total number of mutations, whereas T > A mutations constitute only about 10% of the total. (b) Each of the three signatures contributes a different number of mutations to the actual catalog, represented in the 'pie chart'. In this example, Signature 1 contributes 1,000 mutations, Signature 2 contributes 1,500, and Signature 3 contributes 750. The 1,000 mutations from Signature 1 are allocated according to the bar chart that represents the proportions of different types of mutations in this signature. In this case, Signature 1 would contribute approximately 50% × 1,000 = 500C > T mutations. Signature 2 would contribute approximately 9% × 1,500 = 135C > T mutations. Signature 3 would contribute approximately 10% × 750 = 75C > T mutations. The total number of C > T mutations in the reconstructed catalog would be 500 + 135 + 75 = 710. The reconstruction of the (d) actual catalog is approximate, and in this example, the reconstruction does not account for 65 mutations, approximately 2% of the total in the actual mutation catalog - the gray noisy line in (c). This figure is a simplification; in fact, in references [8,75,76], signatures are composed of nucleotide mutations in their trinucleotide contexts, as shown in Figures 3c,d,h and 4a-d. The mathematical procedures for approximating observed catalogs from mixtures of trinucleotide signatures are the same, but the trinucleotide context provides far more useful information: for example, the spikes in AA-exposed UTUCs show that the AA- induced A > T mutations tend to occur in a (C\|T)AG trinucleotide context. Reproduced from [76] with permission from Elsevier. (b) Signature 3 Signature 1 Signature 2 750 1,000 1,500 Signature 3 had it been present. This would suggest that still other important environmental exposures were not repre- sented among the 7,042 tumors. Because environmental exposures vary widely by geography, it will be important to determine somatic mutation catalogs from a diversity of geographic regions. For example, we previously showed that different genes are mutated in cholangiocar- cinomas from different geographical regions and with different etiologies [10,11]. In addition, it is crucially im- portant to have detailed clinical information associated with somatic mutation catalogs. It is possible that the mutagenic exposures responsible for some signatures in previous studies [8] could not be identified because the relevant clinical information was not available. Mixtures of signatures In its mathematical formulation, the collection of mutation catalogs (Figure 5d) is the approximate product of the matrix representing the mutation signatures (Figure 5a) and the matrix represent- ing the contributions of each signature to each tumor (Figure 5b). In other words, Figure 5a and Figure 5b are factors that, when multiplied, yield an approximation of Figure 5d. These factors are constrained to be non- Despite the power conferred by analysis of mutation signatures across the 7,042 tumors, the environmental or biological factors underlying 10 of the 21 signatures could not be identified, and indeed only three signatures were linked to exogenous exposures [8]. Furthermore, over two-thirds of the cancer types studied harbored sig- natures of unknown source. Thus, there is a large gap in our understanding of the environmental exposures and mutational processes that contribute to common human cancers. Conversely, there are mutagens with well- studied biochemistry - for example, aflatoxins [30], ben- zene [66], and AA - that were not detected in these tumors. Possibly none or few of the 7,042 tumors ana- lyzed had been exposed to these mutagens. Indeed, it seems likely that none were exposed to AA, which has a very distinctive signature that would have been detected Page 10 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 (a) Mutation signatures (b) Number of mutations contributed by each signature (c) (d) Mutations not present in the reconstructed catalog Observed somatic mutation catalog of a tumor genome 50% 40% 30% 20% 10% 0% C>A C>G C>T T>A T>C T>G Signature 1 Signature 3 50% 40% 30% 20% 10% 0% C>A C>G C>T T>A T>C T>G 50% Signature 3 Signature 1 Signature 2 65 mutations (2%) Number of mutations 0 200 400 600 800 1000 750 1,000 1,500 40% 30% 20% 10% 0% C>A C>G C>T T>A T>C T>G C>A C>G C>T T>A T>C T>G Signature 2 x + = (a) Mutation signatures 50% 40% 30% 20% 10% 0% C>A C>G C>T T>A T>C T>G Signature 1 50% 40% 30% 20% 10% 0% C>A C>G C>T T>A T>C T>G Signature 2 Figure 5 Reconstructing the catalog of somatic mutations in a cancer genome as superimposed mutation signatures at varying levels of exposure. (a) Each signature is represented by one of the bar charts, and consists of the relative proportions of different types of mutations in that signature. Mixtures of signatures For ex- ample, exposures to compounds such as aflatoxins would probably not be captured in clinical records. It is also possible that the mutation signatures of some expo- sures were not detected because the trinucleotide con- text and other characteristics of the mutations have not been determined from biochemical studies. (c) (d) Observed somatic mutation catalog of a tumor genome Number of mutations 0 200 400 600 800 1000 C>A C>G C>T T>A T>C T>G The examples of signatures described above focus on single-nucleotide mutations within trinucleotide con- texts as the main distinguishing features of signatures. However, other characteristics of mutation catalogs can also be included as features of mutation signatures and analyzed by NMF [8,76]. For example, strand bias could Page 11 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Page 11 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 be included by considering the two strands separately for each class of mutation in transcribed regions; in this case one would consider C > T on the transcribed strand to be distinct from G > A (the complementary mutation). Other types of mutations, including small insertions and deletions and dinucleotide mutations such as those that occur as a result of UV exposure (CC > TT mutations), can also be included as features of mutation signatures. The framework can also be expanded to consider more bases adjacent to the mutated nucleotide - for example, a pentanucleotide rather than a trinucleotide context. The framework can also be applied to specific regions of the genome. For example, the APOBEC signature (Fig- ure 4a) shows strand bias in exons, but not in introns [76]. Given that both exons and introns are transcribed, the exonic strand bias does not seem to be the result of transcription-coupled repair, and the underlying mech- anism remains unknown. However, by distinguishing mutations according to whether they occur in exons or introns, this information could be used to generate a more informative mutation signature. The utility of these possible extensions remains untested, but is likely to increase as additional tumor genomes, which capture about 50 times more mutation information than exomes, are sequenced. tumors studied. This implies that the signatures of many exposures have yet to be captured in sequenced tumor exomes or genomes. Thus, the analysis of mutation signa- tures in catalogs of somatic mutations from tumors is promising but in its infancy. Mixtures of signatures To realize this promise, we must extend our knowledge in two aspects. The first is to expand the diversities of tumor types and of their geographical origins. There is already rapid growth in the number of sequenced cancer genomes and their cat- alogs of somatic mutations. An important advantage of next-generation sequencing in this endeavor is that it is based on an inexpensive, commodity technology, the price of which will continue to drop. In addition, next-generation sequencing provides direct readouts of the mutations that actually occur in tumors. In this context, we note that using whole-exome or whole-genome sequencing to detect muta- tions (rather than sequencing targeted, cancer-related genes) ensures that most mutations detected are selectively inconsequential passengers. Even though a few somatic mutations in whole-exome or whole-genome sequence are drivers, they are so few that they have negligible influence on the signature. Finally, the large amount of data gener- ated by whole-exome and especially whole-genome sequen- cing provides optimal statistical power to tease apart the signatures of different mutational processes or exposures. The second aspect in which we must extend our knowledge consists of establishing connections between specific mutagens and their mutation signatures. This is likely to require experimental exposure of cells or ani- mals to mutagens or their biochemically active metabo- lites, followed by next-generation sequencing of either clonal populations of exposed cells or of tumors that de- velop in exposed animals. Sequencing of the exposed ge- nomes will connect specific mutagens to their mutation signatures in far more detail than is currently available. When mutation signatures cannot be found among the signatures of known mutagens, this would suggest the effects of an unknown exposure or mutational process, and point to the need for further epidemiological, toxi- cological, or biological research. Mutation signatures for surveillance and prevention Much of cancer is associated with exogenous exposures, and therefore in principle amenable to control by avoid- ance of those exposures. Examples include tobacco smoke, UV light, and many infectious exposures, such as hepatitis B and C, human papilloma virus, and Helicobacter pylori [77-79]. IARC lists 422 known or likely exogenous carcinogens [80]. Indeed, prevention by avoidance of ex- ogenous carcinogenic exposures has been an effective long- term strategy for the control of cancer, with tobacco smok- ing as the most salient example [49,81]. However, evidence from recent work [8] indicates that many exogenous expo- sures remain unidentified. Notably, as described earlier, of the 21 mutation signatures identified in [8], 10 lacked any known underlying mutational process or exposure, and over two-thirds of cancer types were affected by signatures due to unknown causes. Furthermore, only three exogen- ous mutagens were identified: tobacco smoking (12% of all tumors), UV light (5% of all tumors), and temozolomide (0.5% of all tumors), and the cause of Signature 5 (found in 14% of all tumors) is unknown. Some cancers were dispro- portionately affected by signatures with unknown causes. For example, 89% of HCCs showed Signature 12, and 90% showed Signature 16, both with unknown causes. Con- versely, the signatures of some well-known mutagens were not detected (Table 1), suggesting that cancers due to these mutagens were rare or non-existent among the 7,042 To our knowledge, there has been little work toward this goal, and our work on the mutation signature of AA and its application to detect AA exposure in HCC is an example [16]. References 1. Pott P: Cancer Scrot. In Chirurgical Observations Relative to the Cataract, the Polypus of the Nose, Cancer of the Scrotum, Different Kinds of Ruptures, and the Mortification of the Toes and Feet. London: Hawes, Clarke, Collins; 1775. 2 Y i K I hik K E i t l t d f th th i f 1. Pott P: Cancer Scrot. In Chirurgical Observations Relative to the Cataract, the Polypus of the Nose, Cancer of the Scrotum, Different Kinds of Ruptures, and the Mortification of the Toes and Feet. London: Hawes, Clarke, Collins; 1775. 2. Yamagiwa K, Ichikawa K: Experimental study of the pathogenesis of carcinoma. J Cancer Res 1918, 3:1–21. 2. Yamagiwa K, Ichikawa K: Experimenta carcinoma. J Cancer Res 1918, 3:1–21 3. Cook JW, Hewett CL, Hieger I: The isolation of a cancer-producing hydro- carbon from coal tar. Parts I, II, and II. J Chem So (Resumed) 1933, 395–405 3. Cook JW, Hewett CL, Hieger I: The isolation of a cancer-producing hydro- carbon from coal tar. Parts I, II, and II. J Chem So (Resumed) 1933, 395–405 4. Carrell CJ, Carrell TG, Carrell HL, Prout K, Glusker JP: Benzo[a]pyrene and its analogues: structural studies of molecular strain. Carcinogenesis 1997, 18:415–422. 4. Carrell CJ, Carrell TG, Carrell HL, Prout K, Glusker JP: Benzo[a]pyrene and its analogues: structural studies of molecular strain. Carcinogenesis 1997, 18:415–422. 5. Penning TM: Chemical Carcinogenesis. New York: Humana Press; 2011. 6. Ikehata H, Ono T: The mechanisms of UV mutagenesis. J Radiat Res 2011, 52:115–125. 5. Penning TM: Chemical Carcinogenesis. New York: Humana Press; 2011. 6. Ikehata H, Ono T: The mechanisms of UV mutagenesis. J Radiat Res 2011, 52:115–125. 7. Mortelmans K, Zeiger E: The Ames Salmonella/microsome mutagenicity assay. Mutat Res 2000, 455:29–60. 7. Mortelmans K, Zeiger E: The Ames Salmonella/microsome mutagenicity assay. Mutat Res 2000, 455:29–60. 8. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV, Bignell GR, Bolli N, Borg A, Børresen-Dale AL, Boyault S, Burkhardt B, Butler AP, Caldas C, Davies HR, Desmedt C, Eils R, Eyfjörd JE, Foekens JA, Greaves M, Hosoda F, Hutter B, Ilicic T, Imbeaud S, Imielinski M, Jäger N, Jones DT, Jones D, Knappskog S, Kool M, et al: Signatures of mutational processes in human cancer. Nature 2013, 500:415–421. References The second part of the vision, the experimental elu- cidation of signatures and the investigation of possible causes of signatures with unknown causes, will re- quire concerted effort. There will surely be challenges in understanding the signatures of complex mutagens such as tobacco smoke, and challenges in understand- ing the differences in the mutagens' metabolisms and mutagenic activity across different tissues and cell types. Nevertheless, in the near term it will be pos- sible to dissect and refine the worldwide repertoire of signatures and to assign some of these signatures to known causes as experimental studies advance. Of course, not all cancer is due to mutagenic exposures, but linking somatic mutation catalogs generated by next-generation sequencing to specific exposures via the mutation signa- tures of these exposures could substantially reduce the burden of avoidable cancer. 9. Nikolaev SI, Rimoldi D, Iseli C, Valsesia A, Robyr D, Gehrig C, Harshman K, Guipponi M, Bukach O, Zoete V, Michielin O, Muehlethaler K, Speiser D, Beckmann JS, Xenarios I, Halazonetis TD, Jongeneel CV, Stevenson BJ, Antonarakis SE: Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma. Nat Genet 2012, 44:133–139. 10. Ong CK, Subimerb C, Pairojkul C, Wongkham S, Cutcutache I, Yu W, McPherson JR, Allen GE, Ng CC, Wong BH, Myint SS, Rajasegaran V, Heng HL, Gan A, Zang ZJ, Wu Y, Wu J, Lee MH, Huang D, Ong P, Chan-on W, Cao Y, Qian CN, Lim KH, Ooi A, Dykema K, Furge K, Kukongviriyapan V, Sripa B, Wongkham C, et al: Exome sequencing of liver fluke-associated cholan- giocarcinoma. Nat Genet 2012, 44:690–693. 11. Chan-On W, Nairismägi ML, Ong CK, Lim WK, Dima S, Pairojkul C, Lim KH, McPherson JR, Cutcutache I, Heng HL, Ooi L, Chung A, Chow P, Cheow PC, Lee SY, Choo SP, Tan IB, Duda D, Nastase A, Myint SS, Wong BH, Gan A, Rajasegaran V, Ng CC, Nagarajan S, Jusakul A, Zhang S, Vohra P, Yu W, Huang D, et al: Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infection-related bile duct cancers. Nat Genet 2013, 45:1474–1478. 12. Author details 1 1Laboratory of Cancer Epigenome, Division of Medical Sciences, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore. 2Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore. 3Duke-NUS Centre for Computational Biology, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169857, Singapore. 4Division of Cellular and Molecular Research, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore. 5Cancer Science Institute of Singapore, National University of Singapore, Centre for Life Sciences, 28 Medical Drive, Singapore 117456, Singapore. 6Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. The first part of this vision, the assembly of a com- pendium of mutation signatures from ever more cancer genomes, seems certain to happen because of the plum- meting cost of sequencing and the many ongoing ef- forts to sequence tumor genomes. Nevertheless, there are many open questions on how best to deploy NMF or NMF-related procedures to assemble this compendium. For example, what factors determine the power of these procedures to distinguish similar mutation signatures? As the number of genome-wide somatic mutation catalogs increases, will it become worthwhile to include additional information, such as strand bias or pentanucleotide context, in mutation signatures? Fortunately, NMF- related procedures are an active area of machine learning research. For example, enhanced NMF procedures that prefer sparser solutions - solutions in which the mutation catalog of a given tumor is modeled as the mixture of a relatively small number of signatures - have been recently proposed [82-85]. Other proposed enhanced NMF proce- dures could favor solutions with fewer mutation signatures contributing to each tumor, leading to more interpretable results [85-87]. Acknowledgements W h k I C We thank Ioana Cutcutache, Weng Khong Lim, and Iain Beehuat Tan for comments on the manuscript. References Zang ZJ, Cutcutache I, Poon SL, Zhang SL, McPherson JR, Tao J, Rajasegaran V, Heng HL, Deng N, Gan A, Lim KH, Ong CK, Huang D, Chin SY, Tan IB, Ng CC, Yu W, Wu Y, Lee M, Wu J, Poh D, Wan WK, Rha SY, So J, Salto-Tellez M, Yeoh KG, Wong WK, Zhu YJ, Futreal PA, Pang B, et al: Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell ad- hesion and chromatin remodeling genes. Nat Genet 2012, 44:570–574. Abbreviations AA: aristolochic acid; HCC: hepatocellular carcinoma; IARC: International Agency for Research on Cancer; NMF: non-negative matrix factorization, UTUC, upper urinary-tract urothelial cancer; UV: ultraviolet. 13. Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, Kemp Z, Spain SL, Guarino E, Salguero I, Sherborne A, Chubb D, Carvajal-Carmona AA: aristolochic acid; HCC: hepatocellular carcinoma; IARC: International Agency for Research on Cancer; NMF: non-negative matrix factorization, UTUC, upper urinary-tract urothelial cancer; UV: ultraviolet. 12. Zang ZJ, Cutcutache I, Poon SL, Zhang SL, McPherson JR, Tao J, Rajasegaran V, Heng HL, Deng N, Gan A, Lim KH, Ong CK, Huang D, Chin SY, Tan IB, Ng CC, Yu W, Wu Y, Lee M, Wu J, Poh D, Wan WK, Rha SY, So J, Salto-Tellez M, Yeoh KG, Wong WK, Zhu YJ, Futreal PA, Pang B, et al: Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell ad- hesion and chromatin remodeling genes. Nat Genet 2012, 44:570–574. Conclusions and future directions We envision that the groundbreaking technical advances for detection of signatures in genome- and exome-wide catalogs of somatic mutations from thousands of tumors will enable the assembly of a wide-ranging compendium of mutation signatures from diverse cancer types and multiple geographical regions. This compendium would contain many more whole-genome catalogs of somatic mutations (as opposed to exome catalogs) than are Page 12 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Page 12 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Competing interests The authors declare that they have no competing interests. currently available, and would encompass tumors from many more geographical regions, thus capturing a much wider range of mutagenic exposures. This compendium could be combined with experimental determination of the extended signatures of known and suspected muta- gens, including, when necessary, their signatures in dif- ferent tissues or cell types. Signatures with known causes would represent future opportunities for preven- tion. Signatures with unknown causes would point to the need for further investigation of exogenous muta- gens or endogenous mutation processes. Competing interests Competing interests The authors declare that they have no competing interests. Abbreviations AA i l hi Bannasch P, Khoshkhou NI, Hacker HJ, Radaeva S, Mrozek M, Zillmann U, Kopp-Schneider A, Haberkorn U, Elgas M, Tolle T, et al: Synergistic hepato- carcinogenic effect of hepadnaviral infection and dietary aflatoxin B1 in woodchucks. Cancer Res 1995, 55:3318–3330. 15. Nagarajan N, Bertrand D, Hillmer AM, Zang ZJ, Yao F, Jacques PE, Teo AS, Cutcutache I, Zhang Z, Lee WH, Sia YY, Gao S, Ariyaratne PN, Ho A, Woo XY, Veeravali L, Ong CK, Deng N, Desai KV, Khor CC, Hibberd ML, Shahab A, Rao J, Wu M, Teh M, Zhu F, Chin SY, Pang B, So JB, Bourque G, et al: Whole-genome reconstruction and mutational signatures in gastric cancer. Genome Biol 2012, 13:R115. 35. Wild CP, Montesano R: A model of interaction: aflatoxins and hepatitis viruses in liver cancer aetiology and prevention. Cancer Lett 2009, 286:22–28. 36. Johnson BE, Mazor T, Hong C, Barnes M, Aihara K, McLean CY, Fouse SD, Yamamoto S, Ueda H, Tatsuno K, Asthana S, Jalbert LE, Nelson SJ, Bollen AW, Gustafson WC, Charron E, Weiss WA, Smirnov IV, Song JS, Olshen AB, Cha S, Zhao Y, Moore RA, Mungall AJ, Jones SJ, Hirst M, Marra MA, Saito N, Aburatani H, Mukasa A, et al: Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 2014, 343:189–193. 16. Poon SL, Pang ST, McPherson JR, Yu W, Huang KK, Guan P, Weng WH, Siew EY, Liu Y, Heng HL, Chong SC, Gan A, Tay ST, Lim WK, Cutcutache I, Huang D, Ler LD, Nairismägi ML, Lee MH, Chang YH, Yu KJ, Chan-On W, Li BK, Yuan YF, Qian CN, Ng KF, Wu CF, Hsu CL, Bunte RM, Stratton MR, et al: Genome- wide mutational signatures of aristolochic acid and its application as a screening tool. Sci Transl Med 2013, 5:197ra101. 37. Yip S, Miao J, Cahill DP, Iafrate AJ, Aldape K, Nutt CL, Louis DN: MSH6 mutations arise in glioblastomas during temozolomide therapy and mediate temozolomide resistance. Clin Cancer Res 2009, 15:4622–4629. 17. Chen CH, Dickman KG, Moriya M, Zavadil J, idorenko VS, Edwards KL, Gnatenko DV, Wu L, Turesky RJ, Wu XR, Pu YS, Grollman AP: Aristolochic acid-associated urothelial cancer in Taiwan. Proc Natl Acad Sci U S A 2012, 109:8241–8246. 18. Abbreviations AA i l hi 13. Palles C, Cazier JB, Howarth KM, Domingo E, Jones AM, Broderick P, Kemp Z, Spain SL, Guarino E, Salguero I, Sherborne A, Chubb D, Carvajal-Carmona Page 13 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 30. Smela ME, Currier SS, Bailey EA, Essigmann JM: The chemistry and biology of aflatoxin B: from mutational spectrometry to carcinogenesis. Carcinogenesis 2001, 22:535–545. LG, Ma Y, Kaur K, Dobbins S, Barclay E, Gorman M, Martin L, Kovac MB, Hum- phray S, CORGI Consortium; WGS500 Consortium, WGS500 Consortium, Lucassen A, Holmes CC, Bentley D, Donnelly P, Taylor J, Petridis C, Roylance R, Sawyer EJ, Kerr DJ, et al: Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet 2013, 45:136–144. LG, Ma Y, Kaur K, Dobbins S, Barclay E, Gorman M, Martin L, Kovac MB, Hum- phray S, CORGI Consortium; WGS500 Consortium, WGS500 Consortium, Lucassen A, Holmes CC, Bentley D, Donnelly P, Taylor J, Petridis C, Roylance R, Sawyer EJ, Kerr DJ, et al: Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet 2013, 45:136–144. LG, Ma Y, Kaur K, Dobbins S, Barclay E, Gorman M, Martin L, Kovac MB, Hum- phray S, CORGI Consortium; WGS500 Consortium, WGS500 Consortium, Lucassen A, Holmes CC, Bentley D, Donnelly P, Taylor J, Petridis C, Roylance 31. Bailey EA, Iyer RS, Stone MP, Harris TM, Essigmann JM: Mutational properties of the primary aflatoxin B1-DNA adduct. Proc Natl Acad Sci U S A 1996, 93:1535–1539. 14. Imielinski M, Berger AH, Hammerman PS, Hernandez B, Pugh TJ, Hodis E, Cho J, Suh J, Capelletti M, Sivachenko A, Sougnez C, Auclair D, Lawrence MS, Stojanov P, Cibulskis K, Choi K, de Waal L, Sharifnia T, Brooks A, Greulich H, Banerji S, Zander T, Seidel D, Leenders F, Ansén S, Ludwig C, Engel-Riedel W, Stoelben E, Wolf J, Goparju C, et al: Mapping the hallmarks of lung adenocar- cinoma with massively parallel sequencing. Cell 2012, 150:1107–1120. 32. Shen HM, Ong CN: Mutations of the p53 tumor suppressor gene and ras oncogenes in aflatoxin hepatocarcinogenesis. Mutat Res 1996, 366:23–44. 33. Chen CJ, Wang LY, Lu SN, Wu MH, You SL, Zhang YJ, Wang LW, Santella RM: Elevated aflatoxin exposure and increased risk of hepatocellular carcinoma. Hepatology 1996, 24:38–42. 34. Abbreviations AA i l hi Hoang ML, Chen CH, Sidorenko VS, He J, Dickman KG, Yun BH, Moriya M, Niknafs N, Douville C, Karchin R, Turesky RJ, Pu YS, Vogelstein B, Papadopoulos N, Grollman AP, Kinzler KW, Rosenquist TA: Mutational signature of aristolochic acid exposure as revealed by whole-exome sequencing. Sci Transl Med 2013, 5:197ra102. 38. Gaskell M, McLuckie KI, Farmer PB: Comparison of the repair of DNA damage induced by the benzene metabolites hydroquinone and p- benzoquinone: a role for hydroquinone in benzene genotoxicity. Carcinogenesis 2005, 26:673–680. 39. Saberi Hosnijeh F, Christopher Y, Peeters P, Romieu I, Xun W, Riboli E, Raaschou-Nielsen O, Tjonneland A, Becker N, Nieters A, Trichopoulou A, Bamia C, Orfanos P, Oddone E, Luján-Barroso L, Dorronsoro M, Navarro C, Barricarte A, Molina-Montes E, Wareham N, Vineis P, Vermeulen R: Occupa- tion and risk of lymphoid and myeloid leukaemia in the European Pro- spective Investigation into Cancer and Nutrition (EPIC). Occup Environ Med 2013, 70:464–470. 19. Hollstein M, Moriya M, Grollman AP, Olivier M: Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid. Mut Res 2013, 753:41–49. 20. Gokmen MR, Cosyns JP, Arlt VM, Stiborova M, Phillips DH, Schmeiser HH, Simmonds MS, Cook HT, Vanherweghem JL, Nortier JL, Lord GM: The epidemiology, diagnosis, and management of aristolochic acid nephropathy: a narrative review. Ann Intern Med 2013, 158:469–477. 40. Vlaanderen J, Lan Q, Kromhout H, Rothman N, Vermeulen R: Occupational benzene exposure and the risk of chronic myeloid leukemia: a meta- analysis of cohort studies incorporating study quality dimensions. Am J Ind Med 2012, 55:779–785. 21. Berger MF, Hodis E, Heffernan TP, Deribe YL, Lawrence MS, Protopopov A, Ivanova E, Watson IR, Nickerson E, Ghosh P, Zhang H, Zeid R, Ren X, Cibulskis K, Sivachenko AY, Wagle N, Sucker A, Sougnez C, Onofrio R, Ambrogio L, Auclair D, Fennell T, Carter SL, Drier Y, Stojanov P, Singer MA, Voet D, Jing R, Saksena G, Barretina J, et al: Melanoma genome sequencing reveals frequent PREX2 mutations. Nature 2012, 485:502–506. 41. Burns MB, Temiz NA, Harris RS: Evidence for APOBEC3B mutagenesis in multiple human cancers. Nat Genet 2013, 45:977–983. 42. Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B, Refsland EW, Kotandeniya D, Tretyakova N, Nikas JB, Yee D, Temiz NA, Donohue DE, McDougle RM, Brown WL, Law EK, Harris RS: APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 2013, 494:366–370. 22. Abbreviations AA i l hi Drobetsky EA, Grosovsky AJ, Glickman BW: The specificity of UV-induced mutations at an endogenous locus in mammalian cells. Proc Natl Acad Sci U S A 1987, 84:9103–9107. 23. Besaratinia A, Li H, Yoon JI, Zheng A, Gao H, Tommasi S: A high- throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens. Nucleic Acids Res 2012, 40:e116. 43. Vanherweghem JL, Depierreux M, Tielemans C, Abramowicz D, Dratwa M, Jadoul M, Richard C, Vandervelde D, Verbeelen D, Vanhaelen-Fastre R, Van- haelen M: Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 1993, 341:387–391. 24. Pleasance ED, Stephens PJ, O'Meara S, McBride DJ, Meynert A, Jones D, Lin ML, Beare D, Lau KW, Greenman C, Varela I, Nik-Zainal S, Davies HR, Ordoñez GR, Mudie LJ, Latimer C, Edkins S, Stebbings L, Chen L, Jia M, Leroy C, Marshall J, Menzies A, Butler A, Teague JW, Mangion J, Sun YA, McLaughlin SF, Peckham HE, Tsung EF, et al: A small-cell lung cancer genome with complex signatures of tobacco exposure. Nature 2010, 463:184–190. 44. Bhattacharjee P, Bhattacharyya D: Characterization of the aqueous extract of the root of Aristolochia indica: evaluation of its traditional use as an antidote for snake bites. J Ethnopharmacol 2013, 145:220–226. 45. Debelle FD, Vanherweghem JL, Nortier JL: Aristolochic acid nephropathy: a worldwide problem. Kidney Int 2008, 74:158–169. 25. Pfeifer GP, Denissenko MF, Olivier M, Tretyakova N, Hecht SS, Hainaut P: Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers. Oncogene 2002, 21:7435–7451. 46. Heinrich M, Chan J, Wanke S, Neinhuis C, Simmonds MS: Local uses of Aristolochia species and content of nephrotoxic aristolochic acid 1 and 2–a global assessment based on bibliographic sources. J Ethnopharmacol 2009, 125:108–144. 26. Phillips DH: Smoking-related DNA and protein adducts in human tissues. Carcinogenesis 2002, 23:1979–2004. 27. Alberg AJ, Brock MV, Ford JG, Samet JM, Spivack SD: Epidemiology of lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest 2013, 143:e1S–e29S. 47. Moriya M, Slade N, Brdar B, Medverec Z, Tomic K, Jelakovic B, Wu L, Truong S, Fernandes A, Grollman AP: TP53 Mutational signature for aristolochic acid: an environmental carcinogen. Int J Cancer 2011, 129:1532–1536. 48. Arlt VM, Stiborova M, Schmeiser HH: Aristolochic acid as a probable human cancer hazard in herbal remedies: a review. Mutagenesis 2002, 17:265–277. 28. 49. US Department of Health and Human Services: The Health Consequences of Smoking - 50 Years of Progress, A Report of the Surgeon General 2014. Rockville, MD: Public Health Service Office of the Surgeon General; 2014. Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 Abbreviations AA i l hi 57. Friedman HS, Kerby T, Calvert H: Temozolomide and treatment of malignant glioma. Clin Cancer Res 2000, 6:2585–2597. malignant glioma. Clin Cancer Res 2000, 6:2585–2597. 58. Payne MJ, Pratap SE, Middleton MR: Temozolomide in the treatment of solid tumours: current results and rationale for dosing/scheduling. Crit Rev Oncol Hematol 2005, 53:241–252. 82. Guan N, Huang X, Lan L, Luo Z, Zhang X: Graph based semi-supervised non-negative matrix factorization for document clustering. In 2012 11th International Conference on Machine Learning and Applications (ICMLA), Volume 1. Institute of Electrical and Electronics Engineers; 2012, 404–408. 59. Newlands ES, Stevens MF, Wedge SR, Wheelhouse RT, Brock C: Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. Cancer Treat Rev 1997, 23:35–61. 83. Hillebrand M, Krebel U, Wohler C, Kummert F: Traffic sign classifier adaption by semi-supervised co-training. Artificial Neural Networks Pattern Recognition 2012:193–200. 60. Trivedi RN, Almeida KH, Fornsaglio JL, Schamus S, Sobol RW: The role of base excision repair in the sensitivity and resistance to temozolomide- mediated cell death. Cancer Res 2005, 65:6394–6400. 84. Lefevre A, Bach F, Fevotte C: Semi-supervised NMF with time-frequency annotations for single-channel source separation. ISMIR 2012: 13th International Society for Music Information Retrieval Conference [http:// ismir2012.ismir.net/event/papers/115-ismir-2012.pdf]. 61. Margison GP, Santibanez Koref MF, Povey AC: Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine. Mutagenesis 2002, 17:483–487. 62. Caporali S, Falcinelli S, Starace G, Russo MT, Bonmassar E, Jiricny J, D'Atri S: DNA damage induced by temozolomide signals to both ATM and ATR: role of the mismatch repair system. Mol Pharmacol 2004, 66:478–491. 85. Morikawa Y, Yukawa M: A sparse optimization approach to supervised NMF based on convex analytic method. In Proceedings of the 38th IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP): May 2013. Vancouver, Canada: Institute of Electrical and Electronics Engineers; 2013:6078–6082. 63. A review of human carcinogens: Chemical agents and related occupations. IACR Monographs 2012, 100F. 64. Cogliano VJ, Baan R, Straif K, IARC Monographs programme staff: Updating IARC's carcinogenicity assessment of benzene. Am J Ind Med 2011, 54:165–167. 86. Chen M, Chen WS, Chen B, Pan B: Non-negative sparse representation based on block NMF for face recognition. Lecture Notes Comput Sci 2013, 8232:26–33. 65. Whysner J, Reddy MV, Ross PM, Mohan M, Lax EA: Genotoxicity of benzene and its metabolites. Mutat Res 2004, 566:99–130. 87. Sindhwani V, Ghoting A: Large-scale distributed non-negative sparse coding and sparse dictionary learning. Abbreviations AA i l hi Hecht SS: Lung carcinogenesis by tobacco smoke. Int J Cancer 2012, 131:2724–2732. 29. Ross RK, Yuan JM, Yu MC, Wogan GN, Qian GS, Tu JT, Groopman JD, Gao YT, Henderson BE: Urinary aflatoxin biomarkers and risk of hepatocellular carcinoma. Lancet 1992, 339:943–946. 49. US Department of Health and Human Services: The Health Consequences of Smoking - 50 Years of Progress, A Report of the Surgeon General 2014. Rockville, MD: Public Health Service Office of the Surgeon General; 2014. Page 14 of 14 Poon et al. Genome Medicine 2014, 6:24 http://genomemedicine.com/content/6/3/24 50. Talikka M, Sierro N, Ivanov NV, Chaudhary N, Peck MJ, Hoeng J, Coggins CR, Peitsch MC: Genomic impact of cigarette smoke, with application to three smoking-related diseases. Crit Rev Toxicol 2012, 42:877–889. 76. Alexandrov LB, Nik-Zainal S, Wedge DC, Campbell PJ, Stratton MR: Deci- phering signatures of mutational processes operative in human cancer. Cell Rep 2013, 3:246–259. 51. Hecht SS: Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer 2003, 3:733–744. 77. Moudgil V, Redhu D, Dhanda S, Singh J: A review of molecular mechanisms in the development of hepatocellular carcinoma by aflatoxin and hepatitis B and C viruses. J Environ Pathol Toxicol Oncol 2013, 32:165–175. 52. Bedard LL, Massey TE: Aflatoxin B1-induced DNA damage and its repair. Cancer Lett 2006, 241:174–183. 78. Micu G, Staniceanu F, Zurac S, Bastian A, Gramada E, Nichita L, Popp C, Sticlaru L, Andrei R, Socoliuc C: Carcinogenesis and infection with Helicobacter pylori. Rom J Intern Med 2010, 48:299–306. 53. Eaton DL, Gallagher EP: Mechanisms of aflatoxin carcinogenesis. Annu Rev Pharmacol Toxicol 1994, 34:135–172. 54. Aflatoxin. IARC Monographs 1985, 3–87. 79. Deligeoroglou E, Christopoulos P, Aravantinos L, Papadias K: Human papilloma virus molecular profile and mechanisms of cancerogenesis: a review. Eur J Gynaecol Oncol 2009, 30:128–132. 55. Ozturk M: p53 mutation in hepatocellular carcinoma after aflatoxin exposure. Lancet 1991, 338:1356–1359. 56. Gouas D, Shi H, Hainaut P: The aflatoxin-induced TP53 mutation at codon 249 (R249S): biomarker of exposure, early detection and target for ther- apy. Cancer Lett 2009, 286:29–37. 80. Review of human carcinogens. IARC Monographs 2012, 100. 81. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E, Wu XC, Eheman C, Anderson R, Ajani UA, Kohler B, Edwards BK: Annual report to the nation on the status of cancer, 1975–2005, featuring trends in lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst 2008, 100:1672–1694. doi:10.1186/gm541 Cite this article as: Poon et al.: Mutation signatures of carcinogen exposure: genome-wide detection and new opportunities for cancer prevention. Genome Medicine 2014 6:24. Abbreviations AA i l hi In Proceedings of the 18th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining: August 2012. Beijing, China: Association for Computing Machinery; 2012:489–497. 66. Nakayama A, Noguchi Y, Mori T, Morisawa S, Yagi T: Comparison of mutagenic potentials and mutation spectra of benzene metabolites using supF shuttle vectors in human cells. Mutagenesis 2004, 19:91–97. 67. Roberts SA, Lawrence MS, Klimczak LJ, Grimm SA, Fargo D, Stojanov P, Kiezun A, Kryukov GV, Carter SL, Saksena G, Harris S, Shah RR, Resnick MA, Getz G, Gordenin DA: An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat Genet 2013, 45:970–976. 68. Fishel R, Kolodner RD: Identification of mismatch repair genes and their role in the development of cancer. Curr Opin Genet Dev 1995, 5:382–395. 69. Peto J: Cancer epidemiology in the last century and the next decade. Nature 2001, 411:390–395. 70. Edwards BK, Howe HL, Ries LA, Thun MJ, Rosenberg HM, Yancik R, Wingo PA, Jemal A, Feigal EG: Annual report to the nation on the status of cancer, 1973–1999, featuring implications of age and aging on U.S. cancer burden. Cancer 2002, 94:2766–2792. 71. Frank SA: Dynamics of Cancer Incidence, Inheritance, and Evolution. Princeton (NJ): Princeton University Press; 2007. 72. Hoeijmakers JH: DNA damage, aging, and cancer. N Engl J Med 2009, 361:1475–1485. 73. Lombard DB, Chua KF, Mostoslavsky R, Franco S, Gostissa M, Alt FW: DNA repair, genome stability, and aging. Cell 2005, 120:497–512. 74. Greenman C, Stephens P, Smith R, Dalgliesh GL, Hunter C, Bignell G, Davies H, Teague J, Butler A, Stevens C, Edkins S, O'Meara S, Vastrik I, Schmidt EE, Avis T, Barthorpe S, Bhamra G, Buck G, Choudhury B, Clements J, Cole J, Dicks E, Forbes S, Gray K, Halliday K, Harrison R, Hills K, Hinton J, Jenkinson A, Jones D, et al: Patterns of somatic mutation in human cancer genomes. Nature 2007, 446:153–158. 75. Nik-Zainal S, Alexandrov LB, Wedge DC, Van Loo P, Greenman CD, Raine K, Jones D, Hinton J, Marshall J, Stebbings LA, Menzies A, Martin S, Leung K, Chen L, Leroy C, Ramakrishna M, Rance R, Lau KW, Mudie LJ, Varela I, McBride DJ, Bignell GR, Cooke SL, Shlien A, Gamble J, Whitmore I, Maddison M, Tarpey PS, Davies HR, Papaemmanuil E, et al: Mutational processes molding the genomes of 21 breast cancers. Cell 2012, 149:979–993.
https://openalex.org/W2973088202	https://europepmc.org/articles/pmc6736980?pdf=render	English	null	High Fluence Chromium and Tungsten Bowtie Nano-antennas	Scientific reports	2,019	cc-by	8,674	OPEN Received: 6 September 2018 Accepted: 19 August 2019 Published: xx xx xxxx Nano-antennas are replicas of antennas that operate at radio-frequencies, but with considerably smaller dimensions when compared with their radio frequency counterparts. Noble metals based nano-antennas have the ability to enhance photoinduced phenomena such as localized electric fields, therefore-they have been used in various applications ranging from optical sensing and imaging to performance improvement of solar cells. However, such nano-structures can be damaged in high power applications such as heat resisted magnetic recording, solar thermo-photovoltaics and nano-scale heat transfer systems. Having a small footprint, nano-antennas cannot handle high fluences (energy density per unit area) and are subject to being damaged at adequately high power (some antennas can handle just a few milliwatts). In addition, given that nano-antennas are passive devices driven by external light sources, the potential damage of the antennas limits their use with high power lasers: this liability can be overcome by employing materials with high melting points such as chromium (Cr) and tungsten (W). In this article, we fabricate chromium and tungsten nano-antennas and demonstrate that they can handle 110 and 300 times higher fluence than that of gold (Au) counterpart, while the electric field enhancement is not significantly reduced. The invention of radio antennas opened new opportunities to transmit information through free space. In recent years, very small replicas of radio antennas were invented at optical frequencies, so called nano-antennas. These antennas can boost the interaction of light with nano-scale matters by coupling and localizing the freely propa- gating visible and infrared optical radiation in a sub-wavelength region1–3. Key properties of the devices include localization and confinement of light below the diffraction limit4 and high electric field enhancement, which are generated by the excitation of localized surface plasmon resonances (LSPRs)5,6. These unique characteristics unlock a vast potential for different applications such as single molecule detection7, enhancement of the efficiency of photo-detection8, near-field optical trapping9, creation of nanoscale light sources10, solar energy harvesting11, heat transfer12, single algae cell detection13, surface-enhanced infrared absorption (SEIRA)14 and enhancement of surface enhanced Raman spectroscopy (SERS) signals15. The excitation of antennas is a non-resonant effect that allows the enhancement of the electric field at its extremities by exploiting the current appearing at the antenna surface and the lightning rod-effect. In general, electromagnetic waves in a large spectral range can be coupled to the antenna-however, it is a non-resonant coupling1. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports High Fluence Chromium and Tungsten Bowtie Nano-antennas Monir Morshed1, Ziyuan Li2, Benjamin C. Olbricht3, Lan Fu2, Ahasanul Haque1, Li Li4, Ahmmed A. Rifat 5, Mohsen Rahmani5, Andrey E. Miroshnichenko 1 & Haroldo T. Hattori1 PEN Received: 6 September 2018 Accepted: 19 August 2019 Published: xx xx xxxx Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y Results i Description of the structures. To compare how nano-antennas fabricated from different materials work at high fluences, a standard bow-tie shape is chosen for all materials. The antennas are fabricated on top of a quartz (SiO2) substrate, as shown in Fig. 1. The geometrical parameters of the antennas are as follows: the length of each trapezoid is l, width of the trapezoid is w, apex angle is α, and the gap width between two trapezoid is g. The yellow parts indicate metallic regions, which may be gold, chromium or tungsten, while the substrate material is silica in all cases. The electric field enhancement factor and absorption of the nano-antennas are calculated by optimizing the parameters at the wavelength of 1053 nm. The optimum length for gold, chromium, and tungsten nano-antennas are 190 nm, 145 nm, and 130 nm, respectively. The gap width, thickness and apex angle are always fixed to be 50 nm, 100 nm and 90° for antennas, respectively. It is noted that the electric field enhancement of the antenna can increase by reducing the gap size25. Electric field enhancement and absorption by nano-antennas. Firstly, an finite-difference time-domain (FDTD) method is used to numerically calculate the electric field enhancement factor and absorp- tion of the antennas with the optimized parameters (please see the Methods section). Figure 2(a) shows the electric field enhancement factor for all the three types of antennas at the optimum parameters and electric field intensity distribution along the vertical direction of nano-antennas are illustrated in Fig. 2(b–d) at the wave- length of 1053 nm. Figure 2(a) shows that the electric field enhancement at sub-wavelength gap of the gold bowtie antenna is relatively higher than the other two materials due to combined effects of this material-localized sur- face plasmon mode and gap plasmon mode. In addition, from Fig. 2(b), it is observed that the electric field is not uniform for gold antennas because much more light is concentrated at the interface between gold and silica due to larger refractive index compare to air. Whereas, in case of Cr and W, the two types of modes are also present-however, the surface plasmon resonance or resonance effect is reduced due to the high losses in both materials. This is confirmed by the electric field profiles of Fig. 2(c,d), where the electric field enhancement is weaker and quasi-constant across the gap between both nano-antennas. OPEN p g However, the intraband transition of metals can lead to large photon absorption and, consequently, increase the local temperature of the antennas when they are irradiated by external laser sources16,17. Although the gen- eration of localized heat regions in the antenna (hot-spots) may be useful for the treatment of cancer18, heat transfer12, and hot vapor generation19, the accumulation of heating and relaxation processes can produce unex- pected effects on some applications such as imaging, sensing, and spectroscopy20. For example, the performance of the nano-antennas can be affected by the melting of the devices, resulting in a change of their morphology21. Therefore, the resonance may be either blue or red shifted, with significantly reduced electric field enhance- ment22,23. Given their small footprints, even small amounts of power can destroy nano-antennas since they have low thermal emittance in the mid to far infrared wavelength range20. 1School of Engineering and Information Technology, University of New South Wales, Canberra, ACT 2610, Australia. 2Department of Electronic Materials Engineering, Research School of Physics and Engineering, The Australian National University, Canberra, ACT 2601, Australia. 3Coupled Optics LLC, 36 Wenark Drive, Delaware, Nework, 19713, USA. 4Australian National Fabrication Facility, The Australian National University, Canberra, ACT 2601, Australia. 5Nonlinear Physics Centre, Research School of Physics and Engineering, The Australian National University, Acton, Canberra, 2601, Australia. Correspondence and requests for materials should be addressed to M.M. (email: Monir.Morshed@student.adfa.edu.au) or Z.L. (email: ziyuan.li@anu.edu.au) Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ Figure 1. Schematic diagram of single bow-tie nano-antenna. Figure 1. Schematic diagram of single bow-tie nano-antenna. To overcome the above constraints, new materials are in demand for antennas to work under harder condi- tions. Guler et al.24 said that refractory plasmonic materials could be used to replace gold or silver in high power laser applications. Moreover, Mironov et al.25 stated that a proper selection of materials can help to create devices that can work with high fluences without melting. In recent years, distinctive typologies of nano-antennas are created such as dipole26, bowtie4, Yagi-Uda27, spiral28, and log-periodic antennas28 based on the gold material. However, the gold antennas can melt at a few microwatts of power or low fluence (e.g. 250 μW power or 0.059 J/m2 fluence)25. OPEN Therefore, new materials that can operate under higher fluences are needed to allow nano-antennas to work with high power lasers.f g p In this article, we demonstrate the performance of the bowtie nano-antennas made of different metals: gold (Au), chromium (Cr) and tungsten (W) under high fluences. Bowtie nano-antennas are chosen because these nano-antennas have high electric field confinement and enhancement due to the near-field coupling across the gap5,29. In addition, they are suitable for broadband operation28 and single molecule detection7. Our experiments show that chromium and tungsten antennas are capable of handling about 110 and 300 times higher fluence than that of the gold counterpart, respectively. Results i The electric field enhancement factor of (a) gold for the optimum parameters of l = 190 nm, and w = 380 nm, chromium for the optimum parameters of l = 145 nm, and w = 290 nm, and tungsten for the optimum parameters of l = 130 nm, and w = 260 nm, while the gap width g, thickness H and apex angle α are always kept constant to 50 nm, 100 nm and 90° for all nano-antennas and electric field intensity profile in the y − z (Einc − kinc) plane of (b) gold, (c) chromium and (d) tungsten nano-antennas for the optimized parameters at the wavelength of 1053 nm. One of the main problems in the field of thermo-plasmonics is that the continuous laser illumination induces temperature rise in the nano-antennas, which could eventually destroy the nano-antennas. The electromagnetic wave from the laser that strikes the metals in the nano-antennas can be converted into heat due to absorption of light or increasing of current density within the antenna30. The generation of heat in the nano-antennas depends on the physical properties of materials such as absorption that is related to the imaginary part of the refractive index31. To better understand the thermal behavior of these antennas, we have calculated the absorption of power and absorption cross section using Lumerical FDTD software which are shown in Fig. 3. Figure 3(a) shows the absorptance as a function of wavelength while Fig. 3(b) shows the absorption cross-section area as a function of wavelength. Although the figures show a peak at 950 nm, which differs from the peak of the electric field in the gap of nano-antenna because the energy distribution is not uniform along the plane of the antenna and while the absorptance is lower at 1053 nm, the electric field at the antenna edges is the highest which is clear from Fig. 2(b–d). We also observe from this figure that chromium has higher absorption than the other two materials at the wavelength of 1053 nm because chromium has comparatively higher imaginary dielectric constants at this wavelength32–34. In addition, chromium has lower thermal conductivity35 than gold and tungsten-therefore, it will generally produce more heat than gold and tungsten. It should be noted that the adhesive layer titanium can boost-up the temperature in the structures (even for a 2 nm thickness)36, i.e. it can add significant amount of heat to our antennas. Results i From the zoomed version of these figures we can see that the resonance exists but with a significant lower quality factor due to the metal losses. It is noted that, the electric field enhancement factor (calculated from Eq. 1) for gold, chromium, and tungsten are 7.5, 4.15, and 3.10, respectively. Since both tungsten and chromium absorb more power than reflective gold, the electric field is more uniform for chromium and significantly more uniform for tungsten when compared with gold, resulting in better uniformity of energy distribution in chromium and tungsten. The more uniform distribution of energy in the chromium and tungsten based nano-antennas can more uniformly distribute the temperature rise in the antennas. Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ 0 2 4 6 8 10 850 950 1050 1150 1250 1350 1450 Au Cr W Wavelength (nm) Electric field enhancement factor (a) (b) Au (c) Cr (d) W Figure 2. The electric field enhancement factor of (a) gold for the optimum parameters of l = 190 nm, and w = 380 nm, chromium for the optimum parameters of l = 145 nm, and w = 290 nm, and tungsten for the optimum parameters of l = 130 nm, and w = 260 nm, while the gap width g, thickness H and apex angle α are always kept constant to 50 nm, 100 nm and 90° for all nano-antennas and electric field intensity profile in the y − z (Einc − kinc) plane of (b) gold, (c) chromium and (d) tungsten nano-antennas for the optimized parameters at the wavelength of 1053 nm. (b) Au 0 2 4 6 8 10 850 950 1050 1150 1250 1350 1450 Au Cr W Wavelength (nm) Electric field enhancement factor (a) Wavelength (nm) (c) Cr (d) W Figure 2. Results i However, in terms of damage, the antennas shape will not be changed until temperature rise in the metals exceeds their Tamman temperature. When the temperature in the surface of antennas higher than the Tamman temperature, the atoms diffusion and mobility increases significantly, resulting sintering or morphology changing of antennas37,38. The bulk melting points for gold, chromium, and tungsten is 1064.18 °C, 1907 °C, and 3422 °C, respectively. It is true that dielectric based antennas could reduce the power absorption by the antennas, Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ Figure 3. (a) Comparison of the absorbed power (heat genereation) and (b) numerically calculated absorption cross-section for three different nano-antennas. Figure 3. (a) Comparison of the absorbed power (heat genereation) and (b) numerically calculated absorption cross-section for three different nano-antennas. Figure 4. (a) 3D and (b) 2D view of the temperature distribution in gold nano-antennas at the power of 150 μW. Figure 4. (a) 3D and (b) 2D view of the temperature distribution in gold nano-antennas at the power of 150 μW. but they generally have lower electric field enhancement39 and, in some applications, the nano-structures such as nano-antenna are used to convert optical power into heat12. Th f h l d h l f h ll h d fl 25 30 40 h The main factor that leads to the melting of the metallic regions is the energy density or fluence25,30,40. When light reaches the antenna, heat is produced at the metal surfaces from the current density induced by light. In our experiments, we shall consider the fluence as the key parameter that can damage the nano-antennas. The laser fluence is calculated from the equations explained in the Methods section. Furthermore, we have analyzed the structures using the Lumerical device software package to see the thermal distribution in the nano-antennas, and as an example the distribution of temperature for gold antenna is presented in Fig. 4. Coppens et al.30 have also investigated the temperature distribution in nano-structures using the FDTD based software and claimed good approximation with the experiments.l For any fluence, the temperature is higher in the metallic and gap regions that than of the surrounding air and quartz substrate as can be observed in Fig. 4. Results i It is because the thermal conductivity of air (0.0257 W.m−1.K−1) and Quartz (1.3 W.m−1.K−1)) is comparatively low-so that, the heat generated by nano-antennas cannot totally dissipate, therefore, they concentrates in the antennas region. Fabrication and characterization of nano-antennas. The antennas are fabricated by firstly depositing different metals using electron-beam evaporator or sputter system and the patterns are made by focused ion beam(FIB) as described in the Methods section. Firstly, the gold bow-tie antenna is fabricated and characterized, which is shown in Fig. 5. Figure 5(a–c) shows SEM images of a gold bowtie nano-antennas with no laser exposure. In order to examine the damage threshold, the antennas are exposed to different fluences: we start from a low fluence of 0.01 J/m2, then we increase the fluence by 0.012 J/m2 (step size) until the gold antenna is damaged. From Fig. 5(d) it shows that the antenna starts to damage at the fluence of 0.0523 J/m2, However- they are completely damaged for the fluence of 0.054 J/m2 and 0.056 J/m2, as shown in Fig. 5(e,f). Besides that, the cumulative heating which is raised from the absorption of power may lead to the shape deformation when the melting temperature Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ 500 nm 500 nm 500 nm 500 nm 500 nm Gold at the fluence of 0.0523 J/m2 Gold at the fluence of 0.054 J/m2 Gold at the fluence of 0.056 J/m2 )c( ) b ( ) a ( )f( )e( ) d ( 500 nm Figure 5. SEM images of the studied gold nano-antennas (a–c) without laser exposure and (d–f) under the fluence of 0.0523 J/m2, 0.054 J/m2, and 0.056 J/m2, respectively which clearly indicates the melting of nano- antennas. 500 nm )c( 500 nm ) b ( 500 nm ) a ( 500 nm ) b ( ) b ( 500 nm Gold at the fluence of 0.056 J/m2 )f( 500 nm Gold at the fluence of 0.054 J/m2 )e( Gold at the fluence of 0.0523 J/m2 ) d ( 500 nm ) d ( Figure 5. SEM images of the studied gold nano-antennas (a–c) without laser exposure and (d–f) under the fluence of 0.0523 J/m2, 0.054 J/m2, and 0.056 J/m2, respectively which clearly indicates the melting of nano- antennas. Discussion h b From the above results, we can see that the fluences for gold, chromuim, and tungsten are 0.054 J/m2, 6.16 J/m2, and 16.16 J/m2, respectively. Therefore, in comparison with gold and chromium counterparts, tungstens thresh- old damage fluence is 2.73 times and 300 times higher than that of chromium and gold antennas, respectively. In addition, chromium and tungsten has 6 and 7.22 times higher magnitude of electric field intensity in the gap than that of gold counterpart when they operate at their threshold fluence. Since, the incident electric field Einc is proportional to the square root of the fluence of a laser where, spot size and repetition rate are constant, so, we can easily calculate the magnitude of electric field intensity in the gap for different antennas from Eq. 1 (see Methods) for given electric field enhancement factors. The possible reason behind the improved performance of tungsten antenna could be its lower absorbance (see Fig. 3), higher thermal conductivity than chromium and significantly higher melting point. Moreover, since tungsten has higher dissipative loss than gold, more light penetrates into the metal, therefore less power is localized on the surface of tungsten nano-antennas. Also, we can clearly see from Fig. 2(c) that the electric field is uniformly distributed in the gap. Although gold is more reflective and less absorbing than tungsten, tungsten has almost 3.2 times than gold’s melting point.h g g g g g The temperature on the antenna surface is one of the most important parameters that eventually lead to the destruction of the devices. To better understand the temperature dependence of the optical properties of different materials and how they affect the electric field enhancement factors, we have used another setup (see Methods section). The transmission and refractive indices of gold, chromium and tungsten thin films at different temper- atures are shown in Fig. 8(a–c), and (d–i) respectively. From Fig. 8(a), it is observed that the transmission of the gold film decreases with increased temperature and, from Fig. 8(d,g), it can be inferred that, although the real part of the refractive index does not change much, the imaginary part increases significantly with increased temper- ature. Therefore, the absorption of films can increase at high temperatures. We have also calculated the electric field enhancement factors based on the measured temperature dependent refractive indices as shown in Fig. 8(j), which match the theoretically simulated results, confirming the accuracy of our calculations. Results i 500 nm ) a ( 500 nm ) b ( 500 nm )c( ) b ( )c( Tungsten at the fluence of 16.37 J/m2 500 nm (f) Tungsten at the fluence of 16.16 J/m2 500 nm (e) Tungsten at the fluence of 15.9 J/m2 500 nm (d) (d) (e) Figure 7. SEM images of the studied tungsten nano-antennas (a–c) without laser exposure and (d–f) under the fluence of 15.9 J/m2, 16.16 J/m2, and 16.37 J/m2, respectively which clearly indicating melting of nano-antennas. antennas geometry can be optimized by using the electron beam lithography (EBL) technique and related lif techniques. Nevertheless, the antennas presented in this work are sufficient to be used for the laser fluence te 2l q pfil We start the exposure of chromium from the gold threshold fluence of 0.054 J/m2, and increase the fluence until the antenna is damaged. The devastated chromium nano-antennas are appeared in Fig. 6(d–f) and the flu- ence of 6.16 J/m2 is considered as the threshold damage fleunce for chromium. we can also see from this figure that the metallic regions are mostly damaged due to the higher absorptance of chromium.fl We start the exposure of chromium from the gold threshold fluence of 0.054 J/m2, and increase the fluence until the antenna is damaged. The devastated chromium nano-antennas are appeared in Fig. 6(d–f) and the flu- ence of 6.16 J/m2 is considered as the threshold damage fleunce for chromium. we can also see from this figure that the metallic regions are mostly damaged due to the higher absorptance of chromium.fl Lastly, we apply different fluences for tungsten nano-antennas starting from the chromium threshold damage fluence as a reference. The fluence that damages the antennas is about 16.16 J/m2. Figure 7(d–f) shows the SEM image for the damaged antenna. This figure shows that the antenna starts to melt at a fluence of 15.9 J/m2, and is completely damaged at the fluence of 16.16 J/m2 and 16.37 J/m2, respectively. Now, the threshold fluence of tung- sten antenna is considered as 16.16 J/m2. Results i 500 nm ) b ( ( 500 nm ) a ( 500 nm (c) (a) 500 nm 500 nm 500 nm 500 nm 500 nm 500 nm Chromium at the fluence of 5.6 J/m2 Chromium at the fluence of 5.9 J/m2 Chromium at the fluence of 6.16 J/m2 ) b ( ) a ( (d) (e) (f) (c) Figure 6. SEM images of the studied chromium nano-antennas (a–c) without laser exposure and (d–f) under the fluence of 5.6 J/m2, 5.9 J/m2, and 6.16 J/m2, respectively, which clearly indicates the melting of nano- antennas. 500 nm Chromium at the fluence of 5.6 J/m2 (d) 500 nm Chromium at the fluence of 6.16 J/m2 (f) Figure 6. SEM images of the studied chromium nano-antennas (a–c) without laser exposure and (d–f) under the fluence of 5.6 J/m2, 5.9 J/m2, and 6.16 J/m2, respectively, which clearly indicates the melting of nano- antennas. is exceeded. We also observe some surface damaging because we used 4 nm Ti layer as a adhesive layer that may also be damaged at threshold fluence of gold. l We have also fabricated chromium and tungsten nano-antennas, which are shown in Figs 6(a–c) and 7(a–c). The SEM images show that the shapes of the fabricated antennas are not as well-defined as the gold counter- parts. The main reason is that chromium and tungsten are both hard materials which are harder to remove by the Gallium ions. The contamination of Ga ions may increase some losses and thus lead to a broadening of the resonance. However, the effect is minor and will not affect much the near field behaviour of the antennas41. The Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ Tungsten at the fluence of 15.9 J/m2 Tungsten at the fluence of 16.16 J/m2 Tungsten at the fluence of 16.37 J/m2 500 nm 500 nm 500 nm 500 nm 500 nm 500 nm )c( ) b ( ) a ( (e) (f) (d) Figure 7. SEM images of the studied tungsten nano-antennas (a–c) without laser exposure and (d–f) under the fluence of 15.9 J/m2, 16.16 J/m2, and 16.37 J/m2, respectively which clearly indicating melting of nano-antennas. Discussion h b 00 1350 23⁰C 200⁰C (nm) 00 1350 23⁰C 200⁰C (nm) m 00 1350 23⁰C 200⁰C nm) 00 1350 23⁰C 200⁰C h (nm) 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 2.8 3 3.2 3.4 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) 2.5 2.7 2.9 3.1 3.3 3.5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Wavelength (nm) Electric field enhancement factor )c( ) b ( (e) (f) (h) (i) (k) (l) r C W mentally optical properties measurement of gold, chromium and tungsten, respectively for ures: (a–c) transmission, (d–f) real part of index, (g–i) imaginary part of index, (j–l) electric t factor calculated from experimental data. Discussion h b 2 4 6 8 Transmittance, T (%) 0 1 2 3 4 5 Imaginary Part (k) 2 3 3 Electric field enhancement factor 2 2 2 3 3 3 Real Part (n) 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Wavelength (nm) Electric field enhancement factor ) b ( (e) (h) (k) r C al properties measurement of gold, chromium and nsmission, (d–f) real part of index, (g–i) imaginar t d f i t l d t 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) ) b ( r C 0 20 40 60 80 100 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) )a( u A 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) )c( W Transmittance, T (%) 0 0.2 0.4 0.6 0.8 1 900 1050 1200 1350 23⁰C 200⁰C Wavelength (nm) Real Part (n) 0 5 10 15 20 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 30 nm 3 4 5 6 7 8 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) 0 900 1050 1200 1350 Tr Wavelength (nm) 0 900 1050 1200 1350 Tra Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 2.8 3 3.2 3.4 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) 2.5 2.7 2.9 3.1 3.3 3.5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 900 1050 1200 1350 Tra Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Wavelength (nm) Electric field enhancement factor (d) (e) (f) (g) (h) (i) (j) (k) (l) Figure 8. Discussion h b In Fig. 8(j), it is clear that the electric field enhancement decreases, and the resonance blue-shifts with increased temperature. For chromium, it can be observed, from Fig. 8(b,e,h) that the transmission and refractive indices change slightly with Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ 0 0.2 0.4 0.6 0.8 1 900 1050 1200 1350 23⁰C 200⁰C Wavelength (nm) Real Part (n) 0 5 10 15 20 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 30 nm 3 4 5 6 7 8 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) 0 20 40 60 80 100 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 2.8 3 3.2 3.4 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) 2.5 2.7 2.9 3.1 3.3 3.5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 20 40 60 80 900 1050 1200 1350 23⁰C 200⁰C Transmittance, T (%) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Wavelength (nm) Electric field enhancement factor )c( ) b ( )a( (d) (e) (f) (g) (h) (i) (j) (k) (l) r C u A W Figure 8. Experimentally optical properties measurement of gold, chromium and tungsten, respectively for different temperatures: (a–c) transmission, (d–f) real part of index, (g–i) imaginary part of index, (j–l) electric field enhancement factor calculated from experimental data. Discussion h b Experimentally optical properties measurement of gold, chromium and tungsten, respectively for different temperatures: (a–c) transmission, (d–f) real part of index, (g–i) imaginary part of index, (j–l) electric field enhancement factor calculated from experimental data. Wavelength (nm) Wavelength (nm) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) (e) 2.5 2.7 2.9 3.1 3.3 3.5 900 1050 1200 1350 23⁰C 200⁰C Real Part (n) Wavelength (nm) (f) Imaginary Part (k) Wavelength (nm) Wavelength (nm) 0 1 2 3 4 5 900 1050 1200 1350 23⁰C 200⁰C Imaginary Part (k) Wavelength (nm) g ( ) (i) Imaginary Part (k) 0 1 2 3 4 900 1050 1200 1350 23 C 200⁰C Imaginary Part (k) Wavelength (nm) 2.8 3 3.2 3.4 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) (i) (l) Imaginary Part (k) 2.8 3 3.2 3.4 900 1050 1200 1350 23⁰C 200⁰C Electric field enhancement factor Wavelength (nm) (l) Figure 8. Experimentally optical properties measurement of gold, chromium and tungsten, respectively for different temperatures: (a–c) transmission, (d–f) real part of index, (g–i) imaginary part of index, (j–l) electric field enhancement factor calculated from experimental data. temperature. Similarly, the properties of tungsten also change slightly with temperature. Moreover, the electric field spectra have no resonance shift (Fig. 8(l)). Therefore, we may conclude that tungsten is more stable than gold and chromium at high temperatures, and can operate at high powers when compared with the other materials. It is noted that the nano-structures continuously heat up when irradiated by external laser sources: the electric field intensity and dissipated power increase the temperature of nano-antennas42,43.i temperature. Similarly, the properties of tungsten also change slightly with temperature. Moreover, the electric field spectra have no resonance shift (Fig. 8(l)). Therefore, we may conclude that tungsten is more stable than gold and chromium at high temperatures, and can operate at high powers when compared with the other materials. It is noted that the nano-structures continuously heat up when irradiated by external laser sources: the electric field intensity and dissipated power increase the temperature of nano-antennas42,43.i y p p p In the above paragraph, we have just explained indirect way to measure the electric field enhancement factor of different nano-antennas and how they may be affected by the generation of heat from high power. Methodsh Since electric field in the gap of nano-antenna is enhanced, the relative electric field enhancement factor can be calculated as, = \| \| \| \| F E E (1) rel gap peak inc peak , , (1) where, \|Egap,peak\| is the magnitude of the electric field calculated in subwavelength gap of the nano-antenna, and \|Einc,peak\| is the magnitude of electric field of the incoming light.hlhl \| ,p \| gi g g The laser fluence is calculated from the following equations.The energy and fluence for a single pulse of a Q-switched laser are given by25, ∫ τ = = − W P t dt P ( ) (2) single pulse t t peak eff 1 2 π = − − F W r 4 (3) single pulse single pulse spot 2 ∫ τ = = − W P t dt P ( ) (2) single pulse t t peak eff 1 2 ∫ τ = = − W P t dt P ( ) (2) single pulse t t peak eff 1 2 4 (2) π = − − F W r 4 (3) single pulse single pulse spot 2 (3) where, P(t) is the instantaneous value of the power, t1 and t2 are the arbitrary instants when the pulse is not negli- gible, τeff is the effective duration of the pulse, and rspot represent the spot size of the laser beam.hif where, P(t) is the instantaneous value of the power, t1 and t2 are the arbitrary instants when the pulse is not negli- gible, τeff is the effective duration of the pulse, and rspot represent the spot size of the laser beam.hif f The antennas were fabricated by firstly depositing different metals using electron-beam evaporator or sputter system. The thickness of the metals in each of the three samples was taken as 100 nm and for the gold sample, the metal was deposited by Temescal BJD-2000 E-beam/Thermal Evaporator system at the rate of 5 nm/s. A 2 nm titanium layer was used to provide good adhesion between gold and the substrate. On the other hand, the chro- mium and tungsten were deposited by Sputter Coater system-AJA. However, the deposition rate of the metals was different: 2.58 nm/min and 6 nm/min for Chromium and Tungsten, respectively. www.nature.com/scientificreports/ some research groups have measured the electric field profile of nano-antennas4, they have stated that it was very difficult to accurately measure the electric field intensity in the antennas since the probe (e.g. near-field scanning optical microscopy (NSOM) probe) would strongly interfere with the measurement at the nanometer scale, mak- ing their measurements rather qualitative. g q Some groups have simulated the rise of temperature in nano-antennas43 and they have shown that the tem- perature really modifies the electric field enhancement and absorption of plasmonic devices. In another theoret- ical paper by Downes et al.42, they stated that NSOM could detect scattered power with atomic resolution, but very thin tips (less than 30 nm diameter) would be needed. The measurement of local temperature at nanoscale remains difficult because the tips are generally much larger than the nano-device, meaning that the measured temperature is the average temperature of a much larger areas than the device. Based on the reports, the local temperature in nano-antennas and the local electric field enhancement remain hard to be directly measured.i pi y We have indirectly measured the electric field enhancement of nano-antennas by using Raman spectroscopy (the Raman intensity is proportional to the fourth power of the electric field), but only at room temperature44. The Raman equipment used has an internal laser and optical parts that could be damaged at high temperatures and, moreover, the equipment does not have enough room to accommodate heaters to control the temperature of the antennas. Conclusions In outline, for high power laser operation, we have proposed a new methodology to design high fluence bowtie nano-antennas based on diverse materials such as gold, chromium and tungsten. The results show that in spite of the fact that tungsten have 58.7% lower electric field enhancement than gold under the same laser excitation, it can work at 300 times higher fluence as well as tungsten can accomplish 7.22 times higher magnitude of electric field than that of gold counterpart when they work at their threshold fluences-therefore, they may be valuable to work with high power lasers. Moreover, we measured the optical properties for gold, chromium and tungsten at 200 °C temperature and our result shows that the real part of index for all materials remain almost intact, however, the imaginary part for gold changes dramatically, and for tungsten still shows almost similar value to that at room temperature. Therefore, tungsten may perform more stably under high power laser application compared with gold. Discussion h b Although Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y Methodsh The numerical analyses of the optical properties of our nano-antennas are conducted by using commercial three-dimensional FDTD software45 with perfectly matched layer boundary conditions. We have used three dif- ferent materials such as gold, chromium, and tungsten in our nano-antennas. In the simulations for Figs 2 and 3, the refractive indices of different materials were set by using the Lumerical software’s default data, while for Fig. 8 the indices were measured by using an ellipsometer at different temperatures and used as an input parameter to the Lumerical FDTD software. The whole structure is surrounded by air and the incident wave is assumed to be a quasi-plane wave. The spot size diameter of the source is much larger than the computational area of the structure. The mesh size is chosen as Δx = Δy = Δz = 5 nm. Methodsh For tungsten Ti was used as adhesion layer to the substrate, whereas for chromium due to its good adhesive property, no titanium adhesion layer was used. The nano-antenna patterns were milled by FEI Helios NanoLab 600 dual beam focused ion beam (FIB) system. All the scanning electron microscope (SEM) images were taken using either FIB or Electron Beam Lithography (EBL-Raith 150) system. g p y ( ) y For the characterization of our devices, a Lastek Q-switched commercial laser with pulse duration of 10 ns was used, which operates at the wavelength of 1053 nm. Generally, the spot size of the laser is 2 mm with a maximum Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ Power supply Sample holder Microscope objective lens F= 10.6 mm 1.4 kHz signal Q-switched laser =1053 nm Signal generator Variable attenuator 0.4 dB- 60 dB Power supply Polarizer Sample 9.6 mm Figure 9. The schematic diagram of the laser setup for the characterization process of nano-antennas. Microscope objective lens F= 10.6 mm Polarizer Sample Q-switched laser =1053 nm 9.6 mm Signal generator Variable attenuator 0.4 dB- 60 dB Figure 9. The schematic diagram of the laser setup for the characterization process of nano-antennas. Detector Mirror/CCD Focusing Lens Shutter Focusing lens Spectrum Analyzer Power supply Sample in Ceramic Chamber Halogen Light Source Figure 10. Schematic diagram of experimental setup to measure the transmission of thin film. Sample in Ceramic Chamber Shutter Figure 10. Schematic diagram of experimental setup to measure the transmission of thin film. energy of 50 μJ and the calculated maximum fluence of this laser is 0.785 J/m2. However, the spot diameter of laser can be changed using Olympus 10x infinity plan microscope objective lens that helps to increase the fluence (energy density). The estimated maximum fluence of this laser using the microscopic lens is 83.055 J/m2. The normal working distance of that lens is 10.6 mm, however-the light start ti diverge beyond this distance. In our experiment, we fixed the sample 9.6 mm away from lens. The whole characterization process is shown in Fig. 9, where a 1.4 kHz repetition rate signal is used.fi Finally, to assess how heat generation in nano-antennas can affect the electric field enhancement factor, we apply different temperatures with our home-made setup and measure the temperature dependent optical prop- erties of the metals. We have used two different setups. Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y Methodsh To measure the transmission, a home-built white-light spectroscopy setup in confocal configuration is used which is shown in Fig. 10. The sample is illuminated by a white-light source (fiber coupled tungsten halogen light bulb) and an infrared (IR) spectrometer is used. For transmission spectra measurement at different temperatures, a Mitutoyo M Plan NIR 10x NA 0.26 objective is used, with the light transmitted through the sample being measured at different temperatures, then collected and directed to the spectrometer. A modified home-made heater with two electrodes is used control the sample tem- peratures. To perform this experiment 15 nm film thickness is used for all metals, which are deposited by a sput- ter system. The temperature in the surface of thin films is measured by an IR thermo-meter. On the other hand the refractive indices at different temperatures are measured using a Variable Angle Spectroscopic Ellipsometer 9 Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y www.nature.com/scientificreports/ (VASE). It is noted that in order to apply various temperatures in the sample under the same condition while measuring the optical properties, a same voltage dependent heater is mounted onto the VASE. Then, these exper- imental data (refractive indices) for three materials are used as input parameters to Lumerical FDTD software to calculate the electric field enhancement factor. (VASE). It is noted that in order to apply various temperatures in the sample under the same condition while measuring the optical properties, a same voltage dependent heater is mounted onto the VASE. Then, these exper- imental data (refractive indices) for three materials are used as input parameters to Lumerical FDTD software to calculate the electric field enhancement factor. References Gonzalez-Rubio, G., Guerrero-Maritnez, A. & Liz-Marzan, L. y p 22. Roxworthy, B. J., Bhuiya, A. M., Inavalli, V. K., Chen, H. field optical intensity. Nano letters 14, 4687–4693 (2014).i 22. Roxworthy, B. J., Bhuiya, A. M., Inavalli, V. K., Chen, H. & Toussaint, K. C. Jr. Multifunctional plasmonic film for recording near- field optical intensity. Nano letters 14, 4687–4693 (2014).i y y field optical intensity. Nano letters 14, 4687–4693 (2014). i p y ( ) 23. Sun, Y. et al. Approach for fine-tuning of hybrid dimer antennas via laser melting at the nanoscale. Annalen der Physik 529, 1600272 (2 i 23. Sun, Y. et al. Approach for fine-tuning of hybrid dimer antenn i 23. Sun, Y. et al. Approach for fine-tuning of hybrid dimer antennas via laser melting at the nanoscale. An i tasseva, A. & Shalaev, V. M. Refractory plasmonics. Science 344, 263 25. Mironov, E. G. et al. Titanium nano-antenna for high-power pulsed operation. J. Light. Technol. 31, 2459–2466 (2013). 26. Hattori, H. T., Li, Z., Liu, D., Rukhlenko, I. D. & Premaratne, M. Coupling of light from microdisk lasers into plasmonic antennas. Opt. Express 17, 20878–20884 (2009). p p 7. Kosako, T., Kadoya, Y. & Hofmann, H. F. Directional control of light by a nano-optical yagi–uda antenna. Nat. Photonics 4, 312 (2010). 8. González, F. & Boreman, G. Comparison of dipole, bowtie, spiral and log-periodic ir antennas. Infrared Phys. & Technol. 46, 418–428 (2005). (2005). 29. Hatab, N. A. et al. Free-standing optical gold bowtie nanoantenna with variable gap size for enhanced raman spectroscopy. Nano letters 10 4952–4955 (2010) 9. Hatab, N. A. et al. Free-standing optical gold bowtie nanoantenna with variable gap size for enhanced raman spectroscopy. Nano letters 10, 4952–4955 (2010). 0. Coppens, Z. J., Li, W., Walker, D. G. & Valentine, J. G. Probing and controlling photothermal heat generation in plasmonic nanostructures. Nano letters 13, 1023–1028 (2013). . Govorov, A. O. & Richardson, H. H. Generating heat with metal n Richardson, H. H. Generating heat with metal nanoparticles. Nano 32. Johnson, P. & Christy, R. Optical constants of transition metals: Ti, v, cr, mn, fe, co, ni, and pd. Phys. Rev. B 9, 5056 (1974 y y 33. Ordal, M. A., Bell, R. J., Alexander, R. W., Newquist, L. A. & Querry, M. R. Optical properties of al, fe, ti, ta, w, and mo at submillimeter wavelengths. Appl. optics 27, 1203–1209 (1988). Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y References & Brongersma, M. L. Optical antenna thermal em . Schuller, J. A., Taubner, T. & Brongersma, M. L. Optical antenna t g 3. Deng, Y.-L. & Juang, Y.-J. Black silicon sers substrate: effect of surface morphology on sers detection and application of single alga cell analysis. Biosens. Bioelectron. 53, 37–42 (2014). 4. Neubrech, F., Huck, C., Weber, K., Pucci, A. & Giessen, H. Surface-enhanced infrared spectroscopy using resonant nanoantennas Chem. reviews 117, 5110–5145 (2017). 15. Nie, S. & Emory, S. R. Probing single molecules and single nanoparticles by surface-enhanced raman scattering. science 275, 1102–1106 (1997).fi 6. Chou, C.-H., Chen, C.-D. & Wang, C. C. Highly efficient, wavelength-tunable, gold nanoparticle based optothermal nanoconvertors The journal physical chemistry B 109, 11135–11138 (2005). fi The journal physical chemistry B 109, 11135–11138 (2005). h 17. Govorov, A. O. et al. Gold nanoparticle ensembles as heaters and actuators: melting and collective plasmon resonances. Nanoscale Res. Lett. 1, 84 (2006). ( ) 18. Chen, J. et al. Gold nanocages as photothermal transducers for cancer treatment. Small 6, 811–817 (2010). g p 19. Fang, Z. et al. Evolution of light-induced vapor generation at a liquid-immersed metallic nanoparticle. Nano letters 13, 1736–1742 (2013). g p 19. Fang, Z. et al. Evolution of light-induced vapor generation at a liquid-immersed metallic nanoparticle. Nano letters 13, 1736–1742 (2013). 20. Boriskina, S. V., Weinstein, L. A., Tong, J. K., Hsu, W.-C. & Chen, G. Hybrid optical–thermal antennas for enhanced light focusing d l l l h ( ) t al. Evolution of light-induced vapor generation at a liquid-immersed . Fang, Z. et al. Evolution of light-induced vapor generation at a liquid 19. Fang, Z. et al. Evolution of light induced vapor generation at a liquid immersed metallic nanoparticle. Nano letters 13, 1736 1742 (2 20. Boriskina, S. V., Weinstein, L. A., Tong, J. K., Hsu, W.-C. & Chen, G. Hybrid optical–thermal antennas for enhanced light foc and local temperature control. ACS Photonics 3, 1714–1722 (2016). 20. Boriskina, S. V., Weinstein, L. A., Tong, J. K., Hsu, W.-C. & Chen, G. Hybrid optical–thermal antennas and local temperature control. ACS Photonics 3, 1714–1722 (2016). p 1. Gonzalez-Rubio, G., Guerrero-Maritnez, A. & Liz-Marzan, L. M. Reshaping, fragmentation, and assembly of gold nanoparticle assisted by pulse lasers. Accounts chemical research 49, 678–686 (2016).i p 21. Gonzalez-Rubio, G., Guerrero-Maritnez, A. & Liz-Marzan, L. M. Reshaping, fragm assisted by pulse lasers. Accounts chemical research 49, 678–686 (2016). 21. References References 1. Dorfmuller, J. et al. Plasmonic nanowire antennas: Experiment, simulation, and theory. Nano Lett. 10, 3596–3603, https://doi org/10.1021/nl101921y PMID: 20726567 (2010). g y 2. Bharadwaj, P., Deutsch, B. & Novotny, L. Optical antennas. Adv. Opt. Photon. 1, 438–483, https://doi.org/10.1364/AOP.1.000438 (2009). 2. Bharadwaj, P., Deutsch, B. & Novotny, L. Optical antennas. Adv. Opt. Photon. 1, 438–483, https://doi.org/10.1364/AOP.1.000438 (2009). 3. Alda, J., Rico-García, J. M., López-Alonso, J. M. & Boreman, G. Optical antennas for nano-photonic applications. Nanotechnology (2009). 3. Alda, J., Rico-García, J. M., López-Alonso, J. M. & Boreman, G. Optical antennas for nano-photonic applications. Nanotechnology 16, S230 (2005). ( ) 3. Alda, J., Rico-García, J. M., López-Alonso, J. M. & Boreman, G. Optical antennas for nano-photonic applications. Nanotechn 16, S230 (2005). ( ) 4. Yu, N. et al. Bowtie plasmonic quantum cascade laser antenna. Opt. Express 15, 13272–13281 (2007). p q p p 5. Fromm, D. P., Sundaramurthy, A., Schuck, P. J., Kino, G. & Moerner, W. E. Gap-dependent optical coupling of single “b nanoantennas resonant in the visible. Nano Lett. 4, 957–961, https://doi.org/10.1021/nl049951r (2004). p g 6. Novotny, L. & Van Hulst, N. Antennas for light. Nat. photonics 5, 83 (2011). y g p 7. Zhao, C., Liu, Y., Yang, J. & Zhang, J. Single-molecule detection and radiation control in solutions at high concentrations heterogeneous optical slot antenna. Nanoscale 6, 9103–9109 (2014). 7. Zhao, C., Liu, Y., Yang, J. & Zhang, J. Single-molecule detection and radiation c heterogeneous optical slot antenna. Nanoscale 6, 9103–9109 (2014). g p 8. Cao, L., Park, J.-S., Fan, P., Clemens, B. & Brongersma, M. L. Resonant germanium nanoantenna photodetectors. Nano letter 1229–1233 (2010). , , , J , , , , g , g p 1229–1233 (2010). 9 Roxworthy B J et al Application of plasmonic bowtie nanoantenna arrays for optical trapping stacking and sorting Nano letter 9. Roxworthy, B. J. et al. Application of plasmonic bowtie nanoantenna arrays for optical trapping, stacking, and sorting. Nano letter 12, 796–801 (2012).fi 0. Eggleston, M. S. & Wu, M. C. Efficient coupling of an antenna-enhanced nanoled into an integrated inp waveguide. Nano letters 15 3329–3333 (2015). ( ) 11. Briones, E. et al. Seebeck nanoantennas for solar energy harvesting. Appl. Phys. Lett. 105, 093108 (2014). gy g pp y 12. Schuller, J. A., Taubner, T. & Brongersma, M. L. Optical antenna thermal emitters. Nat. Photonics 3, 658 (2009).f gy g pp J. A., Taubner, T. Acknowledgementsh g The authors gratefully acknowledge the fabrication facilities provided by Australian National Fabrication Facility (ANFF ACT node, Australia). We would acknowledge the financial support from UNSW Canberra, Australia. We also would like to thank The Asian Office of Aerospace Research and Development (AOARD US Air Force FA2386-15-1-4084), Australian Research Council (ARC LP160100253, DP170103778 and DE190100413) to provide the funding. References g pp p ( ) 34. Johnson, P. B. & Christy, R.-W. Optical constants of the noble metals. Phys. review B 6, 4370 (1972). 35. Solutions, F. Engineering toolbox. Inc., https://www.engineeringtoolbox.com/thermal-conductivity-metals-d-858.html. (2019). g g p g g y 36. Ma, H., Tian, P., Pello, J., Bendix, P. M. & Oddershede, L. B. Heat generation by irradiated complex composite nanostructures. N Lett. 14, 612–619, https://doi.org/10.1021/nl403798j PMID: 24392799 (2014). P., Pello, J., Bendix, P. M. & Oddershede, L. B. Heat generation by 36. Ma, H., Tian, P., Pello, J., Bendix, P. M. & Oddershede, L. B. Heat generation by irradiated co Lett. 14, 612–619, https://doi.org/10.1021/nl403798j PMID: 24392799 (2014). p g j 37. Moulijn, J., van Diepen, A. & Kapteijn, F. Catalyst deactivation: is it predictable?: What to do? Appl. Catal. A: Gen. 212, 3–16, ht doi.org/10.1016/S0926-860X(00)00842-5 Catalyst Deactivation (2001).h g y 8. Veith, G. M. et al. Thermal stability and catalytic activity of gold nanoparticles supported on silica. J. Catal. 262, 92–101, https://doi org/10.1016/j.jcat.2008.12.005 (2009). g j j ( ) 9. Kuznetsov, A. I., Miroshnichenko, A. E., Brongersma, M. L., Kivshar, Y. S. & Luk’yanchuk, B. Optically resonant dielectric nanostructures. 354, https://doi.org/10.1126/science.aag2472 (2016).h p g g 40. Ekici, O. et al. Thermal analysis of gold nanorods heated with femtosecond laser pulses. J. Phys. D: Appl. Phys. 41, 185501 (2008). ff l b d h f l f p g g et al. Thermal analysis of gold nanorods heated with femtosecond l 40. Ekici, O. et al. Thermal analysis of gold nanorods heated with fe ,h y g p J y pp y , ( 41. Hoffmann, B. et al. Fabrication and characterization of plasmonic nanocone antennas for strong spontaneous emi enhancement. Nanotechnology 26, 404001, https://doi.org/10.1088/0957-4484/26/40/404001 (2015).if gy g 42. Downes, A., Salter, D. & Elfick, A. Heating effects in tip-enhanced optical microscopy. Opt. Express 14, 5216–5222, https://doi. org/10.1364/OE.14.005216 (2006). 43. Alabastri, A. et al. Molding of plasmonic resonances in metallic nanostructures: Dependence of the non-linear electric permittivity on system size and temperature. Materials 6, 4879–4910 (2013).i y p ( ) 44. Khaleque, A. et al. Integration of bow-tie plasmonic nano-antennas on tapered fibers. Opt. Express 25, 8986–8996 (2017). 45. Solutions, F. Lumerical solutions. Inc., http://www.lumerical.com (2016). 10 www.nature.com/scientificreports/ Author Contributions M.M. performed the numerical simulation, fabrication and characterization of antennas. Z.Y.L. supervised the fabrication and characterization of antennas. A.H. helped to process the characterization, L.L. helped with the fabrication, M.M., A.A.R. and M.R. measured the transmission and optical properties of thin films. L.F., B.C.O. and A.M. helped for the interpretation of the results. M.M., H.H. and Z.Y. Li prepared the manuscript. H.H. devised and supervised the whole project. All authors contributed critical revision of paper and gave final approval of the manuscript for submission. Additional Informationh Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports \| (2019) 9:13023 \| https://doi.org/10.1038/s41598-019-49517-y
https://openalex.org/W4200500883	https://digital.csic.es/bitstream/10261/308958/1/Subsurface_iron_Bressac.pdf	English	null	Subsurface iron accumulation and rapid aluminum removal in the Mediterranean following African dust deposition	Biogeosciences	2,021	cc-by	17,014	sac1,2, Thibaut Wagener3, Nathalie Leblond4, Antonio Tovar-Sánchez5, Céline Ridame6, 1Sorbonne Université, CNRS, Laboratoire d’Océanographie de Villefranche, LOV, 06230 Villefranche-sur-Mer, France 2Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Tasmania, Australia 3Aix Marseille Univ., CNRS, IRD, Université de Toulon, MIO UMR 110, 13288 Marseille, France 4Sorbonne Université, CNRS, Institut de la Mer de Villefranche, IMEV, 06230 Villefranche-sur-Mer, France 5Department of Ecology and Coastal Management, Institute of Marine Sciences of Andalusia (ICMAN-CSIC), 07190 Puerto Real, Spain 6Sorbonne Université, LOCEAN, 4 Place Jussieu – 75252 Paris Cedex 05, France 7Department of Environmental and Earth Sciences, University of Milano–Bicocca, Milan, Italy 8Laboratoire des Sciences du Climat et de l’Environnement (LSCE), UMR 8212 CEA-CNRS-UVSQ, Institut Pierre-Simon Laplace, Université Paris-Saclay, 91191 Gif-sur-Yvette, France 9Laboratoire Interuniversitaire des Systèmes Atmosphériques (LISA), UMR7583 CNRS, Université de Paris, Université Paris-Est Créteil, Institut Pierre-Simon Laplace, 75013 Paris, France Correspondence: Matthieu Bressac (matthieu.bressac@imev-mer.fr) Received: 2 April 2021 – Discussion started: 13 April 2021 Revised: 3 November 2021 – Accepted: 18 November 2021 – Published: 15 December 2021 Received: 2 April 2021 – Discussion started: 13 April 2021 Revised: 3 November 2021 – Accepted: 18 November 2021 – Received: 2 April 2021 – Discussion started: 13 April 2021 Revised: 3 November 2021 – Accepted: 18 November 2021 – Published: 15 December 2021 Abstract. Mineral dust deposition is an important supply mechanism for trace elements in the low-latitude ocean. Our understanding of the controls of such inputs has been mostly built on laboratory and surface ocean studies. The lack of di- rect observations and the tendency to focus on near-surface waters prevent a comprehensive evaluation of the role of dust in oceanic biogeochemical cycles. In the frame of the PEACETIME project (ProcEss studies at the Air-sEa Inter- face after dust deposition in the MEditerranean sea), the re- sponses of the aluminum (Al) and iron (Fe) cycles to two dust wet deposition events over the central and western Mediter- ranean Sea were investigated at a timescale of hours to days using a comprehensive dataset gathering dissolved and sus- pended particulate concentrations, along with sinking ﬂuxes. Dissolved Al (dAl) removal was dominant over dAl re- leased from dust. The Fe/Al ratio of suspended and sinking particles revealed that biogenic particles, and in particular diatoms, were key in accumulating and exporting Al rela- tive to Fe. By combining these observations with published Al/Si ratios of diatoms, we show that adsorption onto bio- Abstract. sac1,2, Thibaut Wagener3, Nathalie Leblond4, Antonio Tovar-Sánchez5, Céline Ridame6, Mineral dust deposition is an important supply mechanism for trace elements in the low-latitude ocean. Our understanding of the controls of such inputs has been mostly built on laboratory and surface ocean studies. The lack of di- rect observations and the tendency to focus on near-surface waters prevent a comprehensive evaluation of the role of dust in oceanic biogeochemical cycles. In the frame of the PEACETIME project (ProcEss studies at the Air-sEa Inter- face after dust deposition in the MEditerranean sea), the re- sponses of the aluminum (Al) and iron (Fe) cycles to two dust wet deposition events over the central and western Mediter- ranean Sea were investigated at a timescale of hours to days using a comprehensive dataset gathering dissolved and sus- pended particulate concentrations, along with sinking ﬂuxes. genic particles, rather than active uptake, represents the main sink for dAl in Mediterranean waters. In contrast, system- atic dissolved Fe (dFe) accumulation occurred in subsurface waters (∼100–1000 m), while dFe input from dust was only transient in the surface mixed layer. The rapid transfer of dust to depth, the Fe-binding ligand pool in excess to dFe in sub- surface (while nearly saturated in surface), and low scaveng- ing rates in this particle-poor depth horizon are all important drivers of this subsurface dFe enrichment. At the annual scale, this previously overlooked mechanism may represent an additional pathway of dFe supply for the surface ocean through diapycnal diffusion and vertical mix- ing. However, low subsurface dFe concentrations observed at the basin scale (< 0.5 nmol kg−1) cause us to question the residence time for this dust-derived subsurface reservoir and hence its role as a supply mechanism for the surface ocean, stressing the need for further studies. Finally, these contrast- ing responses indicate that dAl is a poor tracer of dFe input in the Mediterranean Sea. Dissolved Al (dAl) removal was dominant over dAl re- leased from dust. The Fe/Al ratio of suspended and sinking particles revealed that biogenic particles, and in particular diatoms, were key in accumulating and exporting Al rela- tive to Fe. By combining these observations with published Al/Si ratios of diatoms, we show that adsorption onto bio- Research article Research article Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Subsurface iron accumulation and rapid aluminum removal in the Mediterranean following African dust deposition Matthieu Bressac1,2, Thibaut Wagener3, Nathalie Leblond4, Antonio Tovar-Sánchez5, Céline Ridame6, Vincent Taillandier1, Samuel Albani7,8, Sophie Guasco3, Aurélie Dufour3, Stéphanie H. M. Jacquet3, François Dulac Karine Desboeufs9, and Cécile Guieu1 1 Introduction provided a unique opportunity to directly observe the biogeochemical effects of two min- eral dust wet deposition events of contrasting intensity that occurred during late spring 2017 in the central and western open Mediterranean Sea (Guieu et al., 2020). The presence of the R/V before, during, and/or a few days after deposition allowed us to investigate (1) the parameters and processes shaping the contrasting distributions of dAl and dFe, (2) the importance of the timescale considered when assessing the ﬂux of bioavailable Fe to the surface ocean, and (3) the rele- vance of using dAl to constrain dFe input from dust. African dust deposition events have long been known to impact trace element concentrations and ﬂuxes in the upper water column of the Mediterranean (e.g., Buat-Ménard et al., 1989; Davies and Buat-Ménard, 1990; Quétel et al., 1993; Guerzoni et al., 1999; Heimbürger et al., 2011). Our under- standing of the role of dust in marine biogeochemical cycles remains limited, however, partly resulting from the difﬁculty in quantifying atmospheric dust ﬂuxes to the surface ocean at short timescales. In the absence of direct assessments of atmospheric inputs, marine concentrations of tracers such as aluminum (Al) have been widely used to constrain these ﬂuxes (e.g., Measures and Brown, 1996; Han et al., 2008; Anderson et al., 2016; Menzel Barraqueta et al., 2019). Al is predominantly of crustal origin and is characterized by a sim- ilar fractional solubility to Fe with a longer residence time in seawater. Al could thus be used to constrain the integrated input of dust Fe over seasonal timescales (Dammshäuser et al., 2011). However, the fact that the distribution of Al can itself be controlled by biological activity (e.g., Mackenzie et al., 1978; Moran and Moore, 1988a; Li et al., 2013; Middag et al., 2015; Rolison et al., 2015) causes us to question its quality as a tracer. In addition, since dust deposition is highly episodic in time and spatially patchy (Donaghay et al., 1991; Guieu et al., 2014a; Vincent et al., 2016), direct observations at sea are extremely challenging and hence sparse (e.g., Croot et al., 2004; Rijkenberg et al., 2008). To overcome this limitation, a variety of small-volume enclosed systems have been used to quantify Fe solubility from dust. 1 Introduction demonstrated the pivotal role played by the in situ biogeo- chemical conditions at the time of deposition in controlling post-depositional processes (i.e., dissolution, organic com- plexation, adsorption, colloidal aggregation) and their inter- play (Wagener et al., 2010; Bressac and Guieu, 2013; Wuttig et al., 2013). The most striking and unexpected consequence is that upon deposition, dust can act as a net sink of dFe through scavenging (Wagener et al., 2010; Ye et al., 2011). Second, the large range in Fe solubility observed in seawater depending on the season reveals that oceanic rather than at- mospheric conditions determine the ﬂux of “truly” bioavail- able Fe to the surface ocean (Bressac and Guieu, 2013). However, these ﬁndings are only valid in the ﬁrst meters of the water column, and direct observations of the whole water column are needed if we are to fully understand the role of dust in the oceanic iron cycle. Globally, iron (Fe) supply to the surface ocean sets the productivity of major phytoplankton groups (Moore et al., 2013). Among the multiple sources of Fe, atmospheric depo- sition of mineral dust represents an important supply mech- anism in the low-latitude ocean (Duce et al., 1991; Jickells et al., 2005; Conway and John, 2014) and plays a key role in the functioning of low- nutrient, low-chlorophyll (LNLC) systems (e.g., Guieu et al., 2014a). Despite widespread at- tention over the last 3 decades, large uncertainties remain in the factors controlling aerosol Fe solubility (Meskhidze et al., 2019). Consequently, poorly constrained controls of Fe solubility partly explain the substantial inter-model differ- ence in the atmospheric dFe input ﬂux to the global ocean (∼1–30 Gmol yr−1; Tagliabue et al., 2016) and hinder accu- rate predictions of the impact of dust on ocean productivity. For this purpose, the Mediterranean Sea is a particularly relevant region. This semi-enclosed basin, characterized by a west-to-east gradient in oligotrophy, receives some of the largest dust inputs of the ocean (Guerzoni et al., 1999), mostly in the form of wet deposition in the central and west- ern part of the basin, and a few intense events may account for the bulk of the annual deposition (Loÿe-Pilot and Mar- tin, 1996; Vincent et al., 2016). The PEACETIME project (ProcEss studies at the Air-sEa Interface after dust deposition in the MEditerranean sea) and oceanographic campaign on board the R/V Pourquoi Pas? M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6436 1 Introduction Although yielding im- portant insights into atmospheric trace element solubilities (Baker and Croot, 2010, and references therein), these sys- tems do not fully simulate in situ conditions (de Leeuw et al., 2014), motivating the development of larger volume ex- periments (> 100 L), where dust particles are free to sink and interact with dissolved and particulate organic matter while sinking (Bressac and Guieu, 2013; Guieu et al., 2014b; Herut et al., 2016; Gazeau et al., 2021). Two key ﬁndings emerged from these large-volume experiments. First, they 2.1 Oceanographic cruise The PEACETIME cruise (https://doi.org/10.17600/15000900) was conducted during late spring conditions in May and June 2017 aboard the R/V Pourquoi Pas? in the central and western Mediterranean Sea. In total, 10 short stations (∼8 h) and 3 long stations located in the Tyrrhenian Sea (TYR; occupation = 4 d), the Ionian Sea (ION; 4 d), and in the western Algerian basin (FAST; 5 d) were occupied (Fig. 1). FAST was an opportunistic station dedicated to investigating the biogeochemical effects of a dust deposition event by combining atmospheric and oceanographic in situ measurements before, during, and after deposition (Guieu et al., 2020). At all stations, a 2.3 Suspended particulate trace metals standard CTD rosette with 24 Niskin bottles (12 L) equipped with a Sea-Bird SBE9 CTD, transmissometer, chlorophyll ﬂuorescence (Chelsea Acquatracka), photosynthetically active radiation and oxygen (SBE43) sensors, and a trace metal clean (TMC) titanium rosette were deployed to sample the water column for biological and chemical parameters. Samples for aluminum and iron analyses were collected using the TMC titanium rosette mounted with GO-FLO bottles deployed on a Kevlar cable with a dedicated clean winch, while samples for particulate Al (pAl) determination were also collected at all the stations from the standard rosette (see Sect. 2.3 and Supplement Table S1). Just prior to sampling for particulate trace metals (pTMs), GO-FLO bottles were gently mixed, and pTMs were sam- pled directly online from the pressurized (0.2 µm ﬁltered N2) GO-FLO bottles onto acid-cleaned 25 mm diameter Su- por 0.45 µm polyethersulfone ﬁlters mounted on Swinnex polypropylene ﬁlter holders (Millipore), following GEO- TRACES recommendations. Filtration was stopped when the ﬁlter clogged or the bottle was empty. On average, each par- ticulate concentration was obtained by ﬁltering 4.8 L (range 1.1–10.2 L). When the ﬁltration was complete, ﬁlter hold- ers were transferred under a laminar ﬂow hood, and residual seawater was removed using a polypropylene syringe. Fil- ters were stored in acid-cleaned petri slides, left open un- der the laminar ﬂow hood for ∼24 h to allow the ﬁlters to dry. Particulate samples were digested (10 % HF / 50 % HNO3 (v/v)) following the protocol described in the GEO- TRACES “Cookbook” (Cutter et al., 2010) and Planquette and Sherrell (2012). Procedural blanks consisted of unused acid-cleaned ﬁlters. Analyses were performed on a HR-ICP- MS (high-resolution inductively coupled plasma mass spec- trometer; Element XR, Thermo-Fisher Scientiﬁc). The accu- racy of the measurements was established using the certiﬁed reference materials (CRMs) MESS-4 and PACS-3 (marine sediments, National Research Council Canada) (Supplement Table S2). The https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6437 0.2 and 0.5 nmol kg−1. The reagent blank determined by measuring acidiﬁed ultrapure water varied between 0.9 and 1.7 nmol kg−1. Figure 1. Sampling locations during the PEACETIME cruise (ﬁlled circles). The cruise track consisted of 10 short stations and 3 long stations (TYR, ION, and FAST). Open circles correspond to the sta- tions 64PE370_12 (orange), 64PE374_13 (red), and 64PE374_12 (green) from the GEOTRACES GA04 section (Rolison et al., 2015; Gerringa et al., 2017) used for comparison in Fig. 3. Note that the same color code is used in Figs. 3, 6, and 7. g Dissolved Fe concentrations were measured (mostly on board in the class-100 clean laboratory) using an auto- mated ﬂow injection analysis (FIA) with online precon- centration and chemiluminescence detection (Bonnet and Guieu, 2006). The stability of the analysis was assessed by analyzing an internal acidiﬁed seawater standard daily. On average, the DL was 15 pmol kg−1 (3 times the SD of the concentration measured ﬁve times from the same dFe-poor seawater), and the accuracy of the method was controlled by analyzing the GEOTRACES seawater stan- dards SAFe D1 (0.64 ± 0.13 nmol kg−1 (n = 19), consensus value 0.67 ± 0.04 nmol kg−1), GD (1.04 ± 0.10 nmol kg−1 (n = 10), consensus value 1.00 ± 0.10 nmol kg−1), and GSC (1.37 ± 0.16 nmol kg−1 (n = 4), consensus value 1.50 ± 0.11 nmol kg−1) on a regular basis. Figure 1. Sampling locations during the PEACETIME cruise (ﬁlled circles). The cruise track consisted of 10 short stations and 3 long stations (TYR, ION, and FAST). Open circles correspond to the sta- tions 64PE370_12 (orange), 64PE374_13 (red), and 64PE374_12 (green) from the GEOTRACES GA04 section (Rolison et al., 2015; Gerringa et al., 2017) used for comparison in Fig. 3. Note that the same color code is used in Figs. 3, 6, and 7. 2.2 Dissolved Al and Fe concentrations Immediately after recovery, the GO-FLO bottles were trans- ferred inside a class-100 clean laboratory container. Seawa- ter samples were directly ﬁltered from the GO-FLO bottles through acid-cleaned 0.2 µm capsule ﬁlters (Sartorius Sarto- bran P capsule 0.45/0.2 µm). Dissolved Fe and Al samples were stored in acid-washed low-density polyethylene bottles and immediately acidiﬁed to pH 1.8 (quartz-distilled HCl) under a laminar ﬂow hood. Dissolved Al analyses were conducted on board using the ﬂuorometric method described by Hydes and Liss (1976). Brieﬂy, the samples were buffered to pH 5 with ammonium acetate, and the reagent lumogallion was added. The samples were then heated to 80 ◦C for 1.5 h to accelerate the com- plex formation. The ﬂuorescence of the sample was mea- sured with a Jasco FP-2020 Plus spectroﬂuorometer (ex- citation wavelength 495 nm, emission wavelength 565 nm). Calibration was realized with additions of Al standard solu- tion in seawater. The detection limit (DL; 3 times the stan- dard deviation (SD) of the concentrations measured from the dAl-poor seawater used for calibration) varied between In addition, pAl concentrations were also obtained at all the stations from the standard rosette (Supplement Ta- ble S1). This additional pAl dataset already published by Jacquet et al. (2021) was obtained according to the sam- pling, processing, and analysis methods described in Jacquet al. (2015). Brieﬂy, 4 to 6 L of seawater collected with the Niskin bottles was ﬁltered onto acid-cleaned 47 mm poly- carbonate ﬁlters (0.4 µm porosity). Filters were rinsed with Milli-Q grade water and dried at 50 ◦C. A total digestion https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6438 Table 1. Mean ﬂux values for total mass (dry weight), particulate organic carbon (POC), particulate inorganic carbon (PIC), particulate organic nitrogen (PON), biogenic silica (BSi), lithogenic silica (LSi), and particulate aluminum and iron collected at TYR, ION, and FAST (data not available at 500 m depth at TYR due to technical problems with the sediment trap). Values in parentheses correspond to the standard deviation of the arithmetic mean of the 4 (TYR and ION) and 5 d (FAST) deployment period. 2.2 Dissolved Al and Fe concentrations Latitude Longitude Depth Mass POC PIC PON BSi LSi Al Fe ◦N ◦E [m] [mg m−2 d−1] [µmol m−2 d−1] [µmol m−2 d−1] [µmol m−2 d−1] [µmol m−2 d−1] [µmol m−2 d−1] [µmol m−2 d−1] [µmol m−2 d−1] TYR 39.34 12.59 222 64.5 (54.3) 340.7 (215.1) 157.1 (109.1) 40.2 (13.4) 88.2 (78.7) 339.6 (308.9) 100.6 (101.9) 25.3 (25.6) 500 – – – – – – – – 1056 76.0 (21.4) 185.2 (49.1) 225.2 (51.9) 16.8 (4.5) 86.0 (27.4) 458.2 (192.1) 135.6 (40.0) 34.9 (11.6) ION 35.49 19.80 265 34.1 (19.1) 315.6 (146.5) 56.5 (29.9) 31.3 (14.4) 64.4 (53.1) 126.8 (94.3) 34.0 (28.3) 8.5 (6.7) 560 30.1 (10.2) 144.2 (22.9) 108.3 (37.2) 12.6 (1.2) 48.3 (15.6) 125.7 (39.5) 46.2 (18.3) 11.9 (4.4) 1097 30.1 (9.3) 115.7 (16.5) 98.7 (56.6) 7.6 (2.0) 36.3 (12.1) 126.8 (36.5) 45.7 (15.7) 12.3 (3.9) FAST 37.95 2.91 259 29.1 (14.1) 368.2 (139.5) 67.6 (62.0) 45.8 (20.6) 90.8 (58.9) 72.8 (48.6) 26.5 (19.9) 6.1 (4.5) 498 33.9 (10.5) 403.8 (150.3) 116.6 (85.0) 56.7 (26.0) 60.7 ( 42.3) 68.6 (64.3) 27.1 (22.2) 6.3 (5.2) 1004 36.4 (17.7) 379.0 (318.5) 74.1 (44.2) 53.0 (50.7) 59.3 (29.8) 82.4 (57.0) 38.0 (19.6) 9.1 (4.8) https://doi.org/10.5194/bg-18-6435-20 of the membranes was performed using a tri-acid mixture (0.5 mL HF / 1.5 mL HNO3 / 1 mL HCl), and analyses were performed on the same HR-ICP-MS. A good agreement was obtained when comparing pAl concentrations obtained with the TMC and standard rosettes at ION and FAST (differ- ence in sampling time at TYR prevents quantitative compar- ison; see Sect. 4.1) (Supplement Fig. S1), demonstrating the absence of contamination for pAl when using the standard rosette. 2.5 Inventories and Kd The extension and dynamics of this dust trans- port event were reasonably well forecasted by the various existing regional dust transport models available during the campaign (Supplement Fig. S3), including the set of mod- els of the World Meteorological Organization Sand and Dust Storm Warning Advisory and Assessment System operated by the Barcelona Supercomputing Center (BSC; Huneeus et al., 2016), the SKIRON/Dust model operated by the Atmo- spheric Modeling and Weather Forecasting Group of the Na- tional University of Athens (Spyrou et al., 2010), the CAMS model operated by the European Centre for Medium-Range Weather Forecasts (ECMWF; Flemming et al., 2015), the NCEP/GFS model operated by the U.S. National Weather Service (Han et al., 2017), the NAAPS model modiﬁed from that of Christensen (1997) and operated by the U.S. Naval Research Laboratory, and the TAU/DREAM8 model oper- ated by the Weather Research Centre of the Tel Aviv Uni- versity (TAU; Kishcha et al., 2008). The dust plume exten- sion in the cloudy area on 11 May is illustrated by Sup- plement Fig. S3a and b. Most meteorological models pre- dicted signiﬁcant precipitation over the Tyrrhenian Sea on 11 May (Supplement Fig. S3c). Dust transport models pro- ducing dust deposition ﬂuxes generally forecasted dust wet deposition on 11 May between Tunisia and Italy but with sig- niﬁcant variability on the location, extent, and schedule. The NMMB/BSC and SKIRON models predicted a signiﬁcant At the FAST station, the partitioning coefﬁcient be- tween the particulate and dissolved phases (Kd; [particu- late] / [dissolved]) was used to investigate exchanges be- tween dissolved and particulate pools of Al and Fe. Follow- ing the relative change over time of this metric allowed po- tential artifacts related to change in water masses driven by lateral advection to be excluded (Guieu et al., 2020). 2.5 Inventories and Kd Discrete measurements at different depths were used to cal- culate the water-column integrated Al and Fe inventories (µmol m−2) by trapezoidal integration. The concentration measured nearest to the surface was assumed to be constant up to 0 m. At FAST, six replicate measurements of dAl and dFe were performed at 5 and 400 m depth from two sets of six GO-FLO bottles. The CVs obtained at 5 and 400 m depths were used to determine the uncertainties in the 0–20 and 0– 200 m inventories, respectively. Variability among replicates was higher for dFe (CV = 11.3 % and 6.9 % at 5 and 400 m depth, respectively) than for dAl (CV = 5.3 % and 1.1 % at 5 and 400 m depth, respectively), potentially reﬂecting a small- scale variability in the dFe distribution. The impact in the water column of two dust deposition events of contrasting magnitudes was studied during the cruise. They occurred in the area of the TYR and FAST stations (Fig. 1), on 11–12 May and 3–5 June, respectively. The ﬁrst deposition event in the southern Tyrrhenian Sea was not di- rectly observed but hypothesized based on satellite observa- tions of intense dust plume transport and water-column Al inventory presented in the following. The combined analy- sis of time series of quick-looks of operational aerosol prod- ucts from MSG/SEVIRI (Fig. 2a) and from meteorologi- cal and dust transport models available during the campaign (Guieu et al., 2020; Supplement Fig. S3) allowed us to sus- pect that a red rain event likely occurred over the south- ern Tyrrhenian Sea on 11 May and possibly early on 12 May. The daytime daily mean aerosol optical depth (AOD) product over oceanic areas (Thieuleux et al., 2005) shows that a large dust plume was exported from the Tunisian and Libyan coasts towards southern Italy and Greece from 10 May (Fig. 2a). Up to 1.5–1.6 AOD at 550 nm (AOD550) (i.e., about 2 g m−2 of dust in the column assuming a speciﬁc extinction cross-section of dust of 0.77 m2 g−1 following Du- lac et al., 1992) was observed on 12 May north of Sicily in the area of the Tyrrhenian stations, a rather high value relatively unusual in this area (Gkikas et al., 2016). On 11 May, clouds developed over most of the area affected by dust, whereas clear (turbid) sky was again present during daytime on 12 May. 2.4 Export ﬂuxes and composition Sinking particles were collected at ∼200, 500, and 1000 m depth using PPS5 sediment traps (Technicap, France; 1 m2 collection area) deployed on a free-drifting mooring for 4 (TYR and ION) and 5 d (FAST). Cups were ﬁlled with ﬁl- tered seawater and buffered formaldehyde (2 % ﬁnal concen- tration) as a biocide. Once recovered, each cup representing 24 h of collection was stored in the dark at 4 ◦C until pro- cessed. Samples were treated following the standard proto- col followed at the “Cellule Pièges”, a national service of the French INSU-CNRS (Guieu et al., 2005), following the JGOFS’ protocol. After removing the swimmers, the remain- ing sample was rinsed three times with ultrapure water in or- der to remove salt and then freeze-dried. The total amount of material collected was weighed to quantify the total exported ﬂux. Several aliquots were then used to measure the follow- ing components: total and organic carbon, particulate Al and Fe, and lithogenic and biogenic silica (LSi and BSi, respec- tively). Total carbon, particulate organic carbon (POC) (after removing inorganic carbon by acidiﬁcation with HCl 2N), and particulate organic nitrogen (PON) were measured on a CHN elemental analyzer (2400 Series II CHNS/O Elemen- tal Analyzer, Perkin Elmer). For one sample (TYR 1000 m), ﬁve aliquots were analyzed, yielding a coefﬁcient of vari- ation (CV) of 6 %. Particulate inorganic carbon (PIC) was quantiﬁed by subtracting POC from total particulate carbon. Particulate Fe and Al concentrations were determined by an ICP-AES (inductively coupled plasma atomic emission spec- trometer, Spectro ARCOS Ametek) after acid digestion fol- lowing the protocol described in Ternon et al. (2010). Blanks were negligible (< 0.8 % of the lowest Al and Fe concentra- tions of the digested aliquots), and the efﬁciency of the acid digestion was established using the CRM GBW-07313 (ma- rine sediment, National Research Center for CRMs, China) (Supplement Table S2). Samples for BSi and LSi (two or three aliquots) were digested (NaOH at 95 ◦C and HF at am- bient temperature, respectively) and concentrations analyzed by colorimetry (Analytikjena Specor 250 Plus spectropho- tometer) (Nelson et al. 1989; Mullin and Riley, 1955). Mean export ﬂuxes and composition of exported material are pre- sented in Table 1. https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 6439 3.3.1 Central Mediterranean Sea Dust deposition ﬂux over the Tyrrhenian Sea was estimated from the Alexcess inventories corresponding to the differ- ence between the measured 0–1000 m pAl inventories and a background 0–1000 m pAl inventory. In the absence of pre-depositional observations and historic pAl data (to the best of our knowledge), the median pAl vertical proﬁle ob- tained during the cruise at the other stations not impacted by this event (bold grey line on Fig. 3a–d), similar or slightly higher than pAl data available for the open Mediterranean Sea (e.g., Sarthou and Jeandel, 2001), was used as a back- ground level. The comparison between the measured pAl vertical proﬁles and this background level revealed a marked excess in pAl south of Sardinia (ST04) and in the southern Tyrrhenian (ST05, TYR, and ST06; Fig. 3a–d and Table 2). This spatial extent is in good agreement with the maps of precipitation and dust wet deposition provided for 11 May by the ARPEGE, SKIRON, and NMMB/BSC models (Supple- ment Fig. S3). The obtained Alexcess inventories were further corrected for the loss of pAl associated with the sinking ﬂux using the pAl downward ﬂux measured at 1000 m depth at the TYR station (assuming a constant ﬂux over the 3 to 10 d period after deposition). Assuming that Al represents 7.1 % of the dust in mass (Guieu et al., 2002), and further assuming that Alexcess resulted from a single dust event, a dust deposi- tion ﬂux ranging between 1.7 (ST06) and 9.2 g m−2 (ST04) was derived from these Alexcess inventories (Table 2). Large During the early June deposition event in the western Al- gerian basin, precipitation was directly observed in the area of the R/V and even sampled on board (Desboeufs et al., 2021), associated with a dust transport event of moderate ex- tent and intensity over the southwestern Mediterranean basin. The AOD550 peaked at about 0.40 in the area of the FAST station (Desboeufs et al., 2021), corresponding to a maxi- mum columnar dust load < 0.4 g m−2, assuming a non-dust background AOD550 in the boundary layer of 0.10–0.15, as observed north of the plume or the day before the plume arrived. This dust plume encountered a massive rain front covering ∼80 000 km2 and moving eastward from Spain and North Africa regions (Desboeufs et al., 2021). 3.3 Reconstruction of the dust deposition ﬂuxes The absence of direct measurement of the dust deposition ﬂux over the Tyrrhenian Sea and the limited spatial cover- age of collection of atmospheric dust and rain at the FAST station call for an alternative approach to estimate dust depo- sition ﬂuxes. For this purpose, we used the water-column Al inventory. We acknowledge that this approach involves un- certainties, as do all the observational approaches employed so far to quantify deposition (Anderson et al., 2016). Caveats include (1) other sources of pAl and (2) some uncertainties into the derived dust ﬂuxes that could come from the sam- pling method (Twining et al., 2015a), the time lag between deposition and sampling favoring dispersion of dust by lat- eral mixing, and, to a lesser extent, the limited vertical reso- lution below 500 m depth (Fig. 3a–d). M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal However, the half-hourly, 0.1◦resolution Global Precipita- tion Measurement (GPM) mission Integrated Multi-satellitE Retrievals for GPM (IMERG) ﬁnal run images of the rain- fall rate (GPM_3IMERGHH_v06 product; Huffman et al., 2015) report the occurrence of light rains in the Tyrrhenian Sea in the early morning of 11 May, especially around 12◦E and between 39 and 40◦N (< 1 mm of accumulated pre- cipitation, with a large error of up to several millimeters; Fig. 2b). The study of the TRMM-3B42-v6 product, a former multi-satellite, 3-hourly precipitation product in the western Mediterranean region has shown that the detection of light rainfall is difﬁcult when compared to rain gauge observa- tions, with many occurrences missed by the satellite product (Sarrand et al., 2012). Indeed, the minimum corresponding random error image is 0.238 mm h−1 for pixels without de- tected rain, which can be assimilated to a lower detection limit. During the whole day, some light rain cells remain vis- ible near Sardinia, Sicily, or southern Italy. It is therefore well possible that a much larger area of the Tyrrhenian Sea than that reported by the GPM IMERG images was affected by light precipitation on that day. For simplicity, 11 May 2017, at 18:00 UTC, will be considered as the time of deposition, that is approximately 3 to 10 d before our sampling of the area. boeufs et al., 2021). This second rain event was characterized by a dust signature clearly revealed by the chemical com- position of the rain. From the total (dissolved + particulate) Al concentration measured in this rainwater sample, a dust ﬂux of 65 ± 18 mg m−2 was derived (Desboeufs et al., 2021). This sampled ﬂux, considered as relatively modest compared to the multi-year record in this area (Vincent et al., 2016), was likely in the lower range of the total dust deposition ﬂux that affected the whole area between 3 and 5 June. 3.1 Biogeochemical conditions The PEACETIME cruise took place in late spring when the stratiﬁcation of the upper water column was well established with the mixed layer depth ranging between ∼10 and 20 m along the cruise track (Van Wambeke et al., 2020). Sur- face chlorophyll a concentrations (< 0.1 µg L−1) were typi- cal of oligotrophic conditions (Guieu et al., 2020). A diatom- dominated deep chlorophyll maximum (DCM) that coin- cided with a maximum in biomass and primary production was well developed and observed all along the cruise track (Marañón et al., 2021). POC downward ﬂuxes measured at 200 m depth were similar at the three long stations, while downward ﬂuxes of Al and LSi, two proxies for dust, were maximum at TYR (Table 1). At the surface, dAl distribution was characterized by a marked west-to-east increasing gra- dient (Supplement Fig. S2b), driven by advective mixing be- tween (dAl poor) Atlantic and Mediterranean waters and by the accumulation of dust and reﬂected by a strong relation- ship between surface dAl concentrations and salinity (Guer- zoni et al., 1999; Rolison et al., 2015). All along the tran- sect, dFe concentrations were high in the upper 100 m (up to 2.7 nmol kg−1) and decreased to levels < 0.5 nmol kg−1 below the euphotic layer (Supplement Fig. S2c). Subsurface patches of high dFe concentrations previously observed in the eastern Mediterranean basin, and attributed to hydrother- mal activity and mud-volcanoes (Gerringa et al., 2017), were not observed along our cruise track. https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum remov M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6440 wet deposition ﬂux of dust, with at least 1.5 g m−2 over 6 h in the area of our stations ST05, TYR, and ST06 in the after- noon of 11 May (Supplement Fig. S3d and e). The DREAM model versions operated by the BSC and TAU, however, forecasted much lower values or even no dust wet deposi- tion in the Tyrrhenian stations area (Supplement Fig. S3f). However, model reanalyses now available do not show any precipitation in the area between 10 and 13 May (Fig. 2b). However, the half-hourly, 0.1◦resolution Global Precipita- tion Measurement (GPM) mission Integrated Multi-satellitE Retrievals for GPM (IMERG) ﬁnal run images of the rain- fall rate (GPM_3IMERGHH_v06 product; Huffman et al., 2015) report the occurrence of light rains in the Tyrrhenian Sea in the early morning of 11 May, especially around 12◦E and between 39 and 40◦N (< 1 mm of accumulated pre- cipitation, with a large error of up to several millimeters; Fig. 2b). The study of the TRMM-3B42-v6 product, a former multi-satellite, 3-hourly precipitation product in the western Mediterranean region has shown that the detection of light rainfall is difﬁcult when compared to rain gauge observa- tions, with many occurrences missed by the satellite product (Sarrand et al., 2012). Indeed, the minimum corresponding random error image is 0.238 mm h−1 for pixels without de- tected rain, which can be assimilated to a lower detection limit. During the whole day, some light rain cells remain vis- ible near Sardinia, Sicily, or southern Italy. It is therefore well possible that a much larger area of the Tyrrhenian Sea than that reported by the GPM IMERG images was affected by light precipitation on that day. For simplicity, 11 May 2017, at 18:00 UTC, will be considered as the time of deposition, that is approximately 3 to 10 d before our sampling of the area. wet deposition ﬂux of dust, with at least 1.5 g m−2 over 6 h in the area of our stations ST05, TYR, and ST06 in the after- noon of 11 May (Supplement Fig. S3d and e). The DREAM model versions operated by the BSC and TAU, however, forecasted much lower values or even no dust wet deposi- tion in the Tyrrhenian stations area (Supplement Fig. S3f). However, model reanalyses now available do not show any precipitation in the area between 10 and 13 May (Fig. 2b). 3.3.1 Central Mediterranean Sea Direct atmo- spheric and oceanographic observations of this event were possible thanks to a dedicated “fast action” strategy (see Guieu et al., 2020, for details). Two rain periods concomi- tant with the dust plume transported in altitude (1 to 4 km) allowed below-cloud deposition of dust in the FAST station area, as conﬁrmed by onboard lidar records (Desboeufs et al., 2021). The ﬁrst rain period occurred on 3 June in the neighboring area of the R/V, and the second one occurred from 4 (22:00 UTC) to 5 June (09:00 UTC) and was sampled on board the R/V on 5 June from 00:36 to 01:04 UTC (Des- https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6441 Figure 2. (a) MSG/SEVIRI-derived daily (daytime) mean aerosol optical depth; the white ellipse on 11 May image includes the location of the ﬁve Tyrrhenian stations reported in Fig. 1; (b) Left: time-averaged map of the GPM mission multi-satellite precipitation ﬁnal run estimate with gauge calibration (mm h−1) over 04:00–05:30 UTC on 11 May 2017 in the western Mediterranean region, from the half-hourly, 0.1◦ resolution GPM_3IMERGHH v06 product (Huffmann et al., 2015). Right: corresponding random error (mm h−1). After images produced by the Giovanni online data system (Acker and Lepkouth, 2007). Figure 2. (a) MSG/SEVIRI-derived daily (daytime) mean aerosol optical depth; the white ellipse on 11 May image includes the location of the ﬁve Tyrrhenian stations reported in Fig. 1; (b) Left: time-averaged map of the GPM mission multi-satellite precipitation ﬁnal run estimate with gauge calibration (mm h−1) over 04:00–05:30 UTC on 11 May 2017 in the western Mediterranean region, from the half-hourly, 0.1◦ resolution GPM_3IMERGHH v06 product (Huffmann et al., 2015). Right: corresponding random error (mm h−1). After images produced by the Giovanni online data system (Acker and Lepkouth, 2007). part of the basin. By assuming that the deposition was spa- tially homogeneous over the southern Tyrrhenian, an Al ex- port ﬂux of more than 4000 µmol m−2 d−1 is needed to ex- plain the difference in the Alexcess inventory observed be- tween ST04, ST05, and TYR (i.e., ∼3.6 to 8.4 d after depo- sition). 3.3.1 Central Mediterranean Sea This is an order-of-magnitude difference compared to the Al export ﬂux measured at TYR ∼5 to 8 d after depo- sition (136 ± 40 µmol m−2 d−1; Table 1), indicating that the observed spatial variability was primarily driven by the pre- cipitation patchiness rather than related to the time lag be- tween deposition and observations. uncertainties are associated with these dust ﬂux estimates, partly due to potential additional sources of pAl that are sus- pected for ﬂuxes derived from ST04 and ST05 but unlikely for TYR and ST06 (see Sect. 4.1). Nevertheless, the approach remains valuable to estimate the magnitude of this dust event. This range of dust deposition ﬂux is of similar magni- tude to the annual ﬂux observed during former periods in the west central (7.4 g m−2 yr−1; Vincent et al., 2016) and north- western Mediterranean Sea (11.4 g m−2 yr−1; Ternon et al., 2010), highlighting the remarkable magnitude of this event. Furthermore, this comparison with annual ﬂuxes conﬁrms that the annual deposition of African dust in the Mediter- ranean region is generally driven by only a few intense events (Loÿe-Pilot and Martin, 1996; Guerzoni et al., 1999; Kubilay et al., 2000; Desboeufs et al., 2018). The strong spatial vari- ability of these dust ﬂux estimates, with a marked west-to- east gradient, might result from the varying time lag between deposition and sampling of the water column at these dif- ferent stations (Table 2) but also from the patchiness of the rainfalls associated with the rain front (Supplement Fig. S3). Indeed, Vincent et al. (2016) showed that high-deposition events in the western Mediterranean are often limited spa- tially, although the associated dust plumes may affect a large M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6442 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum remov Figure 3. Vertical distribution of pAl (a, b, c, d), dAl (e, f, g, h), and dFe concentrations (i, j, k, l) obtained during the PEACETIME cruise (ﬁlled circles) at ST04 (a, e, i), ST05 (b, f, j), TYR (c, g, k), and ST06 (d, h, l). Particulate Al concentrations in (c) correspond to the vertical proﬁles TYR_2 and TYR_3 (Fig. 5). Previously published vertical proﬁles of dAl (Rolison et al., 2015) and dFe (Gerringa et al., 2017) obtained at similar locations (Fig. 1) are superimposed (open circles). The median pAl vertical proﬁle (bold lines) is used as a background level (see Sect. 3.3.1 for details), and the ﬁrst and third quantiles (grey shaded areas) are represented in (a)–(d). Figure 3. Vertical distribution of pAl (a, b, c, d), dAl (e, f, g, h), and dFe concentrations (i, j, k, l) obtained during the PEACETIME cruise (ﬁlled circles) at ST04 (a, e, i), ST05 (b, f, j), TYR (c, g, k), and ST06 (d, h, l). Particulate Al concentrations in (c) correspond to the vertical proﬁles TYR_2 and TYR_3 (Fig. 5). Previously published vertical proﬁles of dAl (Rolison et al., 2015) and dFe (Gerringa et al., 2017) obtained at similar locations (Fig. 1) are superimposed (open circles). The median pAl vertical proﬁle (bold lines) is used as a background level (see Sect. 3.3.1 for details), and the ﬁrst and third quantiles (grey shaded areas) are represented in (a)–(d). Desboeufs et al., 2021). Based on the increase in the 0–20 m total Al inventory, and assuming 7.1 % Al in the dust (Guieu et al., 2002), a total dust input of 55 mg m−2 was derived. Although direct collection of atmospheric dust aerosols rep- resents the most straightforward approach for quantifying the dust ﬂux, it only has a limited spatial coverage. In con- trast, the upper water-column inventory integrated most of the patchy rainfalls associated with this large rain front. This difference in time and space integrations is best illustrated by the ∼70 % increase in the 0–20 m pAl and pFe invento- ries observed on 4 June (Fig. 4c), i.e., several hours before the rainfall collected onboard the R/V and probably asso- ciated with surrounding precipitation. 3.3.2 Western Mediterranean Sea At the FAST station, dissolved and particulate Al and Fe con- centrations were measured at high temporal and vertical res- olutions before, during, and after the wet deposition of dust (Supplement Fig. S4). About 6 h after deposition, the total (dissolved + particulate) Al and Fe inventories within the up- per 20 m increased by ∼145 and 48 µmol m−2, respectively (Fig. 4a and c). This increase in the 0–20 m inventories was consistent but higher than the atmospheric Al and Fe ﬂuxes collected on the R/V (∼98 and 25 µmol m−2, respectively; https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 4 Discussion in particular the resuspension of sediments and their advec- tive transport from continental margins (e.g., Misic et al., 2008). The Strait of Sicily, characterized by high turbidity values (Gdaniec et al., 2018), represents a zone of formation for nepheloid layers. Levantine intermediate waters can then act as a conveyor belt that accumulates and transports parti- cles from the eastern to western basin of the Mediterranean Sea (Taillandier et al., 2020). Stations ST04 and ST05, lo- cated in the southwestern sector of the Tyrrhenian Sea (i.e., M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6443 Table 2. Estimates of the input of pAl, dAl, and dust south of Sardinia (ST04) and over the Tyrrhenian Sea (ST05, TYR, and ST06) based on the 0–1000 m Alexcess inventories. The relatively low vertical resolution at TYR_1 precludes accurate estimates. Table 2. Estimates of the input of pAl, dAl, and dust south of Sardinia (ST04) and over the Tyrrhenian Sea (ST05, TYR, and ST06) based on the 0–1000 m Alexcess inventories. The relatively low vertical resolution at TYR_1 precludes accurate estimates. Time since Al2excess Al loss3 pAl input4 dAl input5 dust ﬂux6 deposition1 [d] [µmol m−2] [µmol m−2] [µmol m−2] [µmol m−2] [g m−2] ST04 3.6 23 684 485 24 169 363 9.2 ST05 4.5 17 773 610 18 382 276 6.9 TYR_1 5.6 – – – – – TYR_2 6.4 3591 874 4465 67 1.7 TYR_3 8.4 4837 1143 5980 90 2.3 ST06 10.5 3126 1423 4549 68 1.7 1 11 May 2017 18:00 UTC is considered as the time of deposition. 2 Difference between the measured and median 0–1000 m pAl inventories (see Sect. 3.3.1 for details). 3 Estimates based on the downward Al ﬂux (1000 m depth; TYR) and assuming a constant ﬂux over time. 4 Corresponds to Alexcess corrected for Al loss. 5 Estimates based on an Al fractional solubility of 1.5 % (Wuttig et al., 2013). 6 Estimates based on an Al composition of the dust of 7.1 % (Guieu et al., 2002). 1 11 May 2017 18:00 UTC is considered as the time of deposition. 2 Difference between the measured and median 0–1000 m pAl inventories (see Sect. 3.3.1 for details). 3 Estimates based on the downward Al ﬂux (1000 m depth; TYR) and assuming a constant ﬂux over time. 4 Corresponds to Alexcess corrected for Al loss. 5 Estimates based on an Al fractional solubility of 1.5 % (Wuttig et al., 2013). 6 Estimates based on an Al composition of the dust of 7.1 % (Guieu et al., 2002). 1 11 May 2017 18:00 UTC is considered as the time of deposition. 2 Difference between the measured and median 0–1000 m pAl inventories (see Sect. 3.3.1 for details). 3 Estimates based on the downward Al ﬂux (1000 m depth; TYR) and assuming a constant ﬂux over time. 4 Corresponds to Alexcess corrected for Al loss. 5 Estimates based on an Al fractional solubility of 1.5 % (Wuttig et al., 2013). M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6 Estimates based on an Al composition of the dust of 7.1 % (Guieu et al., 2002). Figure 4. Temporal evolution of the dissolved (a, b) and particulate (c, d) Al (black) and Fe (orange) inventories within the 0–20 (a, c, e) and 0–200 m (b, d, f) depth ranges at the FAST station. Only dissolved inventories were measured on 3 June. The relative change in the coefﬁcient of partitioning between the particulate and dissolved fractions (Kd) for Al and Fe is presented in (e) and (f). Note that 0–200 m inventories measured on 4 June (instead of 2 June) were used for the initial Kd in (f) (see Sect. 4.1 for details). Grey vertical bars indicate the two dusty rain events that occurred in the FAST station area. The dotted grey vertical line corresponds to the time of the dusty rainfall sampled on board the R/V (Desboeufs et al., 2021). Figure 4. Temporal evolution of the dissolved (a, b) and particulate (c, d) Al (black) and Fe (orange) inventories within the 0–20 (a, c, e) and 0–200 m (b, d, f) depth ranges at the FAST station. Only dissolved inventories were measured on 3 June. The relative change in the coefﬁcient of partitioning between the particulate and dissolved fractions (Kd) for Al and Fe is presented in (e) and (f). Note that 0–200 m inventories measured on 4 June (instead of 2 June) were used for the initial Kd in (f) (see Sect. 4.1 for details). Grey vertical bars indicate the two dusty rain events that occurred in the FAST station area. The dotted grey vertical line corresponds to the time of the dusty rainfall sampled on board the R/V (Desboeufs et al., 2021). M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal It must be noted that the water-column approach is also subject to uncertainties, and we cannot exclude an underestimation of the deposi- tion ﬂux due to the rapid sinking of the largest dust particles (e.g., Bressac et al., 2012). However, no evidence of these fast-sinking particles was found deeper in the water column (Fig. 4d), nor within the sediment traps (not shown). Desboeufs et al., 2021). Based on the increase in the 0–20 m total Al inventory, and assuming 7.1 % Al in the dust (Guieu et al., 2002), a total dust input of 55 mg m−2 was derived. Although direct collection of atmospheric dust aerosols rep- resents the most straightforward approach for quantifying the dust ﬂux, it only has a limited spatial coverage. In con- trast, the upper water-column inventory integrated most of the patchy rainfalls associated with this large rain front. This difference in time and space integrations is best illustrated by the ∼70 % increase in the 0–20 m pAl and pFe invento- ries observed on 4 June (Fig. 4c), i.e., several hours before the rainfall collected onboard the R/V and probably asso- https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 4.2 Dust dynamic in the water column In the Tyrrhenian Sea, deposition of dust was evidenced by the Al and LSi downward ﬂuxes measured at 200 and 1000 m depth ∼5 to 9 d after deposition that are > 3 times higher relative to those measured at ION and FAST at the same depths (Table 1). At TYR, Al and LSi ﬂuxes both increased by 35 % between 200 and 1000 m depth, suggesting that a signiﬁcant fraction of the dust particles were rapidly trans- ferred to depth. This trend is consistent with the pAl verti- cal proﬁles at the four stations likely impacted by this event, as a subsurface maximum was depicted between ∼200 and 500 m depth (Fig. 3a–d). In addition, three pAl vertical pro- ﬁles performed at TYR over ∼72 h showed a continuous de- crease in surface pAl concentration of 20 µmol m−2 d−1 that was accompanied by subsequent increases within the ∼150– 500 m depth layer (Fig. 5). It is worth noting that ∼5.6 d after the event, a remarkably high pAl concentration was observed at 1000 m depth (∼260 nmol kg−1 (TYR_1); not shown), which could indicate that dust particles were sinking at a rate of ∼180 m d−1. This ﬁnding conﬁrms that dust parti- cles can be rapidly transferred to depth either alone (Bressac et al., 2012) or incorporated into biogenic aggregates (e.g., Hamm, 2002; Bressac et al., 2014; Laurenceau-Cornec et al., 2020; van der Jagt et al., 2018). Together, these observations demonstrate the atmospheric origin of pAl observed in the southern Tyrrhenian (rather than sediment resuspension or advective inputs) and conﬁrm that a signiﬁcant fraction of the dust particles (coarse fraction) can rapidly leave the sur- face mixed layer when the stratiﬁcation is strong (Croot et al., 2004; Ternon et al., 2010; Nowald et al., 2015), while the remaining fraction (small-sized particles) likely accumulates along the thermocline until the disruption of the stratiﬁcation (Migon et al., 2002). Figure 5. Time evolution of the pAl vertical distribution measured at three different times over ∼72 h at the TYR station. Light shaded areas correspond to the uncertainty in pAl measurement. The orange (TYR_1), red (TYR_2), and blue (TYR_3) vertical proﬁles were measured approximately 5.6, 6.4, and 8.4 d after the dust deposition event, respectively. tion (Guieu et al., 2020). 4.2 Dust dynamic in the water column Deeper in the water column, the trend is more complicated to interpret with a 40 % decrease (∼2000 µmol m−2) of the 0–200 m pAl inventory that oc- curred before/during deposition (Fig. 4d). This unexpected decrease cannot be explained by the vertical transfer of pAl, as only ∼130 µmol m−2 of pAl was exported out of the upper 200 m over 5 d (data not shown). On the other hand, a south- westward ﬂow disrupted the water column in the ∼25–100 m depth range from 3 June bringing water masses of distinct properties (Guieu et al., 2020). Therefore, it is likely that the water mass sampled before deposition (2 June) was different from the one sampled during the rest of the time series. For this reason, inventories obtained on 4 June (instead of 2 June) were used as a background level to investigate the temporal evolution of Kd(Al) and Kd(Fe) in the 0–200 m depth range (Fig. 4f). M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal ubsurface iron accumulation and rapid aluminum removal Figure 5. Time evolution of the pAl vertical distribution measured at three different times over ∼72 h at the TYR station. Light shaded areas correspond to the uncertainty in pAl measurement. The orange (TYR_1), red (TYR_2), and blue (TYR_3) vertical proﬁles were measured approximately 5.6, 6.4, and 8.4 d after the dust deposition event, respectively. 6444 the branch of circulation between the Strait of Sicily and the Sardinian Channel), could be potentially impacted by parti- cles driven by this mechanism, contributing to the excess in pAl observed at those stations. Conversely, the central part of the Tyrrhenian Sea is characterized by low turbidity val- ues relative to the rest of the Mediterranean Sea (Gdaniec et al., 2018). During PEACETIME, lateral advection was negligible (Andrea Doglioli, personal communication, 2020) at stations TYR and ST06 precluding any contribution of lithogenic particles other than from dust atmospheric depo- sition. Evidence of a recent dust deposition event over the Tyrrhenian Sea, traced by the excess in pAl, is discussed in the next section. 4.1 Advective transport from continental margins in the central Mediterranean Sea In the absence of direct atmospheric measurements, large un- certainties are associated with the estimates of the dust de- position ﬂux over the Tyrrhenian Sea. These uncertainties are partly driven by potential additional sources of pAl and https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6445 Figure 6. Dissolved Al concentrations versus salinity measured in the upper 30 m of the Mediterranean Sea during the PEACETIME (ﬁlled circles), 64PE370, and 64PE374 cruises (open circles; Roli- son et al., 2015). Yellow, orange, blue, red, and green circles cor- respond to data obtained at FAST, ST04, ST05, TYR, and ST06, respectively (Fig. 1). Grey circles correspond to data obtained dur- ing the PEACETIME cruise at the other stations. dAl input ranging between 68 and 363 µmol m−2 (Table 2). Further assuming an homogeneous distribution within the 0– 20 m mixed layer, this dust event represented a dAl input of 3.3–17.7 nmol kg−1. However, the absence of noticeable anomalies in the long-recognized relationship between sur- face dAl concentrations and salinity, when compared with published data (Rolison et al., 2015), reveals a limited or transient impact of this event on surface dAl concentrations (Fig. 6). Several mechanisms can be invoked here to explain the absence of dAl signal in the upper water column fol- lowing the deposition. First, high surface dAl concentrations (> 20 nmol kg−1) might mask any additional input. At ST06, the putative dAl input of 3.3 nmol kg−1 (Table 2) is within the range of variability of both published and observed sur- face concentrations (30–43 nmol kg−1; Fig. 3h). At the other end (ST04), the situation is different with a dAl input of 17.7 nmol kg−1 that would represent more than 55 % of pre- depositional surface dAl concentrations (31–32 nmol kg−1; Rolison et al., 2015). Considering the short time lag between deposition and observations at ST04 (∼3.6 d), and the low horizontal advection at the TYR station area (Andrea Dogli- oli, personal communication, 2020), it is unlikely that advec- tive mixing diluted any elevated dAl signal from this event. Deeper in the water column, no clear trend was obtained with subsurface dAl concentrations lower (ST04; Fig. 3e) or slightly higher than background levels (TYR; Fig. 3g). Sim- ilarly, no noticeable increase in dAl could be observed at the FAST station in the mixed layer (Figs. 4a and 6). In contrast to Kd(Fe), Kd(Al) was still higher than the pre-depositional value 4 d after deposition (Fig. 4e), potentially reﬂecting a lower fractional solubility for dust-derived Al relative to Fe and/or a higher removal rate for dAl. Below the mixed layer, Kd(Al) remained relatively constant and similar to initial value (Fig. 4f). M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal Together, these observations indicate that wet deposition of dust over the FAST station area had a limited impact on the dAl inventory. Figure 6. Dissolved Al concentrations versus salinity measured in the upper 30 m of the Mediterranean Sea during the PEACETIME (ﬁlled circles), 64PE370, and 64PE374 cruises (open circles; Roli- son et al., 2015). Yellow, orange, blue, red, and green circles cor- respond to data obtained at FAST, ST04, ST05, TYR, and ST06, respectively (Fig. 1). Grey circles correspond to data obtained dur- ing the PEACETIME cruise at the other stations. et al., 2015b; Wuttig et al., 2013; Liu et al., 2019). To in- vestigate the respective role of particle adsorption and bio- logical uptake in removing dAl, Al was compared to Fe – a particle-reactive and bioactive element (Tagliabue et al., 2017) predominantly of crustal origin in the Mediterranean Sea – through the Fe/Al content of suspended and sinking particles collected at different depth horizons (Fig. 7). For suspended particles, the median Fe/Al ratio was max- imum within the surface mixed layer and minimum at the DCM (60–100 m; Fig. 7a), highlighting a strong contrast in Fe/Al between the diatom-dominated particle assemblage at the DCM (Marañón et al., 2021) and detrital and lithogenic particles in the rest of the water column. This contrast sup- ports the signiﬁcant role played by phytoplankton, and in par- ticular diatoms, in accumulating Al via active uptake (Gehlen et al., 2002; Liu et al., 2019) and/or adsorption onto cell membranes (Dammshäuser and Croot, 2012; Twining et al., 2015b). Note that we cannot exclude atmospheric input of BSi associated with dust particles (e.g., Folger et al., 1967). Regarding sinking particles, the relationship between the Fe/Al ratio and the relative proportion of LSi and BSi was relatively well represented by a two-component model, with BSi having a ﬁxed Fe/Al ratio of 0.21 mol mol−1 (value ob- served in the diatom-dominated DCM; Fig. 7a) and LSi hav- ing a Fe/Al ratio ranging between 0.26 (dusty rainwater sam- pled at FAST; Desboeufs et al., 2021) and 0.30 mol mol−1 (Saharan dust end-member; Guieu et al., 2002) (Fig. 7b). Sparse Al/Si ratios available for natural diatom communities range between ∼1 and 10 µmol mol−1 (van Bennekom et al., 1989; Gehlen et al., 2002; Koning et al., 2007). Using this 4.3.1 Absence of dAl anomalies A relatively large range in Al fractional solubility (1 %–15 %; deﬁned as the fraction of dust-derived Al that dissolves in rainwater or seawater) has been reported (e.g., Orians and Bruland, 1986; Baker et al., 2006; Measures et al., 2010; Han et al., 2012; Shelley et al., 2018). Assuming a conser- vative Al fractional solubility of 1.5 % in seawater (Wuttig et al., 2013), dust deposition over the Tyrrhenian Sea led to a At the FAST station, a dust deposition ﬂux that is 2 or- ders of magnitude lower (∼55 mg m−2) led to an increase of the 0–20 m pAl inventory by 78 % (Fig. 4c). About 24 h af- ter deposition, only ∼40 % of this signal was still present in the mixed layer. This is consistent with a short resi- dence time in surface water for a signiﬁcant fraction of the dust, although we cannot exclude the effect of lateral advec- Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal The two dotted black curves correspond to a two- component model, with BSi having a ﬁxed Fe/Al molar ratio of 0.21 mol mol−1 (i.e., value observed in the DCM in a) and LSi having a ﬁxed Fe/Al molar ratio of 0.26 (lower curve; Desboeufs et al., 2021) or 0.30 mol mol−1 (upper curve; Guieu et al., 2002). Yellow shaded areas in (a) and (b) represent the range in Fe/Al molar ratio proposed for Saharan dust, with the lower limit corresponding to dust aerosols collected at FAST (0.26 mol mol−1; Desboeufs et al., 2021) and the upper limit to the Saharan dust end-member (0.30 mol mol−1; Guieu et al., 2002). (TYR) (Fig. 3i–l). These observations made ∼3 to 10 d after deposition indicate that this event had no impact on dFe in the surface mixed layer at a timescale of days. At a shorter timescale, sampling performed at a high temporal resolution at the FAST station revealed two distinct increases of the 0–20 m dFe inventory that occurred during (+13 µmol m−2) and about 6 h after deposition (+15 µmol m−2; Fig. 4a). These ∼50 % increases were only transient, and the pre- depositional level was rapidly recovered. Considering that Fe cycling in this LNLC system is dominated by physico- chemical rather than biological processes, our ﬁndings are consistent with rapid scavenging of dFe in surface Mediter- ranean waters following dust deposition, as already reported in some mesocosm and minicosm dust addition experiments (Wagener et al., 2010; Wuttig et al., 2013; Bressac and Guieu, 2013). Overall, the Fe-binding ligand pool is nearly saturated in surface Mediterranean waters (Gerringa et al., 2017). As a consequence, any new input of dFe will tend to precipitate, pointing to the importance of the initial dFe and Fe-binding ligand concentrations in setting the net effect of dust input on dFe in the surface mixed layer (Ye et al., 2011; Wagener et al., 2010; Wuttig et al., 2013). range of values, the Al downward ﬂux at 200 m depth driven by (and incorporated into) BSi would represent only 0.1– 1.1 µmol m−2 d−1, i.e., ∼0.2 %–3.3 % of the total Al ﬂux. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal This difference with our conservative estimates of dAl in- puts over the Tyrrhenian Sea, by 2 orders of magnitude (68– 371 µmol m−2; Table 2), indicates that adsorption onto bio- genic particles (including BSi), rather than active uptake by diatoms, was likely the main sink for dAl in that region. 4.4.1 Transient dFe increase in the surface mixed layer The absence of pre-depositional observations in the Tyrrhe- nian Sea is more problematic for Fe compared to Al, as no clear longitudinal trend has been reported in the Mediter- ranean Sea for that element. Dissolved Fe vertical proﬁles were thus compared to previously published data that were obtained at similar locations (Fig. 1) and at the same period of the year for ST04 (mid-April) but about 2 months later at TYR and ST06 (early August) (Gerringa et al., 2017). Conse- quently, this approach ignores interannual and seasonal vari- abilities in dFe and cannot be used to strictly quantify dFe input but remains valuable to investigate its magnitude and vertical distribution. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6446 Figure 7. (a) Box-and-whisker plot of the Fe/Al molar ratio (mol mol−1) for suspended particles collected at TYR, ION, and FAST. The Fe/Al median values are 0.27 (n = 37; 0–20 m), 0.24 (n = 13; 20–60 m), 0.21 (n = 17; 60–100 m), 0.24 (n = 12; 100–200 m), 0.24 (n = 9; 200–500 m), and 0.24 (n = 10; 500-3000 m). For clarity, two outliers (Fe/Al = 0.50 and 1.02) observed in the 0–20 m depth range are not represented. (b) Fe/Al versus LSi/BSi molar ratios (mol mol−1) of sinking particles collected at ∼200 m (circles), 500 m (squares), and 1000 m depth (triangles) at the stations TYR (red), ION (purple), and FAST (yellow). The two dotted black curves correspond to a two- component model, with BSi having a ﬁxed Fe/Al molar ratio of 0.21 mol mol−1 (i.e., value observed in the DCM in a) and LSi having a ﬁxed Fe/Al molar ratio of 0.26 (lower curve; Desboeufs et al., 2021) or 0.30 mol mol−1 (upper curve; Guieu et al., 2002). Yellow shaded areas in (a) and (b) represent the range in Fe/Al molar ratio proposed for Saharan dust, with the lower limit corresponding to dust aerosols collected at FAST (0.26 mol mol−1; Desboeufs et al., 2021) and the upper limit to the Saharan dust end-member (0.30 mol mol−1; Guieu et al., 2002). Figure 7. (a) Box-and-whisker plot of the Fe/Al molar ratio (mol mol−1) for suspended particles collected at TYR, ION, and FAST. The Fe/Al median values are 0.27 (n = 37; 0–20 m), 0.24 (n = 13; 20–60 m), 0.21 (n = 17; 60–100 m), 0.24 (n = 12; 100–200 m), 0.24 (n = 9; 200–500 m), and 0.24 (n = 10; 500-3000 m). For clarity, two outliers (Fe/Al = 0.50 and 1.02) observed in the 0–20 m depth range are not represented. (b) Fe/Al versus LSi/BSi molar ratios (mol mol−1) of sinking particles collected at ∼200 m (circles), 500 m (squares), and 1000 m depth (triangles) at the stations TYR (red), ION (purple), and FAST (yellow). 4.3.2 Drivers of the rapid removal of dAl An Al fractional solubility of 5 % was measured in rainwater for dust aerosols collected at the FAST station (Desboeufs et al., 2021), i.e., well above the conservative value of 1.5 % ob- tained for a dust ﬂux of 10 g m−2 (Wuttig et al., 2013), and used to estimate dAl inputs over the Tyrrhenian Sea. This conﬁrms that dust loading partly controls the Al fractional solubility (e.g., Shelley et al., 2018). In addition, a biologi- cal control on dAl distribution in the Mediterranean Sea has been proposed to explain the strong coupling between dAl and orthosilicic acid (Si(OH)4) in subsurface waters (Chou and Wollast, 1997; Rolison et al., 2015). Several labora- tory and ﬁeld studies have demonstrated that marine phy- toplankton, in particular diatoms (mainly incorporated into the frustules; Gehlen et al., 2002), can take up and/or scav- enge dAl (Mackenzie et al., 1978; Orians and Bruland, 1986; Moran and Moore, 1988b; Loucaides et al., 2010; Twining Biogeosciences, 18, 6435–6453, 2021 Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 5 Conclusions During the PEACETIME cruise performed in May–June 2017 in the western and central Mediterranean, we observed two atmospheric wet deposition events while measuring Al and Fe water-column distributions, providing important in- sights into the timescale and pattern of dAl and dFe inputs from African dust in the remote Mediterranean Sea. The water-column Al inventories were successfully utilized to assess dust deposition ﬂuxes, complementing atmospheric measurements and the “fast-action” strategy used during the campaign to directly sample dusty rain events. Our obser- vations show that dAl removal through adsorption onto bio- genic particles was dominant over dAl released from dust at a timescale of hours to days. While surface dAl concen- trations reﬂect seasonal changes and large-scale patterns in dust deposition, this ﬁnding indicates that this tracer may not be appropriate to trace the imprint of a single dust de- position event in highly dust-impacted areas. Furthermore, dust deposition only represented a signiﬁcant input of dFe in the surface mixed layer on a timescale of hours. On a longer timescale (days/weeks), dFe inputs occurred primarily below the surface mixed layer extending to 1000 m depth where the Fe-binding ligand pool likely in excess of dFe allows any ad- ditional input of dFe to be stabilized. This mechanism may represent an additional pathway of dFe resupply for the sur- face ocean (through vertical mixing and diapycnal diffusion), although the residence time of this dust-derived dFe reservoir still needs to be investigated. To account for this dFe excess below the surface mixed layer, dust-bearing Fe must continue to dissolve as dust particles settle through the mixed layer and reach the mesopelagic. The short residence time for dust in surface water (Sect. 4.1) and the presence of a “refractory” Fe pool within dust particles that dissolves over several days (Wa- gener et al., 2008) conﬁrm that dust dissolution can occur in subsurface. It is also likely that the low particle concentra- tion encountered at these depths relative to the particle-rich surface waters at the time of deposition prevented rapid re- moval of dFe (e.g., Spokes et al., 1994; Bonnet and Guieu, 2004). Furthermore, the Fe-binding ligand pool is pivotal in setting the Fe fractional solubility (Rijkenberg et al., 2008; Wagener et al., 2008, 2010; Ye et al., 2011; Fishwick et al., 2014), and its magnitude, composition, and distribution likely shape patterns of dFe supply. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6447 By feeding the subsurface dFe reservoir, dust deposition could represent an indirect supply route for the surface ocean through vertical mixing and diapycnal diffusion (e.g., Tagli- abue et al., 2014). However, the residence time of this dust- derived reservoir remains an open question. Relatively low subsurface dFe concentrations observed at the basin-scale (< 0.5 nmol kg−1; Supplement Fig. S2c), compared to At- lantic waters for instance (Gerringa et al., 2017), support the argument in favor of a short residence time. Scavenging by sinking (dust) particles (e.g., Wagener et al., 2010; Bressac et al., 2019) and bacterial removal of humic-like ligands (Du- laquais et al., 2018; Whitby et al., 2020) represent two po- tential sinks for this subsurface dFe reservoir that need to be explored. input of dFe mainly below the mixed layer, as revealed by the opposite trends in Kd(Fe) observed in the 0–20 m and 0–200 m depth ranges (Fig. 4e–f). This increase in dFe rel- ative to pFe was persistent on a timescale of days (Fig. 4f) and was primarily driven by dust dissolution (Fig. 4b) rather than ballasting of preexisting pFe (Fig. 4d), as evidenced by the low-export Fe ﬂux collected at 200 m depth (1.7– 12.3 µmol m−2 d−1). This systematic excess in dFe observed below the mixed layer and extending to 1000 m suggests that the mechanisms involved are independent of the dust ﬂux – that differed by 2 orders of magnitude – and timescale con- sidered (hours to weeks). Such dust-related subsurface en- richment in dFe (without enhanced surface dFe concentra- tions) has already been observed in the subarctic Paciﬁc and tropical North Atlantic. This feature was attributed either to low oxygen levels, allowing Fe(II) to stay in solution (Schal- lenberg et al., 2017), or to remineralization of organic mat- ter formed in the dust-laden surface ocean (Measures et al., 2008; Fitzsimmons et al., 2013); two mechanisms that cannot be invoked here considering the oxygen levels in subsurface (170–200 µM) are the short timescale considered and the low mesopelagic Fe regeneration efﬁciency (Bressac et al., 2019). 4.4.2 Enrichment in dFe below the surface mixed layer Assuming a Fe content of 4.45 % in dust (Guieu et al., 2002), this dust event over the Tyrrhenian Sea represented a Fe input of ∼1300–7000 µmol m−2 (with a short retention time within the sea surface microlayer; Tovar-Sánchez et al., 2020). Yet, dFe concentrations within the surface mixed layer were at background levels (ST04 and ST06) or slightly below A key feature in the southern Tyrrhenian was the system- atic subsurface excess in dFe observed from ∼40 (ST04) and 200 m depth (TYR and ST06) (Fig. 3i–l) and mirror- ing the vertical distribution of Alexcess (Fig. 3a–d). Similarly, wet dust deposition over the FAST station area led to a net Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 References Acker, J. G. and Leptoukh, G.: Online analysis enhances use of NASA Earth science data, Eos Trans. AGU, 88, 14–17, https://doi.org/10.1029/2007EO020003, 2007. Anderson, R. F., Cheng, H., Edwards, R. L., Fleisher, M. Q., Hayes, C. T., Huang, K.-F., Kadko, D., Lam, P. J., Landing, W. M., Lao, Y., Lu, Y., Measures, C. I., Moran, S. B., Morton, P. L., Ohnemus, D. C., Robinson, L. F., and Shelley, R. U.: How well can we quantify dust deposition to the ocean?, Philos. T. R. Soc. A, 374, 20150285, https://doi.org/10.1098/rsta.2015.0285, 2016. Competing interests. The contact author has declared that neither they nor their co-authors have any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Baker, A. R. and Croot P. L.: Atmospheric and marine controls on aerosol iron solubility in seawater, Mar. Chem., 120, 4–13, https://doi.org/10.1016/j.marchem.2008.09.003, 2010. Baker, A. R., Jickells, T. D., Witt, M., and Linge, K. L.: Trends in the solubility of iron, aluminium, manganese and phosphorus in aerosol collected over the Atlantic Ocean, Mar. Chem., 98, 43– 58, https://doi.org/10.1016/j.marchem.2005.06.004, 2006. Special issue statement. This article is part of the special issue “At- mospheric deposition in the low-nutrient–low-chlorophyll (LNLC) ocean: effects on marine life today and in the future (ACP/BG inter- journal SI)”. It is not associated with a conference. Bonnet, S. and Guieu, C.: Dissolution of atmospheric iron in seawater, Geophy. Res. Lett., 31, L03303, https://doi.org/10.1029/2003GL018423, 2004. Bonnet, S. and Guieu, C.: Atmospheric forcing on the annual iron cycle in the Mediterranean Sea. A one-year survey, J. Geophys. Res., 111, C09010, https://doi.org/10.1029/2005JC003213, 2006. Acknowledgements. We thank the captain and crew of the R/V Pourquoi Pas?, the DT INSU for the design and preparation of the mooring line, and Nagib Bhairy, Guillaume De Liège, and Gilles Rougier for their help with the mooring. Hélène Ferré and the AERIS/SEDOO service are acknowledged for real-time collection during the cruise of maps produced from operational satellites and models used in this study, with appreciated contributions of EU- METSAT and ICARE for MSG/SEVIRI products, Météo-France for ARPEGE model outputs, the WMO SDS-WAS operated by the Barcelona Supercomputing Center (BSC) for DREAM and NMMB model outputs, and the AM&WFG of the University of Athens for SKIRON model outputs. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum remov Review statement. This paper was edited by Silvia Becagli and re- viewed by Thomas Holmes and one anonymous referee. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-18-6435-2021-supplement. Author contributions. MB, TW, and CG designed the study. MB wrote the manuscript. MB, TW, NL, ATS, CR, VT, SA, SG, and AD collected and/or analyzed the samples. FD and KD interpreted the atmospheric data. All the authors commented on and contributed to the improvement of the manuscript. 5 Conclusions While nearly saturated in surface, the Fe-binding ligand pool is in relatively large excess to dFe in subsurface Mediterranean waters (Gerringa et al., 2017) and hence available to stabilize new dFe. Impor- tantly, this subsurface pool is constantly replenished by bac- terial degradation of sinking biogenic particles (Boyd et al., 2010; Velasquez et al., 2016; Bressac et al., 2019; Whitby et al., 2020). Thus, there is a permanent resetting of the ligand pool while dust particles settle (Bressac et al., 2019), and, conceptually, we can imagine that the binding equilibrium between available ligands and Fe is rarely reached at these depths and timescale. This fundamental difference in the sur- face waters (and batch experiments) could explain the high Fe fractional solubility of 4.6 %–13.5 % derived in the south- ern Tyrrhenian from the increase in the 0–1000 m dFe in- ventories (relative to published data; Fig. 3i–l) and assuming 4.45 % Fe in the dust (Guieu et al., 2002). Data availability. Underlying research data are being used by re- searcher participants of the PEACETIME campaign to prepare other papers, and therefore data are not publicly accessible at the time of publication. Data will be accessible once the special issue is com- pleted (http://www.obs-vlfr.fr/proof/php/PEACETIME/peacetime. php, last access 13 December 2021). The policy of the database is detailed here: http://www.obs-vlfr.fr/proof/dataconvention.php, last access 13 December 2021 and https://www.seanoe.org/data/00645/ 75747/ (last access: 13 December 2021, Guieu et al., 2020). https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 6448 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6449 Mediterranean Sea, Deep-Sea Res. Pt. II, 44, 741–768, https://doi.org/10.1016/S0967-0645(96)00092-6, 1997. Mediterranean Sea, Deep-Sea Res. Pt. II, 44, 741–768, https://doi.org/10.1016/S0967-0645(96)00092-6, 1997. J. J., Tsunogai, S., Wollast, R., and Zhou, M.: The atmospheric input of trace species to the world ocean, Global Biogeochem. Cy., 5, 193–259, https://doi.org/10.1029/91GB01778, 1991. Christensen, J. H.: The Danish eulerian hemispheric model – A three-dimensional air pollution model used for the Arctic, At- mos. Environ., 31, 4169–4191, https://doi.org/10.1016/S1352- 2310(97)00264-1, 1997. Dulac, F., Tanré, D., Bergametti, G., Buat-Ménard, P., Des- bois, M., and Sutton, D.: Assessment of the African air- borne dust mass over the western Mediterranean Sea us- ing Meteosat data, J. Geophys. Res.-Atmos., 97, 2489–2506, https://doi.org/10.1029/91JD02427, 1992. Conway, T. M. and John, S. G.: Quantiﬁcation of dissolved iron sources to the North Atlantic Ocean, Nature, 511, 212–215, https://doi.org/10.1038/nature13482, 2014. Dulaquais, G., Waeles, M., Gerringa, L. J., Middag, R., Ri- jkenberg, M. J., and Riso, R.: The biogeochemistry of electroactive humic substances and its connection to iron chemistry in the North East Atlantic and the Western Mediterranean Sea, J. Geophys. Res.-Ocean., 123, 5481–5499, https://doi.org/10.1029/2018JC014211, 2018. Croot, P. L., Streu, P., and Baker, A. R.: Short residence time for iron in surface seawater impacted by atmospheric dry deposi- tion from Saharan dust events, Geophys. Res. Lett., 31, L23S08, https://doi.org/10.1029/2004GL020153, 2004. Cutter, G. A., Andersson, P., Codispoti, L., Croot, P., Francois, R., Lohan, M., Obata, H., and Rutgers van der Loeff, M.: Sampling and Sample-handling Protocols for GEOTRACES Cruises, 2010 GEOTRACES Standards and Intercalibration Committee, 2010. Fishwick, M. P., Sedwick, P. N., Lohan, M. C., Worsfold, P. J., Buck, K. N., Church, T. M., and Ussher, S. J.: The im- pact of changing surface ocean conditions on the dissolu- tion of aerosol iron, Global Biogeochem. Cy., 28, 1235–1250, https://doi.org/10.1002/2014GB004921, 2014. Dammshäuser, A. and Croot, P. L.: Low colloidal associa- tions of aluminium and titanium in surface waters of the tropical Atlantic, Geochim. Cosmochim. Ac., 96, 304–318, https://doi.org/10.1016/j.gca.2012.07.032, 2012. Fitzsimmons, J. N., Zhang, R., and Boyle, E. A.: Dissolved iron in the tropical North Atlantic Ocean, Mar. Chem., 154, 87–99, https://doi.org/10.1016/j.marchem.2013.05.009, 2013. Dammshäuser, A., Wagener, T., and Croot, P. L.: Surface water dis- solved aluminum and titanium: Tracers for speciﬁc time scales of dust deposition to the Atlantic?, Geophys. Res. Lett., 38, L24601, https://doi.org/10.1029/2011GL049847, 2011. Flemming, J., Huijnen, V., Arteta, J., Bechtold, P., Beljaars, A., Blechschmidt, A.-M., Diamantakis, M., Engelen, R. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal J., Gaudel, A., Inness, A., Jones, L., Josse, B., Katragkou, E., Marecal, V., Peuch, V.- H., Richter, A., Schultz, M. G., Stein, O., and Tsikerdekis, A.: Tropospheric chemistry in the Integrated Fore- casting System of ECMWF, Geosci. Model Dev., 8, 975–1003, https://doi.org/10.5194/gmd-8-975-2015, 2015. Davies, J. E. and Buat-Ménard, P.: Impact of atmospheric depo- sition on particulate manganese and aluminium distribution in northwestern Mediterranean surface water, Glob. Planet. Change, 3, 35–45, https://doi.org/10.1016/0921-8181(90)90054-G, 1990. de Leeuw, G., Guieu, C., Arneth, A., Bellouin, N., Bopp, L., Boyd, P. W., Denier van der Gon, H. A. C., Desboeufs, K. V., Dulac, F., Cristina Facchini, M. C., Gantt, B., Langmann, B., Mahowald, N. M., Marañón, E., O’Dowd, C., Olgun, N., Pulido-Villena, E., Ri- naldi, M., Stephanou, E.G., and Wagener, T.: Ocean-Atmosphere interactions of particles, in: Ocean-Atmosphere Interactions of Gases and Particles, edited by: Liss, P. and Johnson, M., Springer Earth System Sciences, Springer Berlin Heidelberg, 171–246, https://doi.org/10.1007/978-3-642-25643-1_4, 2014. Folger, D. W., Burckle, L. H., and Heezen, B. C.: Opal phy- toliths in a North Atlantic dust fall, Science, 155, 1243–1244, https://doi.org/10.1126/science.155.3767.1243, 1967. Gazeau, F., Ridame, C., VanWambeke, F., Alliouane, S., Stolpe, C., Irisson, J.-O., Marro, S., Grisoni, J.-M., De Liège, G., Nunige, S., Djaoudi, K., Pulido-Villena, E., Dinasquet, J., Obernos- terer, I., Catala, P., and Guieu, C.: Impact of dust addition on Mediterranean plankton communities under present and future conditions of pH and temperature: an experimental overview, Biogeosciences, 18, 5011–5034, https://doi.org/10.5194/bg-18- 5011-2021, 2021. Desboeufs, K., Bon Nguyen, E., Chevaillier, S., Triquet, S., and Dulac, F.: Fluxes and sources of nutrients and trace metal at- mospheric deposition in the northwestern Mediterranean, Atmos. Chem. Phys., 18, 14477–14492, https://doi.org/10.5194/acp-18- 14477-2018, 2018. Gdaniec, S., Roy-Barman, M., Foliot, L., Thil, F., Dapoigny, A., Burckel, P., Garcia-Orellana, J., Masqué, P., Mörth, C.- M., and Andersson, P. S.: Thorium and protactinium iso- topes as tracers of marine particle ﬂuxes and deep water cir- culation in the Mediterranean Sea, Mar. Chem., 199, 12–23, https://doi.org/10.1016/j.marchem.2017.12.002, 2018. Desboeufs, K., Fu, F., Bressac, M., Tovar-Sánchez, A., Triquet, S., Doussin, J.-F., Giorio, C., Chazette, P., Disnaquet, J., Feron, A., Formenti, P., Maisonneuve, F., Rodri´guez-Romero, A., Zapf, P., Dulac, F., and Guieu, C.: Wet deposition in the remote western and central Mediterranean as a source of trace met- als to surface seawater, Atmos. Chem. Phys. Discuss. [preprint], https://doi.org/10.5194/acp-2021-624, in review, 2021. Gehlen, M., Beck, L., Calas, G., Flank, A.-M., Van Bennekom, A. J., and Van Beusekom, J. E. References This study is a contribution to the PEACE- TIME project (http://peacetime-project.org; last access: 25 Octo- ber 2021), a joint initiative of the MERMEX and ChArMEx com- ponents supported by CNRS-INSU, IFREMER, CEA, and Météo- France as part of the decadal program MISTRALS coordinated by INSU. PEACETIME was endorsed as a process study by GEO- TRACES and is also a contribution to IMBER and SOLAS inter- national programs. The constructive comments during the review process of Thomas Holmes, an anonymous referee, Silvia Becagli, and Christine Klass are much appreciated. Boyd, P. W., Ibisanmi, E., Sander, S. G., Hunter, K. A., and Jackson, G. A.: Remineralization of upper ocean particles: Implications for iron biogeochemistry, Limnol. Oceanogr., 55, 1271–1288, https://doi.org/10.4319/lo.2010.55.3.1271, 2010. Bressac, M. and Guieu, C.: Post-depositional processes: What really happens to new atmospheric iron in the ocean surface?, Global Biogeochem. Cy., 27, 859–870, https://doi.org/10.1002/gbc.20076, 2013. Bressac, M., Guieu, C., Doxaran, D., Bourrin, F., Obolensky, G., and Grisoni, J. M.: A mesocosm experiment coupled with opti- cal measurements to assess the fate and sinking of atmospheric particles in clear oligotrophic waters, Geo.-Mar. Lett., 32, 153– 164, https://doi.org10.1007/s00367-011-0269-4, 2012. Bressac, M., Guieu, C., Doxaran, D., Bourrin, F., Desboeufs, K., Leblond, N., and Ridame, C.: Quantiﬁcation of the lithogenic carbon pump following a simulated dust-deposition event in large mesocosms, Biogeosciences, 11, 1007–1020, https://doi.org/10.5194/bg-11-1007-2014, 2014. Bressac, M., Guieu, C., Ellwood, M. J., Tagliabue, A., Wagener, T., Laurenceau Cornec, E. C., Whitby, H., Sarthou, G., and Boyd, P. W.: Resupply of mesopelagic dissolved iron controlled by particulate iron composition, Nat. Geosci., 12, 995–1000, https://doi.org/10.1038/s41561-019-0476-6, 2019. Financial support. This research has been supported by the Hori- zon 2020 (IRON-IC (grant no. 626734)) and the Horizon 2020 (DUSC3 (grant no. 708119)). The project leading to this publication received funding from CNRS-INSU, IFREMER, CEA, and Météo- France as part of the program MISTRALS coordinated by INSU and from the European FEDER fund (grant no. 1166-39417). Buat-Ménard, P., Davies, J., Remoudaki, E., Miquel, J. C., Berga- metti, G., Lambert, C. E., Ezat, U., Quetel, C., La Rosa, J., and Fowler, S. W.: Non-steady-state biological removal of at- mospheric particles from Mediterranean surface waters, Nature, 340, 131–134, https://doi.org/10.1038/340131a0, 1989. Chou, L. and Wollast, R.: Biogeochemical behavior and mass balance of dissolved aluminum in the western Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal Riffault, V., Rougier, G., Rousselet, L., Roy-Barman, M., Saiz- Lopez, A., Schmechtig, C., Sellegri, K., Siour, G., Taillandier, V., Tamburini, C., Thyssen, M., Tovar-Sanchez, A., Triquet, S., Uitz, Herut, B., Rahav, E., Tsagaraki, T. M., Giannakourou, A., Tsi- ola, A., Psarra, S., Lagaria, A., Papageorgiou, N., Mihalopou- los, N., Theodosi, C. N., Violaki, K., Stathopoulou, E., Scoul- los, M., Krom, M. D., Stockdale, A., Shi, Z., Berman-Frank, I., Meador, T. B., Tanaka, T., and Paraskevi, P.: The poten- tial impact of Saharan dust and polluted aerosols on microbial populations in the East Mediterranean Sea, an overview of a mesocosm experimental approach, Front. Mar. Sci., 3, p. 226, https://doi.org/10.3389/fmars.2016.00226, 2016. Guerzoni, S., Chester, R., Dulac, F., Herut, B., Loÿe-Pilot, M. D., Measures, C., Migon, C., Molinaroli, E., Moulin, C., Rossini, P., Saydam, C., Soudine, A., and Ziveri, P.: The role of atmospheric deposition in the biogeochemistry of the Mediterranean Sea, Prog. Oceanogr., 44, 147–190, https://doi.org/10.1016/S0079- 6611(99)00024-5, 1999. Guieu, C., Loÿe-Pilot, M.D., Ridame, C., and Thomas, C.: Chemi- cal characterization of the Saharan dust end-member; some bio- logical implications for the western Mediterranean, J. Geophys. Res., 107, 4258, https://doi.org/10.1029/2001JD000582, 2002. Huffman, G. J., Bolvin, D. T., Nelkin, E. J., and Tan, J.: Integrated Multi-satellitE Retrievals for GPM (IMERG) technical docu- mentation, NASA/GSFC Code, 612, 2015. Guieu, C., Roy-Barman, M., Leblond, N., Jeandel, C., Souhaut, M., Le Cann, B., Dufour, A., and Bournot, C.: Vertical particle ﬂux in the northeast Atlantic Ocean (POMME experiment), J. Geophys. Res.-Ocean., 110, CO7S18, https://doi.org/10.1029/2004JC002672, 2005. Huneeus, N., Basart, S., Fiedler, S., Morcrette, J.-J., Benedetti, A., Mulcahy, J., Terradellas, E., Pérez García-Pando, C., Pejanovic, G., Nickovic, S., Arsenovic, P., Schulz, M., Cuevas, E., Bal- dasano, J. M., Pey, J., Remy, S., and Cvetkovic, B.: Forecasting the northern African dust outbreak towards Europe in April 2011: a model intercomparison, Atmos. Chem. Phys., 16, 4967–4986, https://doi.org/10.5194/acp-16-4967-2016, 2016. p g Guieu, C., Aumont, O., Paytan, A., Bopp, L., Law, C. S., Mahowald, N., Achterberg, E. P., Marañón, E., Salihoglu, B., Crise, A., Wa- gener, T., Herut, B., Desboeufs, K., Kanakidou, M., Olgun, N., Peters, F., Pulido-Villena, E., Tovar-Sanchez, A., and Völker, C.: The signiﬁcance of episodicity in atmospheric deposition to Low Nutrient Low Chlorophyll regions, Global Biogeochem. Cy., 28, 1179–1198, https://doi.org/10.1002/2014GB004852, 2014a. Hydes, D. and Liss, P.: Fluorimetric method for the determination of low concentrations of dissolved aluminium in natural waters, Analyst, 101, 922–931, https://doi.org/10.1039/AN9760100922, 1976. Jacquet, S. H. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal E. : Unraveling the atomic structure of biogenic silica: evidence of the structural association of Al and Si in diatom frustules, Geochim. Cosmochim. Ac., 66, 1601– 1609, https://doi.org/10.1016/S0016-7037(01)00877-8, 2002. Donaghay, P. L., Liss, P. S., Duce, R. A., Kester, D. R., Hanson, A. K., Villareal, T., Tindale, N. W., and Gifford, D. J.: The role of episodic atmospheric nutrient inputs in the chemical and biolog- ical dynamics of oceanic ecosystems, Oceanography, 4, 62–70, https://doi.org/10.5670/oceanog.1991.04, 1991. Gerringa, L. J. A., Slagter, H. A., Bown, J., van Haren, H., Laan, P., De Baar, H. J. W., and Rijkenberg, M. J. A.: Dis- solved Fe and Fe-binding organic ligands in the Mediter- ranean Sea–GEOTRACES G04, Mar. Chem., 194, 100–113, https://doi.org/10.1016/j.marchem.2017.05.012, 2017. Duce, R. A., Liss, P. S., Merrill, J. T., Atlas, E. L., Buat-Menard, P., Hicks, B. B., Miller, J. M., Prospero, J. M., Arimoto, R., Church, T. M., Ellis, W., Galloway, J. N., Hansen, L., Jickells, T. D., Knap, A. H., Reinhardt, K. H., Schneider, B. Soudine, A., Tokos, Gkikas, A., Basart, S., Hatzianastassiou, N., Marinou, E., Amiridis, V., Kazadzis, S., Pey, J., Querol, X., Jorba, O., Gassó, S., and Baldasano, J. M.: Mediterranean intense desert dust outbreaks Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6450 and their vertical structure based on remote sensing data, At- mos. Chem. Phys., 16, 8609–8642, https://doi.org/10.5194/acp- 16-8609-2016, 2016. face ocean dissolved Al, Global Biogeochem. Cy., 22, 2, https://doi.org/10.1029/2007GB002975, 2008. face ocean dissolved Al, Global Biogeochem. Cy., 22, 2, https://doi.org/10.1029/2007GB002975, 2008. Han, Q., Zender, C., Moore, J. K., Buck, C. S., Chen, Y., Jo- hansen, A., and Measures, C.: Global estimates of mineral dust aerosol iron and aluminum solubility that account for particle size using diffusion-controlled and surface-area con- trolled approximations, Global Biogeochem. Cy., 26, GB2038, https://doi.org/10.1029/2011GB004186, 2012. Guieu, C., Desboeufs, K., Albani, S., Alliouane, S., Aumont, O., Barbieux, M., Barrillon, S., Baudoux, A.-C., Berline, L., Bhairy, N., Bigeard, E., Bloss, M., Bressac, M., Brito, J., Carlotti, F., de Liege, G., Dinasquet, J., Djaoudi, K., Doglioli, A., D’Ortenzio, F., Doussin, J.-F., Duforet, L., Dulac, F., Dutay, J.-C., Engel, A., Feliu-Brito, G., Ferre, H., Formenti, P., Fu, F., Garcia, D., Garel, M., Gazeau, F., Giorio, C., Gregori, G., Grisoni, J.-M., Guasco, S., Guittonneau, J., Haëntjens, N., Heimburger, L.-E., Helias, S., Jacquet, S., Laurent, B., Leblond, N., Lefevre, D., Mallet, M., Marañón, E., Nabat, P., Nicosia, A., Obernosterer, I., Perez, L. M., Petrenko, A., Pulido-Villena, E., Raimbault, P., Ridame, C., Riffault, V., Rougier, G., Rousselet, L., Roy-Barman, M., Saiz- Lopez, A., Schmechtig, C., Sellegri, K., Siour, G., Taillandier, V., Tamburini, C., Thyssen, M., Tovar-Sanchez, A., Triquet, S., Uitz, J., Van Wambeke, F., Wagener, T., and Zaencker, B.: BIOGEO- CHEMICAL dataset collected during the PEACETIME cruise, SEANOE [data set], https://doi.org/10.17882/75747, 2020. Han, J., Wang, W., Kwon, Y. C., Hong, S. Y., Tallapragada, V., and Yang, F.: Updates in the NCEP GFS cumulus con- vection schemes with scale and aerosol awareness, Weather Forecast., 32, 2005–2017, https://doi.org/10.1175/WAF-D-17- 0046.1, 2017. Brito, G., Ferre, H., Formenti, P., Fu, F., Garcia, D., Ga M., Gazeau, F., Giorio, C., Gregori, G., Grisoni, J.-M., Guasco, , , , , , g , , , , , S., Guittonneau, J., Haëntjens, N., Heimburger, L.-E., Helias, S., Jacquet, S., Laurent, B., Leblond, N., Lefevre, D., Mallet, M., Marañón, E., Nabat, P., Nicosia, A., Obernosterer, I., Perez, L. M., Petrenko, A., Pulido-Villena, E., Raimbault, P., Ridame, C., Heimbürger, L. E., Migon, C., and Cossa, D.: Impact of atmospheric deposition of anthropogenic and natural trace metals on Northwestern Mediterranean surface waters: A box model assessment, Environ. Pollut., 159, 1629–1634, https://doi.org/10.1016/j.envpol.2011.02.046, 2011. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6451 dust, ocean biogeochemistry, and climate, Science, 308, 67–71, https://doi.org/10.1126/science.1105959, 2005. lantic Ocean CLIVAR-CO2 repeat hydrography A16N tran- sect: extensive linkages between atmospheric dust and upper ocean geochemistry, Global Biogeochem. Cy., 22, GB1005, https://doi.org/10.1029/2007GB003042, 2008. Kishcha P., Nickovic S., Ganor E., Kordova L., and Alpert P.: Sa- haran Dust over the Eastern Mediterranean: Model Sensitivity, in: Air Pollution Modeling and Its Application XIX, edited by: Borrego, C. and Miranda, A. I., NATO Science for Peace and Security Series Series C: Environmental Security, Springer, Dor- drecht, https://doi.org/10.1007/978-1-4020-8453-9_39, 2008. Measures, C., Sato, T., Vink, S., Howell, S., and Li, Y.: The frac- tional solubility of aluminium from mineral aerosols collected in Hawaii and implications for atmospheric deposition of biogeo- chemically important trace elements, Mar. Chem., 120, 144–153, https://doi.org/10.1016/j.marchem.2009.01.014, 2010. Koning, E., Gehlen, M., Flank, A.-M., Calas, G., and Epping, E.: Rapid post-mortem incorporation of alu- minum in diatom frustules: evidence from chemical and structural analyses, Mar. Chem., 106, 208–222, https://doi.org/10.1016/j.marchem.2006.06.009, 2007. Menzel Barraqueta, J. L., Klar, J. K., Gledhill, M., Schlosser, C., Shelley, R., Planquette, H. F., Wenzel, B., Sarthou, G., and Achterberg, E. P.: Atmospheric deposition ﬂuxes over the At- lantic Ocean: a GEOTRACES case study, Biogeosciences, 16, 1525–1542, https://doi.org/10.5194/bg-16-1525-2019, 2019. Kubilay, N., Nickovic, S., Moulin, C., and Dulac, F.: An illus- tration of the transport and deposition of mineral dust onto the eastern Mediterranean, Atmos. Environ., 34, 1293–1303, https://doi.org/10.1016/S1352-2310(99)00179-X, 2000. Meskhidze, N., Völker, C., Al-Abadleh, H. A., Barbeau, K., Bres- sac, M., Buck, C., Bundy, R. M., Croot, P., Feng, Y., Ito, A. Jo- hansen, A. M., Landing, W. M., Mao, J., Myriokefalitakis, S., Ohnemus, D., Pasquier, B., and Ye, Y.: Perspective on identifying and characterizing the processes controlling iron speciation and residence time at the atmosphere-ocean interface, Mar. Chem., 217, 103704, https://doi.org/10.1016/j.marchem.2019.103704, 2019. Laurenceau-Cornec, Le Moigne, F. A. C., Gallinari, M., Moriceau, B., Toullec, J., Iversen, M. H., Engel, A., and De La Rocha, C. L.: New guidelines for the application of Stokes’ models to the sinking velocity of marine aggregates, Limnol. Oceanogr., 65, 1264–1285, https://doi.org/10.1002/lno.11388, 2020. Li, F., Ren, J., Yan, L., Liu, S., Liu, C., Zhou, F., and Zhang, J.: The biogeochemical behavior of dissolved aluminum in the south- ern Yellow Sea: Inﬂuence of the spring phytoplankton bloom, Chin. Sci. Bull., 58, 238–248, https://doi.org/10.1007/s11434- 012-5512-5, 2013. Middag, R., van Hulten, M. M. P., van Aken, H. M., Rijkenberg, M. J. A., Gerringa, L. J. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal A., Laan, P., and de Baar, H. J. W.: Dissolved aluminium in the ocean conveyor of the West Atlantic Ocean: mirror image of the biological cycle?, Mar. Chem., 177, 69–86, https://doi.org/10.1016/j.marchem.2015.02.015, 2015. Liu, Q., Zhou, L., Liu, F., Fortin, C., Tan, Y., Huang, L., and Campbell, P. G.: Uptake and subcellular distribution of alu- minum in a marine diatom, Ecotox. Environ. Safe., 169, 85–92, https://doi.org/10.1016/j.ecoenv.2018.10.095, 2019. Migon, C., Sandroni, V., Marty, J. C., Gasser, B., and Miquel, J. C.: Transfer of atmospheric matter through the euphotic layer in the northwestern Mediterranean: seasonal pattern and driving forces, Deep-Sea Res. Pt. II, 49, 2125–2141, https://doi.org/10.1016/S0967-0645(02)00031-0, 2002. Loucaides, S., Michalopoulos, P., Presti, M., Koning, E., Behrends, T., and Van Cappellen, P.: Seawater-mediated interactions be- tween diatomaceous silica and terrigenous sediments: results from long-term incubation experiments, Chem. Geol., 270, 68- 79, https://doi.org/10.1016/j.chemgeo.2009.11.006, 2010. Misic, C., Castellano, M., Ruggieri, N., and Harriague, A. C.: Variations in ectoenzymatic hydrolytic activ- ity in an oligotrophic environment (Southern Tyrrhe- nian Sea, W Mediterranean), J. Mar. Syst., 73, 123–137, https://doi.org/10.1016/j.jmarsys.2007.10.003, 2008. Loÿe-Pilot, M.-D. and Martin, J.-M.: Saharan dust input to the western Mediterranean: an eleven years record in Corsica, in: The Impact of Desert Dust Across the Mediterranean, edited by: Guerzoni, S. and Chester, R., Environmental Sci- ence and Technology Library, Springer, Dordrecht, 11, 191–199, https://doi.org/10.1007/978-94-017-3354-0_18, 1996. p g j j y , Moore, C. M., Mills, M., Arrigo, K., Berman-Frank, I., Bopp, L., Boyd, P. W., Galbraith, E. D., Geider, R. J., Guieu, C., Jaccard, S. L., Jickells, T. D., La Roche, J., Lenton, T. M., Mahowald, N. M., Marañón, E., Marinov, I., Moore, J. K., Nakatsuka, T.,Oschlies, A., Saito, M. A., Thingstad, T. F., Tsuda, A., and Ulloa, O.: Pro- cesses and patterns of oceanic nutrient limitation, Nat. Geosci., 6, 701–710, https://doi.org/10.1038/ngeo1765, 2013. Mackenzie, F. T., Stoffyn, M., and Wollast, R.: Aluminum in sea- water: control by biological activity, Science, 199, 680–682, https://doi.org/10.1126/science.199.4329.680, 1978. Moran, S. B. and Moore, R. M.: Temporal variations in dis- solved and particulate aluminium during a spring bloom, Estuar. Coast. Shelf Sci., 27, 205–215, https://doi.org/10.1016/0272- 7714(88)90090-X,1988a. Marañón, E., Van Wambeke, F., Uitz, J., Boss, E. S., Dimier, C., Dinasquet, J., Engel, A., Haëntjens, N., Pérez-Lorenzo, M., Taillandier, V., and Zäncker, B.: Deep maxima of phy- toplankton biomass, primary production and bacterial produc- tion in the Mediterranean Sea, Biogeosciences, 18, 1749–1767, https://doi.org/10.5194/bg-18-1749-2021, 2021. Moran, S. B. and Moore, R. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M., Dehairs, F., Lefèvre, D., Cavagna, A. J., Planchon, F., Christaki, U., Monin, L., André, L., Closset, I., and Cardinal D.: Early Spring Mesopelagic Carbon Rem- ineralization and Transfer Efﬁciency in the Naturally Iron- Fertilized Kerguelen Area, Biogeosciences, 12, 1713–1731, https://doi.org/10.5194/bg-12-1713-375, 2015. Guieu, C., Ridame, C., Pulido-Villena, E., Bressac, M., Des- boeufs, K., and Dulac, F.: Impact of dust deposition on car- bon budget: a tentative assessment from a mesocosm approach, Biogeosciences, 11, 5621–5635, https://doi.org/10.5194/bg-11- 5621-2014, 2014b. Guieu, C., D’Ortenzio, F., Dulac, F., Taillandier, V., Doglioli, A., Petrenko, A., Barrillon, S., Mallet, M., Nabat, P., and Des- boeufs, K.: Introduction: Process studies at the air–sea inter- face after atmospheric deposition in the Mediterranean Sea– objectives and strategy of the PEACETIME oceanographic campaign (May–June 2017), Biogeosciences, 17, 5563—5585, https://doi.org/10.5194/bg-17-5563-2020, 2020. Jacquet, S. H. M., Tamburini, C., Garel, M., Dufour, A., Van Vam- beke, F., Le Moigne, F. A. C., Bhairy, N., and Guasco, S.: Particulate biogenic barium tracer of mesopelagic carbon rem- ineralization in the Mediterranean Sea (PEACETIME project), Biogeosciences, 18, 5891–5902, https://doi.org/10.5194/bg-18- 5891-2021, 2021. Jickells, T. D., An, Z. S., Andersen, K. K., Baker, A. R., Berga- metti, G., Brooks, N., Cao, J. J., Boyd, P. W., Duce, R. A., Hunter, K. A., Kawahata, H., Kubilay, N., laRoche, J., Liss, P. S., Mahowald, N., Prospero, J. M., Ridgwell, A. J., Tegen, I., and Torres, R.: Global iron connections between desert Hamm, C. E.: Interactive aggregation and sedimentation of diatoms and clay-sized lithogenic material, Limnol. Oceanogr., 47, 1790– 1795, https://doi.org/10.4319/lo.2002.47.6.1790, 2002. Han, Q., Moore, J. K., Zender, C., Measures, C., and Hy- des, D.: Constraining oceanic dust deposition using sur- https://doi.org/10.5194/bg-18-6435-2021 https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M., Maignan, F., Poitou, J., and Tanré, D.: Remote sensing of aerosols over the oceans using MSG/SEVIRI imagery, Ann. Geophys., 23, 3561–3568, https://doi.org/10.5194/angeo-23-3561-2005, 2005. Rolison, J. M., Middag, R., Stirling, C. H., Rijkenberg, M. J. A., and De Baar, H. J. W.: Zonal distribution of dissolved alu- minium in the Mediterranean Sea, Mar. Chem., 177, 87–100, https://doi.org/10.1016/j.marchem.2015.05.001, 2015. Tovar-Sánchez, A., Rodríguez-Romero, A., Engel, A., Zäncker, B., Fu, F., Marañón, E., Pérez-Lorenzo, M., Bressac, M., Wa- gener, T., Triquet, S., Siour, G., Desboeufs, K., and Guieu, C.: Characterizing the surface microlayer in the Mediterranean Sea: trace metal concentrations and microbial plankton abundance, Biogeosciences, 17, 2349–2364, https://doi.org/10.5194/bg-17- 2349-2020, 2020. Sarrand, B., Dulac, F., Baldi, M., Bargaoui, Z., Cindri´c, K., Ducrocq, V., Labiadh, M., Schiavone, J., de Silvestri, L., Somot, S., and Tovar-Sánchez, A.: Precipitation in the Mediterranean basin as seen from the 2000–2010 TRMM-3B42-v6 database, EGU General Assembly Conference Abstracts, Viena, Austria, 22–27 April 2012, 11965, 2012. Twining, B. S., Rauschenberg, S., Morton, P. L., Ohnemus, D. C., and Lam, P. J.: Comparison of particulate trace element concen- trations in the North Atlantic Ocean as determined with discrete bottle sampling and in situ pumping, Deep-Sea Res. Pt. II, 116, 273–282, https://doi.org/10.1016/j.dsr2.2014.11.005, 2015a. Sarthou, G. and Jeandel, C.: Seasonal variations of iron con- centrations in the Ligurian Sea and iron budget in the Western Mediterranean Sea, Mar. Chem., 74, 115–129, https://doi.org/10.1016/S0304-4203(00)00119-5, 2001. Twining, B. S., Rauschenberg, S., Morton, P. L., and Vogt, S.: Metal contents of phytoplankton and labile particulate material in the North Atlantic Ocean, Progr. Oceanogr., 137, 261–283, https://doi.org/10.1016/j.pocean.2015.07.001, 2015b. Schallenberg, C., Ross, A. R., Davidson, A. B., Stewart, G. M., and Cullen, J. T.: Temporal variability of dissolved iron species in the mesopelagic zone at Ocean Station PAPA, J. Mar. Syst., 172, 128–136, https://doi.org/10.1016/j.jmarsys.2017.03.006, 2017. Van Bennekom A. J., Jansen F., Van Der Gaast S., Van Iperen J., and Pieters J.: Aluminum-rich opal: An intermediate in the preservation of biogenic silica in the Zaire (Congo) deep-sea fan, Deep-Sea Res., 36, 173–190, https://doi.org/10.1016/0198- 0149(89)90132-5, 1989. Shelley, R. U., Landing, W. M., Ussher, S. J., Planquette, H., and Sarthou, G.: Regional trends in the fractional solubility of Fe and other metals from North Atlantic aerosols (GEOTRACES cruises GA01 and GA03) following a two-stage leach, Biogeosciences, 15, 2271–2288, https://doi.org/10.5194/bg-15-2271-2018, 2018. van der Jagt, H., Friese, C., Stuut, J. B. W., Fischer, G., and Iversen, M. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6452 ship to hydrography during late summer, Deep-Sea Res. Pt. I, 36, 191–209, https://doi.org/10.1016/0198-0149(89)90133-7, 1989. Ocean sustained by deep winter mixing, Nat. Geosci., 7, 314– 320, https://doi.org/10.1038/ngeo2101, 2014. Ocean sustained by deep winter mixing, Nat. Geosci., 7, 314– 320, https://doi.org/10.1038/ngeo2101, 2014. Tagliabue, A., Aumont, O., DeAth, R., Dunne, J. P., Dutkiewicz, S., Galbraith, E., Misumi, K., Moore, J. K., Ridgwell, A., Sher- man, E., Stock, C., Vichi, M., Völker, C., and Yool, A.: How well do global ocean biogeochemistry models simulate dis- solved iron distributions?, Global Biogeochem. Cy., 30, 149– 174, https://doi.org/10.1002/2015GB005289, 2016. Nowald, N., Iversen, M. H., Fischer, G., Ratmeyer, V., and Wefer, G.: Time series of in-situ particle properties and sediment trap ﬂuxes in the coastal upwelling ﬁlament off Cape Blanc, Mauritania, Progr. Oceanogr. Pt. A, 137, 1–11, https://doi.org/10.1016/j.pocean.2014.12.015, 2015. Orians, K. J. and Bruland, K. W.: The biogeochemistry of alu- minum in the Paciﬁc Ocean, Earth Planet. Sc. Lett., 78, 397–410, https://doi.org/10.1016/0012-821X(86)90006-3, 1986. Tagliabue, A., Bowie, A. R., Boyd, P. W., Buck, K. N., Johnson, K. S., and Saito, M. A.: The integral role of iron in ocean biogeochemistry, Nature, 543, 51–59, https://doi.org/10.1038/nature21058, 2017. Planquette, H. and Sherrell, R. M.: Sampling for particulate trace el- ement determination using water sampling bottles: methodology and comparison to in situ pumps, Limnol. Oceanogr.-Method., 10, 367–388, https://doi.org/10.4319/lom.2012.10.367, 2012. Taillandier, V., Prieur, L., d’Ortenzio, F., Ribera d’Alcalà, M., and Pulido-Villena, E.: Proﬁling ﬂoat observation of ther- mohaline staircases in the western Mediterranean Sea and impact on nutrient ﬂuxes, Biogeosciences, 17, 3343–3366, https://doi.org/10.5194/bg-17-3343-2020, 2020. Quétel, C. R., Remoudaki, E., Davies, J. E., Miquel, J. C., Fowler, S. W., Lambert, C. E., Bergametti, G., and Buat-Ménard, P.: Impact of atmospheric deposition on particulate iron ﬂux and distribu- tion in northwestern Mediterranean waters, Deep-Sea Res. Pt. I, 40, 989–1002, https://doi.org/10.1016/0967-0637(93)90085- H, 1993. Ternon, E., Guieu, C., Loÿe-Pilot, M.-D., Leblond, N., Bosc, E., Gasser, B., Miquel, J.-C., and Martín, J.: The impact of Saha- ran dust on the particulate export in the water column of the North Western Mediterranean Sea, Biogeosciences, 7, 809–826, https://doi.org/10.5194/bg-7-809-2010, 2010. Rijkenberg, M., Powell, C., Dall’Osto, M., Nielsdottir, M., Patey, M., Hill, P., Baker, A., Jickells, T., Harrison, R., and Achterberg, E.: Changes in iron speciation following a Saharan dust event in the tropical North Atlantic Ocean, Mar. Chem., 110, 56–67, https://doi.org/10.1016/j.marchem.2008.02.006, 2008. Thieuleux, F., Moulin, C., Bréon, F. M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal M.: Evidence from mesocosm studies for biological removal of dissolved aluminium from sea water, Nature, 335, 706–708, https://doi.org/10.1038/335706a0, 1988b. Mullin, J. and Riley, J. P.: The colorimetric determination of silicate with special reference to sea and natural waters, Anal. Chim. Ac., 12, 162–176, https://doi.org/10.1016/S0003-2670(00)87825-3, 1955. Measures, C. I. and Brown, E. T.: Estimating Dust Input to the Atlantic Ocean Using Surface Water Aluminium Concentra- tions, in: The Impact of Desert Dust Across the Mediterranean, edited by: Guerzoni, S. and Chester, R., Environmental Sci- ence and Technology Library, Springer, Dordrecht, 11, 301–311, https://doi.org/10.1007/978-94-017-3354-0_30, 1996. Nelson, D. M., Smith Jr, W. O., Muench, R. D., Gordon, L. I., Sul- livan, C. W., and Husby, D. M.: Particulate matter and nutrient distributions in the ice-edge zone of the Weddell Sea: relation- Measures, C. I., Landing, W. M., Brown, M. T., and Buck, C. S.: High-resolution Al and Fe data from the At- https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal H.: The ballasting effect of Saharan dust deposition on ag- gregate dynamics and carbon export: Aggregation, settling, and scavenging potential of marine snow, Limnol. Oceanogr., 63, 1386–1394, https://doi.org/10.1002/lno.10779, 2018. Spokes, L. J., Jickells, T. D., and Lim, B.: Solubilisation of aerosol trace metals by cloud processing: A laboratory study, Geochim. Cosmochim. Ac., 58, 3281–3287, https://doi.org/10.1016/0016- 7037(94)90056-6, 1994. Spyrou, C., Mitsakou, C., Kallos, G., Louka, P., and Vlastou, G.: An improved limited area model for describing the dust cycle in the atmosphere, J. Geophys. Res., 115, D17211, https://doi.org/10.1029/2009JD013682, 2010 Van Wambeke, F., Taillandier, V., Deboeufs, K., Pulido-Villena, E., Dinasquet, J., Engel, A., Marañón, E., Ridame, C., and Guieu, C.: Inﬂuence of atmospheric deposition on biogeochemical cycles in an oligotrophic ocean system, Biogeosciences, 18, 5699–5717, https://doi.org/10.5194/bg-18-5699-2021, 2021. Tagliabue, A., Sallée, J. B., Bowie, A. R., Lévy, M., Swart, S., and Boyd, P. W.: Surface-water iron supplies in the Southern Velasquez, I. B., Ibisanmi, E., Maas, E. W., Boyd, P. W., Nod- der, S., and Sander, S. G.: Ferrioxamine siderophores de- Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021 https://doi.org/10.5194/bg-18-6435-2021 Biogeosciences, 18, 6435–6453, 2021 M. Bressac et al.: Subsurface iron accumulation and rapid aluminum removal 6453 tected amongst iron binding ligands produced during the rem- ineralization of marine particles, Front. Mar. Sci., 3, p. 172, https://doi.org/10.3389/fmars.2016.00172, 2016. Whitby, H., Bressac, M., Sarthou, G., Ellwood, M. J., Guieu, C., and Boyd, P. W.: Contribution of electroactive humic sub- stances to the iron-binding ligands released during microbial remineralization of sinking particles, Geophys. Res. Lett., 47, e2019GL086685, https://doi.org/10.1029/2019GL086685, 2020. p g Vincent, J., Laurent, B., Losno, R., Bon Nguyen, E., Roullet, P., Sauvage, S., Chevaillier, S., Coddeville, P., Ouboulmane, N., di Sarra, A. G., Tovar-Sánchez, A., Sferlazzo, D., Massanet, A., Triquet, S., Morales Baquero, R., Fornier, M., Coursier, C., Desboeufs, K., Dulac, F., and Bergametti, G.: Variability of mineral dust deposition in the western Mediterranean basin and south-east of France, Atmos. Chem. Phys., 16, 8749–8766, https://doi.org/10.5194/acp-16-8749-2016, 2016. Wuttig, K., Wagener, T., Bressac, M., Dammshäuser, A., Streu, P., Guieu, C., and Croot, P. L.: Impacts of dust deposition on dissolved trace metal concentrations (Mn, Al and Fe) dur- ing a mesocosm experiment, Biogeosciences, 10, 2583–2600, https://doi.org/10.5194/bg-10-2583-2013, 2013. Ye, Y., Wagener, T., Völker, C., Guieu, C., and Wolf-Gladrow, D. A.: Dust deposition: iron source or sink? A case study, Bio- geosciences, 8, 2107–2124, https://doi.org/10.5194/bg-8-2107- 2011, 2011. Wagener, T., Pulido-Villena, E., and Guieu, C.: Dust iron dis- solution in seawater: Results from a one-year time-series in the Mediterranean Sea, Geophys. Res. Lett., 35, L16601, https://doi.org/10.1029/2008GL034581, 2008. Wagener, T., Guieu, C., and Leblond, N.: Effects of dust deposition on iron cycle in the surface Mediterranean Sea: results from a mesocosm seeding experiment, Biogeosciences, 7, 3769–3781, https://doi.org/10.5194/bg-7-3769-2010, 2010. Biogeosciences, 18, 6435–6453, 2021 Biogeosciences, 18, 6435–6453, 2021 https://doi.org/10.5194/bg-18-6435-2021
https://openalex.org/W2910037404	https://vestnik.sibadi.org/jour/article/download/780/552	Russian	null	SAFETY OF REINFORCED CONCRETE BRIDGES WITH SPANS STRUCTURES	Vestnik SibADI	2,019	cc-by	5,866	РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА УДК 624.21.012 Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) О БЕЗОПАСНОСТИ ЖЕЛЕЗОБЕТОННЫХ МОСТОВ С ПЛИТНЫМИ ПРОЛЕТНЫМИ СТРОЕНИЯМИ Ю.В. Краснощеков ФГБОУ ВО «СибАДИ», г. Омск, Россия uv1942@mail.ru РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА АННОТАЦИЯ Введение. Приведены результаты анализа нормирования надежности конструктивных си- стем плитных пролетных строений мостовых сооружений. стем плитных пролетных строений мостовых сооружений. Материалы и методы. Выполнен анализ опубликованных материалов с целью использования их при проектировании железобетонных мостов в связи с изменениями норм проектирования и ужесточением нормативных требований по безопасности. Результаты. Результаты обследований плитных пролетных строений свидетельствуют о надежности и долговечности типовых конструкций, эксплуатируемых в обычных условиях, однако в аварийных ситуациях существует опасность прогрессирующего обрушения. В таких у Материалы и методы. Выполнен анализ опубликованных материалов с целью использования их при проектировании железобетонных мостов в связи с изменениями норм проектирования и ужесточением нормативных требований по безопасности. Результаты. Результаты обследований плитных пролетных строений свидетельствуют о надежности и долговечности типовых конструкций, эксплуатируемых в обычных условиях, однако в аварийных ситуациях существует опасность прогрессирующего обрушения. В таких условиях разрезные схемы многопролетных строений недопустимы. Сформулированы задачи исследований, которые необходимо решать для обеспечения живучести плитных пролетных строений. В частности, поставлена задача разработки метода расчета на живучесть при отказе одной из опор неразрезного пролетного строения с учетом динамического эффекта, сопровождающего обрушение. Обсуждение и заключение. Изменения в нормах проектирования конструкций плитных про- летных строений мостовых сооружений вызывают необходимость исследований надежности и живучести в аварийных расчетных ситуациях. КЛЮЧЕВЫЕ СЛОВА: железобетонные мосты, плитные пролетные строения, прогрессирую- щее обрушение, живучесть, надежность. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) 922 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal Контент доступен под лицензией Creative Commons Attribution 4.0 License. © Ю.В. Краснощеков Контент доступен под лицензией Creative Commons Attribution 4.0 License. РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА SAFETY OF REINFORCED CONCRETE BRIDGES WITH SPANS STRUCTURES Y.V. Krasnoshchekov Siberian State Automobile and Highway University, Omsk, Russia uv1942@mail.ru ABSTRACT Introduction. The author presents the results of the reliability analysis of the structural systems in spans of bridges. g Materials and methods. The analysis of published materials for the purpose of using them in the design of reinforced concrete bridges, in connection with changes in design standards and tightening of regulatory requirements for safety is made by the author. g y q y y Results. The results of surveys of slab superstructures indicate the reliability and durability of typical structures operated under normal conditions, but in emergency situations there is a danger of progressive collapse. Therefore, in such conditions, split schemes of multi-span structures are unacceptable. The research tasks to ensure the durability of slab superstructures are formulated. In particular, the task of developing the method of durability calculation in case of one of the pillars failure in the continuous span structure, taking into account the dynamic effect is illustrated. g yf Discussion and conclusion. Changes in design of slab structures of bridge cause the necessity to study the reliability and failure in emergency situations. KEYWORDS: reinforced concrete bridges, slab superstructures, progressive collapse, durability, li bilit KEYWORDS: reinforced concrete bridges, slab superstructures, progressive collapse, durability, reliability. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) 923 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal Content is available under the license Creative Commons Attribution 4.0 License. © Y.V. Krasnoshchekov Content is available under the license Creative Commons Attribution 4.0 License. chekov © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) ОСНОВНЫЕ ПОЛОЖЕНИЯ низация отечественных норм проектирования с европейскими нормами (EN 1990). В раздел расчета несущих конструкций и оснований ак- туализированной редакции СНиП 2.05.03-84* (СП 35.13330.2011) также введено требование к конструктивным схемам мостовых сооруже- ний, которые не должны допускать возможно- сти прогрессирующего обрушения при выходе из строя одного или нескольких элементов в случае экстремальных природных или техно- генных воздействий. низация отечественных норм проектирования с европейскими нормами (EN 1990). В раздел расчета несущих конструкций и оснований ак- туализированной редакции СНиП 2.05.03-84* (СП 35.13330.2011) также введено требование к конструктивным схемам мостовых сооруже- ний, которые не должны допускать возможно- сти прогрессирующего обрушения при выходе из строя одного или нескольких элементов в случае экстремальных природных или техно- генных воздействий. 1. За истекшее время в нормировании конструкций произошли существенные изме- нения, связанные в основном с повышением безопасности сооружений. Изменения ориен- тируют проектировщиков на применение но- вых строительных материалов и технологий и требуют тщательного анализа конструкций железобетонных мостов, возводимых в насто- ящее время. р 2. В результате анализа конструктивных решений серии 3.503.1-108 «Пролетные стро- ения из пустотных плит пролетом от 12 до 18 м для мостов и путепроводов на автомобиль- ных дорогах», разработанной институтом «Со- юздорпроект» в 1992–1999 гг., установлена их достаточная надежность при условии расчета по предельным состояниям. В связи с введе- нием ограничений по срокам службы и необхо- димости учета редких аварийных воздействий требуются исследования по уточнению рас- четных значений температурных, ветровых, сейсмических нагрузок. р 2. В результате анализа конструктивных решений серии 3.503.1-108 «Пролетные стро- ения из пустотных плит пролетом от 12 до 18 м для мостов и путепроводов на автомобиль- ных дорогах», разработанной институтом «Со- юздорпроект» в 1992–1999 гг., установлена их достаточная надежность при условии расчета по предельным состояниям. В связи с введе- нием ограничений по срокам службы и необхо- димости учета редких аварийных воздействий требуются исследования по уточнению рас- четных значений температурных, ветровых, сейсмических нагрузок. д В декабре 2016 г. был издан приказ Мин- строя России № 879 об утверждении Изме- нения №1 к СП 35.13330.2011, в котором ряд положений норм проектирования железобе- тонных мостов получили развитие и стали обязательными для выполнения. В частности, уточнено требование о необходимости удов- летворения проектной долговечности мостов, в связи с чем рекомендованы минимальные сроки службы от 50 до 100 лет железобетон- ных конструкций до первого ремонта. С вре- менным фактором связан расчетный пара- метр модуля деформативности бетона при продолжительном действии нагрузки, который рекомендуется определять по формуле 3. ОСНОВНЫЕ ПОЛОЖЕНИЯ Для обеспечения живучести мостов с плитными пролетными строениями при проек- тировании возможны две модели живучести: детерминированная и вероятностная. Приме- нение вероятностных моделей требует нор- мирования индексов надежности и живучести (установления предельных значений для раз- личных ситуаций). 3. Для обеспечения живучести мостов с плитными пролетными строениями при проек- тировании возможны две модели живучести: детерминированная и вероятностная. Приме- нение вероятностных моделей требует нор- мирования индексов надежности и живучести (установления предельных значений для раз- личных ситуаций). Ebt = Eb/(1 + CnEb), (1) (1) где Сп – нормативное значение деформации ползучести бетона. Снижение значения модуля деформатив- ности бетона в несколько раз по сравнению с начальным модулем упругости Eb может ока- зать влияние на степень перераспределения нагрузки в пространственных расчетах желе- зобетонных конструкций. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) ВВЕДЕНИЕ СТРОИТЕЛЬСТВО И АРХИТЕКТУРА Рисунок 1 – Схема моста с пролетными строениям из пустотных плит Figure 1 – Bridge scheme with span structures of hollow plates Источник: составлено автором на основе анализа конструкций мостов с балочными пролетными строениями Рисунок 2 – Расчетные схемы (разрезная – а, неразрезная – б) и фрагмент сечения пролетного строения из пустотных плит Figure 2 – Design schemes (split-a, non-cut-b) and a section fragment of the superstructure of hollow plates Источник: составлено автором на основе анализа конструкций плитных пролетных строений Рисунок 1 – Схема моста с пролетными строениям из пустотных плит Figure 1 – Bridge scheme with span structures of hollow plates Рисунок 1 Схема моста с пролетными строениям из пустотных плит Рисунок 1 – Схема моста с пролетными строениям из пустотных плит Figure 1 – Bridge scheme with span structures of hollow plates Рисунок 1 – Схема моста с пролетными строениям из пустотных плит Figure 1 – Bridge scheme with span structures of hollow plates Источник: составлено автором на основе анализа конструкций мостов с балочными пролетными строениями = 6, 9, 12, 15 и 18 м для мостов и путепроводов на автомобильных дорогах (рисунок 1). пятидневки) и сейсмичности районов стро- ительства, для эксплуатации в которых они предназначены. Совместная работа сборных железобетонных плит обеспечивается шпо- ночным соединением из монолитного бетона класса В35. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) ВВЕДЕНИЕ За последние 30 лет в стране возводи- ли мосты из железобетона, проекты которых были разработаны на основе СНиП 2.05.03-84* «Мосты и трубы». В этих нормах проектирова- ния предусмотрено требование по обеспече- нию надежности, долговечности сооружений, а также безопасности транспортных средств и пешеходов. Надежность несущих конструкций мостов, согласно ГОСТ 27751, обеспечивалась расчетом по методу предельных состояний. Коэффициенты надежности метода предель- ных состояний за этот период практически не изменялись, хотя был выполнен значительный объем исследований надежности [1–6]. Впервые в нормы введен термин «живу- честь» как обеспеченность от прогрессирую- щего разрушения и приведены рекомендации по проверке на живучесть мостов. В частно- сти, при проверке мостового сооружения на живучесть должны быть рассмотрены случаи появления пластического шарнира в сечениях одного из пролетов, обрушение опоры и др. Изменения №1 ориентируют проектиров- щиков на применение новых строительных ма- териалов и технологий и требуют тщательного анализа конструкций железобетонных мостов, возводимых в настоящее время. В первую очередь это касается типовых решений, для изменения которых необходимы предвари- тельные исследования и соответствующие согласования. В качестве примера в статье выполнен анализ типовых конструкций про- летных строений из пустотных плит длиной L За истекшее время в нормировании кон- струкций произошли существенные измене- ния, связанные в основном с повышением без- опасности сооружений. В частности, в ГОСТ 27751 включено требование по исключению прогрессирующего обрушения конструкций ответственных сооружений. Основанием для появления такого требования явилась гармо- 924 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal РАЗДЕЛ III. РАЗДЕЛ III. МАТЕРИАЛЫ И МЕТОДЫ Наиболее совершенными следует считать конструктивные решения, которые получили отражение в серии 3.503.1-108 «Пролетные строения из пустотных плит пролетом от 12 до 18 м для мостов и путепроводов на автомо- бильных дорогах», разработанной институтом «Союздорпроект» в 1992–1999 гг. Серия вклю- чает рабочие чертежи пустотных плит длиной 12, 15 и 18 м для мостов, расположенных на автомобильных дорогах общего пользования и разработанных в соответствии со СНиП 2.05.03-84 (с изменениями до 1992 г). Пред- усмотрены варианты пустотных плит в зави- симости от климатических условий (темпера- туры наружного воздуха наиболее холодной Конструкции пролетных строений приняты на основании расчетов, выполненных в два этапа. Примеры расчетных схем пролетных строений показаны на рисунке 2. На первом этапе проведены квазистатиче- ские (с коэффициентами динамичности) про- странственные расчеты пролетных строений по методу Б.Е. Улицкого с учетом перерас- пределения нагрузок, соответствующих типо- вым транспортным воздействиям [7]. Ввиду шпоночного соединения плит, не способно- го работать на изгиб, принято условие, что в поперечном направлении передача времен- ной нагрузки между плитами осуществляется 925 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal р у (Vol. 15, no. 6. 2018. Continuous issue – 64) Том 15, № 6. 2018. Сквозной номер выпуска – 64 РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА началось с 1958 г. в основном на Украине [8]. Надежность конструкций проверялась деталь- ными исследованиями, в процессе которых была определена оптимальная конфигурация шпоночных швов. Конструкция шпоночного шва типовых изделий серии 3.503.1-108 изо- бражена на рисунке 4. Швы армируются про- волочными спиралями. только посредством перерезывающих сил. Случайный характер нагрузок и соответствую- щих усилий в элементах пролетных строений, определенных пространственным расчетом, учитывался коэффициентами надежности по нагрузкам. На втором этапе производился кон- структивный расчет по двум группам предель- ных состояний. Случайный характер свойств материалов и условности расчетных моделей учитывали коэффициентами надежности по материалу и условий работы. р В работе [8] описаны результаты испыта- ния пролетных строений мостов с пролетами по 6 м (семь плит в каждом пролете) и 12 м (по семь, девять плит) статической нагрузкой из двух и четырех груженных автомобилей массой по 25 т. В процессе испытания измеря- ли прогибы и деформации бетона на нижних гранях плит в середине пролета и раскрытие швов понизу. Наибольшие усилия в элементах мостов от испытательной нагрузки, опреде- ленные пространственным расчетом, не пре- вышали расчетных усилий по образованию трещин. Измеренные прогибы наиболее за- груженных плит оказались в 1,5 раза меньше теоретических. При испытании отмечена гори- зонтальная раздвижка плит на опорах на 0,45 мм и вертикальные деформации швов на 0,1 – 0,3 мм. Анализ деформаций бетона показал, что условие трещиностойкости (отсутствие трещин) при испытании в целом соблюдалось. Необходимая жесткость и трещиностой- кость типовых плит обеспечивается предвари- тельным напряжением стержневой и канатной арматуры. Канатная арматура (канаты К-7) применена при пролетах более 12 м. Усред- ненные размеры сечений пустотных плит по- казаны на рисунке 3. В серии 3.503.1-108 вы- сота плит длиной до 18 м h = 630 мм, длиной 18 м h = 780 мм, высота овальных пустот до 455 мм, ширина 325 мм. Следует отметить, что надежность и дол- говечность пролетных строений из пустотных плит подтверждена примерами их многолет- ней эксплуатации в СССР и за рубежом (Вели- кобритания, Италия, Венгрия, Чехословакия). В СССР строительство балочных мостов с раз- резными пролетными строениями такого типа Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА Continuous issue – 64) 926 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal Рисуноук 3 – Сечения пустотных плит пролетного строения Figure 3 – Cross sections of hollow plates Источник: составлено автором на основе анализа типовых плит пролетных строений Рисуноук 3 – Сечения пустотных плит пролетного строения © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА Рисуноу 4 – Конструкция шпоночного шва Figure 4 – Design of the keyed seam Источник: составлено автором на основе анализа типовых решений плитных пролетных строений Рисуноу 4 – Конструкция шпоночного шва Рисуноу 4 – Конструкция шпоночного шва Figure 4 – Design of the keyed seam Источник: составлено автором на основе анализа типовых решений плитных пролетных строений Источник: составлено автором на основе анализа типовых решений плитных пролетных строений Источник: составлено автором на основе анализа типовых решений плитных пролетных строений плитных пролетных строений. До сих пор ме- тод Б.Е. Улицкого не вызывал особых возра- жений, так как результаты расчета обеспечи- вали достаточный запас надежности. Однако для оценки реальных резервов и эффектив- ности конструктивных решений необходим бо- лее точный теоретический аппарат. Основной недостаток метода Б.Е. Улицкого – сложность учета изменения жесткости плитных элемен- тов при проявлении ползучести бетона и в ре- зультате появления и развития трещин. На не- обходимость учета влияния ползучести бетона при длительном действии нагрузки указано в Изменениях №1 к СП 35.13330.2011. Непонят- но только, как обеспечить выполнение этого требования при совместном действии на всех плитах пролетного строения постоянных на- грузок и кратковременным загружением транс- портом отдельных элементов. Что касается влияния на перераспределение усилий нерав- номерного развития неупругих деформаций материалов и трещин, особенно в стадии пе- ред разрушением и закритических ситуациях при аварийных воздействиях, то эта проблема вообще мало изучена. В результате испытаний не обнаружено разли- чий в работе армированных и неармирован- ных швов. При обследовании мостов с плитными пролетными строениями видимых трещин в бетоне плит не выявлено. Отдельные повреж- дения являются следствием некачественного изготовления (обычно заниженные защитные слои бетона) или монтажа (наличие влаги в пустотах) и несоблюдением правил эксплу- атации сооружений, связанных в основном с нарушениями ограничений транспортной на- грузки. По результатам испытаний отмечена нецелесообразность применения резиновых опорных частей для плит пролетом 15 и 18 м из-за опасности расстройства межплитных швов [8]. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) РЕЗУЛЬТАТЫ В связи с введением ограничений по сро- кам службы и необходимости учета редких аварийных воздействий необходимы соответ- ствующие исследования по уточнению расчет- ных значений температурных, ветровых, сейс- мических нагрузок. у В работе [9] отмечены многочисленные исследования, посвященные развитию про- странственных методов расчета пролетных строений, в том числе с использованием ко- нечных элементов. Кроме метода Б.Е. Улицко- Значительные расхождения результатов расчета и испытаний вызывают сомнения в совершенстве метода пространственного расчета, который принят при проектировании 927 Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal РАЗДЕЛ III. ованной безопа имость требова Д СТРОИТЕЛЬСТВО И АРХИТЕКТУРА имость требования живучести подтверждаетс ы кадры видеозаписи обрушения двух проле а) б) Рисунок 5 – Этапы обрушения автодорожного моста в г. Чит Figure 5 – Stages of the bridge collapse in Chi Источник: фрагменты видеокадров из сети Интернет Рисунок 5 – Этапы обрушения автодорожного моста в г. Чите Figure 5 – Stages of the bridge collapse in Chita Источник: фрагменты видеокадров из сети Интернет а) б) б) а) ок 5 – Этапы обрушения автодорожного моста в г. Чит Figure 5 – Stages of the bridge collapse in Chi Источник: фрагменты видеокадров из сети Интерне Рисунок 5 – Этапы обрушения автодорожного моста в г. Чите Figure 5 – Stages of the bridge collapse in Chita Источник: фрагменты видеокадров из сети Интернет очник: фрагменты видеокадров из сети Интерне Источник: фрагменты видеокадров из сети Интернет го отмечены работы М.Е. Гибшмана, И.А. Три- фонова, Г.Н. Азизова и др., в которых также имеются определенные недостатки. арактер (см. рисунок 5, а) и без человеч ролетных строений в аварийной ситуации по с Различают две модели живучести: детерми Д ( ) существующая практика конструирования не учитывает возможность прогрессирующего разрушения [12]. Идея проектирования кон- струкций с учетом катастрофических воздей- ствий сформулирована относительно недавно [13, 14]. ких жертв, более соответствующими работ еме гибкой нити. рованную и вероятностную. дель, реализованная в методе предельных со ормированного состояния конструктивной си разрушении одного или нескольких несущи ) П б Основное условие проектирования кон- струкций – это обеспечение безопасности. В последнее время с целью разработки правил проектирования зданий и сооружений широ- ко обсуждается проблема живучести, т.е. на- дежности при катастрофических нагрузках. По определению проф. В.Д. РЕЗУЛЬТАТЫ Райзера, живучесть – это свойство конструкций сохранять при ава- рийных воздействиях способность к выполне- нию основных функций, не допуская лавино- образного (каскадного) развития возмущений и отказов [10]. Это определение соответствует требованиям, приведенным в Изменениях №1 к СП 35.13330.2011. Детерминированная (полувероятностная) м тояний, предполагает анализ напряженно-де темы с оценкой прочности и устойчивости п лементов (моделирование возможной ситуа для третьей группы предельных состояний (по Критериями вероятностных моделей являю им показателем может быть, например, инд пределяется по формуле ( ) F R − = β де R и F – математические ожидания несущ есущей способности и нагрузки. [ , ] Очевидно, что обеспечить живучесть мно- гопролетного строения при разрушении одной из опор при разрезных схемах невозможно. Для выполнения требования живучести кон- струкции пролетного строения каждая пара пролетов должна быть способна к работе по схеме гибкой нити или по неразрезной схеме (см. рисунок 1, б). Существуют разные спосо- бы объединения отдельных пролетов в нераз- резную систему, однако требуется анализ этих способов применительно к условиям аварий- ного отказа опор. Подобная задача решалась при проектировании каркасных зданий, когда в качестве аварийного воздействия рассматри- вается отказ одной из колонн. Основное вни- мание в этом случае уделяют расчету элемен- тов перекрытий, пролеты которых значительно увеличиваются. Поэтому расчетная схема перекрытия над удаленной колонной рассма- тривается в виде мембраны или гибкой нити (струны). При этом отмечена необходимость учета динамического эффекта, вызываемого внезапным удалением из расчетной схемы опоры [15]. и разрушения). Подобную модель предлага вучести) В.Д. Райзер [16]. ся показатели надежности (безотказности). Та с надежности метода двух моментов, которы 2 2 F R s s + , (2 способности и нагрузки; 2 R s и 2 F s – дисперси Причинами прогрессирующего (лавиноо- бразного) разрушения объектов строительства являются локальные разрушения конструктив- ных элементов при воздействии аварийных и чрезвычайных ситуаций, к которым относятся взрывные, ударные и сейсмические динами- ческие воздействия. Чтобы минимизировать возможность прогрессирующего разрушения, сооружение следует проектировать таким об- разом, чтобы в случае отказа любого отдель- ного элемента весь объект или его наиболее ответственная часть сохраняла работоспособ- ность в течение периода времени, достаточно- го для принятия срочных мер (например эваку- ации людей и транспорта). Безотказность этих элементов должна обеспечивать строитель- ный объект от полного разрушения при ава- рийных воздействиях, даже если его дальней- шее использование по назначению окажется невозможным без капитального ремонта [11]. Актуальность проблемы обусловлена тем, что Обеспечение живучести ответственных со- оружений несомненно приводит к увеличению единовременных затрат на строительство. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА рисунке 5 показаны кадры видеозаписи обру- шения двух пролетов общей длиной 24 м авто- дорожного моста в результате подмыва одной из опор паводковыми водами в июле 2018 г. разрушению. Расчет с выключенными элемен- тами выполняется на втором этапе с учетом физической и геометрической нелинейности на действие нагрузки от усилия, определен- ного на первом этапе с увеличением на коэф- фициент, учитывающий динамический эффект локального разрушения. По мнению авторов, такой расчет является компьютерным модели- рованием процесса приспособления конструк- ции к новой расчетной ситуации. разрушению. Расчет с выключенными элемен- тами выполняется на втором этапе с учетом физической и геометрической нелинейности на действие нагрузки от усилия, определен- ного на первом этапе с увеличением на коэф- фициент, учитывающий динамический эффект локального разрушения. По мнению авторов, такой расчет является компьютерным модели- рованием процесса приспособления конструк- ции к новой расчетной ситуации. Если живучесть моста была бы достаточ- ной, то повреждения имели бы ограниченный характер (см. рисунок 5, а) и без человеческих жертв, более соответствующими работе про- летных строений в аварийной ситуации по схеме гибкой нити. ц р у ц В работе [18] показано, что в каркасных зда- ниях с безбалочными железобетонными пере- крытиями при превышении определенных раз- меров сетки колонн определяющим является расчет против прогрессирующего разрушения с учетом пластических деформаций при пре- дельных нагрузках. При этом принимаются во внимание только особые сочетания нагрузок, включающие постоянные и длительные вре- менные нагрузки с коэффициентами сочета- ния и надежности равными единице, а также наиболее опасные схемы локального разру- шения. Величины перемещений (прогибов) и ширина раскрытия трещин в конструкциях не регламентируются, а устойчивость должна быть обеспечена при минимальной жестко- сти конструктивных элементов и узловых со- единений, соответствующих максимально до- пустимым деформациям бетона и арматуры. Критерии несущей способности в этом случае те же, что и в обычных расчетах по предель- ным состояниям. Различают две модели живучести: детер- минированную и вероятностную. Детерминированная (полувероятностная) модель, реализованная в методе предельных состояний, предполагает анализ напряжен- но-деформированного состояния конструктив- ной системы с оценкой прочности и устойчи- вости при разрушении одного или нескольких несущих элементов (моделирование возмож- ной ситуации разрушения). Подобную модель предлагал для третьей группы предельных со- стояний (по живучести) В.Д. Райзер [16]. Критериями вероятностных моделей явля- ются показатели надежности (безотказности). РЕЗУЛЬТАТЫ Эти затраты должны быть компенсированы гарантированной безопасностью их эксплуа- тации в условиях аварийной опасности. Прак- тическая значимость требования живучести подтверждается конкретными примерами. На 928 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) ОБСУЖДЕНИЕ И ЗАКЛЮЧЕНИЕ Изменения в нормах проектирования кон- струкций плитных пролетных строений мосто- вых сооружений вызывают необходимость ис- следований надежности и живучести мостов в аварийных расчетных ситуациях. 13. Abrams D.P. Consequence-based engineering approaches for reducing loss in mid- America. Conference on Apr 4, 2002 at Notre- Dame University. 2002. РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА 8. Штильман Е.И., Березецкий В.И. Про- летные строения мостов из пустотных плит. М.: Транспорт, 1972. 81 с. должна быть рассмотрена аварийная расчет- ная ситуация, соответствующая исключитель- ным условиям работы сооружения, которые могут привести к существенным социальным, экологическим и экономическим потерям. 9. Кожушко В.П. О развитии пространствен- ных методов расчета пролетных строений ав- тодорожных мостов // Наука та прогрес транс- порту. 2008. №21. С. 128-129. В настоящее время намечается постепен- ный переход к вероятностным методам расче- та. Об этом свидетельствуют принципы норми- рования воздействий и свойств материалов, принятые в европейских нормах проектирова- ния конструкций различных сооружений (EN 1990). Эффективность вероятностных мето- дов проявляется в основном при проектирова- нии сооружений на заданную надежность [20]. В настоящее время намечается постепен- ный переход к вероятностным методам расче- та. Об этом свидетельствуют принципы норми- рования воздействий и свойств материалов, принятые в европейских нормах проектирова- ния конструкций различных сооружений (EN 1990). Эффективность вероятностных мето- дов проявляется в основном при проектирова- нии сооружений на заданную надежность [20]. 10. Райзер В.Д. К проблеме живучести зда- ний и сооружений // Строительная механика и расчет сооружений. 2012. №5. С. 77-78. 11. Перельмутер А.В. Избранные проблемы надежности и безопасности строительных кон- струкций. М.: Издательство АСВ, 2007. – 256 с. 12. Starossek U., Wolff M. Design of collapse- resistant structures [Конструирование структур, устойчивых к обрушению]. JCSS and IABSE workshop on Robustness of structures, building research establishment, Garston, Watford, UK. 2005. РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА Таким показателем может быть, например, ин- декс надежности метода двух моментов, кото- рый определяется по формуле ( ) 2 2 / F R s s F R + − = β , (2) (2) где R и F – математические ожидания несу- щей способности и нагрузки; 2 R s и 2 F s – дис- персии несущей способности и нагрузки. В настоящее время надежность сооруже- ний обеспечивают расчетом по методу пре- дельных состояний. В последней редакции стандарта (ГОСТ 27751) кроме первой и вто- рой групп предельных состояний предусмо- трены предельные состояния, возникающие при особых воздействиях и ситуациях, превы- шение которых приводит к разрушению соору- жений с катастрофическими последствиями. В.Д. Райзер предлагает для оценки живуче- сти использовать индекс живучести в виде D INT INT I β − β β = , (3) (3) где βINT, βD – индексы надежности неповре- жденной и поврежденной конструкции. Особые воздействия подразделяют на нор- мируемые (например сейсмические) и аварий- ные, возникающие при отказе работы несуще- го элемента конструктивной системы. В работе [19] отмечается, что большая часть аварийных ситуаций, приведших к обрушению мостов, связана с грубыми ошибками, допущенными при их возведении, или с ударами в процессе эксплуатации. Особые воздействия включа- ются в особые сочетания нагрузок, в которых допускается не учитывать кратковременные нагрузки. Считается, что особые нагрузки и воздействия создают аварийные ситуации. Поэтому при расчете на особые воздействия Применение вероятностных моделей тре- бует нормирования индексов надежности и живучести (установления предельных значе- ний для различных ситуаций). Модель В.Д. Райзера, на наш взгляд, удачно характеризует отличие понятий надежности и живучести. В общем случае расчет на живучесть сво- дится к расчету устойчивости сооружения про- тив прогрессирующего разрушения с учетом пластических деформаций при предельных нагрузках. В работе [17] предлагается выпол- нять расчет на живучесть в 2 этапа. На пер- вом этапе производится расчет в эксплуатаци- онной стадии, предшествующей локальному © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal 929 БИБЛИОГРАФИЧЕСКИЙ СПИСОК y 14. Клюева Н.В., Бухтиярова А.С., Колчу- нов С.И. Исследование живучести железобе- тонных рамно-стержневых пространственных конструкций в запредельных состояниях // Промышленное и гражданское строительство. 2012. №2. С. 55-59. 1. Underwater bridge inspection. Publication No FHWA-NH1-10-027. Prepared by Collins Engineers, Inc. 123 North Wacker Drive, Suite 300 Chicago, Illinois 60606. 2010. 224 p. 1. Underwater bridge inspection. Publication No FHWA-NH1-10-027. Prepared by Collins Engineers, Inc. 123 North Wacker Drive, Suite 300 Chicago, Illinois 60606. 2010. 224 p. 2. Савчинский Б.В. Некоторые аспекты на- дежности железобетонных пролетных строе- ний автодорожных мостов // Мосты и тоннели : теория, исследования, практика, 2012. С. 163- 166. 15. Краснощеков Ю.В. Расчет каркасного здания на прогрессирующее обрушение при аварийном отказе колонны // Строительная механика и расчет сооружений. 2017. № 1. С. 54-58. 3. Медведев К.В., Яцко Ф.В. Прочность, надежность и долговечность железобетонных элементов автодорожных мостов // Мосты и тоннели: теория, исследования, практика, 2013. Вып. 4. С. 52-59. 16. Райзер В.Д. Теория надежности соору- жений. М.: Издательство АСВ, 2010. 384 с. 17. Назаров Ю.П., Городецкий А.С., Сим- биркин В.Н. К проблеме обеспечения живуче- сти строительных конструкций при аварийных воздействиях // Строительная механика и рас- чет сооружений. 2009. №4. С. 5-9. 4. Шестовицкий Д.А. Прогнозирование сро- ка службы железобетонных мостов // Мосты и тоннели: теория, исследования, практика, 2013. Вып. 4. С. 120-129. ру 18. Тихонов И.Н., Козелков М.М. Расчет и конструирование железобетонных монолит- ных перекрытий зданий с учетом защиты от прогрессирующего обрушения // Бетон и желе- зобетон. 2009. №3. С. 2-8. 5. Овчинников И.И., Овчинников И.Г., Шеин А.А., Грацинский В.Г., Вдовин К.М. Особенно- сти подводного обследования транспортных сооружений. 2. Характерные повреждения опор мостовых сооружений // Интернет-жур- нал «Науковедение». 2013. Вып. 6. 19. Руководство для проектировщиков к Ев- рокоду 1: Воздействия на сооружения. Стан- дарты EN 1991-1-1 и 1-3-1-7; пер. с англ. / Х. Гульванесян, П. Формичи, Ж.-А. Калгаро при участии Дж. Хординга. М.: МГСУ, 2011. 340 с. 19. Руководство для проектировщиков к Ев- рокоду 1: Воздействия на сооружения. Стан- дарты EN 1991-1-1 и 1-3-1-7; пер. с англ. / Х. Гульванесян, П. Формичи, Ж.-А. Калгаро при участии Дж. Хординга. М.: МГСУ, 2011. 340 с. 6. Николенко М.А., Минасов А.Г., Савинова Е.И. Новые методы проектирования железо- бетонных пролетных строений // Инженерный вестник Дона. 2017. № 2(45). 20. Краснощеков Ю.В., Заполева М.Ю. Основы проектирования конструкций зданий и сооружений. М.: Инфра-Инженерия, 2018. 296 с. 20. Краснощеков Ю.В., Заполева М.Ю. Основы проектирования конструкций зданий и сооружений. М.: Инфра-Инженерия, 2018. 296 с. 7. Улицкий Б.Е. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) 9. Кожушко В.П. О развитии пространствен- ных методов расчета пролетных строений ав- тодорожных мостов // Наука та прогрес транс- порту. 2008. №21. С. 128-129. БИБЛИОГРАФИЧЕСКИЙ СПИСОК Пространственные расчеты мостов (с использованием ЭЦВМ). М.: Транс- порт, 1967. 406 с. 930 © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal REFERENCES 10. Raizer V.D. K problem zhivuchesti zdaniy I sooruzheniy [To the problem of the survivability of buildings and structures]. Stroitel’naya mechanika I raschet sooruzheniy , 2012, no 5, Pp. 77-78. (in Russian) 1. Underwater bridge inspection. Publication No FHWA-NH1-10-027. Prepared by Collins Engineers, Inc. 123 North Wacker Drive, Suite 300 Chicago, Illinois 60606. 2010. 224 p. 1. Underwater bridge inspection. Publication No FHWA-NH1-10-027. Prepared by Collins Engineers, Inc. 123 North Wacker Drive, Suite 300 Chicago, Illinois 60606. 2010. 224 p. 11. Perel’muter A.V. Izbrannye problem nadezhnosti I bezopasnosti stroitel’nych konstruktsy [Selected problems of reliability and safety of building structures]. Moscow. Publisher ASV, 2007. 256 p. (in Russian) 2. Savchinskiy B.V. Nekotorye aspekty nadezhnosti zhelezobetonnykh proletnykh stroeniy avtodorozhnykh mostov [Some aspects of reliability of reinforced concrete span structures of road bridges]. Mosty I tonneli: teoriya, issledovaniya, praktika, 2012, Pp. 163-166. (in Russian) 12. Starossek U., Wolff M. Design of collapse- resistant structures [Design of structures resistant to destruction]. JCSS and IABSE workshop on Robustness of structures, building research establishment, Garston, Watford, UK. 2005. 3. Medvedev К.V., Yatsko F.V. Prochnost’, nadezhnost’ I dolgovechnost’ zhelezobetonnykh elementov avtodorozhnych mostov [Strength, reliability and durability of reinforced concrete elements of road bridges]. Mosty I tonneli: teoriya, issledovaniya, praktika, 2013, no. 4, Pp. 52-59. (in Russian) 3. Medvedev К.V., Yatsko F.V. Prochnost’, nadezhnost’ I dolgovechnost’ zhelezobetonnykh elementov avtodorozhnych mostov [Strength, reliability and durability of reinforced concrete elements of road bridges]. Mosty I tonneli: teoriya, issledovaniya, praktika, 2013, no. 4, Pp. 52-59. (in Russian) 13. Abrams D.P. Engineering approaches based on the investigation to reduce losses, in Central America]. Conference on Apr 4, 2002 at Notre-Dame University. 2002. 14. Klyueva N.V., Bukhtiyarova A.S., Kolchunov S.I. Issledovanie zhivuchesti zhelezobetonnykh ramno-sterzhnevych prostranstvennych konstruktsiy v zapredel’nych sostoyaniyach [Investigation of the survivability of reinforced concrete frame-rod spatial structures in out-of- bound states]. Promyshlennoe I grazhdanskoe stroitel’stvo, 2012, no 2, Pp. 55-59. (in Russian) 4. Shestovitskiy D.А. Prognosirovanie sroka sluzhby zhelezobetonnykh mostov [Forecasting of the service life of reinforced concrete bridges]. Mosty I tonneli: teoriya, issledovaniya, praktika, 2013, no. 4, Pp. 120-129. (in Russian) 5. Ovchinnikov I.I., Ovchinnikov I.G., Shein A.A., Gratsinskiy V.G., Vdovin K.M. Osobennosti podvodnogo obsledovaniya transportnych soorugeniy. 2. Charakternye povrezhdeniya opor mostovych soorugeniy [Features of underwater inspection of transport facilities. 2. The characteristic damage to the supports of bridge structures]. Internet-zhurnal «Naukovedenie», 2013, no. 6. (in Russian) 15. Krasnoshchekov Yu.V. РАЗДЕЛ III. СТРОИТЕЛЬСТВО И АРХИТЕКТУРА Поступила 25.07.2018, принята к публи- кации 21.12.2018. 8. Shtil’man E.I., Berezetsky V.I. Proletnye stroeniya mostov iz pustotnych plit [Span structures of bridges from hollow slabs]. Moscow. Transport, 1972. 81 p. (in Russian) Автор прочитал и одобрил оконча- тельный вариант рукописи. 9. Kozhushko V.P. О razvitii prostranstvennych metodov rascheta proletnych stroeniy avtodorozhnych mostov [On the development of spatial methods for calculating the span structures of road vehicles]. Nauka ta progres transport, 2008, no. 21, Pp. 128-129. (in Russian) Прозрачность финансовой деятельно- сти: автор не имеет финансовой заинте- ресованности в представленных матери- алах или методах. Конфликт интересов отсутствует. Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) Том 15, № 6. 2018. Сквозной номер выпуска – 64 (Vol. 15, no. 6. 2018. Continuous issue – 64) REFERENCES Raschet karkasnogo zdaniya na progressiruyushchee obrushenie pri avariynom otkaze kolonny [Calculation of the frame building for a progressive collapse in the event of an emergency failure of the column]. Stroitel’naya mechanika I raschet sooruzheniy, 2017, no 1, Pp. 54-58. (in Russian) 6. Nikolenko М.А., Minasov A.G., Savinova E.I. Novye metody proektirovaniya zhelezobetonnykh proletnykh stroeniy [New methods of the reinforced concrete superstructures design]. Inzhenerny vestnik Dona, 2017, no 2(45). (in Russian) 16. Raizer V.D. Teoriya nadezhnosti sooruzheniy [Theory of the structures reliability]. Moscow. Publisher ASV. 2010. 384 p. (in Russian) ( ) 17. Nazarov Yu.P., Gorodetsky A.S., Simbirkin V.N. К problem obespecheniya zhivuchesti stroitel’nych konstruktsy pri avariynych vozdeystviyach [To the problem of ensuring the survivability of building structures under emergency influences]. Stroitel’naya mechanika i raschet sooruzheniy, 2009, no 4, pp. 5-9. (in Russian) 17. Nazarov Yu.P., Gorodetsky A.S., Simbirkin V.N. К problem obespecheniya zhivuchesti stroitel’nych konstruktsy pri avariynych vozdeystviyach [To the problem of ensuring the survivability of building structures under emergency influences]. Stroitel’naya mechanika i raschet sooruzheniy, 2009, no 4, pp. 5-9. (in Russian) 7. Ulitsky B.E. Prostranstvennye raschety mostov (s ispol’zovaniem EZVM) [Spatial calculations of bridges (using an electronic digital computer)]. Moscow. Transport, 1967. 406 p. (in Russian) © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal 931 ИНФОРМАЦИЯ ОБ АВТОРЕ 18. Tikhonov I.N., Kozelkov M.M. Raschet I konstruirovanie zhelezobetonnykh monolitnych perekrytiy zdaniy s uchetom zashchity ot progressiruyushchego obrusheniya [Calculation and construction of reinforced concrete monolithic ceilings, taking into account protection against progressive collapse]. Beton I zhelezobeton, 2009, no 3, Pp. 2-8. Краснощеков Юрий Васильевич – д-р техн. наук, доц., кафедра «Строительные кон- струкции», «Сибирский государственный ав- томобильно-дорожный университет (СибА- ДИ)», ORCID 0000-0002-6695-1648 (644080, г. Омск, пр. Мира, д. 5, e-mail: uv1942@mail.ru). p 19. Guidance for designers to Eurocode 1: Impacts on structures. Standards EN 1991-1-1 and 1-3-1-7; trans. with English. / H. Gulvanesyan, P. Formici, J.-A. Kalgaro with the participation of J.Hording. Moscow: MGSU, 2011. - 340 p. 20. Krasnoshchekov Yu.V., Zapoleva M.Yu. Osnovy proektirovaniya konstruktsiy zdaniy I sooruzheniy [Fundamentals of building structures design]. Moscow. Infra-Inzheneriya, 2018. 296 с. (in Russian) INFORMATION ABOUT THE AUTHOR Krasnoshchekov Y.V. – Doctor of Technical Science, Associate Professor of the Building Construction Department, Siberian State Automobile and Highway University (SibADI), ORCID 0000-0002-6695-1648 (644080, 5, Mira Ave., е-mail: uv1942@mail.ru). Krasnoshchekov Y.V. – Doctor of Technical Science, Associate Professor of the Building Construction Department, Siberian State Automobile and Highway University (SibADI), ORCID 0000-0002-6695-1648 (644080, 5, Mira Ave., е-mail: uv1942@mail.ru). 20. Krasnoshchekov Yu.V., Zapoleva M.Yu. Osnovy proektirovaniya konstruktsiy zdaniy I sooruzheniy [Fundamentals of building structures design]. Moscow. Infra-Inzheneriya, 2018. 296 с. (in Russian) © 2004–2018 Вестник СибАДИ The Russian Automobile and Highway Industry Journal 932
https://openalex.org/W2122252204	https://escholarship.org/content/qt40w814wj/qt40w814wj.pdf?t=nbspk1	English	null	MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells	Molecular cancer	2,010	cc-by	11,105	UC Irvine UC Irvine Previously Published Works Title MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells Permalink https://escholarship.org/uc/item/40w814wj Journal Molecular Cancer, 9(1) ISSN 1476-4598 Authors Liu, Feng Singh, Amarinder Yang, Zhen et al. Publication Date 2010 DOI 10.1186/1476-4598-9-214 Copyright Information This work is made available under the terms of a Creative Commons Attribution Licens availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells Permalink https://escholarship.org/uc/item/40w814wj Journal Molecular Cancer, 9(1) ISSN 1476-4598 Authors Liu, Feng Singh, Amarinder Yang, Zhen et al. Publication Date 2010 DOI 10.1186/1476-4598-9-214 Copyright Information This work is made available under the terms of a Creative Commons Attribution Licens availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells Permalink https://escholarship.org/uc/item/40w814wj Journal Molecular Cancer, 9(1) ISSN 1476-4598 Authors Liu, Feng Singh, Amarinder Yang, Zhen et al. Publication Date 2010 DOI 10.1186/1476-4598-9-214 Copyright Information This work is made available under the terms of a Creative Commons Attribution Licens availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells Title MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells Permalink https://escholarship.org/uc/item/40w814wj Journal Molecular Cancer, 9(1) Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ * Correspondence: liufe@uci.edu 1Department of Medicine, University of California-Irvine School of Medicine, Orange, CA 92868, USA Full list of author information is available at the end of the article RESEARCH Open Access © 2010 Liu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells Feng Liu1,2, Amarinder Singh3, Zhen Yang1,2,4, Angela Garcia2, Yu Kong2,5, Frank L Meyskens Jr1,2,3 Powered by the California Digital Library University of California eScholarship.org Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Abstract Finally, cell lines with high levels of MiTF expression showed higher resistance to UVC-induced cell death than those with low-level MiTF. These data sug- gest that MiTF mediates a survival signal linking Erk1/2 activation and p21WAF1/CIP1 regulation via phosphorylation on serine 73, which facilitates cell cycle arrest. In addition, our data also showed that exposure to different wave- lengths of UV light elicited different signal pathways involving MiTF. Abstract As a survival factor for melanocytes lineage cells, MiTF plays multiple roles in development and melanomagenesis. What role MiTF plays in the DNA damage response is currently unknown. In this report we observed that MiTF was phosphorylated at serine 73 after UVC radiation, which was followed by proteasome-mediated degradation. Unlike after c-Kit stimulation, inhibiting p90RSK-1 did not abolish the band shift of MiTF protein, nor did it abolish the UVC-mediated MiTF degradation, suggesting that phosphorylation on serine 73 by Erk1/2 is a key event after UVC. Furthermore, the MiTF-S73A mutant (Serine 73 changed to Alanine via site-directed mutagenesis) was unable to degrade and was continuously expressed after UVC exposure. Compared to A375 melanoma cells expressing wild- type MiTF (MiTF-WT), cells expressing MiTF-S73A mutant showed less p21WAF1/CIP1 accumulation and a delayed WAF1/CIP1 f l ll h d f As a survival factor for melanocytes lineage cells, MiTF plays multiple roles in development and melanomagenesis. What role MiTF plays in the DNA damage response is currently unknown. In this report we observed that MiTF was phosphorylated at serine 73 after UVC radiation, which was followed by proteasome-mediated degradation. Unlike after c-Kit stimulation, inhibiting p90RSK-1 did not abolish the band shift of MiTF protein, nor did it abolish the UVC-mediated MiTF degradation, suggesting that phosphorylation on serine 73 by Erk1/2 is a key event after UVC. Furthermore, the MiTF-S73A mutant (Serine 73 changed to Alanine via site-directed mutagenesis) was unable to degrade and was continuously expressed after UVC exposure. Compared to A375 melanoma cells expressing wild- type MiTF (MiTF-WT), cells expressing MiTF-S73A mutant showed less p21WAF1/CIP1 accumulation and a delayed p21WAF1/CIP1 recovery after UVC. Consequently, cells expressing MiTF-WT showed a temporary G1 arrest after UVC, but cells expressing MiTF-S73A mutant or lack of MiTF expression did not. Finally, cell lines with high levels of MiTF expression showed higher resistance to UVC-induced cell death than those with low-level MiTF. These data sug- gest that MiTF mediates a survival signal linking Erk1/2 activation and p21WAF1/CIP1 regulation via phosphorylation on serine 73, which facilitates cell cycle arrest. In addition, our data also showed that exposure to different wave- lengths of UV light elicited different signal pathways involving MiTF. p21WAF1/CIP1 recovery after UVC. Consequently, cells expressing MiTF-WT showed a temporary G1 arrest after UVC, but cells expressing MiTF-S73A mutant or lack of MiTF expression did not. Background preventing apoptosis via activating melanoma inhibitor of apoptosis (ML-IAP) [11], inhibiting invasion via acti- vating DIAPH-1 [12], and promoting survival after ele- vation of cellular reactive oxygen species via activating Ape/Ref-1 [13]. A recent study using mouse melano- cytes with various MiTF doses indicated that MiTF dose was a primary determinant for murine melanocytes survival after UVR [14]; however, the mechanism(s) by which this occurred was not clear. MiTF plays a critical role in melanocyte lineage differ- entiation and survival [1], as well as melanomagenesis [2]. The MiTF gene is amplified in about 20% of mela- nomas and is capable of transforming normal melano- cytes in certain genetic environments, therefore it has been suggested that MiTF can function as an oncogene [3,4]. However, re-expression of MiTF in BRAF-expres- sing human melanocytes inhibited cell proliferation, suggesting that MiTF represses cell cycle progression [5]. This is consistent with reports showing that MiTF activates the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p16INK4A [6,7]. More and more evi- dence indicates that MiTF plays multiple roles in mela- nomagenesis including stimulating angiogenesis via activating Hif1a [8], enhancing cell proliferation via activating transcription of Bcl-2 and CDK2 [9,10], A genetic hallmark of human melanoma is mutually exclusive mutations of BRAF and NRAS, which are found in more than 90% of tumors [15]. Oncogenic BRAF or NRAS mutations activate cell proliferation pathway through downstream mitogen-activated kinases Mek1/2 and extracellular signal-regulated kinase (Erk1/ 2) [16]. BRAF or NRAS activation leads to Mek1/2 acti- vation which in turn activates Erk1/2 which directly phosphorylates MiTF at serine 73 [17,18]. Activated Erk1/2 can further activate its downstream kinase p90- RSK1 which can also phosphorylate MiTF at serine 409 [19]. Phosphorylation at both sites triggered by c-Kit Correspondence: liufe@uci.edu 1Department of Medicine, University of California-Irvine School of Medicine, Orange, CA 92868, USA Full list of author information is available at the end of the article Page 2 of 12 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 suggesting that phosphorylation of MiTF was not a sig- nal for recruiting DNA repair proteins to DNA-damage sites, nor was it a signal for translocation to cytoplasm. Background MiTF phosphorylation was examined 1 hour after var- ious doses of UVC radiation; as low as 1 mJ/cm2 of radiation led to MiTF phosphorylation in c83-2C cells (Fig 1D). stimulation leads to a signal cascade for pigment cell development [19]. This dual phosphorylation results in a transient increase of MiTF trans-activation activity and a subsequent degradation; however, the biological conse- quence of this transient activation and degradation is not clear. Recently in vivo studies indicated that muta- tion at serine 73 completely rescued mouse coat color [20], suggesting this mutation may have other functions than melanocyte development, among which participat- ing in the DNA damage response is one of the possibili- ties [21]. Whether MiTF plays a role in DNA damage response has not been previously reported and is the subject of this study. MiTF phosphorylation is via Erk1/2 mitogen-activated protein kinases (MAPK) and is required for its subsequent proteasome-dependent degradation To investigate the upstream signal for MiTF phosphory- lation, three kinase inhibitors were incubated with NHMs before they were exposed to UVC (3 mJ/cm2): MEK inhibitor U0126 which leads to Erk1/2 inhibition (10 μM), the p38 MAPK inhibitor SB203580 (20 μM), and wortamannin (20 μM), an inhibitor of phosphatidy- linositol-3 kinase, Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinase. Cells were exposed to UVC (3 mJ/cm2) and collected 1 hour later to examine MiTF phosphorylation. As shown in Fig 2A, top panel, among these kinase inhibitors, only U0126 inhibited UVC-mediated MiTF phosphorylation, sug- gesting that Erk1/2 is the upstream kinase. This obser- vation was further confirmed in c83-2C melanoma cells. The c83-2C cells were pre-treated with U0126 (10 μM), c-Jun N-terminal kinase inhibitor SP600125 (10 μM), RSK1/2 inhibitor SL0101 (70 μM) and another Erk1/2 kinase inhibitor PD98059 ((20 μM), and then exposed to UVC (2.5 mJ/cm2) and allowed to recover for 1 hour. Both U0126 and PD98059 inhibited UVC-mediated MiTF phosphorylation, while SP600125 and SL0101 did not (Fig 2A, bottom panel). Erk1/2 activation upon UVC radiation and its inhibition by U0126 was con- firmed by western blot using phospho-Erk-specific anti- bodies (Fig 2B). In this study, we report that the DNA damaging agent UVC radiation leads to Erk1/2 mediated phosphorylation of MiTF at serine 73, which in turn leads to proteasome- mediated MiTF degradation. Erk1/2 phosphorylation of MiTF played a critical role in activating p21WAF1/CIP1 transcription and a temporary G1 cell cycle arrest, which enhanced cell survival after UVC radiation. These results suggest a novel function of MiTF in linking Erk1/2 acti- vation and p21WAF1/CIP1 regulation after UVC radiation in normal human melanocytes and melanoma cells. MiTF is phosphorylated and transiently degraded after UVC in NHMs and some melanoma cells To examine whether MiTF plays a role in DNA damage response, two normal human melanocyte (NHM) cell lines were exposed to potent DNA damaging agent UVC (3 mJ/cm2) and allowed them to recover for var- ious periods of time. As shown in Fig 1A, MiTF at base- line was detected as a doublet band on western blot: the lower band represented unphosphorylated and the top band the phosphorylated form of MiTF [19]. One hour after UVC, all the MiTF was shifted to the top band (Fig 1A). The phosphorylation continued for 2 hours after UVC, followed by a decrease of MiTF protein (both forms) at 4 and 6 hours. After that, MiTF protein started to recover 9 hours post radiation and nearly completely recovered to its pre-treatment levels 12 to 24 hours after UVC (Fig 1A). Next we examined whether the Erk1/2-mediated phos- phorylation was required for MiTF degradation after UVC. Pre-treatment with U0126 in c83-2C cells abol- ished MiTF phosphorylation, as well as its subsequent degradation (Fig 2C, top panel). A similar result was also observed in Malme-3 M melanoma cells pre-treated with U0126 (Fig 2C, bottom panel). These data suggest that phosphorylation of MiTF by Erk1/2 was necessary for its degradation. The two NHMs were isolated from neonatal foreskin of a Caucasian (top) and an African black baby (bottom) respectively. There was no significant difference in their response to UVC. A similar response was observed in c83-2C melanoma cells (Fig 1B). MiTF degradation was further confirmed by immunofluorescence (Fig 1C). c83- 2C cells were exposed to UVC and fixed for immuno- fluorescence staining at various time points. Consistent with its nuclear localization, the fluorescence signal for MiTF was mainly observed in nuclei (Fig 1C). However, no specific foci were observed, nor was there a dramatic re-localization of the protein at 1 hour post radiation, It was previously reported that the c-Kit signal trig- gered dual-phosphorylation of MiTF, one at serine 73 by Erk2 and the other on serine 409 by Erk1/2 down- stream kinase p90 RSK-1. To examine whether UVC also exhibited a similar effect on MiTF through p90 RSK-1, we pre-treated c83-2C cells with RSK-1 inhibitor SL0101 before UVC radiation, MiTF degradation was still observed (Fig 2D), suggesting that p90 RSK-1 phos- phorylation of MiTF was not a critical event under this Liu et al. MiTF is phosphorylated and transiently degraded after UVC in NHMs and some melanoma cells MiTF-S73A did not show any band shift nor degrada- tion after UVC (Fig 3C), while MiTF-WT was phos- phorylated and degraded (Fig 3D). To investigate whether poly-ubiquitination is involved in MiTF regu- lation after UVC radiation, NHMs were exposed to 3 mJ/cm2 of UVC and then collected 2 hours later for immunoprecipitation. As shown in Fig 3E, UVC dra- matically enhanced poly-ubiquitination of MiTF pro- tein (compare Lanes 1 and 2). Anti-GFP antibody was used as a negative control for anti-MiTF antibody (Lane 3). Taken together, these results suggest that Erk1/2-mediated MiTF phosphorylation on serine 73 is required for MiTF degradation after UVC. These results are consistent with previous observation that phosphorylation on serine 73 is essential for MiTF poly-ubiquitination and degradation [22]. MiTF is phosphorylated and transiently degraded after UVC in NHMs and some melanoma cells Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Page 3 of 12 Figure 1 MiTF was phosphorylated and degraded after UVC. A, Normal human melanocytes were exposed to 3 mJ/cm2 of UVC and collected at various time points for western blot analysis. NHMs from a white individual (top panel) and a black individual (bottom panel) showed a similar response. B, human melanoma c83-2C cells were subjected to 3 mJ/cm2 of UVC and collected for western blot at the indicated time points. C, c83-2C cells were treated with UVC and fixed at the indicated time and subjected to immunofluorescence detection of MiTF protein. DAPI staining was used to show nucleus. D, dose-dependent phosphorylation of MiTF in c83-2C cells. In western blot, either p84 or a-tubulin was used as a loading control. Figure 1 MiTF was phosphorylated and degraded after UVC. A, Normal human melanocytes were exposed to 3 mJ/cm2 of UVC and collected at various time points for western blot analysis. NHMs from a white individual (top panel) and a black individual (bottom panel) showed a similar response. B, human melanoma c83-2C cells were subjected to 3 mJ/cm2 of UVC and collected for western blot at the indicated time points. C, c83-2C cells were treated with UVC and fixed at the indicated time and subjected to immunofluorescence detection of MiTF protein. DAPI staining was used to show nucleus. D, dose-dependent phosphorylation of MiTF in c83-2C cells. In western blot, either p84 or a-tubulin was used as a loading control. condition, and Erk1/2 was the major kinase for UVC- triggered MiTF phosphorylation and degradation. MiTF-S73A did not show any band shift nor degrada- tion after UVC (Fig 3C), while MiTF-WT was phos- phorylated and degraded (Fig 3D). To investigate whether poly-ubiquitination is involved in MiTF regu- lation after UVC radiation, NHMs were exposed to 3 mJ/cm2 of UVC and then collected 2 hours later for immunoprecipitation. As shown in Fig 3E, UVC dra- matically enhanced poly-ubiquitination of MiTF pro- tein (compare Lanes 1 and 2). Anti-GFP antibody was used as a negative control for anti-MiTF antibody (Lane 3). Taken together, these results suggest that Erk1/2-mediated MiTF phosphorylation on serine 73 is required for MiTF degradation after UVC. These results are consistent with previous observation that phosphorylation on serine 73 is essential for MiTF poly-ubiquitination and degradation [22]. Phosphorylation on serine 73 is responsible for proteasome-mediated MiTF degradation To confirm that MiTF degradation is mediated by pro- teasome pathway, c83-2C cells were treated with MG132, a proteasome inhibitor and then exposed to UVC. MiTF exhibited an unchanged expression under these conditions (Fig 3A). Next we expressed MiTF-WT and MiTF-S73A in MiTF-negative A375 melanoma cells, and examined their accumulation after UVC. As shown in Fig 3B, MiTF-WT showed on western blot as a doublet band, MiTF-S73A, on the other hand, exhibited a single band that corresponded to the faster moving band. Liu et al. Molecular Cancer 2010, 9:214 Page 4 of 12 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 http://www.molecular-cancer.com/content/9/1/214 Figure 2 MiTF was phosphorylated by Erk1/2 kinase after UVC. A various kinase inhibitors and then exposed to UVR at 3 mJ/cm2, and co Erk1/2 activation and U0126 effect on Erk1/2 inhibition was examined b Figure 2 MiTF was phosphorylated by Erk1/2 kinase after UVC. A, NHM (top panel) or c83-2C cells (bottom panel) were pre-treated with various kinase inhibitors and then exposed to UVR at 3 mJ/cm2, and collected 1 hour post-radiation for western blot analysis. B, UVC-mediated Erk1/2 activation and U0126 effect on Erk1/2 inhibition was examined by western blot. NHM cells were pre-treated with U0126, exposed to UVC and collected for western blot 1 hour post-radiation. C, c83-2C cells (top panel) and Malme-3 M cells (bottom) were pre-treated with U0126 and then subjected to UVR at 3 mJ/cm2, and collected at the indicated time points for western blot analysis. D, NHM were pre-treated with p90 RSK- 1 inhibitor SL0101 and subjected to UVC, and collected at the indicated time for western blot analysis. Figure 2 MiTF was phosphorylated by Erk1/2 kinase after UVC. A, NHM (top panel) or c83-2C cells (bottom panel) were pre-treated with various kinase inhibitors and then exposed to UVR at 3 mJ/cm2, and collected 1 hour post-radiation for western blot analysis. B, UVC-mediated Erk1/2 activation and U0126 effect on Erk1/2 inhibition was examined by western blot. NHM cells were pre-treated with U0126, exposed to UVC and collected for western blot 1 hour post-radiation. C, c83-2C cells (top panel) and Malme-3 M cells (bottom) were pre-treated with U0126 and then subjected to UVR at 3 mJ/cm2, and collected at the indicated time points for western blot analysis. D, NHM were pre-treated with p90 RSK- 1 inhibitor SL0101 and subjected to UVC, and collected at the indicated time for western blot analysis. Phosphorylation on serine 73 is responsible for proteasome-mediated MiTF degradation Expression of MiTF-WT led to a temporary G1 arrest and enhanced cell survival in A375 cells but expression of MiTF-S73A did not were no significant changes in cells expressing MiTF- S73A or GFP. At 24 hours post radiation, the G1 popu- lation decreased significantly in all three groups of cells due to cell death (data not shown). Sub-G1 population was then quantified. 21.4% of sub-G1 cells were present in control cells expressing GFP, while only 12.1% of sub-G1 cells were found in cells expressing MiTF-WT (Fig 4B). In cells expressing MiTF-S73A, the sub-G1 population was 25.7%, more than 2 fold higher than that in MiTF-WT-expressing cells and close to what was observed in control GFP cells (Fig 4B). Cells normally undergo cell cycle arrest after UVC expo- sure to allow enough time for DNA damage repair [23]. To investigate the role of MiTF in UVC-mediated DNA damage response and cell cycle control, A375 cells which carry a wild-type p53 gene were transfected with QCXIP-GFP (control vector), QCXIP-MiTF-WT or QCXIP-MiTF-S73A and then exposed to UVR (3 mJ/ cm2). Cell cycle distribution was analyzed by fluores- cence-activated cell sorting at various time points after staining with Propidium Iodide (PI). About 40% of cells were in G1 phase when un-irradiated in all three groups. Eight hours after UVR, G1 population in MiTF-WT- expressing cells increased to 68% (Fig 4A), while there The above results suggested that expression of MiTF- WT caused a temporary G1 arrest after UVC, which enhanced cell survival. To further confirm this observa- tion, colony formation assay was used to measure cell survival rate after UVC. A375 cells were again Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Page 5 of 12 Figure 3 MiTF phosphorylation on serine 73 by Erk1/2 kinase was required for its proteasome-mediated degradation after UVR. A, c83-2C cells were exposed to UVC in the presence of MG132 and collected for western blot analysis at the indicated time points. B, transient expression of MiTF-WT, MiTF-S73A in A375 melanoma cell line. C83-2C cells served as a positive control for MiTF western blot. GFP is the control cells transfected with GFP in the same QCXIP vector that carried MiTF-WT or MiTF-S73A coding sequence. C, Cells expressing MiTF-S73A were exposed to UVC and collected for western blot analysis. D, Cells expressing MiTF-WT were exposed to UVC and collected for western blot analysis. E, MiTF was poly-ubiquitinated after UVC radiation. Phosphorylation on serine 73 is responsible for proteasome-mediated MiTF degradation NHMs were exposed to UVC (3 mJ/cm2) and collected for immunoprecipitation by anti-MiTF antibodies (un-irradiated cells and anti-GFP antibodies were used as controls), then probed with anti-ubiquitin antibodies (top panel). The membrane was stripped and blotted with anti-MiTF antibodies transfected with QCXIP-GFP, QCXIP-MiTF-WT or QCXIP-MiTF-S73A and were irradiated with 3 mJ/cm2 of UVC 24 hours after transfection. Colonies were counted 2 weeks later. The relative survival rates were normalized to that of GFP-expressing control cells and the results are shown in Fig 4C. MiTF-WT increased cell survival after UVR, but MiTF-S73A did not. transfected with QCXIP-GFP, QCXIP-MiTF-WT or QCXIP-MiTF-S73A and were irradiated with 3 mJ/cm2 of UVC 24 hours after transfection. Colonies were counted 2 weeks later. The relative survival rates were normalized to that of GFP-expressing control cells and the results are shown in Fig 4C. MiTF-WT increased cell survival after UVR, but MiTF-S73A did not. MiTF-negative melanoma cells are more sensitive to UVC To investigate whether MiTF confers to a survival advantage in other melanoma cell lines, we exposed dif- ferent melanoma cell lines with different MiTF accumu- lation levels to 3 mJ/cm2 of UVC and examined the cell survival 24 hours later by Propidium Iodide staining and FACS analysis. As shown in Fig 4D, three melanoma cell lines (A375, 1205Lu and WM3211) which accumu- lated undetectable MiTF protein [13] showed higher cell death (19% to 26%) as compared to three MiTF-positive melanoma cell lines (Malme-3 M, SK-Mel-28 and c83- 2C) (4% to 10%) (Fig 4D and 4E). The difference between these two groups was significant (two tailed p value from an unpaired t test is 0.003). To further confirm that MiTF plays a key role in cell survival after UVC radiation, MiTF was knocked down in SK-Mel-28 melanoma cell line by 2 different shRNA constructs Mish1 and Mish2 (Fig 5E); cells were exposed to 2 and 4 mJ/cm2 of UVC, and colonies were counted 2 weeks later. The results indicated that Mish1 and Mish2 trans- duced cells showed decreased colony formation after UVC as compared to control parental SK-Mel-28, as well as SK-Mel-28 cells transduced with pGIPZ empty vector (Fig 4F). MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 Because p16INK4A is often lost in melanoma cells, we examined accumulation of CDK inhibitors p21WAF1/CIP1 and p27KIP1, both of which are downstream of MiTF. MiTF directly activates p21WAF1/CIP1 expression and indirectly activates p27 [6,12]. The basal level of p27KIP1 was not significantly altered in these three groups of cells (Fig 5A). However, p21WAF1/CIP1 level was elevated in cells expressing MiTF-WT as compared to cells expressing MiTF-S73A, which showed a slightly elevated level of p21WAF1/CIP1 as compared to cells expressing GFP (Fig 5A). Figure 3 MiTF phosphorylation on serine 73 by Erk1/2 kinase was required for its proteasome-mediated degradation after was required for its proteasome-mediated degradation after UVR. A, c83-2C cells were exposed to UVC in the presence of MG132 and collected for western blot analysis at the indicated time points. B, transient expression of MiTF-WT, MiTF-S73A in A375 melanoma cell line. C83-2C cells served as a positive control for MiTF western blot. GFP is the control cells transfected with GFP in the same QCXIP vector that carried MiTF-WT or MiTF-S73A coding sequence. C, Cells expressing MiTF-S73A were exposed to UVC and collected for western blot analysis. D, Cells expressing MiTF-WT were exposed to UVC and collected for western blot analysis. E, MiTF was poly-ubiquitinated after UVC radiation. NHMs were exposed to UVC (3 mJ/cm2) and collected for immunoprecipitation by anti-MiTF antibodies (un-irradiated cells and anti-GFP antibodies were used as controls), then probed with anti-ubiquitin antibodies (top panel). The membrane was stripped and blotted with anti-MiTF antibodies for a loading control (bottom). The IgG label indicates antibody heavy chain of IgG proteins. was required for its proteasome mediated degradation after UVR. A, c83-2C cells were exposed to UVC in the presence of MG132 and collected for western blot analysis at the indicated time points. B, transient expression of MiTF-WT, MiTF-S73A in A375 melanoma cell line. C83-2C cells served as a positive control for MiTF western blot. GFP is the control cells transfected with GFP in the same QCXIP vector that carried MiTF-WT or MiTF-S73A coding sequence. C, Cells expressing MiTF-S73A were exposed to UVC and collected for western blot analysis. D, Cells expressing MiTF-WT were exposed to UVC and collected for western blot analysis. E, MiTF was poly-ubiquitinated after UVC radiation. NHMs were exposed to UVC (3 mJ/cm2) and collected for immunoprecipitation by anti-MiTF antibodies (un-irradiated cells and anti-GFP antibodies were used as controls), then probed with anti-ubiquitin antibodies (top panel). MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 The membrane was stripped and blotted with anti-MiTF antibodies for a loading control (bottom). The IgG label indicates antibody heavy chain of IgG proteins. g To confirm that the regulation of p21WAF1/CIP1 by MiTF was indeed via transcriptional regulation, mRNA from A375 cells expressing MiTF-WT, MiTF-S73A and GFP was isolated and p21WAF1/CIP1 mRNA level deter- mined by quantitative RT-PCR. As shown in Fig 5B, MiTF-WT increased p21WAF1/CIP1 mRNA to about 5 fold that in control GFP-expressing cells, while MiTF- S73A also increased p21WAF1/CIP1 mRNA to about Liu et al. Molecular Cancer 2010, 9:214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Page 6 of 12 http://www.molecular-cancer.com/content/9/1/214 Figure 4 Expression of MiTF-WT enabled a temporary G1 arrest for improving cell survival after UVR. A, A375 cells were transfected with GFP, MiTF-WT or MiTF-S73A and then exposed to UVC at 3 mJ/cm2, and fixed 8 hours later for FACS analysis after Propidium Iodide staining. B, sub-G1 population of cells treated in A were calculated by FACS analysis and graphed 24 hours after UVC treatment. C, cells in A were seeded and exposed to UVC, then incubated for colony formation assay. Colonies formed 2 weeks after were counted, normalized to that in GFP- expressing cells and graphed. D, A375, WM3211, 1205Lu, Malme-3 M, SK-Mel-28 and c83-2C cells were exposed to UVC at 3 mJ/cm2, and cells were then collected 24 hours later for FACS analysis. E, Percentage of cell death before and after UVC were calculated and graphed F, knockdown of MiTF decreased cell survival after UVC. MiTF was knocked down by Mish1 and Mish2 shRNA (see Fig 5E) and exposed to 3 mJ/ cm2 of UVC. Colony formation was analyzed about 2 weeks post-radiation. Figure 4 Expression of MiTF-WT enabled a temporary G1 arrest for improving cell survival after UVR. A, A375 ce Figure 4 Expression of MiTF-WT enabled a temporary G1 arrest for improving cell survival after UVR. A, A375 cells were transfected with GFP, MiTF-WT or MiTF-S73A and then exposed to UVC at 3 mJ/cm2, and fixed 8 hours later for FACS analysis after Propidium Iodide staining. B, sub-G1 population of cells treated in A were calculated by FACS analysis and graphed 24 hours after UVC treatment. C, cells in A were seeded and exposed to UVC, then incubated for colony formation assay. Colonies formed 2 weeks after were counted, normalized to that in GFP- expressing cells and graphed. MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 Quantitative RT-PCR (left) and western blot (right) analysis of p21WAF1/CIP1 expression in control SK-Mel-28 cells (Mel-28), cells transduced with empty lentivirus vector pGIPZ (GIPZ), and cells transduced with lentivirus carrying MiTF shRNA constructs (Mish1 and Mish2). Again a-tubulin was used as a loading control. F, top: p27KIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP; bottom: p21WAF1/CIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP. The p53 served as a positive control for UVC radiation, a-tubulin served as a loading control. The western was repeated three times and a representative blot is shown; G, the p21 protein levels in the western blot were quantified by a densitometry, normalized to a-tubulin levels and then normalized to that in cells without irradiation and graphed. H, qRT-PCR analysis of p21WAF1/CIP1 mRNA accumulation after UVC in A375 cells expressing MiTF-WT or MiTF-S73A. Figure 5 MiTF-S73A is less potent in activating p21WAF1/CIP1 transcription. A, p21WAF1/CIP1 and p27KIP1 protein accumulation in A375 cells expressing MiTF-WT, MiTF-S73A and GFP were analyzed by western blot. B, transcripts of p21WAF1/CIP1 and MiTF were analyzed by qRT-PCR in the above cells, a-tubulin was the reference for both genes. C, p21WAF1/CIP1 promoter reporter analysis in cells co-transcfected with MiTF-WT or MiTF-S73A mutant constructs. D, p21WAF1/CIP1 protein accumulation decreased when MiTF phosphorylation was inhibited. NHMs were treated with 20 μM of U0126 for 24 hours and collected for western blot analysis. E, knockdown MiTF led to decreased p21WAF1/CIP1 expression. Quantitative RT-PCR (left) and western blot (right) analysis of p21WAF1/CIP1 expression in control SK-Mel-28 cells (Mel-28), cells transduced with empty lentivirus vector pGIPZ (GIPZ), and cells transduced with lentivirus carrying MiTF shRNA constructs (Mish1 and Mish2). Again a-tubulin was used as a loading control. F, top: p27KIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP; bottom: p21WAF1/CIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP. The p53 served as a positive control for UVC radiation, a-tubulin served as a loading control. The western was repeated three times and a representative blot is shown; G, the p21 protein levels in the western blot were quantified by a densitometry, normalized to a-tubulin levels and then normalized to that in cells without irradiation and graphed. H, qRT-PCR analysis of p21WAF1/CIP1 mRNA accumulation after UVC in A375 cells expressing MiTF-WT or MiTF-S73A. MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 D, A375, WM3211, 1205Lu, Malme-3 M, SK-Mel-28 and c83-2C cells were exposed to UVC at 3 mJ/cm2, and cells were then collected 24 hours later for FACS analysis. E, Percentage of cell death before and after UVC were calculated and graphed F, knockdown of MiTF decreased cell survival after UVC. MiTF was knocked down by Mish1 and Mish2 shRNA (see Fig 5E) and exposed to 3 mJ/ cm2 of UVC. Colony formation was analyzed about 2 weeks post-radiation. Figure 4 Expression of MiTF-WT enabled a temporary G1 arrest for improving cell survival after UVR. A, A375 cells were transfected with GFP, MiTF-WT or MiTF-S73A and then exposed to UVC at 3 mJ/cm2, and fixed 8 hours later for FACS analysis after Propidium Iodide staining. B, sub-G1 population of cells treated in A were calculated by FACS analysis and graphed 24 hours after UVC treatment. C, cells in A were seeded and exposed to UVC, then incubated for colony formation assay. Colonies formed 2 weeks after were counted, normalized to that in GFP- expressing cells and graphed. D, A375, WM3211, 1205Lu, Malme-3 M, SK-Mel-28 and c83-2C cells were exposed to UVC at 3 mJ/cm2, and cells were then collected 24 hours later for FACS analysis. E, Percentage of cell death before and after UVC were calculated and graphed F, knockdown of MiTF decreased cell survival after UVC. MiTF was knocked down by Mish1 and Mish2 shRNA (see Fig 5E) and exposed to 3 mJ/ cm2 of UVC. Colony formation was analyzed about 2 weeks post-radiation. 2 fold of that in control cells. MiTF expression levels were also examined in these cells by qRT-PCR. The control A375 GFP cells expressed very low levels of MiTF, nearly undetectable, which is consistent with our previous observation that no MiTF protein was detect- able in A375 cells. In cells transfected with either MiTF- WT or MiTF-S73A constructs the mRNA of MiTF accumulated to approximately 90 fold that in control cells. To further confirm that this regulation is via dif- ferential transcriptional activities on the p21WAF1/CIP1 promoter, MiTF-WT or MiTF-S73A constructs were co-transfected with p21WAF1/CIP1 promoter-luciferase reporter plasmid. We observed that expression of MiTF- WT led to about 2 fold of p21WAF1/CIP1 promoter activ- ity as compared to expression of MiTF-S73A mutant (Fig 5C). Further more, treating the NHMs with U0126 Liu et al. Molecular Cancer 2010, 9:214 Liu et al. MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Page 7 of 12 http://www.molecular-cancer.com/content/9/1/214 (20 μM) caused a decrease on MiTF phosphorylation 5E both shRNA knocked down MiTF to about 30% of Figure 5 MiTF-S73A is less potent in activating p21WAF1/CIP1 transcription. A, p21WAF1/CIP1 and p27KIP1 protein accumulation in A375 cells expressing MiTF-WT, MiTF-S73A and GFP were analyzed by western blot. B, transcripts of p21WAF1/CIP1 and MiTF were analyzed by qRT-PCR in the above cells, a-tubulin was the reference for both genes. C, p21WAF1/CIP1 promoter reporter analysis in cells co-transcfected with MiTF-WT or MiTF-S73A mutant constructs. D, p21WAF1/CIP1 protein accumulation decreased when MiTF phosphorylation was inhibited. NHMs were treated with 20 μM of U0126 for 24 hours and collected for western blot analysis. E, knockdown MiTF led to decreased p21WAF1/CIP1 expression. Quantitative RT-PCR (left) and western blot (right) analysis of p21WAF1/CIP1 expression in control SK-Mel-28 cells (Mel-28), cells transduced with empty lentivirus vector pGIPZ (GIPZ), and cells transduced with lentivirus carrying MiTF shRNA constructs (Mish1 and Mish2). Again a-tubulin was used as a loading control. F, top: p27KIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP; bottom: p21WAF1/CIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP. The p53 served as a positive control for UVC radiation, a-tubulin served as a loading control. The western was repeated three times and a representative blot is shown; G, the p21 protein levels in the western blot were quantified by a densitometry, normalized to a-tubulin levels and then normalized to that in cells without irradiation and graphed. H, qRT-PCR analysis of p21WAF1/CIP1 mRNA accumulation after UVC in A375 cells expressing MiTF-WT or MiTF-S73A. Figure 5 MiTF-S73A is less potent in activating p21WAF1/CIP1 transcription. A, p21WAF1/CIP1 and p27KIP1 protein accumulation in A375 cells expressing MiTF-WT, MiTF-S73A and GFP were analyzed by western blot. B, transcripts of p21WAF1/CIP1 and MiTF were analyzed by qRT-PCR in the above cells, a-tubulin was the reference for both genes. C, p21WAF1/CIP1 promoter reporter analysis in cells co-transcfected with MiTF-WT or MiTF-S73A mutant constructs. D, p21WAF1/CIP1 protein accumulation decreased when MiTF phosphorylation was inhibited. NHMs were treated with 20 μM of U0126 for 24 hours and collected for western blot analysis. E, knockdown MiTF led to decreased p21WAF1/CIP1 expression. Discussion MiTF is a lineage-specific transcription factor; how it is regulated after DNA damage has not been reported, although it was evident that MiTF dose was correlated with cell survival after UVR [14]. Here we show that the action of MiTF was downstream of Erk1/2 kinase and that phosphorylation on serine 73 played a key role in its trans-activation activity on p21WAF1/CIP1 promoter under these conditions. The Erk1/2 phosphorylation led to proteasome-mediated MiTF degradation, which was concomitant with a temporary G1 cell cycle arrest. Although it was previously known that both Erk1/2 and p21WAF1/CIP1 was activated by UVC [26], a direct link between these two factors was not elucidated. Our data suggest that MiTF participates in G1 cell cycle arrest after UVC via Erk1/2 kinase and p21WAF1/CIP1 regula- tion, and hence provides a direct link between Erk1/2 kinase and p21WAF1/CIP1 activation. , p y p level in MiTF-S73A-expressing cells was already lower than that in MiTF-WT cells. This slower recovery of p21WAF1/CIP1 may also result from less effective activa- tion of p21WAF1/CIP1 by MiTF-S73A mutants. The p21WAF1/CIP1 protein level showed a similar slower recovery in control cells expressing GFP (Fig 5F, bottom panel). The kinetics of p21WAF1/CIP1 protein levels from these western blots were quantified by a densitometer and normalized to the untreated cells, and graphed in Fig 5G. The kinetics of p21WAF1/CIP1 mRNA following UVC radiation was determined by qRT-PCR, normalized to a-tubulin mRNA, and the results are shown in Fig 5H. Interestingly, the mRNA levels of p21WAF1/CIP1 remained basically unchanged during the first 4 hours of recovery, but then it was induced dramatically and rapidly in MiTF-WT cells but to a lesser extend in MiTF-S73A cells (Fig 5H). It was previously reported that Erk2 directly phos- phorylated MiTF at serine 73 [18], and this phosphory- lation occurred under the condition of c-Kit stimulation, which also triggered a second phosphorylation on serine 409 by p90 RSK-1, leading to a transient increase of its trans-activation activity and subsequent proteasome- mediated MiTF degradation [19]. We observed that under UVC stress, inhibition of Mek1/2 kinase activity led to MiTF stabilization while inhibition of p90 RSK-1 activity did not, suggesting that phosphorylation on ser- ine 73 was the key signaling event after UVC. This was further confirmed by MiTF-S73A mutation which was not degraded after UVC. Discussion The degradation was inhibited by proteasome inhibitor MG132, suggesting that the sig- naling pathways via Erk1/2 activation after UVC and after c-Kit stimulation were distinct from each other. MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 In cells expressing MiTF-S73A protein, p21WAF1/CIP1 also degraded 2 to 4 hours post UVC; however, at 8 and 12 hour post radiation, it remained at 25% and 42% of that in untreated cells, respectively. Note that the p21WAF1/CIP1 MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 (20 μM) caused a decrease on MiTF phosphorylation, which was concomitant with reduced p21WAF1/CIP1 pro- tein levels (Fig 5D). To further confirm regulation of p21WAF1/CIP1 by MiTF, MiTF was knocked down in SK- Mel-28 cells by lentivirus mediated shRNA Mish1 and Mish2 (2 different shRNA constructs). As shown in Fig 5E, both shRNA knocked down MiTF to about 30% of its original protein levels (Fig 5E, right panel), the con- trol lentivirus vector GIPZ did not affect MiTF expres- sion. Both p21WAF1/CIP1 mRNA and protein levels decreased when MiTF was knocked down (Fig 5E). A known MiTF target Bcl2 protein accumulation was also 5E, both shRNA knocked down MiTF to about 30% of its original protein levels (Fig 5E, right panel), the con- trol lentivirus vector GIPZ did not affect MiTF expres- sion. Both p21WAF1/CIP1 mRNA and protein levels decreased when MiTF was knocked down (Fig 5E). A known MiTF target Bcl2 protein accumulation was also Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Page 8 of 12 4 hours post-radiation. This degradation was not inhib- ited by U0126 (Fig 6B), suggesting that there were dis- tinct signal transduction pathways involved in MiTF regulation after UVC and UVA radiation. To further understand this difference, we examined Erk1/2 activa- tion 1 hour after UVA radiation. In fact Erk1/2 did not show substantial activation at this time (Fig 6C). In con- trast, MiTF did not exhibit any changes in terms of accumulation levels or phosphorylation status after UVB radiation (Fig 6D). 25 mJ/cm2 of UVB did not affect MiTF accumulation or phosphorylation up to 24 hours (Fig 6D, top panel); Up to 75 mJ/cm2 of UVB radiation did not trigger MiTF phosphorylation at 1 hour after radiation (Fig 6D, bottom panel). As a positive control, p53 up-regulation was observed (Fig 6D). reduced in Mish1 and Mish2 transduced cells (Fig 5E), which may help to explain in part why MiTF knock- down led to decreased cell survival after UVC (Fig 4F). which may help to explain in part why MiTF knock- down led to decreased cell survival after UVC (Fig 4F). Next we examined the kinetics of p21WAF1/CIP1 and p27KIP1 after UVC. MiTF participates in G1 arrest via its regulation of p21WAF1/CIP1 The p27KIP1 protein showed a rapid degradation after UVC in all cells examined and no dif- ference was observed in these three groups of cells (Fig 5F, top panel), suggesting that p27KIP1 was not responsi- ble for the observed temporary G1 arrest in MiTF-WT- expressing cells. The p21WAF1/CIP1 protein degraded transiently after UVC as previously reported [24] at 2 to 4 hours, and followed by a rapid re-accumulation (Fig 5F, bottom panel). In cells expressing MiTF-WT pro- tein, p21WAF1/CIP1 degraded to less than 20% of its origi- nal level 2 to 4 hours post-UVC and recovered to about 50% at 8 hour, over 60% at 12 hour. In cells expressing MiTF-S73A protein, p21WAF1/CIP1 also degraded 2 to 4 hours post UVC; however, at 8 and 12 hour post radiation, it remained at 25% and 42% of that in untreated cells, respectively. Note that the p21WAF1/CIP1 level in MiTF-S73A-expressing cells was already lower than that in MiTF-WT cells. This slower recovery of p21WAF1/CIP1 may also result from less effective activa- tion of p21WAF1/CIP1 by MiTF-S73A mutants. The p21WAF1/CIP1 protein level showed a similar slower recovery in control cells expressing GFP (Fig 5F, bottom panel). The kinetics of p21WAF1/CIP1 protein levels from these western blots were quantified by a densitometer and normalized to the untreated cells, and graphed in Fig 5G. The kinetics of p21WAF1/CIP1 mRNA following UVC radiation was determined by qRT-PCR, normalized to a-tubulin mRNA, and the results are shown in Fig 5H. Interestingly, the mRNA levels of p21WAF1/CIP1 remained basically unchanged during the first 4 hours of recovery, but then it was induced dramatically and rapidly in MiTF-WT cells but to a lesser extend in MiTF-S73A cells (Fig 5H). Next we examined the kinetics of p21WAF1/CIP1 and p27KIP1 after UVC. The p27KIP1 protein showed a rapid degradation after UVC in all cells examined and no dif- ference was observed in these three groups of cells (Fig 5F, top panel), suggesting that p27KIP1 was not responsi- ble for the observed temporary G1 arrest in MiTF-WT- expressing cells. The p21WAF1/CIP1 protein degraded transiently after UVC as previously reported [24] at 2 to 4 hours, and followed by a rapid re-accumulation (Fig 5F, bottom panel). In cells expressing MiTF-WT pro- tein, p21WAF1/CIP1 degraded to less than 20% of its origi- nal level 2 to 4 hours post-UVC and recovered to about 50% at 8 hour, over 60% at 12 hour. Differential response of MiTF to different wavelengths of UV radiation B, NHMs were pre-treated with U0126 and then exposed to UVA and collected for western blot analysis at the indicated time points. C, No obvious Erk1/2 activation after UVA radiation. NHM was irradiated with either UVA (2.5 J/cm2) or UVC (3 mJ/ cm2) and collected for western blot analysis 1 hour post-radiation. D, NHMs were exposed to UVB (25 mJ/cm2) and collected for western blot at the indicated time points (top panel) or 1 hour post-UVB at various doses (bottom panel). Again the p53 serves as a positive control and a- tubulin serve as a loading control. Figure 6 Distinct responses of MiTF to UVA and UVB radiation. A, NHMs were exposed to UVA (2.5 J/cm2) and collected for western blot at the indicated time points (top panel) or 4 hours post-UVA at various doses (bottom panel). The p53 serves as a positive control and p84 or a-tubulin serve as loading controls. B, NHMs were pre-treated with U0126 and then exposed to UVA and collected for western blot analysis at the indicated time points. C, No obvious Erk1/2 activation after UVA radiation. NHM was irradiated with either UVA (2.5 J/cm2) or UVC (3 mJ/ cm2) and collected for western blot analysis 1 hour post-radiation. D, NHMs were exposed to UVB (25 mJ/cm2) and collected for western blot at the indicated time points (top panel) or 1 hour post-UVB at various doses (bottom panel). Again the p53 serves as a positive control and a- tubulin serve as a loading control. [6,27,28]. While it is well established that p21WAF1/CIP1 inhibits CDK activities and therefore inhibits cell cycle progression, p21WAF1/CIP1 is also important for DNA replication initiation by binding to proliferating cell nuclear antigen (PCNA) [29,30]. Therefore the precise role of p21WAF1/CIP1 in cell cycle progression is more complicated and remains to be clarified. In A375 mela- noma cell lines we observed a transient degradation of p21WAF1/CIP1 and then a rapid recovery of this protein 12 hours after UVC. The early degradation event may serve the purpose of releasing PCNA from replication fork [31] and therefore initiating a G1 arrest, and the subsequent recovery may serve the purpose of inhibiting CKD activities for further maintaining the G1 arrest. CDK inhibitor p27Kip1 usually increases when cell cycle is arrested in G1 phase [32], yet in our experiment we observed that p27Kip1 degraded 8 to 12 hours post-UVC radiation. Differential response of MiTF to different wavelengths of UV radiation Although UVC is a strong carcinogen and elicits a dis- tinct DNA damage response, UVA and UVB are more directly relevant to melanomagenesis. A large amount of data indicates that these different wavelengths of UV radiation each triggers different signaling cascades upon radiation [25]. We examined how MiTF responded to UVA and UVB radiation. After UVA radiation, MiTF was degraded 4 to 6 hours after radiation without a dis- tinct phase of phosphorylation (Fig 6A, top panel). MiTF protein was restored to its pre-radiation level 9 hours after radiation. The p53 protein accumulation increased from 4 hours post-radiation and served as a positive control for the treatment. The bottom panel of Fig 6A shows the dose-dependent degradation of MiTF We observed that re-expression of MiTF-WT in the A375 melanoma cell line restored a temporary G1 arrest after UVC, while control cells expressing GFP or MiTF- S73A cells did not, suggesting that degradation of MiTF Liu et al. Molecular Cancer 2010, 9:214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Page 9 of 12 http://www.molecular-cancer.com/content/9/1/214 Figure 6 Distinct responses of MiTF to UVA and UVB radiation. A, NHMs were exposed to UVA (2.5 J/cm2) and collected for western blot at the indicated time points (top panel) or 4 hours post-UVA at various doses (bottom panel). The p53 serves as a positive control and p84 or a-tubulin serve as loading controls. B, NHMs were pre-treated with U0126 and then exposed to UVA and collected for western blot analysis at the indicated time points. C, No obvious Erk1/2 activation after UVA radiation. NHM was irradiated with either UVA (2.5 J/cm2) or UVC (3 mJ/ cm2) and collected for western blot analysis 1 hour post-radiation. D, NHMs were exposed to UVB (25 mJ/cm2) and collected for western blot at the indicated time points (top panel) or 1 hour post-UVB at various doses (bottom panel). Again the p53 serves as a positive control and a- tubulin serve as a loading control. ct responses of MiTF to UVA and UVB radiation. A, NHMs Figure 6 Distinct responses of MiTF to UVA and UVB radiation. A, NHMs were exposed to UVA (2.5 J/cm2) and collected for western blot at the indicated time points (top panel) or 4 hours post-UVA at various doses (bottom panel). The p53 serves as a positive control and p84 or a-tubulin serve as loading controls. Differential response of MiTF to different wavelengths of UV radiation Intriguingly, while p21WAF1/CIP1 was degraded rapidly 2 to 4 hours post-radiation, p27Kip1 maintained a relatively unchanged level (Fig 5F); when p27Kip1 was degraded 8 hours post-radiation, p21WAF1/CIP1 levels started to restore. It seems these two CDK inhibitors are orchestrated to ensure a G1 arrest in MiTF- expressed A375 cells. after UVC may ensure a proper G1 cell cycle arrest and therefore allow DNA repair and enhance cell survival. In fact we observed that cells expressing MiTF-WT showed better overall survival after UVC. Although MiTF-S73A mutant was present constantly after UVC, it was unable to trigger the G1 arrest. As our data shows, part of the reason may be the weak activation on p21WAF1/CIP1 pro- moter by this mutant. However, it is also possible that there are other downstream genes differentially regu- lated by MiTF-WT and MiTF-S73A, therefore affecting the cell cycle progression. The temporary G1 arrest mediated by MiTF-WT seemed to enhance cell survival after UVC, as the cell death was decreased to about half of that in cells expressing MiTF-S73A or control GFP protein. This result was further confirmed in different melanoma cell lines expressing different levels of MiTF. Cell lines with high levels of MiTF accumulation survived better than cells with lower or un-detectable level of MiTF. This result is consistent with a recent finding that MiTF dose was correlated with cell survival after broad-band UV radiation [14]. As a tumor suppressor playing versatile roles in many aspects of cell cycle progression and DNA replication, p21WAF1/CIP1 is subjected to regulation of multiple tran- scription factors including p53, Rb, c-Myc and MiTF Previously we showed that MiTF was temporarily degraded after elevation of cellular reactive oxygen Page 10 of 12 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 species levels [13], a process that was also mediated by Erk1/2 kinase. Considering that both UVC and ROS causes similar DNA damages and therefore may employ similar repair pathways [33], the Erk1/2-mediated phos- phorylation and degradation of MiTF may reflect a gen- eral mechanism of MiTF-mediated survival pathways which is outlined in Fig 7. Upon UVR or ROS stress, MAP kinase is activated which leads to phosphorylation of MiTF on serine 73 and subsequent degradation of MiTF protein. Conclusions In summary, our data indicated that MiTF played an active role in response to UVC radiation by directly linking Erk1/2 and p21WAF1/CIP1 activation. Erk1/2 kinase is downstream of BRAF and NRAS pathways, which are frequently mutated in human melanomas [35]. Recently it was reported that the MiTF pathway was also frequently mutated in human melanomas [36-38]. Taken together, mutations in these pathways may compromise the cellular defense mechanisms against UV-mediated DNA damage and therefore increase the genome instability, eventually leading to melanomagenesis. In response to UVB radiation, MiTF levels were not changed at the examined dose and time range, nor its phosphorylation status (Fig 6D). However, MiTF was degraded without obvious band shifting after UVA treat- ment (Fig 6A). Pre-treatment with U0126 also did not prevent MiTF degradation after UVA radiation, suggest- ing that after UVA MiTF was not phosphorylated by Erk1/2 kinase, nor was the degradation mediated by phosphorylation. These data indicate that signaling path- ways after UVA, UVB and UVC are different, which is Cell lines and cell culture Normal human melanocytes were isolated from new-born foreskin followed the procedure by Eisinger and Marco [39], and cultured in MCDB153 medium (Sigma, St. Louis, MO) containing 2% FCS, 0.3% bovine pituitary extract (Cambrex Bio Science Walkersville, Inc., Walkersville, MD), 10 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA), 2 mmol/L CaCl2, 5 μg/mL insulin, and 0.1 mmol/L IBMX (Sigma) (Yang et al.: 2005). Melanoma Malme-3 M cells were cultured in IMDM media containing 20% FBS and 1% penicillin and streptomycin. The c83-2C, A375, SK-Mel-28 or SK-Mel-5 cells were cultured in F10, DMEM, EMEM or AMEM media; each supplied with 5% FBS, 5% new born bovine sera, and 2% penicillin and streptomycin. All cells were kept at 37°C in 5% CO2 incubator. Figure 7 MiTF mediates signal transduction from Erk1/2 to p21WAF1/CIP1 after UVC and ROS stress. UVC and ROS trigger Erk1/2 activation, which leads to MiTF phosphorylation and degradation, and enables a temporary G1 cell cycle arrest and subsequent cell survival. Differential response of MiTF to different wavelengths of UV radiation The temporary degradation was corre- lated with a temporary G1 cell cycle arrest, correspond- ing with p21WAF1/CIP1 degradation and re-activation, which allows sufficient time for DNA damage repair and ensure of a better cell survival (Fig 7). consistent with previous observations that different wavelengths of UV light trigger different cellular responses [34]. The UVA-MiTF signaling pathway is still under intensive investigation in our laboratory. Acknowledgements We thank Dr. David Fisher (Massachusetts General Hospital, Harvard Medical School) for his gift of MiTF-S73A mutant construct, Drs. Anand Ganesan and Sun Yang (University of California at Irvine) for their critiques and suggestions. This research is supported in part by CA62230 to F.L.M and the Waltmar and Oxnard Foundations. 1Department of Medicine, University of California-Irvine School of Medicine, Orange, CA 92868, USA. 2Chao Family Comprehensive Cancer Center, University of California-Irvine School of Medicine, Orange, CA 92868, USA. Immunofluorescence Cells were seeded on cover slips and treated as indi- cated, then fixed in 4% formaldehyde solution in 1× PBS at room temperature for 30 minutes. After three washes in 1× PBS, cells were treated with 0.05% Saponin at room temperature. Cells were washed in 1× PBS again for 3 times, and incubated with 10% normal goat serum for 1 hour at room temperature. Cells were further incubated with primary antibody C5 (1:50 dilution in 10% goat serum) at 4°C overnight. After 5 brief wash with 1× PBS plus 0.01% NP-40, cells were incubated with Texas-Red labeled anti-mouse secondary antibody in dark for 1 hour at room temperature. 1 μg/ml DAPI was added into staining solution at the last 10 minutes of incubation for the secondary antibody. Cells were then washed and mounted to a slide for viewing under a Zeiss fluorescence microscope. Colony formation assay y y Cells were irradiated and then returned to incubator with fresh media. Culture media was changed every three days for 2 weeks. Plates were stained with 0.5% crystal violet solution in 25% methanol. Only colonies with more than 50 cells were counted. DNA constructs and lentivirus transduction Wild-type MiTF cDNA was cloned into expression vec- tor QCXIP (Clontech, Mountain View, CA) via EcoR I and Apa I sites. MiTF-S73A mutant was a gift from Dr. David Fisher (Harvard Medical School, MA), and was also cloned into QCXIP vector via the same restriction enzyme sites. MiTF-S409A mutant was generated using site-directed mutagenesis kit from Stratagen following the manufacturer’s instruction, with the following primers: S409-r, 5′-TCCGTC TCTTCC ATGCTC ATAGCG CTCCTC CGGCTG CTTGTT-3′, and S409- f, 5′-AACAAG CAGCCG GAGGAG CGCTAT GAG- CAT GGAAGA GAC GGA-3′. All mutations were con- firmed by DNA sequencing. The QCXIP-GFP vector was generated by ligating GFP coding sequence from pEGFP-N1 (Nhe I and BamH I fragment) into the BamH I site on QCXIP vector. The p21WAF1/CIP1 pro- moter construct (pWWP-Luc, containing about 2.4 kb promoter region) was a kind gift from Dr. Wafik El- Deiry (University of Pennsylvania). The Mish1 and Mish2 shRNA plasmids were purchased from Open Bio- systems. These plasmids were co-transfected with pMD2G and pSPAX2 plasmids into 293T cells for virus production http://tronolab.epfl.ch/. Transduction was performed in the presence of 10 μg/ml of protamine, using the filtered 293T media as virus source. cDNAs, we used primers AT1 (5′-GCG TGA TGG TGG GCA TGG GTC AG-3′) and AT2 (5′-AGG GGG GCC TCG GTC AGC AGC AC-3′) and primers p21-f (5′-GAA GAA GGG TAG CTG GGG CT -3′) and p21-r (5′-CTC TAA GGT TGG GCA GGG TG -3), respec- tively. The primers For MiTF were mi6a (5′-CCA ACC GGC ATT TGT TGC TCA-3′) and mi2b (5′-GTT GTT GAA GGT GAT GGT GCC-3′). Promoter reporter ana- lysis was carried out using dual luciferase assay system from Promega. Renilla luciferase driven by SV40 early promoter (pSV40-RL) was used as an internal control. Cell lysate and western blot analysis Cell pellet was lysed in a lysis 250 buffer [40] and quan- tified by the Bradford protein assay method (Bio-Rad, Richmond, CA). Western blot was performed using antibodies against MiTF C5 plus D5 (MS-773-P, Lab Vision, Fremont, CA), p21 (Santa Cruz), p27 (Santa Cruz), p53 DO-1 (Santa Cruz), p84 (Abcam, Cambridge, MA) and a-tubulin (T9026, Sigma, St. Louis, MO), ubi- quitin (ab7780, Abcam). All western blots were repeated at least twice, one representative blot is shown in figures. Quantitative Real Time RT-PCR and promoter reporter analysis Quantitative Real Time RT-PCR and promoter reporter analysis Quantitative Real Time RT-PCR and promoter reporter analysis remained in 37°C incubator during the 20 minutes treat- ment. Culture media were then removed and cells were exposed to UVR. Fresh media was added into irradiated cells without further washing to leave residue kinase inhibitors in the media. One microgram of total RNA isolated from cells was used for first-strand DNA synthesis with random pri- mers. One-twentieth of the total cDNA was subjected to real time PCR amplification in an iCycler iQ5 Real Time PCR Instrument using iQ SYBR Green Supermix from Bio-Rad (170-8882). For the a-tubulin and p21CIP1/WAF1 One microgram of total RNA isolated from cells was used for first-strand DNA synthesis with random pri- mers. One-twentieth of the total cDNA was subjected to real time PCR amplification in an iCycler iQ5 Real Time PCR Instrument using iQ SYBR Green Supermix from Bio-Rad (170-8882). For the a-tubulin and p21CIP1/WAF1 cDNAs, we used primers AT1 (5′-GCG TGA TGG TGG GCA TGG GTC AG-3′) and AT2 (5′-AGG GGG GCC TCG GTC AGC AGC AC-3′) and primers p21-f (5′-GAA GAA GGG TAG CTG GGG CT -3′) and p21-r (5′-CTC TAA GGT TGG GCA GGG TG -3), respec- tively. The primers For MiTF were mi6a (5′-CCA ACC GGC ATT TGT TGC TCA-3′) and mi2b (5′-GTT GTT GAA GGT GAT GGT GCC-3′). Promoter reporter ana- lysis was carried out using dual luciferase assay system from Promega. Renilla luciferase driven by SV40 early promoter (pSV40-RL) was used as an internal control. Acknowledgements We thank Dr. David Fisher (Massachusetts General Hospital, Harvard Medical School) for his gift of MiTF-S73A mutant construct, Drs. Anand Ganesan and Sun Yang (University of California at Irvine) for their critiques and suggestions. This research is supported in part by CA62230 to F.L.M and the Waltmar and Oxnard Foundations. Flow-cytometry and cell cycle analysis Cells were trypsinized and washed once with 1× PBS, fixed in cold 70% ethanol overnight or until use. Cells were incubated in Propidium Iodide (PI) staining solu- tion in dark for 30 minutes: 50 μg/ml PI, 0.1% sodium citrate, 50 μg/ml RNase A, 0.03% NP-40 in 1× PBS. 10,000 total events were counted for each sample. Cell populations from each phase were calculated according to CellQuest instructions (BD Biosciences). UV radiation and cell treatment Cells were grown to about 70% confluence and media was removed completely for UVB and UVC radiation. For UVA radiation, 5 ml of 1× PBS was added to one 10-cm dish of cells and ice cubes were placed next to dishes for absorbing the heat generated by UVA. UVC radiation was performed in a tissue culture hood with genotoxic UVC lamp (peak wavelength 254 nm). UVB radiation was performed in a Stratagen crosslinker with peak wavelength at 312 nm; and UVA radiation was also performed in a Stratagen crosslinker with lamps with peak wavelength at 350 nm. The UV intensity was measured by a radiometer with proper probes. The cul- ture media was returned to cells after radiation and cells were returned to 37°C incubator for recovering. For kinase inhibitor treatment, inhibitors were added into culture media 20 minutes before radiation; cells Page 11 of 12 Page 11 of 12 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 References 1. Widlund HR, Fisher DE: Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival. Oncogene 2003, 22(20):3035-41. 27. el-Deiry WS, et al: WAF1, a potential mediator of p53 tumor suppression. Cell 1993, 75(4):817-25. 28. Gartel AL, Radhakrishnan SK: Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Res 2005, 65(10):3980-5. 2. Mitra D, Fisher DE: Transcriptional regulation in melanoma. Hematol Oncol Clin North Am 2009, 23(3):447-65, viii. 29. Prives C, Gottifredi V: The p21 and PCNA partnership: a new twist for an old plot. Cell Cycle 2008, 7(24):3840-6. 3. Garraway LA, et al: Integrative genomic analyses identify MITF as a 3. Garraway LA, et al: Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005, 436(7047):117-22. 3. Garraway LA, et al: Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma Nature lineage survival oncogene amplified in malignant melanoma. Nature 2005, 436(7047):117-22. 30. Soria G, et al: p21 differentially regulates DNA replication and DNA- repair-associated processes after UV irradiation. J Cell Sci 2008, 121(Pt 19):3271-82. 4. Joo A, et al: STAT3 and MITF cooperatively induce cellular transformation through upregulation of c-fos expression. Oncogene 2004, 23(3):726-34. 31. Soria G, et al: P21Cip1/WAF1 downregulation is required for efficient PCNA ubiquitination after UV irradiation. Oncogene 2006, 25(20):2829-38. 5. Wellbrock C, Marais R: Elevated expression of MITF counteracts B-RAF- stimulated melanocyte and melanoma cell proliferation. J Cell Biol 2005, 170(5):703-8. 32. Sgambato A, et al: Multiple functions of p27(Kip1) and its alterations in tumor cells: a review. J Cell Physiol 2000, 183(1):18-27. 6. Carreira S, et al: Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature 2005, 433(7027):764-9. 33. Zhang X, et al: Identification of possible reactive oxygen species involved in ultraviolet radiation-induced oxidative DNA damage. Free Radic Biol Med 1997, 23(7):980-5. 7. Loercher AE, et al: MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A. J Cell Biol 2005, 168(1):35-40. 34. Placzek M, et al: Effect of ultraviolet (UV) A, UVB or ionizing radiation on the cell cycle of human melanoma cells. Br J Dermatol 2007, 156(5):843-7. 35. Dankort D, et al: Braf(V600E) cooperates with Pten loss to indu metastatic melanoma. Nat Genet 2009, 41(5):544-52. 8. Busca R, et al: Hypoxia inducible factor 1a is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells. Authors’ contributions 20. Bauer GL, et al: The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue. Genetics 2009, 183(2):581-94. FL conceived of and designed the study, and carried out most of the experiments and drafted the manuscript. AR carried out the UV-mediated cell survival analysis. AG and YK carried out MiTF shRNA knockdown experiment and colony formation analysis. ZY participated in the western blot experiments. FLM participated in the study design, coordination and direction, and edited the manuscript. All authors read and approved the final manuscript. 21. Bertolotto C, Ballotti R: Functional role of MITF phosphorylation. In vivo veritas? Pigment Cell Melanoma Res 2009, 22(6):703-4. 22. Xu W, et al: Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9. Exp Cell Res 2000, 255(2):135-43. y 23. Sanchez Y, Elledge SJ: Stopped for repairs. Bioessays 1995, 17(6):545-8. Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 18. Hemesath TJ, et al: MAP kinase links the transcription factor Microphthalmia to c-Kit signalling in melanocytes. Nature 1998, 391(6664):298-301. 3Department of Biological Sciences, University of California-Irvine, Irvine, CA 92617, USA. 4Shandong Provincial Hospital, Jingwu Road, Shandong 3Department of Biological Sciences, University of California-Irvine, Irvine, CA 92617, USA. 4Shandong Provincial Hospital, Jingwu Road, Shandong Province, 250021, China. 5Department of Chemistry, School of Life Science, Xi’an Jiao Tong University, Xi’an, Shaanxi Province, 710049, China. 3Department of Biological Sciences, University of California-Irvine, Irvine, CA 92617, USA. 4Shandong Provincial Hospital, Jingwu Road, Shandong 3Department of Biological Sciences, University of California-Irvine, Irvine, CA 92617, USA. 4Shandong Provincial Hospital, Jingwu Road, Shandong Province, 250021, China. 5Department of Chemistry, School of Life Science, Xi’an Jiao Tong University, Xi’an, Shaanxi Province, 710049, China. Province, 250021, China. 5Department of Chemistry, School of Life Science, Xi’an Jiao Tong University, Xi’an, Shaanxi Province, 710049, China. Province, 250021, China. 5Department of Chemistry, School of Life Science, Xi’an Jiao Tong University, Xi’an, Shaanxi Province, 710049, China. 19. Wu M, et al: c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi. Genes Dev 2000, 14(3):301-12. References Med Sci (Paris) 2006, 22(1):10-3. 36. Yokoyama S, Salma N, Fisher DE: MITF pathway mutations in melanoma. Pigment Cell Melanoma Res 2009, 22(4):376-7. 36. Yokoyama S, Salma N, Fisher DE: MITF pathway mutations in melanoma. Pigment Cell Melanoma Res 2009, 22(4):376-7. 9. Du J, et al: Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF. Cancer Cell 2004, 6(6):565-76. 37. Cronin JC, et al: Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res 2009, 22(4):435-44. 37. Cronin JC, et al: Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res 2009, 22(4):435-44. 38. Jonsson G, et al: Genomic profiling of malignant melanoma using tiling- resolution arrayCGH. Oncogene 2007, 26(32):4738-48. 38. Jonsson G, et al: Genomic profiling of malignant melanoma using tiling- resolution arrayCGH. Oncogene 2007, 26(32):4738-48. 10. McGill GG, et al: Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 2002, 109(6):707-18. 39. Eisinger M, Marko O: Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA 1982, 79(6):2018-22. 11. Dynek JN, et al: Microphthalmia-associated transcription factor is a critical transcriptional regulator of melanoma inhibitor of apoptosis in melanomas. Cancer Res 2008, 68(9):3124-32. 40. Liu F, Lee WH: CtIP activates its own and cyclin D1 promoters via the E2F/RB pathway during G1/S progression. Mol Cell Biol 2006, 26(8):3124-34. 12. Carreira S, et al: Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Dev 2006, 20(24):3426-39. 13. Liu F, Fu Y, Meyskens FL Jr: MiTF regulates cellular response to reactive oxygen species through transcriptional regulation of APE-1/Ref-1. J Invest Dermatol 2009, 129(2):422-31. doi:10.1186/1476-4598-9-214 Cite this article as: Liu et al.: MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells. Molecular Cancer 2010 9:214. 14. Hornyak TJ, et al: Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation. Pigment Cell Melanoma Res 2009, 22(3):307-18. 15. Omholt K, et al: NRAS and BRAF mutations arise early during melanoma pathogenesis and are preserved throughout tumor progression. Clin Cancer Res 2003, 9(17):6483-8. 16. Meier F, et al: The RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways present molecular targets for the effective treatment of advanced melanoma. Front Biosci 2005, 10:2986-3001. 17. Competing interests 24. Bendjennat M, et al: UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell 2003, 114(5):599-610. The authors declare that they have no competing interests. Received: 19 January 2010 Accepted: 11 August 2010 Published: 11 August 2010 Received: 19 January 2010 Accepted: 11 August 2010 Published: 11 August 2010 25. Molho-Pessach V, Lotem M: Ultraviolet radiation and cutaneous carcinogenesis. Curr Probl Dermatol 2007, 35:14-27. 26. Bode AM, Dong Z: Mitogen-activated protein kinase activation in UV- induced signal transduction. Sci STKE 2003, 2003(167):RE2. Author details 1 1Department of Medicine, University of California-Irvine School of Medicine, Orange, CA 92868, USA. 2Chao Family Comprehensive Cancer Center, University of California-Irvine School of Medicine, Orange, CA 92868, USA. Page 12 of 12 Page 12 of 12 References Molina DM, Grewal S, Bardwell L: Characterization of an ERK-binding domain in microphthalmia-associated transcription factor and differential inhibition of ERK2-mediated substrate phosphorylation. J Biol Chem 2005, 280(51):42051-60.
https://openalex.org/W4320059539	https://journals2.ums.ac.id/index.php/warta/article/download/1095/392	English	null	Empowerment of Posyandu Cadres in Detecting and Preventing Stunting	Warta LPM	2,022	cc-by	5,327	Kata Kunci: Ketahanan pangan, Pos Pelayanan Terpadu, Stunting Jurnal Warta LPM Vol. 25, No. 4, Oktober 2022, hlm. 514-522 p-ISSN: 1410-9344; e-ISSN: 2549-5631 homepage: http://journals.ums.ac.id/index.php/warta Jurnal Warta LPM Vol. 25, No. 4, Oktober 2022, hlm. 514-522 p-ISSN: 1410-9344; e-ISSN: 2549-5631 homepage: http://journals.ums.ac.id/index.php/warta Article Info Medan City is one of the North Sumatra areas with stunting problems. Pulo Brayan Kota, West Medan District, is included in the ten villages with the highest stunting cases. Strengthening integrated service posts by improving cadres’ skills is essential to monitoring toddlers’ growth every month. This research was carried out at the same time as community service, which was carried out from June to August 2021. The activity began with a preliminary survey, training for 12 cadres of integrated service posts, and conducting food security training for 40 Family Cards. Cadre training is carried out by presenting and practicing measuring the height of toddlers with a pacemaker and recording the measurement results on the Towards Health Card. Food security activities are carried out by submitting and practicing catfish farming in buckets and growing vegetables vertically using bottles. The results obtained include improving the skills of Integrated Service Post cadres in measuring the height of toddlers and the community to be skilled in catfish farming and growing vegetables vertically, the results of which can be used to strengthen food security in households to reduce the growth rate of stunting. Submitted: 29 August 2022 Revised: 23 September 2022 Accepted: 25 October 2022 Published: 11 November 2022 Submitted: 29 August 2022 Revised: 23 September 2022 Accepted: 25 October 2022 Published: 11 November 2022 Keywords: Food security, Integrated Service Post, Stunting Keywords: Food security, Integrated Service Post, Stunting Empowerment of Posyandu Cadres in Detecting and Preventing Stunting 1Ecia Meilonna Koka, 2Etti Sudaryati, 3Heru Santosa Faculty of Public Health, University of North Sumatra email : 1meilonna@usu.ac.id; 2etti@usu.ac.id; 3heru_php2@yahoo.com Jurnal Warta LPM Vol. 25, No. 4, Oktober 2022, hlm. 514-522 p-ISSN: 1410-9344; e-ISSN: 2549-5631 homepage: http://journals.ums.ac.id/index.php/warta 1. INTRODUCTION Stunting is a national problem experienced by children under five, including in Medan City. Stunting is a condition where children experience a height lower than usual according to the child’s age (Setiawan, et al., 2018). Stunting can be experienced from the beginning of life; when the child is born, the length is less than 48 cm (Nurmalasari, et al., 2017). V arious interrelated factors can cause the problem of stunting. The issue of stunting does not stand alone and is not only related to health problems. Poor nutrition factors are the main factors that cause stunting, but far from it, how parental parenting represents and educates children needs to be considered because how a child will be very dependent on his parents. Various nutritious feeding programs through Puskemas have been carried out, but activities emphasizing charity can only solve problems for a short time(Hilmi, et al., n.d.). Poor nutrition factors are the main factors that cause stunting, but far from it, how parental parenting represents and educates children needs to be considered because how a child will be very dependent on his parents. Various nutritious feeding programs through Puskemas have been carried out, but activities emphasizing charity can only solve problems for a short time(Hilmi, et al., n.d.). Riset Kesehatan Data (Riskesdas) in 2018 stated that stunting cases in Indonesia are still high (30.8%), there has been a decrease from 2013 (37.2%), as well as stunting in North Sumatra Province is still higher than in Indonesia (32.4% in 2018; 42.5% in 2013). The stunting limit set by WHO (2005) is not to exceed 20% (Badan Penelitian dan Pengembangan Kesehatan, 2019) . The city of Medan, an area in North Sumatra, is also experiencing stunting problems; even in 2020, it is a locus area for stunting prevention. One of the areas in Medan that is experiencing stunting is Pulo Brayan (IniMedan.com, 2020). Integrated Service Post is a health service activity organized by and for the community (Mahyarni, 2016). hrough integrated service, post activities can increase community participation to develop health activities so that healthy communities are prosperous (Hafifah & Abidin, 2020). Implementing the Integrated Service Post also helps toddlers’ growth to be well monitored regularly. Suppose the community fully participates in bringing children to the Integrated Service Post to be periodically monitored for weight and height. In that case, nutritional problems such as stunting can be prevented. Abstrak The village size is 0.68 km2 with a population of more than 7000 people, so this village is classified as a reasonably densely populated area. This area is also a pioneer of the business center in the city of Medan. Therefore, all the streets are filled with shops. This resulted in the region being frequently hit by traffic jams. The majority of people’s livelihoods are self-employed and traders. Many residents do business making boiled fish that is supplied throughout the city of Medan. The average daily production is about 3 tons per day (Yefni, 2019). The existing health facilities are 1 Puskesmas, 1 Poly Clinic, 1 Maternity Clinic, three dental clinics, 9 Integrated Service Posts, 5 Pharmacies, and 2 Drug Stores. During the current Covid-19 pandemic, not all Integrated Service Posts actively participate in activities. Integrated Service Post activities are divided into 2 Integrated Service Posts spread across wards one and 23. Before the pandemic, the Integrated Service Posts in this village were 9 Integrated Service Posts located in the I, II, IV, VI, IX, XIII, XV, XVII, and XXIV wards, with a total of 45 cadres. However, during the pandemic in 2020, it opened only 2 Integrated Service Posts. Abstrak Kota Medan merupakan salah satu daerah di Sumatera Utara yang memiliki masalah stunting. Pulo Brayan Kota, Kecamatan Medan Barat, masuk dalam sepuluh desa dengan kasus stunting tertinggi. Penguatan pos pelayanan terpadu dengan meningkatkan keterampilan kader dinilai penting untuk memantau pertumbuhan balita setiap bulannya. Penelitian ini dilakukan sekaligus dengan pengabdian masyarakat yang dilaksanakan mulai bulan Juni hingga Agustus 2021. Kegiatan diawali dengan survei pendahuluan, pelatihan bagi 12 kader pos pelayanan terpadu, dan melakukan pelatihan ketahanan pangan bagi 40 Kartu Keluarga. Pelatihan kader dilakukan dengan mempresentasikan dan berlatih mengukur tinggi badan balita dengan alat pacu jantung dan mencatat hasil pengukuran pada Kartu Menuju Sehat. Kegiatan ketahanan pangan dijalankan dengan cara pengajuan dan praktik budidaya ikan lele dalam ember dan menanam sayuran 514 Koka, dkk - Empowerment of Posyandu Cadres ... Koka, dkk - Empowerment of Posyandu Cadres ... secara vertikal menggunakan botol. Hasil yang diperoleh diantaranya meningkatkan keterampilan kader Pos Pelayanan Terpadu dalam mengukur tinggi badan balita dan masyarakat menjadi terampil dalam budidaya ikan lele dan menanam sayuran secara vertikal yang hasilnya dapat digunakan untuk memperkuat ketahanan pangan dalam rumah tangga sehingga dapat menekan laju pertumbuhan stunting. Jl. Bilal and Helvetia, West Medan. The village size is 0.68 km2 with a population of more than 7000 people, so this village is classified as a reasonably densely populated area. This area is also a pioneer of the business center in the city of Medan. Therefore, all the streets are filled with shops. This resulted in the region being frequently hit by traffic jams. The majority of people’s livelihoods are self-employed and traders. Many residents do business making boiled fish that is supplied throughout the city of Medan. The average daily production is about 3 tons per day (Yefni, 2019). The existing health facilities are 1 Puskesmas, 1 Poly Clinic, 1 Maternity Clinic, three dental clinics, 9 Integrated Service Posts, 5 Pharmacies, and 2 Drug Stores. During the current Covid-19 pandemic, not all Integrated Service Posts actively participate in activities. Integrated Service Post activities are divided into 2 Integrated Service Posts spread across wards one and 23. Before the pandemic, the Integrated Service Posts in this village were 9 Integrated Service Posts located in the I, II, IV, VI, IX, XIII, XV, XVII, and XXIV wards, with a total of 45 cadres. However, during the pandemic in 2020, it opened only 2 Integrated Service Posts. Jl. Bilal and Helvetia, West Medan. 1. INTRODUCTION Pulo Brayan Village is included in the West Medan District and consists of 25 environments, including six domains; there are cases of stunting, namely I, I, II, IV, VI, IX, and XXIV. This village is located 5 km from the center of Medan, in the Jalan KL.Yos Sudarso and its surroundings, bordering other areas such as The problem of stunting can occur due to the child’s insufficient intake and the presence 515 Warta LPM, Vol. 25, No. 4, Oktober 2022 Koka, dkk - Empowerment of Posyandu Cadres ... of infectious diseases suffered by children under five, which are direct factors (Tria & Endah, 2021). Meanwhile, an indirect factor is food availability in the family, parenting, and health services (Handayani, 2017). In Pulo Brayan Kota Village, dense population density and environmental hygiene problems cause diseases to develop rapidly, mainly affecting growing children. The illness toddlers in this village often suffer from diarrhea, the ten most common diseases. Children affected by diarrhea can have their child’s growth rate disturbed. Moreover, repeated cases occur. purchasing power of the community is related to the fulfillment of food, especially in households with toddlers. This causes the child’s risk of suffering from stunting to be more significant. Strengthening food security in homes is very important; that is, families can provide food at the household level without having to buy or at least save expenses in meeting food needs. The innovation is that people can use a small amount of land to grow crops, raise fish in barrels/ buckets for daily essentials, and use used bottles to grow vegetables vertically. The novelty carried out in this service activity is that in the KMS, there is a round (Buble) which makes it easier for cadres to provide marking of the results of measuring children’s height and the existence of information about the provision of Complementary Breast Milk (MP- ASI) on the KMS return sheet. In addition, on the spur measuring board where the child’s height is measured, the results obtained not only know the child’s measurement but can determine the child’s height it should achieve according to his age to achieve a standard size. Besides holding community training, other innovations need to be equipped with long-term knowledge, namely catfish farming in buckets and growing vegetables vertically by utilizing used bottles. By being equipped with knowledge and skills in food security, it is hoped to reduce the stunting rate. Koka, dkk - Empowerment of Posyandu Cadres ... Koka, dkk - Empowerment of Posyandu Cadres ... Koka, dkk - Empowerment of Posyandu Cadres ... 2. METHOD This stage of community service activities begins with the management of permits and the delivery of training plans to the Pulo Brayan Village Head of West Medan District and socialization to other partners, namely the Head of the environment, the Head of the West Medan District Health Center and the Integrated Service Post cader to support this activity. Community Health Service support is needed to help the community understand the importance of nutrition in the First 1000 Days of Life in monitoring toddlers’ growth. This research uses a qualitative approach with a descriptive method. The descriptive qualitative (QD) research method moves on a simple qualitative approach with an inductive flow (Paembonan, 2022). This inductive flow means that qualitative descriptive research (QD) begins with an explanatory process or event it can finally be drawn, a generalization that is a conclusion of the process or event (Yuliani, 2018) Descriptively describes institutional strengthening activities as one of the efforts to prevent stunting problems. The location of this service was carried out in Pulo Brayan, West Medan District. The main data source is primary data. Primary data are taken by researchers in the field (Cintya et al., 2013). Primary data is generally used to generate information that reflects the truth according to factual conditions (Pramiyati, et al., 2017). The data was collected through field notes, documentation, observations, and interviews. The Service Time has been held from June to August 2022. Furthermore, training was carried out on measuring the height of toddlers using measuring spur boards and KMS Buble to the Kader integrated service posts, catfish farming training using buckets, and planting vegetables vertically by utilizing used bottles and plastic cups in the community in 4 Lingungan Pulo Brayan Village, West Medan District. The results of the activities of this service program are in the form of a descriptive narrative that interprets the implementation of the training program. The sampling technique used is purposive sampling. The population in this study was cadres of integrated service posts and conducted food security training for 40 Family cards in Pulo Brayan. Cadre training is carried out only to cadres of Integrated Service Posts who are active in activities. In contrast, catfish and vegetable cultivation training are carried out in environments with stunting cases represented by domains II, VI, VI, and IX. 1. INTRODUCTION The existence of a market in Pulo Brayan Village illustrates the availability of food or easy access to food for the surrounding community living in the village. Still, the problem is the level of purchasing power of the community related to the fulfillment of food at home, especially in households with toddlers. Food affordability at the household level, especially during the Covid-19 pandemic in the past year, has had an impact due to the disrupted economy. Economic disruption due to restrictions on movement in the community makes family income uncertain, especially in families with odd jobs. The giant picture of people’s work in this village is traders with irregular incomes, so food fulfillment at home can not be guaranteed daily. This causes the risk of children suffering from stunting, so strengthening household food security is very important by providing food that does not have to buy. In addition, strengthening the Integrated Service Post by improving cadres’ skills is also very important in inviting the community to regularly participate in integrated service post activities so that children under five’s growth can be monitored every month. The expected output is that the Cadres of the Integrated Service Post will be skilled in measuring the height of toddlers with measuring spur boards and KMS Buble to monitor and reduce the stunting rate. In addition, in terms of food security to fulfill daily nutrition, the community becomes skilled in vegetable farming and catfish farming as a fulfillment of fish and vegetable consumption at the household level, especially for families with pregnant women and toddlers. In responding to the problem, it’s necessary to conduct training on integrated service post cadres so that cadres have skills in monitoring and measuring the height of toddlers with new media, namely measuring boards and Cards Towards Health (KMS). In addition, food security training at the household level is also needed, namely by cultivating catfish in buckets and growing vegetables vertically by utilizing used bottles. The benefit of this community service is that the community becomes skilled in detecting the characteristics of stunting in toddlers by measuring the height of toddlers with measuring boards and KMS Buble and being proficient in catfish farming and growing vegetable plants to fulfill daily consumption. Pulo Brayan Kota Village is classified as a densely populated area, and the lack of 516 Warta LPM, Vol. 25, No. 4, Oktober 2022 516 3. RESULTS AND DISCUSSION This community service activity begins with the management of permits. The delivery of training plans to the Pulo Brayan Village Head of West Medan District and socialization to other partners, namely the Head of the environment, the Head of the West Medan District Health Center, and the cadres of the 517 Warta LPM, Vol. 25, No. 4, Oktober 2022 Figure 1. Stages of Activity Figure 1. Stages of Activity 517 Warta LPM, Vol. 25, No. 4, Oktober 2022 517 Warta LPM, Vol. 25, No. 4, Oktober 2022 Koka, dkk - Empowerment of Posyandu Cadres ... during the Covid-19 pandemic was opened. The selected cadres totaled 12 people. Integrated Service Post, to support this activity and can continue in stunting prevention efforts through strengthening family food security. And strengthening of Integrated Service Posts. Puskesmas support is needed to help the community understand the importance of nutrition in the First 1000 Days of Life (HPK) in monitoring toddlers› growth. Integrated Service Post, to support this activity and can continue in stunting prevention efforts through strengthening family food security. And strengthening of Integrated Service Posts. Puskesmas support is needed to help the community understand the importance of nutrition in the First 1000 Days of Life (HPK) in monitoring toddlers› growth. Cadres are given an understanding of stunting, monitoring toddlers’ growth, and training in measuring body length and height using a spur measuring board. This tool can detect the body size that should be by the child’s age. In addition, cadres are also taught to plot the measurement results into a record called KMS buble. On the back sheet of the KMS bubble is information about the stages of feeding babies and children aged 0-24 months. KMS Buble is a recording tool that is an accessible innovation in understanding the growth of toddlers who have previously been tested through dissertation research by alums of S3 Public Health Sciences. Furthermore, training Integrated Service Post cadres and catfish farming using buckets and planting vegetables vertically by utilizing used bottles and plastic cups in the community in 4 Lingungan Pulo Brayan Village, West Medan District. The results are expected to be used as an additional family food source. Before and after the training, pre-tests and Post-Test were carried out on Posyandu Cadres. The questions consist of 15 questions related to monitoring the growth of toddlers. 3. RESULTS AND DISCUSSION The correct answer result is given a score of 1 and the wrong answer is given a score of 0. The results of the pre-test and post-test can be seen in table 1. Integrated Service Post cadres trained in bookkeeping toddlers’ height and length using a spur measuring board can find out how much toddlers should achieve length or height. This information can be conveyed to mothers of toddlers so that mothers pay more attention to their child’s nutritional intake. In addition to measurements using measuring spur boards, Cadres are also trained to use KMS buble in terms of correctly plotting and filling in measurement results. After the training, the service team distributed 600 KMS Buble A3 sizes to people with toddlers. In the KMS Buble return sheet, there’s information about the stages of feeding the child so that the KMS that has been shared can be read by the mother of the toddler to be applied correctly. Cadre training activities have been carried out on July 7, 2021. Community Empowerment through Improving Catfish Cultivation skills and Vegetable Planting in Pulo Brayan Village The food security of the family influences the nutritional intake of the family. A food-secure family is a family that can provide sufficient quantity and quality food, has good access to food, and can use nutrition in daily family consumption. Family income is a determining factor in food security in the household. Short children come from low-income families, so the nominal costs that can be spent to obtain food are only a tiny amount (Sihadi & Djaiman, 2011). Figure 3. Training to Record Measurement Results into a Card Towards Health Forming groups for catfish farming (1 group consisting of 10 Family Cards) in 4 environments with stunting cases will create four catfish farming groups. Each group will be given 200 catfish fries and two plastic buckets with a capacity of 100 liters. Catfish farming is relatively easy to carry out, preferred by most people, and contains high nutrients. The selection of a location that will be used for manufacturing catfish ponds in the form of residents’ yards. Purchase of tools and materials used to create catfish ponds. Conducting catfish farming coaching using plastic buckets with four predetermined groups. The stages of making catfish ponds are(Febri et al., 2019): The role of the Integrated Service Post is more of a preventive or preventive measure against stuntig (Khosiah & Muhardini, 2019). his preventive action is carried out by taking anthropometric measurements, including measuring the height and weight of toddlers to determine their nutritional status. In addition, there are also various counseling provided by the Integrated Service Post regarding nutritional health to increase awareness of mothers of toddlers regarding nutritional adequacy in toddlers, as well as the understanding of cadres in filling KMS is also essential so that there are no mistakes in plotting the results of the calculation. 1. Preparations make a pond by hollowing out the top. After that, the plastic bucket is washed thoroughly; the next step is to fill the pool with clean water as high as 1/2 of the drum height or a maximum of 3/4 of the size of the bucket, precipitate water for approximately one week, and pour the EM 4 solution to fix water quality in fish ponds. Optimizing the role of integrated service posts by training cadres is expected to reduce stunting rates in Pulo Brayan Kota Village so that they do not continue to increase. Empowerment of Integrated Service Posts through Improving the Skills of Integrated Service Post Cadres in Pulo Brayan Village Strengthening integrated service posts is improving cadres’ skills in correctly taking height measurements in monitoring the growth of children under five for stunting prevention. The cadres trained are cadres involved in the activities of the Integrated Service Post, which 518 Warta LPM, Vol. 25, No. 4, Oktober 2022 Table 1. Pre-Test and Post-Test Scores of Posyandu Cadres No Pre-test score Post-test score 1 10 10 2 8 12 3 10 11 4 9 14 5 10 12 6 7 9 7 9 10 8 8 11 9 8 9 10 9 11 11 10 13 12 10 14 Table 1. Pre-Test and Post-Test Scores of Posyandu Cadres Figure 2. Training on Measuring Baby Body Length with a Spur Measuring Board Figure 2. Training on Measuring Baby Body Length with a Spur Measuring Board 518 Warta LPM, Vol. 25, No. 4, Oktober 2022 Koka dkk Empowerment of Posyandu Cadres Koka, dkk - Empowerment of Posyandu Cadres ... Figure 3. Training to Record Measurement Results into a Card Towards Health Community Empowerment through Improving Catfish Cultivation skills and Vegetable Planting in Pulo Brayan Village One of the roles of the Integrated Service Post is to measure height and weight and provide counseling and health counseling related to nutrition. Increasing the knowledge and skills of Integrated Service Post cadres can be done by holding coaching and training regularly and consistently with the hope that Integrated Service Post cadres can have the ability to conduct good nutritional health counseling and counseling so that there is a change in behavior that is more both from mothers of toddlers related to childcare to prevent stunting. A behavior change will encourage realizing a nutrition awareness movement in the community so that stunting treatment can be more effective and efficient (Novianti, R., et al, 2021). 2. After the bucket is filled with water, the catfish seedlings are ready to be put in. One such bucket can be filled with 100 catfish seedlings; further water is supplemented according to the growth of the catfish. The formation of catfish cultivation groups and planting of vegetables vertically through training that was carried out on August 11, 2021 519 Warta LPM, Vol. 25, No. 4, Oktober 2022 Koka, dkk - Empowerment of Posyandu Cadres ... Koka, dkk - Empowerment of Posyandu Cadres ... Figure 5. Catfish Harvest with the People of Pulo Brayan Village Figure 4. Training to Spread Catfish Seedlings into Buckets Figure 4. Training to Spread Catfish Seedlings into Buckets Figure 5. Catfish Harvest with the People of Pulo Brayan Village Catfish are harvested within 2.5 months to 3 months. Harvesting catfish is not carried out simultaneously for the entire bucket since catfish growth is not uniform. The fish first harvested was about four buckets, each weighing approximately 20-30 heads and 1-1.5 kg. You can gather again next month until the 4th month of maintenance. The catfish harvested are then distributed to people with toddlers to be consumed as an additional protein intake for children and families (Masyitoh, Sudaryanti, Munawar, & Rahmawati, 2020). 4. Preparing the planting medium in the form of soil that has been mixed with fertilize 5. Filling the soil into used bottles 6. Sprinkling vegetable seeds into used bottles and plastic cups 7. Vegetables can be harvested in approximately one month On average, the initial amount of vegetable harvest is one bunch in two buckets and one in 18 plastic bottles. Community Empowerment through Improving Catfish Cultivation skills and Vegetable Planting in Pulo Brayan Village The amount of vegetable harvest will be further reduced when it enters the third and fourth months of maintenance. When the amount of vegetable harvest has begun to decrease, it can be replanted through alternation with new seedlings. The success of growing vegetables in buckets and plastic bottles’ lids depends largely on sunlight exposure. Vegetables that are sufficiently exposed to sunlight will be more fertile than those not exposed to sunlight. This cultivation method is expected to be a solution for fulfilling family nutrition on a household scale and a solution to preventing stunting. Fulfillment of food can be done in pregnant and lactating women and the first 1000 days of life. The vegetable growing activity began with an initial survey in Pulo Brayan Kota Village to see the yard of the house that will be used to grow vegetables vertically. Furthermore, it provides 12 packs of vegetable seeds, six packs of spinach seeds, and six packs of kale vegetable seeds. The reason for choosing spinach and kale vegetables to grow is that both vegetables are vegetables commonly consumed by the public and have a high nutritional content in the form of vitamins and minerals that are good for the body, such as iron, vitamin C, calcium, magnesium, fiber, and miscellaneous. Figure 6. Training on Growing Vegetables in Used Bottles and Plastic Cups Vegetable seeds were distributed to 4 groups living in 4 neighborhoods with stunting cases. The selected family is the KK, who also participated in catfish farming activities. Fostering vegetable planting using plastic bottles and cups with four selected groups by doing the following stages: Figure 6. Training on Growing Vegetables in Used Bottles and Plastic Cups Warta LPM, Vol. 25, No. 4, Oktober 2022 520 Koka, dkk - Empowerment of Posyandu Cadres ... Figure 7. Result of Growing Vegetables Vertically with Plastic Bottles and Cups then recorded into the KMS buble. In addition, mothers of toddlers are trained to fill out the Towards Health Card (KMS) and explain the information contained in the return sheet of the Card Towards Health (KMS), which is information about the stages of feeding babies and children aged 0-24 months. Training on catfish cultivation and planting vegetables vertical provides enormous benefits for the community because they can take advantage of the yard for catfish cultivation and farming. The needs for protein, vitamins, minerals, and fiber can be met independently without buying. Community Empowerment through Improving Catfish Cultivation skills and Vegetable Planting in Pulo Brayan Village The availability of adequate food can reduce the rate of stunting. Figure 7. Result of Growing Vegetables Vertically with Plastic Bottles and Cups Fish farming in buckets and planting vegetables vertically is one way to meet food needs in densely populated areas with little yards, such as urban areas. Vegetable & fish farming does not require a large area or place beside it; it is easy to do, requires a small medium, is flexible, and does not require electricity. In addition to functioning for fish farming, it can also be planted with vegetables such as mustard greens, kale, spinach, and lettuce using a planting medium in the form of charcoal. It can also use chaff, placed in a plastic cup and tied above the water’s surface in a bucket (Zen, Camellia, Noor, & Asih, 2020). 5. ACKNOWLEDGEMENTS We thank Allah SWT for all His gifts and mercy so that we can complete the skim community service of the assisted village entitled “ Empowerment of Posyandu Cadres in Detecting and Preventing Stunting” On this occasion, we would like to thank the Rector of the University of North Sumatra, the Community Service Institute of the University of North Sumatra, the Faculty of Public Health, the University of North Sumatra, the entire Team of Assisted Villages of the University of North Sumatra and the community groups of Pulo Brayan Village, West Medan District, as partners in activities that have given an opportunity to carry out these activities. Finally, we hope this community service activity can contribute positively and is valuable to all parties. 4. CONCLUSION The training carried out on cadres of integrated service posts can improve the skills of cadres in measuring body length and height by using a spur measuring board, where the tool can detect the size of the body that should be achieved according to the age of the child and REFERENCE Badan Penelitian dan Pengembangan Kesehatan. (2019). Hasil Utama Riskesdas 2018 . https://www. litbang.kemkes.go.id/hasil-utama-riskesdas-2018/ Cintya, S., Sinolungan, B. J. S. v, Program, R. S. H., Keperawatan, S. I., Kedokteran, F., Sam, U., & Manado, R. (2013). Hubungan Pengetahuan dan Sikap Perawat dengan Pelaksanaaan Keselamatan Pasien (Patient Safety) di Ruang Rawat Inap RSUD Liun Kendage Tahuna. Jurnal Keperawatan, 1(1). https://doi.org/10.35790/JKP.V1I1.2237 Febri, S. P., Alham, F., & Afriani, A. (2019). Pelatihan BUDIKDAMBER (Budidaya Ikan dalam Ember) di Desa Tanah Terban Kecamatan Karang Baru Kabupaten Aceh Tamiang. Prosiding Seminar Nasional Politeknik Negeri Lhokseumawe, 3(1). http://e-jurnal.pnl.ac.id/semnaspnl/article/ view/1786 521 Warta LPM, Vol. 25, No. 4, Oktober 2022 521 Koka, dkk - Empowerment of Posyandu Cadres ... Hafifah, N., & Abidin, Z. (2020). Peran Posyandu dalam Meningkatkan Kualitas Kesehatan Ibu dan Anak di Desa Sukawening, Kabupaten Bogor. In Jurnal Pusat Inovasi Masyarakat Juli (Vol. 2020, Issue 5). Handayani, R. (2017). Faktor-Faktor yang Berhubungan dengan Status Gizi pada Anak Balita. Jurnal Endurance : Kajian Ilmiah Problema Kesehatan, 2(2), 217–224. https://doi.org/10.22216/JEN. V2I2.1742 Hilmi, M. I., Rahmawati, I., Indrianti, D. T., Sekolah, P. L., Jember, U., & Keperawatan, I. (n.d.). Penguatan Kelembagaan Posyandu Dalam Penanganan Masalah Stunting. 2(1). https://faktualnews. co/2018/08/25/puluhan-balita-di-jelbuk-jember-alami- IniMedan.com. (2020). Pemko Medan Tetapkan 10 Kelurahan Jadi Lokus Rembuk Stunting. IniMedan. Com. https://www.inimedan.com/pemko-medan-tetapkan-10-kelurahan-jadi-lokus-rembuk- stunting/ Khosiah, K., & Muhardini, S. (2019). Pengembangan Sumberdaya Manusia (PSDM) Unsur Perangkat Desa Dan Kader Pembangunan Manusia Se-Kecamatan Aikmel Kabupaten Lombok (Stunting dan Konvergensi Pencegahan Stunting). JUPE : Jurnal Pendidikan Mandala, 4(5). https://doi. org/10.36312/JUPE.V4I5.985 Mahyarni, M. (2016). Penyuluhan Sosial Bagi Para Kader Pos Pelayanan Terpadu untuk Meningkatkan Gizi Balita di Kecamatan Kuok. Kutubkhanah, 18(2), 176–186. https://doi.org/10.24014/ KUTUBKHANAH.V18I2.1480 Masyitoh, N., Sudaryanti, D. S., Munawar, A. H., & Rahmawati, M. (2020). Optimalisasi Kawasan Rumah Pangan Lestari dengan Budikdamber untuk Meningkatkan Ketahanan Pangan Masyarakat di Masa Pandemic Covid-19 di Desa Sukamahi Kecamatan Sukaratu Kabupaten Tasikmalaya. Kommas: Jurnal Pengabdian Kepada Masyarakat, 1(3), 111–122. http://openjournal.unpam. ac.id/index.php/kommas/article/view/8602 Nurmalasari, R. G., Widyastuti, Y., & Margono. (2017). Hubungan Panjang Badan Lahir Dengan Perkembangan Anak Usia 3-24 Bulan di Kabupaten Gunungkidul Tahun 2017. http:// poltekkesjogja.ac.id Paembonan, S. (2022). Aplikasi Pendaftaran Pasien berbasis Android pada Poli Anak Rumah Sakit Umum Andi Djemma Masamba. Kesatria : Jurnal Penerapan Sistem Informasi (Komputer Dan Manajemen), 3(1), 18–25. https://doi.org/10.30645/KESATRIA.V3I1.93 Pramiyati, T., Jayanta, J., & Yulnelly, Y. (2017). Peran Data Primer pada Pembentukan Skema Konseptual yang Faktual (Studi Kasus: Skema Konseptual Basisdata Simbumil). Simetris: Jurnal Teknik Mesin, Elektro Dan Ilmu Komputer, 8(2), 679–686. REFERENCE https://doi.org/10.24176/SIMET.V8I2.1574 Setiawan, E., Machmud, R., & Masrul, M. (2018). Faktor-Faktor yang Berhubungan dengan Kejadian Stunting pada Anak Usia 24-59 Bulan di Wilayah Kerja Puskesmas Andalas Kecamatan Padang Timur Kota Padang Tahun 2018. Jurnal Kesehatan Andalas, 7(2), 275–284. https://doi. org/10.25077/JKA.V7I2.813 Tria, A., & Endah, P. (2021). Pengaruh Pola Asuh Pembrian Makan terhadap Kejadian Stunting pada Balita. Jurnal Kesehatan Masyarakat Andalas, 14(2), 3–11. https://doi.org/10.24893/JKMA. V14I2.527 Yuliani, W. (2018). Metode Penelitian Deskriptif Kualitatif dalam Perspektif Bimbingan dan Konseling. QuantA, 2(2), 83–91. https://doi.org/10.22460/Q.V2I2P83-91.1641 Zen, S., Kamelia, M., Noor, R., & Asih, T. (2020). SNPPM-2 (Seminar Nasional Penelitian dan Pengabdian kepada Masyarakat) Tahun 2020. 522 Warta LPM, Vol. 25, No. 4, Oktober 2022 522
https://openalex.org/W2340102376	https://scholarship.claremont.edu/cgi/viewcontent.cgi?article=1193&context=aliso	English	null	Rarity in Astragalus: a California Perspective	Aliso	2,015	cc-by	8,549	Rarity in Astragalus: a California Perspective Rarity in Astragalus: a California Perspective Philip W. Rundel University of California, Los Angeles Follow this and additional works at: https://scholarship.claremont.edu/aliso Follow this and additional works at: https://scholarship.claremont.edu/aliso Part of the Botany Commons, and the Ecology and Evolutionary Biology Commons Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany Volume 33 Issue 2 Article 4 2015 Rarity in Astragalus: a California Perspective Rarity in Astragalus: a California Perspective Philip W. Rundel University of California, Los Angeles Thomas R. Huggins University of California, Los Angeles Barry A. Prigge University of California, Los Angeles M. Rasoul Sharifi University of California, Los Angeles Follow this and additional works at: https://scholarship.claremont.edu/aliso Part of the Botany Commons, and the Ecology and Evolutionary Biology Commons Recommended Citation Recommended Citation Rundel, Philip W.; Huggins, Thomas R.; Prigge, Barry A.; and Rasoul Sharifi, M. (2015) "Rarity in Astragalus: a California Perspective," Aliso: A Journal of Systematic and Floristic Botany: Vol. 33: Iss. 2, Article 4. Available at: https://scholarship.claremont.edu/aliso/vol33/iss2/4 Aliso: A Journal of Systematic and Floristic Botany Aliso: A Journal of Systematic and Floristic Botany Volume 33 Issue 2 Volume 33 Issue 2 Article 4 2015 Rarity in Astragalus: a California Perspectiv Rarity in Astragalus: a California Perspectiv Philip W. Rundel University of California, Los Angeles Thomas R. Huggins University of California, Los Angeles Barry A. Prigge University of California, Los Angeles M. Rasoul Sharifi University of California, Los Angeles 2015 Rarity in Astragalus: a Cali Rarity in Astragalus: a Cali Philip W. Rundel University of California, Los Angeles Thomas R. Huggins University of California, Los Angeles Barry A. Prigge University of California, Los Angeles M. Rasoul Sharifi University of California, Los Angeles Rundel, Philip W.; Huggins, Thomas R.; Prigge, Barry A.; and Rasoul Sharifi, M. (2015) "Rarity in Astragalus: a California Perspective," Aliso: A Journal of Systematic and Floristic Botany: Vol. 33: Iss. 2, Article 4. Available at: https://scholarship.claremont.edu/aliso/vol33/iss2/4 INTRODUCTION life history traits or membership in rapidly speciating lineages, are positively associated with the persistence of naturally rare species (Harrison et al. 2008). Issues of conservation management of rare species are particularly critical in California, a global hotspot of biodiversity (Norman 2003), where the large flora contains a high proportion of endangered, threatened, and rare species. An understanding of rare plants and their relation to specific habitats of occurrence form a fundamental component of biodiversity management. Although considerable effort has gone into cataloguing rare taxa using a variety of criteria, we nevertheless lack the unifying principles and underlying critical data for the practical management of rare taxa and an understanding of the traits that separate them from common taxa (Kunin and Gaston 1997; Me´dail and Verlaque 1997; Bevill and Louda 1999; Domı´nguez Lozano and Schwartz 2005). While there is a large body of published literature describing individual rare taxa, there is far less research on the nature of microhabitats and microclimates that support rare local endemics. The high frequency of rarity in regional floras leads to the question of how these naturally rare species have persisted through evolutionary time and avoided extinction in spite of what are often three- way problems of small geographic ranges, low population numbers, and specific habitat conditions (Rabinowitz 1981). Recent studies have suggested that benign climates with spatially extensive specialized habitats, rather than specific The legume genus Astragalus L. with an estimated 3270 species of largely herbaceous perennials is the largest genus of vascular plant in the world (Frodin 2004) and presents classic examples of rarity. The distribution of Astragalus is widespread in semi-arid, arid, and cool temperate regions of the northern hemisphere, with very high species diversity in a band extending across the mountain steppes of the eastern Mediterranean Basin across south-central Asia to the western Himalayan Plateau, where there are estimated to be about 1500–2000 species. Examples of this extreme diversity can be seen in Afghanistan with 380 species (Breckle and Rafiqpoor 2010), Turkey with 391 species (Gu¨nar et al. 2001), China with 401 species (Xu et al. 2010), and Iran with 451 species (Mahmoodi et al. 2009). However, western North America is a secondary center of evolution of Astragalus with about 400–450 species (Barneby 1964). There is a small disjunct species complex in tropical East Africa (Gillett 1964). ’ 2015, The Author(s), CC-BY. This open access article is distributed under a Creative Commons Attribution License, which allows unrestricted use, distribution, and reproduction in any medium, provided that the original author(s) and source are credited. Articles can be downloaded at http://scholarship.claremont.edu/aliso/. ABSTRACT Astragalus (Fabaceae), the largest genus of plants in the world with an estimated 3270 species, is known for large numbers of rare endemic species. An inventory of patterns of climatic, topographic, and edaphic diversity of Astragalus taxa in California (98 native species and 144 named taxa) provides a means to understand the occurrence of rarity in relation to climatic equitability and regional species richness of congeneric taxa. Most taxa in the genus have relatively small ranges of distribution, with 50% restricted geographically to a single Jepson Bioregion. The California Native Plant Society lists 51 Astragalus taxa (35% of the native Astragalus taxa) as rare, threatened, or endangered (RTE). Climate characteristics of geographic regions such as rainfall and temperature extremes show no obvious relationship to species richness or the proportion of listed taxa. Species richness is highest in the arid Great Basin (35 species and 53 taxa) combining both its components, followed by 29 species and 39 taxa in the Sierra Nevada East region that includes the White and Inyo Mountains. The Mojave Desert is also high in diversity with 32 species and 39 taxa, but in contrast the Sonoran Desert region is low with only 12 species and 14 taxa. Despite ranking highest in the number of Astragalus taxa present, the Great Basin regions are low in their proportion of RTE taxa (17%) compared to the South Coast Region (39.5%) and Mojave Desert (32%). Strong edaphic specialization is associated with the majority but not all RTE taxa. While no single ecophysiological adaptation can explain this pattern, it is significant that Astragalus taxa have the potential ability to develop symbiotic nitrogen fixation, and this trait is key to success in soils not conducive to growth of many potential competitors. Land use changes, alien grass invasion and grazing, among other threats, are increasing fragmentation of habitats for many rare taxa with consequent impacts on gene flow. The continued survival of rare and locally endemic taxa will require improved knowledge of their individual demographic traits and long-term population dynamics. Key words: Astragalus, California biodiversity, edaphic specialization, nitrogen fixation, rarity. Recommended Citation Recommended Citation Rundel, Philip W.; Huggins, Thomas R.; Prigge, Barry A.; and Rasoul Sharifi, M. (2015) "Rarity in Astragalus: a California Perspective," Aliso: A Journal of Systematic and Floristic Botany: Vol. 33: Iss. 2, Article 4. Available at: https://scholarship.claremont.edu/aliso/vol33/iss2/4 Aliso, 33(2), pp. 111–120 ISSN 0065-6275 (print), 2327-2929 (online) Aliso, 33(2), pp. 111–120 ISSN 0065-6275 (print), 2327-2929 (online) INTRODUCTION South America is home to about 100 species, with their diversity centered in the southern and central Andean region. These South American species represent two distinct clades that arrived independently by long-distance dispersal from North America (Scherson et al. 2008). 112 Rundel, Huggins, Prigge, and Sharifi ALISO ALISO The geographic center of diversity and presumed origin of Astragalus, as with most of its close relatives in the tribe Galegeae Dumort., lies in arid and semi-arid regions of south- central Asia (Polhill 1981). The Galegeae clade is entirely an Old World group with the exception of the genera Astragalus and sister group Oxytropis DC. which also became established in North America. Although species-rich in northern temperate latitudes, Astragalus has relatively few arctic species, a trait that it shares with other large temperate genera such as Carex L., Senecio L., and Vaccinium L. (Hoffmann and Ro¨ser 2009). of several scales of rarity, including those taxa listed either federally or by the state of California as Rare, Threatened, or Endangered (RTE). The database also includes a Rare Plant Rank, with a designation of 1B signifying taxa that are rare, threatened, or endangered (RTE) in California and elsewhere. Plants listed in category 1B are further divided into three threat ranks. Plants constituting California Rare Plant Rank 1B meet the definitions of California Endangered Species Act and are eligible for state listing. It is mandatory that they be fully considered during preparation of environmental docu- ments relating to the California Environmental Quality Act (CEQA). One of the characteristic biogeographic features of Astra- galus over its range of occurrence is a remarkable level of edaphic specialization, often leading to highly restricted geographic ranges of distribution and rarity. This population trait is not only characteristic of the Irano-Turkish region and Himalayan Plateau in floras from those regions, but is also expressed in the Astragalus flora of the western United States. The Great Basin and Mojave Desert are notable for diverse examples of local endemism in Astragalus associated with edaphic specialization. These geographic patterns of isolated populations coupled with limited dispersal ability of seeds have been hypothesized to promote rapid local differentiation and geographic speciation over the Quaternary (Sanderson and Wojciechowski 1996). Biogeography and Diversity of California Astragalus The California flora includes 98 native Astragalus species with a total of 144 described taxa (Baldwin et al. 2012, plus one new record as described above). Following the Jepson Manual’s designations of bioregions, the greatest richness of Astragalus is found in the combined Great Basin Bioregion with 35 species and 53 taxa (Table 1). Much of this diversity was present in the Sierra Nevada East Bioregion with 29 species and 39 taxa. Diversity was also high in the Mojave Desert with 33 species and 41 taxa. The high species richness of these arid regions does not extend to the Sonoran Desert which is relatively low in diversity with 12 species and 14 taxa. This low diversity reflects in part its smaller size compared with the Mojave Desert and also its more limited distribution of azonal edaphic habitats. Only six taxa are shared with the Mojave Desert. Our objectives in this study are to examine the bio- geographic and ecological patterns of rarity in the diverse taxa of Astragalus present in the California flora as a stimulus to future research. Are there patterns in the climatic, topographic, and edaphic diversity of California ecosystems that provide a useful template to predict the occurrence of rarity across these gradients? Do higher precipitation levels with less climatic stress favor the presence of rare species (Harrison et al. 2008)? A better understanding of geographic, and more specifically climatic correlates of rarity in Astragalus, could be significant in setting conservation strategies for rare species. In addition, this knowledge would provide a stimulus for expanding investigations of the associated genetic struc- ture, life history, and demography of rare species. The next-richest geographic regions are the South Coast Bioregion, including the Transverse and Peninsular Ranges (30 species and 38 taxa), and the Sierra Nevada (23 species and 30 taxa). There is a clear pattern showing the higher-elevation areas of the Sierra Nevada to be home to a higher diversity of Astragalus than lower-elevation areas. The Sierra Nevada foothills have only eight species and nine taxa, compared to the higher montane, subalpine, and alpine areas with 19 species and 24 taxa. There are eight Astragalus taxa that reach to alpine habitats of 3300 m or more in the range (Rundel 2011). INTRODUCTION Available information on RTE species was gathered from our own field experience, the published literature, gray literature in government reports including Federal Register publications, and the California Native Plant Society’s online Inventory of Rare and Endangered Plants (CNPS 2015). This inventory includes considerable information on habitat, geo- graphic and elevational distribution, population numbers, and threats, as available. MATERIALS AND METHODS The second edition of the Jepson Manual (Baldwin et al. 2012) was used to determine the species biogeography of Astragalus taxa with a natural distribution in California, based on the Jepson bioregions of California. One new record for California, A. nyensis, was added. The Jepson Manual divides California into ten bioregions (Fig. 1): the northwest coast and Klamath Ranges (NW), Cascade Range (CaR), Sierra Nevada (SN), Great Valley (GV), Central Coast Ranges (CW), Southwest Coast and Ranges (SW), Modoc Plateau (MP), Great Basin/Sierra Nevada East (SNE), Mojave Desert (DMoj), and Sonoran Desert (DSon). Distribution was recorded for each Astragalus taxon within these geographic regions of California and their major subregions. The two floristically and climatically related Great Basin regions were merged into a single unit to produce nine regions for analysis. The Natural Diversity Database of the California De- partment of Fish and Wildlife provides a classification system for vascular plants of special concern in the state (CNDDB 2015). This broad list categorizes taxa of concern on the basis Taxa of Astragalus in California are largely restricted in their geographic distribution. Of the 144 taxa present, 86 taxa (60%) are restricted to a single bioregion (lumping the two Great Basin Bioregions). Another 39 taxa are present in only two bioregions. Only five Astragalus taxa have moderately broad ranges of distribution within California such that they occur in four or more of the major bioregions. Astragalus purshii Douglas var. tinctus M.E. Jones is present throughout eight of the nine bioregions of California treated here, missing only from the Central Valley, and extends into Oregon and Nevada. The species with all of its varieties occurs widely across the western US and Canada. The Natural Diversity Database of the California De- partment of Fish and Wildlife provides a classification system for vascular plants of special concern in the state (CNDDB 2015). This broad list categorizes taxa of concern on the basis 113 VOLUME 33(2) Rarity in Astragalus cies and taxa (in parentheses) in the Jepson Manual Bioregions of California (Baldwin et al. 2012).—2–8. Rare taxa of California Astragalus lentiginosus var. coachellae (photo by Brent Miller).—4. Astragalus albens (photo by Chris Wagner, SBNF).—5. yus var. lanosissimus (photo by Nicholas Jensen).—7. Astragalus jaegerianus (photo by Duncan Bell).—8. Astragalus brauntonii Fig. 1–8. California Astragalus.—1. Distribution of Astragalus species and taxa (in parentheses) in the Jepson Manual Bioregions of California (Baldwin et al. 2012).—2–8. MATERIALS AND METHODS Rare taxa of Californ Astragalus.—2. Astragalus agnicidus (photo by Jennifer L. Kalt).—3. Astragalus lentiginosus var. coachellae (photo by Brent Miller).—4. Astragalus albens (photo by Chris Wagner, SBNF).— Astragalus claranus (photo by Jake Ruygt).—6. Astragalus pycnostachyus var. lanosissimus (photo by Nicholas Jensen).—7. Astragalus jaegerianus (photo by Duncan Bell).—8. Astragalus braunto (photo by Michael Charters). Fig. 1–8. California Astragalus.—1. Distribution of Astragalus species and taxa (in parentheses) in the Jepson Manual Bioregions of California (Baldwin et al. 2012).—2–8. Rare taxa of Califo Astragalus.—2. Astragalus agnicidus (photo by Jennifer L. Kalt).—3. Astragalus lentiginosus var. coachellae (photo by Brent Miller).—4. Astragalus albens (photo by Chris Wagner, SBNF).— Astragalus claranus (photo by Jake Ruygt).—6. Astragalus pycnostachyus var. lanosissimus (photo by Nicholas Jensen).—7. Astragalus jaegerianus (photo by Duncan Bell).—8. Astragalus braunt (photo by Michael Charters). Fi 1 8 C lif i A l 1 Di t ib ti f A l 114 ALISO Rundel, Huggins, Prigge, and Sharifi Table 1. Occurrence of Astragalus species and described taxa within the major Jepson bioregions and subregions of California. The number and proportion of listed rare, threatened and endangered taxa (RTE) based on classification by the California Native Plant Society (CNPS 2015). Totals include one species added since Baldwin et al. (2012). The column for dominant vegetation gives a broad characterization of each bioregion and subregion. Table 1. Occurrence of Astragalus species and described taxa within the major Jepson bioregions and subregions of California. The number and proportion of listed rare, threatened and endangered taxa (RTE) based on classification by the California Native Plant Society (CNPS 2015). Totals include one species added since Baldwin et al. (2012). The column for dominant vegetation gives a broad characterization of each bioregion and subregion. MATERIALS AND METHODS Sheld. var. gambelianus (Gambel milkvetch) is present in all six of the non-desert bioregions of California and extends into southwestern Oregon and north- western Baja California. Astragalus pachypus Greene var. pachypus (thick-pod milkvetch) is present in six bioregions, skipping only the northern bioregions and Great Basin. Astragalus didymocarpus Hook. & Arn. var. didymocarpus (dwarf white milk vetch) is reported in five bioregions of the state, including the Mojave Desert Bioregion and adjacent Nevada, but like A. pachypus it is absent from the northern bioregions and Great Basin. Adding in the range of the other three varieties does not expand its range into other bioregions. three rare. Interestingly, only four taxa of Astragalus are designated on both the federal and state lists of protected species—A. claranus, A. magdalenae var. peirsonii, A. pycno- stachyus var. lanosissimus, and A. tener var. titi. Rare (RTE) taxa are disproportionately represented in California Astragalus compared to the flora as a whole. Of the 144 taxa present, 51 taxa (35%) are listed in the California Rare Plant Ranking as rare, threatened, or endangered (RTE). This designation is defined by those species in categories 1B.1 (18 taxa), 1B.2 (27 taxa), and 1B.3 (6 taxa). Another 12 taxa are listed as rare in California but more common elsewhere, and 19 listed species have limited distribution and are classified as deserving of monitoring. At the species level, the most widespread species present in California is Astragalus canadensis L. (Canadian milkvetch), which occurs throughout the United States and Canada in many habitats from wetlands to woodlands and prairies. California has A. canadensis var. brevidens (Gand.) Barneby, which occurs only in Great Basin areas and the northern high Sierra Nevada. Also very widespread over its range of varieties is A. lentiginosus which occurs across the western United States, north into Canada, and south into northern Mexico. Lumping all of the varieties of this polymorphic species together, it is present in seven of the bioregions of California, being absent from only the northern two regions (North Coast/ Klamath and Cascade). Astragalus lentiginosus var. variabilis Barneby is the most widely distributed variety in California with a range that includes the Central Valley, Sierra Nevada, Great Basin, and Mojave Desert Bioregions. There are no obvious patterns linking the proportion of RTE taxa and their diversity with bioregion and associated climate regime or geographic position. MATERIALS AND METHODS Biogeographic region Species Taxa RTE taxa % listed taxa Dominant vegetation Northwest Coast/Klamath 15 16 3 18.8 Wet conifer forest Cascade Range 9 9 1 11.1 Conifer forest Sierra Nevada 23 30 8 26.7 Conifer forest Foothills 8 9 0 0 Woodland, chaparral High Sierra 19 24 8 33.3 Conifer forest, alpine Central Coast 18 22 4 18.2 Woodland, chaparral South Coast and Southern California Ranges 30 38 15 39.5 Chaparral, conifer forest Transverse Ranges 20 24 5 20.8 Conifer forest Peninsular Ranges 14 16 4 25.0 Conifer forest South Coast 10 13 4 30.8 Chaparral, woodland Channel Islands 8 8 2 25.0 Chaparral, sage scrub Great Basin 35 53 9 17.0 Cold Desert Modoc/Warner Mountains 17 22 4 18.2 Sagebrush, pinyon/juniper Sierra Nevada East 29 39 6 15.4 Conifer forest Mojave Desert 33 41 13 31.7 Warm desert Sonoran Desert 12 14 3 21.4 Warm desert Astragalus gambelianus E. Sheld. var. gambelianus (Gambel milkvetch) is present in all six of the non-desert bioregions of California and extends into southwestern Oregon and north- western Baja California. Astragalus pachypus Greene var. pachypus (thick-pod milkvetch) is present in six bioregions, skipping only the northern bioregions and Great Basin. Astragalus didymocarpus Hook. & Arn. var. didymocarpus (dwarf white milk vetch) is reported in five bioregions of the state, including the Mojave Desert Bioregion and adjacent Nevada, but like A. pachypus it is absent from the northern bioregions and Great Basin. Adding in the range of the other three varieties does not expand its range into other bioregions. At the species level, the most widespread species present in California is Astragalus canadensis L. (Canadian milkvetch), which occurs throughout the United States and Canada in many habitats from wetlands to woodlands and prairies. California has A. canadensis var. brevidens (Gand.) Barneby, which occurs only in Great Basin areas and the northern high Sierra Nevada. Also very widespread over its range of varieties is A. lentiginosus which occurs across the western United States, north into Canada, and south into northern Mexico. Lumping all of the varieties of this polymorphic species together, it is present in seven of the bioregions of California, being absent from only the northern two regions (North Coast/ Klamath and Cascade). Astragalus lentiginosus var. variabilis Barneby is the most widely distributed variety in California with a range that includes the Central Valley, Sierra Nevada, Great Basin, and Mojave Desert Bioregions. Astragalus gambelianus E. MATERIALS AND METHODS Although the Great Basin Bioregion of California has the highest number of Astragalus taxa present, it ranks relatively low in proportion of RTE taxa with 17.0% (Table 1). The Mojave Desert Bioregion was higher at 31.7%, while the Sonoran Desert Bioregion relatively low at 21.4%. The highest proportion of RTE species was found in the South Coast Bioregion (39.5%). However, the Central Coast Bioregion had only 18.2% RTE taxa. Within the Sierra Nevada Bioregion the proportion of RTE taxa was relatively high for the montane and higher elevations at 33.3%, but no RTE taxa were present in the foothills. Low rates of RTE taxa were present in the Cascade Range Bioregion (11.1%) and Northwest Coast/Klamath Bioregion (18.8%). Table 2 lists known edaphic specialization for the 51 RTE Astragalus taxa in California. This edaphic specialization takes many forms, indicating that no single ecophysiological adaptation to specialized soil conditions can explain this pattern. Edaphic conditions point to a tolerance of diverse substrates within Astragalus including carbonate soils, volca- nic clays, sedimentary clays, serpentines, alkaline wetlands, vernal pools, pebble plains, pumices, granite barrens, desert dunes, coastal dunes, and volcanic hot spring sites. DISCUSSION trait facilitates establishment and survival of nodulated plants on oligotrophic soils and habitats with regular disturbance that promote primary succession. There are examples in both Europe and the western United States of extremely rare species of Astragalus found to be restricted in occurrence to disturbed and heavily grazed sites (e.g., Sauer et al. 1979; Nordhag 1991). This trait is also present in California, with A. agnicidus Barneby as a prominent example (see below), and many species are best known from roadside verges where road maintenance is thought to present a threat (Table 2). It is worth noting that two other genera of California legumes with diverse species and local endemics, Lupinus and Acmispon (formerly Lotus), also are highly successful on oligotrophic soils and disturbed sites. There are 103 Lupinus taxa and 37 Acmispon taxa in California, with many more unresolved taxa of the former (Jepson eFlora, http://ucjeps.berkeley.edu/IJM. html). These genera, however, lack the frequency of rarity present in Astragalus taxa. There are 21 taxa of Lupinus and 7 taxa of Acmispon with RTE status in California (CNPS 2011). Rarity and Life History Traits Rarity and Life History Traits Rarity and Life History Traits As described by Rabinowitz (1981), there are multiple components of rarity. Rarity can be determined on the basis of geographic range (wide vs. narrow), habitat requirements (generalist vs. specialist), and population size (large vs. small). Thus, rarity can be classified into seven categories representing the combinations of the three components, with the eighth category of widespread, generalist species with large popula- tion size classified as common. Most rare taxa in Astragalus have special conservation significance in that they fit the category of the most extreme rarity in having limited geographic range, highly specialized habitat requirements, and small population sizes. Several features of the life history and population genetics of Astragalus have been hypothesized to promote high rates of diversification and speciation in other plants. These include the herbaceous growth form and associated short generation time (Doyle and Donoghue 1993), isolated population structure with restricted gene flow (Niklas et al. 1985), short seed dispersal distances (Morris et al. 2002; Becker 2010), and large amounts of chromosomal variability (Levin and Wilson 1976). DISCUSSION However, Sanderson and Wojciechowski (1996) dismiss the last of these as a likely factor by demonstrating that the causal factors of high rates of diversification in Astragalus may be related to basal traits associated with the entire astragalean clade, which lacks much of this chromosome variability. This clade has notably radiated in arid and semi-arid regions and is largely composed of herbaceous perennials that exhibit high levels of local endemism. Oxytropis (300 species, Eurasian and North American temperate) and Colutea L. (200 species, Eurasian temperate) are other diverse genera within this lineage (Lewis et al. 2005). It has been hypothesized that rarity and local endemism may be associated with the most climatically favorable sites within a region having unusual soil conditions. Rarity in California serpentine endemics is positively associated with regions with higher mean annual precipitation, suggesting that less compet- itive taxa may be more likely to survive where climatic stress is reduced (Harrison et al. 2008). However, the patterns of local endemism and rarity in Astragalus do not support this hypothesis. There is no obvious pattern of relationship between climate regime and the proportions of Astragalus rarity present across the diversity bioregions of California. High proportions of rarity are present in a range of climatic regimes throughout California ranging from arid desert bioregions, coastal regions of moderate climate conditions, high mountains, or the humid northwestern coast and Klamath Ranges. The recurring pattern of ecological restriction and/or edaphic specialization present in the astragalean clade suggests that there may be key adaptive innovations associated with this specialization (Barneby 1964; Spellenberg 1976). However, vegetative traits of growth form and leaf morphology show no such obvious innovation. Instead, they are common features that have evolved countless times in parallel through evolutionary time, presenting a large but finite number of variations on the same structural plan (Polhill 1981 et al.). Rarity in California Astragalus On a national basis of designated rarity, the genus Astragalus appears prominently with 21 taxa that are federally listed as endangered or threatened and an additional five candidate taxa. Ten of these federally listed taxa, eight endangered and two threatened, are present in California. Formal state designations of protection in California extend to nine taxa, with five listed as endangered, one threatened, and VOLUME 33(2) 115 Rarity in Astragalus ALISO 116 Table 2. Vegetation association, edaphic specialization, and major threats to Astragalus taxa listed as rare, threatened, or endangered by the California Native Plant Society (CNPS 2015). See text for explanation of rare plant rank categories. Formal listings of rank include FE (federal endangered), FT (federal threatened), SE (state endangered), ST (state threatened), and SR (state rare). Threat information based on CNPS (2015). Scientific name Rank Vegetation Edaphic specialization Threats Astragalus agnicidus Barneby 1B.1, SE Northwest conifer Disturbed soils Logging, eradication Astragalus albens Greene 1B.1, FE Mojave desert scrub, pinyon-juniper Carbonates Mining Astragalus anxius Meinke & Kaye 1B.3 Great Basin scrub, pinyon- juniper Volcanic Grazing in past Astragalus atratus S. Watson var. mensanus M.E. Jones 1B.1 Great Basin scrub, Mojave, pinyon-juniper Volcanic clay Grazing Astragalus austiniae A. Gray 1B3 Alpine and subalpine rock fields Boulder fields ? Astragalus bernardinus M.E. Jones 1B.2 Joshua tree, pinyon-juniper Granite or carbonate Mining, urbanization, grazing Astragalus brauntonii Parish 1B.1, FE Chaparral Calcareous Urbanization, fire regime, non-native species Astragalus cimae M.E. Jones var. cimae 1B.2 Great Basin scrub, pinyon- juniper Clay Grazing Astragalus cimae var. sufflatus Barneby 1B.3 Great Basin scrub, pinyon- juniper Calcareous Grazing? Astragalus claranus Jeps. 1B.1, FE, ST Chaparral/woodland Serpentine/volcanic Urbanization, recreation, non-native plants Astragalus deanei (Rydb.) Barneby 1B.1 Chaparral/woodland Sandy wash Urbanization, fire regime, non-native plants Astragalus didymocarpus Hook. & Arn. var. milesianus (Rydb.) Jeps. 1B.2 Coastal scrub Clay Urbanization Astragalus douglasii (Torr. & A. Gray) A. Gray var. perstrictus (Rydb.) Munz & McBurney 1B.2 Chaparral/woodland, pinyon-juniper Rocky Urbanization, non-native plants Astragalus ertterae Barneby & Shevock 1B.3 Pinyon-juniper Granitic ? Astragalus funereus M.E. Jones 1B.2 Mojave desert scrub Calcareous Grazing? Astragalus gilmanii Tidestr. 1B.2 Great Basin scrub, pinyon- juniper Gravel Grazing, mining Astragalus hornii A. Gray var. hornii 1B.1 Valley grassland Alkaline wetlands Eradication Astragalus jaegerianus Munz 1B.1, FE Desert scrub Granite barrens Military training, non-native plants Astragalus johannis-howellii Barneby 1B.2, SR Great Basin scrub Hot spring soils Grazing, mining, vehicles Astragalus lemmonii A. Gray 1B.2 Great Basin scrub Wet meadow, seeps Habitat loss, pipeline Astragalus lentiformis A. Gray 1B.2 Great Basin scrub, conifer Volcanic Road maintenance, logging Astragalus lentiginosus Douglas var. antonius Barneby 1B.3 Conifer Granitic Urbanization, non-native plants, road maintenance Astragalus lentiginosus var. coachellae Barneby 1B.2, FE Sonoran desert scrub Desert dunes Urbanization, vehicles Astragalus lentiginosus var. kernensis (Jeps.) Barneby 1B.2 Subalpine conifer Wet sandy ? Astragalus lentiginosus var. Examples of Local Endemism The notable pattern of rarity in Astragalus can be seen in many California endemic taxa that are restricted to unusual substrates in local areas. The following examples show the diversity of geographic regions and edaphic conditions which may be associated with the evolution of these rare taxa. Astragalus agnicidus (Humboldt County milkvetch, Fig. 2), known from only a small number of sites in the outer North Coast Ranges of Humboldt and Mendocino counties in northwestern California, is an early successional species that prefers disturbed sites such as logged ridges, open canopy wooded areas, and scarified ground. Although never common, it was subjected to a deliberate program of eradication in the 1930s and 1940s when it was considered to be poisonous to livestock. It was thought to be extinct until it was rediscovered in 1987 on a recently logged and highly disturbed site. It is listed as endangered by the State of California. There have long been studies comparing the breeding systems, levels of inbreeding depression, and genetic structure in widespread and local species of many lineages, with the hypothesis that taxa with restricted ranges and small population sizes will more likely exhibit self-compatibility, low levels of inbreeding depression, low levels of genetic polymorphism than widespread abundant species (Lande and Schemske 1985; Schemske and Lande 1985). The results of research on Astragalus have shown uneven support for these hypotheses, as self-compatibility and low inbreeding may be present in widespread as well as restricted range species (Karron 1989), and the organization of genetic variation may not differ between these groups (Karron et al. 1988; Walker and Metcalf 2008). One of the most interesting examples of taxonomic di- vergence and rarity in Astragalus can be seen in Astragalus lentiginosus (freckled milkvetch). This species has 34 named subspecific taxa, more than any other species in the entire flora of the United States (Knaus 2010). The overall species distribution of A. lentiginosus extends throughout the arid regions of western North America where it occupies both pristine and disturbed soils, saline sites, dunes, and other While it is not sufficient to explain the pattern of endemism and rarity, Astragalus and most other genera of Papilionoideae have the potential to fix atmospheric nitrogen in root tissues through a symbiotic relationship with rhizobium bacteria. This Rundel, Huggins, Prigge, and Sharifi ALISO Its habitat is an area of granite barrens with relatively low plant cover. Individual plants are herbaceous perennials that resprout annually from their base and develop trailing stems that clamber into the canopy of a low-growing host plant. Populations numbers have declined dramatically due to drought from a high of more than 5000 thousand plants surveyed a decade ago (Huggins et al. 2010; Sharifi et al. 2011). Astragalus brauntonii (Braunton’s milkvetch, Fig. 8), a fed- erally endangered species, occurs in a small number of scattered populations in coastal ranges and foothill areas of Ventura, Los Angeles, and Orange counties, generally on carbonate soils. It is a tall perennial that can reach a subshrub form up to 1.5 m in height, but with a short lifespan of 2–3 years (Fotheringham and Keeley 1998). Soil seed pools are known to germinate after fires, indicating that altered fire regimes and perhaps invasive species as well as urbanization may be critical issues in its survival. Astragalus jaegerianus (Lane Mountain milkvetch, Fig. 7) is a federally listed endangered species restricted in distribution to small fragmented populations occurring over an area of less than 24 km2 in the central Mojave Desert northwest of Barstow. Its habitat is an area of granite barrens with relatively low plant cover. Individual plants are herbaceous perennials that resprout annually from their base and develop trailing stems that clamber into the canopy of a low-growing host plant. Populations numbers have declined dramatically due to drought from a high of more than 5000 thousand plants surveyed a decade ago (Huggins et al. 2010; Sharifi et al. 2011). Astragalus brauntonii (Braunton’s milkvetch, Fig. 8), a fed- erally endangered species, occurs in a small number of scattered populations in coastal ranges and foothill areas of Ventura, Los Angeles, and Orange counties, generally on carbonate soils. It is a tall perennial that can reach a subshrub form up to 1.5 m in height, but with a short lifespan of 2–3 years (Fotheringham and Keeley 1998). Soil seed pools are known to germinate after fires, indicating that altered fire regimes and perhaps invasive species as well as urbanization may be critical issues in its survival. Astragalus albens Greene (Cushenbury milkvetch, Fig. 4), federally listed as an endangered species, is restricted in occurrence to a narrow belt of carbonate soils derived from decomposing limestone along rocky washes on the northern slopes of the San Bernardino Mountains. ALISO micans Barneby 1B.2 Mojave desert scrub Desert dunes Vehicles Astragalus lentiginosus var. piscinensis Barneby 1B.1, FT Great Basin scrub Alkaline wetlands Hydrology, non-native plants, grazing Astragalus lentiginosus var. sesquimetralis (Rydb.) Barneby 1B.1, SE Desert scrub Alkaline wetlands Trampling VOLUME 33(2) Rarity in Astragalus 117 Scientific name Rank Vegetation Edaphic specialization Threats Astragalus lentiginosus var. sierrae M.E. Jones 1B.2 Mojave desert scrub, pinyon-juniper Moist gravelly flats Urbanization, mining, fire regime Astragalus leucolobus S. Watson ex M.E. Jones 1B.2 Conifer Pebble plains Urban, recreation, vehicles Astragalus magdalenae Greene var. peirsonii (Munz & McBurney) Barneby 1B.2, FT, SE Sonoran scrub Desert dunes Vehicles Astragalus mohavensis S. Watson var. hemigyrus (Clokey) Barneby 1B.1 Desert scrub, Joshua tree Carbonates Mining Astragalus monoensis Barneby 1B.2, SR Great Basin scrub, conifer Pumice Road maintenance, vehicles, grazing Astragalus nevinii A. Gray 1B.2 Coastal scrub Coastal dunes Grazing Asragalus nyensis Barneby 1B1 Mojave scrub Alkaline soils Solar development, non-native spp. Astragalus oocarpus A. Gray 1B.2 Chaparral/woodland ? Urbanization, road maintenance, recreation Astragalus oophorus S. Watson var. lavinii Barneby 1B.2 Great Basin scrub, pinyon- juniper ? ? Astragalus pachypus Greene var. jaegeri Munz & McBurney 1B.1 Chaparral/woodland Sandy/rocky Urbanization Astragalus preussii A. Gray var. laxiflorus A. Gray 1B.1 Desert chenopod scrub Clay Urbanization Astragalus pseudiodanthus Barneby 1B.2 Great Basin scrub Stabilized desert dunes Grazing Astragalus pulsiferae A. Gray var. pulsiferae 1B.2 Pinyon-juniper, conifer, Great Basin scrub, Granitic Grazing, urbanization Astragalus pulsiferae var. suksdorfii (Howell) Barneby 1B.2 Pinyon-juniper, conifer, Great Basin scrub, Volcanic Grazing mining, logging Astragalus pycnostachyus A. Gray var. lanosissimus (Rydb.) Munz & McBurney 1B.1, FE, SE Coastal scrub Sand/coastal dunes Urbanization, non-native plants Astragalus pycnostachyus var. pycnostachyus 1B.2 Coastal scrub Coastal dunes Grazing, erosion, non-native plants Astragalus rattanii A. Gray var. jepsonianus Barneby 1B.2 Chaparral/woodland Serpentine Road maintenance, energy development Astragalus ravenii Barneby 1B.3 Alpine, subalpine conifer Fellfield ? Astragalus shevockii Barneby 1B.3 Conifer Granitic ? Astragalus tener A. Gray var. ferrisiae Liston 1B.1 Valley grassland Vernal alkaline meadows Agriculture non-native plants, urbanization Astragalus tener var. tener 1B.2 Valley grassland Alkaline wetlands Agriculture Astragalus tener var. titi (Eastw.) Barneby 1B.1, FE, SE Coastal scrub Coastal dunes Urbanization, non-native plants Astragalus traskiae Eastw. 1B.2, SR Coastal scrub Coastal dunes, bluffs Military training Astragalus tricarinatus A. Gray 1B.2, FE Sonoran desert scrub, Joshua tree Sandy, gravelly Pipeline, vehicles Astragalus webberi A. ALISO These soils occur at elevations of 1500–2000 m in Joshua tree and pinyon-juniper woodland habitat. Astragalus albens is known today from just 19 sites with a few thousand plants. Populations experience extreme fluctuations related to amounts of annual precipita- tion, with maximum numbers of no more than 5000–10,000 individuals. Nevertheless, isozyme research has shown a sur- prisingly high degree of heterozygosity for a local endemic species with small population size (Neel 2008). Threats to the survival of A. albens come principally from limestone mining, with off-road vehicles and rural developments as secondary concerns. This local belt of carbonate soils is also home to four other rare and endangered taxa—Erigeron parishii A. Gray, Eriogonum ovalifolium Nutt. var. vineum (Small) A. Nelson, Lesquerella kingii (S. Watson) S. Watson subsp. bernardina (Munz) Munz, and Oxytheca parishii Parry var. goodmaniana Ertter (Gonella and Neel 1995). ALISO Gray 1B.2 Conifer Wet meadow Mining, non-native plants, road maintenance Rundel, Huggins, Prigge, and Sharifi 118 ALISO ALISO marginal habitats. Although many of the named varieties were originally described as species, further research found in- termediate forms that led to the reduction of these species to varieties of a single morphologically plastic species. Astragalus lentiginosus has been viewed as a mosaic of taxa with morphological diversity representing a clinal response to the ecologically heterogeneous climates rather than to patterns of geographic occurrence (Knaus 2010). from a few scattered collections from coastal sites in Los Angeles and Ventura counties, a limited area but broader than its single site of occurrence today. More recently it was thought to be extinct until 1997 when a population was rediscovered in a degraded coastal dune system near Oxnard. This rediscovered population consisted of fewer than 300 individual plants located within an area of less than 0.2 ha, and in subsequent years has dipped in population to as low as 29 plants. Habitat destruction over an already limited range is the cause of its extreme rarity today, and it is both federally and state-listed as endangered. The type variety pycnostachyus has a non-overlapping and broad range along the central and northern California coast but is also classified as rare. The California flora includes 19 varieties of Astragalus lentiginosus, with six of these classified as RTE taxa by the state, and two federally listed as endangered. There is no simple pattern of habitat preference expressed by these RTE taxa. Astragalus lentiginosus var. coachellae (Coachella Valley milkvetch, federally listed as endangered, Fig. 3) and A. lentiginosus var. micans (shining milkvetch, Death Valley milkvetch) are specialists on local areas of desert dunes, while sandy soils of subalpine meadows are the favored habitat for A. lentiginosus var. kernensis (Kern Plateau milkvetch). Two rare varieties, A. lentiginosus var. piscinensis (Fish Slough milkvetch, federally listed as threatened) and A. lentiginosus var. sesquimetralis (Sodaville milkvetch, state-listed as endan- gered), occur only in alkaline desert wetlands and playas. The sixth RTE taxon, A. lentiginosus var. antonius (San Antonio milkvetch), is restricted to montane pine forests of the San Gabriel Mountains. Astragalus jaegerianus (Lane Mountain milkvetch, Fig. 7) is a federally listed endangered species restricted in distribution to small fragmented populations occurring over an area of less than 24 km2 in the central Mojave Desert northwest of Barstow. CONCLUSIONS FOX, L. R., H. N. STEELE, K. D. HOLL, AND M. H. FUSARI. 2006. Contrasting demographies and persistence of rare annual plants in highly variable environments. Pl. Ecol. 183: 157–170. It remains clear that despite considerable effort in recent years, our knowledge of the causal factors of rarity in Astragalus remains poor. There are certainly indications that the survival of rare and locally endemic herbaceous species likely depends heavily on demographic traits and population dynamics (Schemske et al. 1994; Fox et al. 2006) as well as genetic structure (see Lande 1988). There is cause for concern that land use changes and continued fragmentation of rare populations are leading to low levels of gene flow, even between geographically close populations (Walker and Metcalf 2008; Breinholt et al. 2009). We know little about the genetic makeup of most extant populations of rare taxa, although such information should be considered in management decisions such as new population establishment. The informed manage- ment and the persistence of these taxa will require not only a mitigation of major threats, but also a greater understanding of individual life history traits and long-term demography. The conservation of rarity requires better means of assessing risk, with these biological traits as well as geography and history as important factors (Knapp 2011). FRODIN, D. G. 2004. History and concepts of big plant genera. Taxon 53: 753–776. GILLETT, J. B. 1964. Astragalus L. (Leguminosae) in the highlands of tropical Africa. Kew Bull. 17: 413–423. GONELLA, M. P. AND M. C. NEEL. 1995. Characterization of rare plant habitat for restoration in the San Bernardino National Forest, pp. 81–93. In B. A. Roundy, E. D. McArthur, J. S. Haley, and D. K. Mann [eds.], Proceedings: wildland shrubs and arid land restoration symposium, 19–21 Oct 1993, Las Vegas, NV, U.S. Department of Agriculture, Forest Service, Intermountain Research Station, General Technical Report INT-GTR-315, Ogden, Utah. GU¨ NER, A., N. OZHATAY, T. EKIM, K. HUSNU, C. BASER, AND I. HEDGE (editors). 2001. Flora of Turkey and the East Aegean Islands, vol. 11. Edinburgh University Press, Edinburgh, UK. HARRISON, S., J. H. VIERS, J. H. THORNE, AND J. B. GRACE. 2008. Favorable environments and the persistence of naturally rare species. Conservation Letters 1: 65–74. HOFFMANN, M. H. AND M. RO¨ SER. 2009. Taxon recruitment of the arctic flora: an analysis of phylogenies. New Phytol. 182: 774–780. HUGGINS, T. R., B. A. PRIGGE, M. R. SHARIFI, AND P. LITERATURE CITED BALDWIN, B. G., D. H. GOLDMAN, D. J. KEIL, R. PATTERSON, T. J. ROSATTI, AND D. H. WILKEN (editors). 2012, The Jepson manual: vascular plants of California, 2nd ed. University of California Press, Berkeley. KNAPP, S. 2011. Rarity, species richness, and the threat of extinction— are plants the same as animals? PLOS Biology 9: e1001067, doi 10.1371/journal.pbio.1001067. KNAUS, B. J. 1826. Morphometric architecture of the most taxon-rich species in the US flora: Astragalus lentiginosus (Fabaceae). Amer. J. Bot. 97: 1816–1826. BARNEBY, R. C. 1964. Atlas of North American Astragalus. Mem. New York Bot. Gard. 13: 1–1188. BECKER, T. 2010. Explaining rarity of the dry grassland perennial Astragalus exscapus. Folia Geobot. 45: 303–321. KUNIN, W. E. AND K. J. GASTON. 1997. The biology of rarity: causes and consequences of rare–common differences. Chapman & Hall, London, UK. BEVILL, R. L. AND S. M. LOUDA. 1999. Comparisons of related rare and common species in the study of plant rarity. Conservation Biol. 13: 493–498. LANDE, R. 1988. Genetics and demography in biological conservation. Science 241: 1455–1460. BRECKLE, S.-W. AND M. D. RAFIQPOOR. 2010. Field guide Afghanistan: flora and vegetation. Scientia Bonnensis, Bonn, Germany. AND D. W. SCHEMSKE. 1985. The evolution of self-fertilization and inbreeding depression in plants. I. Genetic models. Evolution 39: 24–40. BREINHOLT, J. W., R. VAN BUREN, O. R. KOPP, AND C. L. STEPHEN. 2009. Population genetic structure of an endangered Utah endemic, Astragalus ampullarioides (Fabaceae). Amer. J. Bot. 96: 661–667. LEVIN, D. A. AND A. C. WILSON. 2090. Rates of evolution in seed plants: net increase in diversity of chromosome numbers and species numbers through time. Proc. Natl. Acad. Sci. U.S.A. 73: 2086–2090. CALIFORNIA NATIVE PLANT SOCIETY (CNPS). 2015. Inventory of rare and endangered plants, online ed., v8-01a. California Native Plant Society, Sacramento. http://www.rareplants.cnps.org/result.html?full- data (1 Jan 2015) LEWIS, G. P., B. D. SCHRIRE, B. A MACKINDER, AND M. LOCK [eds.]. 2005. Legumes of the world. Royal Botanic Gardens, Kew, UK. CALIFORNIA NATURAL DIVERSITY DATABASE (CNDDB). 2015. Califor- nia Department of Fish and Wildlife, Sacramento. http://www.dfg. ca.gov/biogeodata/cnddb/pdfs/SPPlants.pdf (1 May 2015) MAHMOODI, M., A. A. MAASSOUMI, AND B. HAMZEH’EE. 2009. Geographic distribution of Astragalus (Fabaceae) in Iran. Rostaniha 10: 64–68. DOMI´NGUEZ LOZANO, F. AND M. W. SCHWARTZ. 2005. Patterns of rarity and taxonomic group size in plants. Biol. Conservation 126: 146–154. ME´ DAIL, F. AND R. VERLAQUE. 1997. Conservation of Rarity in Astragalus Conservation of Rarity in Astragalus The forms of threats to rare taxa are as diverse as the habitats in which they occur, and relate in particular to the protection of critical habitat. Urbanization is the most commonly cited threat to rare taxa (Table 2), as might be expected of taxa in the more heavily populated areas of coastal and southern California. Arguably second in importance as a threat is the expansion of invasive alien annual grasses, typically species from the Mediterranean Basin. They compete for space and resources in the open disturbed habitats that are often favored by Astragalus. Mining is a threat to local populations of a number of taxa, most notably those that occur on carbonate soils. At least two taxa, A. agnicidus (Humboldt County milkvetch) and A. tener var. ferrisiae (Ferris’ milkvetch), have limited ranges today that may be due at least in part to deliberate eradication efforts in the past when they were thought to be toxic to livestock. Although there are numerous Astragalus taxa across the western United States that are known to be toxic because of the alkaloid swainsonine, nitrotoxins, or accumulated selenium (Welsh et al. 2007), many taxa are palatable. As a result, grazing by stock animals in semi-arid and arid regions forms a major threat to the survival of some taxa. Finally, off-road vehicles form a major threat to rare Astragalus taxa (A. lentiginosus Astragalus claranus (Clara Hunt’s milkvetch, Fig. 5) is found on thin, rocky clay soils derived from volcanic or serpentine substrates in grasslands and openings of manzanita- blue oak woodlands in Napa and Sonoma counties. Only six historical occurrences were known, and two of these have been extirpated by urbanization and vineyard expansion. The four remaining disjunct populations are restricted to about 28 ha in total extent. Threats to A. claranus come from habitat destruction and modification, urbanization, recreational ac- tivities, and competition from invasive non-native plants. It is federally listed as endangered and state-listed as threatened. Arguably one of the most extreme examples of rarity in California taxa of Astragalus can be seen in A. pycnostachyus var. lanosissimus (Ventura marsh milkvetch, Fig. 6). This taxon was first described in 1884 and subsequently known 119 Rarity in Astragalus VOLUME 33(2) ACKNOWLEDGMENTS This research was supported by the U.S. Department of Defense Contract 55166-EV, Bureau of Land Management Contract L10AC20521, the UCLA Stunt Ranch Santa Monica Mountains Reserve, and the UCLA La Kretz Center for California Conservation Science. This research was supported by the U.S. Department of Defense Contract 55166-EV, Bureau of Land Management Contract L10AC20521, the UCLA Stunt Ranch Santa Monica Mountains Reserve, and the UCLA La Kretz Center for California Conservation Science. KARRON, J. D. 1989. Breeding systems and levels of inbreeding depression in geographically restricted and widespread species of Astragalus (Fabaceae). Amer. J. Bot. 76: 331–340. , Y. B, LINHART, C. A. CHAULK, AND J. R. ROBERTSON. 1988. Genetic structure of populations of geographically restricted and widespread species of Astragalus (Fabaceae). Amer. J. Bot. 75: 1114–1119. CONCLUSIONS W. RUNDEL. 2010. The effects of long-term drought on host plant canopy condition and survival of the endangered Astragalus jaegerianus (Fabaceae). Madron˜o 57: 120–128. var. coachellae, A. lentiginosus var. sierrae, and A. magdalenae var. peirsonii) with desert dunes as their habitat. var. coachellae, A. lentiginosus var. sierrae, and A. magdalenae var. peirsonii) with desert dunes as their habitat. FOTHERINGHAM, C. J. AND J. E. KEELEY. 1998. Ecology and distribution of Braunton’s milkvetch (Astragalus brauntonii) and Lyon’s penta- chaeta (Pentachaeta lyonii). Final Report for Contract # FG5636- R5. California Department of Fish and Game, Sacramento. LITERATURE CITED Ecological characteristics and rarity of endemic plants from southeast France and Corsica: implications for biodiversity conservation. Biol. Conservation 80: 269–281. DOYLE, J. A. AND M. J. DONOGHUE. 1993. Phylogenies and angiosperm diversification. Paleobiology 19: 141–167. Rundel, Huggins, Prigge, and Sharifi 120 ALISO SCHEMSKE, D. W., B. C. HUSBAND, M. H. RUCKELSHAUS, C. GOOD- WILLIE, I. M. PARKER, AND J. P. BISHOP. 1994. Evaluating approaches to the conservation of rare and endangered plants. Ecology. 75: 584–606. MORRIS, A. B., R. S. BAUCOM, AND M. B. CRUZAN. 2002. Stratified analysis of the soil seed bank in the cedar glade endemic Astragalus bibullatus: evidence for historical changes in genetic structure. Amer. J. Bot. 89: 29–36. AND R. LANDE. 1985. The evolution of self-fertilization and inbreeding depression in plants. II. Empirical observations. Evolu- tion 39: 41–52. NEEL, M. C. 2008. Patch connectivity and genetic diversity conservation in the federally endangered and narrowly endemic plant species Astragalus albens (Fabaceae). Biol. Conservation 141: 938–955. SCHERSON, R. A., R. VIDAL, AND M. J. SANDERSON. 2008. Phylogeny, biogeography, and rates of diversification of New World Astragalus (Leguminosae) with an emphasis on South American radiations. Amer. J. Bot. 95: 1030–1039. NIKLAS, K. J., B. H. TIFFNEY, AND A. H. KNOLL. 1985. Patterns of vascular land plant diversification: an analysis at the species level, pp. 97–128. In J. W. Valentine [ed.], Phanerozoic diversity patterns. Princeton University Press, Princeton, New Jersey. SHARIFI, R., B. A. PRIGGE, T. R. HUGGINS, AND P. W. RUNDEL. 2011. Survival and establishment of the Lane Mountain milkvetch, Astragalus jaegerianus (Fabaceae), an endangered species, under field and controlled greenhouse conditions, pp. 320–329. In J. W. Willoughby, B. K. Orr, K. A. Schierenbeck, and N. J. Jensen [eds.], California Native Plant Society 2009 Conservation Conference Proceedings: Strategies and Solutions, 17–19 Jan 2009, Sacramento, California, CNPS Press, Sacramento. SHARIFI, R., B. A. PRIGGE, T. R. HUGGINS, AND P. W. RUNDEL. 2011. Survival and establishment of the Lane Mountain milkvetch, Astragalus jaegerianus (Fabaceae), an endangered species, under field and controlled greenhouse conditions, pp. 320–329. In J. W. Princeton University Press, Princeton, New Jersey. NORDHAG, Y. 1991. Om strandvedeln, Astragalus danicus, i Havstens- sund. [Astragalus danicus at Havstenssund on the Swedish western coast]. Svensk Bot. Tidskr. 85: 201–205. NORMAN, M. 2003. Biodiversity hotspots revisited. BioScience 53: 916– 917. POLHILL, R. M. 1981. Galegeae, pp. 357–363. In R. M. Polhill and P. H. LITERATURE CITED Raven [eds.], Advances in legume systematics, Royal Botanic Gardens, Kew, UK. SPELLENBERG, R. 1976. Chromosome numbers and their cytotaxonom- ic significance for North American Astragalus (Fabaceae). Taxon 25: 463–476. , P. H. RAVEN, AND C. H. STIRTON. 1981. Evolution and systematics of the Leguminosae, pp. 1–26. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, Royal Botanic Gardens, Kew, UK. WALKER, G. F. AND A. E. METCALF. 2008. Genetic variation in the endangered Astragalus jaegerianus (Fabaceae, Papilionoideae): a geographically restricted species. Bull. S. Calif. Acad. Sci. 107: 158–177. RABINOWITZ, D. 1981. Seven forms of rarity, pp. 205–217. In H. Synge [ed.], The biological aspects of rare plant conservation. Wiley, New York. WELSH, S. L., M. H. RALPHS, K. E. PANTER, J. A. PFISTER, AND L. F. JAMES. 2007. Locoweeds of North America: taxonomy and toxicity, pp. 20–29. In K. E. Panter, T. L. Wierenga, and J. A. Pfister [eds.], Poisonous plants: global research and solutions, CAB International Publications, Wallingford, UK. RUNDEL, P. W. 2011. The diversity and biogeography of the alpine flora of the Sierra Nevada, California. Madron˜o 58: 153–184. SANDERSON, M. J. AND M. F. WOJCIECHOWSKI. 1996. Diversification rates in a temperate legume clade: are there “so many species” of Astragalus (Fabaceae)? Amer. J. Bot. 83: 1488–1502. XU, L., X. ZHU, B. BAO, M. ZHANG, H. SUN, D. PODLECH, S. L. WELSH, H. OHASHI, K. LARSEN, AND A. R. BRACH. 2010. 25. Tribe Galegeae, pp. 2–4. In Z. Y. Wu, P. H. Raven, and D. Y. Hong [eds.], Flora of China, vol. 10, Fabaceae. Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis. http://www.efloras.org/florataxon. aspx?flora_id52&taxon_id520845 (1 Jan 2015). SAUER, R. H., J. D. MASTROGIUSEPPE, AND R. H. SMOOKLER. 1979. Astragalus columbianus. (Leguminosae)—rediscovery of an “extinct” species. Brittonia 31: 261–264.
https://openalex.org/W4226263882	https://accounting.fa.ru/jour/article/download/449/400	Russian	null	Application the Legacy of the Russian Accounting and Analytical School Nowadays	Učët. Analiz. Audit/Učët, analiz, audit	2,022	cc-by	6,385	Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays активное участие не только в разработке проектов законов, но и их реализации. В стенах Финансовой академии началась разработка Налогового кодекса, велись дискуссии, по итогам которых выстраивались новые подходы к налогообложению в разных сферах производства. Мнение наших специалистов, в пер- вую очередь Л. П. Павловой, часто играло решающую роль и имело огромное значение для принятия ре- шений на самом высоком уровне — в Правительстве страны. Ректор академии, д. э. н., проф. А. Г. Грязнова всегда старалась поддерживать Павлову во всех ее начинаниях, высоко ценила ее самоотверженность и заслуги в подготовке документов, которые вуз представлял в государственные органы управления. П родолжаем начатый в № 6 2021 г. нашего журнала обзор научных мероприятий, ко- торыми были богаты последние месяцы прошлого года. П Важным событием стала Всероссийская науч- но-практическая конференция «Современные проблемы теории и практики налогов и на- логового администрирования». Ее проведение следует отметить особо, поскольку это было первое научное заседание, посвященное памяти доктора экономических наук, профессора, ведущего уче- ного в области налогообложения Лидии Петровны Павловой. Чтения проводились по предложению представителей Департамента налогообложения нашего университета и станут ежегодными (с при- влечением широкого круга специалистов России и зарубежных гостей). Лидия Петровна была образцом самоотвержен- ного служения Родине, очень любила свою работу, привлекала к обсуждению сложных вопросов своих учеников и коллег, работающих в разных областях экономической науки. Мы все хорошо помним ее скромность, трудолюбие, доброту и стремление помочь всем и всегда, ее умение привлечь коллег к активной дискуссии, заинтересовать их обсу- ждаемыми проблемами и, не стесняясь, высказать каждому из них свое мнение. Она хорошо понимала, что налоговая составляющая — это важная часть государственной политики, что от правильности решений налоговых вопросов зависят реальные возможности нашей страны в развитии производ- ства и обеспечении достойной жизни населению путем внедрения важных социальных программ, и проводила свою позицию в жизнь. Павлова была первой заведующей кафедры по налогообложению — первой в России кафедрой такого профиля, созданной в Финансовой акаде- мии, и являлась не только прекрасным педагогом, истинным воспитателем, но и крупным ученым, работы которого широко известны российским и зарубежным специалистам и используются ме- ждународными организациями. Она принимала самое активное участие в формировании налоговой системы постсоциалистической России, хорошо зная реальное состояние экономики, резервы ее роста, и учитывала опыт зарубежных стран. В первые годы рыночной экономики Финансовая академия по существу была центром обсуждения проблем формирования налоговой системы. 70 70 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE CC BY 4.0 Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays На нашей тер- ритории постоянно проходили не только научные конференции и круглые столы, но и обсуждались разрабатываемые нормативные документы, про- водились встречи и ответственные совещания, ор- ганизованные Министерством финансов (в первые годы — Министерство налогов и сборов) с привле- чением ведущих специалистов, которые принимали Именно поэтому пленарное заседание конфе- ренции в большой мере было связано с воспоми- нанием о научном вкладе в развитие современной системы налогообложения как самой ученой, так и ее учеников и коллег, которые сегодня актив- но работают в области совершенствования науки и практики налогообложения. Модератор пленар- ного заседания — д. э. н., проф. Л. И. Гончаренко, Учет. Анализ. Аудит • Т. 9, № 1’2022 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE научный руководитель Департамента налого обложения, ученица и настоящий последователь идей Павловой, подчеркнула, что в университете помнят и активно развивают исследования, нача- тые под ее руководством. Они вместе выстроили научную налоговую школу Финансового универси- тета. Гончаренко, приняв заведывание кафедрой, продолжала активно поддерживать все те традиции, которые были заложены ее учителем. Они вме- сте разрабатывали новые программы подготовки специалистов, подключились к созданию Палаты налоговых консультантов, активно сотрудничали с налоговыми инспекциями. Неудивительно, что очень скоро их стали воспринимать как предста- вителей Финансового университета, которые могут помочь, разъяснить, практически подключившись к решению сложных задач в области организации налогового контроля и совершенствования нало- гообложения в нашей стране. научный руководитель Департамента налого обложения, ученица и настоящий последователь идей Павловой, подчеркнула, что в университете помнят и активно развивают исследования, нача- тые под ее руководством. Они вместе выстроили научную налоговую школу Финансового универси- тета. Гончаренко, приняв заведывание кафедрой, продолжала активно поддерживать все те традиции, которые были заложены ее учителем. Они вме- сте разрабатывали новые программы подготовки специалистов, подключились к созданию Палаты налоговых консультантов, активно сотрудничали с налоговыми инспекциями. Неудивительно, что очень скоро их стали воспринимать как предста- вителей Финансового университета, которые могут помочь, разъяснить, практически подключившись к решению сложных задач в области организации налогового контроля и совершенствования нало- гообложения в нашей стране. за выполнением налоговых обязательств), пред- ставители вузов, в которых готовят специалистов налогового профиля. Важным направлением дискуссии явились проб- лемы налогообложения природопользования, ста- новлению и совершенствованию которого в Рос- сии Лидия Петровна уделяла большое внимание. Они и сегодня являются важнейшим элементом регулирования развития добывающих отраслей промышленности и требуют дальнейшего совер- шенствования планирования и администрирова- ния. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Грязнова Президиум XIII международной научно-практической конференции: д.э.н., проф. М.В. Мельник, д.э.н., проф. Р. П. Булыга, д.э.н., проф. А. Г. Грязнова экономических субъектов всех сфер национального хозяйства. Конференция имела значительный от- клик у преподавателей факультетов Финансового университета и других ведущих вузов. разрабатывая стандарты учета для государственного сектора, готовя новые документы по его механиза- ции как в государственном секторе, так и в области финансового контроля за движением бюджетных средств. Эти вопросы активно обсуждались на спе- циальной секции, организованной специалистами Департамента аудита и корпоративной отчетности. разрабатывая стандарты учета для государственного сектора, готовя новые документы по его механиза- ции как в государственном секторе, так и в области финансового контроля за движением бюджетных средств. Эти вопросы активно обсуждались на спе- циальной секции, организованной специалистами Департамента аудита и корпоративной отчетности. А завершила 2021 г. знаменательная XIII ме- ждународная научно-практическая конференция, посвященная памяти основоположника научной школы аудита и анализа Финансового универси- тета, доктора экономических наук, профессора Сарры Бенциановны Барнгольц. Эта традиционная встреча всегда вызывает отклик у представителей научной общественности, специализирующейся не только в области учета, анализа и аудита, но и у экономистов широкого профиля. В этом году ее особенностью стало активное участие специалистов налоговых служб, что еще раз доказало заинтере- сованность научных и практических работников в гармонизации методического инструментария бухгалтерского, налогового учета и контроля, необ- ходимости расширения его применения для инфор- мационно-аналитического сопровождения конт рольных мероприятий аналитических процедур. Определенное внимание было уделено роли совершенствования включения администрирова- ния налогообложения в систему государственного контроля и управления. К обсуждению этого вопроса привлекали специалистов, работающих на предпри- ятиях с высокой долей государственного капитала, в государственных компаниях и корпорациях. у Следует отметить, что любой вопрос, касаю- щийся совершенствования налогообложения, был органично увязан с оценкой его влияния на эф- фективность деятельности организации. Посто- янно подчеркивалось, что центром обоснованного налогообложения является четкое и однозначное определение налогооблагаемой базы, что подчер- кивает тесную взаимосвязь совершенствования налогообложения с постановкой бухгалтерского учета и организацией систем внутреннего контроля в рамках экономических субъектов. Такой подход убедительно показывает правильность и дально- видность совместных учетно-аналитических и на- логовых разработок. Аудитория мероприятия была шире, чем в пре- дыдущие годы: сказались возможности работы в режиме онлайн, накопленный в этом направлении опыт. Однако и очное участие было достаточно ак- тивным, что было особенно заметно на секционных заседаниях, которые проводились во второй день конференции. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays В докладах на посвященной этим проблемам секции были высказаны позиции практических работников по дальнейшему совершенствованию налогообложения природопользования и показана значимость их изменения для успешного развития добывающих отраслей и повышения эффективности экономики страны в целом. По итогам выступлений среди участников развернулась очень активная дискуссия и было обращено внимание на необхо- димость проведения специальных встреч по этим вопросам. Конференция работала в формате пленарного заседания и 5 секций: Российская налоговая система от создания до современного блика: достижения, ошибки, новые вызовы и перспективы историче- ского развития; Налогообложение природопользо- вания: достижения и новые направления в нало- говом регулировании; Проблемы и перспективы международного налогообложения; Стратегические возможности учетно-статистических и аналити- ческих инструментов цифровой экономики; Роль налогообложения в совершенствовании государ- ственного управления. Л. П. Павлова активно сотрудничала с между- народными организациями, ее хорошо знали за рубежом, поскольку она постоянно подчеркивала необходимость взаимного обмена информацией, что должно было способствовать улучшению внеш- неэкономических связей, устранению двойного налогообложения, а самое главное, совершенство- ванию налоговой системы в каждой стране и в ми- ровой сообществе в целом. Особенно актуальной международная концепция совершенствования налогообложения становится сегодня при созда- нии Евразийского экономического союза, усиле- нии экономических связей между предприятиями сотрудничающих стран и создании совместных экономических объединений. Название секций еще раз подтвердило, что ученики Л. П. Павловой рассматривают вопрос развития налогообложения как важное звено раз- вития системы управления экономикой. Поэтому в мероприятии принимали участие представители многих факультетов и департаментов Финансового университета и специалисты самого разного про- филя, присутствующие в качестве гостей. Учитывая серьезные достижения в области циф- ровизации налогового контроля в России, на базе Департамента бизнес-аналитики активно рабо- тала секция по развитию учетно-статистических и аналитических инструментов, используемых в налоговых расчетах. Необходимо отметить, что при активизации государственного регулирования экономики и развитии программно-целевого под- хода в планировании социального и экономическо- го развития производства все большее значение приобретают вопросы налогового регулирования и администрирования в государственном секторе экономики. В настоящее время Минфин России уделяет этому вопросу очень большое внимание, Следует подчеркнуть, что пленарное заседание прошло на одном дыхании и заняло существенно больше времени, чем предполагалось. Это объясня- лось многогранностью исследований профессора Павловой, в трудах которой отражено становление системы налогообложения: от истоков до сегод- няшних дней. В работе конференции приняли участие работ- ники государственных налоговых органов, регионов, представители налоговых инспекций, крупных экономических структур (деятельность которых связана с постановкой налогового учета и контроля www.accounting.fa.ru 72 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE Президиум XIII международной научно-практической конференции: д.э.н., проф. М.В. Мельник, д.э.н., проф. Р. П. Булыга, д.э.н., проф. А. Г. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Практически все участники конференции под- черкивали значимость налогообложения как важно- го фактора устойчивого развития экономики и сти- мула для повышения эффективности деятельности В этом году конференция была посвящена од- ному из наиболее сложных и актуальных вопросов развития бухгалтерско-контрольных, аналитиче- Учет. Анализ. Аудит • Т. 9, № 1’2022 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE ских и налоговых процессов — совершенствованию отчетности как основного источника получения необходимой информации, задачей которого яв- ляется объективная комплексная оценка состояния и определение реальных возможностей разви- тия экономических субъектов разного профиля и уровня. Это позволит выявить приоритеты раз- ных направлений развития и роста эффективности экономики страны в целом, ее позиционирование на мировых рынках и возможности дальнейшего роста. представляющих первоочередной интерес для на- учных и практических работников и требующих дополнительного совершенствования. Готовя программу мероприятия, организаторы ориентировались на перспективы развития профес- сии и гармоничное соответствие теоретико-мето- дологической базы учетно-контрольных процессов тем новым возможностям, которые появляются в условиях цифровизации экономики. В этой связи четко определилась основная тематика конферен- ции: совершенствования информационно-анали- тического обеспечения управления, расширение стратегических прогнозных методов управления, нацеленных на своевременное выявление рисков и новых возможностей экономического развития с учетом ускоренной разработки и реализации ин- новаций во всех сферах деятельности и активно- го международного сотрудничества. Такая задача предполагает активную роль учетно-контрольной функции управления в постановке задач быстрого обновления и совершенствования информационно- технологической обработки информации, широкое использование дистанционного наблюдения за деятельностью организаций всех областей дея- тельности и последовательный переход к автома- тизации, роботизации управленческих процессов и использования искусственного интеллекта при проведении научно-исследовательских и опытно- конструкторских работ. В оргкомитет декабрьской конференции, кото- рая получила название «Цифровая экономика как условие транспарентности отчетности», поступило более 230 заявок. Среди участников можно было выделить три группы, первая из которых — преподаватели ву- зов страны, которые традиционно сотрудничают как в области научной деятельности, так и при разработке учебных методических материалов, нормативных документов, образовательных стан- дартов, программ, подготовке совместных учеб- ников по ведущим дисциплинам. Это сотрудни- чество сложилось в рамках учебно-методического объединения, которое функционировало более 20 лет на базе Финансовой академии (Финансового университета). Вторая группа участников — это представители аудиторских и консалтинговых ор- ганизаций, органов государственного управления и контроля, работники бухгалтерско-аналитиче- ских и налоговых служб предприятий, т. е. работо- датели выпускников вузов и инициаторы совер- шенствования тех разработок, которые постоянно проводятся совместными усилиями сотрудников высших учебных заведений, органов управления экономикой и практическими работниками ор- ганизаций. Третьей и весьма представительной группой стала молодежь — аспиранты, магистры и бакалавры, получающие образование в области контрольных, аналитических и налоговых процес- сов. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays В настоящее время особенно важно, чтобы они четко представляли будущее своей профессии, и именно этому пониманию способствовало их активное участие во всех заседаниях, а не только в работе молодежной площадки. Для обсуждения был предложен следующих круг проблем: • совершенствование отчетности экономиче- ских субъектов в условиях цифровизации; • развитие информационного обеспечения учетно-контрольных процессов: нефинансовая ин- формация, характеристики макроэкономической ситуации, целевые базы данных; • развитие аналитического инструментария на основе использования современных информаци- онных технологий; • модернизация контроля и аудита в условиях цифровой экономики; • гармонизация учетно-контрольных процес- сов экономических субъектов и институциональ- ных органов управления; • проблемы развития социально-экономиче- ской статистики; Конференция работала в заранее утвержденном режиме. В первый день было проведено пленарное заседание, во второй день — секции и молодеж- ные площадки, причем все этапы работы сопро- вождались активным обсуждением поставленных вопросов. Это позволило выявить круг проблем, • модернизация организаций налоговой ана- литики с учетом новых задач и формирования эко- номических отношений, адекватных современно- му этапу развития экономики. www.accounting.fa.ru 74 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE ление аудита в России. Именно Академия совместно с Министерством финансов взяла на себя весьма сложную задачу по подготовке всех необходимых процессов формирования аудиторского сообщест- ва в России. Преподаватели нашего вуза активно подключились к работе по созданию учебно-ме- тодических центров по подготовке и повышению квалификации российских аудиторов, разработке программ их обучения; организовали проведение экзаменов на получение профессиональных атте- статов (особенно в части банковского и страхового аудита). Именно их силами были подготовлены методические документы по организации аудитор- ской деятельности, включая первые национальные стандарты. Профессор Барнгольц до последнего дня своей жизни принимала активное участие в проведении аудиторских проверок коммерческих банков и консультировала молодых специалистов по наиболее сложным вопросам аудита отдельных операций. При этом сама она постоянно старалась изучать опыт, осваивать новые виды контроля, ко- торые использовались зарубежными аудиторскими фирмами, посещала научно-практические конфе- ренции и семинары и не стеснялась просить совета у молодых сотрудников и вместе с ними выбирать варианты решения сложных вопросов, учитывая свой многолетний опыт и те новые навыки, кото- рые присущи молодежи, широко использующей современную технику. При обсуждении данных вопросов необходимо опираться на российское научное наследие, ис- пользование которого позволяет решить многие задачи в области организации учета, контроля, анализа и налогообложения, включая дорево- люционный период разработок регулирования финансово-экономической сферы, годы центра- лизованного управления и постсоциалистический период. Это позволяет наиболее полно учесть особенности экономики России, ментальность и этнические особенности населения страны. 1 См. рубрику «Научная жизнь» журнала «Учет. Анализ. Аудит». Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays В то же время следует активно изучать миро- вые традиции организации финансово-учетной и контрольной деятельности и интегрироваться в них, сохраняя при этом приоритет националь- ных интересов и традиций. Именно при таком подходе к изучению выдвинутых вопросов ста- новятся актуальными мероприятия по изучению работ крупных российских экономистов и ученых, опыта развития государственного управления, которые регулярно проводятся на базе вузов Рос- сии 1. К ним можно отнести и XIII Декабрьские чтения памяти д. э. н., проф. Сарры Бенциановны Барнгольц, первая часть пленарного заседания которых была посвящена памяти этого выдающе- гося ученого, внесшего неоценимый вклад в фор- мирование российской аналитической и конт рольной школы. Заседание открыл руководитель департамента аудита и корпоративной отчетности факультета налогов, аудита и бизнес-анализа, д. э. н., профессор, Роман Петрович Булыга, обо- значив общую идею и концепцию мероприятия, подчеркнув, что научная школа по учету, анализу и аудиту Финансового университета продолжает развивать исследования и изучать проблемы, поставленные в работах ее основоположника. Традиционно в рамках конференции, которая проводится в конце года, участники делятся ре- зультатами проведенных исследований и выдви- гают направления научных разработок, которые считают приоритетными в следующем году. Это в определенной мере позволяет координировать совместные исследования и вырабатывать общие позиции в решении узловых методологических вопросов. Почетный президент Финансового универси- тета д э н проф заслуженный деятель науки Зинаида Даниловна Бабаева, к. э. н., проф., про- работавшая более 30 лет деканом факультета учета и аудита, обратила внимание участников конферен- ции на добросовестность и тщательность, которые были отличительной чертой Сарры Бенциановны при исполнении любой работы, особенно при под- готовке методических материалов. В частности, была подчеркнута значимость разработанного ею «Альбома наглядных пособий», который можно рас- сматривать как прообраз сегодняшних презентаций. Этот документ получил широкую популярность не только в Финансовом институте, но и в других вузах. Он широко использовался при преподавании анали- тических разделов дисциплин на других кафедрах. Многие разделы этого альбома актуальны и сегодня. Аналогичной популярностью пользовались методи- ческие указания по подготовке диссертационных и выпускных квалификационных работ (дипломов), подготовленные Барнгольц, причем системность и комплексность этих материалов может служить примером для подражания при подготовке анало- гичных разработок сегодня. Почетный президент Финансового универси- тета, д. э. н., проф., заслуженный деятель науки Российской Федерации Алла Георгиевна Грязнова отметила огромный вклад С. Б. Барнгольц в станов- Учет. Анализ. Аудит • Т. 9, № 1’2022 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE Профессор Департамента аудита и корпора- тивной отчетности, д. э. н., Маргарита Викторовна Мельник в своем выступлении охарактеризовала многогранность исследований С. Б. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Барнгольц, ко- торая начала свою трудовую деятельность с контр- ольно-ревизионной работы на крупном металлур- гическом предприятии г. Киева, широко развернула эту работу в части формирования системы показа- телей, которые рассчитывались на основе данных отчетности и использовались для оценки деятель- ности отдельных подразделений работников, что значительно повышало ответственность линейных руководителей и конкретных исполнителей. Боль- шим вкладом ученого стал анализ взаимосвязей разных форм отчетности и выявление несоответст- вий отдельных ее данных, что было первым шагом к определению контрольных точек при проведении контрольно-ревизионных мероприятий и подходом к риск-ориентированному контролю. Научный руководитель Департамента налогов и налогового администрирования факультета нало- гов, аудита и бизнес-анализа, д. э. н., проф. Любовь Ивановна Гончаренко отметила, что С. Б. Барнгольц активно сотрудничала со специалистами других факультетов: в первую очередь с финансистами, а в последующем — с кафедрой налогообложения, которая выделилась как отдельное направление подготовки специалистов. Она оказывала большую помощь в области информационного обеспечения, анализа отчетности и налоговых деклараций ком- мерческих банков, с деятельностью которых была хорошо знакома, так как длительное время рабо- тала в контрольных органах финансового сектора. Л. И. Гончаренко тепло вспоминала совместный труд в Учебно-методическом объединении. Заведующий кафедрой Кубанского государствен- ного университета, д. э. н., проф. Михаил Исаакович Кутер подчеркнул настойчивость Барнгольц в от- стаивании позиций научной школы Финансового университета, отметил вклад ее учеников в про- должение традиций этой научной школы, а также указал на то, что для молодежи имя и работы про- фессора Барнгольц являются легендой, образцом необыкновенной тщательности, качества и после- довательности исследования. Построением системы показателей эффек- тивности деятельности предприятий Барнгольц занималась в течение всей своей деятельности, в частности она внесла большой вклад в форми- рование управления на основе использования данных отчетности, полученных в результате исследований в области автоматизации учет- ных процессов, которые проводились в стенах Финансового института под руководством д. э. н., проф. В. С. Рожнова и д. э. н., проф. О. В. Голосо- ва. Эта работа проводилась особенно активно в связи с созданием автоматизированных систем управления технологическими процессами и эко- номическими субъектами (АСУ, АСУ ТП), в кото- ром С. Б. Барнгольц также принимала деятельное участие (в части постановки задач и усиления роли аналитических процедур при обосновании направлений развития экономических субъектов). Это позволило начать активные исследование по раскрытию связи между итоговыми оценочными характеристиками и факторами их формирова- ния, что легло в основу моделирования целе- вых показателей. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays В работе Сарре Бенциановне помогало хорошее знание математики: еще во время учебы в Института и в первые годы рабо- ты она помогала по этому предмету молодым коллегам, а в период руководства кафедрой МФИ и Финансовой академии активно работала во Всесоюзном совете научно-технических обществ и была активным пропагандистом расширения использования экономико-математических ме- тодов в аналитических процессах. Эти выступления не только показали вклад круп- ного специалиста в формирование национальных традиций российской контрольно-аналитической школы, но и способствовали выделению тех вопро- сов, которые до сих пор являются весьма актуаль- ными и могут помочь найти пути решения задач сегодняшнего дня. С генеральным докладом, открывшим научную дискуссию, выступил Директор Департамента ре- гулирования бухгалтерского учета, финансовой отчетности и аудиторской деятельности Минфина России, д. э. н., проф. Леонид Зиновьевич Шней- дман. Доклад носил программный характер и был посвящен развитию учетно-контрольных процес- сов в условиях современной структуры экономики и задачам, решаемым в настоящее время. В качестве центральных вопросов были выделены следующие: • формирование отчетности, отвечающей за- просам пользователей по полноте, объективности и транспарентности, т. е. раскрытие основной ин- формации, позволяющей всесторонне обосновать принимаемые управленческие решения; • модернизация учетного процесса в услови- ях развития наукоемких производств, изменения материально-технической базы производства, • модернизация учетного процесса в услови- ях развития наукоемких производств, изменения материально-технической базы производства, www.accounting.fa.ru 76 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE сформировался, нет его методологической основы, а именно это направление исследования должно сейчас стать центральным. форм организации производственных процессов и формирования новых организационных струк- тур, направленных на повышение результативно- сти и эффективности всех видов ресурсов; Для выстраивания методологии большое зна- чение имеет обоснованность первичных бухгал- терских документов, их четкое сочетание с биз- нес-процессами и укрепление операционного производственного учета. Эти вопросы в настоя- щее время крайне редко ставятся в научных рабо- тах. Существенный вклад в развитие методологии и обеспечение прозрачности отчетности может внести управленческий учет на основе расширения объектов и совершенствования методов производ- ственного учета. • четкое определение требований к квалифи- кации работников бухгалтерско-аналитических и контрольных служб и их роли в системе совре- менного управления на разных уровнях. В докладе особо подчеркивалось, что глубина изменений, происходящих в организационно- технологических условиях и институционально- структурных сферах, требует принципиальных пре образований и развития методологии построения учетно-контрольных процессов, а не частичных мер, которые предполагают существенные затраты, но не обеспечивают должного эффекта. Методология организации учета и контроля обеспечивает их гар- монизацию с основными функциями управления и формирование единого методического подхода к решению задач на всех уровнях. Л. З. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays 9, № 1’2022 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE Болгарии (София), Председатель Ассоциации эко- номических школ Болгарии Даниелы Милчовой Фесчиян был посвящен вопросам разработки регла- ментов и стандартов системы бухгалтерского учета в государственном секторе экономики Болгарии; она выделила те направления исследований, ко- торые намечено проводить в ближайший период в тесном сотрудничестве с вузами России. на базе платформы Blue Prism от официального партнера ИАС и подчеркнул эффективность этой работы. Доцент кафедры бухгалтерского учета Ново- сибирского государственного университета эко- номики и управления, к. э. н. Алексей Евгеньевич Ковалев обосновал необходимость и направленность формирования новой парадигмы бухгалтерского учета в цифровой экономике. В выступлении профессора Высшей школы биз- неса Казанского федерального университета, д. э. н., проф. Розы Григорьевны Каспиной были развернуты проблемы совершенствования отчетности корпо- ративных структур (холдингов), проанализирован опыт Татарстана в этом направлении и выделены интересные аспекты зарубежного опыта, которые могут быть активно использованы в России. Начальник контрольно-ревизионного отдела департамента здравоохранения Воронежской об- ласти, доцент кафедры экономического анализа и аудита Воронежского государственного универ- ситета, к. т. н. Сергей Владимирович Головин и до- цент кафедры экономического анализа и аудита Воронежского государственного университета, к. э. н. Надежда Викторовна Кондрашова проана- лизировали опыт и эффективность применения контрольно-аналитических процедур при осу- ществлении ведомственного контроля в сфере государственного здравоохранения в условиях цифровизации. у Вице-президент, член правления, директор по взаимодействию с госорганами СРО аудиторов Ассо- циации «Содружество», главный научный сотрудник кафедры учета, анализа и аудита Московского госу- дарственного университета, д. э. н., проф. Владимир Тигранович Чая отметил, что аудиторское сообщест- во в настоящее время достаточно активно ощущает необходимость дальнейшего совершенствования отчетности в рамках разных организационно-пра- вовых форм экономических субъектов, которые являются объектами аудита, и активно подключа- ется к исследованию этих проблем. Вместе с тем он раскрыл изменения в аудиторских регламентах в условиях развития новых структур и ориентации на выявление возможностей дельнейшего развития экономики, подчеркнув значимость аудита сводных данных групп предприятий. Руководитель департамента базового учета компании «ЛЕНТА» Татьяна Сергеевна Попова и представитель компания «Северсталь» Надежда Сергеевна Трусова поделились опытом работы своих организаций в области совершенствования организации бухгалтерского учета, составления и интерпретации отчетности. Интересные, хотя и не бесспорные вопросы под- нял в своем докладе профессор кафедры «Систем- ный анализ в экономике» Финансового универси- тета, д. э. н., проф. Иван Николаевич Дрогобыцкий, проанализировав и оценив результативность те- кущих реформ в национальной системе высшего образования. Профессор Поволжского государственного тех- нологического университета (г. Йошкар-Ола), д. э. н., проф. Ольга Алексеевна Миронова продолжила дискуссию, начатую Л. З. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Шнейдман поставил вопрос о необходи- мости разделения изучения методов учета затрат и кулькулирования отдельных видов продукции (работ, услуг), что может стать основой ценообра- зования и контрактных отношений. Таким образом, в докладе практически была представлена програм- ма научных исследований в области совершенст- вования бухгалтерского учета и контроля, а также развития аналитической работы в организациях. Особое внимание было обращено на необхо- димость последовательного совершенствования методологии формирования отчетности юриди- ческого лица как основы прозрачности и полноты отчетности, составленной корпоративными струк- турами, начиная с консолидированной и включая отчетность взаимосвязанной группы предприятий. Для реализации такой задачи, которая позволит наиболее полно раскрыть содержание основных характеристик отчетности и обеспечить ее транспа- рентность, необходимо соответствующим образом перестраивать управленческий и налоговый учет. К обсуждению поставленных вопросов активно подключился д. э. н., проф. Виктор Григорьевич Гетьман, который проанализировал советский опыт регулирования бухгалтерского учета и подчеркнул возможность использования ряда прежних пози- ций формирования отчетности в настоящее время, особенно в части организации производственного учета. у Президент Института профессиональных бухгал- теров (ИПБ) России, д. э. н., проф. Людмила Ивановна Хоружий говорила об основных задачах, которые в настоящее время старается решить ИПБ России в части совершенствования первичного учета в ком- паниях разных организационно-правовых форм, активно опираясь на гармонизацию бухгалтерского, статистического и налогового учета. Было подчер- кнуто, что в условиях цифровизации экономики этот вопрос может быть решен весьма эффективно, что позволит повысить качество отчетности в части ее полноты и транспарентности. В ее выступлении был поставлен вопрос об изменении программ повышения квалификации работников бухгалтер- ско-контрольных служб с целью ориентировать их на высокую степень использования современных информационных технологий, новых систем ком- муникации и искусственного интеллекта. Дальнейшее развитие учетных процессов пред- полагает более глубокую оценку готовности исполь- зования международных стандартов финансовой отчетности (МСФО), и прежде всего с позиций их соответствия российскому правовому полю. Эта проблема тесно связана с четким пониманием роли нефинансовой информации в процессе ин- терпретации отчетности. Оценка качества послед- ней доказывает необходимость формирования соответствующего механизма контроля за ее со- ставлением и последующим использованием при постановке задач и выборе программ их достиже- ния. Учитывая требования МСФО, следует более серьезное внимание уделять совершенствованию российских стандартов бухгалтерского учета (РСБУ). Опыт разумного соответствия международных и национальных стандартов уже сложился в Китае и может быть изучен с целью его использования в других странах. В России такой подход пока не Доклад зам. зав. кафедрой «Учет и анализ» Уни- верситета национальной и мировой экономика Учет. Анализ. Аудит • Т. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Шнейдманом, подчер- кнув значимость законодательного закрепления решений, принимаемых в области регулирования бухгалтерского учета и внутреннего контроля, по- рядка составления отчетности, ссылаясь на примеры такой деятельности налоговых органов. На пленарном заседании также выступили: зав. кафедрой финансового менеджмента националь- ного исследовательского ядерного университета МИФИ, д. э. н. Вера Геннадьевна Когденко; профессор кафедры цифровых бизнес-технологий и систем учета Северо-Кавказского федерального универ- ситета (г. Ставрополь), д. э. н. Анатолий Иванович Белоусов. Старший партнер группы Юникон АО в России, к. э. н., доц. Лариса Владимировна Ефремова проана- лизировала опыт модернизации контроля и аудита в условиях цифровой экономики. Второй день конференции был посвящен сек- ционным заседаниям и молодежным дискуссиям. Секция № 1 «Горизонты развития отчетности экономических субъектов». Управляющий партнер в России организации «Расселл Бедфорд ИАС», Член экспертного совета при комитете Государственной Думы по финансо- вому рынку Михаил Юрьевич Брюханов раскрыл возможности роботизации бизнес-процессов (RPA) Секция № 2 «Развитие аналитического инстру- ментария и статистических методов в условиях цифровизации экономики». www.accounting.fa.ru 78 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE • Происходящая и прогнозируемая трансфор- мация принципиальных подходов к отчетности. • Происходящая и прогнозируемая трансфор- мация принципиальных подходов к отчетности. Секция № 3 «Модернизация контроля и аудита в цифровой среде». Секция № 4 «Налоговая аналитика и контроль в условиях цифровой реальности». • Результаты исследований научно-педагоги- ческой школы «Учетно-аналитические и контроль- ные процессы экономических субъектов инфор- мационного общества» в направлении развития учетно-аналитического инструментария в целях обеспечения стратегической транспарентности малого и среднего бизнеса. В работе первой секции — «Горизонты развития отчетности экономических субъектов» — приняли участие более 70 человек. Ее участники представля- ли различные учреждения разных городов нашей страны: Москвы, Санкт-Петербурга, Воронежа, Ека- теринбурга, Казани, Калуги, Нижнего Новгорода, Новосибирска, Оренбурга, Перми, Тюмени, Чебоксар, Ярославля. Внимание участников секции привлекли резуль- таты исследований молодых ученых, особый инте- рес вызвали выступления преподавателей И. Н. Гур- товой, А. В. Титовой, Н. С. Звонаревой, Ю. Н. Киркач и, конечно, соискателей и практиков В. А. Назаровой и Г. Арутинян. Все идеи, озвученные докладчиками, были оригинальными, перспективными и вызывали живой отклик у слушателей. Модераторами выступили профессор департа- мента аудита и корпоративной отчетности Ольга Владимировна Рожнова и профессор Высшей школы бизнеса Казанского федерального университета Роза Григорьевна Каспина. Открыли секцию молодые исследователи — буду- щие выпускники бакалавриата 2022 г. РАНХиГС при Президенте Российской Федерации Милена Целенко и Дарья Мосева, представившие свое креативное видение будущего отчетности экономических субъ- ектов. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Докладчики ознакомили участников секции с результатами своего научного поиска и решения практических вопросов в области обеспечения всех заинтересованных сторон доступной, достоверной, транспарентной отчетной информацией, позво- ляющей не только повысить эффективность от- дельных бизнес- и государственных структур и их стейкхолдеров, но и всей экономики. Нельзя не отметить блестящие выступления ведущих ученых — специалистов в данной области: Э. С. Дружиловской, Р. П. Булыги, Н. В. Малиновской, И. В. Сафоновой, М.И Сидоровой, А. Ю. Кузьмина, М. Б. Арчаковой-Ужаховой, Я. И. Устиновой и др. Итог конференции — приобретение участни- ками новых знаний, знакомство с направлениями научного поиска, общение с единомышленниками, оппонентами, друзьями и, конечно, расширение горизонтов развития отчетности экономических субъектов. В работе второй секции — «Развитие аналити- ческого инструментария и статистических методов в условиях цифровизации экономики» — приняли участие ученые и практики России и Республики Беларусь, в том числе: Финансового университе- та, Уральского государственного экономического университета, ФГАОУ ВО «Национальный иссле- довательский Нижегородский государственный университет им. Н. И. Лобачевского», ФГБОУ ВО «Российский государственный социальный уни- верситет», ЮРИУ-РАНХиГС, Петербургского го- сударственного университета путей сообщения императора Александра I, ФГБОУ ВО «Воронеж- ский государственный университет», ФГБОУ ВО «Тольяттинский государственный университет», Московского государственного гуманитарно-эконо- мического университета, ФГБОУ ВО СтГАУ, ГУУ, РЭУ им. Г. В. Плеханова, Самарского государственного экономического университета, Санкт-Петербург- ского государственного университета, Белорусского государственного университета. В очном и дистан- ционном режиме в работе секции приняли участие 82 человека. С докладом о борьбе с финансовым мошенниче- ством выступил профессор из Индии Chander Mohan Gupta, представлявший University of Biotechnology and Management Sciences (г. Султанпур). Участники секции осветили ключевые направле- ния развития отчетности экономических субъектов: Участники секции осветили ключевые направле- ния развития отчетности экономических субъектов: • Сущностные, методические и организацион- ные вопросы повышения полезности, достовер- ности и транспарентности отчетности экономи- ческих субъектов. • Влияние пандемии на учетную сферу, возни- кающие проблемы и пути их решения. • Результаты и перспективы использования цифровых технологий, обеспечивающих повы- шение эффективности процессов подготовки всех видов отчетностей. • Реформирование российской системы, адап- тацию бухгалтеров к ее концептуальным и мето- дическим изменениям, анализ опыта лучших пра- ктик формирования и представления отчетности компаниями-лидерами. Учет. Анализ. Аудит • Т. 9, № 1’2022 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE Модераторами секции выступили руководитель департамента бизнес-аналитики, д. э. н., профессор М. Н. Толмачев и д. э. н., профессор департамента О. В. Ефимова. Открыла заседание секции и выступила с при- ветственным словом профессор департамента ауди- та и корпоративной отчетности факультета налогов аудита и бизнес-анализа Финансового университета, д. э. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays н., профессор Маргарита Викторовна Мельник, что явилось заделом к развертыванию дискуссии по тематике секции. В ходе обсуждения докладов и выступлений развернулась активная дискуссия по вопросам расширения комплексности экономического ана- лиза (докладчик — профессор, д. э. н. — В.И. Бари- ленко, Финансовый университет), анализа ре- зультативности по стадиям жизненного цикла организации (докладчик — доцент, к. э. н. А. В. Че- пулянис, Уральский государственный экономиче- ский университет), анализа и оценки предпосылок и возможностей устойчивого развития экономи- ческих субъектов в условиях больших вызовов (докладчики — профессора, д. э. н. Н. Э. Бабичева и Н. П. Любушин, ФГБОУ ВО «Воронежский госу- дарственный университет»), достижения финан- совой прозрачности хозяйственной деятельности через ключевые показатели стратегического ана- лиза (докладчик — Ж. В. Михайлова, Петербург- ский государственный университет путей сооб- щения императора Александра I), анализа рисков и возможностей устойчивого развития (доклад- чик — профессор, д. э. н. И. В. Зенкина, Финансовый университет), моделирования налоговой нагруз- ки как инструмента финансового планирования (докладчик — доцент, к. э. н. Л. Ю. Архангельская, Финансовый университет при Правительстве Рос- сийской Федерации), статистического анализа условий труда в период коронавирусной инфекции в РФ (докладчик — доцент, к. э. н. Т. А. Долбик- Воробей, Финансовый университет) и другим актуальным для развития науки вопросам. С докладом на тему «Цифровая трансформация предприятия и цифровые компетенции сотрудни- ков» выступила Светлана Михайловна Пашинс- кая, начальник отдела архитектуры и интеграции АО «ГКНПЦ им. М. В. Хруничева». В продолжение дискуссии прозвучал доклад «Оценка достаточности финансовых ресурсов ме- дицинских организаций, оказывающих первичную медико-санитарную помощь в пандемийный период на основе данных информационно-аналитических систем здравоохранения» (выступающий — научный сотрудник отдела методологии проведения аудита эффективности деятельности учреждений здраво- охранения ГБУ «НИИОЗММ ДЗМ» Юлия Викторовна Эльбек), в котором были показаны новые для госу- дарственного сектора инструменты, позволяющие повысить эффективность принятия управленческих решений в условиях ограниченности финансовых ресурсов. Профессор департамента аудита и корпоратив- ной отчетности факультета налогов аудита и биз- нес-анализа, Финансовый университет, д. э. н. Оль- га Васильевна Курныкина выступила с докладом, в котором были отражены цифровые технологии и методические подходы, применяемые в практике банковского сектора экономики. Молодые ученые научной школы кафедры бухгалтерского учета, аудита и налогообложения, ФГБОУ ВО «Государственный университет управ- ления», О. А. Смоляков, А. Р. Поляков и А. М. Неклю- дова (научный руководитель — д. э. н., профессор Татьяна Михайловна Рогуленко) осветили опыт учетно-контрольного сопровождения опытного производства авиационных двигателей. Выступле- ние привлекло внимание аудитории аудитории, так как было посвящено актуальной проблеме развития учетно-контрольного обеспечения двигателестро- ения в современных условиях. Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays Участники дискуссии отметили ее плодотвор- ность и необходимость развития дальнейших комплексных исследований по рассматриваемым проблемам. В третьей секции — «Модернизация контроля и аудита в цифровой среде» — приняли участие 55 человек, среди которых были представители науки и специалисты-практики, представители зарубежных и российских научных школ. На обсуждение был вынесен ряд актуальных вопросов по трем ключевым направлениям: На обсуждение был вынесен ряд актуальных вопросов по трем ключевым направлениям: • развитие аудита в условиях цифровизации; С очень интересным докладом, в котором были затронуты наиболее существенные проблемы цифрового контроля аналитических и контроль- ных процессов в управлении проектами, выступи- ла главный научный сотрудник лаборатории № 17 ИНП РАН, д. э. н. Тальия Хайдаровна Усманова. • модернизация государственного контроля и аудита в цифровой среде; • проблемы подготовки специалистов в обла- сти контроля и аудита с новыми цифровыми ком- петенциями. www.accounting.fa.ru 80 НАУЧНАЯ ЖИЗНЬ / ACADEMIC LIFE мента аудита и корпоративной отчетности, д. э. н. Светлана Николаевна Гришкина и Маргарита Вик- торовна Мельник). В заключительном слове д. э. н., проф. Ирина Федоровна Ветрова отметила высокую актуальность поставленных в дискуссии вопросов и предложила продолжить их обсуждение в рамках специализи- рованных научно-методических семинаров и на страницах научных журналов. Молодежная дискуссионная площадка № 2 — для обучающихся по программам магистратуры по направлению «Аудит и контроль» (модератор — профессор департамента аудита и корпоративной отчетности, д. э. н. Ирина Федоровна Ветрова. Четвертая секция — «Налоговая аналитика и контроль в условиях цифровой реальности» — собрала более 30 человек. Модераторами выступили профессор департамента налогов и налогового администрирования факультета налогов, аудита и бизнес-анализа Финансового университета, д. э. н. Дмитрий Иванович Ряховский и доцент того же Факультета, к. э. н. Наталья Александровна Назарова. Молодежная дискуссионная площадка № 3 — для обучающихся по программам магистратуры по направлению «Учет и отчетность» (модератор — профессор департамента аудита и корпоративной отчетности Ольга Владимировна Рожнова). Молодежная дискуссионная площадка № 4 — для обучающихся по программам бакалавриата по направлению «Учет и аудит» (модератор — доцент департамента аудита и корпоративной отчетности, к. э. н. Ирина Викторовна Сафонова). Молодежная дискуссионная площадка № 4 — для обучающихся по программам бакалавриата по направлению «Учет и аудит» (модератор — доцент департамента аудита и корпоративной отчетности, к. э. н. Ирина Викторовна Сафонова). Учет. Анализ. Аудит • Т. 9, № 1’2022 Использование наследия российской бухгалтерско- аналитической школы в современных условиях / Application the Legacy of the Russian Accounting and Analytical School Nowadays у р р На заседании было заслушано 24 научных до- клада (авторами 18 из них были преподаватели Финансового университета, 6 — сотрудники других вузов страны), в которых были раскрыты вопросы совершенствования теории и практики налогово- го администрирования, организации налогового контроля в условиях цифровой реальности. Зна- чительный интерес и конструктивную дискуссию вызвали доклады: зам. начальника контрольного управления ФНС России, д. э. н. К. В. Новоселова, экономиста, партнера компании «Эрнст энд Янг» А. В. Маленкина, доцента кафедры налогов и налого- обложения Белорусского государственного экономи- ческого университета (г. Минск), к. э. н. И.А. Лукья новой. В докладах представителей вузов большое внимание было уделено вопросам совершенство- вания подготовки кадров для налоговой службы и повышения квалификации служащих налоговых органов с учетом изменений в организации и циф- ровизации их деятельности. Молодежная дискуссионная площадка № 5 — для обучающихся по программам бакалавриата и маги- стратуры по направлению «Анализ» (модератор — профессор департамента бизнес-аналитики, д. э. н. Ольга Владимировна Ефимова). Молодежная дискуссионная площадка № 6 — для обучающихся по программам бакалавриата и магистратуры по направлению «Налоги и налого- вый контроль» (модератор — доцент департамента налогов и налогового администрирования, к. э. н. Александр Владимирович Гурнак). Учитывая тот факт, что С. Б. Барнгольц была не только аналитиком и аудитором, но и замечатель- ным лингвистом, в ходе конференции проводился студенческий конкурс перевода профессионально ориентированных текстов (организатор — старший преподаватель Департамента английского языка и профессиональной коммуникации Ольга Юрь- евна Короткая). Дискуссии молодых специалистов проходили в рамках молодежных площадок. Молодежная дискуссионная площадка № 1 — для аспирантов по специальности «Бухгалтерский учет, статистика» (модераторы — профессора департа- Материал подготовлен членами оргкомитетов конференций. Учет. Анализ. Аудит • Т. 9, № 1’2022 Учет. Анализ. Аудит • Т. 9, № 1’2022
https://openalex.org/W2885633604	http://ijpeds.iaescore.com/index.php/IJPEDS/article/download/12349/11451	English	null	Modeling and Real-Time Simulation of Induction Motor Using RT-LAB	International Journal of Power Electronics and Drive Systems (IJPEDS)	2,018	cc-by-sa	4,230	International Journal of Power Electronics and Drive System (IJPEDS) Vol. 9, No. 4, December 2018, pp. 1476~1485 ISSN: 2088-8694, DOI: 10.11591/ijpeds.v9.i4.pp1476-1485 International Journal of Power Electronics and Drive System (IJPEDS) Vol. 9, No. 4, December 2018, pp. 1476~1485 ISSN: 2088-8694, DOI: 10.11591/ijpeds.v9.i4.pp1476-1485 International Journal of Power Electronics and Drive System (IJPEDS) Vol. 9, No. 4, December 2018, pp. 1476~1485 ISSN: 2088-8694, DOI: 10.11591/ijpeds.v9.i4.pp1476-1485  1476  1476 Modeling and Real-Time Simulation of Induction Motor Using RT-LAB A. Hamed, A. Hazzab Department of Electrical Engineering, Laboratory (COASEE), Tahri Mohamed Bechar University, Algeria A. Hamed, A. Hazzab Department of Electrical Engineering, Laboratory (COASEE), Tahri Mohamed Bechar University, Algeria ABSTRACT This paper presents the modeling and real-time simulation of an induction motor. The RT- LAB simulation software enables the parallel simulation of power drives and electric circuits on clusters of a PC running QNX or RT- Linux operating systems at sample time below 10 µs. Using standard Simulink models including SimPowerSystems models, RT-LAB build computation and communication tasks are necessary to make parallel simulation of electrical systems. The code generated by the Real-Time Workshop of RT- LAB is linked to the OP5600 digital real-time simulator. A case study example of real-time simulation of an induction motor system is presented.This paper discusses methods to overcome the challenges of real- time simulation of an induction motor system synchronizing with a real-time clock. Keyword: RT-LAB Real-Time Induction motor Modeling Simulation Copyright © 2018 Institute of Advanced Engineering and Science. All rights reserved. Corresponding Author: p g A. Hamed, Department of Electrical Engineering, Laboratory (COASEE), Tahri Mohamed Bechar University Algeria , Bechar University, Street Of Independence, BP 417, Bechar, Algeria. Email: hamedali1985@yahoo.fr 1. INTRODUCTION The induction motor is used in many industrial sectors as the main element of converting electrical energy into a mechanical drive because of its low cost, robustness, high degree of reliability and good power- to-weight ratio. Due to its feature sand high applicability in industrial fields, studies for higher efficiency have always been demanded. As testing and validation of induction motor has become more and more important in the design and engineering process, the need for constant improvement of component modeling and for increasing the speed of prototyping the system has also become greater. Conventionally, validation of a induction motor was done by non-real-time simulation of the concept at early stage in the design, and by testing the system once the design is implemented. But this method has some major drawbacks:first, the leap in the design process, from off-line simulation to real prototype is so wide that it is prone to many troubles and problems related to the integration at once of different modules; second, the off-line, non-real-time, simulation may become tediously long for any moderately complex system, especially for motor drives with switching power electronics [1]. Real-time simulation of induction motor is a valuable tool for development and testing of control.Two situations can arise depending on the time required by the simulation platform to complete the computation of state outputs for each time-step (Figure 1): 1) if the execution time, Te, for the simulation of the system is shorter or equal to the selected time-step, the simulation is considered to be real-time (Figure 1 (a)); and 2) if Te is greater than its time-step size for one or more time-steps, overruns occur and the simulation is considered as non-real-time or offline. In the latter case, either the time-step can be increased or the system model can be simplified to run it in Real-Time [2]-[5]. Journal homepage: http://iaescore.com/journals/index.php/IJPEDS  1477 1477 ISSN: 2088-8694  Int J Pow Elec & Dri Syst Figure 1. Illustration of real-time and offline simulation: (a) Real-time simulation. (b) Non-real-time simulation Figure 1. Illustration of real-time and offline simulation: (a) Real-time simulation. (b) Non-real-time simulation This paper presents the modeling and real-time simulation of an induction motor in a power system. Matlab/Simulink software is used to develop the induction motor model. The generated code of the Simulink model is linked to the OP5600 digital real-time simulator in order to simulate the induction motor. 2.1.1. The Command Station The command station is a PC workstation that operates under Windows, and serves as the user interface. The command station allows users to: edit and modify models; see model data; run the original model under its simulation software (Simulink, SystemBuild, etc.); generate and separate code; and control the simulator's Go/Stop sequences [7]. 2.1. Hardware Configuration 2.1. Hardware Configuration RT-LAB software runs on a hardware configuration consisting of command station (host n target nodes, the communication links (real-time and Ethernet), and the I/O boards [7]. 1. INTRODUCTION The paper is divided into six sections, the hardware and software configuration of RT-LAB is presented in section 2, section 3 shows the mathematical model of the induction motor, a detailed modeling of an induction motor in RT-LAB is shown in secion 4. Section 5 illustrates the real time simulation results using RT-LAB, finally the conclusion is drawn in section 6. Modeling and Real-Time Simulation of Induction Motor Using RT-LAB (A. Hamed) 2. RT-LAB CONFIGURATION RT-LAB is an industrial-grade software package for engineers who use mathematical block diagrams for simulation, control and related applications. The software use popular programming tools such as MATLAB/Simulink and works with viewers such as Lab VIEW and programming languages including C++. RT-LAB allows the user to readily convert Simulink Models to real-time simulations, via Real-Time workshop (RTW) and run them over one or more PC processors [1],[4]-[6]. 2.1. Hardware Configuration RT-LAB software runs on a hardware configuration consisting of command station (host node), target nodes, the communication links (real-time and Ethernet), and the I/O boards [7]. 2.3. Opcomm Communication Blocks Once the model is grouped into console and computation subsystems, special blocks called opcomm blocks must be inserted into the subsystems.these are simple feed-through blocks that intercept all incoming signals before sending them to computation blocks within a given subsystem.opcomm blocks serve several purposes. p p When a simulation model runs in the RT-LAB environment,all connections between the main subsystems(SC_,SM_,or SS_)are replaced by hardware communication links .for communication between real-time target nodes(SM_ and SS_)RT-LAB uses adesignated real-time link.for communication between the console(SC_)and the real-time nodes(SM_ or SS_)RT-LAB uses TCP/IP. Because of these communication links between nodes, the simulation may not run the same way in simulink or systemBuild as in RT-LAB. In RT-LAB, a computation subsystem waits for reception of all signals before it is able to start calculating.in Simulink and SystemBuild,on the other hand,computations performed on a signal start as soom as the signal is available,OpComm blocks emulate the behavior of the system as it is run in RT-LAB [10]. 2.4. Recording Data with OpWriteFile To obtain all data for any particular signal, use the OpWriteFile block data recorded with this block is stored on the target real-time node’s hard drive.the data file is automatically transferred to the command station when the model is reset [10]. 2.2. Software Configuration For the above configuration of RT-Lab, the software in the command station (console) is Windows, and the simulation software is Matlab-Simulink to program the simulation and control tasks. The simulation program is coded into C code in the consol unit and transferred to the target node, which has linux operating system. The target unit compiles and executes the C code file in parallel with the simulation program in the console. The data is transferred on-line between the target and console throw communication Ethernet. In the consol station, the program is written in two main blocks (Consol-Master). p g SM_: master subsystem. There is always one and only one master subsystem inside a model.it contains the computational elements of the model. p g SM_: master subsystem. There is always one and only one master subsystem inside a model.it contains the computational elements of the model. SC_: console subsystem. The console subsystem is the subsystem operating on the command station that enables you to interact with the system .it contains all the simulink/systembuild blocks related to acquiring and viewing data (scope, manual switch, to workspace-type blocks, etc).the blocks you need, whether it is during or after the execution of the real-time model, must be included in the console subsystem.the console runs asynchronously from the other subsystems.note that there can only be one console per model [10]-[12]. 2.1.2. Target nodes The target nodes are real-time processing and communication computers that use commerci ocessors interconnected by an Ethernet adapter. The real-time target nodes perform: The target nodes are real-time processing and communication computers that use processors interconnected by an Ethernet adapter. The real-time target nodes perform: a. Real-time execution of the model’s simulation; b. Real-time communication between the nodes and I/Os; c. Acquisition of the model’s internal variables and external outputs through I/O modules [8],[9];  1478  78 ISSN: 2088-8694 Figure 2. Communication between the Console and Target Node Figure 2. Communication between the Console and Target Node The command node and target node are commercial PC’s with different operating system. A PCI- 626 I/O card (from Sensory Company Inc.) is used which satisfies all I/O requirements. Moreover, it is supported by RT- Linux real-time operating system. In this configuration the only communication link used is between the target and command station using Ethernet communication [7]. 2.5. EXtreme High Performance (XHP) Mode The XHP mode is a means for the real-time operating system to disable interrupts.this is done on a per-subsystem (thus per-CPU) basic.not allowing interrupts prevents process switching which removes latencies time for additional computation in the same time slice. The XHP mode is aimed at real-time applications of a given base sample time that cause overruns (overflow their allowed time-step for Int J Pow Elec & Dri Syst, Vol. 9, No. 4, December 2018 : 1476 – 1485 1479 ISSN: 2088-8694  Int J Pow Elec & Dri Syst computation) while running in RT-LAB in the standard mode. In XHP mode, the model waits in an empty loop for its next scheduled step.the next model step is then computed. computation) while running in RT-LAB in the standard mode. In XHP mode, the model waits in an empty loop for its next scheduled step.the next model step is then computed. In this mode, we use the CPU counter as our time reference.because this counter operates at the CPU’s frequency, it offers a very high resolution, even for step-size in the microsecond range.because the counter resides on the CPU and reading its value is done within one CPU cycle, this operation introduces almost no latency [10]. 3. INDUCTION MOTOR MODELING The mathematical models of induction motor in the park frame are written as follows [13]-[19]. Vsd = Rs. Isd + dϕsd dt −Wa. ϕsq Vsq = Rs. Isq + dϕsq dt −Wa. ϕsd Vsd = Rr. Ird + dϕrd dt −Wr. ϕrq Vrq = Rr. Irq + dϕrq dt −Wr. ϕrd (1) Vsd = Rs. Isd + dϕsd dt −Wa. ϕsq Vsq = Rs. Isq + dϕsq dt −Wa. ϕsd Vsd = Rr. Ird + dϕrd dt −Wr. ϕrq Vrq = Rr. Irq + dϕrq dt −Wr. ϕrd (1) The stator and rotor fluxes are related to the current by: he stator and rotor fluxes are related to the current by: ϕsd = Ls. Isd + Lm. Ird ϕsq = Ls. Isq + Lm. Irq ϕrd = Ls. Ird + Lm. Isd ϕrq = Ls. Irq + Lm. Isq (2) ϕrd = Ls. Ird + Lm. Isd ϕrq = Ls. Irq + Lm. Isq (2) (2) The stator and rotor angular velocities are linked by the following relation: The stator and rotor angular velocities are linked by the following relation: Ws = W + Wr (3) Ws = W + Wr (3) (3) Ws = W + Wr The equations of mechanical and electromagnetic torques are: The equations of mechanical and electromagnetic torques are: J dΩ dt = Te −Tm −fΩ (4) Te = P(ϕrqIrd −ϕrdIrq), Ω = W P (5) J dΩ dt = Te −Tm −fΩ Te = P(ϕrqIrd −ϕrdIrq), Ω = W P (5) Te = P(ϕrqIrd −ϕrdIrq), Ω = W P (5) where: Rs is stator resistance, Rr is rotor resistance, Ls and Lr are respectively stator and rotor inductance Lm the mutual inductance, ϕsd and ϕsq are the direct and quadrature stator flux, ϕrd and ϕrq are the direct and quadrature rotor flux, Isd and Isq are the direct and quadrature stator current, Ird and Irq are the direct and quadrature rotor current, P is the number of pair poles, Ws and Wr are the asynchronous and rotor angular frequency, Te and Tm are the electromagnetique and mechanical torque, and F is the viscosity coefficient. 4. MODELING INDUCTION MOTOR IN RT-LAB 4. MODELING INDUCTION MOTOR IN RT-LAB 4. MODELING INDUCTION MOTOR IN RT-LAB In RT-LAB all top-level subsystems must be named with a prefix identifying their function .The prefixes are: SC_ for consol sybsystem and SM_ for master subsystem. Depending on the type of data acquisition, we propose two RT-LAB models; in the first model the data transfer between the target node and the console machine is during the simulation task, however in the second one, the transfer will be done after the end of simulation performed. 3. INDUCTION MOTOR MODELING where: Rs is stator resistance, Rr is rotor resistance, Ls and Lr are respectively stator and rotor inductance Lm the mutual inductance, ϕsd and ϕsq are the direct and quadrature stator flux, ϕrd and ϕrq are the direct and quadrature rotor flux, Isd and Isq are the direct and quadrature stator current, Ird and Irq are the direct and quadrature rotor current, P is the number of pair poles, Ws and Wr are the asynchronous and rotor angular frequency, Te and Tm are the electromagnetique and mechanical torque, and F is the viscosity coefficient. To get a model state in terms of the rotor sizes ( Ird , Irq , ϕrd , ϕrq),We express the stator sizes ( Isd , Isq , ϕsd , ϕsq) in terms of rotor sizes [13]. Isd = ϕrd Lm − Lr Lm Ird Isq = ϕrq Lm − Lr Lm Irq ϕsd = Ls Lm ϕrd + δ Lm Ird ϕsq = Ls Lm ϕrq + δ Lm Irq (6) Isd = ϕrd Lm − Lr Lm Ird Isq = ϕrq Lm − Lr Lm Irq ϕsd = Ls Lm ϕrd + δ Lm Ird ϕsq = Ls Lm ϕrq + δ Lm Irq (6) By using equations (1) and the equations (6) of the IM, we obtain the dynamic model of induction motor having as state vector (Ird , Irq, ϕrd , ϕrq , w): Modeling and Real-Time Simulation of Induction Motor Using RT-LAB (A. Hamed) ISSN: 2088-8694  1480 1480  480  dIrq dt = (Rs Lr+ Rr Ls) δ Lm2 Irq + wa Ird − Rs δ Lm2 ϕrq + Ls δ Lm2 w ϕrd + Vsd δ Lm − Ls δ Lm2 Vrd dϕrd dt = −Rr. Ird + waϕrq −wϕrq + Vrd dϕrq dt = −Rr. Irq + waϕrd −wϕrd + Vrq dw dt = P2 J (ϕrqIrd − ϕrdIrq) + P J (Tm −fw) (7) (7) 4.1. Data Acquisition during Simulation In this case RT-LAB enables signal acquisition from a real-time target during simulation using OpComm block for synchronization between target node and console. Figure 3. RT-Lab Real-Time simulation graphic model of IM Figure 4. Master subsystem of induction mtor with opcomm block Figure 3. RT-Lab Real-Time simulation graphic model of IM Figure 3. RT-Lab Real-Time simulation graphic model of IM Figure 3. RT-Lab Real-Time simulation graphic model of IM Figure 4. Master subsystem of induction mtor with opcomm block Figure 4. Master subsystem of induction mtor with opcomm block Figure 4. Master subsystem of induction mtor with opcomm block Int J Pow Elec & Dri Syst, Vol. 9, No. 4, December 2018 : 1476 – 1485  1481  1481  1481 ISSN: 2088-8694 Int J Pow Elec & Dri Syst Figure 5. Console subsystem of induction motor with opcomm block Figure 5. Console subsystem of induction motor with opcomm block We use OpComm block in master and console subsystems; the consol sends the signal of Resistant Torque to the master subsystem and the latter sends signals of Isa,Isb,Isc,Ids,Iqs,Qrd,Qsd,Ce,Speed to the console subsystem. 4.2. Data Acquisition after the End of Simulation q In this case, we use OpWriteFile block for recording all data in the target node’s hard disk, and when the simulation ends, the target node sends recorded data in matefile format to the console. Figure 6. RT-Lab Real-Time simulation graphic model of IM with OpWriteFile Block Figure 6. RT-Lab Real-Time simulation graphic model of IM with OpWriteFile Block Modeling and Real-Time Simulation of Induction Motor Using RT-LAB (A. Hamed)  1482 ISSN: 2088-8694  82 Figure 7. Master subsystem of induction motor with OpWriteFile Block Figure 7. Master subsystem of induction motor with OpWriteFile Block We use an OpWriteFile block in master subsystem for recording simulation data We use an OpWriteFile block in master subsystem for recording simulation data Figure 8. Console subsystem of induction motor without opcomm block Figure 8. Console subsystem of induction motor without opcomm block The OpComm block is not used in the console subsystem. The OpComm block is not used in the console subsystem. 5. RESULTS AND ANALYSIS All the simulations are carried out on an induction motor whose parameters are listed in the Table 1. SU S N N S S All the simulations are carried out on an induction motor whose parameters are listed in the Table 1 Table 1. Induction motor parameters Parameters Symbols Input values Rotor Resistance Stator Resistance Rtor Inductance Stator Inductance Inductance mutual Phase voltage frequency pair of pole Rr Rs Lr Ls Lm Vph f p 3.81 4.85 0.274 0.274 0.258 220 50 2 We applied a resistant torque to the induction motor at time 2.0 s to 3.0 s. Figure 9 shows the speed response in real-time simulation in different times steps.Through the red curve we note that there is no data Int J Pow Elec & Dri Syst, Vol. 9, No. 4, December 2018 : 1476 – 1485  1483 1483  14  ISSN: 2088-8694 Int J Pow Elec & Dri Syst loss when we use 30 µs as time-step, The response is the same as in offline simulation.In the second case, when we use 20 µs as time-step it is shown clearly in the zoom graph that it started to loss data, However in the last case when we use 10 µs as time-step we observe that data was considerably lost and the signal of the speed has completely changed. Figure 9. Speed respone of induction motor in different time-steps Figure 9. Speed respone of induction motor in different time-steps We conclude that the acquisition data from the target node during simulation leads to the data loss if the time-step is small then 20 µs, because TCP/IP is relatively slow compared with most real-time systems like fireWire-type network. REFERENCES [1] S. Abourida, et al., “Hardware-in-the-loop simulation of finite-element based motor drives with RT-LAB and JMAG,” Industrial Electronics, 2006 IEEE International Symposium on, pp. 2462-2466, 2006. [2] J. Belanger, et al., “The what, where and why of real-time simulation,” Planet RT, vol. 1, pp. 25-29, 2010 g y pp [3] M. O. Faruque, et al., “Real-time simulation technologies for power systems design, testing, and analysis,” IEEE Power and Energy Technology Systems Journal, vol. 2, pp. 63-73, 2015. ] O. Crăciun, et al., “Hardware-in-the-loop testing of PV control systems using RT-Lab simulator,” Pow Electronics and Motion Control Conference (EPE/PEMC), 2010 14th International, pp. S2-1-S2-6, 2010. ] C. Dufour, et al., “Hardware-in-the-loop simulation of power drives with RT-LAB,” Power Electronics and Driv Systems, 2005. PEDS 2005. International Conference on, pp. 1646-1651, 2005. y f pp [6] A. Idir and M. Kidouche, “Rt-lab and dspace: two softwares for real time control of induction motors,” Rev. Roum. Sci. Techn.–Électrotechn. et Énerg, vol. 59, pp. 205-214, 2014. g pp [7] M. A. Geliel, “Real-time implementation of constrained control system on experimental hybrid plant using RT- Lab,” Control and Automation, 2008 16th Mediterranean Conference on, pp. 1060-1065, 2008. ] X. Y. Xu and L. Wang, “RT-LAB Rapid Control Prototyping's Application in Servo System,” Journal of Syste Simulation, vol. 4, pp. 062, 2006. [9] H. l. Zheng, et al., “RT-LAB based real-time simulation of photovoltaic power generation system,” Advanced Technology of Electrical Engineering and Energy, vol. 4, pp. 014, 2010. gy f g g gy pp [10] F. G. Areed, et al., “Adaptive neuro-fuzzy control of an induction motor,” Ain Shams Engineering Journal, vol. 1, pp. 71-78, 2010. [11] C. Dufour, et al., “RT-LAB real time simulation of electric drives and systems,” National Power Electronics Conference–NPEC, 2005. 2] H. Guo, et al., “Real-time simulation of BLDC-based wind turbine emulator using RT-LAB,” Electrical Machin and Systems, 2009. ICEMS 2009. International Conference on, pp. 1-6, 2009. y f pp [13] A. M. Fihakhir and M. Bouhamida, “Nonlinear Control of a Doubly Fed Induction Generator Driven Wind Turbine,” Electrotehnica, Electronica, Automatica, vol. 64, pp. 23, 2016. [14] M. Jannati, et al., “Indirect rotor field-oriented control of fault-tolerant drive system for three-phase induction motor with rotor resistance estimation using EKF,” Indonesian Journal of Electrical Engineering and Computer Science, vol. 12, pp. 6633-6643, 2014. pp [15] M. We conclude that the acquisition data from the target node during simulation leads to the data loss if the time-step is small then 20 µs, because TCP/IP is relatively slow compared with most real-time systems like fireWire-type network. Figure 10 shows the speed response in second proposal where OpWriteFile block is used. In this case the target node does not send data to the console while the simulation is running. After simulation ends, the target node sends the recorded data in hard disque of target as mat file content all signal information to the console. Figure 10.speed respone of induction motor in real-time with opwritefile block Figure 10.speed respone of induction motor in real-time with opwritefile block We note from the curve that although very small time-step is used compared to the previous experiment, no data loss occurred just as in offline simulation. Later, a simulation attempt with time-step as 0,1 µs caused overruns and the simulator showed the following error message Modeling and Real-Time Simulation of Induction Motor Using RT-LAB (A. Hamed)  1484  ISSN: 2088-8694 Figure 11. Overruns detected Figure 11. Overruns detected Figure 11. Overruns detected 6. CONCLUSION In this paper we proposed two RT-LAB models for real-time simulation of the induction motor, in the first, model, data acquisition was during simulation, however in the second model, data acquisition was after the end of simulation, The simulation results show that in the first model we got data loss when we use small time-step because TCP/IP is relatively slow compared with real-time systems like fireWire-type network. However in the second model no data loss occured. REFERENCES Jannati, et al., “Speed sensorless fault-tolerant drive system of 3-phase induction motor using switching extended kalman filter,” Indonesian Journal of Electrical Engineering and Computer Science, vol. 12, pp. 7640- 7649, 2014. [16] V. Kumar, et al., “Implementation of Scalar Control Technique in SVPWM Switched Three-Level Inverter Fed Induction Motor Using DSP Controller,” International Journal of Power Electronics and Drive Systems, vol. 1, pp. 83, 2011. Int J Pow Elec & Dri Syst, Vol. 9, No. 4, December 2018 : 1476 – 1485  1  1485 Int J Pow Elec & Dri Syst ISSN: 2088-8694 [17] L. Lahcen, “Fuzzy Sliding Mode Controller for Induction Machine Feed by Three Level Inverter,” International Journal of Power Electronics and Drive Systems (IJPEDS), vol. 9, 2018. [17] L. Lahcen, “Fuzzy Sliding Mode Controller for Induction Machine Feed by Three Level Inverter,” International Journal of Power Electronics and Drive Systems (IJPEDS), vol. 9, 2018. f y ( ) [18] H. Othmani, et al., “Fuzzy Gain-Scheduling Proportional-Integral control for Improving the speed behavio three-phases induction motor,” International Journal of Power Electronics and Drive Systems, vol. 7, 2016. [19] E. Ramprasath and P. Manojkumar, “Modelling and analysis of induction motor using LabVIEW,” International Journal of Power Electronics and Drive Systems, vol. 5, pp. 344, 2015. [19] E. Ramprasath and P. Manojkumar, “Modelling and analysis of induction motor using LabVIEW,” International Journal of Power Electronics and Drive Systems, vol. 5, pp. 344, 2015. Modeling and Real-Time Simulation of Induction Motor Using RT-LAB (A. Hamed) BIOGRAPHIES OF AUTHORS Hamed Ali was born on 18th december 1985 in bechar, Algeria he received the ingeniorat degree in computer science from the Bechar University, Algeria in 2008 and the master degree in computer science from the Tahri Mohammed Bechar University, Algeria in 2012. In 2014, he was a laboratory membre at, Laboratory of Control Analysis and Optimization of the Electro- Energetic Systems (CAOSEE). His research interrest covers, power electronics electric drives control, real-time simulation and artificial intelligence. e-email : hamedali1985@yahoo.fr Hazzab Abdeldjebar received the state engineer degree in electrical engineering in 1995 from the University of Sciences and Technology of Oran (USTO), Algeria the M.Sc. degree from the Electrical Engineering Institute of the USTO in 1999, and the Ph.D. degree from the Electrical Engineering Institute2 of the USTO in 2006. He is currently professor of electrical engineering at University of Bechar, Bechar, Algeria, where he is Director of the Research Laboratory of Control Analysis and Optimization of the Electro-Energetic Systems. His research interests include power electronics, electric drives control, and artificial intelligence and their applications.
https://openalex.org/W853418146	https://thescipub.com/pdf/jcssp.2015.627.638.pdf	English	null	Novel Prefix Tri-Literal Word Analyser: Rule-Based Approach	Journal of computer sciences/Journal of computer science	2,015	cc-by	8,444	Novel Prefix Tri-Literal Word Analyser: Rule-Based Approach 1Mohammed M. Abu Shquier and 2Khaled M. Alhawiti 1Department of Information Science, University of Tabuk, Tabuk, KSA 2Department of Computer Science, University of Tabuk, Tabuk, KSA 1Mohammed M. Abu Shquier and 2Khaled M. Alhawiti 1Department of Information Science, University of Tabuk, Tabuk, KSA 2Department of Computer Science, University of Tabuk, Tabuk, KSA 1Department of Information Science, University of Tabuk, Tabuk, KSA 2Department of Computer Science, University of Tabuk, Tabuk, KSA Abstract: Arabic stemming is a technique to find the stem or lexical root for Arabic words through the process of eliminating affixes (preffixes, infixes and suffixes) attached to their roots. Several approaches have been implemented to generate the stem of Arabic words according to a certain level of analysis, i.e., root-based approach, stem-based approach and statistical approach. Arabic language is a Semitic language which means that it is a derivational rather than a concatinative language. In this study we designed and implemented an Arabic triliteral Morphological Analyser that is capable of analysing the classical and Modern Standard Arabic (MSA) effectively with the capability of analysing vowelised, semi- vowelised and nonvowelised text. The system is integratable with other applications so that vast number of people can get benfited from. One shortcomming for the developed system is that the output obtained from the morphological analyser may contain several alternative solutions which leads to extraction ambiguity. Article history Received: 31-07-2014 Revised: 29-11-2014 Accepted: 17-5-2015 Corresponding Author: Mohammed M. Abu Shquier Department of Information Science, University of Tabuk, Tabuk, KSA Email: shquier@gmail.com Article history Received: 31-07-2014 Revised: 29-11-2014 Accepted: 17-5-2015 Corresponding Author: Mohammed M. Abu Shquier Department of Information Science, University of Tabuk, Tabuk, KSA Email: shquier@gmail.com Keywords: Morphological Analyser, Stemmer, Semitic, Hamzated, Doubled, Hollow, Defective, Roots, Stems © 2015 Mohammed M. Abu Shquier and Khaled M. Alhawiti. This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Journal Computer Science Journal Computer Science Original Research Paper Introduction Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 guided Arabic rule-based stemmers, it is worthy stressing to mention some of these difficulties; the existence of irregular/broken plurals MــــــNOP ریاTUV., i’lal لXYZ اand ibdal \[الZ ,اthe huge number of Arabic roots, differentiating between affixes and original letters is ambiguous, un-vocalised Arabic representation, the existance of and the semantic ambiguity is also another challenge to the Arabic stemmers. Boudlal et al. (2011) provided a new way to find the system that assigns, for every non-vowel word a unique root depending on the context of the word on the sentence. The proposed system is composed of two modules. These modules start by segmenting the words of the sentence into its elementary morphological units in order to identify its possible roots. Momani and Faraj (2007) proposed a novel algorithm to extract triliteral Arabic roots. The first step of their algorithm was to eliminate the stop words and then the prefixes and suffixes of each word are removed until only three letters remained. Finally, the remaining letters are arranged according to their order in the original word, which form the root of the original word. The researchers tested their algorithm on two types of Arabic text documents. The researchers claimed that the results of both runs were very promising and satisfactory enough to score over 73% of accuracy. An affix is a morpheme that can be added before or after, or inserted within a root or a stem as a prefix, suffix or infix, respectively, to form new words or meanings (Al-Khuli, 1991; Thalouth and Al-Dannan, 1987). Arabic prefixes and suffixes are sets of letters and articles attached to the beginning and the end of the lexical word and written as part of it respectively (Al-Atram, 1990). English has 75 prefixes and about 250 suffixes (Salton, 1989). Arabic has fewer affixes to concatenate with each other in predefined linguistic rules. This feature increases the overall number of affixes (Ali, 1992). The removal of prefixes in English requires further analysis since it can alter the meaning or grammatical function of the word. This is not the case in Arabic, since the removal of prefixes does not usually reverse the meaning of words. Khoja’s stemmer is a root-based Arabic stemmer (Khoja and Garside, 1999). The Khoja’s algorithm removes prefixes, infixes and suffixes and uses patterns to extract the roots using a dictionary. Introduction quadlateral and biliteral origin (Al-Fedaghi and Al- Sadoun, 1990). Arabic language plays a crucial role with the root (C1aC2aC3a) (To clarify the structure of Morphological forms we have used the corresponding CV array of each form alongside. Cns corresponds to radical letters and represent the consonants of ; )>=ــــto add subtle variations to the meaning. Arabic verbs are constructed on the root ; >=ــــthat uses three consonants ع ,فand لthat is know by Arabic grammarians as Morphological Balance (MB), the result of mapping root letters to MB forms is verbal or nominal stems. The stem is used to construct verbs or nouns through prefixing and suffixing inflectional prefixes and suffixes to those stems (Attia, 2008). The Arabic three consonants in the root-verb (; )>=ــــare represented as (C1), (C2) and (C3) respectively, while the supscript followed the consonant represents the sequence of these consonants However, the multifarious vowels and affixes are attached to the root verbs to create the desired inflection of the meaning. Each root can generate a vast number of meanings. Arabic roots can be classified into two classes as shown in Fig. 2; the vowelized roots and non-vowelized Roots (Al-Omari, 1995; Al-Dahdah, 1985). This classification was made in accordance with the availability of the Arabic vowels in the roots. Arabic is considered as one of the Semitic language based on roots. A root is the original form of a word which can not be further analysed. Arabic roots are verbs only. The majority of Arabic roots are triliteral (George, 1990; Al-Najem, 1998; Al-Momani, 2010). Al-Fedaghi and Al-Anzi (1989) claimed that there are around ten thousand independent roots. Each root may be attached to prefixes, suffixes, infixes to derive nouns and adjectives. The addition of infixes is based on certain structurers. Words constructed from the same root are not related semantically in general (Rafea and Shaalan, 1993). Stemmer or morphological analyser are widely used by researchers dealing with languages with complicated Many challenges may face the construction of well The previous studies in the Arabic language research explained that the greater portion of the Arabic root verbs are of trilateral origin, while the remaining are of Mohammed M. Abu Shquier and Khaled M. Literature Review Several methods were developed to represent text in Natural Language Processing (NLP) and Information Retrieval (IR) fields. For Arabic Language, there are three different Stemming approaches: The root-based approach (Khoja and Garside, 1999); the light stemmer approach (Larkey et al., 2002) and the statistical stemmer approach (N-Gram (Khreisat, 2006; Mustafa and Al-Radaideh, 2004)). In this study we propose an algorithm for word analyser that accepts the non-article trilateral words and finds out their roots. The word analyser module is shown in Fig. 1. The word analyser process starts with the prefix/suffix analyser modules that determine whether the particular word is preceded by prefix(es) or attached with suffix(es) or not. The output of this module is the longest prefix/suffix list generaed, then we further invoke the stem generator module that generates all the permutations of the possible stems and then matches template(s) that represent the corresponding stem(s). Afterward, the triliteral root processor recodes the generated root to their original form. Al-Shammari (2010) stated that both Arabic root- based and stem-based algorithms are lacking from generating errors. The removal of prefixes and suffixes generate many errors, especially when the algorithm is expected to distinguish between an extra letter and a root letter. Al-Shammari claimned that stemming process can return with errors known as over-stemming and under- stemming respectively. Introduction Although the algorithm suffers from some issues with proper nouns, broken plurals `abcOP ا TUV and nouns, the Khojas algorithm showed superiority over previous work in root detection algorithms (Khoja and Garside, 1999). Overview of Arabic Affixation Essentially, the Arabic word can be described (Abu Shquier, 2013) as follows: Hawas (2013) presented a novel Arabic words root- extraction approach, he tried to assign a unique root for each Arabic word without having an Arabic roots list, a words patterns list, or even the Arabic words prefixes/suffixes list, his algorithm predicts the letters positions using rules based on the relations between the Arabic word letters and their position in the word. The proposed approach was composed of several corporate modules. Hawas tested the proposed approach using the Holy Quran words and he claimed that the total success ratio for the proposed algorithm was about 93.7% but she considered the root is correct if it has one correct letter. [Prefix1] [prefix1] stem [infixes] suffix1] [suffix2] [Prefix1] [prefix1] stem [infixes] suffix1] [suffix2] The stem is the minimal meaning-bearing unit in a language. Affixes in Arabic can be categorized into three types, prefixes, suffixes (or postfixes) and infixes (Saliba and Al-Dannan, 1990). The prefixes are added at beginning of the stem while the suffixes are attached to the end, Table 1 shows some affix conjugation for the verb ــ`بg. 628 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 DOI: 10.3844/jcssp.2015.627.638 Fig. 1. Arabic word analyser module Fig. 2. Arabic roots classification Table 1. An Arabic affixes example adopted from (Abu Shquier, 2013) Suffixes stem Infex Prefixes Arabic Structure ــ`بg ــ`بg C1aC2aC3 ــ`بg ي ` ب ij ُ ِ yaC1C2iC3u ا ــ`بg ي ر بmij ِ yuC1AC2iC3u n ھ ــ`بg ي np\ `ij ِ yaC1C2iC3uhum n ھ ــ`بg ي سnp\ `iar ِ syaC1C2iC3uhum n اھ ــ`بg ي سnp\ ر ِ یسmــg syuC1AC2iC3uhum nst او ــ`بg ي سnptv\ رmiar ِ syuC1AC2iC3unahum Table 2. Arabic Suffixes for the regular verb hit ــ`بg adopted from (Abu Shquier, 2013) Person, Gender and Suffix Number Features Suffix Category Example Transliteration wــt (3rd, N, S) Verb wx\`ـــــg drabny ك (2nd, N, S) Verb and Noun z\`ــــg drabk ھ (3rd, M, S) Verb and Noun {\`ـــg drabhu m ھ (3rd, F, S) Verb and Noun mp\`ــــg drabha n ھ (3rd, M, P) Verb and Noun np\`ــــg drabhum \|ھ ّ (3rd, F, P) Verb and Noun \|p\`g ّ drabhuna mU ھ (3rd, N, D) Verb and Noun mUp\`ــــg drabahuma n ﮐــ (2nd, M, P) Verb and Noun nN\`ـــــg drabakum \|ﮐ ّ (2nd, F, P) Verb and Noun \|N\`g ّ drabkunna mــU ﮐ (2nd, N, D) Verb and Noun mUN\`ـــــg drabakuma 1st: Denotes the 1st Person (n•ــــــNOUP )ا 2nd: Denotes the 2nd Person (€طــm‚UP )ا 3rd: Denotes the 3rd Person (€ــــƒm„P )ا S b f Si l i li d l li Fig. 1. Arabic word analyser module Fig. 1. Arabic word analyser module Fig. 2. Arabic roots classification Fig. 1. Arabic word analyser module Fig. 1. Arabic word analyser module Fig. 2. Arabic roots classification Fig. 2. Arabic roots classification Fig. 2. Arabic roots classification Table 1. [Prefix1] [prefix1] stem [infixes] suffix1] [suffix2] An Arabic affixes example adopted from (Abu Shquier, 2013) Suffixes stem Infex Prefixes Arabic Structure ــ`بg ــ`بg C1aC2aC3 ــ`بg ي ` ب ij ُ ِ yaC1C2iC3u ا ــ`بg ي ر بmij ِ yuC1AC2iC3u n ھ ــ`بg ي np\ `ij ِ yaC1C2iC3uhum n ھ ــ`بg ي سnp\ `iar ِ syaC1C2iC3uhum n اھ ــ`بg ي سnp\ ر ِ یسmــg syuC1AC2iC3uhum nst او ــ`بg ي سnptv\ رmiar ِ syuC1AC2iC3unahum Table 2. Arabic Suffixes for the regular verb hit ــ`بg adopted from (Abu Shquier, 2013) Person, Gender and Suffix Number Features Suffix Category Example Transliteration wــt (3rd, N, S) Verb wx\`ـــــg drabny ك (2nd, N, S) Verb and Noun z\`ــــg drabk ھ (3rd, M, S) Verb and Noun {\`ـــg drabhu m ھ (3rd, F, S) Verb and Noun mp\`ــــg drabha n ھ (3rd, M, P) Verb and Noun np\`ــــg drabhum \|ھ ّ (3rd, F, P) Verb and Noun \|p\`g ّ drabhuna mU ھ (3rd, N, D) Verb and Noun mUp\`ــــg drabahuma n ﮐــ (2nd, M, P) Verb and Noun nN\`ـــــg drabakum \|ﮐ ّ (2nd, F, P) Verb and Noun \|N\`g ّ drabkunna mــU ﮐ (2nd, N, D) Verb and Noun mUN\`ـــــg drabakuma 1st: Denotes the 1st Person (n•ــــــNOUP )ا 2nd: Denotes the 2nd Person (€طــm‚UP )ا 3rd: Denotes the 3rd Person (€ــــƒm„P )ا S,D,P: Denotes Number features, Singularity, Duality and Plurality M,N,F: Denotes Gender features, Masculine, Nuteral and Feminine Table 1. An Arabic affixes example adopted from (Abu Shquier, 2013) Suffixes stem Infex Prefixes Arabic Structure ــ`بg ــ`بg C1aC2aC3 ــ`بg ي ` ب ij ُ ِ yaC1C2iC3u ا ــ`بg ي ر بmij ِ yuC1AC2iC3u n ھ ــ`بg ي np\ `ij ِ yaC1C2iC3uhum n ھ ــ`بg ي سnp\ `iar ِ syaC1C2iC3uhum n اھ ــ`بg ي سnp\ ر ِ یسmــg syuC1AC2iC3uhum nst او ــ`بg ي سnptv\ رmiar ِ syuC1AC2iC3unahum Table 2. Arabic Suffixes for the regular verb hit ــ`بg adopted from (Abu Shquier, 2013) n`ب ي سnp\ رِیسsyuC1 C2 C3u u nst او ــ`بg ي سnptv\ رmiar ِ syuC1AC2iC3unahum Table 2. [Prefix1] [prefix1] stem [infixes] suffix1] [suffix2] Morphological balance significant derived forms Form Structure Arabic balance Form I yaC1C2aC3 ; =…j َ Form II yuC1aC2C2iC3 ; = …j َ ﱢ Form III yuC1aC2iC3 ; Ym… j ِ ُ Form IV yuC1C2iC3 ; =… j ِ ُ Form V ytaC1aC2C2aC3 ; =…Oj ّ Form VI ytaC1AC2aC3 ; Ym… O j َ َ َ Form VII yanC1aC2iC3 ; = …x j ِ َ َ Form VIII yaC1taC2iC3 T O… j ِ َ َ ل Form IX yaC1taC2iC3 ; =… Ob j ِ َ َ (1) Forms II and IV can have the meaning of carrying out an action to someone/something else (2) Forms II and IV are making the verb transitive or causative (3) Form II can also give a verb the meaning of doing something intensively and/or repeatedly (4) Form III often carries the meaning of doing something with someone else: Or the meaning of trying to do something (Wightwick and Gaffar, 2007) (1) Forms II and IV can have the meaning of carrying out an action to someone/something else (2) Forms II and IV are making the verb transitive or causative ( ) g (3) Form II can also give a verb the meaning of doing something intensively and/or repeatedly (4) Form III often carries the meaning of doing something with someone else: Or the meaning of and Gaffar, 2007) ) g 3) Form II can also give a verb the meaning of doing something intensively and/or repeatedly 4) Form III often carries the meaning of doing something with someone else: Or the meaning of trying to do something (Wightwick d G ff 2007) • Roots with ’waaw’ or ’yaa’ as the first root letter (Mithal roots) (لm‘ــــUP’ــ•ر اP )اا Suffixes in Arabic can be categorized into two basic categories, the suffixes that are attached to the verbs and the suffixes that are added to the nouns (Yusif, 2007). Furthermore, some of the suffixes can be attached to both the noun and verb stem. Nevertheless, Arabic permits the use of up to three suffixes simultaneously to be attached to the end of the same stem (Abu-Ata, 2001). Furthermore, Arabic words are built from roots rather than stems and involve diacritization. Written Arabic is also characterized by the inconsistent and irregular use of punctuation marks (Attia, 2008). Table 2 presents a wide range of suffixes example for the verb hit (ــ`بg). [Prefix1] [prefix1] stem [infixes] suffix1] [suffix2] •Roots with ’waaw’ or’ yaa’ as the second root letter (Hollow roots) (فvV“’•ر اP )ا •Roots with ’weaw’ or ’yaa’ as the third root letter (Defective roots) (”•mـــــxP’ــ•راP )ا •Roots that have two weak letters in their roots (Enfolding roots) (–a… •P’•راPا ّ ) Enfolding roots are categorized into two groups; the first group has a middle and final weak original letters, while the second group has a first and final weak original letters: Arabic language plays a crucial role with the root (C1aC2aC3a) (To clarify the structure of Morphological forms we have used the corresponding CV array of each form alongside. Cns corresponds to radical letters and represent the consonants of )لـــto add subtle variations to the meaning. There are nine significant derived forms (for the singular masculine 3rd person in the present tense) as shown in Table 3: Arabic language plays a crucial role with the root (C1aC2aC3a) (To clarify the structure of Morphological forms we have used the corresponding CV array of each form alongside. Cns corresponds to radical letters and represent the consonants of )لـــto add subtle variations to the meaning. There are nine significant derived forms (for the singular masculine 3rd person in the present tense) as shown in Table 3: • The first group enfolds the definitions of both hollow defective roots, yet it is always treated as a defective only and the middle weak letter is treated as if it were a regular letter i.e., (يvY ، )روي •The second group enfolds the definitions of both Mithal and defective roots. These roots get the dealing of both Mithal and Defective verbs together. i.e., ( )یوق، ویع [Prefix1] [prefix1] stem [infixes] suffix1] [suffix2] Arabic Suffixes for the regular verb hit ــ`بg adopted from (Abu Shquier, 2013) Person, Gender and Suffix Number Features Suffix Category Example Transliteration wــt (3rd, N, S) Verb wx\`ـــــg drabny ك (2nd, N, S) Verb and Noun z\`ــــg drabk ھ (3rd, M, S) Verb and Noun {\`ـــg drabhu m ھ (3rd, F, S) Verb and Noun mp\`ــــg drabha n ھ (3rd, M, P) Verb and Noun np\`ــــg drabhum \|ھ ّ (3rd, F, P) Verb and Noun \|p\`g ّ drabhuna mU ھ (3rd, N, D) Verb and Noun mUp\`ــــg drabahuma n ﮐــ (2nd, M, P) Verb and Noun nN\`ـــــg drabakum \|ﮐ ّ (2nd, F, P) Verb and Noun \|N\`g ّ drabkunna mــU ﮐ (2nd, N, D) Verb and Noun mUN\`ـــــg drabakuma 1st: Denotes the 1st Person (n•ــــــNOUP )ا 2nd: Denotes the 2nd Person (€طــm‚UP )ا 3rd: Denotes the 3rd Person (€ــــƒm„P )ا S,D,P: Denotes Number features, Singularity, Duality and Plurality M,N,F: Denotes Gender features, Masculine, Nuteral and Feminine 629 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Table 3. Morphological balance significant derived forms Form Structure Arabic balance Form I yaC1C2aC3 ; =…j َ Form II yuC1aC2C2iC3 ; = …j َ ﱢ Form III yuC1aC2iC3 ; Ym… j ِ ُ Form IV yuC1C2iC3 ; =… j ِ ُ Form V ytaC1aC2C2aC3 ; =…Oj ّ Form VI ytaC1AC2aC3 ; Ym… O j َ َ َ Form VII yanC1aC2iC3 ; = …x j ِ َ َ Form VIII yaC1taC2iC3 T O… j ِ َ َ ل Form IX yaC1taC2iC3 ; =… Ob j ِ َ َ (1) Forms II and IV can have the meaning of carrying out an action to someone/something else (2) Forms II and IV are making the verb transitive or causative (3) Form II can also give a verb the meaning of doing something intensively and/or repeatedly (4) Form III often carries the meaning of doing something with someone else: Or the meaning of trying to do something (Wightwick and Gaffar, 2007) Table 3. Arabic Roots Classification These classifications are general. In our paper, we conduct more analysis for the roots since roots of the same category may act differently during the morphological process. For instance, the verb promised [Y وwill be changed to promise [یY in the present tense form, while the root facilitated ــ`یr will be to facilitate˜ـــj`ــr in the same derivational form. Thus, the roots classification takes into account the following considerations: First: The category of the root and second: The vowels that are involved in root formulation. During the morphological analysis, a word might be represented in many forms. Arab grammarians Al-Dahdah (1985) classifies Arabic roots as shown below in Fig. 2. Regular roots: The non vowelized roots. This type of roots is sub-divided into the following categories: • Strong roots: The root that contains neither vowels nor ( ءhamzah and its second and third consonants are not identical, i.e., (ــ`عr ، ــ`بg ، €=ـــP) (ع) • Hamzated roots: The root that contain ( ءhamzah i.e., ( ; ، \ـــ`أŒــــr ، •Ž )أ •Doubled roots: the root in which its second and third consonant are identical i.e., ([د ، •[دY ، )•`ر For example, the root لv •ــmay have many derivational forms. Let us shed light on the generation of the hollow verb said for all person, gender and tenses with singularity, duality and plurality conjugational cases respectively as shown in Table 4. Irregular roots: The vowelized roots. This type of roots is classified into four types depending on which of the root letters is affected: 630 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Table 4. Derivation for the second root hollow-verb say لیv •ــadopted from (Abu Shquier, 2013; Abu Shquier et al., 2012) Singular Dual Plural ---------------------------------------- -------------------------------------------- -------------------------------------------------- Features Past Pres Imp Past Pres Imp Past Pres Imp 1st-M ™ ••ـــ لv أ•ــ mـــــx•• لvـــšt mـــــx•• لvـــšt 1st-F ™ ••ـــ لv أ•ــ mـــــx•• لvـــšt mـــــx•• لvـــšt 2nd-M ™•• َ لvــــš› ; •ــmـــــUO•• œvـــــš› œv •ـــnــــــO•• نvــــــPvš› اvـــPv• 2nd-F ™•• ِ wــــــPvš› wــــPv• mـــــUO•• œvـــــš› œv •ـــ \| O•• ّ \|•š› َ \|•• َ 3rd-M لm •ــ لیv •ــ œm •ـــ یœv •ـــ اvPm •ـــ نیvـــPv• 3rd-F ™ـــPm• لvــــš› mـــــOPm• œvـــــš› \|•• َ \|•šj َ Table 5. Arabic Roots Classification Arabic roots representation Form 1st Letter 2nd Letter 3rd Letter Example XXX X X X €ـــــO ﮐ XVX X وX نv ﮐــ XVX X يX ریط XXV X X ؤŸ \ ـــ VXX وX X [V و VXX يX X ــ`یr HXX ءX X ﮐــ;أ X: Denotes the non vowel character, no ( )ا ,و ,يcharacters V: Denotes the vowel ( )ا ,و ,يcharacter H: Denotes the Z character, i.e., ( )أ، ؤ، ئ ation for the second root hollow-verb say لیv •ــadopted from (Abu Shquier, 2013; Abu Shquier et al., 2012) l D l Pl l root hollow-verb say لیv •ــadopted from (Abu Shquier, 2013; Abu Shquier et al., 2012) Table 4. Derivation for the second root hollow-verb say Table 6. Arabic attachable prefixes Prefix Meaning Prefix Meaning لm \ـــin the فand, therefore الthe M >ـــthen will لm >ـــand the, therefore كlike, as لm ﮐــlike the لfor, to ;ــP for the, to the ولand (for, to) ;ــP وand (for the, to the) وand والand the سwill لmـــــ£> therefore in the لœ وand for the لm و\ـــand in the € >ـــand in, therefore لm وﮐــand like the وبand in ولand will ; >ـــand for, therefore ولand for and to بin, with Table 7. Arabic attachable prefixes Prefix Meaning Prefix Meaning لm \ـــin the فand, therefore الthe M >ـــthen will لm >ـــand the, therefore كlike, as الکlike the لfor, to ;ــP for the, to the ولand (for, to) ;ــP وand (for the, to the) وand والand the سwill لmـــــ£> therefore in the لœ وand for the لm و\ـــand in the € >ـــand in, therefore لm وﮐــand like the وبand in ولand will ; >ـــand for, therefore ولand for and to بin, with Table 6. Arabic attachable prefixes From Table 4 above we can conclude that verbs of the form C1awaC3 have the perfective stem patterns C1uC3 and C1uwC3 and the imperfective stem patterns C1uC3 and C1uwC3. For example, qaAl لm( •ــfrom [qawal]) لv •ــto visit has the perfective qul ; •ــand qaAl لm •ــand the imperfective stems qul ; •ــand quwl. لv •ــ E.g.: perfect: Qultu I said and qaAlat she said imperfect: Yaqulna they (fem) said ••ــــ\|یand yaquwlu he says. Arabic Roots Classification one can conclude that based on the person, number and gender; hollow verbs are realised by two stems in both perfect (simple past) and imperfect tenses (simple present, simple future), one long and one short; the long stems occurs with a weak middle letter, while the long stem cause the middle letter to disappear. It is worth stressing at this point that the words that derived from roots contain ( ءhamzah) i.e., ( )أ، ؤ،ء،ئas one of their consonants might also change during the morphological process. For instance, the word to take (S, M, 3rd) •یŽ ؤ is derived from the root •Ž .أIn such cases, we consider all the other forms that might a root appears in, Table 5 categories the trilateral roots based on the position of ء (hamzah), vowel and non-vowel letters. Table 7. Arabic attachable prefixes This classification will be very helpful in identifying the original root form during the analysis process. Table 5 illus- trates a portion of the roots classification that we will adopt. Prefix Extraction Process The prefix analyser starts after matching a certain word against a set of possible patterns to handle its prefix/suffix sequence ambiguity, then we start parsing the word from its beginning to extract the longest- possible-prefix, The process stops when there is no more prefix(es) left for extraction. The output of the prefix analyser will be stored in a separate file for further processing. In Arabic text, the analysis of the word is much more complicated. A word can be pronounced differently based on the chosen possible root, this proves that the absence of diacritics can result in ambiguities. Figure 3 represents the prefix extraction module, the module starts with converting the word to the Arabic encoding system, then we remove all punctuations, diacratics, non letters and the special characters, we continue to replace the hamzated letters, أ، ئ،ؤwith alif ا and replace the Alif Al-Maqsorah یwith يand replace the ةTaa Al-Marbotah with ;هthe remainder of the module is illustrated in the Fig. 3. g y p On the other hand, the suffix analyser parses the word from the end through the beginning of the word, bearing the following condition during the extraction process, first: The suffix has to match the comparable fragment of the word, second: The suffix has to suit the suffix representation of the CFG and third: The suffix should satisfy the prefix/suffix joining rules (Al-Omari, 1995; Abu Shqeer, 2002). Suffixes can be attached to the end of the word according to certain rules. Table 9 represents a sample of the Arabic suffixes combining rules. Arabic Prefix/Suffix Analyser As a preprocess of the prefix/suffix analyser, we have to check whether a word is an article or not. However, when the word is not an article the system passes the word to the word analyser for further analysis. 631 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Fig. 3. Prefix extraction flowchart Fig. 3. Prefix extraction flowchart Table 8. Arabic prefixes joining RULES Prefix Meaning ب ال ک ال ل ب، ل ف ب، ل، ال ، و ، س أ ب، ك ، ل ، ف ، س و لœ ، ;ــP ، ب، ك ، ل ، ال ، و، س This particular process starts by executing the prefix analyser module which determine whether a word is preceeded by prefix(es) or not. Prefixes with Arabic language form a closed list. Arabic allows up to three prefixes to precede the word within certain rules. Table 6 and 7 illustrates these prefixes with their associated meanings. When the prefix analyser processes a word; it requires certain information to decide what to process and where to stop. Table 8 lists the prefixes and their corresponding combining rules based on Table 7. After determining the prefix/suffix that will be extracted, the analyser checks the entry of the previously extracted prefix/suffix to ensure that the order of the extracted prefix/suffix is correct, moreover, the stem generator finds a template that matches the proposed stem and then it checks if the extracted prefix is allowed to be concatenated with the generated stem by a certain template. Suffix Extraction Process This section presents the algorithm embedded in the suffix analyser module (Fig. 4). The algorithm expects a stream of characters as an input. 632 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Table 9. Arabic suffix joining rules Case Suffix Attachable Suffix 1 نی ت ، ي 2 ان ت ، ي 3 ات ت ، ي ، و 4 ون ت ، ي ، و 5 n ﮐــ ان ، ات ، ون ، ت ، ن ، ا ، و 6 n ھ ان ، ات ، ون ، ت ، ن ، ي ، ا ، و 7 ن ،n ،ھn ، ﮐــvــt ، ان ، ات ، ت ، ا ، ك ، ه ، وn ›ـــ 8 ي ، ت ، نm ›ـــ 9 أ ، ت ، ا ، هn ، ›ـــn ،ھn ﮐــ 10 ك ، ت ، ن ، ا ، وvــt ، ان ، ات 11 ه ، ت ، ن ، ي ، ا ، وvــt ، ان ، ات 12 و ، تn ، ›ـــn ،ھn ﮐــ Table 10. Generated triliteral roots representation Case Form Case Form Case Form 1 ;Y أ2 ;Y ؤ3 لŸ >ـــ 4 mY أ5 ؤال6 ءŸ >ـــ 7 vY أ8 وؤل9 لیف 10 wY أ11 ؤلی12 ئیف 13 یأع14 ؤوي15 يیف 16 األ17 أوؤ18 ئیف 19 أول20 ;Y إ21 ـــ˜فj 22 vY إ23 ایف24 ألی 25 ای ل26 >ـــ©ل27 وأا 28 یوع29 لm >ـــ30 أیف 31 wY و32 ءm >ـــ33 © >=ـــ 34 ©Y و35 لv >ـــ36 m >=ـــ 37 ªY و38 يv >ـــ39 T ی>ـــ 40 ;Y و41 ª >=ــــ42 وm >ـــ 43 w >=ــــ44 لیو45 ءv >ـــ 46 v >=ــــ47 ;یY 48 أv >ـــ 49 ءT >ـــ50 یعی51 وŸ >ـــ 52 Ÿ >=ــــ53 ولی54 Ÿ >ـــ 55 « >=ـــ56 الی57 ;ـــــŒ> 58 wŒ >ـــــ59 أول60 ; >=ــــ It produces a list of parameters which express the extracted suffixes. After the extraction of prefixes and suffixes, the remaining part of the word obtained is called the stem. Table 4 exhibts the procedures of extracting the suffix from a certian word. Notice that P+1 means the number of possible prefixes including the null prefix and S+1 denotes the Table 9. Generation of Arabic Roots The root generation algorithm expects three arguments as input: Prefix, suffix and stem. The algorithm finds all the template(s) that are related to the stem according to the rules mentioned in Table 9. As shown in Fig. 6, the root generation process aims to find a template that can represent the stem under certain conditions, first: Both of the template and the stem must be of the same length. Second: The template must be a valid form for the extracted possible prefix and Third: The template is attachable to the associated possible suffix (Al-Omari, 1995). Table 10. Generated triliteral roots representation Case Form Case Form Case Form 1 ;Y أ2 ;Y ؤ3 لŸ >ـــ 4 mY أ5 ؤال6 ءŸ >ـــ 7 vY أ8 وؤل9 لیف 10 wY أ11 ؤلی12 ئیف 13 یأع14 ؤوي15 يیف 16 األ17 أوؤ18 ئیف 19 أول20 ;Y إ21 ـــ˜فj 22 vY إ23 ایف24 ألی 25 ای ل26 >ـــ©ل27 وأا 28 یوع29 لm >ـــ30 أیف 31 wY و32 ءm >ـــ33 © >=ـــ 34 ©Y و35 لv >ـــ36 m >=ـــ 37 ªY و38 يv >ـــ39 T ی>ـــ 40 ;Y و41 ª >=ــــ42 وm >ـــ 43 w >=ــــ44 لیو45 ءv >ـــ 46 v >=ــــ47 ;یY 48 أv >ـــ 49 ءT >ـــ50 یعی51 وŸ >ـــ 52 Ÿ >=ــــ53 ولی54 Ÿ >ـــ 55 « >=ـــ56 الی57 ;ـــــŒ> 58 wŒ >ـــــ59 أول60 ; >=ــــ Form لŸ >ـــ ءŸ >ـــ لیف ئیف يیف ئیف ـــ˜فj ألی وأا أیف © >=ـــ m >=ـــ T ی>ـــ وm >ـــ ءv >ـــ أv >ـــ وŸ >ـــ Ÿ >ـــ ;ـــــŒ> ; >=ــــ Triliteral Root Processor The three letters root processor aims to refer the generated root to their original root form (Arabic Orthography). Previously, we classify the roots according to two characteristics. First: The positions of the vowels and ( ءhamzah). Second: The vowels and the forms of the written ( ءhamzah) which are involved in the formulation of the root. Here, we use these classifications to recode the root to its original root form, however, regular root nPmــــــbP’•راP اis the only type of root that need not any recoding process since it does not contain any vowel or ( ءhamzah). Furthermore, in some cases, a vowel might be converted to a non vowel which cause the root to be recoded. It produces a list of parameters which express the extracted suffixes. After the extraction of prefixes and suffixes, the remaining part of the word obtained is called the stem. Table 4 exhibts the procedures of extracting the suffix from a certian word. Table 10 shows the generated trilliteral root representation, a special recoding process is conducted for each form listed below: We have used the Morphological Balance (MB) (C1aC2aC3a) for all the form representation, the Arabic three consonants ع ,ف and لin the root-verb (; )>=ــــare represented as (C1), (C2) and (C3) respectively, however, vowels and hamzah (ـ ، ا ، و ، ي، ءŒ )ؤ ، ئ ، أ ، ی، ـhave replaced their corresponding consonents ع , فand لin the root-verb (; .)>=ــــFor each form represented in Table 10, there is a corresponding recoding process implemented, we will discuss the usage of Table 10 throughout the following examples. Let us take the word ـــ`بit أas an example. There would be two possible stems for this word ـــ`بit إi.e., (`ـــit )بand ــ`بg .)إ Notice that P+1 means the number of possible prefixes including the null prefix and S+1 denotes the number of possible suffixes including the null suffix. Due to the possibility of the improper prefixes/suffixes extraction. The morphological analyser should be smart enough to generate all possible stems as well as the joining rules of prefixes and templates. Suffix Extraction Process Arabic suffix joining rules The root extraction process matches the stem with the corresponding template based on the verb (C1aC2aC3a) ; .>=ــــThe system will recode the root and then decide whether it is a correct not. An enhanced structure of the Arabic words has been shown in Fig. 5; For example, the word نیفv\`ــــg can be simplified to the following components: Prefixes فroot prefixes يroot ــ`بg (no embedded infix), suffixes ونthere is no root suffixes for the word نیفv\`ــــg. Arabic Roots The Arabic roots can be classified into two classes; the Vowelized roots and Non-Vowelized Roots (Al-Dahdah, 1985). This classification was made in accordance with the availability of the Arabicvowels in the roots. 633 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 q DOI: 10.3844/jcssp.2015.627.638 j p Fig. 4. Suffix extraction flowchart Fig. 5. Enhanced Structure of Arabic word Fig. 6. Root generation flowchart Fig. 4. Suffix extraction flowchart Fig. 4. Suffix extraction flowchart Fig. 5. Enhanced Structure of Arabic word Fig. 6. Root generation flowchart Fig. 6. Root generation flowchart 634 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 The recoding process of ـــ`بit إis shown below: The proposed testing technique of the developed system consists of two main steps to evaluate the performance of the morphological analyser: • Input word: ـــ`بit after removing the prefix • Input word: ـــ`بit after removing the prefix • Prefix: إ • Neither using the roots dictionary nor the root decision table •Stem: ـــ`بit •Stem: ـــ`بit • Template Form: ;…=ـــــt • With using roots dictionary but not the roots decision table • Generated Root: ــ`بg • Recoded Root (1): ــ`بg • Recoded Root (1): ــ`بg • With using both the roots dictionary and the roots decision table As presented above the stem will be analysed and the root ــ`بg will be generated. The root will remain as it is during the recoding process. A. The First Test We may have another result of the word ـــ`بit إto be analysed as follows: In this test, the system is used to process the text using neither dictionary nor the root decision table. However, the system was not able to return the correct analyses of the triliteral words. • Stem: ـــ`بit إ •Template Form: ;…=ـــــt إpast present of the verb …=ـــــ;یt of yanC1aC2iC3 Table 3 - Form 7 • Stem: ـــ`بit إ •Template Form: ;…=ـــــt إpast present of the verb …=ـــــ;یt of yanC1aC2iC3 Table 3 - Form 7 •Template Form: ;…=ـــــt إpast present of the verb …=ـــــ;یt of yanC1aC2iC3 Table 3 - Form 7 After removing the 94 article of the test data, 547 words remaining. In this test the number of analyses returned is 1034 with only 345 correct analysis. Figure 8 shows the percentage of errors obtained from the first test. The second result should be discarded since the word is not used in Arabic despite the correct analysis of the word. The absence of the roots dictionary and the roots decision table are the main reasons behind this result. Another reason might be due to the type of the texts. The texts that contain less vowelized roots will have smaller percentage of errors since vowelized-derived words may have more analyses. Therefore, this factor should be taken into consideration in the evaluation of the system. To reduce the errors we may need the roots dictionary and the roots decision table. Figure 9 shows the analysis of the factors affecting the result. Method Limitation When the system is integrated with some applications like Machine Translation (MT) where the template affects the Part of Speech (POS) (Part of Speech (POS) is the method of classification of words according to their meaning, functions and categories such as noun, verb and adjective. The POS tagging occurs during the Syntactic Analysis phase and it involves assigning of words to their proper part-of-speech tag), in this case the generation of the correct root leads to correct solution, however, in some cases of our method where a particular templates starts with a character that is considered as a prefix. i.e., if the template ; أ>=ــــwas used to derive the word `£ ,أﮐـــــthe analyzer will consider the character أas a prefix and produces the root `£ ﮐـــــthat matches the template ; >=ــــand that cause ambiguity, however, such issues occur when there are more than one correct analysis for a particular word, in other cases we may obtain three correct roots with respect to the morphological process, while semantically, one of them only is correct. As shown above, most of the errors occur due to the absence of the roots dictionary. Some of these errors can also be due to the morphological rules of the system which can be reduced when applying a roots dictionary. Three percent of the errors returned as a result of the misuse of the morphological rules. These rules can be reconstructed to eliminate this percentage. Ten percent of the errors are due to the absence of the roots decision table. The correct roots obtained from this test can be classified into two categories as follows: • Exact root: This occurs when there is only one analysis for a given word. For example, from the word € حی•ــ we will obtain the root ¬ــــ£• from the system Experiments and Results In this section we will be testing the performance of the developed system, we will not be able to conduct a precise evaluation of the system, since the system has not yet been integrated with any other system. However, the test will help in understanding the capabilities of the system better. The test data is taken from one poem ¬ــP ةایاmــــs• ﮐ€ ›=ــــfor Abu Elalaa Al ,أوا ءا ـــــــرى which contains 641 tokens. Figure 7 shows a pie chart for the breakdown of articles and words in the text. • Ambiguous root: This occurs when there is more than one correct analysis for a particular word. For example, from the word ﮐـــ\|یthe system will return three different roots. i.e., (نvن ، ﮐـــm ﮐـــand \| .)ﮐـــThese roots are all correct with respect to the morphological process, while it is only one correct root when considering semantics. Figure 10 shows the analysis of the correct results obtained from the first test. 635 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Fig. 7. Words and articles in poem ¬ــP ةایاmــــs• ﮐ€ ›=ــــ Fig. 8. Percentage of errors retruned from the first test Fig. 9. Analysis of Errors in the first test Fig. 10. Analysis of the correct results Fig. 12. Errors analysis for the second test Fig. 13. Percentage of Errors returned from the Third test Fig. 14. Experiment results The ambiguous analysis can be due to the following factors: • The root types • The Proper usage of the template: Templates that starts with character that can be considered as a prefix. For example, if the template ; أ>=ــــwas used to derive the word doubled `•أ ّ , the analyzer will consider the character أas a prefix and produces the root `• ّ with the template T> ّ which will be matching the template ; >=ــــafter the separation of the doubled letter. Since the system will be integrating with Fig. 7. Words and articles in poem ¬ــP ةایاmــــs• ﮐ€ ›=ــــ Fig. 8. Percentage of errors retruned from the first test Fig. 7. Words and articles in poem ¬ــP ةایاmــــs• ﮐ€ ›=ــــ Fig. 12. Errors analysis for the second test Fig. 12. Errors analysis for the second test Fig. 13. Author’s Contributions •some other morphological rules that are not manipulated in the system The author’s contributions played a significant role in the following categories as shown below: Ethics The manuscript has not been previously published or accepted for publication elsewhere, either in whole (including book chapters) or in part (including paragraphs of text or exhibits), whether in English or another language. Experiments and Results Percentage of Errors returned from the Third test Fig. 12. Errors analysis for the second test Fig. 13. Percentage of Errors returned from the Third test Fig. 14. Experiment results Fig. 12. Errors analysis for the second test Fig. 12. Errors analysis for the second test Fig. 7. Words and articles in poem ¬ــP ةایاmــــs• ﮐ€ ›=ــــ Fig. 8. Percentage of errors retruned from the first test Fig. 13. Percentage of Errors returned from the Third test Fig. 8. Percentage of errors retruned from the first test Fig. 9. Analysis of Errors in the first test Fig. 9. Analysis of Errors in the first test Fig. 10. Analysis of the correct results Fig. 11. Percentage of Errors returned from the second test Fig. 13. Percentage of Errors returned from the Third test Fig. 13. Percentage of Errors returned from the Third test Fig. 9. Analysis of Errors in the first test Fig. 10. Analysis of the correct results Fig. 14. Experiment results The ambiguous analysis can be due to the following factors: Fig. 10. Analysis of the correct results • The root types Fig. 10. Analysis of the correct results Fig. 11. Percentage of Errors returned from the second test • The Proper usage of the template: Templates that starts with character that can be considered as a prefix. For example, if the template ; أ>=ــــwas used to derive the word doubled `•أ ّ , the analyzer will consider the character أas a prefix and produces the root `• ّ with the template T> ّ which will be matching the template ; >=ــــafter the separation of the doubled letter. Since the system will be integrating with other application, such as machine translation, the determination of the correct root is the main part of the correct solution. Fig. 11. Percentage of Errors returned from the second test 636 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 enhancing the pattern-based stemmer. At present, designing a fully-automated Arabic morphological analyser might not be possible. Instead, analysers should be application-oriented or for specific domain. On the other hand, the system has rejected some words due to different reasons these are: • The word was derived from quadrilateral root (e.g., ;ــrنیm•) The Third Test This test has been carried out manually since the roots decision table is not yet available. This test proves that the errors that occurred in the second test can be reduced. Figure 13 shows errors that has been eliminated to 4%. References Abu-Ata, B., 2001. An Arabic stemming algorithm on ERA for information retrieval. PhD. Thesis, Universiti Kebangsaan Malaysia. The Second Test Abu Shquier: Conception, design, analysis and Interpretation. Final approval of the article. Statistical analysis and Overall responsibility. This test considers that there is a roots dictionary as a component of the morphological analyzer. Figure 11 shows the percentage of errors encountered in this test. As we can see, the percentage of errors has been reduced from 67% to 12%. This emphasizes the urgent need for the roots dictionary. The error occurred in this test can be reduced further if the roots decision table is included in the system (as we will see in the third test). Figure 12 shows the analysis of the errors encountered in the second test. Alhawiti: Data collection and Critical revision of the article and obtained funding. Funding Information • The words which have no Arabic root (e.g., اءvــsP )ا •The words written in different way because of letter- dropping grammars (e.g., حm )®ــwhich is originally came from (€¯m )ی®ــ Conclusion Abu Shqeer, O., 2002. Handling agreement and word reordering in English to Arabic machine translation. Master Thesis, University of Sains Malaysia. Stemmers and word analysers usually help in resolving the lexical ambiguity, The goal of this paper is to develop a stemmer for the triliteral words of Arabic Language. However, as we analysed Arabic morphology deeply, we realised that the problem is not just a matter of truncating affixes to obtain the stem; the analysis requires heavy computational processes and the usage of large amount of information; on the other hand, the system might be used as an Arabic morphological analyser for general domain since the database can be updated to cover all the Arabic trilateral roots. The three conducted tests prove that the morphological rules used in the system has reduced the errors by 94% when using both the roots dictionary and the roots decision table that we implement. Figure 14 shows a Bar chart comparing the three tests results. In fact, building practical stemmers or morphological analysers requires fully understanding of the language morphology structure. To enhance the output of the morphological analyser, we recommed conducting the following steps: Reducing the rules number and increasing language coveragance while keeping the same level of performance and functionality. Merging rules is very helpful for Abu Shquier, M. and O. Abu Shqeer, 2012. Words ordering and corresponding verb-subject agreements in English-Arabic machine translation: Hybrid- based approach. Int. J. Soft Comput. Software Eng., 2: 49-60. DOI: 10.7321/jscse.v2.n8.5 Abu Shquier, M.M., 2013. Computational approach to the derivation and inflection of Arabic irregular verbs in English-Arabic machine translation. Int. J. Advance. Comput. Technol., 5: 1-21. Al-Atram, M.A., 1990. Effectiveness of natural language in indexing and retrieving Arabic documents [in Arabic] (King Abdulaziz City for Science and Technology Project number AR-8-47). Riyadh, Saudi Arabia. Al-Dahdah, A., 1985. Arabic language grammar dictionary. Lebanon Library, Lebanon. Al-Fedaghi, S.S. and F. Al-Anzi, 1989. A new algorithm to generate Arabic root-pattern forms. Proceedings of the 11th National Computer Conference and Exhibition, (CCE 89), pp: 391-400. 637 Mohammed M. Abu Shquier and Khaled M. Alhawiti / Journal Computer Science 2015, 11 (4): 627.638 DOI: 10.3844/jcssp.2015.627.638 Larkey, L., L. Ballesteros and M.E. Connell, 2002. Improving stemming for Arabic information retrieval: Light Stemming and co-occurrence analysis. Proceedings of the 25th Annual International ACM SIGIR Conference on Research and Development in Information Retrieval, Aug. 11- 15, ACM, New York, USA, pp: 275-282. Conclusion DOI: 10.1145/564376.564425 Al-Fedaghi, S.S. and H.B. Al-Sadoun, 1990. Morphological compression of Arabic text. Inform. Proc. Manage., 26: 303-316. DOI: 10.1016/0306-4573(90)90033-X Ali, N., 1992. Parsing and automatic diacritization of written Arabic: A breakthrough. Proceedings of the 13th National Computer Conference (NCC’ 92), King Abdul-Aziz City for Science and Technology, Riyadh, KSA, pp: 794-812. Momani, M. and J. Faraj, 2007. A novel algorithm to extract tri-literal Arabic roots. Proceedings of the IEEE/ACS International Conference on Computer Systems and Applications. May 13-16, IEEE Xplore Press, Amman, pp: 309-315. DOI: 10.1109/AICCSA.2007.370899 Al-Khuli, M., 1991. A dictionary of theoretical linguistics: English-Arabic with an Arabic-English glossary. Library of Lebanon. Al-Momani, I., 2010. Does the VP node exist in modern standard Arabic?. J. Langu. Literature, 2: 76-76. Al-Omari, H., 1995. ALMAS: An Arabic language morphological analyzer system. Malaysian J. Comput. Sci., 8: 30-50. Mustafa, S.H. and Q.A. Al-Radaideh, 2004. Using n- grams for Arabic text searching. J. Am. Society Inform. Sci. Technol., 55: 1002-1007. DOI: 10.1002/asi.20051 Al-Shammari, E.T. 2010. Improving Arabic text processing via stemming with application to text mining and web retrieval. PhD Thesis, George Mason University, USA. Rafea, A.A. and K.F. Shaalan, 1993. Lexical analysis of inflected Arabic words using exhaustive search of an augmented transition network. Software Practice Experience, 23: 567-588. DOI: 10.1002/spe.4380230602 Attia, M., 2008. Handling Arabic morphological and syntactic ambiguity within the LFG framework with a view to machine translation. PhD Thesis, University of Manchester. Saliba, B. and A. Al-Dannan, 1990. Automatic morphological analysis of Arabic: A study of content word analysis. Proceedings of the First Kuwait Computer Conference (KCC’ 90), pp: 231-243. Boudlal, A., R. Belahbib, A. Belahbib and A. Mazroui, 2011. A markovian approach for Arabic root extraction. Int. Arab J. Inform., 8: 91-98. Salton, G., 1989. Automatic text processing: The transformation, analysis and retrieval of information by computer. Addison-Wesley, Reading, ISBN-10: 0201122278, pp: 530. George, M., 1990. Al Khaleel: A dictionary of Arabic syntax terms. Beirut: Library of Lebanon. Hawas, F.A., 2013. Exploit relations between the word letters and their placement in the word for Arabic root extraction. Comput. Sci., 14: 327-431. DOI: 10.7494/csci.2013.14.2.327 Thalouth, B. and A. Al-Dannan, 1987. A comprehensive Arabic morphological analyzer/generator. IBM Kuwait Scientific Center, Kuwait. Khoja, S. and R. Garside, 1999. Stemming Arabic text. Lancaster, UK, Department of Computing, Lancaster University. Wightwick, J. and M. Gaffar, 2007. Arabic Verbs and Essintials of Grammar. 2nd Edn. McGraw-Hill, New York, ISBN-10: 0071596038, pp: 160. Conclusion Khreisat, L., 2006. Arabic text classification using N-Gram frequency statistics a comparative study. Proceedings of the International Conference on Data Mining (CDM’ 06), Las Vegas, NV: USCCM, pp: 7882. Yusif, J., 2007. Automatic part of speech tagger for Arabic language using neural network. PhD Thesis, National University of Malaysia. 638
https://openalex.org/W3109269546	https://pilotfeasibilitystudies.biomedcentral.com/track/pdf/10.1186/s40814-021-00770-x	English	null	Determining sample size for progression criteria for pragmatic pilot RCTs: the hypothesis test strikes back!	Pilot and feasibility studies	2,021	cc-by	13,036	Determining sample size for progression criteria for pragmatic pilot RCTs: the hypothesis test strikes back! M. Lewis1,2* , K. Bromley1,2, C. J. Sutton3, G. McCray1,2, H. L. Myers2 and G. A. Lancaster1,2 © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Keywords: Outcome and process assessment, Pilots, Sample size, Statistics * Correspondence: a.m.lewis@keele.ac.uk 1Biostatistics Group, School of Medicine, Keele University, Room 1.111, David Weatherall Building, Keele, Staffordshire ST5 5BG, UK 2Keele Clinical Trials Unit, Keele University, Keele, Staffordshire, UK Full list of author information is available at the end of the article Lewis et al. Pilot and Feasibility Studies (2021) 7:40 https://doi.org/10.1186/s40814-021-00770-x Lewis et al. Pilot and Feasibility Studies (2021) 7:40 https://doi.org/10.1186/s40814-021-00770-x Open Access Abstract Background: The current CONSORT guidelines for reporting pilot trials do not recommend hypothesis testing of clinical outcomes on the basis that a pilot trial is under-powered to detect such differences and this is the aim of the main trial. It states that primary evaluation should focus on descriptive analysis of feasibility/process outcomes (e.g. recruitment, adherence, treatment fidelity). Whilst the argument for not testing clinical outcomes is justifiable, the same does not necessarily apply to feasibility/process outcomes, where differences may be large and detectable with small samples. Moreover, there remains much ambiguity around sample size for pilot trials. Methods: Many pilot trials adopt a ‘traffic light’ system for evaluating progression to the main trial determined by a set of criteria set up a priori. We construct a hypothesis testing approach for binary feasibility outcomes focused around this system that tests against being in the RED zone (unacceptable outcome) based on an expectation of being in the GREEN zone (acceptable outcome) and choose the sample size to give high power to reject being in the RED zone if the GREEN zone holds true. Pilot point estimates falling in the RED zone will be statistically non- significant and in the GREEN zone will be significant; the AMBER zone designates potentially acceptable outcome and statistical tests may be significant or non-significant. Results: For example, in relation to treatment fidelity, if we assume the upper boundary of the RED zone is 50% and the lower boundary of the GREEN zone is 75% (designating unacceptable and acceptable treatment fidelity, respectively), the sample size required for analysis given 90% power and one-sided 5% alpha would be around n = 34 (intervention group alone). Observed treatment fidelity in the range of 0–17 participants (0–50%) will fall into the RED zone and be statistically non-significant, 18–25 (51–74%) fall into AMBER and may or may not be significant and 26–34 (75–100%) fall into GREEN and will be significant indicating acceptable fidelity. Discussion: In general, several key process outcomes are assessed for progression to a main trial; a composite approach would require appraising the rules of progression across all these outcomes. This methodology provides a formal framework for hypothesis testing and sample size indication around process outcome evaluation for pilot RCTs. * Correspondence: a.m.lewis@keele.ac.uk 1Biostatistics Group, School of Medicine, Keele University, Room 1.111, David Weatherall Building, Keele, Staffordshire ST5 5BG, UK 2Keele Clinical Trials Unit, Keele University, Keele, Staffordshire, UK Full list of author information is available at the end of the article Background Sample size may be based on preci- sion of a feasibility parameter [6, 7]; precision of a clinical parameter which may inform main trial sample size—particularly the standard deviation (SD) [8–11] but also event rate [12] and effect size [13, 14]; or, to a lesser degree, for clinical scale evaluation [9, 15]. Billingham et al. [16] reported that the median sample size of pilot and feasibility studies is around 30–36 per group but there is wide variation. Herbert et al. [17] reported that targets within internal as opposed to external pilots are often slightly larger and somewhat different, being based on percentages of the total sample size and timeline ra- ther than any fixed sample requirement. p A common misuse of pilot and feasibility studies has been the application of hypothesis testing for clinical outcomes in small under-powered studies. Arain et al. [19] claimed that pilot studies were often poorly re- ported with inappropriate emphasis on hypothesis test- ing. They reviewed 54 pilot and feasibility studies published in 2007–2008, of which 81% incorporated hy- pothesis testing of clinical outcomes. Similarly, Leon et al. [20] stated that a pilot is not a hypothesis testing study: safety, efficacy and effectiveness should not be evaluated. Despite this, hypothesis testing has been com- monly performed for clinical effectiveness/efficacy with- out reasonable justification. Horne et al. [21] reviewed 31 pilot trials published in physical therapy journals be- tween 2012 and 2015 and found that only 4/31 (13%) carried out a valid sample size calculation on effective- ness/efficacy outcomes but 26/31 (84%) used hypothesis testing. Wilson et al. [22] acknowledged a number of statistical challenges in assessing potential efficacy of complex interventions in pilot and feasibility studies. The CONSORT extension [2] re-affirmed many re- searchers’ views that formal hypothesis testing for effect- iveness/efficacy is not recommended in pilot/feasibility studies since they are under-powered to do so. Sim’s commentary [23] further contests such testing of clinical outcomes stating that treatment effects calculated from pilot or feasibility studies should not be the basis of a sample size calculation for a main trial. The need for a clear directive on sample size of studies is of upmost relevance. Background implications for all key stakeholders (funders, re- searchers, clinicians and patients). The CONSORT ex- tension [2] states that “decision processes about how to proceed needs to be built into the pilot design (which might involve formal progression criteria to decide whether to proceed, proceed with amendments, or not to proceed)” and authors should present “if applicable, the pre-specified criteria used to judge whether or how to proceed with a future definitive RCT; … implications for progression from pilot to future definitive RCT, in- cluding any proposed amendments”. Avery et al. [18] published recommendations for internal pilots empha- sising a traffic light (stop-amend-go/red-amber-green) approach to progression with focus on process assess- ment (recruitment, protocol adherence, follow-up) and transparent reporting around the choice of trial design and the decision-making processes for stopping, amend- ing or proceeding to a main trial. The review of Herbert et al. [17] reported that the use of progression criteria (including recruitment rate) and traffic light stop- amend-go as opposed to simple stop-go is increasing for internal pilot studies. The importance and need for pilot and feasibility studies is clear: “A well-conducted pilot study, giving a clear list of aims and objectives … will encourage methodological rigour … and will lead to higher quality RCTs” [1]. The CONSORT extension to external pilot and feasibility tri- als was published in 2016 [2] with the following key methodological recommendations: (i) investigate areas of uncertainty about the future definitive RCT; (ii) ensure primary aims/objectives are about feasibility, which should guide the methodology used; (iii) include assess- ments to address the feasibility objectives which should be the main focus of data collection and analysis; and (iv) build decision processes into the pilot design whether or how to proceed to the main study. Given that many trials incur process problems during imple- mentation—particularly with regard to recruitment [3– 5]—the need for pilot and feasibility studies is evident. One aspect of pilot and feasibility studies that remains unclear is the required sample size. There is no consen- sus but recommendations vary from 10 to 12 per group through to 60–75 per group depending on the main ob- jective of the study. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Page 2 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies Background Schoenfeld [25] advocates that, even for clinical outcomes, there may be a place for testing at the level of clinical ‘indica- tion’ rather than ‘clinical evidence’. He suggested that preliminary hypothesis testing for efficacy could be con- ducted with high alpha (up to 0.25), not to provide de- finitive evidence but as an indication as to whether a larger study should be conducted. Lee et al. [14] also re- ported how type 1 error levels other than the traditional 5% could be considered to provide preliminary evidence for efficacy, although they did stop short of recommend- ing doing this by concluding that a confidence interval approach is preferable. it aligns with the main aims and objectives of pilot and feasibility studies and with the progression stop-amend- go recommendations of Eldridge et al. [2] and Avery et al. [18]. Progression rules Let E denote the observed point estimate (ranging from 0 to 1 for proportions, or for percentages 0–100%). Sim- ple 3-tiered progression criteria would follow as: pp p Current recommendations for sample sizes of pilot/ feasibility studies vary, have a single rather than a multi- criterion basis, and do not necessarily link directly to formal progression criteria. The purpose of this article is to introduce a simple methodology that allows sample size derivation and formal testing of proposed progres- sion cut-offs, whilst offering suggestions for multi- criterion assessment, thereby giving clear guidance and sign-posting for researchers embarking on a pilot/feasi- bility study to assess uncertainty in feasibility parameters prior to a main trial. The suggestions within the article do not directly apply to internal pilot studies built into the design of a main trial, but given the similarities to external randomised pilot and feasibility studies, many of the principles outlined here for external pilots might also extend to some degree to internal pilots of rando- mised and non-randomised studies. E ≤RUL [P value non-significant (P ≥α)] -> RED (unacceptable—STOP) RUL < E < GLL -> AMBER (potentially acceptable—AMEND) E ≥GLL [P value significant (P < α)] -> GREEN (acceptable—GO) E ≤RUL [P value non-significant (P ≥α)] -> RED (unacceptable—STOP) RUL < E < GLL -> AMBER (potentially acceptable—AMEND) E ≥GLL [P value significant (P < α)] -> GREEN (acceptable—GO) Hypothesis concept Let RUL denote the upper RED zone cut-off and GLL de- note the lower GREEN zone cut-off. The concept is to set up hypothesis testing around progression criteria that tests against being in the RED zone (designating unacceptable feasibility—‘STOP’) based on an alternative of being in the GREEN zone (designating acceptable feasibility—‘GO’). This is analogous to the zero difference (null) and clinic- ally important difference (alternative) in a main superiority trial. Specifically, we are testing against RUL when GLL is hypothesised to be true: Null hypothesis: True feasibility outcome (ε) not greater than the upper “RED” stop limit (RUL) Alternative hypothesis: True feasibility outcome (ε) is greater than RUL The test is a 1-tailed test with suggested alpha (α) of 0.05 and beta (β) of 0.05, 0.1 or 0.2, dependent on the required strength of evidence of the test. An example of a feasibility outcome might be percentage recruitment uptake. Background The CONSORT extension [2] re- ports that “Pilot size should be based on feasibility objec- tives and some rationale given” and states that a “confidence interval approach may be used to calculate and justify the sample size based on key feasibility objec- tive(s)”. Specifically, item 7a (How sample size was de- termined: Rationale for numbers in the pilot trial) qualifies: “Many pilot trials have key objectives related to estimating rates of acceptance, recruitment, retention, or uptake … for these sorts of objectives, numbers required in the study should ideally be set to ensure a desired de- gree of precision around the estimated rate”. Item 7b (When applicable, explanation of any interim analyses and stopping guidelines) is generally an uncommon sce- nario for pilot and feasibility studies and is not given consideration here. However, when the focus of analysis is on confidence interval estimation for process outcomes, this does not give a definitive basis for acceptance/rejection of pro- gression criteria linked to formal powering. The issue in A key aspect of pilot and feasibility studies is to inform progression to the main trial, which has important Page 3 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies this regard is that precision focuses on alpha (α, type I error) without clear consideration of beta (β, type II error) and may therefore not reasonably capture true dif- ferences if a study is under-powered. Further, it could be argued that hypothesis testing of feasibility outcomes (as well as addressing both alpha and beta) is justified on the grounds that moderate-to-large differences (‘process- effects’) may be expected rather than small differences that would require large sample numbers. Moore et al. [24] previously stated that some pilot studies require hy- pothesis testing to guide decisions about whether larger subsequent studies can be undertaken, giving the follow- ing example of how this could be done for feasibility outcomes: asking the question “Is taste of dietary supple- ment acceptable to at least 95% of the target popula- tion?”, they showed that sample sizes of 30, 50 and 70 provide 48%, 78% and 84% power to reject an accept- ance rate of 85% or lower if the true acceptance rate is 95% using a 1-sided α = 0.05 binomial test. Sample size Table 1 displays a quick look-up grid for sample size across a range of anticipated proportions for RUL and GLL for one-sample one-sided 5% alpha with typical 80% and 90% (as well as 95%) power for the normal approxi- mation method with continuity correction (see Appen- dix for corresponding mathematical expression; derived from Fleiss et al. [26]). Table 2 is the same look-up grid relating to the Binomial exact approach with sample sizes derived using GPower version 3.1.9.7 [27]. Clearly, as the difference between proportions RUL and GLL in- creases the sample size requirement is reduced. Methods The proposed approach focuses on estimation and hy- pothesis testing of progression criteria for feasibility out- comes that are potentially modifiable (e.g. recruitment, treatment fidelity/ adherence, level of follow up). Thus, Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Methods Pilot and Feasibility Studies (2021) 7:40 Page 5 of 14 Table 1 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on normal approximation (with continuity correction) (Continued) Table 1 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on normal approximation (with continuity correction) (Continued) RUL GLL α (0.05), β (0.2) α (0.05), β (0.1) α (0.05), β (0.05) % % n Ac (%) n Ac (%) n Ac (%) 65 85 34 26.7 (78.5) 44 33.8 (76.8) 52 39.5 (75.9) 70 80 129 98.9 (76.6) 170 128.8 (75.8) 209 157.2 (75.2) 70 85 56 44.8 (80.1) 72 56.8 (78.9) 87 67.9 (78.1) 75 85 113 92.3 (81.7) 147 118.9 (80.9) 179 143.8 (80.3) 75 90 48 40.9 (85.3) 60 50.5 (84.2) 71 59.3 (83.5) 80 90 93 80.7 (86.8) 119 102.4 (86.0) 143 122.3 (85.5) RUL upper limit of RED zone (expressed as percentage of total sample), GLL lower limit of GREEN zone (expressed as percentage of total sample), AC AMBER- statistical significance threshold (within the AMBER zone) where an observed estimate below the cut-point will result in a non-significant result (p ≥0.05) and figures at or above the cut-point will be significant (p < 0.05) (%, as a percentage of n) Sample sizes were derived using the normal approximation to the binomial distribution (with continuity correction) formula given in the Appendix, which by convention is stable for np > 5 and n(1 −p) > 5. For this approach, AC% is calculated from the 1-sided upper 95% confidence limit for the null proportion: 100% × (RUL + z1−α√((RUL(1 −RUL))/n)) [e.g. for RUL = 20% v GLL = 35%, n = 78, power 90%: AC% = 100% × (0.2 + 1.645√((0.2(1 −0.2))/78)) = 27.5%. In the example this is expressed as a proportion (0.275)] The AC values do not account for the continuity correction (−0.5 deduction) which would need to be applied to the observed count from a study prior to cross- checking against the AC cut-offs provided here Methods Pilot and Feasibility Studies Page 4 of 14 Table 1 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on normal approximation (with continuity correction) Table 1 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on normal approximation (with continuity correction) Table 1 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on normal approximation (with continuity correction) Table 1 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on normal approximation (with continuity correction) RUL GLL α (0.05), β (0.2) α (0.05), β (0.1) α (0.05), β (0.05) % % n Ac (%) n Ac (%) n Ac (%) 10 20 79 12.3 (15.6) 111 16.3 (14.7) 143 20.2 (14.1) 15 25 101 21.1 (20.8) 140 28.0 (20.0) 179 34.7 (19.4) 15 30 49 11.5 (23.4) 68 15.0 (22.1) 87 18.5 (21.3) 20 30 119 31.0 (26.0) 165 41.5 (25.1) 209 51.3 (24.6) 20 35 57 16.4 (28.7) 78 21.4 (27.5) 99 26.3 (26.6) 20 40 34 10.6 (31.3) 46 13.7 (29.7) 59 16.9 (28.6) 25 35 134 41.7 (31.2) 185 55.9 (30.2) 234 69.4 (29.7) 25 40 63 21.4 (34.0) 86 28.1 (32.7) 109 34.7 (31.8) 25 45 37 13.6 (36.7) 51 17.8 (35.0) 64 21.7 (33.9) 25 50 25 9.8 (39.2) 33 12.3 (37.4) 42 15.1 (36.0) 30 40 146 52.9 (36.2) 201 71.0 (35.3) 253 87.9 (34.7) 30 45 68 26.6 (39.1) 93 35.2 (37.8) 117 43.3 (37.0) 30 50 39 16.4 (42.1) 54 21.7 (40.3) 67 26.3 (39.2) 30 55 26 11.6 (44.8) 35 15.0 (42.7) 44 18.2 (41.4) 30 60 18 8.6 (47.8) 25 11.3 (45.1) 30 13.1 (43.8) 35 45 155 64.0 (41.3) 212 85.6 (40.4) 267 106.3 (39.8) 35 50 71 31.5 (44.3) 97 41.7 (43.0) 121 51.0 (42.1) 35 55 41 19.4 (47.3) 56 25.5 (45.5) 69 30.7 (44.4) 35 60 27 13.5 (50.1) 36 17.3 (48.1) 44 20.6 (46.8) 35 65 19 10.1 (53.0) 25 12.7 (50.7) 31 15.2 (49.1) 40 50 160 74.2 (46.4) 219 99.5 (45.4) 275 123.4 (44.9) 40 55 73 36.1 (49.4) 99 47.6 (48.1) 124 58.6 (47.2) 40 60 42 22.0 (52.4) 56 28.4 (50.8) 70 34.7 (49.6) 40 65 27 15.0 (55.5) 36 19.2 (53.4) 44 22.9 (52.1) 40 70 19 11.1 (58.5) 25 14.0 (56.1) 30 16.4 (54.7) 45 55 163 83.8 (51.4) 222 112.1 (50.5) 278 138.7 (49.9) 45 60 74 40.3 (54.5) 100 53.2 (53.2) 124 64.9 (52.3) 45 65 42 24.2 (57.6) 56 31.3 (55.9) 69 37.8 (54.9) 45 70 27 16.4 (60.7) 36 21.1 (58.6) 44 25.2 (57.3) 45 75 19 12.1 (63.8) 24 14.8 (61.7) 29 17.5 (60.2) 50 60 162 91.5 (56.5) 220 122.2 (55.5) 275 151.5 (55.0) 50 65 73 43.5 (59.6) 98 57.1 (58.3) 121 69.5 (57.5) 50 70 41 25.8 (62.8) 55 33.6 (61.1) 67 40.2 (60.0) 50 75 27 17.8 (65.8) 34 21.8 (64.1) 42 26.3 (62.7) 55 65 159 97.8 (61.5) 214 129.7 (60.6) 267 160.2 (60.0) 55 70 71 45.9 (64.7) 94 59.6 (63.4) 117 73.2 (62.6) 55 75 40 27.2 (67.9) 52 34.5 (66.3) 64 41.7 (65.2) 60 70 152 101.1 (66.5) 204 133.9 (65.6) 253 164.6 (65.1) 60 75 68 47.4 (69.8) 89 61.0 (68.5) 109 73.8 (67.7) 60 80 38 27.8 (73.1) 48 34.4 (71.6) 59 41.6 (70.5) 65 75 142 101.6 (71.6) 189 133.6 (70.7) 234 164.1 (70.1) 65 80 63 47.2 (74.9) 81 59.7 (73.7) 99 72.2 (72.9) Lewis et al. Multi-criteria assessment 80% power across five criteria will reduce the power of the combined test to 33%. The final three columns of Table 1 cover the sample sizes required for 95% power, which may address collective multi-criteria assessment when considering keeping a high overall statistical power. We recommend that progression for all key feasibility criteria should be considered separately, and hence over- all progression would be determined by the worst- performing criterion, e.g. RED if at least one signal is RED, AMBER if none of the signals fall into RED but at least one falls into AMBER and GREEN if all signals fall into the GREEN zone. Hence, the GREEN signal to ‘GO’ across the set of individual criteria will give indication that progression to a main trial can take place without any necessary changes. A signal to ‘STOP’ and not proceed to a main trial is recommended if any of the ob- served estimates are ‘unacceptably’ low (i.e. fall within the RED zone). Otherwise, where neither ‘GO’ nor ‘STOP’ are signalled, the design of the trial will need amending by indication of subpar performance on one or more of the criteria. Further expansion of AMBER zone p Within the same sample size framework, the AMBER zone may be further split to indicate whether ‘minor’ or ‘major’ amendments are required according to the sig- nificance of the p value. Consider a 2-way split in the AMBER zone denoted by cut-off AC, which indicates the threshold for statistical significance, where an observed estimate below the cut-point will result in a non- significant result and an estimate at or above the cut- point a significant result. Let AMBERR denote the region of Amber zone adjacent to the RED zone between RUL and AC, and AMBERG denote the region of AMBER zone between AC and GLL adjacent to the GREEN zone. This would draw on two possible levels of amendment (‘major’ AMEND and ‘minor’ AMEND) and the re- configured approach would follow as: Sample size requirements across multi-criteria will vary according to the designated parameters linked to the progression criteria, which may be set at different stages of the study on different numbers of patients (e.g. those screened, eligible, recruited and randomised, allo- cated to the intervention arm, total followed up). The overall size needed will be dictated by the requirement to power each of the multi-criteria statistical tests. Since these tests will yield separate conclusions in regard to the decision to ‘STOP’, ‘AMEND’ or ‘GO’ across all indi- vidual feasibility criteria there is no need to consider a multiple testing correction with respect to alpha. How- ever, researchers may wish to increase power (and hence, sample size) to ensure adequate power to detect ‘GO’ signals across the collective set of feasibility criteria. For example, powering at 90% across three criteria (as- sumed independent) will ensure a collective power of 73% (i.e. 0.93), which may be considered reasonable, but E ≤RUL [P value non-significant (P ≥α)] -> RED (unacceptable—STOP) RUL < E < GLL -> AMBER (potentially acceptable—AMEND) RUL < E < GLL and P ≥α {RUL < E < Ac} -> AMBERR (major AMEND) RUL < E < GLL and P < α { Ac ≤E < GLL} -> AMBERG (minor AMEND) E ≥GLL [P value significant (P < α)] -> GREEN (acceptable—GO) Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Further expansion of AMBER zone Pilot and Feasibility Studies (2021) 7:40 Page 7 of 14 Table 2 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on the binomial exact test (Continued) Table 2 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on the binomial exact test (Continued) g RUL GLL α (0.05), β (0.2) α (0.05), β (0.1) α (0.05), β (0.05) % % n Ac (%) n Ac (%) n Ac (%) 65 85 31 25 (80.6) 42 33 (78.6) 52 40 (76.9) 70 80 119 92 (77.3) 164 125 (76.2) 204 154 (75.5) 70 85 49 40 (81.6) 69 55 (79.7) 85 67 (78.8) 75 85 103 85 (82.5) 139 113 (81.3) 176 142 (80.7) 75 90 45 39 (86.7) 55 47 (85.5) 70 59 (84.3) 80 90 82 72 (87.8) 112 97 (86.6) 135 116 (85.9) RUL upper limit of RED zone (expressed as percentage of total sample), GLL lower limit of GREEN zone (expressed as percentage of total sample), AC AMBER- statistical significance threshold (within the AMBER zone) where an observed estimate below the cut-point will result in a non-significant result (p ≥0.05) and figures at or above the cut-point will be significant (p < 0.05) (%, expressed as a percentage of sample size (n)) RUL upper limit of RED zone (expressed as percentage of total sample), GLL lower limit of GREEN zone (expressed as percentage of total sample), AC AMBER- statistical significance threshold (within the AMBER zone) where an observed estimate below the cut-point will result in a non-significant result (p ≥0.05) and figures at or above the cut-point will be significant (p < 0.05) (%, expressed as a percentage of sample size (n)) In Tables 1 and 2 in relation to designated sample sizes for different RUL and GLL and specified α and β, we show the corresponding cut-points for statistical signifi- cance (p < 0.05) both in absolute terms of sample num- ber (n) [AC] and as a percentage of the total sample sizes [AC%]. gives the sample size calculations for the example sce- nario using the extended 4-tiered approach. Cut-points for the feasibility outcomes relating to the shown sample sizes are also presented to show RED, AMBER and GREEN zones for each of the three progression criteria. Further expansion of AMBER zone Overall sample size requirement should be dictated by the multi-criteria approach. This is illustrated in Table 4 where we have three progression criteria each with a dif- ferent denominator population. For recruitment uptake, the denominator denotes the total number of children screened and the numerator the number of children ran- domised; for follow-up, the denominator is the number of children randomised with the numerator being num- ber of those randomised who are successfully followed up; and lastly for treatment fidelity, the denominator is the number allocated to the intervention arm with the numerator being the number of children who were ad- ministered the treatment correctly by the dietician. In the example in order to meet the individual ≥90% power requirement for all three criteria we would need: (i) for recruitment, the number to be screened to be 78; (ii) for treatment fidelity, the number in the intervention arm to be 34; and (iii) for follow up, the number randomised to be 44. In order to determine the overall sample size for the whole study, we base our decision on the criterion that requires the largest numbers, which is the treatment fidelity criterion which requires 68 to be randomised. We cannot base our decision on the 78 required to be screened for recruitment because this would give only an expected number of 28 randomised (i.e. 35% of 78). If we expect 35% recruitment uptake, then we need to in- flate the total 68 (randomised) to be 195 (1/0.35 × 68) children to be screened (rounded to 200). This would give 99.9%, 90% and 98.8% power for criteria (i), (ii) and (iii), respectively (assuming 68 of the 200 screened are randomised), giving a very reasonable collective 88.8% power of rejecting the null hypotheses over the three cri- teria if the alternative hypotheses (for acceptable feasibil- ity outcomes) are true in each case. Further expansion of AMBER zone Pilot and Feasibility Studies Page 6 of 14 Table 2 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on the binomial exact test RUL GLL α (0.05), β (0.2) α (0.05), β (0.1) α (0.05), β (0.05) % % n Ac (%) n Ac (%) n Ac (%) 10 20 78 13 (16.7) 109 17 (15.6) 135 20 (14.8) 15 25 101 22 (21.8) 136 28 (20.6) 176 35 (19.9) 15 30 48 12 (25.0) 64 15 (23.4) 85 19 (22.4) 20 30 116 31 (26.7) 160 41 (25.6) 204 51 (25.0) 20 35 56 17 (30.4) 77 22 (28.6) 98 27 (27.6) 20 40 35 12 (34.3) 47 15 (31.9) 60 18 (30.0) 25 35 129 41 (31.8) 179 55 (30.7) 230 69 (30.0) 25 40 62 22 (35.5) 83 28 (33.7) 107 35 (32.7) 25 45 36 14 (38.9) 49 18 (36.7) 62 22 (35.5) 25 50 26 11 (42.3) 33 13 (39.4) 42 16 (38.1) 30 40 144 53 (36.8) 193 69 (35.8) 248 87 (35.1) 30 45 67 27 (40.3) 93 36 (38.7) 114 43 (37.7) 30 50 39 17 (43.6) 53 22 (41.5) 67 27 (40.3) 30 55 25 12 (48.0) 36 16 (44.4) 44 19 (43.2) 30 60 17 9 (52.9) 25 12 (48.0) 28 13 (46.4) 35 45 148 62 (41.9) 206 84 (40.8) 262 105 (40.1) 35 50 68 31 (45.6) 96 42 (43.8) 119 51 (42.9) 35 55 41 20 (48.8) 53 25 (47.2) 68 31 (45.6) 35 60 26 14 (53.8) 36 18 (50.0) 45 22 (48.9) 35 65 19 11 (57.9) 24 13 (54.2) 29 15 (51.7) 40 50 158 74 (46.8) 214 98 (45.8) 268 121 (45.1) 40 55 71 36 (50.7) 94 46 (48.9) 119 57 (47.9) 40 60 42 23 (54.8) 56 29 (51.8) 67 34 (50.7) 40 65 28 16 (57.1) 34 19 (55.9) 45 24 (53.3) 40 70 19 12 (63.2) 25 15 (60.0) 28 16 (57.1) 45 55 154 80 (51.9) 220 112 (50.9) 269 135 (50.2) 45 60 70 39 (55.7) 98 53 (54.1) 119 63 (52.9) 45 65 42 25 (59.5) 54 31 (57.4) 68 38 (55.9) 45 70 25 16 (64.0) 36 22 (61.1) 44 26 (59.1) 45 75 16 11 (68.8) 23 15 (65.2) 29 18 (62.1) 50 60 158 90 (57.0) 213 119 (55.9) 268 148 (55.2) 50 65 69 42 (60.9) 93 55 (59.1) 119 69 (58.0) 50 70 37 24 (64.9) 53 33 (62.3) 67 41 (61.2) 50 75 23 16 (69.6) 33 22 (66.7) 42 27 (64.3) 55 65 150 93 (62.0) 210 128 (61.0) 262 158 (60.3) 55 70 70 46 (65.7) 92 59 (64.1) 114 72 (63.2) 55 75 37 26 (70.3) 50 34 (68.0) 62 41 (66.1) 60 70 143 96 (67.1) 197 130 (66.0) 248 162 (65.3) 60 75 62 44 (71.0) 85 59 (69.4) 107 73 (68.2) 60 80 36 27 (75.0) 45 33 (73.3) 60 43 (71.7) 65 75 133 96 (72.2) 180 128 (71.1) 230 162 (70.4) 65 80 55 42 (76.4) 75 56 (74.7) 98 72 (73.5) Table 2 Sample size and significance cut-points for (GLL-RUL) differences for a one-sample test, power (80%, 90%, 95%) and 1-tailed 5% significance level based on the binomial exact test Lewis et al. Results A motivating example (aligned to the normal approxi- mation approach) is presented in Table 3, which illus- trates a pilot trial with three progression criteria. Table 4 presents the sample size calculations for the example scenario following the 3-tiered approach, and Table 5 Table 3 Motivating example—feasibility trial for oral protein energy supplements as flavoured drinks to improve nutritional status in children with cystic fibrosis A feasibility trial is being set up to see whether children aged 2 to 15 years with cystic fibrosis will take oral protein energy supplements as flavoured drinks to improve their nutritional status, compared to receiving dietary advice alone. Children are to be randomised in a 1:1 allocation ratio using a parallel two-arm design. The research team wants to be sure they can meet three feasibility objectives before they go ahead and plan the main trial: reasonable recruitment uptake, high treatment fidelity (i.e. extent to which dietician practitioners comply with the treatment protocol) and adequate retention of children at follow up. The team asks their senior statistician to help them decide on an appro- priate methodology including pilot sample size. The statistician suggests a traffic light approach incorporating hypothesis testing of the feasibility outcomes. Together, the team devise three progression criteria that should be met before the main trial can be considered feasible as follows: a. At least 35% of the children screened as eligible should be recruited but the trial will not be feasible if recruitment uptake is 20% or less. b. A high level of treatment fidelity should be maintained with 75% or more children being given the correct treatment plan by the dietician, but if 50% or less children are given the plan as specified in the protocol then the trial is not feasible. c. 85% or more of the children should be retained in the study at follow up, with 65% or less retention indicating that the main trial is not feasible. c. 85% or more of the children should be retained in the study at follow up, with 65% or less retention indicating that the main trial is not feasible. The decision criteria and required sample size around these are detailed through two possible approaches within Table 4 (simple 3-tier ap- proach) and Table 5 (extended 4-tier approach). The statistician is to use the normal approximation method (with continuity correction) for the sample size calculation and analysis. Results Page 8 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 (2021) 7:40 Table 5 Case illustration (re-visited using 4-tiered approach) 0.65 × 68 = 44.2, 0.745 × 68 = 50.66, 0.85 × 68 = 57.8} [Note: The continuity correction (-0 5 deduction) needs to be applied to Taking each of the objectives in turn, we re-express the progression cri- teria for the three objectives according to the 4-tiered approach, as follows: A two-arm parallel design (1:1 allocation to intervention and control arms) with three key feasibility objectives, to assess (i) recruitment up- take (percent of screened patients recruited), (ii) treatment fidelity and (iii) participant retention (follow up). Hypothesis testing incorporates α (1-sided) = 5% and power = 90%. The normal approximation method is used. Taking each of the objectives in turn, we re-express the progression cri- teria for the three objectives according to the 4-tiered approach, as follows: (i) Recruitment uptake [expected ns = 200] (i) Recruitment uptake [expected ns = 200] p p • E ≤0.2 [P ≥0.05] -> RED (STOP) • E ≤0.2 [P ≥0.05] -> RED (STOP) • 0.2 < E < 0.35 -> AMBER (AMEND)//{Ac = 0.247 (i.e. 0.2 + 1.645√(0.2 × 0.8/200))} Assume the progression criteria (and affiliated sample size requirements) for each are as follows: Assume the progression criteria (and affiliated sample size requirements) for each are as follows: ))} o 0.2 < E < 0.247 [P ≥0.05] -> AMBERR (AMEND-major) o 0.2 < E < 0.247 [P ≥0.05] -> AMBERR (AMEND-major) (i) Recruitment uptake ≤20% (RED zone) and ≥35% (GREEN zone) {RUL = 20%, GLL = 35%} (i) Recruitment uptake ≤20% (RED zone) and ≥35% (GREEN zone) {RUL = 20%, GLL = 35%} • E ≥0.35 [P < 0.05] -> GREEN (GO) →Required sample size n = 78 [total screened patients] →Required sample size n = 78 [total screened patients] (ii) Treatment fidelity ≤50% (RED zone) and ≥75% (GREEN zone) {R →Required sample size n = 78 [total screened patients] (ii) Treatment fidelity ≤50% (RED zone) and ≥75% (GREEN zone) {RUL = 50%, GLL = 75%} →Required sample size n = 34 [intervention arm only] (iii) Follow up: ≤65% (RED zone), ≥85% (GREEN zone) {RUL = 65%, GLL = 85%} →Required sample size n = 44 (total randomised participants with 22 per arm) j o 0.641 ≤E < 0.75 [P < 0.05] -> AMBERG (AMEND-mino The sample sizes across criteria (i)-(iii) are at different levels—(i) is at the level of screened patients, whereas (ii)–(iii) are at the level of randomised patients. RUL upper limit of RED zone, GLL lower limit of GREEN zone, ns number of screened patients who are eligible to being randomised, nr number of eligible patients randomised, ni number of patients randomised to the intervention arm Table 5 Case illustration (re-visited using 4-tiered approach) (1/0.2)x nr)] o 0.65 < E < 0.745 [P ≥0.05] -> AMBERR (AMEND-major) o 0.745 ≤E < 0.85 [P < 0.05] -> AMBERG (AMEND-minor) • E ≤0.2 [P ≥0.05] -> RED (STOP) • 0.2 < E < 0.35 -> AMBER (AMEND) Signals for nr = 68: • E ≥0.35 [P < 0.05] -> GREEN (GO) 0 to 44.2 (RED), > 44.2 to < 50.66 (AMBERR), 50.66 to < 57.8 (AMBERG) and 57.8 to 70 (GREEN) {i.e. 0.65 × 68 = 44.2, 0.745 × 68 = 50.66, 0.85 × 68 = 57.8} Signals for expected ns = 200: g p s 0 to 40 (RED), > 40 to < 70 (AMBER) and 70 to 200 (GREEN) {i.e. 0.2 × 200 = 40; 0.35 × 200 = 70} (ii) Treatment fidelity [ni = 34 (intervention arm only)] [Note: The continuity correction (-0.5 deduction) needs to be applied to the observed count from the study for each criterion prior to assessing into which signal band it falls] y • E ≤0.5 [P ≥0.05] -> RED (STOP) • 0.5 < E < 0.75 -> AMBER (AMEND) In accordance with the multi-criteria aim, the decision to proceed would be based on the worst signal (as in Table 4) • E ≥0.75 [P < 0.05] -> GREEN (GO) Signals for ni = 34: g 0 to 17 (RED), > 17 to < 25.5 (AMBER) and 25.5 to 34 (GREEN) {i.e. 0.5 × 34 = 17; 0 75 × 34 = 25 5} g 0 to 17 (RED), > 17 to < 25.5 (AMBER) and 25.5 to 34 (GREEN) {i.e. 0.5 × ns number of screened patients who are eligible to being randomised, nr number of eligible patients randomised, ni number of patients randomised to the intervention arm 34 = 17; 0.75 × 34 = 25.5} (iii) Follow up [nr = 68 (intervention and control arms)] • E ≤0.65 [P ≥0.05] -> RED (STOP) AC is calculated from the 1-sided upper 95% confidence limit for the null proportion: RUL + z1−α√((RUL(1 −RUL))/n) where z1−α = 1.645 (for 1-sided 5% significance test) • 0.65 < E < 0.85 -> AMBER (AMEND) • E ≥0.85 [P < 0.05] -> GREEN (GO) Signals for nr = 68: 0 to 44.2 (RED), > 44.2 to < 57.8 (AMBER) and 57.8 to 68 (GREEN) {i.e. Table 5 Case illustration (re-visited using 4-tiered approach) To meet criteria (i), we need ns ≥78 (although we will recruit ns = 200 (i.e. (1/0.35) × nr (rounded up to 200)) where 0.35 is the expected proportion uptake of the total number screened), and for (ii)–(iii), we need nr = 68 (34 per arm, based on (ii)). Taking each of the objectives in turn (and the updated sample sizes to The sample sizes across criteria (i)-(iii) are at different levels—(i) is at the level of screened patients, whereas (ii)–(iii) are at the level of • E ≥0.75 [P < 0.05] -> GREEN (GO) p randomised patients. To meet criteria (i), we need ns ≥78 (although we will recruit ns = 200 (i.e. (1/0.35) × nr (rounded up to 200)) where 0.35 is the expected proportion uptake of the total number screened), and for (ii)–(iii), we need nr = 68 (34 per arm, based on (ii)). Signals for ni = 34: 0 to 17 (RED), > 17 to < 21.79 (AMBERR), 21.79 to < 25.5 (AMBERG) and 25.5 to 34 (GREEN) {i.e. 0.5 × 34 = 17, 0.641 × 34 = 21.794, 0.75 × 34 = 25.5} will recruit ns = 200 (i.e. (1/0.35) × nr (rounded up to 200)) where 0.35 is the expected proportion uptake of the total number screened), and for (ii)–(iii), we need nr = 68 (34 per arm, based on (ii)). (iii) Follow up [nr = 68 (intervention and control arms)] Taking each of the objectives in turn (and the updated sample sizes to meet the multi-criteria objectives), we express progression criteria for the three objectives as follows: p r • E ≤0.65 [P ≥0.05] -> RED (STOP) • 0.65 < E < 0.85 -> AMBER (AMEND)//{Ac = 0.745 (i.e. 0.65 + 1.645x√(0.65 × 0.35/68))} (i) Recruitment uptake [required ns ≥78; expected ns = 200; maximum (i) Recruitment uptake [required ns ≥78; expected ns = 200; maximum n = 340 (i e (1/0 2)x n )] ns = 340 (i.e. ns number of screened patients who are eligible to being randomised, nr number of eligible patients randomised, ni number of patients randomised to the intervention arm Table 5 Case illustration (re-visited using 4-tiered approach) 0.65 × 68 = 44.2, 0.745 × 68 = 50.66, 0.85 × 68 = 57.8} [Note: The continuity correction (-0.5 deduction) needs to be applied to the observed count from the study for each criterion prior to assessing Taking each of the objectives in turn, we re-express the progression cri- teria for the three objectives according to the 4-tiered approach, as follows: (i) Recruitment uptake [expected ns = 200] • E ≤0.2 [P ≥0.05] -> RED (STOP) • 0.2 < E < 0.35 -> AMBER (AMEND)//{Ac = 0.247 (i.e. 0.2 + 1.645√(0.2 × 0.8/200))}* o 0.2 < E < 0.247 [P ≥0.05] -> AMBERR (AMEND-major) o 0.247 ≤E < 0.35 [P < 0.05] -> AMBERG (AMEND-minor) • E ≥0.35 [P < 0.05] -> GREEN (GO) Signals for ns = 200: 0 to 40 (RED), > 40 to < 49.4 (AMBERR), 49.4 to < 70 (AMBERG) and 70 to 200 (GREEN) {i.e. 0.2 × 200 = 40, 0.247 × 200 = 49.4, 0.35 × 200 = 70} (ii) Treatment fidelity [ni = 34 (intervention arm only)] • E ≤0.5 [P ≥0.05] -> RED (STOP) • 0.5 < E < 0.75 -> AMBER (AMEND)//{AC = 0.641 (i.e. 0.5 + 1.645√(0.5 × 0.5/34))—as shown in Table 1}* o 0.5 < E < 0.641 [P ≥0.05] -> AMBERR (AMEND-major) o 0.641 ≤E < 0.75 [P < 0.05] -> AMBERG (AMEND-minor) • E ≥0.75 [P < 0.05] -> GREEN (GO) Signals for ni = 34: 0 to 17 (RED), > 17 to < 21.79 (AMBERR), 21.79 to < 25.5 (AMBERG) and 25.5 to 34 (GREEN) {i.e. 0.5 × 34 = 17, 0.641 × 34 = 21.794, 0.75 × 34 = 25.5} (iii) Follow up [nr = 68 (intervention and control arms)] • E ≤0.65 [P ≥0.05] -> RED (STOP) • 0.65 < E < 0.85 -> AMBER (AMEND)//{Ac = 0.745 (i.e. 0.65 + 1.645x√(0.65 × 0.35/68))}* o 0.65 < E < 0.745 [P ≥0.05] -> AMBERR (AMEND-major) o 0.745 ≤E < 0.85 [P < 0.05] -> AMBERG (AMEND-minor) • E ≥0.85 [P < 0.05] -> GREEN (GO) Signals for nr = 68: 0 to 44.2 (RED), > 44.2 to < 50.66 (AMBERR), 50.66 to < 57.8 (AMBERG) and 57.8 to 70 (GREEN) {i.e. Table 5 Case illustration (re-visited using 4-tiered approach) Table 5 Case illustration (re visited using 4 tiered approach) Taking each of the objectives in turn, we re-express the progression cri- teria for the three objectives according to the 4-tiered approach, as follows: (i) Recruitment uptake [expected ns = 200] • E ≤0.2 [P ≥0.05] -> RED (STOP) • 0.2 < E < 0.35 -> AMBER (AMEND)//{Ac = 0.247 (i.e. 0.2 + 1.645√(0.2 × 0.8/200))}* o 0.2 < E < 0.247 [P ≥0.05] -> AMBERR (AMEND-major) o 0.247 ≤E < 0.35 [P < 0.05] -> AMBERG (AMEND-minor) • E ≥0.35 [P < 0.05] -> GREEN (GO) Signals for ns = 200: 0 to 40 (RED), > 40 to < 49.4 (AMBERR), 49.4 to < 70 (AMBERG) and 70 to 200 (GREEN) {i.e. 0.2 × 200 = 40, 0.247 × 200 = 49.4, 0.35 × 200 = 70} (ii) Treatment fidelity [ni = 34 (intervention arm only)] • E ≤0.5 [P ≥0.05] -> RED (STOP) • 0.5 < E < 0.75 -> AMBER (AMEND)//{AC = 0.641 (i.e. 0.5 + 1.645√(0.5 × 0.5/34))—as shown in Table 1}* o 0.5 < E < 0.641 [P ≥0.05] -> AMBERR (AMEND-major) o 0.641 ≤E < 0.75 [P < 0.05] -> AMBERG (AMEND-minor) • E ≥0.75 [P < 0.05] -> GREEN (GO) Signals for ni = 34: 0 to 17 (RED), > 17 to < 21.79 (AMBERR), 21.79 to < 25.5 (AMBERG) and 25.5 to 34 (GREEN) {i.e. 0.5 × 34 = 17, 0.641 × 34 = 21.794, 0.75 × 34 = 25.5} (iii) Follow up [nr = 68 (intervention and control arms)] • E ≤0.65 [P ≥0.05] -> RED (STOP) • 0.65 < E < 0.85 -> AMBER (AMEND)//{Ac = 0.745 (i.e. 0.65 + 1.645x√(0.65 × 0.35/68))}* o 0.65 < E < 0.745 [P ≥0.05] -> AMBERR (AMEND-major) o 0.745 ≤E < 0.85 [P < 0.05] -> AMBERG (AMEND-minor) • E ≥0.85 [P < 0.05] -> GREEN (GO) Signals for nr = 68: 0 to 44.2 (RED), > 44.2 to < 50.66 (AMBERR), 50.66 to < 57.8 (AMBERG) and 57.8 to 70 (GREEN) {i.e. AC is calculated from the 1-sided upper 95% confidence limit for the null proportion: RUL + z1−α√((RUL(1 −RUL))/n) where z1−α = 1.645 (for 1-sided 5% significance test) Table 5 Case illustration (re-visited using 4-tiered approach) E, observed point estimate; RUL, upper limit of RED zone; GLL, lower limit of GREEN zone; Ac, cut-off for statistical significance (at the 1-sided 5% level); α, type I error; β, type II error Page 10 of 14 Page 10 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Probability of 0.4 of it falling in the AMBERG zone (i.e. area under the curve to the right of AC but below GLL) analysis around the set null value (at RUL) when the hypothesised alternative (GLL) is true. The figure empha- sises the need for a large enough sample to safeguard against under-powering of the pilot analysis (as shown in the last plot which has a wider bell-shape than the first two plots and where the size of the beta probability is increased). Probability of 0.5 of the estimate falling in the GREEN zone (i.e. GLL and above). If RUL (the null) holds true (i.e. true feasibility outcome (ε) = RUL), there would be the following: Figure 2 plots the probabilities of making each type of traffic light decision as functions of the true parameter value (focused on the recruitment uptake example from Table 5 (i)). Additional file 1 presents the R code for re- producing these probabilities and enables readers to in- sert different parameter values. A probability of 0.05 (one-tailed type I error probability α) of the statistic/estimate falling in the AMBERG/GREEN zones (i.e. pink shaded area under the curve to the right of AC where the test result will be significant (p < 0.05) as shown within Fig. 1) Table 5 Case illustration (re-visited using 4-tiered approach) 0.65 × 68 = 44.2; 0.85 × 68 = 57.8} [Note: The continuity correction (−0.5 deduction) needs to be applied to the observed count from the study for each criterion prior to assessing into which signal band it falls] set a lower GREEN zone limit of GLL = 0.75 (“accept- able” (hypothesised alternative value)) and an upper RED zone limit of RUL = 0.5 (“not acceptable” (hypothesised null value)) for rejecting the null for this criterion based on 90% power and a 1-sided 5% significance level (alpha). Figure 1 presents the nor- mal probability density functions for ε, for the null and alternative hypotheses. In the illustration this would imply through normal sampling theory that if GLL holds true (i.e. true recruitment uptake (ε) = GLL) there would be the following: g g In accordance with the multi-criteria aim, the decision to proceed would be based on the worst signal ➢If signal = RED for (i) or (ii) or (iii) -> overall signal is RED ➢Else, if no signal is RED but signal = AMBER for (i) or (ii) or (iii) -> overall signal is AMBER ➢Else, if signals = GREEN for (i) and (ii) and (iii) -> overall signal is GREEN A probability of 0.1 (type II error probability β) of the estimate falling within RED/AMBERR zones (i.e. blue shaded area under the curve to the left of AC where the test result will be non-significant (p ≥0.05)) Inherent in our approach are the probabilities around sample size, power and hypothesised feasibility parame- ters. For example, taking the cut-offs from treatment fi- delity as a feasibility outcome from Table 4 (ii), we Page 9 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Fig. 1 Illustration of power using the 1-tailed hypothesis testing against the traffic light signalling approach to pilot progression. E, observed point estimate; RUL, upper limit of RED zone; GLL, lower limit of GREEN zone; Ac, cut-off for statistical significance (at the 1-sided 5% level); α, type I error; β, type II error Fig. 1 Illustration of power using the 1-tailed hypothesis testing against the traffic light signalling approach to pilot progression. Discussion A statistically significant find- ing in this context will at least provide evidence to reject RED (signifying a decision to STOP) and in the 4-tiered case it would fall above AMBERR (decision to major- AMEND); hence, the estimate will fall into AMBERG or GREEN (signifying a decision to minor-AMEND or GO, respectively). The importance of adequately powering the pilot trial to address a feasibility criterion can be simply illustrated. For example, if we take RUL as 50% and GLL as 75% but with two different sample sizes of n = 25 and n = 50; the former would have 77.5% power of rejecting RED on the basis of a 1-sided 5% alpha level whereas the larger sample size would have 97.8% power of rejecting RED. So, if GLL holds true, there would be 20% higher probability of rejecting the null and being in the AMBERG/GREEN zone for the larger sample giving an increased chance of progressing to the main trial. It will be necessary to carry out the hypothesis test for the extended 4-tier approach if the observed statistic (E) falls in the AMBER zone to determine statistical significance or not, which will inform whether the result falls into the ‘minor’ or ‘major’ AMBER sub-zones. beta parameters (rather than alpha alone), the method ensures rigour and capacity to address the progression criteria by ensuring there is adequate power to detect an acceptable threshold for moving forward to the main trial. As several key process outcomes are assessed in parallel and in combination, the method embraces a composite multi-criterion approach that appraises sig- nals for progression across all the targeted feasibility measures. The methodology extends beyond the require- ment for ‘sample size justification but not necessarily sample size calculation’ [28]. The focus of the strategy reported here is on process outcomes, which align with the recommended key objec- tives of primary feasibility evaluation for pilot and feasi- bility studies [2, 24] and necessary targets to address key issues of uncertainty [29]. The concept of justifying pro- gression is key. Charlesworth et al. [30] developed a checklist for intended use in decision-making on whether pilot data could be carried forward to a main trial. Our approach builds on this philosophy by introdu- cing a formalised hypothesis test approach to address the key objectives and pilot sample size. Discussion Though the suggested sample size derivation focuses around the key process objectives, it may also be the case that other ob- jectives are also important, e.g. assessment of precision of clinical outcome parameters. In this case, researchers may also wish to ensure that the size of the study suit- ably covers the needs of those evaluations, e.g. to esti- mate the SD of the intended clinical outcome, then the overall sample size may be boosted to cover this add- itional objective [10]. This tallies with the review by Blatch-Jones et al. [31] who reported that testing recruit- ment, determining the sample size and numbers avail- able, and the intervention feasibility were the most commonly used targets of pilot evaluations. j We provide recommended sample sizes within a look- up grid relating to perceived likely progression cut- points to aid quick access and retrievable sample sizes for researchers. For a likely set difference in proportions between hypothesised null and alternative parameters of 0.15 to 0.25 when α = 0.05 and β = 0.1 the correspond- ing total sample size requirements for the approach of normal approximation with continuity correction take the range of 33 to 100 (median 56) [similarly these are 33–98 (median 54) for the binomial exact method]. Note, for treatment fidelity/adherence/compliance par- ticularly, the marginal difference could be higher, e.g. ≥ 25%, since in most situations we would anticipate and hope to attain a high value for the outcome whilst being prepared to make necessary changes within a wide inter- val of below par values (and providing the value is not unacceptably low). As this relates to an arm-specific ob- jective (relating to evaluation of the intervention only), then a usual 1:1 pilot will require twice the size; hence, the arm-specific sample size powered for detecting a ≥ 25% difference from the null would be about 34 (or Hypothesis testing in pilot studies, particularly in the context of effectiveness/efficacy of clinical outcomes, has been widely criticised due to the improper purpose and lack of statistical power of such evaluations [2, 20, 21, 23]. Hence, pilot evaluations of clinical outcomes are not expected to include hypothesis testing. Since the main focus is on feasibility the scope of the testing reported here is different and importantly relates back to the rec- ommended objectives of the study whilst also aligning with nominated progression criteria [2]. Discussion g Probability of 0.45 of it falling in the AMBERR zone (i.e. to the left of AC but above RUL) The methodology introduced in this article provides an innovative formal framework and approach to sample size derivation, aligning sample size requirement to pro- gression criteria with the intention of providing greater transparency to the progression process and full engage- ment with the standard aims and objectives of pilot/ feasibility studies. Through the use of both alpha and Probability of 0.5 of the estimate falling in the RED zone (i.e. RUL and below) Figure 1 also illustrates how changing the sample size affects the sampling distribution and power of the Fig. 2 Probability of traffic light given true underlying probability of an event using the example from Table 5 (i). Two plots are presented: a relating to normal approximation approach and b relating to binomial exact approach. Based on n = 200, RUL = 40 and GLL = 70 Fig 2 Probability of traffic light given true underlying probability of an event using the example from Table 5 (i) Two plots are presented: a Fig. 2 Probability of traffic light given true underlying probability of an event using the example from Table 5 (i). Two plots are presented: a relating to normal approximation approach and b relating to binomial exact approach. Based on n = 200, RUL = 40 and GLL = 70 Page 11 of 14 Page 11 of 14 Page 11 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 feasibility outcome gives this approach the extra rigour over the confidence interval approach. It is this sample size-power linkage that is key to the determination of the respective probabilities of falling into the different zones and is a fundamental underpinning to the meth- odological approach. In the same way as for a key clin- ical outcome in a main trial where the emphasis is not just on alpha but also on beta thereby addressing the capacity to detect a clinically significant difference, simi- larly, our approach is to ensure there is sufficient cap- acity to detect a meaningful signal for progression to a main trial if it truly exists. Discussion Beyond our pro- posed 4-tier approach, other ways of providing an indi- cation of level of amendment could include evaluation and review of the point and interval estimates or by evaluating posterior probabilities via a Bayesian ap- proach [14, 32]. lower)—as depicted from our illustration (Table 4 (ii), equating to n ≤68 overall for a 1:1 pilot; intervention and control arms). Hence, we expect that typical pilot sizes of around 30–40 randomised per arm [16] would likely fit with the proposed methodology within this manuscript (the number needed for screening being ex- trapolated upward of this figure) but if a smaller mar- ginal difference (e.g. ≤15%) is to be tested then these sample sizes may fall short. We stress that the overall re- quired sample size needs to be carefully considered and determined in line with the hypothesis testing approach across all criteria ensuring sufficiently high power. In our paper, we have made recommendations regarding various sample sizes based on both the normal approxi- mation (with continuity correction) and binomial exact approaches; these are conservative compared to the Nor- mal approximation (without continuity correction). Importantly, the methodology outlines the necessary multi-criterion approach to the evaluation of pilot and feasibility studies. If all progression criteria are perform- ing as well as anticipated (highlighting ‘GO’ according to all criteria), then the recommendation of the pilot/feasi- bility study is that all criteria meet their desired levels with no need for adjustment and the main trial can proceed without amendment. However, if the worst sig- nal (across all measured criteria) is an AMBER signal, then adjustment will be required against those criteria that fall within that signal. Consequently, there is the possibility that the criteria may need subsequent re- assessment to re-evaluate processes in line with updated performance for the criteria in question. If one or more of the feasibility statistics fall within the RED zone then this signals ‘STOP’ and concludes that a main trial is not feasible based on those criteria. This approach to collect- ively appraising progression based on the results of all feasibility outcomes assessed against their criteria will be conservative as the power of the collective will be lower than the individual power of the separate tests; hence, it is recommended that the power of the individual tests is set high enough (for example, 90–95%) to ensure the collective power is high enough (e.g. Discussion Hence, there is clear justification for this approach. Further, for the sim- ple 3-tiered approach hypothesis testing is somewhat hypothetical: there is no need to physically carry out a test since the zonal positioning of the observed sample statistic estimate for the feasibility outcome will deter- mine the decision in regard to progression; thus adding to the simplicity of the approach. The link between the sample size and need to ad- equately power the study to detect a meaningful Page 12 of 14 Page 12 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies further data recruitment methods, significant reconfigur- ation of exclusions, major change to the method of de- livery of trial intervention to ensure enhanced treatment fidelity/adherence, enhanced measures to systematically ensure greater patient compliance with allocated treat- ment, additional mode(s) of collecting and retrieving data (e.g. use of electronic data collection methods in addition to postal questionnaires). Minor amendments include small changes to the protocol and methodology, e.g. addition of one or two sites for attaining a slightly higher recruitment rate, use of occasional reminders in regard to treatment protocol and adding a further re- minder process for boosting follow up. For the most likely parametrisation of α = 0.05/β = 0.1, the AMBER zone division will be roughly at the midpoint. However, researchers can choose this point (the major/minor cut- point) based on decisive arguments around how major and minor amendments would align to the outcome in question. This should be factored within the process of sample size determination for the pilot. In this regard, a smaller sample size will move AC upwards (due to in- creased standard error/reduced precision) and hence in- crease the size of the AMBERR zone in relation to AMBERG (whereas a larger sample size will shift AC downwards and do the opposite, increasing the ratio of AMBERG:AMBERR). From Table 1, for smaller sample sizes (related to 80% power) the AMBERR zone makes up 56–69% of the total amber zone across presented sce- narios, whereas this falls to 47–61% for samples (related to 90% power) and 41–56% for larger samples (related to 95% power) for the same scenarios. Conclusions We propose a novel framework that provides a paradigm shift towards formally testing feasibility progression cri- teria in pilot and feasibility studies. The outlined ap- proach ensures rigorous and transparent reporting in line with CONSORT recommendations for evaluation of STOP-AMEND-GO criteria and presents clear progres- sion sign-posting which should help decision-making and inform stakeholders. Targeted progression criteria are focused on recommended pilot and feasibility objec- tives, particularly recruitment uptake, treatment fidelity and participant retention, and these criteria guide the methodology for sample size derivation and statistical testing. This methodology is intended to provide a more definitive and rounded structure to pilot and feasibility design and evaluation than currently exists. Sample size recommendations will be dependent on the nature and cut-points for multiple key pre-defined progression cri- teria and should ensure a sufficient sample size for other feasibility outcomes such as review of the precision of clinical parameters to better inform main trial size. Issues relating to progression criteria for internal pilots may be different to those for external pilots and non- randomised feasibility studies. The consequence of a ‘stop’ within an internal pilot may be more serious for stake- holders (researchers, funders, patients) as it would bring an end to the planned continuation into the main trial phase, whereas there would be less at stake for a negative external pilot. By contrast, the consequence of a ‘GO’ sig- nal may work the other way with a clear and immediate gain for the internal pilot whereas for an external pilot, the researchers would still need to apply and get the ne- cessary funding and approvals to undertake an intended main trial. The chances of falling into the different traffic light zones are likely to be quite different between the two designs. Possibly external pilot and feasibility studies are more likely to have estimates falling in and around the RED zone than for internal pilots, reflecting the greater uncertainty in the processes for the former and greater confidence in the mechanisms for trial delivery for the lat- ter. However, to counter this, there are often large chal- lenges with recruitment within internal pilot studies where the target population is usually spread over more diverse sites than may be expected for an external pilot. Despite this possible imbalance, the interpretation of zonal indications remains consistent for external and internal pilot studies. Appendix Mathematical formulae for derivation of sample size Mathematical formulae for derivation of sample size The required sample size may be derived using normal approximation to binary response data—using a continu- ity correction, via Fleiss et al. [26] if the convention of np > 5 and n(1 −p) > 5 holds true: n ¼ z1 −∝ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ RUL 1 −RUL ð Þþ p z1 −β ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ GLL 1 −GLL ð Þ p GLL −RUL ð Þ !2 þ 1 GLL −RUL j j where RUL = upper limit of RED zone; GLL = lower limit of GREEN zone; z1−α = one-sided statistical significance level (type I error probability); z1−β = beta (type II error probability) where RUL = upper limit of RED zone; GLL = lower limit of GREEN zone; z1−α = one-sided statistical significance level (type I error probability); z1−β = beta (type II error probability) Supplementary Information Supplementary Information pp y The online version contains supplementary material available at https://doi. org/10.1186/s40814-021-00770-x. pp y The online version contains supplementary material available at https://doi. org/10.1186/s40814-021-00770-x. Additional file 1. R codes used for Fig. 2. Additional file 1. R codes used for Fig. 2. Conclusions As such, our focus with regard to the recom- mendations in this article are aligned to requirements for external pilots, though application of this methodology to Discussion For ex- ample, in regard to the choice of centres (and hence prac- titioners and participants), a common concern is that the selection of feasibility trial centres might not be a fair and representative sample of the ‘population’ of centres to be used for the main trial. It may be that the host centre (likely used in pilot studies) recruits far better than others (positive bias), thus exaggerating the signal to progress and subsequent recruitment to the main trial. Beets et al. [33] ‘define “risk of generalizability biases” as the degree to which features of the intervention and sample in the pilot study are NOT scalable or generalizable to the next stage of testing in a larger, efficacy/effectiveness trial … whether aspects like who delivers an intervention, to whom it is de- livered, or the intensity and duration of the intervention during the pilot study are sustained in the larger, efficacy/ effectiveness trial.’ As in other types of studies, safeguards regarding bias should be addressed through appropriate pilot study design and conduct. Discussion at least 70 or 80%) to detect true ‘GO’ signals across all the feasibility criteria. The methodology illustrated here focuses on feasibility outcomes presented as percentages/proportions, which is likely to be the most common form for progression criteria under consideration. However, the steps that have been introduced can be readily adapted to any feasibility outcomes taking a numerical format, e.g. rate of recruitment per month per centre, count of centres taking part in the study. Also, we point out that in the examples presented in the paper (recruitment, treatment fidelity and percent follow-up), high proportions are ac- ceptable and low ones not. This would not be true for, say, adverse events where a reverse scale is required. In this article, we also expand the possibilities for pro- gression criterion and hypothesis testing where the AMBER zone is sub-divided arbitrarily based on the sig- nificance of the p value. This may work well when the AMBER zone has a wide range and is intended to pro- vide a useful and workable indication of the level of amendment (‘minor’ (non-substantive) or ‘major’ (sub- stantive)) required to progress to the main trial. Exam- ples of substantial amendments include study re-design with possible re-appraisal and change of statistical pa- rameters, inclusion of several additional sites, adding Biased sample estimates are a concern as they may re- sult in a wrong decision being made. This systematic error is over-and-above the possibility of an erroneous decision being made on the basis of sampling error; the latter may be reduced through an increased pilot sample size. Any positive bias will inflate/overestimate the Page 13 of 14 Page 13 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies a degree may similarly hold for internal pilots (and further, to non-randomised studies that can include progression criteria—including longitudinal observational cohorts with the omission of the treatment fidelity criterion). feasibility sample estimate in favour of progressing whereas a negative bias will deflate/underestimate it to- wards the null and stopping. Both are problematic for op- posite reasons; for example, the former may inform researchers that the main trial can ‘GO’ ahead when in fact it will struggle to meet key feasibility targets, whereas the latter may caution against progression when in reality the feasibility targets of a main trial would be met. Additional file 1. R codes used for Fig. 2. References On the use of a pilot sample for sample size determination. Stat Med. 1995;14:1933–40. Consent for publication Not applicable. 20. Leon AC, Davis LL, Kraemer HC. The role and interpretation of pilot studies in clinical research. J Psychiatr Res. 2011;45(5):626–9. 21. Horne E, Lancaster GA, Matson R, Cooper A, Ness A, Leary S. Pilot trials in physical activity journals: a review of reporting and editorial policy. Pilot Feasibility Stud. 2018;4:125. References 27. Faul F, Erdfelder E, Lang AG, Buchner A. GPower 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39:175–91. 1. Lancaster GA, Dodd S, Williamson PR. Design and analysis of pilot studies: recommendations for good practice. J Eval Clin Pract. 2004;10(2):307–12. 2. Eldridge SM, Chan CL, Campbell MJ, Bond CM, Hopewell S, Thabane L, et al. CONSORT 2010 statement: extension to randomised pilot and feasibility trials. Pilot Feasibility Stud. 2016;2:64. 2. Eldridge SM, Chan CL, Campbell MJ, Bond CM, Hopewell S, Thabane L, et al. CONSORT 2010 statement: extension to randomised pilot and feasibility trials. Pilot Feasibility Stud. 2016;2:64. 28. Julious SA. Pilot studies in clinical research. Stat Methods Med Res. 2016; 25(3):995–6. 29. Lancaster GA. Pilot and feasibility studies come of age! Pilot Feasibility Stud. 2015;1(1):1. 3. McDonald AM, Knight RC, Campbell MK, Entwistle VA, Grant AM, Cook JA, et al. What influences recruitment to randomised controlled trials? A review of trials funded by two UK funding agencies. Trials. 2006;7:9. 30. Charlesworth G, Burnell K, Hoe J, Orrell M, Russell I. Acceptance checklist for clinical effectiveness pilot trials: a systematic approach. BMC Med Res Methodol. 2013;13:78. 4. Sully BG, Julious SA, Nicholl J. A reinvestigation of recruitment to randomised, controlled, multicenter trials: a review of trials funded by two UK funding agencies. Trials. 2013;14:166. 31. Blatch-Jones AJ, Pek W, Kirkpatrick E, Ashton-Key M. Role of feasibility and pilot studies in randomised controlled trials: a cross-sectional study. BMJ Open. 2018;8(9):e022233. 5. Walters SJ, Bonacho Dos Anjos Henriques-Cadby I, Bortolami O, Flight L, Hind D, Jacques RM, et al. Recruitment and retention of participants in randomised controlled trials: a review of trials funded and published by the United Kingdom Health Technology Assessment Programme. BMJ Open. 2017;7(3):e015276. 32. Willan AR, Thabane L. Bayesian methods for pilot studies. Clin Trials 2020; 17(4):414-9. 33. Beets MW, Weaver RG, Ioannidis JPA, Geraci M, Brazendale K, Decker L, et al. Identification and evaluation of risk of generalizability biases in pilot versus efficacy/effectiveness trials: a systematic review and meta-analysis. Int J Behav Nutr Phys Act. 2020;17:19. 6. Julious SA. Sample size of 12 per group rule of thumb for a pilot study. Pharm Stat. 2005;4:287–91. 7. Thabane L, Ma J, Chu R, Cheng J, Ismaila A, Rios LP, et al. A tutorial on pilot studies: the what, why and how. BMC Med Res Methodol. 2010;10:1. 8. Browne RH. Abbreviations Alpha (α): Significance level (Type I error probability); AMBERG: AMBER sub- zone split adjacent to the GREEN zone (within 4-tiered approach); AMBERR: AMBER sub-zone split adjacent to the RED zone (within 4-tiered ap- proach); AC: AMBER-statistical significance threshold (within the AMBER zone) where an observed estimate below the cut-point will result in a non- Page 14 of 14 Page 14 of 14 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 Lewis et al. Pilot and Feasibility Studies (2021) 7:40 significant result (p ≥0.05) and figures at or above the cut-point will be sig- nificant (p < 0.05); AC%: AC expressed as a percentage of the sample size; Beta (β): Type II error probability; E: Estimate of feasibility outcome; ε: True feasibility parameter; GLL: Lower Limit of GREEN zone; n: Sample size (ns = number of patients screened; nr = number of patients randomised; ni = number of patients randomised to the intervention arm only); Power = 1-Beta: (1 – Type II error probability); RUL: Upper Limit of RED zone significant result (p ≥0.05) and figures at or above the cut-point will be sig- nificant (p < 0.05); AC%: AC expressed as a percentage of the sample size; Beta (β): Type II error probability; E: Estimate of feasibility outcome; ε: True feasibility parameter; GLL: Lower Limit of GREEN zone; n: Sample size (ns = number of patients screened; nr = number of patients randomised; ni = number of patients randomised to the intervention arm only); Power = 1-Beta: (1 – Type II error probability); RUL: Upper Limit of RED zone 10. Sim J, Lewis M. The size of a pilot study for a clinical trial should be calculated in relation to considerations of precision and efficiency. J Clin Epidemiol. 2012;65(3):301–8. 11. Whitehead AL, Julious SA, Cooper CL, Campbell MJ. Estimating the sample size for a pilot randomised trial to minimise the overall trial sample size for the external pilot and main trial for a continuous outcome variable. Stat Methods Med Res. 2016;25(3):1057–73. 12. Teare MD, Dimairo M, Shephard N, Hayman A, Whitehead A, Walters SJ. Sample size requirements to estimate key design parameters from external pilot randomised controlled trials: a simulation study. Trials. 2014;15:264. Acknowledgements f We thank Professor Julius Sim, Dr Ivonne Solis-Trapala, Dr Elaine Nicholls and Marko Raseta for their feedback on the initial study abstract. 13. Cocks K, Torgerson DJ. Sample size calculations for pilot randomized trials: a confidence interval approach. J Clin Epidemiol. 2013;66(2):197–201. 14. Lee EC, Whitehead AL, Jacques RM, Julious SA. The statistical interpretation of pilot trials: should significance thresholds be reconsidered? BMC Med Res Methodol. 2014;14:41. Author details 1 1Biostatistics Group, School of Medicine, Keele University, Room 1.111, David Weatherall Building, Keele, Staffordshire ST5 5BG, UK. 2Keele Clinical Trials Unit, Keele University, Keele, Staffordshire, UK. 3Centre for Biostatistics, School of Health Sciences, University of Manchester, Manchester, Staffordshire, UK. 23. Sim J. Should treatment effects be estimated in pilot and feasibility studies? Pilot Feasibility Stud. 2019;5:107. 24. Moore CG, Carter RE, Nietert PJ, Stewart PW. Recommendations for planning pilot studies in clinical and translational research. Clin Transl Sci. 2011;4(5):332–7. 25. Schoenfeld D. Statistical considerations for pilot studies. Int J Radiat Oncol Biol Phys. 1980;6(3):371–4. Received: 23 April 2020 Accepted: 7 January 2021 Received: 23 April 2020 Accepted: 7 January 2021 26. Fleiss JL, Levin B, Paik MC. Statistical methods for rates and proportions, Third Edition. New York: John Wiley & Sons; 2003. p. 32. Competing interests The authors declare that they have no competing interests. 22. Wilson DT, Walwyn RE, Brown J, Farrin AJ, Brown SR. Statistical challenges in assessing potential efficacy of complex interventions in pilot or feasibility studies. Stat Methods Med Res. 2016;25(3):997–1009. Authors’ contributions ML and CJS conceived the original methodological framework for the paper. ML prepared draft manuscripts. KB and GMcC provided examples and illustrations. All authors contributed to the writing and provided feedback on drafts and steer and suggestions for article updating. All authors read and approved the final manuscript. 15. Johanson GA, Brooks GP. Initial scale development: sample size for pilot studies. Edu Psychol Measurement. 2010;70(3):394–400. 15. Johanson GA, Brooks GP. Initial scale development: sample size for pilot studies. Edu Psychol Measurement. 2010;70(3):394–400. 16. Billingham SA, Whitehead AL, Julious SA. An audit of sample sizes for pilot and feasibility trials being undertaken in the United Kingdom registered in the United Kingdom Clinical Research Network database. BMC Med Res Methodol. 2013;13:104. Availability of data and materials Not applicable. Availability of data and materials Not applicable. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. 19. Arain M, Campbell MJ, Cooper CL, Lancaster GA. What is a pilot or feasibility study? A review of current practice and editorial policy. BMC Med Res Methodol. 2010;10:67. Funding g KB was supported by a UK 2017 NIHR Research Methods Fellowship Award (ref RM-FI-2017-08-006). KB was supported by a UK 2017 NIHR Research Methods Fellowship Award (ref RM-FI-2017-08-006). 17. Herbert E, Julious SA, Goodacre S. Progression criteria in trials with an internal pilot: an audit of publicly funded randomised controlled trials. Trials. 2019;20(1):493. 18. Avery KN, Williamson PR, Gamble C, O’Connell Francischetto E, Metcalfe C, Davidson P, et al. Informing efficient randomised controlled trials: exploration of challenges in developing progression criteria for internal pilot studies. BMJ Open. 2017;7(2):e013537. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 9. Hertzog MA. Considerations in determining sample size for pilot studies. Res Nurs Health. 2008;31(2):180–91.
https://openalex.org/W2000923919	https://europepmc.org/articles/pmc3605056?pdf=render	English	null	Optimal Vaccine Allocation for the Early Mitigation of Pandemic Influenza	PLOS computational biology/PLoS computational biology	2,013	cc-by	14,655	Optimal Vaccine Allocation for the Early Mitigation of Pandemic Influenza Laura Matrajt1¤, M. Elizabeth Halloran2,3, Ira M. Longini, Jr.4,5 Abstract This is an open-access article distributed under the terms of the Creative Comm unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was partially supported by National Institute of General Medical Sciences MIDAS grant U01-GM070749 and National Institute of Allergy and Infectious Diseases grant R37-AI32042. LM was partially supported by Consejo Nacional de Ciencia y Tecnologia, Mexico, scholarship 196221. (http://www.nigms. nih.gov/Research/FeaturedPrograms/MIDAS/, http://www.niaid.nih.gov/Pages/default.aspx, http://www.conacyt.gob.mx/Paginas/default.aspx). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: laurama@uw.edu ¤ Current address: Department of Medicine, University of Washington, Seattle, Washington, United States of America. In this highly connected world, people travel fast, and new strains of influenza can travel with them. Indeed, the airline transportation network can accelerate the diffusion of new strains of influenza. For example, the pandemic influenza A(H1N1) 2009 (2009H1N1P) was first detected in Mexico in April 2009, and only two weeks later, more than twenty countries reported their first cases of 2009H1N1P, with most of these cases being imported via airline travel [6,7]. This highlights the necessity of an extremely fast global response to a new strain of pandemic influenza, on the order of days. Laura Matrajt1¤, M. Elizabeth Halloran2,3, Ira M. Longini, Jr.4,5 1 Department of Applied Mathematics, University of Washington, Seattle, Washington, United States of America, 2 Center for Statistics and Quantitative Infectious Diseases, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 3 Department of Biostatistics, School of Public Health, University of Washington, Seattle, Washington, United States of America, 4 Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America, 5 Department of Biostatistics, Colleges of Medicine, and Public Health and Health Professions, University of Florida, Gainesville, Florida, United States of America Abstract With new cases of avian influenza H5N1 (H5N1AV) arising frequently, the threat of a new influenza pandemic remains a challenge for public health. Several vaccines have been developed specifically targeting H5N1AV, but their production is limited and only a few million doses are readily available. Because there is an important time lag between the emergence of new pandemic strain and the development and distribution of a vaccine, shortage of vaccine is very likely at the beginning of a pandemic. We coupled a mathematical model with a genetic algorithm to optimally and dynamically distribute vaccine in a network of cities, connected by the airline transportation network. By minimizing the illness attack rate (i.e., the percentage of people in the population who become infected and ill), we focus on optimizing vaccine allocation in a network of 16 cities in Southeast Asia when only a few million doses are available. In our base case, we assume the vaccine is well-matched and vaccination occurs 5 to 10 days after the beginning of the epidemic. The effectiveness of all the vaccination strategies drops off as the timing is delayed or the vaccine is less well-matched. Under the best assumptions, optimal vaccination strategies substantially reduced the illness attack rate, with a maximal reduction in the attack rate of 85%. Furthermore, our results suggest that cooperative strategies where the resources are optimally distributed among the cities perform much better than the strategies where the vaccine is equally distributed among the network, yielding an illness attack rate 17% lower. We show that it is possible to significantly mitigate a more global epidemic with limited quantities of vaccine, provided that the vaccination campaign is extremely fast and it occurs within the first weeks of transmission. Citation: Matrajt L, Halloran ME, Longini IM, Jr (2013) Optimal Vaccine Allocation for the Early Mitigation of Pandemic Influenza. PLoS Comput Biol 9(3): e1002964. doi:10.1371/journal.pcbi.1002964 Editor: Neil Ferguson, Imperial College London, United Kingdom Editor: Neil Ferguson, Imperial College London, United Kingdom Received July 13, 2012; Accepted January 16, 2013; Published March 21, 2013 Received July 13, 2012; Accepted January 16, 2013; Published March 21, 2013 Copyright: 2013 Matrajt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Matrajt et al. PLOS Computational Biology \| www.ploscompbiol.org March 2013 \| Volume 9 \| Issue 3 \| e1002964 Author Summary In the past, the emergence of new strains of influenza has been sometimes responsible for large and deadly pan- demics. With a very high mortality rate, (i.e., about 60% of the reported cases), H5N1AV influenza, commonly known as bird flu, is thought to be an important potential threat for a new pandemic. Because of this, several vaccines have been developed, but only a few million doses are readily available. Other zoonotic influenza strains, particularly in pigs, also threaten, and vaccines are being produced for them as well. In the event of an influenza pandemic, utilizing these resources optimally could make the difference between dealing with a serious infectious disease at a global scale and reducing it to a highly localized and controlled outbreak. In this paper, we address this issue by developing a mathematical model of influenza transmission on a network of cities. We couple the model with an optimization algorithm to allocate vaccine in time and space through the network. We find that our optimal allocation strategies can mitigate a pandemic, provided that vaccination occurs quickly, within the first weeks of a potential pandemic. In addition, our analysis highlights the importance of cooperative and coordinated vaccine distribution, if we want to mitigate a pandemic. Results for a single batch of vaccine These cities are highly connected (fig. 1) and were previously shown to form a available prior to an epidemic. In the present work, we focus on optimizing vaccine allocation in a network of cities when only a few million doses are available, and we search for their optimal distribution by minimizing the illness attack rate (i.e., the percentage of people in the population who became infected and ill). We couple a mathematical infection transmission model with two age-groups, children and adults, with a fast genetic algorithm [22] to optimally and dynamically distribute vaccine through a network of cities, connected by the airline transportation network. Since Southeast Asia has seen more than half of the H5N1AV influenza related deaths worldwide, we consider this region for our model. We selected the most highly populated 16 cities in Southeast Asia for which we could find reliable airline transportation data (fig. 1). This represents 10 different countries and a total population of 70,980,365 people (table 1). These cities are highly connected (fig. 1) and were previously shown to form a All the simulations presented below started in the same city, Jakarta, with 10 infectious people. Fig. S1 shows the epidemic curves when no vaccination is applied. We also started our simulations in Hong Kong and Taipei. The transmissibility of an infectious disease is often characterized by the basic reproduction number, R0, defined as the expected number of secondary infections resulting from a single typical infectious person in a completely susceptible population. In the results presented here, R0~1:5, corresponding to a virus roughly as transmissible as the 2009H1N1P [24,25]. We also considered a less (R0~1:2) and a more (R0~1:8) transmissible virus. Figure 1. Network representation of 16 cities in Southeast Asia used for the simulations. An edge connecting two cities represents daily travel between those cities. The size of the nodes in (A) correspond to the population size relative to the total population in the network. The size of the nodes in (B) correspond to the flux of passengers traveling through each city relative to the total number of flights in the network. The base case simulations were started in Jakarta, which accounts for 12.8% of the total population of the network but only 7% of the total daily travel goes through it. doi:10.1371/journal.pcbi.1002964.g001 Figure 1. Network representation of 16 cities in Southeast Asia used for the simulations. Results for a single batch of vaccine Because we are interested in investigating the optimal use of vaccine for a quick response, we concentrate most of this work on optimizing vaccine delivered in a single batch at the beginning of the epidemic when only a few million doses are available. We considered four vaccination days very early in transmission, at either 5, 10, 15, or 30 days after its start, and two vaccination days later on at either 60 or 90 days after the beginning of transmission. Since we cannot know how much vaccine production will be ready at the time of an epidemic, for each of these control days, we consider allocating two, four, five, six, seven or ten million doses corresponding to vaccinating 2.8, 5.6, 7.0, 8.5, 9.9 or 14.1% of the total population, respectively (table 2). For each possible vaccination day and coverage combination, we compare the best vaccine allocation given by the genetic algorithm, denoted as the optimal strategy, to a baseline scenario, where no vaccine is available, and two other possible allocations. The first is the pro rata strategy, where we distribute vaccine to each age-group in each city proportional to the age-group’s size. For example, if adults in city k correspond to 20% of the total population in the network, then we assign 20% of the available resources to the adults in city k. The second strategy is the children-only pro rata strategy, where vaccine is distributed only to the children in each city proportional to the children’s population size. available prior to an epidemic. In the present work, we focus on optimizing vaccine allocation in a network of cities when only a few million doses are available, and we search for their optimal distribution by minimizing the illness attack rate (i.e., the percentage of people in the population who became infected and ill). We couple a mathematical infection transmission model with two age-groups, children and adults, with a fast genetic algorithm [22] to optimally and dynamically distribute vaccine through a network of cities, connected by the airline transportation network. Since Southeast Asia has seen more than half of the H5N1AV influenza related deaths worldwide, we consider this region for our model. We selected the most highly populated 16 cities in Southeast Asia for which we could find reliable airline transportation data (fig. 1). This represents 10 different countries and a total population of 70,980,365 people (table 1). Introduction Highly pathogenic avian influenza A(H5N1AV) emerged in the 1990s in Southeast Asia with new cases arising in different parts of the world [1]. Recent studies have shown that a few mutations can make the H5N1AV influenza virus transmissible in ferrets [2,3], reminding us that the possibility of a mutated H5N1AV influenza strain capable of infecting humans might not be a remote one. With a very high mortality ratio (i.e., about 60% of the reported cases), the threat of a H5N1AV influenza pandemic remains one of the biggest public health fears. Many pharmaceutical and non- pharmaceutical interventions can be implemented during a pandemic, but vaccination, when available, is the most effective intervention. Several vaccines are being produced specifically for H5N1AV [4] , but their production is still very limited [5]. In the event of a H5N1AV pandemic, utilizing these vaccines optimally at the beginning of transmission could make the difference between reducing transmission to negligible levels or dealing with a deadly infectious disease on a global scale. Mathematical models are useful tools to explore different pandemic scenarios and possible interventions, and they are particularly well-suited for determining optimal vaccine distribu- tion. With pioneering work starting in the 1970s, [8,9] and more recently [10,11,12,13,14,15,16,17,18,19,20,21], important prog- ress has been made in investigating the optimal resource allocation for a given population. Most of this work has been centered around a single population, and assumes that all the vaccine, enough to cover a significant fraction of the population, will be March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 1 Optimal Vaccine Allocation for Pandemic Influenza transmission cluster in the global network of cities [23]. We show that the vaccine allocation proposed by our optimizer could greatly reduce the attack rate. Author Summary Results for a single batch of vaccine An edge connecting two cities represents daily travel between those cities. The size of the nodes in (A) correspond to the population size relative to the total population in the network. The size of the nodes in (B) correspond to the flux of passengers traveling through each city relative to the total number of flights in the network. The base case simulations were started in Jakarta, which accounts for 12.8% of the total population of the network but only 7% of the total daily travel goes through it. doi:10.1371/journal.pcbi.1002964.g001 g doi:10.1371/journal.pcbi.1002964.g001 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org PLOS Computational Biology \| www.ploscompbiol.org 2 Optimal Vaccine Allocation for Pandemic Influenza Table 1. Population values. City Population Percentage of the total populationNation Percent under 20a Bandung 2,510,982 3.5 Indonesia 38.32 Bangkok 6,704,000 9.4 Thailand 29.95 Denpasar 405,923 0.5 Indonesia 38.32 Haiphong 2,614,764 3.7 Vietnam 35.23 Ho Chi Minh City 5,314,000 7.5 Vietnam 35.23 Hong Kong 7,206,000 10.2 China 19.30 Jakarta 9,125,000 12.9 Indonesia 38.32 Kaohsiung 1,526,575 2.2 Taiwan 22.56 Kuala Lumpur 1,887,674 2.7 Malaysia 41.39 Manila 11,100,000 15.6 Philippines 44.24 Medan 2064719 2.9 Indonesia 38.32 Phnom Penh 2,009,264 2.8 Cambodia 45.78 Rangoon 4,477,638 6.3 Burma 41.52 Singapore 4,436,000 6.3 Singapore 25.65 Surabaya 2,845,000 4.0 Indonesia 38.32 Taipei 6,752,826 9.5 Taiwan 22.56 TOTAL 70,980,365 100 aThe percentages for each city were computed from [58] using the countr y’s percentage of children under 20 years old. Taiwan’s percentage was obtained from [59]. doi:10.1371/journal.pcbi.1002964.t001 aThe percentages for each city were computed from [58] using the countr y’s percentage of children under 20 years old. Taiwan’s percentage was obtained from [59]. doi:10 1371/journal pcbi 1002964 t001 aThe percentages for each city were computed from [58] using the countr y’s percentage of children under 20 years old. Taiwan’s percentage was obtained from [59]. doi:10.1371/journal.pcbi.1002964.t001 The optimal strategy shows a modest decrease in the attack rate when very little vaccine is available, provided vaccination occurs during the first five days of the epidemic (i.e., 27% reduction in the attack rate relative to the baseline for two million doses, fig. 2A). As more vaccine becomes available, the optimal strategy greatly reduces the attack rate, although this effect is attenuated as vaccination starts later in the epidemic. Results for a single batch of vaccine Million doses Percentage of the total population 2 2.8 4 5.6 5 7.0 6 8.5 7 9.9 10 14.1 doi:10.1371/journal.pcbi.1002964.t002 PLOS Computational Biology \| www.ploscompbiol.org Table 2. Vaccine coverages considered. Million doses Percentage of the total population 2 2.8 4 5.6 5 7.0 6 8.5 7 9.9 10 14.1 doi:10.1371/journal.pcbi.1002964.t002 PLOS Computational Biology \| www.ploscompbiol.org Results for a single batch of vaccine For example, if vaccination occurs on day five with five million doses, 3.5% of the population would become infected, with an 85% reduction in the attack rate compared to the baseline case. But if vaccination occurs on day 30, then 14.9% of the population would become infected with only 41% reduction compared to baseline (fig. 2C). Once 10 million doses are available, the optimal strategy can interrupt transmission as long as vaccination occurs before or on day 30 (fig. 2F). peaking when five and 10 million doses are available (17% and 16% difference in the attack rate, respectively, fig. 2C and 2F). Our results suggest that for the vaccine coverages considered, the pro rata strategy is somewhat insensitive to the timing of the intervention, while the other two strategies considered are not. This would further imply that the pro rata strategy has little indirect effects of herd immunity, but still protects the individuals being vaccinated. When compared to the children-only pro rata strategy, the optimal strategy performs better only when vaccination occurs early in the epidemic and there are few doses of vaccine available (fig. 2B–D). As more vaccine becomes available, the optimal strategy and the children-only pro rata strategy yield very similar attack rates, and for some cases the optimal strategy is in fact the children-only pro rata strategy (for example fig. 2F, vaccination on day 30, 60 or 90). The optimal strategy outperforms the pro rata strategy for all scenarios considered. With only two million doses available, there is a slight difference in the attack rate with early vaccination (5% difference), but this difference tends to disappear as we start vaccination later in the epidemic (fig. 2A). As more vaccine becomes available, this difference becomes more noticeable, We next investigate the capacity of a strategy to mitigate a large epidemic (where more than 1% of the population in each city got infected and ill) from occurring. To this end, we calculate for each solution the epidemic prevention potential (EPP) [26], defined as one minus the ratio of the probability of an epidemic given a particular intervention over the probability of an epidemic given no intervention (fig. 3). Mathematically, Table 2. Vaccine coverages considered. Table 2. Vaccine coverages considered. Table 2. Vaccine coverages considered. Sensitivity analysis two strategies considered fail to mitigate the epidemics in all of the scenarios considered when less than 10 million doses are available.With 10 million doses, the optimal strategy mitigates over 90% of the epidemics as long as vaccination occurs during the first 15 days. If vaccination occurs during the first ten days, children-only pro rata is also able to mitigate the majority of the epidemics with ten million doses. When the epidemic starts in Hong Kong, applying the optimal strategy results in a lower EPP and a lower reduction in the attack rate compared to baseline, specially noticeable when only a few million doses of vaccine are available (fig. S2 and S3). Similar results were obtained when the epidemic started in Taipei (figs. S4 and S5). Only 7% of the daily travel through the network goes through Jakarta. In contrast, 17% of the daily flux of travelers occurs (fig. 1B and table 3) in Hong Kong (16% of the daily flux occurs in Taipei). Our results suggest that if an epidemic starts in a city with a more important flux of travelers, it will spread more rapidly and will be more difficult to mitigate. Our results suggest that with limited quantities of vaccine, the geographical allocation of vaccine is key in stopping the epidemic if vaccination occurs early on, with most of the vaccine going only to a few cities (figs. S2A, S3A). However, as the epidemic progresses, or more vaccine becomes available, allocating resources more evenly, to the high transmission groups, here the children, becomes a predominant feature (figs. 4C–E and 5C–E). For late vaccination, allocating vaccine in children becomes less relevant and again, geographical location is more important, with the optimal strategy favoring cities where either where the epidemic has not peaked yet or it is possible to reach a high proportion of children vaccinated (figs. 4F, 5F). This is in agreement with previous work [27,28,20], which have suggested that vaccinating children is important early in the epidemic, but that there is a threshold after which other groups might benefit more from limited quantities of vaccine. Our results were not sensitive to changes in the basic reproductive number. When the epidemic is less transmissible, all three strategies perform better, with the optimal strategy being slightly better than the children-only pro rata, both yielding to substantial reductions in the attack rate (fig. S6). EPP~1{ Pr(EpidemicD intervention) Pr(EpidemicDno intervention) : ð1Þ ð1Þ The optimal strategy is able to mitigate epidemics with very low quantities of vaccine, provided that vaccination starts very early in the epidemic. With as little as four million doses, the optimal strategy mitigates 58% of the epidemics if vaccination starts on day 5, but only 21% if vaccination starts on day 10 (fig. 3B). As more vaccine becomes available, the optimal strategy is able to mitigate a higher proportion of epidemics (fig. 3C–F). In contrast, the other PLOS Computational Biology \| www.ploscompbiol.org March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 3 Optimal Vaccine Allocation for Pandemic Influenza Figure 2. Attack rate with 95% bootstrapped CI with the epidemic starting in Jakarta. This figure shows the results for a single intervention for six different vaccination days considered and six different vaccination coverages. Each panel represents a given number of vaccine doses available to distribute in the entire network: A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. The baseline scenario (red) indicates no vaccination. For each vaccination coverage and day combination, the optimal strategy considerably outperformed the pro rata strategy. When vaccination occurs early in the epidemic, and few doses are available, the optimal strategy also outperforms the children-only pro rata strategy. doi:10.1371/journal.pcbi.1002964.g002 Figure 2. Attack rate with 95% bootstrapped CI with the epidemic starting in Jakarta. This figure shows the results for a single intervention for six different vaccination days considered and six different vaccination coverages. Each panel represents a given number of vaccine doses available to distribute in the entire network: A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. EPP~1{ Pr(EpidemicD intervention) Pr(EpidemicDno intervention) : ð1Þ The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. The baseline scenario (red) indicates no vaccination. For each vaccination coverage and day combination, the optimal strategy considerably outperformed the pro rata strategy. When vaccination occurs early in the epidemic, and few doses are available, the optimal strategy also outperforms the children-only pro rata strategy. doi:10.1371/journal.pcbi.1002964.g002 PLOS Computational Biology \| www.ploscompbiol.org March 2013 \| Volume 9 \| Issue 3 \| e1002964 Sensitivity analysis With as few as 4 million doses, the optimal strategy can mitigate as many as 57% of the epidemics. doi:10.1371/journal.pcbi.1002964.g003 prominent than for R0~1:5, suggesting that when the virus is more transmissible allocating the resources optimally is more important, at least at the beginning of the epidemic (see Text S1 for details). vaccination occurred during the first days of the epidemic (see Text S1). We also varied the values of the vaccine efficacies. We considered a poorly-matched vaccine (VES~0:13, VEI~0:15 and VEP~0:25) and a moderately-matched vaccine (VES~0:27, VEI~0:30, and VEP~0:50). These values corre- spond to one-third and two-thirds of the values used for the original analysis, which assumed that the vaccine would be as efficacious as seasonal vaccines. As expected, if the vaccine is poorly-matched, all the strategies perform poorly yielding high attack rates (fig. S13A), and all of them fail to prevent the epidemics (fig. S14A). If the vaccine is moderately-matched, the results are close to the original results, with the optimal strategy yielding clear reductions in the attack rates (fig. S13B) and a significant proportion of the epidemics prevented, when vaccina- tion occurs early on (fig. S14B). Furthermore, we performed sensitivity analysis to the assumption that vaccine is administered at once, and repeated the optimization assuming that the vaccination would be completed in 10 days (fig. S15, see Text S1 for details). While the optimal strategy still performs better than the other strategies considered, the difference in the attack rate (panel A) is not as marked as before. The optimal strategy in this case could prevent 36% of the epidemics but only if vaccination started on day 5 (panel B). Our model assumed that infectious symptomatic individuals will reduce their probability of traveling by 25%. We performed sensitivity analysis with respect to this parameter (fig. S10 and S11). We also performed sensitivity analysis with respect to the reduction of the travel probability of an infected child compared to the travel probability of an infected adult (fig. S12). As expected, if more infected symptomatic people travel through the network (10% reduction in the probability of travel) the attack rates are slightly higher (fig. S10A), and the EPP is not as good (fig. S11A). This makes sense since in this case the epidemic process is accelerated. Sensitivity analysis This is expected, as we know from previous studies [29] that for a low R0 as this one, the threshold in deterministic models for the number of children and adults needed to be vaccinated to bring R0 below 1 is much lower. The optimal strategy could mitigate most of the epidemics with as little as two million doses of vaccine (fig. S7). For a higher R0, the optimal strategy still outperforms the pro rata strategy. If vaccination occurs early on, this difference is more March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 4 Optimal Vaccine Allocation for Pandemic Influenza Figure 3. Epidemic prevention potential (EPP) with 95% bootstrapped CI with the epidemic starting in Jakarta. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. Each point in each graph corresponds to the EPP for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. When fewer than 10 million doses of vaccine are available, only the optimal strategy is capable of mitigating a significant fraction of the epidemics if vaccination starts early. With as few as 4 million doses, the optimal strategy can mitigate as many as 57% of the epidemics. doi:10.1371/journal.pcbi.1002964.g003 Figure 3. Epidemic prevention potential (EPP) with 95% bootstrapped CI with the epidemic starting in Jakarta. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. Each point in each graph corresponds to the EPP for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. When fewer than 10 million doses of vaccine are available, only the optimal strategy is capable of mitigating a significant fraction of the epidemics if vaccination starts early. PLOS Computational Biology \| www.ploscompbiol.org Sensitivity analysis These results suggest that the geographical allocation of vaccine is important early in the beginning of the epidemic, when the optimal strategy allocates most of vaccine in Jakarta, but then it is better to distribute vaccine evenly among children up to a certain threshold in time when it becomes important to allocate the vaccine to those cities where either the epidemic has not peaked yet or it is possible to reach a higher proportion of children vaccinated. doi:10.1371/journal.pcbi.1002964.g004 five million doses and the second one with either five, 10, or 15 million doses. We further consider allocating vaccine on either days 10 and 30, days 10 and 60, or days 30 and 90 after the beginning of the epidemic. Here, the optimal strategy basically coincides with the children-only pro rata strategy for all scenarios considered but one, where the optimal strategy has a modest 2% lower attack rate (Fig. 6A). For all the scenarios considered, these strategies perform much better than the pro rata strategy, particularly when vaccination occurs on days 10 and 30, with a maximal reduction of 17% in the attack rate (Fig. 6B). Finally, we performed the optimization assuming that only children could receive vaccine. Here, the optimal strategy also outperforms the other two strategies considered (fig. S16A), and yields an EPP similar to the one obtained when the optimization was performed using the entire population (fig. S16B, see Text S1 for details). These results suggest that there might be a tradeoff between vaccinating specific geographical locations and vaccinat- ing high-transmission groups. For some vaccine coverages or vaccination dates, it might be better to vaccinate children and adults in a city to guarantee that the reproduction number is below 1 in as many cities as possible, but for other coverages or dates it might be better to concentrate the effort in the children (the high- transmission group). This is consistent with previous work [30,12,27,31,32,33], which have proposed tradeoffs between vaccinating the high-transmission groups and the high-risk groups. With seven or ten million doses (both batches included), all the strategies considered fail to mitigate the epidemic (Fig. 7A–B). With 15 million doses of vaccine, the optimal strategy and the children-only pro rata strategy mitigate over 95% of the epidemics if vaccination occurs at days 10 and 30 or 10 and 60, but cannot mitigate any epidemic if vaccination occurs at days 30 and 90 (Fig. 7C). Sensitivity analysis On the other hand, if the reduction in the travel probability is higher (75% reduction) then less infected symptom- atic people are traveling through the network. Here, the attack rates are lower and the optimal strategy can prevent epidemics for more days (figs. S10B, S11B, and S12). Our conclusions were not sensitive to changes in the probability of travel for symptomatic infectious people (both adults and children), or in changes in the probability of travel for symptomatic infectious children in the sense that the optimal strategy outperformed the other two strategies considered. The biggest difference was still when March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 5 Optimal Vaccine Allocation for Pandemic Influenza Figure 4. Optimal vaccine distribution when four million doses are available and the epidemic is started in Jakarta. Each panel corresponds to allocating vaccine on a different day: either 5 days (A), 10 days (B), 15 days (C), 30 days (D), 60 days (E), or 90 days (F) after the beginning of the epidemic. Each bar corresponds to the percentage of children (red) or adults (blue) vaccinated in each city. These results suggest that the geographical allocation of vaccine is important early in the beginning of the epidemic, when the optimal strategy allocates most of vaccine in Jakarta, but then it is better to distribute vaccine evenly among children up to a certain threshold in time when it becomes important to allocate the vaccine to those cities where either the epidemic has not peaked yet or it is possible to reach a higher proportion of children vaccinated. doi:10.1371/journal.pcbi.1002964.g004 Figure 4. Optimal vaccine distribution when four million doses are available and the epidemic is started in Jakarta. Each panel corresponds to allocating vaccine on a different day: either 5 days (A), 10 days (B), 15 days (C), 30 days (D), 60 days (E), or 90 days (F) after the beginning of the epidemic. Each bar corresponds to the percentage of children (red) or adults (blue) vaccinated in each city. Sensitivity analysis These results are expected, since we found that for a single intervention, the EPP was zero for all the strategies considered if vaccination started after day 30. Results for two batches of vaccine In this section we present the results when vaccine is considered to be available in two batches, the first one carrying only two or PLOS Computational Biology \| www.ploscompbiol.org March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 6 Optimal Vaccine Allocation for Pandemic Influenza Figure 5. Optimal vaccine distribution when seven million doses are available and the epidemic is started in Jakarta. Each panel corresponds to allocating vaccine on a different day: either 5 days (A), 10 days (B), 15 days (C), 30 days (D), 60 days (E), or 90 days (F) after the beginning of the epidemic. Each bar corresponds to the percentage of children (red) or adults (blue) vaccinated in each city. When vaccination occurs early in the epidemic, the optimal strategy is to allocate most of the available vaccine in Jakarta. As vaccination occurs later on, the optimal strategy switches to favor a more evenly distribution of vaccine amongst children. For late vaccination, allocating vaccine evenly in children becomes less relevant but instead the optimal strategy allocates vaccine to fewer cities where the epidemic has not peaked yet or it is possible to reach a high proportion of children vaccinated. doi:10.1371/journal.pcbi.1002964.g005 Figure 5. Optimal vaccine distribution when seven million doses are available and the epidemic is started in Jakarta. Each panel corresponds to allocating vaccine on a different day: either 5 days (A), 10 days (B), 15 days (C), 30 days (D), 60 days (E), or 90 days (F) after the beginning of the epidemic. Each bar corresponds to the percentage of children (red) or adults (blue) vaccinated in each city. When vaccination occurs early in the epidemic, the optimal strategy is to allocate most of the available vaccine in Jakarta. As vaccination occurs later on, the optimal strategy switches to favor a more evenly distribution of vaccine amongst children. For late vaccination, allocating vaccine evenly in children becomes less relevant but instead the optimal strategy allocates vaccine to fewer cities where the epidemic has not peaked yet or it is possible to reach a high proportion of children vaccinated. doi:10 1371/journal pcbi 1002964 g005 doi:10.1371/journal.pcbi.1002964.g005 versus other types of optimal policies [34]. Dimitrov et al. optimized antiviral allocation for the 2009 influenza epidemic in the United States [11]. Results for two batches of vaccine More recently, Klepac and colleagues [36] developed a model of two coupled populations to incorporate economic costs in finding optimal vaccination thresholds. Keeling and Shattock [13] used the final epidemic size to compute optimal vaccine distribution for two interacting communities. The present study contributes to the body of knowledge by incorporating stochastic components in the model and by allowing the possibility of dynamic allocation of resources, both before and after the beginning of an epidemic. In addition, our work can easily be adapted for other infectious diseases. For example, our method- ology could be used for the novel reassortant influenza A(H3N2) virus, originating from swine, avian, and human viruses which has been spreading from swine to humans, and then among humans in the United States [37]. Indeed, because wider spread of this virus is possible, plans are underway to make limited quantities of this vaccine if needed. PLOS Computational Biology \| www.ploscompbiol.org Optimal Vaccine Allocation for Pandemic Influenza While genetic algorithms have the advantage of being fast and adaptable, they are not guaranteed to converge. We have reported here our best solutions and shown that even if these solutions are only nearly optimal, they still perform much better than the status quo strategies, which are usually pro rata. Table 3. Daily flux of passengers through the network. Table 3. Daily flux of passengers through the network. City Percentage of the total travel Bandung 0.03 Bangkok 11.52 Denpasar 3.95 Haiphong 0.05 Ho Chi Minh 2.31 Hong Kong 17.36 Jakarta 7.47 Kaohsiung 8.85 Kuala Lumpur 7.23 Manila 4.13 Medan 1.1 Phnom Penh 1.06 Rangoon 0.66 Singapore 15.33 Surabaya 2.32 Taipei 16.61 Percentage of the total number of daily passengers traveling through each city considered in the network. doi:10.1371/journal.pcbi.1002964.t003 Percentage of the total number of daily passengers traveling through each city considered in the network. doi:10.1371/journal.pcbi.1002964.t003 Furthermore, the results presented here emphasize the impor- tance of confronting a global problem with a global solution, rather than many individualistic ones. Indeed, when little vaccine is available, sharing resources and optimally vaccinating early in the outbreak results in mitigating the epidemic most of the time, while the pro rata strategy always results in an epidemic with high attack rates. This is in agreement with previous work by Colizza et al. [38], in which they compared three different strategies for antiviral use and found that cooperative strategies perform better than non cooperative strategies. Our study also highlights the importance of having a good global surveillance system. In our results, most of the epidemics could be mitigated with as little as five million doses of vaccine provided that we act fast, during the first days of an epidemic, but no mitigation is likely to succeed after just a few weeks (30 days). In brief, our results suggest that a global stockpile of vaccine as the one established by WHO [39] would be fundamental to stop an H5N1AV influenza pandemic. Many of the current governmental or institutional guidelines for vaccine allocation are indeed based in a prorated type strategy, where vaccine is distributed to states or countries according to their population (e.g [40,41]). While this is presumably the most fair strategy, it could also be, depending on the objective function used, not the optimal use of resources. Optimal Vaccine Allocation for Pandemic Influenza Optimal Vaccine Allocation for Pandemic Influenza present in the city. We could not find reliable data regarding the probability of an infected person suspending a trip due to illness, or differential rates for traveling stratified by age. In this sense, our model is conservative. Better data is urgently needed to create more realistic models. Clearly, the simultaneous use of multiple interventions would be more efficient in controlling an epidemic, so an optimization routine that considers a portfolio of interven- tions points the direction for future work. We assumed that the vaccines for H5N1AV influenza virus would be as efficacious as the seasonal vaccines. However, the current available vaccines for avian influenza might not be well-matched to a new avian influenza virus. Clearly, the effectiveness of any vaccination policy relies on the efficacy of the vaccine used. Indeed, if the vaccine were poorly-matched, then the optimal solution, while still being better than the other two strategies considered, would not be very efficacious. This highlights the necessity to recognize the influenza strain quickly, so that the efficacy of the vaccine can be rapidly evaluated, and additional targeted mitigation measures can be implemented if necessary. In addition, our model does not account for antibody buildup. Adding this feature would probably yield results similar to those when vaccine is completed in several days. Our sensitivity analysis showed that the attack rates and the EPP were considerably lower when vaccination was completed in several days. This highlights the importance of a fast and aggressive vaccination campaign. In addition, our results were sensitive to the knowledge of the exact point in the epidemic (calculated starting from the introduction of the first infected individuals) when vaccination was applied, specially during the first days of an epidemic. This could become problematic in a real situation, since inferring the exact date of the beginning of an epidemic from epidemiological data might be very difficult to achieve. The comparison between one and two batches of vaccine showed that the results were sensitive to the assumption of an a- priori knowledge of the vaccination times. As the H1N1 2009 influenza epidemic showed us, different problems can arise during the production of a vaccine leading to delays in its delivery. In this sense, our conclusions are optimistic. The results presented here were obtained using a genetic algorithm. Discussion We propose here a mathematical model of influenza transmission coupled with a fast genetic algorithm, which provides strategies for vaccine allocation in a network of cities, rather than in an individual population. Our results show that the optimal strategy found by our genetic algorithm always outperforms a pro rata strategy, both in reducing the total final attack rate and in increasing the probability of mitigating an epidemic, with differences in the attack rates as high as 16% of the population. This conclusion is consistent with previous findings [34]. Substantial study has been done to optimally allocate vaccine within a country or a population [11,12,8,9,15,16,17,18,19, 20,35,21]. Furthermore, significant advances have been made in optimizing resources for an epidemic in a network: Wu et al. considered a metapopulation model of the continental US in 2007, and they compared vaccine allocation in a prorated schedule March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 7 PLOS Computational Biology \| www.ploscompbiol.org Optimal Vaccine Allocation for Pandemic Influenza Optimal strategies depend heavily in the objective function used, and different objective functions can give rise to contradicting vaccination policies [42,13,34]. While our optimal strategy significantly reduces the overall attack rate, it creates an inequitable distribution of burden of disease, with some cities having no epidemic at all and others experiencing a big epidemic. An objective function with constraints in the distribution of vaccine would be more fair. Also, other objective functions could be used instead. For example, one could set the objective function that allocates vaccine in fewer populations but guarantees to attain critical coverage in them, so that R0v1, or one could set an objective function that maximizes the EPP, thereby maximizing the probability of a strategy to mitigate an epidemic. Our results suggest that vaccinating only children in a pro rated fashion can be a very efficient solution, and it is more likely to be accepted by the population. A strategy for The framework presented here has several limitations. Once the epidemic starts in a city, our model becomes deterministic, and this comes with simplifying assumptions. While in the present study the objective function was set to minimize the overall illness attack rate, the optimal solution depends heavily on attaining the critical vaccination coverage in each city: indeed, under the deterministic paradigm, an epidemic will not take off in a city if R0ƒ1. This threshold will be achieved once a significant fraction of children is vaccinated [29]. In reality, even if R0ƒ1, a small epidemic exceeding the 1% threshold we set for EPP could possibly occur. Thus our EPP values could be overly optimistic. The network used here is a closed network, which does not allow for any immigration or emigration of passengers from other cities and we did not consider any flow of passengers through ground transportation. Any epidemic will probably spread beyond this network, potentially affecting the effectiveness our results. The beginning of an epidemic in each city was approximated by a simple birth-death process, and it does not differentiate between the number of infected children and the number of infected adults March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 8 Optimal Vaccine Allocation for Pandemic Influenza Figure 6. Attack rate for vaccine allocation in two batches on two different days. Optimal Vaccine Allocation for Pandemic Influenza Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. Here, the epidemic was seeded in Jakarta. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. Here, the optimal strategy and the children-only pro rata strategy yield similar attack rates for all scenarios considered except for one. When seven million doses of vaccine are available at days 10 and 30, the optimal strategy yield a slightly lower attack rate than the children-only pro rata strategy. doi:10.1371/journal.pcbi.1002964.g006 Figure 6. Attack rate for vaccine allocation in two batches on two different days. Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. Here, the epidemic was seeded in Jakarta. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. Here, the optimal strategy and the children-only pro rata strategy yield similar attack rates for all scenarios considered except for one. When seven million doses of vaccine are available at days 10 and 30, the optimal strategy yield a slightly lower attack rate than the children-only pro rata strategy. doi:10.1371/journal.pcbi.1002964.g006 prioritizing high-risk people and health workers together with children might be a good starting point for discussion. Optimal Vaccine Allocation for Pandemic Influenza Choosing a vaccination strategy is inherently a difficult process, as one needs to balance concepts like ethics, equality or fairness, together with practical and logistical implications, and economic considerations. We hope to provide decision-makers with the tools to find optimal resource distribution, so that once the goals are established, the available resources can be used at their best. stochastic effect during an epidemic was the transportation of newly infected people between cities and on the initialization of an epidemic in a city. We then assumed a deterministic course of the epidemic once established in a city. Table 4 summarizes the parameter values used in the model. The base case scenario had an epidemic with a basic reproduction number R0 of 1.5 on average. We assumed that vaccine efficacies would be similar to those for the seasonal influenza vaccine, and used the estimates given in [44]. The population in each city is divided into children (age v20 years) or adults (age §20 years), where the proportion of people in each age-group for each city is given by the proportion of people in that age-group for that country (see table 1). The contact rates were computed based on [45]. The data for the airline transportation network was taken from [46] and [23]. A full description of the implementation can be found in the Text S1. Mathematical model In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. Here, the epidemic was seeded in Jakarta. All strategies fail to mitigate the epidemics if seven or 10 million doses are available in two batches. With 15 million doses, the optimal strategy and the children-only pro rata strategy mitigate over 95% of the epidemics when the first batch of vaccine is delivered on day 10 and the second one is delivered either on day 30 or on day 60. doi:10.1371/journal.pcbi.1002964.g007 Figure 7. Epidemic prevention potential for vaccine allocation in two batches on two days. Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. Here, the epidemic was seeded in Jakarta. All strategies fail to mitigate the epidemics if seven or 10 million doses are available in two batches. With 15 million doses, the optimal strategy and the children-only pro rata strategy mitigate over 95% of the epidemics when the first batch of vaccine is delivered on day 10 and the second one is delivered either on day 30 or on day 60. doi:10.1371/journal.pcbi.1002964.g007 Each day, a random number rkl of infected travelers going from city k to city l is computed for each of the eight infected classes (children or adults, asymptomatic vaccinated or unvaccinated, symptomatic vaccinated or unvaccinated). For the asymptomatic classes, module written for Python, Pyevolve [22], to find nearly optimal solutions to this problem. module written for Python, Pyevolve [22], to find nearly optimal solutions to this problem. Stochastic importation of infectious people. Mathematical model We developed a semi-discrete model [43] of a network of K cities connected by means of the airline transportation network, similar to [11]. Namely, the model in each city is a continuous compartmental deterministic model, but we stochastically pulse the populations in the model every day to account for the travel between the cities and to determine if an epidemic will start or not in a susceptible city. We assumed that the most important Our model is written in Python 2.7 (http://www.python.org) and Cython (http://cython.org, [47]) using the modules of Scipy and Numpy [48]. This allowed us to use a fast genetic algorithm March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 9 Optimal Vaccine Allocation for Pandemic Influenza Figure 7. Epidemic prevention potential for vaccine allocation in two batches on two days. Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. Here, the epidemic was seeded in Jakarta. All strategies fail to mitigate the epidemics if seven or 10 million doses are available in two batches. With 15 million doses, the optimal strategy and the children-only pro rata strategy mitigate over 95% of the epidemics when the first batch of vaccine is delivered on day 10 and the second one is delivered either on day 30 or on day 60. doi:10.1371/journal.pcbi.1002964.g007 Figure 7. Epidemic prevention potential for vaccine allocation in two batches on two days. Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. Mathematical model C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively. Here, the epidemic was seeded in Jakarta. All strategies fail to mitigate the epidemics if seven or 10 million doses are available in two batches. With 15 million doses, the optimal strategy and the children-only pro rata strategy mitigate over 95% of the epidemics when the first batch of vaccine is delivered on day 10 and the second one is delivered either on day 30 or on day 60. doi:10.1371/journal.pcbi.1002964.g007 Figure 7. Epidemic prevention potential for vaccine allocation in two batches on two days. Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. In each panel, three combinations of vaccination days are considered: vaccination on day 10 and day 30, day 10 and day 60, or day 30 and day 90. The optimal, pro rata and children-only pro rata strategies are shown in blue, green and orange respectively Here the epidemic was seeded in Jakarta All strategies fail to mitigate the epidemics if seven or 10 million doses are available in two Figure 7. Epidemic prevention potential for vaccine allocation in two batches on two days. Three vaccine coverages are considered: A) Seven million doses of vaccine total, with two million available on the first day and five million available on the second day. B) 10 million doses of vaccine total, with five million doses of vaccine available on each day. C) 15 million doses of vaccine total, five million doses available on the first day and 10 million doses available on the second day. Optimization Once an epidemic has started in a city, it follows the dynamics given by a deterministic compartmental model developed in [29]. Specifically, the popu- lation in each city is divided into two age-groups, children and adults. The population in each city is further divided in susceptibles, infectious asymptomatic, infectious symptomatic, and recovered. Members of each class can be vaccinated or unvaccinated. A full description of the deterministic model can be found in the Text S1. X 2K k~1 X 2 i~1 uki(tm)Nkiƒq(tm), for j~1, . . . ,M: ð7Þ ð7Þ This represents the fact that at any control time tm, there is a finite amount of vaccine q(tm) to be used that cannot be exceeded. Let F be the set of all feasible solutions, and denote by LF the boundary of F. Vaccination. Vaccine distribution to each city and age-group is dictated by the optimizer. Because it is difficult to track susceptible and infectious individuals, especially given that a fraction of the latter are asymptomatic, we assume that only a fraction of the vaccine given to a particular group is used, and we consider the rest of it to be wasted. So if we have M doses of vaccine available for age-group i in city k, only a fraction of these will be delivered to the susceptible individuals in that group (see Text S1 for details). The vaccine is assumed to be delivered all at once, in a single day. We also assume that vaccinated people are immediately protected. Our aim is to minimize the expected number of people who become infected and ill, denoted by the objective function f(u). Hence, we wish to find solutions to the following optimization problem: min u[F f(u)~ min (u(t1),...,u(tM)) E½ X K k~1 X 2 i~1 X 1 j~0 Rijk ð8Þ ð8Þ subject to the constraint (7). Rijk represents the number of recovered symptomatic in subgroup i (i~1 for children and i~2 for adults) and vaccinated status j (j~0 for unvaccinated and j~1 for vaccinated). Complete model. A run of the model consists of simulating an epidemic over the network of cities for 250 days. All cities are assumed to have different populations and different percentages of children, but the same contact rate matrix and the same basic parameters for the influenza transmission. Optimization We consider the following optimization problem: given limited quantities of vaccine available at given times, what is the optimal vaccine distribution such that the final illness attack rate is minimized? Formally, suppose q(t1),q(t2), . . . ,q(tM) doses of vaccine are available at control times t1,t2, . . . ,tM. Both the control times and the quantities of vaccine available at each control time are known in advance. Recall that we are considering a network of K cities, and let Nki represent the children (i~1) or adults (i~2) in city k. Define a control vector u(tm) to be a vector in R2K, Pk(t)~ 1{( 1 R0 )Itot k (t) if there are no vaccinated people in city k and R0w1 1{( 1 Rf )Itot k (t) if a fraction f of the population has been vaccinated in city k and Rf w1 0 if R0ƒ1 or Rf ƒ1, 8 > > > > > > > < > > > > > > > : ð( u(tm)~(u11(tm),u12(tm),u21(tm),u22(tm), . . . ,uK1(tm),uK2(tm)), ð5Þ ð5Þ ð4Þ (4) where uki(tm) represents the fraction of the population i to be vaccinated in city k at control time tm. A solution u to the optimization problem is a vector of control vectors, each of these corresponding to a given control time, where uki(tm) represents the fraction of the population i to be vaccinated in city k at control time tm. A solution u to the optimization problem is a vector of control vectors, each of these corresponding to a given control time, if R0ƒ1 or Rf ƒ1, u~(u(t1), . . . ,u(tM)) ð6Þ where Itot k (t) represents the total number of imported infectious people (both symptomatic and asymptomatic) present in city k on day t. We repeat this process for each city until an epidemic starts in this city (or the end of the simulation is reached). where Itot k (t) represents the total number of imported infectious people (both symptomatic and asymptomatic) present in city k on day t. We repeat this process for each city until an epidemic starts in this city (or the end of the simulation is reached). ð6Þ such that 0ƒuki(t)ƒ1 for all k[1 . . . K and i[f1,2g. We define a feasible solution as a solution of the form u given above that satisfies, in addition, the constraint Transmission within cities. Optimal Vaccine Allocation for Pandemic Influenza 25%. Infectious people travel to a new city and stay there for a period of six days, after which they are assumed to be recovered and return to their original city. for the deterministic model. If an epidemic has already started in the city, the model still exports and imports infectious travelers, effectively changing the initial conditions of the deterministic model for the next day. The optimizer predeter- mines the cities where vaccine will be applied, as well as the quantity of vaccine to be given to a particular city and age- group. If the given day is a vaccination day, then the susceptible populations in those cities are vaccinated as described in the previous section. Stochastic initialization of an epidemic. Let R0 be the basic reproduction number, that is, the expected number of secondary new infections that a single typical infected individual would produce in a completely susceptible population. Let Rf be the effective reproduction number, that is, the expected number of secondary infections that a single typical infected individual would produce in a population where a fraction f of the population is vaccinated and no natural infection has yet occurred. Each day, the model computes either R0 orRf for each city where the epidemic has not started, using the approach given in [49] and [50,51]. The basic reproduction number will be computed if no vaccine has been distributed to that particular city, whereas the effective reproduction number will be computed when a fraction of the population was previously vaccinated. We approximate the beginning of the epidemic in each city by a standard birth-death process [52], and hence compute the probability Pk(t) of an epidemic in city k starting on day t to be Mathematical model Let M be a matrix representing the airline transportation network (M~(mkl)), where mkl represents the mean number of travelers per day going from city k to city l. Define P to be a matrix of travel probabilities (P~(pkl)), where pkl is defined as the probability that a person in city k will travel to city l. We compute pkl from mkl as rklBinomial(A, pkl): ð3Þ ð3Þ where A represents the number (rounded to the nearest integer) of infectious asymptomatic people in that class (children or adults, vaccinated or unvaccinated) in city k on day t and pkl was computed above. The number of symptomatic infectious travelers is computed in a similar way, but the probability is reduced by pkl~ mkl Nl ð2Þ pkl~ mkl Nl ð2Þ pkl~ mkl Nl ð2Þ ð2Þ where Nk is the total population in city k, with k,l~1, . . . ,K. Table 4. Parameter values. Parameter Description Value Reference c recovery rate 0.25 [60] r fraction of symptomatic 2/3 [60] m reduction of infectiousness for asymptomatics 0.5 [60] c11,c12,c21,c22 contact rates 1,0.1155, 0.1155, 0.4744 calculated. VES,VEI,VEP vaccine efficacies for susceptibility, infectiousness and pathogenicity 0.4, 0.45, 0.75 [44] p probability of transmission 0.4527 calculateda aThis probability of transmission gives rise to a basic reproduction number of R0 = 1.5. doi:10.1371/journal.pcbi.1002964.t004 PLOS Computational Biology \| www.ploscompbiol.org 10 March 2013 \| Volume 9 \| Issue 3 \| e1002964 Table 4. Parameter values. March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 10 Optimal Vaccine Allocation for Pandemic Influenza Optimization Every day, for each susceptible city (i.e a city where no epidemic has started yet), the model counts the number of imported infections in the city and determines if a new epidemic will start or not, using the probability Pk(t), eq. (4). If it does start a new epidemic, it does so with the number of imported infections as initial conditions Optimal Vaccine Allocation for Pandemic Influenza The baseline scenario (red) indicates no vaccination. For early vaccination, an epidemic starting in Hong Kong yield to a higher attack rates for the optimal and children only pro rata solutions, this is due to the fact that the flux of daily travelers through Hong Kong is much higher than the flux through Jakarta. (TIF) particular solution consists of a pro rata distribution of resources. Here, we distribute the available vaccine among all the cities and all age-groups, proportionally to the size of that age-group. The second particular solution consists of a children-only pro rata distribution. Here, we distribute vaccine among children only, proportionally to the number of children with respect to the total child population in the network. This procedure ensures that our optimizer has a variety of strategies to choose from, and that it takes into account the observation that prioritizing vaccination in children can lead to the optimal use of resources [12,55,27,15,56,57]. Figure S3 Epidemic prevention potential (EPP) starting in Hong Kong with 95% bootstrapped CI. Three different allocations are shown in each panel. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. Each point in each graph corresponds to the EPP for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age-group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. When less than 10 million doses are available, the EPP for an epidemic starting in Hong Kong is considerably lower for than if the epidemic starting in Jakarta, highlighting the fact that it is more difficult to mitigate an epidemic if it starts in a more connected city. (TIF) In each generation, we use the objective function f to determine the fitness of each solution. To ensure that each generation has better (more fit) individuals than its predecessor, we carry over the best 25 chromosomes from one generation directly to the next one. Optimal Vaccine Allocation for Pandemic Influenza We then use a crossover method to create the remaining set of 25 new chromosomes. Finally, the chromosomes undergoe a muta- tion. Because the new chromosome likely will not satisfy constraint (7), we incorporate an extra step in the genetic algorithm. At this stage, the chromosome is transformed just before its evaluation by mapping each gene to the boundary of its feasible region. 2K Define the transformation T : (0,12K?LF by T(u(tm))~T(u11(tm),u12(tm),u21(tm),u22(tm), . . . ,uK1(tm),uK2(tm)) ~am(u11(tm),u12(tm),u21(tm),u22(tm), . . . ,uK1(tm),uK2(tm)) ð9Þ where am is given by am~min 1, qm N11u11(tm)zN12u12(tm)z . . . zNK1uK1(tm)zNK2uK2(tm) : ð10Þ Figure S4 Attack rate with 95% bootstrapped CI for a single intervention for six different vaccination days considered and six different vaccination coverages for an epidemic starting in Taipei. Each panel represents a given number of vaccine doses available to distribute in the entire network: A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Three different allocations are shown in each panel. The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age-group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. The baseline scenario (red) indicates no vaccination. The attack rates under this scenario are similar to those when the epidemic is seeded in Hong Kong. ð10Þ This transformation maps radially each vector to its corre- sponding vector on the boundary of the feasible region. The minimum is taken to ensure that no transformed gene has a coordinate greater than one, which would imply in our case vaccinating more than 100% of the population. for vaccinated). Genetic algorithm. We use a genetic algorithm [53,54] to compute the optimal strategies. In our case, a chromosome represents a solution to our optimization problem, and we can think of a gene as a particular control vector. We initialize the genetic algorithm by randomly generating 48 feasible solutions (Text S1). In addition, we add two particular solutions. The first March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 11 Optimal Vaccine Allocation for Pandemic Influenza Supporting Information Figure S1 Epidemic curves for the 16 cities considered in the baseline case. The epidemic is started in Jakarta, with 10 infectious individuals. (TIF) (TIF) Figure S5 Epidemic prevention potential (EPP) starting in Taipei with 95% bootstrapped CI. Three different allocations are shown in each panel. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. Each point in each graph corresponds to the EPP for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age-group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. The EPP here is very similar to the one obtained when the epidemic starts in Hong Kong. Figure S2 Attack rate with 95% bootstrapped CI for a single intervention for six different vaccination days considered and six different vaccination coverages for an epidemic starting in Hong Kong. Each panel represents a given number of vaccine doses available to distribute in the entire network: A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Three different allocations are shown in each panel. The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age-group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 12 Optimal Vaccine Allocation for Pandemic Influenza Figure S6 Attack rate with 95% bootstrapped CI for a single intervention for 6 different vaccination days considered and 6 different vaccination coverages for an epidemic with R0~1:2. Here, the epidemic was seeded in Jakarta. (TIF) Each panel represents a given number of vaccine doses available to distribute in the entire network: A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Three different allocations are shown in each panel: The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age-group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. The baseline scenario (red) indicates no vaccination. As expected, a low R0 requires few doses of vaccine to mitigate an epidemic: with four million of doses of vaccine, all strategies considered yield an attack rate of less than 2% of the total population. (TIF) million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. Each point in each graph corresponds to the EPP for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Three different allocations are shown in each panel: The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age- group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. Here, the EPP is much lower than in the base case scenarios (R0~1:5). The optimal strategy is the only strategy able to mitigate some of the epidemics for all days and coverages considered. (TIF) Figure S10 Attack rate with 95% bootstrapped CI for five million doses with different travel probabilities. A) An infectious symptomatic individual is 10% less likely to travel than an asymptomatic individual. B) An infectious symptomatic individual is 75% less likely to travel than an asymptomatic individual. , (TIF) Figure S11 Epidemic prevention potential (EPP) with 95% bootstrapped CI for five million doses with different travel probabilities. A) An infectious symptomatic individual is 10% less likely to travel than an asymptomatic individual. B) An infectious symptomatic individual is 75% less likely to travel than an asymptomatic individual. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. (TIF) Figure S12 Results for five million doses when children have a 50% reduction in their probability of travel. A) Attack rates with 95% bootstrapped CI. B) EPP with 95% bootstrapped CI. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. (TIF) Figure S8 Attack rate with 95% bootstrapped CI for a single intervention for 6 different vaccination days considered and 6 different vaccination coverages for an epidemic with R0~1:8. Here, the epidemic was seeded in Jakarta. Each panel represents a given number of vaccine doses available to distribute in the entire network: A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Three different allocations are shown in each panel: The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age- group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. The baseline scenario (red) indicates no vaccination. (TIF) Figure S13 Attack rate with 95% bootstrapped CI for five million doses with lower vaccine efficacies. A) One- third of their original values (VES~0:13, VEI~0:15 and VEP~0:25). B) Two-thirds of their original values (VES~0:27, VEI~0:30, and VEP~0:50). For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. (TIF) For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. (TIF) Figure S7 Epidemic prevention potential (EPP) for R0~1:2 with 95% bootstrapped CI. Here, the epidemic was seeded in Jakarta. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two million doses. B) Four million doses. C) Five million doses. D) Six million doses. E) Seven million doses. F) Ten million doses. Each point in each graph corresponds to the EPP for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Three different strategies are shown in each panel. The optimal strategy (blue) is the one given by our method. The pro rata strategy (green) consists of distributing vaccine to each age-group in each city proportional to the age- group population size. The children-only pro rata strategy (orange) consists of distributing vaccine only to children in each city proportional to the children’s population size. The optimal and the children-only pro rata strategy can mitigate most of the epidemics with as few as four million doses. (TIF) References Tennenbaum S (2008) Simple criteria for finding (nearly) optimal vaccination strategies. Journal of Theoretical Biology 250: 673–683. 42. Hollingsworth TD, Klinkenberg D, Heesterbeek H, Anderson RM (2011) Mitigation strategies for pandemic influenza a: balancing conflicting policy objectives. PLoS Computational Biology 7: e1001076. 18. Tennenbaum S (2008) Simple criteria for finding (nearly) optimal vaccination strategies. Journal of Theoretical Biology 250: 673–683. 18. Tennenbaum S (2008) Simple criteria for finding (nea strategies. Journal of Theoretical Biology 250: 673–683 j p gy 43. Mailleret L, Lemesle V (2009) A note on semi-discrete modelling in the life sciences. Philosophical Transactions of the Royal Society A 367: 4779–4799. strategies. Journal of Theoretical Biology 250: 673 19. Tuite A, Fisman DN, Kwong JC, Greer A (2010) Optimal pandemic influenza vaccine allocation strategies for the canadian population. PLoS Currents Influenza : RRN1144. 44. Basta N, Halloran ME, Matrajt L, Longini IM (2008) Estimating influenza vaccine efficacy from challenge and community-based study data. American Journal of Epidemiology 168: 1343–52. 20. Wallinga J, van Boven M, Lipsitch M (2010) Optimizing infectious disease interventions during an emerging epidemic. PNAS 107: 923–928. 45. Wallinga J, Teunis P, Kretzschmar M (2006) Using data on social contacts to estimate age-specific transmission parameters for respiratory-spread infectious agents. American Journal of Epidemiology 164: 936–944. 21. Yaesoubi R, Cohen T (2011) Dynamic health policies for controlling the spread of emerging infections: influenza as an example. PLoS One 6: e24043. of emerging infections: influenza as an example. PLoS One 6: e2404 46. Epstein JM, Goedecke DM, Yu F, Morris RJ, Wagener DK, et al. (2007) Controlling pandemic flu: The value of international air travel restrictions. PLoS ONE 2: e401. 22. Perone CS (2009) Pyevolve: a Python open-source framework for genetic algorithms. Newsletter ACM SIGEVOlution Volume 4. 23. Kenah E, Chao DL, Matrajt L, Halloran ME, Longini IM Jr (2011) The global transmission and control of influenza. PLoS ONE 6: e19515. 47. Behnel S, Bradshaw R, Citro C, Dalcin L, Seljebotn D, et al. (2011) Cython: The best of both worlds. Computing in Science Engineering 13: 31–39. 24. Fraser C, Donnelly CA, Cauchemez S, Hanage WP, Van Kerkhove MD, et al. (2009) Pandemic potential of a strain of influenza A (H1N1): early findings. Science 324: 1557–1561. 48. Jones E, Oliphant T, Peterson P (2001). SciPy: Open source scientific tools for Python. Available: http://www.scipy.org/. 25. Yang Y, Sugimoto JD, Halloran ME, Basta NE, Chao DL, et al. We thank Archis Ghate and Dennis Chao for helpful discussions. We thank Archis Ghate and Dennis Chao for helpful discussions. Author Contributions Conceived and designed the experiments: LM MEH IML. Performed the experiments: LM. Analyzed the data: LM. Wrote the paper: LM MEH IML. References 27. Matrajt L, Longini IM Jr (2010) Optimizing vaccine allocation at different points in time during an epidemic. PLoS ONE 5: e13767. 1. WHO (2012). Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO. Available: http://www.who.int/ influenza/human_animal_interface/H5N1_cumulative_table_archives/en/. Last accesed June 1st, 2012. 28. Mylius SD, Hagenaars TJ, Lugner AK,Wallinga J (2008) Optimal allocation of pandemic influenza vaccine depends on age, risk and timing. Vaccine 26: 3742–3749. 2. Herfst S, Schrauwen EJA, Linster M, Chutinimitkul S, de Wit E, et al. (2012) Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets. Science 336: 1534–1541. 29. Matrajt L, Longini Jr IM (2012) Critical immune and vaccination thresholds for determining multiple influenza epidemic waves. Epidemics 4: 22–32. 30. Bansal S, Pourbohloul B, Meyers LA (2006) A comparative analysis of influenza vaccination programs. PLoS Medicine 3: e387. 3. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, et al. (2012) Experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets. Nature 486: 420–428. 31. Medlock J, Meyers LA (2009) Optimizing allocation for a delayed influenza vaccination campaign. PLoS Currents Influenza : RRN1134. 4. WHO (2012). Availability of a new A(H5N1) candidate vaccine virus (clade 2.3.2.1). Available: http://www.who.int/influenza/vaccines/virus/candidates_ reagents/a_h5n1_idcdc-rg-30/en/index.html. Accessed February 2012. 32. Gojovic MZ, Sander B, Fisman D, Krahn MD, Bauch CT (2009) Modelling mitigation strategies for pandemic (H1N1) 2009. Canadian Medical Association Journal 181: 673–680. g g y 5. WHO (2008). Reports by the Director-General. Available: www.who.int/gb/ pip/pdf_files/PIP_IGM_11-en.pdf. Accesed April 29, 2009. 33. Sypsa V, Pavlopoulou I, Hatzakis A (2009) Use of an inactivated vaccine in mitigating pandemic influenza A(H1N1) spread: a modelling study to assess the impact of vaccination timing and prioritisation strategies. Euro Surveillance 14: 19356. 5. WHO (2008). Reports by the Director-General. Available: 6. WHO (2009). Influenza A(H1N1) - update 14. Available: http://www.who.int/ csr/don/2009_05_04a/en/index.html. 34. Wu JT, Riley S, Leung GM (2007) Spatial considerations for the allocation of pre-pandemic influenza vaccination in the United States. Proceedings of the Royal Society B-Biological Sciences 274: 2811–2817. 7. Khan K, Arino J, HuW, Raposo P, Sears J, et al. (2009) Spread of a novel influenza a (h1n1) virus via global airline transportation. N Engl J Med 361: 212–214. 35. Wood J, McCaw J, Becker N, Nolan T, Macintyre RM (2009) Optimal Dosing and Dynamic Distribution of Vaccines in an Influenza Pandemic. American Journal of Epidemiology 169: 1517–1524. 8. References Hethcote H (1978) An immunization model for an heterogeneous population. Theoretical population biology 14: 338–349. p p gy 9. Longini IMJ, Ackerman E, Elveback LR (1978) Optimization model for Influenza A Epidemics. Mathematical Biosciences 38: 141–157. 36. Klepac P, Laxminarayan R, Grenfell BT (2011) Synthesizing epidemiological and economic optima for control of immunizing infections. PNAS 108: 14366–14370. 10. Araz O, Galvani A, Meyers L (2012) Geographic prioritization of distributing pandemic influenza vaccines. Health Care Manag Sci 15(3): 175–87. 37. Centers for Disease Control and Prevention (2012). Human Infections with Novel Reassortant Influenza A(H3N2)v Viruses, United States, 2011. Available: http://wwwnc.cdc.gov/eid/article/18/5/11-1922_article.htm. Last accesed May 19th, 2012. 11. Dimitrov NB, Goll S, Hupert N, Pourbohloul B, Meyers LA (2011) Optimizing tactics for use of the U.S. antiviral strategic national stockpile for pandemic influenza. PLoS One 6: e16094. 38. Colizza V, Barrat A, Barthelemy M, Valleron AJ, Vespignani A (2007) Modeling the worldwide spread of pandemic influenza: baseline case and containment interventions. PLoS Medicine 4: e13. 12. Goldstein E, Apolloni A, Lewis B, Miller JC, Macauley M, et al. (2010) Distribution of vaccine/antivirals and the ‘least spread line’ in a stratified population. Journal of the Royal Society Interface 7: 755–764. 39. WHO (2007). Options for the use of human H5N1 influenza vaccines and the WHO H5N1 vaccine stockpile. Available: http://www.who.int/csr/resources/ publications/WHO_HSE_EPR_GIP_2008_1/en/. 13. Keeling MJ, Shattock A (2012) Optimal but unequitable prophylactic distribution of vaccine. Epidemics 4: 78–85. p 14. Lee S, Golinski M, Chowell G (2012) Modeling optimal age-specific vaccination strategies against pandemic influenza. Bull Math Biol 74: 958–980. 40. Centers for Disease Control and Prevention (2009). Allocation and Distribution Q and A. Available: http://www.cdc.gov/h1n1flu/vaccination/statelocal/ centralized_distribution_qa.htm. Last accesed August 5th 2011. strategies against pandemic influenza. Bull Math Biol 74: 958–98 15. Medlock J, Galvani AP (2009) Optimizing Influenza Vaccine Distribution. Science 325: 1705–1708. 41. WHO (2009). Production and availability of pandemic influenza A (H1N1) vaccines. Available: www.who.int/csr/disease/swineflu/frequently_asked_ questions/vaccine_preparedness/production_availability/en/index.html. Ac- cesed July 28th, 2009. 16. Patel R, Longini Jr IM, Halloran ME (2005) Finding optimal vaccination strategies for pandemic influenza using genetic algorithms. Journal of Theoretical Biology 234: 201–212. gy 17. Riley S, Wu JT, Leung GM (2007) Optimizing the dose of pre-pandemic influenza vaccines to reduce the infection attack rate. PLoS Medicine 4: e218. gy 17. Riley S, Wu JT, Leung GM (2007) Optimizing the dose of pre-pandemic influenza vaccines to reduce the infection attack rate. PLoS Medicine 4: e218. 18. Text S1 Complete mathematical model and sensitivity analysis. bootstrapped CI. B) EPP with 95% bootstrapped CI. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. (TIF) bootstrapped CI. B) EPP with 95% bootstrapped CI. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Text S1 Complete mathematical model and sensitivity analysis. (PDF) (TIF) Figure S14 Epidemic prevention potential (EPP) with 95% bootstrapped CI for five million doses with lower vaccine efficacies. A) One-third of their original values (VES~0:13, VEI~0:15 and VEP~0:25). B) Two-thirds of their original values (VES~0:27, VEI~0:30, and VEP~0:50). For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. Figure S9 Epidemic prevention potential (EPP) for R0~1:8 with 95% bootstrapped CI and the epidemic was seeded in Jakarta. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two Figure S9 Epidemic prevention potential (EPP) for R0~1:8 with 95% bootstrapped CI and the epidemic was seeded in Jakarta. Each panel represents a given number of vaccine doses available to distribute in the network. A) Two (TIF) Figure S15 Results for five million doses when vaccina- tion is completed in 10 days. A) Attack rates with 95% March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org 13 Optimal Vaccine Allocation for Pandemic Influenza Acknowledgments Figure S16 Results for five million doses when vaccines are given to children only. A) Attack rates with 95% bootstrapped CI. B) EPP with 95% bootstrapped CI. For each panel, each point in the graph corresponds to the attack rate for a single vaccination day, either on day 5, 10, 15, 30, 60, or 90 after the beginning of the epidemic. References (2009) The transmissibility and control of pandemic influenza A (H1N1) virus. Science 326: 729–33. 49. Diekmann O, Heesterbeek J, Metz JA (1990) On the definition and computation of the basic reproduction ratio R0 in models for infectious-diseases in heterogeneous populations. Journal of Mathematical Biology 28: 365–382. 26. Halloran ME, Longini IM, Cowart DM, Nizam A (2002) Community interventions and the epidemic prevention potential. Vaccine 20: 3254–3262. 50. Brauer F, van den Driessche P, Wu J, editors(2008) Mathematical Epidemiology, volume 1945 of Lecture Notes in Mathematics. Springer. March 2013 \| Volume 9 \| Issue 3 \| e1002964 March 2013 \| Volume 9 \| Issue 3 \| e1002964 14 PLOS Computational Biology \| www.ploscompbiol.org years, 5 to 9 years, and 10 to 18 years of age in a community-based, nonrandomized, open-label trial. Pediatrics 116: e397–407. Optimal Vaccine Allocation for Pandemic Influenza years, 5 to 9 years, and 10 to 18 years of age in a community-based, nonrandomized, open-label trial. Pediatrics 116: e397–407. 51. van den Driessche P, Watmough J (2002) Reproduction numbers and sub- threshold endemic equilibria for compartmental models of disease transmission. Mathematical Biosciences 180: 29–48. years, 5 to 9 years, and 10 to 18 years of age in a community-based, nonrandomized, open-label trial. Pediatrics 116: e397–407. 57. Reichert TA, Sugaya N, Fedson DS, Glezen WP, Simonsen L, et al. (2001) The japanese experience with vaccinating schoolchildren against influenza. New England Journal Medicine 344: 889–896. 52. Chiang CL (1978) Introduction to Stochastic Processes and Their Applications. Krieger Pub Co. g 53. Goldberg DE (1989) Genetic Algorithms in Search, Optimization and Machine Learning. Boston, MA: Kluwer Academic Publishers. g J 58. UN (2008). Table 7. population by age, sex and urban/rural residence: latest available year, 1999–2008. Available: http://unstats.un.org/unsd/ demographic/sconcerns/popsize/size2.htm#PVSR. g 53. Goldberg DE (1989) Genetic Algorithms in Search, Optimization and Machine g 53. Goldberg DE (1989) Genetic Algorithms in Search, Optim Learning. Boston, MA: Kluwer Academic Publishers. 53. Goldberg DE (1989) Genetic Algorithms in Search, Op Learning. Boston, MA: Kluwer Academic Publishers. g 54. Holland JH (1975) Adaptation in Natural and Artificial Systems. Ann Arbor: University of Michigan Press. g p p p 59. US Census Bureau. U.S. Census Bureau, International Data Base. Available: http://www.census.gov/population/international/data/idb/country.php. Last accesed July 17th 2011. y g 55. Longini IMJ, Halloran ME (2005) Strategy for distribution of influenza vaccine to high-risk groups and children. American Journal of Epidemiology 161: 303– 306. 60. Longini IMJ, Halloran ME, Nizam A, Yang Y (2004) Containing Pandemic Influenza with Antiviral Agents. American Journal of Epidemiology 159: 623– 633. 56. Piedra PA, Gaglani MJ, Riggs M, Herschler G, Fewlass C, et al. (2005) Live attenuated influenza vaccine, trivalent, is safe in healthy children 18 months to 4 March 2013 \| Volume 9 \| Issue 3 \| e1002964 PLOS Computational Biology \| www.ploscompbiol.org PLOS Computational Biology \| www.ploscompbiol.org 15
https://openalex.org/W2533894073	https://europepmc.org/articles/pmc5066227?pdf=render	English	null	A large-scale chloroplast phylogeny of the Lamiaceae sheds new light on its subfamilial classification	Scientific reports	2,016	cc-by	17,566	Bo Li1,, Philip D. Cantino2,, Richard G. Olmstead3,, Gemma L. C. Bramley4, Chun-Lei Xiang5, Zhong-Hui Ma6, Yun-Hong Tan7 & Dian-Xiang Zhang8 received: 18 April 2016 accepted: 13 September 2016 Published: 17 October 2016 received: 18 April 2016 accepted: 13 September 2016 Published: 17 October 2016 Lamiaceae, the sixth largest angiosperm family, contains more than 7000 species distributed all over the world. However, although considerable progress has been made in the last two decades, its phylogenetic backbone has never been well resolved. In the present study, a large-scale phylogenetic reconstruction of Lamiaceae using chloroplast sequences was carried out with the most comprehensive sampling of the family to date (288 species in 191 genera, representing approximately 78% of the genera of Lamiaceae). Twelve strongly supported primary clades were inferred, which form the phylogenetic backbone of Lamiaceae. Six of the primary clades correspond to the current recognized subfamilies Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, and Symphorematoideae, and one corresponds to a portion of Viticoideae. The other five clades comprise: 1) Acrymia and Cymaria; 2) Hymenopyramis, Petraeovitex, Peronema, and Garrettia; 3) Premna, Gmelina, and Cornutia; 4) Callicarpa; and 5) Tectona. Based on these results, three new subfamilies— Cymarioideae, Peronematoideae, and Premnoideae—are described, and the compositions of other subfamilies are updated based on new findings from the last decade. Furthermore, our analyses revealed five strongly supported, more inclusive clades that contain subfamilies, and we give them phylogenetically defined, unranked names: Cymalamiina, Scutelamiina, Perolamiina, Viticisymphorina, and Calliprostantherina. The circumscriptions of Lamiaceae and Verbenaceae have changed dramatically in the past 25 years as a conse- quence of the discovery that both families were polyphyletic as traditionally circumscribed (e.g., by Bentham1 and Briquet2 for Lamiaceae and by Briquet3 for Verbenaceae; see Cantino4 for a summary of traditional classifications of Lamiaceae). The polyphyly of Lamiaceae was first proposed based on gynoecial morphology5, palynology6,7, and phylogenetic analyses of non-DNA data4,8 and subsequently corroborated by molecular research9,10. Based on these studies, the traditionally circumscribed family Verbenaceae was thought to be paraphyletic (as also implied earlier by Cronquist11 using different terms), but more recent molecular studies of Lamiales12–15 have shown that Verbenaceae as traditionally circumscribed were polyphyletic, with genera such as Vitex L., Clerodendrum L., and Callicarpa L. being more closely related to the traditional Lamiaceae than they are to Verbenaceae s. str. In an attempt to delimit monophyletic families, Cantino8 resurrected Junell’s5 proposed transfer of about 50 genera (in subfamilies Caryopteridoideae, Chloanthoideae, Viticoideae, and tribe Monochileae) of Verbenaceae to Lamiaceae, leaving only subfamily Verbenoideae in the reconstituted Verbenaceae. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 18 April 2016 accepted: 13 September 2016 Published: 17 October 2016 A large-scale chloroplast phylogeny of the Lamiaceae sheds new light on its subfamilial classification PEN Bo Li1,, Philip D. Cantino2,, Richard G. Olmstead3,, Gemma L. C. Bramley4, Chun-Lei Xiang5, Zhong-Hui Ma6, Yun-Hong Tan7 & Dian-Xiang Zhang8 Bo Li1,, Philip D. Cantino2,, Richard G. Olmstead3,, Gemma L. C. Bramley4, Chun-Lei Xiang5, Zhong-Hui Ma6, Yun-Hong Tan7 & Dian-Xiang Zhang8 Wagstaff et al.10 addition- ally found that Congea Roxb., a representative of subfamily Symphorematoideae of Verbenaceae, which was not transferred to Lamiaceae by Cantino8, should be included in Lamiaceae. Harley et al.16 adopted the expansion of 1College of Agronomy, Jiangxi Agricultural University, Nanchang, 330045, Jiangxi, P. R. China. 2Department of Environmental and Plant Biology, Ohio University, Athens, Ohio 45701-2979, USA. 3Department of Biology and Burke Museum, University of Washington, Box 355325, Seattle, Washington 98195-5325, USA. 4Herbarium, Royal Botanic Gardens Kew, Richmond, Surrey, TW9 3AE, UK. 5Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, P. R. China. 6College of Agriculture, Guangxi University, Nanning 530004, Guangxi, P. R. China. 7Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, Yunnan, P. R. China. 8South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, Guangdong, P. R. China. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to D.-X.Z. (email: dx-zhang@scbg.ac.cn) Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 www.nature.com/scientificreports/ Lamiaceae and proposed a subfamilial classification of the family, which is the first global, genus-level treatment of the entire family in more than a century (since Briquet2). y y q As presently circumscribed16, Lamiaceae are the largest family-level clade within Lamiales17, an order com- prising 26 families and over 20,000 species18. They are cosmopolitan in distribution and occur as herbs, shrubs, lianas, and trees. Economically important products include teak wood (Tectona), oil of peppermint (Mentha) and patchouli (Pogostemon), and various culinary herbs—e.g., rosemary (Rosmarinus), thyme (Thymus), basil (Ocimum), oregano (Origanum), sage (Salvia), and both spearmint and peppermint (Mentha). Recent phyloge- netic studies of angiosperms19, and especially Lamiales13,15, place both Lamiaceae and Verbenaceae within a large clade called “core Lamiales”13, where Lamiaceae are sister to a well-supported clade comprising Orobanchaceae and several small families (Mazaceae, Paulowniaceae, Phrymaceae, Rehmanniaceae), and Verbenaceae are sis- ter to the small African family Thomandersiaceae. The early misunderstanding of the proper division between Lamiaceae and Verbenaceae relied on macroscopic features of the ovary, such as the degree to which it is divided and the placement of the style. Bo Li1,, Philip D. Cantino2,, Richard G. Olmstead3,, Gemma L. C. Bramley4, Chun-Lei Xiang5, Zhong-Hui Ma6, Yun-Hong Tan7 & Dian-Xiang Zhang8 However, what Junell5 recognized and Cantino4 later brought to the attention of botanists, is the fundamental distinction between where the ovules attach to the ovary wall relative to the false septa that divide each carpel into two single-seeded chambers; ovules in Lamiaceae attach to the sides of an inrolled carpel wall, whereas ovules in Verbenaceae attach directly to the margins of the false carpel septa. In addition, the inflorescence is fundamentally cymose in Lamiaceae versus racemose in Verbenaceae, but this is not a consistent distinction because a recemoid inflorescence has independently evolved in several subgroups of Lamiaceae. In general, Lamiaceae can be recognized by a combination of traits, including opposite leaves, bilaterally symmetric flowers with four stamens, and ovaries consisting of two fused carpels, each divided into one-seeded chambers. However, most Verbenaceae also exhibit these traits, albeit with much less variation in floral form, hence the long-standing belief that the two families are each other’s closest relatives. Only the advent of molecular phylogenetic studies9,10,15 showed conclusively that this was not the case. p y g y In Lamiaceae, Harley et al.16 recognized 236 genera (comprising more than 7000 species), 226 of which were assigned to seven subfamilies: Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, Symphorematoideae and Viticoideae. Ten genera that could not be placed in a subfamily were listed as incertae sedis: Acrymia Prain, Callicarpa, Cymaria Benth., Garrettia Fletch., Holocheila (Kudo) S. Chow, Hymenopyramis Wall. ex Griff., Ombrocharis Hand.-Mazz., Peronema Jack, Petraeovitex Oliv., and Tectona L. A decade later, Harley et al.’s16 classification has been widely adopted, and new evidence has incrementally improved the classification. The monophyly of five of the seven subfamilies (Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, and Scutellarioideae) has been supported by molecular studies20–26. Within subfamilies, intergeneric relation- ships have been illuminated to varying degrees in Ajugoideae22,27, Lamioideae21,23,25,26,28–33, Nepetoideae34–43, Prostantheroideae44–46, and Scutellarioideae24, and tribal subdivisions in Lamioideae have been proposed and updated21,23,26. However, the monophyly of two subfamilies, Symphorematoideae and Viticoideae, still has not been satisfactorily examined. The former is well characterized by morphological characters (e.g., woody climbing stems, capitate inflorescences surrounded by an involucre of 3–6 bracteoles, polysymmetric flowers, and ovary with incomplete septum), some of which are probably synapomorphies, but its monophyly has not been tested with DNA data because no molecular study has included more than one representative9,10,21,23–25,47. In contrast, there is now strong evidence that subfamily Viticoideae, as circumscribed by Harley et al.16, is not monophy- letic. Bo Li1,, Philip D. Cantino2,, Richard G. Olmstead3,, Gemma L. C. Bramley4, Chun-Lei Xiang5, Zhong-Hui Ma6, Yun-Hong Tan7 & Dian-Xiang Zhang8 Its members fall into two clades that are not sister groups10,23–25,43,47, with Vitex (grouped with Paravitex H. R. Fletcher, Petitia Jacq., Teijsmanniodendron Koord., Tsoongia Merr., and Viticipremna H. J. Lam) being the largest genus in one clade and Premna (grouped with Cornutia L. and Gmelina L.) in the other. Based on these results, Paravitex, Tsoongia, and Viticipremna were reduced to synonymy with Vitex47, and Cornutia, Gmelina, and Premna were assigned to a provisional subfamily, “Premnoideae”18,48. g p y Of the ten genera considered to be incertae sedis by Harley et al.16, Holocheila has since been shown to be a member of tribe Pogostemoneae in subfamily Lamioideae25, and Ombrocharis has been shown to be part of tribe Elsholtzieae in subfamily Nepetoideae43. The other eight genera have still not been placed in subfamilies, but sev- eral molecular studies have shed light on their relationships: Acrymia and Cymaria form a moderately supported clade23,25,26; Hymenopyramis, Peronema and Petraeovitex25,47 or Hymenopyramis, Petraeovitex and Garrettia25 group together; Callicarpa is sister to the rest of the family21,23 or groups with subfamily Prostantheroideae15; Tectona emerges in various positions21,23,47. g p Although all ten genera incertae sedis and all seven subfamilies have been included in molecular stud- ies cited above, no single study has included all of them, and the phylogenetic backbone of the family remains poorly resolved. The present study employs the most broadly comprehensive sampling of the family to date, including representatives of every subfamily and tribe, all ten of the genera incertae sedis of Harley et al.16, and a substantially larger sample of the mainly tropical and subtropical taxa Premna, Callicarpa, Gmelina, Tectona, and Symphorematoideae than in previous studies. We are using five plastid DNA regions to infer a large-scale phylogeny of the whole family with four objectives, to: (1) increase resolution of the phylogenetic backbone of Lamiaceae, (2) determine the phylogenetic positions of the genera incertae sedis, (3) assess relationships among subfamilies, and (4) test the monophyly of Symphorematoideae. Resultsh Comparison of support values for subfamilial or above nodes in the different analyses. The numbers in matK, ndhF, rbcL, rps16 and trnL-F were bootstrap support values in ML and MP analysis respectively, with gaps treated as simple indels. The numbers in D270 and D155 were posterior probabilities values in BI analysis, and bootstrap support values in ML and MP analysis, respectively. “−” Indicates support values of less than 50% in MP or ML analysis, and posterior probabilities value less than 0.90 in BI analysis. Table 1. Properties of data partitions used in this study and tree statistics. Table 2. Comparison of support values for subfamilial or above nodes in the different analyses. The numbers in matK, ndhF, rbcL, rps16 and trnL-F were bootstrap support values in ML and MP analysis respectively, with gaps treated as simple indels. The numbers in D270 and D155 were posterior probabilities values in BI analysis, and bootstrap support values in ML and MP analysis, respectively. “−” Indicates support values of less than 50% in MP or ML analysis, and posterior probabilities value less than 0.90 in BI analysis. Based on the combined data sets D270 and D155, all MP, ML and BI analyses yielded very similar topol- ogies, and this was true regardless of whether gaps were treated as simple indels or as missing data. Twelve well-supported primary clades were obtained in all analyses (Figs 1–4; Supplementary Figs S6–S12). A simplified phylogenetic tree shows the phylogenetic backbone of Lamiaceae (Fig. 1), and the 50% majority-rule consen- sus tree from the BI analysis of the combined D270 data set with simple gap coding shows detailed relation- ships (Figs 2 and 3). Six of the 12 primary clades in Fig. 1 correspond to subfamilies Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, and Symphorematoideae, as recognized by Harley et al.16 and Olmstead18, and one corresponds to subfamily Viticoideae s. str. as recognized by Bramley et al.47. The monophyly of Symphorematoideae was confirmed for the first time, with all three genera sampled in one study (Fig. 2 and 3). Besides these subfamilial clades, the other five primary clades comprise: 1) Acrymia and Cymaria; 2) Hymenopyramis, Petraeovitex, Garrettia, and Peronema; 3) Premna, Gmelina, and Cornutia; 4) Callicarpa; and 5) Tectona. The relationships among these 12 clades were inferred with varying degrees of support. Resultsh The number of sequences, new sequences generated in this study, aligned length of sequences, proportion of missing data, parsimony informative characters and indels, tree length, consistency index (CI), retention index (RI), and evolutionary model, for separate and combined data sets are summarized in Table 1. MP and ML analyses of separate data sets (matK, ndhF, rbcL, rps16, and trnL-F) did not yield fully resolved gene trees for the whole family. Generally, MP and ML analyses of the same data set yielded similar supported clades (Supplementary Figs S1–S5). Trees generated from different data sets had variable topological structure, but there were several comparable clades among these different trees (Table 2). Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 2 www.nature.com/scientificreports/ Data matrix Number of sequences New reported sequences Number of aligned positions Number of informative substitutions Number of indels Number of informative indels Proportion of missing data Tree length Consistency index (CI) Retention index (RI) Model matK 202 54 1578 613 19 10 25.54% 2384 0.53 0.87 TVM + I + G ndhF 160 83 2108 765 23 9 19.13% 3891 0.43 0.75 GTR + I + G rbcL 170 59 1400 251 2 0 11.57% 1282 0.34 0.75 TVM + I + G rps16 181 57 926 375 89 56 1.62% 1602 0.55 0.88 GTR + G trnL-F 259 88 918 398 123 64 1.67% 1801 0.52 0.88 GTR + G D270 270 6930 2402 256 139 39.65% 11084 0.47 0.83 GTR + I + G D155 155 6930 2168 218 123 23.51% 9381 0.51 0.80 GTR + I + G Table 1. Properties of data partitions used in this study and tree statistics. Table 1. Properties of data partitions used in this study and tree statistics. Resultsh Clade matK ndhF rbcL rps16 trnL-F D270 D155 gaps treated as simple indels gaps treated as missing data gaps treated as simple indels gaps treated as missing data Ajugoideae 100, 98 100, 100 85, 61 100, 98 98, 88 1.00, 100, 100 1.00, 100, 100 1.00, 100, 100 1.00, 100, 100 Callicarpa 99, 94 100, 100 100, 92 100, 92 99, 79 1.00, 100, 100 1.00, 100, 100 100, 100, 1.00 1.00, 100, 100 Calliprostantherina −, − 95, 62 −, − −, − −, − 1.00, 91, 60 1.00, 88, 59 0.96, 75, − 0.93, 67, − Cymalamiina 100, 97 100, 99 90, 81 100, 98 98, 87 1.00,100, 99 1.00,100, 99 100, 100, 1.00 100, 100, 1.00 Cymarioideae −, − 95, 74 −, − 56, 51 89, 75 1.00, 93, 83 1.00, 89, 79 1.00, 97, 86 0.98, 87, 80 Lamioideae −, − 100, 95 −, − 95, 90 79, 74 100, 100, 1.00 1.00, 100, 99 100, 100, 1.00 1.00, 100, 99 Nepetoideae 100, 100 100, 100 100, 100 100, 100 100, 100 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 100, 100, 1.00 Perolamiina −, − 100, 91 −, − 100, 88 76, 56 1.00, 100, 92 1.00, 96, 89 1.00, 100, 90 1.00, 94, 76 Peronematoideae 100, 93 93, 85 100, 99 79, 61 99, 86 100, 100, 1.00 1.00, 100, 99 100, 100, 1.00 1.00, 100, 99 Premnoideae −, − 92, 91 −, − −, − −, − 1.00, 100, 97 1.00, 100, 96 1.00, 97, 91 1.00, 95, 90 Prostantheroideae 100, 100 100, 100 100, 100 100, 100 100, 98 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 1.00, 100, 100 Scutelamiina −, − 100, 93 52, − 98, 85 100, 94 100, 100, 100 1.00, 100, 100 100, 100, 1.00 100, 100, 1.00 Scutellarioideae 100, 100 100, 100 88, 76 100, 100 100, 99 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 1.00, 93, 69 Symphoremoideae 100, 100 100, 100 100, 98 100, 100 100, 100 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 100, 100, 1.00 Tectona 100, 100 100, 100 100, 99 100, 100 100, 100 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 100, 100, 1.00 Viticoideae 100, 99 100, 100 92, 85 100, 100 100, 100 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 100, 100, 1.00 Viticisymphorina 81, 66 −, − 86, 67 98, 78 100, 96 100, 100, 1.00 1.00, 100, 100 100, 100, 1.00 1.00, 100, 99 Table 2. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 Discussion Our phylogenetic reconstruction of Lamiaceae, on the basis of the most comprehensive sampling of Lamiaceae to date, builds on prior studies using chloroplast DNA markers9,10,21–25,27,28,31–41,43,45–47,49–53. All of our analyses of the concatenated datasets revealed 12 highly supported primary clades (Figs 1–4), which are grouped into four mod- erately to highly supported larger clades (Fig. 1: clade I–IV). This set of four clades has not been identified in any previous published analysis but is consistent with the unpublished results of another combined analysis of three cpDNA regions (ycf1 + ycf1–rps15 + trnL-F; B. Drew, pers. comm.). Of the 12 primary clades, five correspond to subfamilies Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, and Scutellarioideae as recognized both by Harley et al.16 and Olmstead18. We have no additional findings on these subfamilies relative to previous molecular studies21–26,43,46, so we will focus our discussion on the other seven primary clades. One of these clades corresponds to Symphorematoideae recognized by Harley et al.16 and Olmstead18, and another corresponds to Viticoideae s. str. recognized by Bramley et al.47. These two clades are sister groups in all our combined-data analy- ses. The other five primary clades comprise Acrymia-Cymaria, Hymenopyramis-Petraeovitex-Peronema-Garrettia, Premna-Gmelina-Cornutia, Callicarpa, and Tectona. Eight genera listed as incertae sedis in Harley et al.16 are included in these five clades. Resolution of their phylogenetic placements makes it possible to improve the sub- familial classification of the Lamiaceae. Calliprostantherina. The clade comprising Callicarpa and Prostantheroideae, which we are naming Calliprostantherina (see Phylogenetic Nomenclature, below), emerged as sister to the remaining Lamiaceae in our phylogeny. This finding agrees with the large-scale phylogenetic analysis of Lamiidae15, while in other analyses, Callicarpa9,21,23 or Symphorematoideae (represented by Congea)10,24,25 was inferred to be sister to the rest of Lamiaceae. Inconsistency among published trees probably reflects taxon sampling or insufficient data, since these conditions could impact the accuracy of phylogenetic analyses54,55. Outgroups used by Scheen et al.21, Bendiksby et al.23, Li et al.24, and Chen et al.25 are distantly related to the Lamiaceae, and Prostantheroideae were not sampled by Schäferhoff et al.13. In the present study, outgroups were selected from Mazaceae, Orobanchaceae, Paulowniaceae, and Phrymaceae, which together form the sister group to Lamiaceae in recent studies of Lamiales12–15, and the ingroup was more comprehensively and densely sampled than in previous studies. Resultsh Callicarpa and Prostantheroideae group together in all of the combined-data analyses, with support ranging from low to high, and the Callicarpa-Prostantheroideae clade usually emerges as sister to the remaining Lamiaceae. Symphorematoideae and Viticoideae s. str. are sister groups in all combined-data analyses with high support. Acrymia-Cymaria, Scutellarioideae, and Hymenopyramis-Petraeovitex-Garrettia-Peronema are successive sister groups to Lamioideae (Fig. 2), with each node highly supported in all combined-data analyses (Fig. 1). Overall, the 12 primary clades cluster into four larger clades. Relationships among the four larger clades are poorly resolved, but each usually received moderate to high support in our analyses (Fig. 1: clade I–IV). Support values for the above-mentioned clades are summarized in Table 2. Phylogenetically defined names (names ending in – ina in Fig. 1) are hereby proposed for five clades that are moderately to strongly supported in our analyses and do not already have genus or subfamily names. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 3 www.nature.com/scientificreports/ Figure 1. Phylogenetic backbone of Lamiaceae based on simplification of trees generated from the analyses of the combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D270. Color-coded circles above branches indicate support values from BI, ML and MP analyses of the combined dataset D270, with or without gaps coded respectively, while those below branches indicate support values from BI, ML and MP analyses of the combined dataset D155, with or without gaps coded respectively. Subfamilies recognized by Olmstead46 (Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, Symphorematoideae, and Viticoideae s. str.) are shown in black bold font, while new subfamilies (Cymarioideae, Peronematoideae and Premnoideae) and two genera (Callicarpa and Tectona) that we do not assign to a subfamily are in red bold font. The arrows show the clade node of Lamiaceae and the nodes of five new clades (Cymalamiina, Scutelamiina, Perolamiina, Viticisymphorina, and Calliprostantherina). Figure 1. Phylogenetic backbone of Lamiaceae based on simplification of trees generated from the analyses of the combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D270. Color-coded circles above branches indicate support values from BI, ML and MP analyses of the combined dataset D270, with or without gaps coded respectively, while those below branches indicate support values from BI, ML and MP analyses of the combined dataset D155, with or without gaps coded respectively. Subfamilies recognized by Olmstead46 (Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, Symphorematoideae, and Viticoideae s. Resultsh str.) are shown in black bold font, while new subfamilies (Cymarioideae, Peronematoideae and Premnoideae) and two genera (Callicarpa and Tectona) that we do not assign to a subfamily are in red bold font. The arrows show the clade node of Lamiaceae and the nodes of five new clades (Cymalamiina, Scutelamiina, Perolamiina, Viticisymphorina, and Calliprostantherina). Discussion Thus, there is reason to have greater confidence in our inference of a Callicarpa-Prostantheroideae clade that is sister to the remaining Lamiaceae than the different inferred positions of these taxa in some previous studies. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 4 www.nature.com/scientificreports/ entificreports/ Figure 2. Bayesian 50% majority-rule consensus tree (box on the top left corner shows the topology) based on the combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D270, with gaps treated as simple indels, showing the taxa from Lamioideae, Cymarioideae, Scutellarioideae, Peronematoideae, Premnoideae, Ajugoideae, and Tectona. The topologies of the ML and MP trees are congruent with the BI tree. Bayesian posterior probability values ≥ 0.90 are marked with bold lines. Bootstrap values ≥ 50% in ML and MP analyses are plotted above the branches, successively, while “-” indicates support values of less than 50%. Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name indicates that the combined sequences pooled from different species of the genus. Subfamilies and tribes recognized by Olmstead18 were covered by gray boxes with different grey level, while new subfamilies and clades proposed in this study were covered by pink boxes and marked in red bold font. Figure 2. Bayesian 50% majority-rule consensus tree (box on the top left corner shows the topology) based on the combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D270, with gaps treated as simple indels, showing the taxa from Lamioideae, Cymarioideae, Scutellarioideae, Peronematoideae, Premnoideae, Ajugoideae, and Tectona. The topologies of the ML and MP trees are congruent with the BI tree. Bayesian posterior probability values ≥ 0.90 are marked with bold lines. Bootstrap values ≥ 50% in ML and MP analyses are plotted above the branches, successively, while “-” indicates support values of less than 50%. Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name indicates that the combined sequences pooled from different species of the genus. Subfamilies and tribes recognized by Olmstead18 were covered by gray boxes with different grey level, while new subfamilies and clades proposed in this study were covered by pink boxes and marked in red bold font. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 5 www.nature.com/scientificreports/ Figure 3. Discussion Bayesian 50% majority-rule consensus tree (box on the top left corner shows the topology) based on the combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D270, with gaps treated as simple indels, showing the taxa from Nepetoideae, Viticoideae s. str., Symphoremoideae, Callicarpa, and Prostantheroideae. The topologies of the ML and MP trees are congruent with the BI tree. Bayesian posterior probability values ≥ 0.90 are marked with bold lines. Bootstrap values ≥ 50% in ML and MP analyses are plotted above the branches, successively, while “-” indicates support values of less than 50%. Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name indicates that the combined sequences pooled from different species of the genus. Subfamilies and tribes recognized by f Figure 3. Bayesian 50% majority-rule consensus tree (box on the top left corner shows the topology) based on the combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D270, with gaps treated as simple indels, showing the taxa from Nepetoideae, Viticoideae s. str., Symphoremoideae, Callicarpa, and Prostantheroideae. The topologies of the ML and MP trees are congruent with the BI tree. Bayesian posterior probability values ≥ 0.90 are marked with bold lines. Bootstrap values ≥ 50% in ML and MP analyses are plotted above the branches, successively, while “-” indicates support values of less than 50%. Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name indicates that the combined sequences pooled from different species of the genus. Subfamilies and tribes recognized by Olmstead18 were covered by gray boxes with different grey level, while new subfamilies and clades proposed in this study were covered by pink boxes and marked in red bold font. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. The Bayesian 50% majority-rule consensus phylogram based on combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D155, with gaps treated as simple indels. The topologies of the ML and MP trees are congruent with the BI tree. Support values displayed on the branches follow the order BI-PP/ML-BS/MP-BS (“−” Indicates support values of less than 0.90 in BI or 50% in ML and MP analyses, respectively). The bold lines indicate that the three support values get full scores simultaneously. Discussion In all of our combined anal- yses, the Premna-Gmelina-Cornutia clade was strongly supported (Table 2). This clade is part of a larger clade that also includes Ajugoideae, Hymenopyramis-Petraeovitex-Garrettia-Peronema, Scutellarioideae, Acrymia-Cymaria and Lamioideae, consistent with the findings in Chen et al.25. Though traditionally placed in Viticoideae3,16, pre- vious studies10,23–25,43,47 as well as the present one have shown that the Premna-Gmelina-Cornutia clade is not sister to the rest of Viticoideae sensu Harley et al.16. Furthermore, these three genera cannot be included in any other established subfamily based on our results. In a paper intended to contrast conventional and phyloge- Premnoideae. This clade, comprising Premna, Gmelina and Cornutia, has been partially recovered in pre- vious molecular phylogenetic analyses10,24,25,43, in which Premna and Gmelina always grouped together. Cornutia was first included by Bendiksby et al.23 and revealed to be sister to a clade containing Premna, Gmelina, and Tectona (though the inclusion of Tectona in that clade conflicts with all other studies). In all of our combined anal- yses, the Premna-Gmelina-Cornutia clade was strongly supported (Table 2). This clade is part of a larger clade that also includes Ajugoideae, Hymenopyramis-Petraeovitex-Garrettia-Peronema, Scutellarioideae, Acrymia-Cymaria and Lamioideae, consistent with the findings in Chen et al.25. Though traditionally placed in Viticoideae3,16, pre- vious studies10,23–25,43,47 as well as the present one have shown that the Premna-Gmelina-Cornutia clade is not sister to the rest of Viticoideae sensu Harley et al.16. Furthermore, these three genera cannot be included in any other established subfamily based on our results. In a paper intended to contrast conventional and phyloge- netic nomenclature, Cantino et al.56 informally referred to the Premna-Gmelina clade (the position of Cornutia being unknown at that time) as Premnoideae under rank-based nomenclature and Premnina under phylogenetic nomenclature, and the former name was provisionally adopted by Olmstead18,48. Below we formally describe the new subfamily Premnoideae B. Li, R. G. Olmstead & P. D. Cantino. Peronematoideae. This clade comprises Petraeovitex, Peronema, Hymenopyramis and Garrettia. The first three of these were inferred to form a well-supported clade25,47 that is sister to the Scutellarioideae-Acrymia-Cymaria- Lamioideae clade25. The same sister position to Scutellarioideae-Acrymia-Cymaria-Lamioideae was found for Garrettia, which was first included by Bendiksby et al.23. When Garrettia, Hymenopyramis, and Petraeovitex were included in the same analysis, they formed a moderately supported clade25. Discussion Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name represented that the combined sequences pooled from different species of the genus. Subfamilies recognized by Olmstead45 were covered by gray boxes with different grey level, while new subfamilies (Cymarioideae, Peronematoideae and Premnoideae) and clades (Callicarpa and Tectona) proposed in this study were covered re 4. The Bayesian 50% majority-rule consensus phylogram based on combined cpDNA h Figure 4. The Bayesian 50% majority-rule consensus phylogram based on combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D155, with gaps treated as simple indels. The topologies of the ML and MP trees are congruent with the BI tree. Support values displayed on the branches follow the order BI-PP/ML-BS/MP-BS (“−” Indicates support values of less than 0.90 in BI or 50% in ML and MP analyses, respectively). The bold lines indicate that the three support values get full scores simultaneously. Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name represented that the combined sequences pooled from different species of the genus. Subfamilies recognized by Olmstead45 were covered by gray boxes with different grey level, while new subfamilies (Cymarioideae, Peronematoideae, and Premnoideae) and clades (Callicarpa and Tectona) proposed in this study were covered by pink boxes and marked in red bold font. Figure 4. The Bayesian 50% majority-rule consensus phylogram based on combined cpDNA (matK + ndhF + rbcL + rps16 + trnL-F) dataset D155, with gaps treated as simple indels. The topologies of the ML and MP trees are congruent with the BI tree. Support values displayed on the branches follow the order BI-PP/ML-BS/MP-BS (“−” Indicates support values of less than 0.90 in BI or 50% in ML and MP analyses, respectively). The bold lines indicate that the three support values get full scores simultaneously. Multiple accessions of the same species are numbered according to Supplementary Table S1. A single generic name represented that the combined sequences pooled from different species of the genus. Subfamilies recognized by Olmstead45 were covered by gray boxes with different grey level, while new subfamilies (Cymarioideae, Peronematoideae, and Premnoideae) and clades (Callicarpa and Tectona) proposed in this study were covered by pink boxes and marked in red bold font. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 7 www.nature.com/scientificreports/ Viticisymphorina. The clade comprising the subfamilies Symphorematoideae and Viticoideae s. Discussion str., which we are naming Viticisymphorina (see Phylogenetic Nomenclature, below), was strongly supported in all our anal- yses, consistent with some previous studies23,47. Each subfamily in this clade was confirmed to be monophyletic for the first time (Figs 1–4, Table 2). Though Symphorematoideae is well characterized by many morphologi- cal characters, its monophyly had never been tested in previous molecular studies because only one represent- ative had been included9,10,21,23–25,47. With all three genera included in the present study, Symphorematoideae was confirmed to be monophyletic (Figs 1–4, Table 2). Viticoideae as circumscribed by Harley et al.16 have been shown to form two clades that are not sister groups10,23,25,43,47, with Vitex (grouped with Paravitex, Petitia, Teijsmanniodendron, Tsoongia, and Viticipremna) being the largest genus in one clade and Premna (grouped with Cornutia and Gmelina) the largest genus in the other. After Premna, Cornutia, and Gmelina were removed from Viticoideae18 and Paravitex, Tsoongia, and Viticipremna were reduced to synonymy with Vitex47, the remaining Viticoideae consist of only four genera: Petitia, Pseudocarpidium Millsp., Teijsmanniodendron, and Vitex. We included representatives of all four of these genera in a single analysis for the first time here and the monophyly of Viticoideae s. str. was strongly supported (Figs 1–4, Table 2). g y pp g Symphorematoideae and Viticoideae s. str. are morphologically distinct from each other. Symphorematoideae are woody climbers with simple leaves, 3–7-flowered capitate inflorescences with accrescent bracteoles, whereas Viticoideae s. str. are generally shrubs or trees that have simple or palmately compound leaves and lack the dis- tinctive inflorescence structure of Symphorematoideae. However, the two subfamilies are putatively connected by several anatomical structures: 1) Ovaries without a false septum are rare in other Lamiaceae, but can be found both in Symphorematoideae and Viticoideae s. str. 2) In Symphorematoideae, orthotropous and pendulous ovules are borne in the free apex of the locules16. Ovules in most Lamiaceae are anatropous or hemianatropous. Junell5 noted that the distinctive placentation in Symphorematoideae could easily be derived from the type of placenta- tion found in many Viticoideae. Premnoideae. This clade, comprising Premna, Gmelina and Cornutia, has been partially recovered in pre- vious molecular phylogenetic analyses10,24,25,43, in which Premna and Gmelina always grouped together. Cornutia was first included by Bendiksby et al.23 and revealed to be sister to a clade containing Premna, Gmelina, and Tectona (though the inclusion of Tectona in that clade conflicts with all other studies). Discussion Br.63, and indications that addi- tional realignments to several genera will be forthcoming44,64 (also T. Wilson, pers. comm.). A probable synapo- morphy is a dry schizocarp that splits into four one-seeded mericarps. This feature also characterizes several other clades within Lamiaceae, but our results indicate that it evolved independently in each. Because this fruit type is found in all members of Westringieae and in Brachysola Rye, which is sister to the rest of Chloantheae4,20,45, it is the most parsimonious assignment to the most recent common ancestor of Prostantheroideae. Callicarpa Linnaeus. Callicarpa contains about 140 species occurring in both temperate and tropical regions16. The plants are small trees or shrubs with actinomorphic, 4–5 (−7)-parted flowers and drupaceous fruits. Despite being one of the largest genera in Lamiaceae, its phylogenetic position had not previously been confirmed. In previous molecular studies, Callicarpa was included with only one or few representatives, and has been inferred to be sister to the rest of the family9,21,23, to group with subfamily Prostantheroideae15,20,43, or to be variably isolated in different positions10. Bramley49 sampled more representatives and indicated that Callicarpa is monophyletic, but she could not infer its phylogenetic position because of poor sampling from the whole family. In the present study, Callicarpa was sampled much more extensively (18 spp.), taking into consideration its morphological and geographic breadth and its infrageneric classification. In all our analyses, the monophyly of Callicarpa was well supported (Figs 1–4; Table 2). A sister relationship between Callicarpa and subfamily Prostantheroideae was moderately to highly supported in analyses of the combined dataset D270 (Figs 1–3, Table 2), as well as in BI and ML analyses of the combined dataset D155 (Fig. 4). Symphorematoideae Briquet. The subfamily has three genera: Congea (ca. 7 species), Sphenodesme Jack (ca. 14 species) and Symphorema Roxb. (3 species). All genera are endemic to continental Asia (India to Indochina and southern and eastern China) and parts of Malesia (Peninsular Malaysia, Sumatra, Borneo, Java, Tanimbar Islands, and the Philippines). Congea tomentosa Roxb. and to a lesser extent C. griffithiana Munir are cultivated as ornamental climbers. There are several morphological traits that unite the three genera. All are climbers with inflorescences of 3–7-flowered capitate cymes. These are usually surrounded by conspicuous brac- teoles, often coloured and accrescent. Flowers of Sphenodesme are 5 or 6-merous; the flowers of Symphorema are 6 to 16 (–18)-merous. Discussion The most recent and widely adopted classifi- cation of Lamiaceae was proposed by Harley et al.16 in the first global, genus-level treatment of the entire fam- ily in more than a century since Briquet2. Harley et al.’s landmark work includes 226 genera assigned to seven subfamilies (Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, Scutellarioideae, Symphorematoideae, and Viticoideae), and ten genera listed as incertae sedis (Acrymia, Callicarpa, Cymaria, Garrettia, Holocheila, Hymenopyramis, Ombrocharis, Peronema, Petraeovitex, and Tectona). A decade later, numerous new findings have improved the classification incrementally. The results reported here provide the basis for a revised subfamilial classification. We take this opportunity to describe three new subfamilies and to update the subfamilial classifi- cation of the family incorporating new findings since Harley et al.16. For each subfamily, we provide a brief sum- mary of its historical classification and presently understood phylogenetic position, generic and species diversity, morphology, synapomorphies, and distribution. Of the ten genera treated as incertae sedis by Harley et al.16, there are only two that we do not assign to a subfamily—Callicarpa and Tectona; these genera are inserted among the subfamilies in positions consistent with the phylogeny in Fig. 1. Prostantheroideae Luerssen. This endemic Australian subfamily includes 17 genera and ca. 300 species belonging to two major clades, Chloantheae (12 genera) and Westringieae (5 genera). Prior to the cladistic study of Cantino4, these tribes were usually placed in Verbenaceae (or Chloanthaceae57,58) and Lamiaceae, respectively (e.g., Briquet2,3). However, based on gynoecial anatomy, Junell5 transferred “Chloanthoideae” (i.e., Chloantheae) to Lamiaceae and suggested that it shares a common origin with “Prostantheroideae” (i.e., Westringieae). Cantino et al.59 first placed the two groups together in a subfamily (“Chloanthoideae”). Monophyly of each tribe (excluding Spartothamnella Briq. and Tectona from Chloantheae, contrary to Munir60 and Cantino et al.59, respectively) and of the combined Prostantheroideae was confirmed by molecular phylogenetic analysis20, which also produced the first evidence that Callicarpa is sister to Prostantheroideae. Phylogenetic studies of Prostantheroideae20,44–46 (as well as from T. Wilson and B. Conn, pers. comm.) have revealed that several genera are not monophyletic as currently circumscribed. Recent and ongoing studies have led to abandonment of the genera Wrixonia F. Muell. (included in Prostanthera Labill.)61 and Mallophora Endl. (included in Dicrastylis J. Drumm. ex W. H. Harvey)62, the reinstatement of Dasymalla Endl. and Quoya Gaudich. and addition of Muniria N. Streiber & B. J. Conn comprising species previously assigned to Pityrodia R. Discussion In our combined analyses, the four genera form a highly supported clade that is sister to a larger clade comprising of Scutellarioideae, Acrymia-Cymaria and Lamioideae (Figs 1–4, Table 2). Olmstead18 suggested that a new name should be pro- vided to accommodate the Hymenopyramis-Petraeovitex-Peronema clade, while Garrettia was still listed as having uncertain subfamilial placement in his A Synoptical Classification of the Lamiales (Version 2.4). Based on the pres- ent phylogeny, the clade comprising Hymenopyramis, Petraeovitex, Garrettia, and Peronema cannot be assigned to any established subfamily, thus we here propose a new subfamilial name: Peronematoideae B. Li, R. G. Olmstead & P. D. Cantino. Formal description of the new subfamily is provided below. Cymarioideae. This small clade comprising Acrymia and Cymaria received high support in all our analyses of combined dataset D270, as well as in BI, ML, and MP analyses of D155 with gaps coded. It was inferred to be sister to subfamily Lamioideae with strong support in all analyses (Figs 1–4, Table 2). Our findings corroborate previous molecular phylogenetic analyses, where Cymaria21 or Acrymia-Cymaria23,25 was revealed to be closely related to Lamioideae. Now that the phylogenetic relationships seem to be well established, a taxonomic decision is needed whether to expand Lamioideae to include these two genera or name a new subfamily to accommodate them. The two approaches are equally consistent with the molecular phylogenetic results; i.e., both Lamioideae s. str. and a broader Lamioideae, expanded to include Acrymia and Cymaria, are well supported in our analyses and previous studies23,25. Bendiksby et al.23 and Chen et al.25 have argued that an expanded Lamioideae would be more morphologically heterogeneous and difficult to diagnose, and they therefore recommended excluding Acrymia and Cymaria from Lamioideae. Because the Acrymia-Cymaria clade was only moderately supported in their analyses, Chen et al.25 noted that if future evidence were to strongly corroborate the existence of this clade, a new subfamily could be named to accommodate them. This hypothesis is confirmed with strong confidence in our analyses (Figs 1–4, Table 2); therefore, we hereby erect the new subfamily Cymarioideae B. Li, R. G. Olmstead & P. D. Cantino and formally describe it below. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 8 www.nature.com/scientificreports/ Taxonomy and Nomenclaturei y An Updated Subfamilial Classification of Lamiaceae. Discussion p Viticoideae are distributed predominantly in the Tropics (Vitex throughout; Teijsmanniodendron in Malesia; Pseudocarpidium in the Caribbean), although there are a few temperate species of Vitex. Madagascar may be home to a number of currently unrecognized species of Vitex67. Analysis of a greater number of viticoid taxa could result in further changes to generic boundaries in this subfamily. Possible synapomorphies for the sub- family are phytochemical (see Pedersen65). There has been no comparative study of morphological or anatomical characters including all of the viticoid genera aside from Junell’s5 work on gynoecial structure. Although Junell noted that Vitex, Petitia, Pseudocarpidium, and the other genera now recognized as Vitex have a very similar ovary structure, he found some differences in Teijsmanniodendron. Further comparative studies including subfamily Viticoideae, particularly focusing on gynoecial structure, may elucidate unifying characters. Nepetoideae (Dumortier) Luerssen. Nepetoideae is the largest subfamily of Lamiaceae, containing almost half of the genera and species. It now contains 118 genera (compared to 105 recognized by Harley et al.16) and ca. 3400 species, which are widely distributed across tropical and temperate regions of the northern and southern hemispheres but with few native species in Australia and New Zealand. Probable synapomorphies for Nepetoideae include hexacolpate and three-celled pollen, investing embryos, myxocarpy, gynobasic style, and the presence of rosmarinic acid4,16,68–71. Three tribes are now recognized within Nepetoideae16: Elsholtzieae, Mentheae, and Ocimeae. The monophyly of each of these tribes is well supported by molecular phylogenetic studies34,37,40,43,72, but there are conflicting findings about relationships among the three tribes.h l gi g p g There have been several genus-level changes since the treatment of the subfamily by Harley et al.16. Bräuchler et al.72 described a new genus Killickia Bräuchler, Heubl & Doroszenko from South Africa. Harley and Pastore73 did a major genus-level revision of Hyptidinae, recognizing 12 genera that were not recognized by Harley et al.16. Nine of them were new (Cantinoa Harley & J. F. B. Pastore, Cyanocephalus (Pohl ex Benth.) Harley & J. F. B. Pastore, Eplingiella Harley & J. F. B. Pastore, Gymneia (Benth.) Harley & J. F. B. Pastore, Leptohyptis Harley & J. F. B. Pastore, Martianthus Harley & J. F. B. Pastore, Medusantha Harley & J. F. B. Pastore, Oocephalus (Benth.) Harley & J. F. B. Pastore, and Physominthe Harley & J. F. B. Pastore), and the other three were resurrected (Condea Adans., Eriopidion Harley, and Mesosphaerum P. Browne). Discussion The corolla of Congea is 2-lipped, but the corollas of Sphenodesme and Symphorema are actinomorphic. The ovaries are incompletely 2-locular, and the ovules are orthotropous and pendulous. Although unique to this subfamily, the ovary type was interpreted by Junell5 as being derived from that found in genera of “Viticoideae” (in which Junell included not only Viticoideae s. str. but also Callicarpa, Tectona, Premnoideae, Peronematoideae, Cymarioideae, and Ajugoideae in our classification). The fruit in all three genera is indehiscent, weakly drupaceous or dry, and is 1 (−2) -seeded by abortion. Viticoideae Briquet. Viticoideae, as circumscribed here, includes only three genera: Vitex (ca. 250 spp.), Teijsmanniodendron (23 spp.), and Pseudocarpidium (9 spp.). In contrast, Viticoideae sensu Harley et al.16 included ten genera (viz., Petitia, Cornutia, Premna, Viticipremna, Tsoongia, Paravitex, Vitex, Teijsmanniodendron, Gmelina, and Pseudocarpidium). Molecular studies, initially by Wagstaff and Olmstead10, had identified two dis- tinct clades, one centered on Vitex and another including Gmelina, Cornutia, and Premna. Phytochemical studies65 hinted at the same relationships, finding that phenolic compounds present in Premna or Gmelina were absent in Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 9 www.nature.com/scientificreports/ Vitex, Petitia, and Teijsmanniodendron. Because several smaller viticoid genera were not included in these early analyses, the circumscription of the subfamily was not altered by Harley et al.16. Bramley et al.47 further elucidated the relationships among the viticoid genera, focusing particularly on Southeast Asian taxa traditionally allied to Vitex. Based on their results, Viticipremna, Tsoongia, and Paravitex were included in Vitex, reducing the number of viticoid genera to seven. The generic status of Petitia and Pseudocarpidium was unchanged, because of poor support for the position of the former, lack of any data for the latter, and poor sampling among Neotropical taxa. In the present study, we find sufficient evidence to include Petitia, but not Pseudocarpidium, in Vitex. Similarly, the generic status of Teijsmanniodendron remains problematic. Neither the analyses of Bramley et al.47 nor our analyses provide convincing support to include Teijsmanniodendron in Vitex. Identification of species in these two genera is often confused; the sole morphological character that can be used to delimit them in most cases is a swelling present at the base and apex of the petiole in Teijsmanniodendron. Traditionally, Teijsmanniodendron species were also recognised by their capsule-like rather than drupaceous fruit that is 1-seeded (by abortion)66, but a reduction in the number of mature seeds can also occur in Vitex species. Discussion Drew et al.42 synonymized Chaunostoma Donn. Sm. and Neoeplingia Ramam., Hiriart & Medrano with Lepechinia Willd. Chen et al.43 resurrected Keiskea Miq. (included in Collinsonia L. by Harley et al.16) and showed that Ombrocharis (unassigned to subfamily by Harley et al.16) is sister to Perillula Maxim. within tribe Elsholtzieae. Drew and Sytsma37 found Heterolamium C. Y. Wu to be nested within Meehania Britton, but Deng et al.74 found that the specimen of Heterolamium studied by Drew and Sytsma41 was misidentified and was in fact a member of Meehania. Thus, the systematic position of Heterolamium within Nepetoideae is still uncertain. Tectona Linnaeus f. Tectona is a genus of large trees comprising three species distributed from India to southeast Asia. The large drupaceous fruits contain a hard four-celled endocarp and are enclosed in an enlarged persistent calyx. Tectona has been included in several molecular studies9,10,21,23,47, but its phyloge- netic position has never been determined definitively. It has been inferred to be sister to a clade comprising Hymenopyramis-Petraeovitex-Peronema and Premna-Gmelina47, to a large clade containing Ajugoideae, Lamioideae, Scutellarioideae, Peronema-Petraeovitex, and Premna-Gmelina-Cornutia (B. Drew, pers. comm.), to another larger clade comprising Ajugoideae, Lamioideae, Premna-Gmelina, Prostantheroideae, Scutellarioideae, and Vitex-Petitia10, or to group with Gmelina21,23. In our more comprehensive analyses, Tectona is inferred to be sister to a large clade comprised of Lamioideae, Acrymia-Cymaria, Scutellarioideae, Hymenopyramis-Petraeovitex-Garrettia-Peronema, Ajugoideae, and Premna-Gmelina-Cornutia, with moderate to strong support in BI and ML analyses of both datasets D270 and D155, with or without gaps coded (Figs 1–4). The distinct morphology of Tectona including an actinomorphic 5–7-lobed calyx and corolla, greatly enlarged and inflated persistent calyx, and 4-celled endocarp with small central cavity between the cells16, contributed to the difficulty of placing it in previous classifications. Our results suggest that Tectona is an early diverging lineage from the major clade IV (Figs 1–4). Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 Peronematoideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Peronema Jack in Malayan Misc. 2 (7): 18. 1822. Peronematoideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Peronema Jack in Malayan Misc. 2 (7): 18. 1822. Shrubs, trees, and lianas. Leaves opposite, petiolate, simple or ternately, biternately, or pinnately compound. Inflorescence cymose, axillary, and/or terminal, highly variable in form. Calyx actinomorphic, 4–5-lobed, and usually accrescent (not accrescent in Peronema) in fruit. Corolla white to yellow, 4–5-lobed, and nearly actino- morphic to zygomorphic. Stamens 4 or posterior pair reduced to staminodes (Peronema), equal or didynamous, included or exserted, thecae parallel to divaricate, usually separate (confluent in Garrettia) at dehiscence; pollen tricolpate, tectate-perforate. Ovary unlobed, stigma 2-lobed, equal or unequal; disc absent or poorly developed. Fruit dry, globose or turbinate, glabrous or pubescent to villous, indehiscent or breaking into two or four meri- carps, abscission-scar as long as the mericarp.h This new subfamily comprises four small, mostly tropical Asian genera that were treated as incertae sedis by Harley et al.16: Garrettia (1 sp., southwest China, Thailand, and Indonesia), Hymenopyramis (7 spp., India, China, and Indo-China), Peronema (1 sp., Thailand to Malaysia and western Indonesia), and Petraeovitex (8 spp., Burma, Thailand, Malaysia, Indonesia, Philippines, New Guinea, and Melanesia). Previously, Hymenopyramis, Peronema, and Petraeovitex have been placed in Caryopteridoideae3 or transferred to Viticoideae5, or Hymenopyramis was retained in Viticoideae but Peronema and Petraeovitex were transferred to Teucrioideae59. Garrettia was always placed in Caryopteridoideae92,93 before being transferred to Ajugoideae59. The four genera of Peronematoideae differ greatly in morphology and have never been linked in any previous classification, but Chen et al.25 has found some traits in common, including woody stems (small or climbing shrubs, lianas or large trees), white to yellow- ish corolla, unlobed ovary, nectar disc poorly developed or absent, and dry fruit. Each of these traits is probably either synapomorphic at a more inclusive level within Lamiaceae or plesiomorphic in the family as a whole25. Scutellarioideae (Dumortier) Caruel. A taxon centered on Scutellaria was recognized as a distinct ele- ment within Lamiaceae in early classifications (e.g., Bentham1; Briquet2), often comprising only Scutellaria and the segregate genera, Perilomia Kunth and Salazaria Torr., now included within Scutellaria16,94. Early phylogenetic studies based on morphology4,8 and DNA sequences10 expanded this clade to include Renschia Vatke, Tinnea Kotschy ex Hook. f., and Holmskioldia Retz., the latter formerly assigned to Verbenaceae. The rediscovery of the extremely rare Wenchengia C. Y. Wu & S. Premnoideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Premna L. in Mant. ii, 154 771 Trees, shrubs, lianas, or rarely small herbs. Leaves simple, opposite, usually aromatic. Inflorescence cymose, usually terminal, variable in form. Calyx tubular or campanulate, truncate or 4–5-toothed, often obscurely 2-lipped. Corolla blue, purple-violet, mauve, yellow, brownish or white, infundibular or hypocrateriform, 4–5-lobed, ±2-lipped or occasionally actinomorphic. Stamens 4 or posterior pair reduced to staminodes, didy- namous or equal, included or slightly exserted, thecae separate, parallel to widely divergent; pollen usually Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 10 www.nature.com/scientificreports/ tricolpate (4–5-colpate in Cornutia), tectate-perforate, psilate or suprareticulate. Ovary unlobed, stigma 2-lobed, equal or unequal; disc well developed (Cornutia) or absent (Premna, Gmelina). Fruit drupaceous, exocarp fleshy, pyrene hard, 4-seeded (sometimes3–1-seeded by abortion). y y This new subfamily contains three genera: Premna (50–200 spp. in tropical to subtropical Asia, Africa, Australia, and the Pacific Islands), Gmelina (31 spp. in tropical and subtropical Asia to Australia and western Pacific Islands), and Cornutia (12 spp. in tropical America). The three genera were traditionally placed in sub- family Viticoideae of Verbenaceae3, and transferred to Lamiaceae together with the subfamily5,16,59, and then excluded from Viticoideae by Olmstead18 based on molecular evidence that Viticoideae is non-monophyletic if they are included23,47. A possible synapomorphy for Premnoideae is a drupaceous fruit with one four-seeded pyrene. However since a similar fruit structure is also found in Tectona and some species of Vitex, it may instead be a synapomorphy at a deeper level in the phylogeny with subsequent reversals. With the number of species estimated from 5075 to 20076, Premna now ranks among the most taxonomically difficult and complicated genera of Lamiaceae. Though some regional revisions of the genus have been done in recent decades77–82, there is no treatment of the genus throughout its range. A global taxonomic revision of Gmelina was published by de Kok83. Ajugoideae Kosteletzky. Ajugoideae contains 26 genera and ca. 760 species and is cosmopolitan in distribu- tion. A series of phylogenetic studies, which collectively included every genus except Monochilus Fisch. & C. A. Mey., have resolved most of the generic boundaries and relationships22,27,84–88. Our results find a small clade compris- ing Karomia Dop and Rotheca Raf. to be sister to the rest of the subfamily. Unpublished results by one of us (C. L. Xiang) indicate that Discretitheca P. D. Cantino and Glossocarya Wall. ex Griff. are close relatives of Rotheca. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 Premnoideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Premna L. in Mant. ii, 154 771 This clade of four genera is distributed from Africa to the Indian subcontinent, Southeast Asia, and Queensland. The rest of Ajugoideae comprises two large clades. One, with ca. 260 species, is primarily temperate and centered on Teucrium L. (this clade remains poorly studied). The other large clade, with ca. 425 species, is centered on Clerodendrum19,24,51,87. The latter clade comprises a primarily tropical clade, which includes Clerodendrum and related genera, and a primarily temperate clade, which includes Ajuga L., Trichostema L., Caryopteris Bunge, and related genera. Molecular analyses22,27,89 have also increased the number of genera accepted from 2416 to 26, with Huxleya Ewart now included in Clerodendrum, Faradaya F. Muell. included in Oxera Labill., and four gen- era (Kalaharia Baill., Ovieda L., Tetraclea A. Gray, and Volkameria L.) segregated from Clerodendrum. Probable synapomorphies of Ajugoideae include pollen exine with supratectal spines, spinules or verrucae, and exine with branched to granular columellae. These character states are widespread in Ajugoideae16,90,91 and rare (branched columellae) or absent (spinules and verrucae) elsewhere in the Lamiaceae. Peronematoideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Peronema Jack in Malayan Misc. 2 (7): 18. 1822. Cymarioideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Cymaria Bentham in dwards’ Bot. Reg. 15: t. 1292. 1830. Cymarioideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Cymaria Bentham in Edwards’ Bot. Reg. 15: t. 1292. 1830. Shrubs and subshrubs. Leaves simple, opposite, petiolate, elliptic or ovate to rhombic, crenate to crenulate or repand. Cymes axillary, lax, long-pedunculate, with secund, monochasial branches, sometimes grading into a terminal paniculiform thyrse. Calyx campanulate, accrescent, broadly campanulate to urceolate or subglobose in fruit, 5-lobed, lobes equal to subequal, triangular. Corolla white to yellowish, 2-lipped, posterior lip entire to deeply 2-lobed, anterior lip with median lobe largest. Stamens 4, didynamous (anterior pair longer), included or exserted, thecae divaricate, confluent at dehiscence; pollen tricolpate, tectate-perforate, suprareticulate, columellae simple to sparsely branched. Ovary shallowly 4-lobed; style sub-terminal, stigma lobes subequal to unequal; disc absent. Nutlets obovoid, reticulately ridged, pubescent, abscission-scar lateral, 0.4–0.6× the length of the nutlet.h This new subfamily consists of two small, tropical Asian genera: Acrymia (1 sp., Peninsular Malaysia) and Cymaria (2–3 spp., Hainan, Indo-China and Malesia)(species numbers and ranges from Harley et al.16). The two genera have been included in subfamily Ajugoideae2,59,96,97 or treated as incertae sedis16. A probable synapomor- phy of Cymarioideae is its inflorescence structure: the cymes are axillary, lax, and long-pedunculate, with secund, monochasial branches16. This form of inflorescence is rare in the family but also occurs in Garrettia, where it apparently evolved independently. y y When describing Cymarioideae, we realized that the type genus of the new subfamily, Cymaria, is currently without a type species. Bentham98 simultaneously named two species, C. dichotoma Benth. and C. elongata Benth., at the same time he described the genus, but he did not designate either as the type. Cymaria was recognized by several subsequent authors, and the two species were always listed in parallel without any type designation1,2,97,99. We take this opportunity to designate C. dichotoma as the type species for Cymaria, because it is more widely distributed and better represented in herbaria. Lamioideae Harley. The largely Old World subfamily Lamioideae is second in size only to Nepetoideae with over 60 genera and ca. 1200 species. Tribe Stachydeae is cosmopolitan in distribution, and Synandreae is endemic to North America. The other eight tribes are largely Eurasian, but four of them include some African species, and Pogostemon also occurs in Australia16,21,23. All Lamioideae have a gynobasic style, a synapomorphy that arose independently in Nepetoideae and Scutellarioideae9,10. Cymarioideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Cymaria Bentham in dwards’ Bot. Reg. 15: t. 1292. 1830. Another possible synapomorphy is the presence of seed oils with an allenic component present4, but this character has been studied in too few species to be fully evalu- ated. Recent phylogenetic studies based on cpDNA sequence data have identified ten clades that have been ranked as tribes, with several genera unassigned to tribe21,23, but the monophyly of some of these tribes is not supported by nuclear DNA (PPR) data26. Further studies of relationships within some tribes (Synandreae—Scheen et al.29; Roy et al.33; Leucadeae—Scheen and Albert100; Phlomideae—Pan et al.52; Mathiesen et al.101; Salmaki et al.30; Lamieae—Bendiksby et al.102; Stachydeae—Salmaki et al.31; Gomphostemmateae—Xiang et al.53) have led to changes in the composition of some genera, acceptance of genera not recognized by Harley et al.16, and elim- ination of some genera that were recognized by Harley et al.16. In addition, Chen et al.25 showed that the for- merly unplaced genus Holocheila belongs in Lamioideae. Besides Holocheila, four genera have been added to Lamioideae since 2004: Rydingia Scheen & V. A. Albert103, Betonica L.21, Acanthoprasium (Benth.) Spenn.23, and Phlomoides Moench101. Six genera recognized by Harley et al.16 are no longer recognized: Alajja Ikonn. (included in Eriophyton Benth.)23, Sulaimania Hedge & Rech. f. (included in Moluccella L.)23, Pseudoeremostachys Popov and Lamiophlomis Kudo (included in Phlomoides)52,101, Bostrychanthera Benth. (included in Chelonopsis Miq.)102, and Stachyopsis Popov & Vved. (included in Eriophyton Benth.)104. As a result of these changes, Lamioideae is now considered to have 62 genera (versus 63 recognized by Harley et al.16). Peronematoideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Peronema Jack in Malayan Misc. 2 (7): 18. 1822. Chow permitted Li et al.24 to confirm its placement in Scutellarioideae by Harley et al.16. A characteristic two lobed, untoothed calyx is shared by a clade of Scutellaria, Renschia, and Tinnea. Holmskioldia is sister to this clade and has an expanded saucer-shaped calyx with five, often indistinct, lobes, which form the dominant part of the floral display, unlike the other genera. Wenchengia is sister to the rest of the clade and has a two-lobed, but five-toothed calyx. Probable synapomorphies for Scutellarioideae include pericarps with tuberculate or elongate processes24, high densities of xylem fibers in the calyces95, and possibly racemose inflorescences (but they are cymose in Holmskioldia and most species of Tinnea, suggesting that inde- pendent origin of racemes within Scutellarioideae may be equally parsimonious). Scutellaria includes approxi- mately 360 species, is cosmopolitan in distribution, occurs in a wide range of habitats, and includes annual and perennial herbs and shrubs. A global taxonomic revision of Scutellaria established infrageneric classification and Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 11 www.nature.com/scientificreports/ reduced Harlanlewisia, Perilomia, and Salazaria to synonymy94. Tinnea includes 19 species, all endemic to Africa. The rest of the clade consists of small, narrowly endemic genera: Renschia (1–2 spp., Somalia), Holmskioldia (1 sp., southern Himalayas), and Wenchengia (1 sp., Hainan island, China). reduced Harlanlewisia, Perilomia, and Salazaria to synonymy94. Tinnea includes 19 species, all endemic to Africa. The rest of the clade consists of small, narrowly endemic genera: Renschia (1–2 spp., Somalia), Holmskioldia (1 sp., southern Himalayas), and Wenchengia (1 sp., Hainan island, China). Cymarioideae B. Li, R. G. Olmstead & P. D. Cantino, subfam. nov. Type: Cymaria Bentham in Edwards’ Bot. Reg. 15: t. 1292. 1830. Phylogenetic Nomenclaturei Given the distri- bution of the character states and its variability within many tribes of Lamioideae, it is not clear whether confluent thecae is a synapomorphy of Cymalamiina with a series of reversals within Lamioideae or, alternatively, that it evolved independently in Cymarioideae, Pogostemoneae, and in scattered other members of Lamioideae. Scutelamiina B. Li, R. G. Olmstead & P. D. Cantino, new clade name. Definition. The smallest crown clade containing Lamium purpureum L. 1753 and Scutellaria galericulata L. 1753. Scutelamiina B. Li, R. G. Olmstead & P. D. Cantino, new clade name. Definition. The smallest rown clade containing Lamium purpureum L. 1753 and Scutellaria galericulata L. 1753. rimary reference phylogeny. Figure 2; see also Fig. 1 (this paper), Bendiksby et al.23 (Fig. 1), Chen et al.25 Fig. 4), and Chen et al.43 (Fig. 3). ary reference phylogeny. Figure 2; see also Fig. 1 (this paper), Bendiksby et al.23 (Fig. 1), Chen et al.25 4), and Chen et al.43 (Fig. 3). Composition. Scutellarioideae, Cymarioideae and Lamioideae. Synapomorphies. A four-lobed ovary appears to be a synapomorphy for Scutelamiina. It is shallowly four lobed in Cymarioideae and the more basal members of Scutellarioideae (though unlobed in Holmskioldia) and deeply lobed in Lamioideae and Scutellaria. A developmentally related feature, a schizocarpic fruit with four mericarps (“nutlets”), may be another synapomorphy of Scutelamiina. However, within its sister group (Peronematoideae), the 4-seeded capsule of Garrettia and Peronema breaks with pressure into four mericarps, possibly representing a stage in the evolution of the schizocarp of Scutelamiina. Another likely synapomorphy is suprareticulate (some- times called bireticulate) pollen, which is found in all genera of Scutellarioideae and Cymarioideae and most genera of Lamioideae90,107–109. However the presence of suprareticulate pollen in Garrettia (Peronematoideae)90,110 and suprarugulose pollen in Peronema110 raises the possibility that this feature may be a synapomorphy for Perolamiina rather than Scutelamiina, though Petraeovitex and Hymenopyramis (the more distal genera within Peronematoideae) have psilate pollen110. Furthermore, suprareticulate sculpturing could be synapomorphic at an even more inclusive level because it occurs in some genera of Premnoideae (Gmelina but not Cornutia or Synapomorphies. A four-lobed ovary appears to be a synapomorphy for Scutelamiina. It is shallowly four lobed in Cymarioideae and the more basal members of Scutellarioideae (though unlobed in Holmskioldia) and deeply lobed in Lamioideae and Scutellaria. A developmentally related feature, a schizocarpic fruit with four mericarps (“nutlets”), may be another synapomorphy of Scutelamiina. Phylogenetic Nomenclaturei However, within its sister group (Peronematoideae), the 4-seeded capsule of Garrettia and Peronema breaks with pressure into four mericarps, possibly representing a stage in the evolution of the schizocarp of Scutelamiina. Another likely synapomorphy is suprareticulate (some- times called bireticulate) pollen, which is found in all genera of Scutellarioideae and Cymarioideae and most genera of Lamioideae90,107–109. However the presence of suprareticulate pollen in Garrettia (Peronematoideae)90,110 g p p p ( ) and suprarugulose pollen in Peronema110 raises the possibility that this feature may be a synapomorphy for Perolamiina rather than Scutelamiina, though Petraeovitex and Hymenopyramis (the more distal genera within Peronematoideae) have psilate pollen110. Furthermore, suprareticulate sculpturing could be synapomorphic at an even more inclusive level because it occurs in some genera of Premnoideae (Gmelina, but not Cornutia or Premna, based on very few species90,110). Comments. Cantino et al.56 applied the name Lamiina to this clade and provided a phylogenetic definition, but the names used in that paper were intended only to illustrate differences between phylogenetic and traditional rank-based nomenclature. They were not intended to have nomenclatural precedence under either system, and the PhyloCode did not yet exist. Perolamiina B. Li, R. G. Olmstead & P. D. Cantino, new clade name. Definition. The smallest crown clade containing Lamium purpureum L. 1753 and Peronema canescens Jack 1822. Primary reference phylogeny. Figure 2; see also Fig. 1 (this paper), Bendiksby et al.23 (Fig. 1, where Garrettia represents Peronematoideae), Chen et al.25 (Fig. 4, where Garrettia, Hymenopyramis and Petraeovitex repre- sent Peronematoideae), and Chen et al.43 (Fig. 3, where Hymenopyramis, Petraeovitex and Peronema represent Peronematoideae). Primary reference phylogeny. Figure 2; see also Fig. 1 (this paper), Bendiksby et al.23 (Fig. 1, where Garrettia represents Peronematoideae), Chen et al.25 (Fig. 4, where Garrettia, Hymenopyramis and Petraeovitex repre- sent Peronematoideae), and Chen et al.43 (Fig. 3, where Hymenopyramis, Petraeovitex and Peronema represent Peronematoideae). Composition. Peronematoideae, Scutellarioideae, Cymarioideae and Lamioideae. Apomorphies. Supareticulate pollen may be a synapomorphy; see Scutelamiina. Viticisymphorina B. Li, R. G. Olmstead & P. D. Cantino, new clade name. Definition. The smallest crown clade containing Vitex agnus-castus L. 1753 and Symphorema involucratum Roxb. 1805 but not Nepeta cataria L. 1753, Tectona grandis L. f. 1782, Premna serratifolia L. 1771, Ajuga reptans L. 1753, and Lamium pur- pureum L. 1753. Primary reference phylogeny. Figure 3; see also Fig. 1 (this paper), Bendiksby et al.23 (Fig. Phylogenetic Nomenclaturei g Our analyses revealed five strongly supported but previously unnamed clades that contain subfamilies (labe- led in Fig. 1). These clades warrant naming to facilitate communication about them, but there is no standard rank between family and subfamily. We therefore have given them unranked names, which are defined below, following the rules and recommendations of the draft PhyloCode105. For readers unfamiliar with phylogenetic nomenclature, the preface of the draft PhyloCode provides a good introduction (https://www.ohio.edu/phy- locode/preface.html). The names of a variety of plant clades have been defined following the draft PhyloCode, including a set of major tracheophyte and angiosperm clades106, but there have been few previous applications of phylogenetic nomenclature to Lamiaceae. Cantino et al.56 provided phylogenetic definitions for some clade names within Lamiaceae to illustrate differences between phylogenetic and traditional nomenclature, but the PhyloCode did not yet exist, and the names and definitions in that paper were not intended to have any formal nomenclatural status. Salmaki et al.31 phylogenetically defined the name Eurystachys to accommodate the clade comprising the paraphyletic genus Stachys and ten other genera that nest within it. Phylogenetic definitions for the names Labiatae and Nepetoideae have been prepared by P. D. Cantino & R. G. Olmstead and will be published in Phylonyms (de Queiroz et al., in prep.). Cymalamiina B. Li, R. G. Olmstead & P. D. Cantino, new clade name. Definition. The smallest crown clade containing Lamium purpureum L. 1753 and Cymaria dichotoma Benth. 1930. Primary reference phylogeny. Figure 2; see also Fig. 1 (this paper), Bendiksby et al.23 (Fig. 1), Chen et al.25 (Fig. 4), Roy and Lind qvist26 (Fig. 1a), and Chen et al.43 (Fig. 3, where Cymaria represents Cymarioideae). Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 12 www.nature.com/scientificreports/ Composition. Cymarioideae and Lamioideae. Synapomorphies. Confluent anther thecae may be a synapomorphy. It is shared by Cymarioideae and one of the two basal subclades of Lamioideae (i.e., Pogostemoneae), but the anthers in the other basal subclade (i.e., the rest of Lamioideae) vary from distinct (e.g., Gomphostemmateae, Synandreae, Galeopsis L.) to confluent (e.g., Colquhounia Wall. and some Stachydeae). The closest outgroups to Cymalamiina (i.e., Scutellarioideae and Peronematoideae) have distinct thecae (except Garrettia in Peronematoideae), as do the more distant outgroups Premnoideae and Tectona; this character varies in Ajugoideae, another more distant outgroup. Phylogenetic Nomenclaturei 1, where Congea represents Symphorematoideae, Vitex and Petitia represent Viticoideae), and Bramley et al.47 (Fig. 1, where Sphenodesme represent Symphorematoideae). Composition. Viticoideae and Symphorematoideae. Composition. Viticoideae and Symphorematoideae. Apomorphies. No non-molecular synapomorphies are known. Comments. We intend this name to be applicable only if Symphorematoideae and Viticoideae are sister groups. Although our analyses strongly support the monophyly of this grouping, it is not supported in some other analyses24,25,43. For this reason, the definition includes external specifiers to make the name inapplicable under many alternative phylogenies. Comments. We intend this name to be applicable only if Symphorematoideae and Viticoideae are sister groups. Although our analyses strongly support the monophyly of this grouping, it is not supported in some other analyses24,25,43. For this reason, the definition includes external specifiers to make the name inapplicable under many alternative phylogenies. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 13 www.nature.com/scientificreports/ Calliprostantherina B. Li, R. G. Olmstead & P. D. Cantino, new clade name. Definition. The smallest crown clade containing Callicarpa americana L. 1753 and Prostanthera lasianthos Labillardiére 1806, but not Vitex agnus-castus L. 1753, Nepeta cataria L. 1753, Tectona grandis L. f. 1782, Premna serratifolia L. 1771, Ajuga reptans L. 1753, and Lamium purpureum L. 1753. Primary reference phylogeny. Figure 3; see also Fig. 1 (this paper), Olmstead et al.20 (Fig. 1), Refulio-Rodriguez and Olmstead15 (Fig. 1A, where Prostanthera represents Prostantheroideae), and Chen et al.43 (Fig. 3, where Prostanthera and Westringia represent Prostantheroideae). Composition. Callicarpa and Prostantheroideae. Composition. Callicarpa and Prostantheroideae. Apomorphies. Branched trichomes and actinomorphic corollas, two characters that are infrequent in Lamiaceae, are shared by Callicarpa and Chloantheae (one of the two basal subclades of Prostantheroideae). Branched trichomes also occur in scattered species of the other subclade (Westringieae), increasing the likelihood that this feature characterizes Calliprostantherina. However, it could be apomorphic at a deeper level in the phylogeny since Symphorematoideae and Tectona also have branched hairs (see Fig. 1 for the relationship of the latter taxa to Calliprostantherina). Actinomorphic corollas also occur in Tectona and some genera of Symphorematoideae; furthermore, all species of Westringieae have zygomorphic flowers, weakening the hypothesis that actinomorphy is a synapomorphy for Calliprostantherina. There are similarities in pollen surface sculpturing between some species of Callicarpa and some genera of Prostantheroideae111, but there is considerable variation in both groups, and the polarity of the character is unknown. Comments. Type designation and new combinations Type designation and new combinations Cymaria Bentham in Edwards’s Bot. Reg. 15: t. 1292. 1830.—Type (here designated): Cymaria dichotoma Bentham in Edwards’ Bot. Reg. 15: t. 1292. 1830. Vitex petitia Bramley, nom. nov. Bas.: Petitia domingensis Jacq. in Enum. Syst. Pl.: 12. 1760.—Lectotype (here designated): Haiti, Jacquin s.n. (BM! fragment, barcode no. BM000992805). Note:—A new name rather than a new combination has been created for Petitia domingensis, since the name Vitex domingensis Urb. & Ekman already exists (=Pseudocarpidium domingense (Urb. & Ekman) Moldenke). The epithet ‘petitia’ has been chosen to retain an obvious link to the original name of the species, which is quite common across the West Indies. The fragment of Jacquin’s specimen housed at the BM has been designated as lectotype, as suggested by annotation on the sheet by H. N. Moldenke according to d’Arcy112. Jacquin’s herbarium was reportedly bought by Banks and incorporated into his collections. Vitex urbanii (Ekman) Bramley, comb. nov. Bas.: Petitia urbanii Ekman in Ark. Bot. 21A(5): 94. 1927. —Lectotype (here designated): Haiti, Ile de la Tortue, steep limestone rocks west of Monillago Anglais, 22 May 1925, E. L. Ekman H4096 (S! sheet number S04-2601; isolectotypes A!, B, F!, G!, K!, NY!, S!, UC!, US!). Vitex urbanii (Ekman) Bramley, comb. nov. Bas.: Petitia urbanii Ekman in Ark. Bot. 21A(5): 94. 1927. —Lectotype (here designated): Haiti, Ile de la Tortue, steep limestone rocks west of Monillago Anglais, 22 May 1925, E. L. Ekman H4096 (S! sheet number S04-2601; isolectotypes A!, B, F!, G!, K!, NY!, S!, UC!, US!). , ( ; yp , , , , , , , , ) Note:—One of the sheets at S [sheet number S04-2601] is designated as lectotype because E. L. Ekman was based there, and it has attached to it a handwritten note “it is a pleasure to dedicate this fine new species to Prof. Urban, the admirable botanist, the never tiring worker, and the best friend a man ever had”. Phylogenetic Nomenclaturei Because the molecular support for this clade is only moderate and the potential morphological syn- apomorphies discussed above are not convincing, the definition is designed to become inapplicable under many alternative phylogenies (e.g., Bendiksby et al.23: Fig. 1). Type designation and new combinations Cymaria Bentham in Edwards’s Bot. Reg. 15: t. 1292. 1830.—Type (here designated): Cymaria dichotoma Bentham in Edwards’ Bot. Reg. 15: t. 1292. 1830. Materials and Methods Choice of markers, taxon sampling and molecular data. Five chloroplast DNA markers—matK, ndhF, rbcL, rps16, and trnL-F—were employed in this study because (1) they have been widely used in phyloge- netic reconstructions of Lamiaceae at generic, tribal or subfamilial level, and (2) many species of Lamiaceae have already been sequenced for these markers in previous molecular studies9,10,21–53,101. No comparable source of data exists for any nuclear DNA region for a broad sample of Lamiaceae.h y g The ingroup sample included representatives of all seven subfamilies and all ten genera incertae sedis recog- nized by Harley et al.16 and all 14 tribes recognized by Olmstead18. Nomenclature of Lamiaceae and Viticoideae s. str. followed Olmstead18 and Bramley et al.47, respectively. Initially, we downloaded data for all taxa of Lamiaceae with sequence information for any of the five gene regions deposited in Genbank as of August 2015. In the five subfamilies whose monophyly is well supported (viz., Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae and Scutellarioideae), sampling was designed to cover their genus-level diversity. Generally, genera with at least two sequenced regions were selected, and each selected genus was represented by one or two species. Particular emphasis was placed on sampling Symphorematoideae, Viticoideae s. str., all genera incertae sedis, and three genera formerly assigned to Viticoideae—Cornutia, Gmelina, and Premna. In three large genera—Callicarpa, Premna, and Vitex, sampling was designed to cover their morphological and geographic breadth. In total, 288 species representing 191 genera were included, representing approximately 78% of the genera of Lamiaceae. Five outgroup species were selected representing the closest relatives to Lamiaceae in Lamiales12–15. They are Lindenbergia philippensis (Cham. & Schltdl.) Benth. and Pedicularis groenlandica Retz. from Orobanchaceae, Paulownia tomentosa (Thunb.) Steud. from Paulowniaceae, Mazus reptans N. E. Br. from Mazaceae and Phryma leptostachya L. from Phrymaceae. Information on sampled taxa and Genbank accession numbers is assembled in Supplementary Table S1. Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 14 www.nature.com/scientificreports/ The five separate molecular data sets matK, ndhF, rbcL, rps16 and trnL-F contained 202, 160, 170, 181, and 259 sequences with 54, 83, 59, 57, and 88 newly reported sequences, respectively. The dataset combining the five markers included 270 taxa (D270), with 39.65 % missing data. Materials and Methods According to investigations by Wiens113 and Wiens and Moen114, the proportion of missing data should not affect the accuracy of the phylogenetic analysis; however, just to make sure, a reduced dataset was assembled including 155 taxa (D155) with at least three of the five regions or 50 % of the total aligned sequence length available for each terminal taxon. The total amount of missing data in D155 was 23.51 %. For most species in the combined datasets, data were available for all five regions, but there were some genera of Ajugoideae, Lamioideae, Nepetoideae, Prostantheroideae, and Scutellarioideae in which different species were used for different gene regions. When data were pooled in this way, generic names, rather than species names, were used to represent the combined sequences in the phylogenetic trees. DNA extraction, amplification, and sequencing. The 2x CTAB method of Doyle and Doyle115 was used to extract total genomic DNA of the samples with silica dried leaf tissue, and DNEasy® Plant Mini Kit (QIAGEN®, Valencia, California, USA) was used for herbarium materials according to the manufacturer’s speci- fications. The DNA extracts were dissolved in TE buffer and preserved at −20 °C for further use.ii ihf p Primer pairs used in Polymerase chain reaction (PCR) amplification of the five regions are listed in Table S2 with their sequences and references. The PCR reaction system and amplification protocol were identical for all five fragments. PCR reactions used 2.5 μL sample DNA, 0.5 μL Dream Taq DNA polymerase, 5 μL 10 × DreamTaq Green Buffer, 5 μL dNTP Mix (2 mM each), 1 μL of bovine serum albumin (BSA, 20 mg/mL), 1 μL of each primer in a final reaction volume of 50 μL. The PCR program was as follows: an initial template denaturation at 94 °C for 5 min, 35 cycles of 30 second denaturation at 94 °C, 1 minute primer annealing at 52 °C, 1.5 min extension at 72 °C, with a final extension of 8 min at 72 °C. Sequencing was done by the Invitrogen sequencing service (Invitrogen, commercial sequencing facility, Guangzhou, China) using the same primers for PCR amplifications. Sequence alignment and phylogenetic analyses. Sequencher v.4.5116 was used to evaluate chromato- grams for base confirmation and to edit contiguous sequences. Materials and Methods All DNA sequences were initially aligned using Clustal X v.2.0.117 and adjusted manually in BioEdit Sequence Alignment Editor v.7.0.0118.hi j y q g The separate data sets were first analyzed using Maximum parsimony (MP) and Maximum likelihood (ML) methods, with gaps treated as simple indels determined by the program Gapcoder119 and added to the matrix as binary presence/absence characters. The combined data sets D270 and D155 were analyzed using MP, ML and Bayesian inference (BI) methods with gaps treated either as missing data or as simple indels. MP analyses were conducted using PAUP* v.4.0b10120 with all characters unordered and equally weighted. Heuristic search was conducted using 1000 random addition sequence replicates, tree-bisection-reconnection (TBR) branch swapping, MulTrees in effect, and steepest descent off. Bootstrap support values (BS) were esti- mated using a heuristic search strategy with 500 bootstrap replicates and 1000 random sequences additions. g gy p p q ML analyses were performed on the web server RAxML Black Box121. Before each submission, the “Maximum likelihood search” and “Estimate proportion ofinvariable sites” options were selected, with a total of 1000 boot- strap replicates performed. p p p BI analysis was executed using MrBayes version 3.2.2122 on the CIPRES Science Gateway123 with the default parameters. The best substitution types (Nst) and rate distribution models (rates) were determined by the Akaike information criterion (AIC) using Model Test v.3.7124 with the hierarchical likelihood ratio tests. Four Markov chain Monte Carlo (MCMC) chains were run, each beginning with a random tree and sampling one tree every 1000 generations for 30 000 000 generations. Mixing, convergence and a suitable burn-in were assessed with the statistics provided by the program and with Tracer v. 1.6125. Post burn-in samples from the four runs were merged using LogCombiner v1.7.5. (available at http://beast.bio.ed.ac.uk/, LogCombiner) prior to the calculation of a 50 % majority-rule consensus tree. References 1. Bentham, G. In Genera plantarum, Vol. 2 (eds Bentham, G. & Hooker, J. D.), Verbenaceae and Labiatae, 1131–1223 (Reeve London, 1876).l 1. Bentham, G. In Genera plantarum, Vol. 2 (eds Bentham, G. & Hooker, J. D.), Verbenaceae and Labiatae, 1131–1223 (Reeve, London, 1876). 2. Briquet, J. In Die natürlichen Pflanzenfamilien, Vol. 4 (3a)(eds Engler, A. & Prantl, K.), Labiatae, 183–375 (Engelmann, Leipzig 1895–1897).l 3. Briquet, J. In Die natürlichen Pflanzenfamilien, Vol. 4 (3a) (eds Engler, A. & Prantl, K.), Verbenaceae, 132–182 (Engelmann, Leipzig 1895).i ) 4. Cantino, P. D. In Advances in Labiate science (eds Harley, R. M. & Reynolds, T.), Toward a phylogenetic classification of the Labiatae, 27–37 (Royal Botanic Gardens, Kew, London, 1992). ) 4. Cantino, P. D. In Advances in Labiate science (eds Harley, R. M. & Reynolds, T.), Toward a phylogenetic classification of the Labiatae, 27–37 (Royal Botanic Gardens, Kew, London, 1992). 5. Junell, S. Zur Gynaceum morphologie und Systematik der Verbenaceen und Labiaten. Symb. Bot. Upsal. 4, 1 219 (1934). 6. Abu-Asab, M. S. Phylogenetic implications of pollen morphology in subfamily Lamioideae (Labiatae) and related taxa (Ph.D Thesis) (Ohio University, USA, 1990). J , y p g y y p , ( ) 6. Abu-Asab, M. S. Phylogenetic implications of pollen morphology in subfamily Lamioideae (Labiatae) and related taxa (Ph.D. Thesis) (Ohio University, USA, 1990). h 7. Abu-Asab, M. S. & Cantino, P. D. In Advances in Labiate science (eds Harley, R. M. & Reynolds, T.), Pollen morphology i subfamily Lamioideae (Labiatae) and its phylogenetic implications, 97–112 (Royal Botanic Gardens, Kew, London, 1992). y y g y 8. Cantino, P. D. Evidence for a polyphyletic origin of the Labiatae. Ann. Missouri Bot. Gard. 79, 361–379 (1992). ff l d h l f d b f d f b 9. Wagstaff, S. J. & Olmstead, R. G. Phylogeny of Lamiaceae and Verbenaceae inferred from rbcL sequences. Syst. Bot. 22, 165–179 (1997).f 10. Wagstaff, S. J., Hickerson, L., Spangler, R., Reeves, P. A. & Olmstead, R. G. Phylogeny of Lamiaceae s. l. inferred from cpDNA sequences. Plant Syst. Evol. 209, 265–274 (1998).il q y 11. Cronquist, A. An integrated system of classification of flowering plants. (Columbia University Press, New York, 1981). O l h ll Ol d G & h d f S h l T q g y f fi f fl g p y 12. Oxelman, B., Kornhall, P., Olmstead, R. G. References Taxon 59, 125–133 (2010). d k b Th b k h d d d d h l d l fi f 23. Bendiksby, M., Thorbek, L., Scheen, A. C., Lindqvist, C. & Ryding, O. An updated phylogeny and classification of Lamiaceae subfamily Lamioideae. Taxon 60, 471–484 (2011). y 24. Li, B. et al. Phylogenetic position of Wenchengia (Lamiaceae): a taxonomicallyenigmatic and critically endangered genus. Taxon 61, 392–401 (2012). 25. Chen, Y. P. et al. Phylogenetic placement of the enigmatic genus Holocheila (Lamiaceae) inferred from plastid DNA sequences Taxon 63, 355–366 (2014). , ( ) 26. Roy, T. & Lindqvist, C. New insights into evolutionary relationships within the subfamily Lamioideae (Lamiaceae) based on pentatricopeptide repeat (PPR) nuclear DNA sequences. Amer. J. Bot. 102, 1721–1735 (2015). 27. Steane, D. A., de Kok, R. P. & Olmstead, R. G. Phylogenetic relationships between Clerodendrum (Lamiaceae) and other Ajugoid genera inferred from nuclear and chloroplast DNA sequence data. Mol. Phylogenet. Evol. 32, 39–45 (2004). 28. Lindqvist, C. & Albert, V. A. Origin of the Hawaiian endemic mints within North American Stachys (Lamiaceae). Am. J. Bot. 89 1709–1724 (2002). 29. Scheen, A. C., Lindqvist, C., Fossdal, C. G. & Albert, V. A. Molecular phylogenetics of tribe Synandreae, a North American lineage of lamioid mints (Lamiaceae). Cladistics 24, 299–314 (2008). 30. Salmaki, Y. et al. Phylogeny of the tribe Phlomideae (Lamioideae: Lamiaceae) with special focus on Eremostachys and Phlomoides: new insights from nuclear and chloroplast sequences. Taxon 61, 161–179 (2012). g p q 31. Salmaki, Y. et al. Molecular phylogeny of tribe Stachydeae (Lamiaceae subfamily Lamioideae). Mol. Phylogenet. Evol. 69, 535–551 (2013). 32. Roy, T., Chang, T. H., Lan, T. Y. & Lindqvist, C. Phylogeny and biogeography of New World Stachydeae (Lamiaceae) with emphasi on the origin and diversification of Hawaiian and South American taxa. Mol. Phylogenet. Evol. 69, 218–238 (2013). l l l h h h l d b d ( ) b d l l l l l 33. Roy, T. et al. Evolutionary relationships within the lamioid tribe Synandreae (Lamiaceae) based on multiple low-copy nuclear loci PeerJ PrePrints 4, e1725v1 (2016). 34. Paton, A. J. et al. Phylogeny and evolution of basils and allies (Ocimeae, Labiatae) based on three plastid DNA regions. Mol Phylogenet. Evol. 31, 277–299 (2004). y g 35. Walker, J. B., Sytsma, K. J., Treutlein, J. & Wink, M. References Salvia (Lamiaceae) is not monophyletic: implications for the systematics radiation, and ecological specializations of Salvia and tribe Mentheae. Am. J. Bot. 91, 1115–1125 (2004). 36. Walker, J. B. & Sytsma, K. J. Staminal evolution in the genus Salvia (Lamiaceae): molecular phylogenetic evidence for multiple origins of the staminal lever. Ann. Bot. 100, 375–391 (2007). g 37. Bräuchler, C., Meimberg, H. & Heubl, G. Molecular phylogeny of Menthinae (Lamiaceae, Nepetoideae, Mentheae)–taxonomy biogeography and conflicts. Mol. Phylogenet. Evol. 55, 501–523 (2010). g g p yl y g 38. Moon, H. K., Smets, E. & Huysmans, S. Phylogeny of tribe Mentheae (Lamiaceae): the story of molecules and micromorphologica characters. Taxon 59, 1065–1076 (2010). 39. Drew, B. T. & Sytsma, K. J. Testing the monophyly and placement of Lepechinia in the tribe Mentheae (Lamiaceae). Syst. Bot. 36 1038–1049 (2011). 40. Drew, B. T. & Sytsma, K. J. Phylogenetics, biogeography, and staminal evolution in the tribe Mentheae (Lamiaceae). Am. J. Bot. 99 933–953 (2012).h 41. Drew, B. T. & Sytsma, K. J. The South American radiation of Lepechinia (Lamiaceae): phylogenetics, divergence times and evolution of dioecy. Bot. J. Linn. Soc. 171, 171–190 (2013).h y 42. Drew, B. T., Cacho, N. I. & Sytsma, K. J. The transfer of two rare monotypic genera, Neoeplingia and Chaunostoma, to Lepechinia (Lamiaceae), and notes on their conservation. Taxon 63, 831–842 (2014). 43. Chen, Y. P. et al. Resolving the phylogenetic position of Ombrocharis (Lamiaceae), with reference to the molecular phylogeny of tribe Elsholtzieae. Taxon 65, 123–136 (2016). d f l h l d ( ) ( ) 44. Guerin, G. R. Evidence for polyphyly in Hemigenia and Microcorys (Lamiaceae: Westringieae). Aust. Syst. Bot. 21, 313–325 (2008) p yp y y g y g y 45. Conn, B. J., Streiber, N., Brown, E. A., Henwood, M. J. & Olmstead, R. G. Infrageneric phylogeny of Chloantheae (Lamiaceae) based on chloroplast ndhF and nuclear ITS sequence data. Aust. Syst. Bot. 22, 243–256 (2009). 46. Wilson, T. C., Conn, B. J. & Henwood, M. J. Molecular phylogeny and systematics of Prostanthera (Lamiaceae). Aust. Syst. Bot. 25 341–352 (2012). 47. Bramley, G. L. C., Forest, F. & De Kok, R. P. J. Troublesome tropical mints: re-examining generic limits of Vitex and relations (Lamiaceae) in South East Asia. Taxon 58, 500–510 (2009).ii 48. Olmstead, R. G. A synoptical classification of the Lamiales, version 2.2. (2010) Available at: http://my-plant.org/sites/default/files/ lamiales_classn.v.2.2_0.pdf. (Accessed: 20 November 2012).h p 49. References & Bremer, B. Further disintegration of Scrophulariaceae. Taxon 54, 411–425 (2005).f il 12. Oxelman, B., Kornhall, P., Olmstead, R. G. & Bremer, B. Further disintegration of Scrophulariaceae. Taxon 54, 411–425 (2005). 13 Schäferhoff B et al Towards resolving Lamiales relationships: insights from rapidly evolving chloroplast sequences BMC Evol 12. Oxelman, B., Kornhall, P., Olmstead, R. G. & Bremer, B. Further disintegration of Scrophulariaceae. Taxon 54, 411–425 (200 13. Schäferhoff, B. et al. Towards resolving Lamiales relationships: insights from rapidly evolving chloroplast sequences. BMC Biol 10 352 (2010) . O e a , ., Ko a , ., O stead, R. G. & e e , . u t e d s teg at o o Sc op u a aceae. axon 5 , 5 ( 005). 13. Schäferhoff, B. et al. Towards resolving Lamiales relationships: insights from rapidly evolving chloroplast sequences. BMC Evol Biol. 10, 352 (2010). 14. Perret, M., Chautems, A., De Araujo, A. O. & Salamin, N. Temporal and spatial origin of Gesneriaceae in the New World inferred from plastid DNA sequences. Bot. J. Linn. Soc. 171, 61–79 (2013). Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 15 www.nature.com/scientificreports/ 15. Refulio-Rodriguez, N. F. & Olmstead, R. G. Phylogeny of Lamiidae. Am. J. Bot. 101, 287–299 (2014). 16. Harley, R. M. et al. In Families and genera of vascular plants, Vol. 7 (eds Kubitzki, K. & Kadereit, J. W.), Labiatae, 167–275 (Springer Berlin, 2004).hi 17. The Angiosperm Phylogeny Group. An update of the Angiosperm Phylogeny Group classification for the orders and families o flowering plants: APG IV. Bot. J. Linn. Soc. 181, 1–20 (2016).i l g p 18. Olmstead, R. G. A synoptical classification of the Lamiales, version 2.4 (2012) Available at: http://depts.washington.edu/phylo Classification.pdf. (Accessed: 5th April 2013). i p p 19. Soltis, D. E. et al. Angiosperm phylogeny: 17-genes, 640 taxa. Am. J. Bot. 98, 704–730 (2011).i g y g y g 20. Olmstead, R. G., Reeves, P. A. & Lepschi, B. J. Confirmation of a monophyletic Chloanthoideae (Lamiaceae) comprising tribes Chloantheae and Prostanthereae. Lamiales Newsletter 6, 7–10 (1998).i 21. Scheen, A. C. et al. Molecular phylogenetics, character evolution, and suprageneric classification of Lamioideae (Lamiaceae). Ann Missouri Bot. Gard. 97, 191–217 (2010).i , ( ) 22. Yuan, Y. W., Mabberley, D. J., Steane, D. A. & Olmstead, R. G. Further disintegration and redefinition of Clerodendrum (Lamiaceae) implications for the understanding of the evolution of an intriguing breeding strategy. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 59. Cantino, P. D., Harley, R. M. & Wagstaff, S. J. In Advances in Labiate science (eds Harley, R. M. & Reynolds, T.), Genera of Lab status and classification, 511–522 (Royal Botanic Gardens, Kew, London, 1992). 59. Ca t o, . ., a ey, R. . & Wagstaf , S. J. dva ces ab ate science (eds a ey, R. . & Rey o ds, .), Ge e a o ab atae status and classification, 511–522 (Royal Botanic Gardens, Kew, London, 1992). f h h ll ( hl h ) d l d d ( ) status and classification, 511–522 (Royal Botanic Gardens, Kew, London, 1992). 60 M i A A A i i i f h S t th ll (Chl h ) J Ad l id B t G d 1 3 25 (1976) i y 60. Munir, A. A. A taxonomic revision of the genus Spartothamnella (Chloanthaceae). J. Adelaide Bot. Gard. 1, 3–25 (1976). g p 1. Wilson, T. C., Henwood, M. J. & Conn, B. J. Status of the genus W 62. Rye, B. L. A taxonomic review of Dicrastylis sect. Corymbosae (Lamiaceae: Chloantheae), incorporating Mallophora as a new synonym. Nuytsia 15, 445–455 (2005). y y y 63. Conn, B. J., Henwood, M. J. & Streiber, N. Synopsis of the tribe Chloantheae and new nomenclatural combinations in Pityrodias lat. (Lamiaceae). Aust. Syst. Bot. 24, 1–9 (2011). y 64. Guerin, G. R. A taxonomic revision of Hemigenia section Malleantha sect. nov. (Lamiaceae: Westringieae). Aust. Syst. Bot. 21, 326–374 (2008). 65. Pedersen, J. A. Distribution and taxonomic implications of some phenolics in the family Lamiaceae determined by ESR spectroscopy. Biochem. Syst. Ecol. 28, 229–253 (2000). spectroscopy. Biochem. Syst. Ecol. 28, 229 253 (2000). 66. Koorders, S. H. Teijsmanniodendron, eine neue Gattung der Verbanaceae im Botanischen Garten von Buitenzorg. Ann. Jard. Bot Buitenzorg 19 19 31 (1904) p py y 66. Koorders, S. H. Teijsmanniodendron, eine neue Gattung der Verbanaceae im Botanischen Garten von Buitenzorg. Ann. Jard. Bot Buitenzorg 19, 19–31 (1904). g , ( ) 67. Callmander, M. W., Philipson, P. B. & Schatz, G. E. Towards a revision of the genus Vitex L. (Lamiaceae) in Madagascar I: a distinctive new species from North-eastern Madagascar. Candollea 69, 141–147 (2014).i p g 68. Cantino, P. D. & Sanders, R. W. Subfamilial classification of Labiatae. Syst. Bot. 11, 163–185 (1986). i 69. Erdtman, G. www.nature.com/scientificreports/ Pollen morphology and plant taxonomy IV: Labiatae, Verbenaceae and Avicenniaceae. Svensk Bot. Tidskr. 39, 279 (1945).h 70. Ryding, O. In Advances in Labiate Science (eds. Harley, R. M. & Reynolds, T.), The distribution and evolution of myx Lamiaceae, 85–96 (Royal Botanic Gardens, Kew, London, 1992). ( y ) 71. Wunderlich, R. Ein Vorschlag zu einer natürlichen Gliederung der. Labiaten. Oesterr. Bot. Z. 114, 383–483 (1967). 72. Bräeuchler, C., Doroszenko, A., Esser, H. J. & Heubl, G. Killickia (Lamiaceae): a new genus from KwaZulu-Natal, South Africa. Bot. J. Linn. Soc. 157, 575–586 (2008). 73. Harley, R. M. & Pastore, J. F. B. A generic revision and new combinations in the Hyptidinae (Lamiaceae), based on molecular and morphological evidence. Phytotaxa 58, 1–55 (2012). p g y 74. Deng, T. et al. Does the Arcto-Tertiary biogeographic hypothesis explain the disjunct distribution of northern hermisphere herbaceous plants? The case of Meehania (Lamiaceae). PLoS ONE 10, e0117171 (2015). g y 74. Deng, T. et al. Does the Arcto-Tertiary biogeographic hypothesis explain the disjunct distribution of northern hermisphere h b l ? Th f h ( ) S O ( ) 74. Deng, T. et al. Does the Arcto-Tertiary biogeographic hypothesis explain the disjunc herbaceous plants? The case of Meehania (Lamiaceae). PLoS ONE 10, e0117171 (2015). 74. Deng, T. et al. Does the Arcto Tertiary biogeographic hypothesis explain the disjun herbaceous plants? The case of Meehania (Lamiaceae). PLoS ONE 10, e0117171 (2015). h 75. Verdcourt, B. In Flora of tropical east Africa (ed. Polhill, R. M.), Verbenaceae, 1–156 (Balkema, Rotterdam, 1992). , f p f ( , ), , ( , , ) 76. Mabberley, D. J. Mabberley’s Plant-Book: a Portable Dictionary of Plants, 3rd ed. (Cambridge University Press, Cambridge, 2008 Mabberley, D. J. Mabberley’s Plant-Book: a Portable Dictionary of P y y y f g y g 77. Munir, A. A. A taxonomic revision of the genus Premna L. (Verbenaceae) in Australia. J. Adelaide Bot. Gard. 7, 1–44 (1984). 77. Munir, A. A. A taxonomic revision of the genus Premna L. (Verbenaceae) in Australia. J. Adel 78. Chen, S. L. & Gilbert, M. G. In Flora of China, Vol. 17 (eds Wu, C. Y. & Raven, P. H.), Verbenaceae, 1–49 (Science Press, Beijing & Missouri Botanical Garden Press, St. Louis, 1994).h Rajendran, A. & Daniel, P. The Indian Verbenaceae (Shiva Press, Dehra Dun, 2002). 79. Rajendran, A. & Daniel, P. www.nature.com/scientificreports/ The Indian Verbenaceae (Shiva Press, Dehra Dun, 2002). jh 80. Mabberley, D. J. & de Kok, R. P. J. In Flore de la Nouvelle-Calédonieet Dépendances, Vol. 25 (eds Morat, P. & Mackee, H. S.), Labiatae, 20–141 (Muséum National D’Histoire Naturelle, Paris, 2005). 81. Leeratiwong, C., Chantaranothai, P. & Paton, A. A synopsis of the genus Premna L. (Lamiaceae) in Thailand. Nat. H Chulalongkorn Univ. 9, 113–142 (2009).h g 82. De Kok, R. P. J. The genus Premna L. (Lamiaceae) in the Flora Malesiana area. Kew Bull. 68, 55–84 (2013). h 83. De Kok, R. P. J. A revision of the genus Gmelina (Lamiaceae). Kew Bull. 67, 293–329 (2012). 84. Steane, D. A. et al. Phylogenetic relationships of Clerodendrum s. l. (Lamiaceae) inferred from chloroplast DNA. Syst. Bot. 22, 229–244 (1997). 85. Steane, D. A., Scotland, R. W., Mabberley, D. J. & Olmstead, R. G. Molecular systematics of Clerodendrum (Lamiaceae): ITS sequences and total evidence. Amer. J. Bot. 86, 98–107 (1999).fl q 86. Cantino, P. D., Wagstaff, S. J. & Olmstead, R. G. Caryopteris (Lamiaceae) and the conflict between phylogenetic and pragmatic considerations in botanical nomenclature. Syst. Bot. 23, 369–386 (1999).h f siderations in botanical nomenclature. Syst. Bot. 23, 369–386 (1999 87. Huang, M. Systematics of Trichostema L. (Lamiaceae) and phylogenetic relationships with its disjunct taxa in Asia. (Ph.D. Thesis (Ohio University, USA, 2002). (Ohio University, USA, 2002). 88. Shi, S. H. et al. Phylogenetic position of Schnabelia, a genus endemic to China: evidence from sequences of cpDNA matK gene and nrDNA ITS regions Chinese Sci Bull 48 1576–1580 (2003) 88. Shi, S. H. et al. Phylogenetic position of Schnabelia, a genus endemic to China: evidence from sequences of cpDNA matK gene and nrDNA ITS regions. Chinese Sci. Bull. 48, 1576–1580 (2003). g 89. Barrabe, L. et al. Recircumscription of Oxera (Lamiaceae: Ajugoideae) to include Faradaya based on molecular and anatomica data. Bot. J. Linn. Soc. 179, 693–711 (2015). 90. Abu-Asab, M. S. & Cantino, P. D. Phylogenetic implications of pollen morphology in tribe Ajugeae (Labiatae). Syst. Bot. 18 100–122 (1993). 91. Abu-Asab, M. S., Cantino, P. D., Nowicke, J. W. & Sang, T. Systematic implications of pollen morphology in Caryopteris (Labiatae). Syst. Bot. 18, 502–515 (1993). 92. Wu, C. Y. In Flora Yunnanica, Vol. 1 (eds. Kunming Institute of Botany, Academia Sinica), Verbenaceae, 390–490 (Science Press Beijing, 1977). j g 93. P’ei, C. & Chen, S. L. www.nature.com/scientificreports/ In Flora Reipublicae Popularis Sinicae, Vol. 65 (eds. P’ei, C. & Chen, S. L.), Verbenaceae, 1–208 (Science P Beijing, 1982). j g 94. Paton, A. A global taxoboic investigation of Scutellaria (Labiatae). Kew Bull. 45, 399–450 (1990) d f l fib l l h l d j g 94. Paton, A. A global taxoboic investigation of Scutellaria (Labiatae). Kew Bull. 45, 399–450 (1990).i 95. Ryding, O. Amount of calyx fibers in Lamiaceae, relation to calyx structure, phylogeny and ecology. Plant Syst. Evol. 268, 45–58 (2007). 95. Ryding, O. Amount of calyx fibers in Lamiaceae, relation to calyx structure, phylogeny and ecol (2007). ( ) 96. Prain, D. Decades Kewenses: XLVII.-XLVIII. Bull. Misc. Inform. Kew 3, 105–116 (1908). n, D. Decades Kewenses: XLVII.-XLVIII. Bull. Misc. Inform. Kew 3 f 97. Keng, H. In Flora Malesiana. Series I, Spermatophyta, Flowering Plants, Vol. 8 (ed. Van Steenis, C. G. G. J.), Labiatae, 301–394 (Sij h ff & N dh ff I i l P bli h Al h d Rij 1978) f g, H. In Flora Malesiana. Series I, Spermatophyta, Flowering Plants, Vol. 8 (ed. Van Steenis, C. G. G. J.), Labiatae, 301–394 hoff & Noordhoff International Publishers, Alphen aan den Rijn, 1978). 97. Keng, H. In Flora Malesiana. Series I, Spermatophyta, Flowering Plants, Vol. 8 (ed. Van Steenis, C. G. G. J.), Labi (Sijthoff & Noordhoff International Publishers, Alphen aan den Rijn, 1978). Keng, H. In Flora Malesiana. Series I, Spermatophyta, Flowering Plants, Vol. 8 (ed. Van Steenis, C. G. G. J.), Labiatae, 301–394 Sijthoff & Noordhoff International Publishers, Alphen aan den Rijn, 1978). g p p y g ( J ) (Sijthoff & Noordhoff International Publishers, Alphen aan den Rijn, 1978). & Noordhoff International Publishers, Alphen aan den Rijn, 1978) jff p j 98. Bentham, G. In Edwards’s Botanical Register, Vol. 15 (ed. Lindley, J.), Cymaria, sub. t. 1292. (James Ridgway, London, 1830). g y y g y 99. Keng, H. Flora Malesianae precursores XLVIII: a revision of Malesian Labiatae. Gard. Bull. Sing. 24, 13–180 (1969). 100. Scheen, A. C. & Albert, V. A. Molecular phylogenetics of the Leucas group (Lamioideae; Lamiaceae). Syst. Bot. 34, 173–181 (2 100. Scheen, A. C. & Albert, V. A. Molecular phylogenetics of the Leucas group (Lamioideae; Lamiaceae). Syst. Bot. 34, 173–181 (2009). 101. Mathiesen, C., Scheen, A. C. & Lindqvist, C. Phylogeny and biogeography of the lamioid genus Phlomis (Lamiaceae). Kew Bull. References Bramley, G. L. C. The genus Callicarpa (Lamiaceae) on Borneo. Bot. J. Linn. Soc. 159, 416–455 (2009). h 50. Zhong, J. S., Li, J., Li, L., Conran, J. G. & Li, H. W. Phylogeny of Isodon (Schrad. ex Benth.) Spach (Lamiaceae) and related genera inferred from nuclear ribosomal ITS, trnL-trnF region, and rps16 intron sequences and morphology. Syst. Bot. 35, 207–219 (2010). f d d f h ( ) d f dh inferred from nuclear ribosomal ITS, trnL-trnF region, and rps16 intron sequences and morphology. Syst. Bot. 35, 207–219 (2010). 51. Huang, M., Crawford, D. J., Freudenstein, J. V. & Cantino, P. D. Systematics of Trichostema (Lamiaceae): evidence from ITS, ndhF, and morphology. Syst. Bot. 33, 437–446 (2008). 51. Huang, M., Crawford, D. J., Freudenstein, J. V. & Cantino, P. D. Systematics of Trichostema (Lamiaceae): evidence from ITS, ndhF, and morphology. Syst. Bot. 33, 437–446 (2008). p gy y 52. Pan, Y. Z., Fang, L. Q., Hao, G., Cai, J. & Gong, X. Systematic positions of Lamiophlomis and Paraphlomis (Lamiaceae) based on nuclear and chloroplast sequences. J. Syst. Evol. 47, 535–542 (2009). 53. Xiang, C. L. et al. Molecular phylogenetics of Chelonopsis (Lamiaceae: Gomphostemmateae) as inferred from nuclear and plastid DNA and morphology. Taxon 62, 375–386 (2013). p gy 54. Hillis, D. M. Taxonomic sampling, phylogenetic accuracy, and investigator bias. Syst. Biol. 47, 3–8 (1998). 55. Heath, T. A., Hedtke, S. M. & Hillis, D. M. Taxon sampling and the accuracy of phylogenetic analyses. J. Syst. Evol. 46, 239 (2008).f 56. Cantino, P. D., Olmstead, R. G. & Wagstaff, S. J. A comparison of phylogenetic nomenclature with the current system: a botanical case study. Syst. Biol. 46, 313–331 (1997).hl y y 57. Hutchinson, J. The families of flowering plants 2nd ed, Vol. 1 (Oxford University Press, London, 1959). 58. Munir, A. A. A taxonomic revision of the genus Chloanthes (Chloanthaceae). J. Adelaide Bot. Gard. 1, 83–106 (1977 y y 57. Hutchinson, J. The families of flowering plants 2nd ed, Vol. 1 (Oxford University Press, London, 1959). 58 M i A A A i i i f h Chl th (Chl h ) J Ad l id B t G d 1 83 106 (1977) Jh f f fl g p ( y ) 58. Munir, A. A. A taxonomic revision of the genus Chloanthes (Chloanthaceae). J. Adelaide Bot. Gard. 1, 83–106 (1977). Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 16 www.nature.com/scientificreports/ d d h f h l l ( ) i 113. Wiens, J. J. Can incomplete taxa rescue phylogenetic analyses from long branch attraction? Sy 114. Wiens, J. J. & Moen, D. Missing data and the accuracy of Bayesian phylogenetics. J. Syst. Evol. 46, 307–314 (2008). 114. Wiens, J. J. & Moen, D. Missing data and the accuracy of Baye 115. Doyle, J. J. & Doyle, J. D. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 1 (1987). ( ) 116. Gene Codes Corporation. Sequencher, version 4.5. (2005) Available at: http://genecodes.com/. (Accessed: 13 March 2011). k A l Cl l d Cl l f ( ) orporation. Sequencher, version 4.5. (2005) Available at: http://gene ene Codes Corporation. Sequencher, version 4.5. (2005) Available a Gene Codes Corporation. Sequencher, version 4.5. (2005) Available at: http://genecodes.com/. (Accessed: 13 March 2011). Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). 116. Gene Codes Corporation. Sequencher, version 4.5. (2005) Available at: http://genecodes.com/. (Accessed: 13 March 2011). 117. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). 117. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). 118. Hall, T. A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98 (1999). y p 19. Young, N. D. & Healy, J. GapCoder automates the use of indel characters in phylogenetic analysis. BMC Bioinformatics 4, 6 (2003) g y J p p y g y f ( ) 120. Swofford, D. L. PAUP: Phylogenetic analysis using parsimony (and other methods), version 4.0b10. (Sinauer, Massachusetts, Sunderland, 2002). 21. Stamatakis, A., Hoover, P. & Rougemont, J. A rapid bootstrap algorithm for the RAxML web-servers. Syst. Biol. 75, 758–771 (2008) 121. Stamatakis, A., Hoover, P. & Rougemont, J. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol. 75, 758 771 (2008). 122. Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012). 122. Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012). 123 Miller M A Pfeiffer W & Schwartz T Creating the CIPRES Science Gateway for inference of large phylogenetic trees in 123. Author Contributions Conceived and designed the experiments: B.L., D.-X.Z., P.D.C. and R.G.O. Collected the materials: B.L., C.-L.X., G.L.C.B., Y.-H.T. and Z.-H.M. Performed the experiments: B.L., C.-L.X., R.G.O. and Z.-H.M. Analyzed the data: B.L. and R.G.O. Wrote the paper: B.L., C.-L.X., D.-X.Z, G.L.C.B., P.D.C. and R.G.O. Conceived and designed the experiments: B.L., D.-X.Z., P.D.C. and R.G.O. Collected the materials: B.L., C.-L.X., G.L.C.B., Y.-H.T. and Z.-H.M. Performed the experiments: B.L., C.-L.X., R.G.O. and Z.-H.M. Analyzed the data: B.L. and R.G.O. Wrote the paper: B.L., C.-L.X., D.-X.Z, G.L.C.B., P.D.C. and R.G.O. Acknowledgementsh g This work was supported by Ministry of Science and Technology of China (grant no. 2013FY111200), National Natural Science Foundation of China (grant no. 31460044), and National Science Foundation of the United States (DEB 1020369). Assistance in the lab was provided by P.A. Reeves and Y.W. Yuan. www.nature.com/scientificreports/ Miller, M. A., Pfeiffer, W. & Schwartz, T. Creating the CIPRES Science Gateway for inference of large phylogenetic tre Proceedings of the Gateway Computing Environments Workshop (GCE), USA 1–8 (New Orleans, LA., 2010). 124. Posada, D. & Crandall, K. A. Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818 (1998). 25. Rambaut, A., Suchard, M. A., Xie, D. & Drummond, A. J. Tracer v.1.6. (2014) Available at: http://beast.bio.ed.ac.uk/Tracer (Accessed: 10 October 2015). Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 www.nature.com/scientificreports/ 66, 83–99 (2011).h 101. Mathiesen, C., Scheen, A. C. & Lindqvist, C. Phylogeny and biogeography of the lamioid genus Phlomis (Lamiaceae). Kew Bull. 66, 83–99 (2011).h 102. Bendiksby, M., Brysting, A. K., Thorbek, L., Gussarova, G. & Ryding, O. Molecular phylogeny and taxonomy of the genus Lamium L. (Lamiaceae): disentangling origins of presumed allotetraploids. Taxon 60, 986–1000 (2011). 03. Scheen, A. C. & Albert, V. A. Nomenclatural and taxonomic changes within the Leucas clade (Lamioideae; Lamiaceae). Syst. Geogr Plants 77, 229–238 (2007).h 104. Bendiksby, M., Salmaki, Y., Bräuchler, C. & Ryding, O. The generic position of Stachys tibetica Vatke and amalgamation of the genera Eriophyton and Stachyopsis (Lamiaceae subfam. Lamioideae). Plant Syst. Evol. 300, 961–971 (2014). 105. Cantino, P. D. & de Queiroz, K. International code of phylogenetic nomenclature, version 4c. (2010) Available at: https://www.ohio. edu/phylocode/. (Accessed: 20 December 2014). p y 106. Cantino, P. D. et al. Towards a phylogenetic nomenclature of Tracheophyta. Taxon 56, 822–846 (2007). 107 Cantino P D & Abu Asab M S A new look at the enigmatic genus Wenchengia (Labiatae) Taxon 42 339 344 (1993) 106. Cantino, P. D. et al. Towards a phylogenetic nomenclature of Tracheophyta. Taxon 56, 822 846 (2007). 107. Cantino, P. D. & Abu-Asab, M. S. A new look at the enigmatic genus Wenchengia (Labiatae). Taxon 42, 339–344 (1993). p y g p y 107. Cantino, P. D. & Abu-Asab, M. S. A new look at the enigmatic genus Wenchengia (Labiatae). Taxon 42, 33 Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 17 www.nature.com/scientificreports/ 08. Abu-Asab, M. S. & Cantino, P. D. Systematic implications of pollen morphology in subfamilies Lamioideae and Pogostemonoideae (Labiatae). Ann. Missouri Bot. Gard. 81, 653–686 (1994).l ( ) , ( ) 109. Hsieh, T. H. & Huang, T. C. Notes on the flora of Taiwan (20)—Scutellaria (Lamiaceae) in Taiwan. Taiwania 40, 35–56 (1995). gl 110. Raj, B. A contribution to the pollen morphology of Verbenaceae. Rev. Palaeobot. Palynol. 39, 343–422 (1983). 111. Ma, Z. H., Bramley, G. L. C. & Zhang, D. X. Pollen morphology of Callicarpa L. (Lamiaceae) from China and its syste implication. Plant Syst. Evol. 302, 67–88 (2015).i p y 112. D’Arcy, W. G. Jacquin names, some notes on their typification. Taxon 19(4), 554–560 (1970). i 113. Wiens, J. J. Can incomplete taxa rescue phylogenetic analyses from long branch attraction? Syst. Biol. 54, 731–742 (2005). Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepihi Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Li, B. et al. A large-scale chloroplast phylogeny of the Lamiaceae sheds new light on its ubfamilial classification. Sci. Rep. 6, 34343; doi: 10.1038/srep34343 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports \| 6:34343 \| DOI: 10.1038/srep34343 18
https://openalex.org/W2223071651	https://seer.ufrgs.br/anos90/article/download/6359/3810	Portuguese	null	A Carta Niemeyer de 1846 e as condições de leitura dos produtos cartográficos	Anos 90	2,004	cc-by	7,267	Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto é Doutor em História Social pela UFRJ. Renato Amado Peixoto é Doutor em História Social pela UFRJ. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura dos produtos cartográficos Renato Amado Peixoto* Resumo. A Carta Niemeyer de 1846 foi a primeira Carta Geral do Brasil e, por meio desta, pretendemos discorrer sobre o emprego e as condições de leitura dos produtos cartográficos pelos historiadores, procurando, ao mesmo tempo, demarcar tanto seus limites quanto suas possibilidades. Palavras-chave: Carta Niemeyer (1846). Produtos cartográficos. Este artigo é um pequeno estudo acerca da Carta Niemeyer de 1846, a primeira Carta Geral brasileira produzida no século XIX, a qual se insere no esforço de construção historiográfica do espaço nacional, incorporada que foi a um projeto de nação construído em A Carta Niemeyer de 1846 e as condições de leitura... torno do Instituto Histórico e Geográfico Brasileiro – IHGB.1 Utilizando tal enfoque, pretendemos discorrer sobre o emprego e as condições de leitura dos produtos cartográficos pelos historiadores, procurando, ao mesmo tempo, demarcar tanto seus limites quanto suas possibilidades. 300 J. B. Harley foi o único autor a propor uma leitura dos produtos cartográficos capaz de ultrapassar os métodos e a interpretação costumeira dos historiadores da cartografia, que visavam, segundo esse autor, apenas investigar e catalogar os mapas segundo suas características técnicas e de produção. Tal atitude, segundo Harley, refletiria a adesão de seus cultores a um “positivismo cartográfico” que deveria ser confrontado e substituído por uma interpretação baseada, por sua vez, numa teoria iconológica e semiológica da natureza dos mapas (Harley, 2001a, 2001b). Para esse fim, Harley proporia a utilização dos conceitos anteriormente desenvolvidos por Erwin Panofsky (1976) para o estudo dos níveis dos temas ou significados na arte, visando, com estes, identificar através dos elementos simbólicos e estruturais dos mapas certas disposições qualificadas como “eminentemente retóricas”, as quais seriam capazes de explicitar relações de “Poder e Saber”, conforme a definição foucaultiana, bem como certos condicionamentos sociais.2 Ainda que reconheçamos a pertinência da teorização de Harley, acreditamos que, por conta da grande abertura e universalidade de seus conceitos, esta deva ter seu uso condicionado a análises e enfoques que, por sua vez, devam estar orientados e direcionados por um método que permita perscrutar o símbolo a partir de uma pesquisa do contexto que envolve a composição cartográfica, entendida aqui como um ato da representação que objetiva a Vontade de certos indivíduos ou grupos. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura dos produtos cartográficos Em nosso entender, esse ato de representação está conectado a propósitos, conveniências e circunstâncias que, para serem alcançados, demandariam tanto a constituição de certas mecânicas de produção, quanto a consecução de certos processos de escolha, cuja compreensão permitiria a leitura dos significados dos elementos e das estruturas técnicas do mapa como participantes de um processo criativo, a composição Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto Renato Amado Peixoto cartográfica, uma vez que sua efetivação está conectada diretamente à objetivação do ato de representação. Portanto, o estudo da composição do mapa pode ser tão ou mais significante quanto a interpretação do mapa em si.3 Esse método permite também trabalharem-se os textos referentes ao esforço da composição cartográfica relacionando-os com seus próprios produtos, o que possibilita leituras que não estão diretamente relacionadas com a utilização do mapa ou com os efeitos de sua divulgação, mas com um contexto partilhado pelas dinâmicas da mecânica de produção ou pelos processos de escolha, o que possibilita ao historiador, por exemplo, inferir a episteme relativa a um determinado período, grupo ou lugar, objetivo também perseguido por Harley (2001d, p.87- 88), ainda que mais pontualmente. 301 Finalmente, em relação às intenções restritas deste estudo, falta- nos definir, utilizando os argumentos anteriores, que, se o mesmo ato de representação está relacionado a determinados propósitos, conveniências e circunstâncias, insertos em determinada condição, esse ato objetiva-se através da competição ou pelo ajustamento a outros atos também objetivados pela motivação, o que, por sua vez, leva a estabelecer, para o historiador, novos lugares para a leitura de contextos e referências (v. Schopenhauer, 2001, § 56-57, 2003, cap. 2). ) A partir de Schopenhauer, podemos compreender essa objetivação da Vontade como uma disputa entre os indivíduos que visam expressar suas Idéias por meio de sua materialização. Contudo, como essa matéria será disputada com o mesmo fim por outros indivíduos, todos tenderão continuamente a usurpá-la, possuindo- a, cada um deles, apenas na medida do que puderam tomar dos outros: constituir-se-á assim, em torno do ato de representação, uma guerra eterna de vida ou de morte, quando o surgimento de obstáculos e impedimentos à objetivação da Vontade se consubstanciará no indivíduo através do sofrimento e da insatisfação. A Carta Niemeyer de 1846 e as condições de leitura dos produtos cartográficos Portanto, o ato de representação dá-se em meio a um competição contínua pela expressão da Idéia, interessando e emocionando a Vontade, daí relacionar-se pela sua satisfação com determinados Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... propósitos, conveniências, eventos e circunstâncias. Por conseguinte, esse mesmo ato da vontade será objetivado ainda que ao custo de sua transformação e do seu ajustamento a outros atos da vontade inclusive alheios e vinculados a outras motivações, não sem lançar o indivíduo novamente no sofrimento e na insatisfação que, por sua vez, o conduzirão a novos atos de vontade, no estado que denominamos de luta de representações (v. Schopenhauer, 2001, § 56-57, 2003, cap. 2). 302 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A cartografia do século XIX e sua leitura O estudo da Carta Niemeyer de 1846 será constituído sobre o método anterior, mas utilizando a premissa, a qual a exiguidade deste trabalho não nos permite desenvolver, de que o esforço da construção do Estado nacional e da produção de sua representação cartográfica valeu-se do material e da estrutura dos produtos cartográficos anteriores, adaptando-os à narração de seu próprio passado e em prol de seus objetivos, visando assim estabelecer uma legitimidade narrativa, por meio da qual o espaço foi sucessivamente apagado e reescrito em torno de um novo eixo de sintaxe: a Ordem e a Civilização (v. Peixoto, 2003, 2005). Ainda é necessário explicar que, durante os séculos XVII e XVIII, a cartografia constituiu-se numa escrita coletiva por excelência, dotada de práticas diversas e complexas, tornando necessário que a leitura do processo de composição dos mapas seja feito através da apreensão de estratégias e táticas que incluem tanto o agenciamento das técnicas e das condições da escrita, quanto a distribuição e atribuição de tarefas. Por conta dessas características, a cartografia tornou-se o lugar por excelência de inscrição da narração territorial, o que nos leva a ter que analisar também os processos de escolha, produção, reprodução e divulgação da cartografia, ou seja, investigar a socialidade dessa escrita tendo em vista que seu sujeito é também um sistema de relações entre seus diversos estratos, compostos pela recepção, compreensão, interação e transformação, o que denominaremos de teatro da narrativa.4 Nesse sentido, entendemos ser necessário distinguir, na Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto Renato Amado Peixoto leitura dos mapas, a existência simultânea de dois processos, um externo, relativo às relações com as Instituições e o Estado, e outro interno, que diz respeito à natureza das práticas e procedimentos cartográficos, ou seja, das classificações, generalizações, hierarquizações, divisões de trabalho e formalização das decisões. 303 Esses dois processos simultâneos distinguir-se-iam do que Harley definiu como poder interno e poder externo, entendidos por esse autor como a contraposição de uma instância de poder local e descentralizado a uma outra, centralizada e concentrada. Para Harley, a convivência entre essas duas instâncias faria parte das relações de poder que penetrariam os interstícios da prática e da representação cartográfica, permitindo assim com que os mapas pudessem ser lidos como textos que legitimariam a teorização Poder-Saber de Foucault (Harley, 2001c, p.111-113). Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A cartografia do século XIX e sua leitura Contudo, entendemos em nossa idéia de processo interno e processo externo, anteriormente expressada, que, além das relações apontadas por Harley, a construção da representação esteja sujeita ainda a ser modificada, alterada ou limitada por circunstâncias inerentes às propriedades e características das técnicas e procedimentos, devendo-se estender ainda essa impressão às leituras daí resultantes. Nesse sentido, deve-se salientar que o agenciamento das técnicas faz parte de um processo de escolhas que não é apenas subjetivo, mas que também constitui-se num procedimento da representação da forma, ligado às estratégias e táticas dos operadores das representações. Estas, por sua vez, estão sujeitas ainda às capacidades técnicas ou operacionais dos últimos e mesmo às finalidades da representação. No caso da apropriação da cartografia por parte da histo- riografia dos séculos XIX e XX, adiantaremos que as finalidades operacionais da narrativa ultrapassaram os procedimentos técnicos dos mapas, fazendo com que o agenciamento das técnicas tornasse- se um medium para a entrada em cena do objeto no mundo da representação. Assim, esse medium agenciado no esforço da repre- sentação do espaço nacional constituiu, ele mesmo, parte dessa operação, transformando a objetivação da representação do espaço esboçada através do debate realizado no teatro da narrativa, ou, Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... utilizando a comparação da Retórica com a representação feita por Schopenhauer, o medium pode constituir o objeto através de uma “dissimulação” de sua forma, uma vez que o objeto é a representação do sujeito mesmo (v. Schopenhauer, 2001, § 47, 2003, p.48-49). Em nossa idéia de processo externo e processo interno, contudo, o medium, seja este uma técnica, seja a Retórica, primeiramente é entendido como condicionado, na medida em que, através do processo externo, a expressão da Idéia compreendeu objeto e sujeito de maneira igual. Em segundo lugar, o medium também é entendido como condicionante, ao certas características do processo interno, seja sua constituição autônoma, sejam suas limitações técnicas, imporem, à expressão da Idéia do processo externo, restrições à sua representação pura, constituindo-a como uma objetividade imperfeita da Vontade, e que, em tese, devesse ser complementada ou substituída por outras representações. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A cartografia do século XIX e sua leitura 304 304 Finalmente em relação ao medium, e novamente remetendo ao caso da apropriação historiográfica da cartografia, se as táticas ou estratégias dos operadores exigirem um sacrifício intencional da forma, ou seja, se as finalidades operacionais ultrapassarem as condições técnicas, pode produzir-se, através do medium, uma alteração do objeto não prevista pelos operadores, constituindo-se essa alteração do objeto, ela mesma, como uma representação mais ou menos independente da objetivação da Vontade, ou no caso, do espaço em produção. Assim, o medium é entendido, em nosso método, como um facilitador da apreensão da Idéia pelos outros, e essa apreensão da Idéia será condicionada pela natureza ou característica do medium e pelo gênio do operador.5 Em respeito à importância do medium para a representação, podemos citar o adendo de Schopenhauer à célebre discussão sobre a razão de não se representar o grito do personagem ferido no grupo escultural de Laocoonte. Enquanto Winckelmann e Lessing atribuíram tal característica, respectivamente, ao estoicismo do personagem ou à incompatibilidade da beleza com a dor, para Schopenhauer a ação de gritar não fora representada “pela simples razão de que o grito é inteiramente rebelde aos meios de imitação Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto da escultura”. Portanto, para Schopenhauer, era impossível tirar do mármore um Laocoonte a gritar, entendendo, assim, existirem limites para a representação, os quais estariam impressos nas possibilidades mesmas do medium (Schopenhauer, 2001, § 46). Portanto, ainda que a apropriação da cartografia pela narrativa historiográfica fosse determinada pela disponibilidade dos objetos cartográficos e derivasse, sobretudo, do esforço do Estado, a inscrição da representação do espaço nacional fez-se no cruzamento de diferentes processos externos e processos internos, com suas dissimulações e alterações, proporcionando distintas leituras do espaço nacional. Estas, por sua vez, ocasionaram a subseqüente necessidade de os operadores da narrativa fazê-las convergir para uma norma da representação cartográfica, a qual se consubstanciaria nas iniciativas visando à composição de uma Carta Geral brasileira. 305 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 O medium cartográfico nos séculos XVII e XVIII Até o século XIX, o método usual para a reprodução de mapas e de atlas era o da gravação em cobre: os mapas manuscritos tinham seus detalhes copiados para uma placa desse material, na qual eram gravados em alto-relevo, gerando-se assim uma matriz de impressão passível de receber alterações e capaz de permitir seguidas reimpressões. Nesse sentido, estima-se que uma matriz de cobre bem cuidada e que recebesse uma manutenção regular do traçado de seu relevo podia ser utilizada até três mil vezes, possuindo comumente uma durabilidade capaz de ultrapassar a centena de anos (Verner, 1975, p.72). Entretanto, a gravação em cobre era um processo caro, trabalhoso e altamente especializado e, por conta dessas caracte- rísticas, o processo cartográfico consolidou-se, nos séculos XVII e XVIII, apenas onde o Estado fosse capaz de arcar com seus custos ou onde existisse um mercado capaz de atrair empreen-dimentos particulares que possibilitassem, sobretudo, a manutenção dos me- lhores profissionais. Nesse período, foi estabelecida uma nova tradição no processo cartográfico, com uma separação e uma estandardização rigorosa Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... das tarefas entre astrônomos, desenhistas, gravadores e impressores, o que consolidou o controle do processo interno nas mãos dos editores (o que pode ser exemplificado, inclusive, através da prevalência do anonimato no processo cartográfico) (Harley, 2001c, p.113-115). Contudo, alguns cartógrafos, como Gerhard Mercator, John Thornton e John Arrowsmith foram capazes de dominar todos as instâncias do processo cartográfico, estabelecendo-se privativamente e disputando o mercado de mapas e atlas com trabalhos de sua autoria (Verner, 1975, p.70). Portanto, uma das principais características da cartografia anterior ao século XIX é a existência de diferentes centros fora do controle direto do processo externo, capazes de produzir em escala e em disputa pelo controle de um mercado, em busca de uma lucratividade que se devia ao fato de os produtos cartográficos não serem apenas utilizados como fonte de informação para o Estado ou para o investidor, mas também como estímulos de sociabilidade e artigos de uma cultura de consumo que se estabeleceram no período. Tais eventos foram impulsionados pelas transformações culturais decorrentes da difusão da tipografia e das notícias das viagens transatlânticas, popularizadas pelas corografias e narrações dos viajantes (v. Mukerji, 1983a, 1983b, p.30-130). Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 O medium cartográfico nos séculos XVII e XVIII O desenvolvimento da gravação em cobre foi decisivo para o estabelecimento das condições desse novo mercado, propiciando que a cartografia se tornasse, durante o século XIX, parte mesmo da cultura material, com seus produtos circulando sob as mais variadas formas, tanto como atlas e mapas de diversos tamanhos, quanto como elemento decorativo em utensílios e vestimentas. Contudo, essa popularização dos produtos cartográficos e corográficos, que compunham uma cultura de elites até o século XVIII, somente se tornou possível pela apropriação, nas estratégias dos processos internos, de uma nova técnica desenvolvida e divulgada nas primeiras décadas do século XIX: a litografia. Renato Amado Peixoto Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A litografia e a cartografia no Brasil A técnica litográfica consistia na escrita direta sobre uma matriz de pedra calcária ou zinco ou no transporte dessa escrita para a pedra através de uma folha especial, quando então se utilizava um processo químico que tornava a superfície capaz de permitir sucessivas impressões. Além de tornar a composição dos mapas mais rápida, pois exigia uma menor especialização de tarefas, ao eliminar, por exemplo, a obrigação de que esses fossem desenhados em reverso como na gravação em cobre, a litografia também possibilitou uma diminuição acentuada dos custos materiais na cartografia. Essas características tornaram possível, no século XIX, disponibilizarem- se os produtos cartográficos a um público imensamente maior e mais diversificado que nos séculos anteriores, ao mesmo tempo em que permitiriam que países sem tradição de produção cartográfica em escala, como era o caso de Portugal e depois do Brasil, pudessem desenvolver uma incipiente produção cartográfica em escala. 307 A criação do Arquivo Militar, já no mesmo ano da chegada da Corte ao Brasil, serve para aferir a existência de uma percepção, no bojo da transferência do Estado português, de que a produção cartográfica em escala poderia coadjuvar a ação do Estado, trazendo vantagens administrativas e servindo como um instrumento prático para a centralização da autoridade. Nesse sentido, essa instituição teria a função de centralizar a guarda, a organização e a classificação dos produtos cartográficos, para que fosse possível então, utilizando- se os critérios da utilidade e da necessidade administrativa, escolher- se o material a ser vulgarizado. O principal objetivo dessa iniciativa foi o de recolher todos as cartas, os mapas topográficos e os planos iconográficos trazidos de Portugal para que fossem juntados aos que se encontravam dispersos no Brasil entre várias repartições, acabando-se assim com a descentralização documental que imperava até então nas secretarias de Estado portuguesas. Entretanto, essa primeira iniciativa de centralização cartográfica no Brasil estaria dada ao fracasso por dois motivos. Primeiro, porque a antiga tradição de descentralização seria Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... paulatinamente retomada, sendo que, durante o Segundo Reinado, se constituiriam outros dois grandes arquivos cartográficos ao lado do Arquivo Militar, um na Secretaria de Estrangeiros e outro na de Obras Públicas. Em segundo lugar, grande parte da documentação que fora reunida no Arquivo Militar retornou a Portugal junto com D. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A litografia e a cartografia no Brasil João VI em 1821, sem que se distinguisse critério algum nesse repatriamento, o que tanto acarretou a permanência no Brasil de muitos produtos cartográficos relativos a Portugal e seus domínios, quanto a ida para Portugal de muito do que fora produzido sobre o Brasil. Esse problema somente seria sanado em 1867 com uma permuta documental efetuada pela Comissão Investigadora de Mapas e Memórias Concernentes ao Brasil, negociada e acompanhada em Portugal diretamente por Duarte da Ponte Ribeiro, que também foi o responsável pela seleção desses documentos nos arquivos dos dois países. Embora se pensasse, quando da criação do Arquivo Militar, em utilizar a gravação em cobre na produção cartográfica em escala, as vantagens da litografia tornar-se-iam óbvias, tanto para o Estado português quanto para seu sucessor, a partir da divulgação dessa técnica no final da segunda década do século XIX. Essa opção consolidou-se na prática com a criação, em 1825, da Oficina Litográfica do Exército, quando se importaria todo o material necessário à sua operação junto com dois técnicos estrangeiros responsáveis por sua utilização, os quais deveriam atuar também como professores junto a um corpo de aprendizes composto por soldados do Exército. Ainda que, com essas iniciativas, o Estado buscasse resguardar para si o controle da vulgarização dos mapas, não foi possível consolidar, junto ao processo externo, a centralização da produção cartográfica, uma vez que, em Portugal, esse processo não havia se transformado em consonância com as mudanças no processo interno que acompanharam o desenvolvimento da reprodução em escala na Europa nos séculos anteriores, ou seja, através de uma especialização e uma estandardização das tarefas cartográficas. Nesse sentido, preservaram-se, no Brasil, as condições tecnológicas e culturais Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto Renato Amado Peixoto herdadas de Portugal, as quais impuseram, ao processo de produção, a composição cartográfica manuscrita, com suas características de individualização, concentração em setores determinados, sigilo e repetição de padrões, em que cada cartógrafo era, acima de tudo, o membro de uma escola e um transmissor de padrões estabelecidos.5 Em conseqüência, a parte mais representativa da produção cartográfica em escala no Brasil durante o século XIX ou foi uma reprodução direta do manuscrito, ou foi uma composição sob as técnicas da reprodução manuscrita, ou seja, submeteu-se o medium litográfico às regras, às limitações e aos condicionamentos culturais do medium manuscrito. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A litografia e a cartografia no Brasil 309 Por outro lado, o controle do processo interno da produção cartográfica pelo processo externo seria dificultado pela constante defecção dos quadros da Oficina para a atividade privada, uma vez que o custo e a adaptabilidade da técnica litográfica a outras tarefas tornavam esse ofício muito lucrativo. Mesmo assim, alguns poucos profissionais bastante qualificados fizeram parte dos quadros da Oficina, como Pedro Torquato Xavier de Brito, autor da redução da Carta do Império de 1856 e Carlos Abeleé, que produziu a Coleção dos figurinos dos uniformes dos corpos do Exército, significativa como demonstração do controle, pela Oficina, da técnica de impressão litográfica em cores, a chamada cromolitografia (Brito, s./d., 1870). Embora criticados, esses profissionais seriam responsáveis pelas reproduções litográficas de bom nível técnico, como, por exemplo, as cartas dos rios Uruguai, Içá e Javari e os mapas provinciais do Rio Grande do Sul, Santa Catarina, Rio de Janeiro, Mato Grosso, Sergipe, Piauí, Ceará, Espírito Santo, Minas Gerais, Maranhão e Paraná. Cabe salientar, entretanto, algumas cifras em relação à Oficina: em primeiro lugar, os mapas e cartas compuseram apenas uma parte muito restrita de sua produção, uma vez que somente cerca de 3% do acervo do Arquivo Militar, no século XIX, era composto por aqueles itens, sendo o restante integrado em pouco mais de 90% por plantas e projetos. Em segundo lugar, pode-se observar, nesse rol, que a participação de documentos anteriores ao século XIX é minoritária, compondo apenas cerca de 10% do total do mesmo Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... acervo. Assim, conclui-se que, no século XIX, embora a produção cartográfica do Exército tenha sido importante, compreendida enquanto tal o somatório dos esforços de seus oficiais engenheiros, do Arquivo Militar e da Oficina Litográfica, ela se concentrou mais na elaboração de plantas e projetos. 310 Em terceiro lugar, a produção do Exército apresentou mais atividade entre 1850 e 1889, com seu apogeu entre 1860 e 1889, data a partir da qual ela foi dividida por províncias, decrescendo entre cinco e até sete vezes. Portanto, dada a natureza dessa produção e se entendermos que sua origem, a criação do Arquivo Militar, foi a necessidade de o Estado utilizar diretamente a produção cartográfica no esforço de governo, podemos deduzir que os seus objetos concentrassem os interesses da administração e os esforços para a centralização da autoridade. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A litografia e a cartografia no Brasil Em defesa dessa tese, observe-se que as províncias do Rio de Janeiro e do Rio Grande do Sul foram o foco da produção cartográfica do Exército, correspondendo, respecti- vamente, por 26% e 16% de todos os projetos e plantas, seguidas de longe pelas províncias da Bahia e do Pará, com 9%.7 Em quarto lugar, constata-se que o esforço de composição e da produção em escala de vários dos mapas provinciais escapou do controle direto do processo externo e passou às mãos de particulares, como, por exemplo, no caso do Mapa da Província do Rio de Janeiro, que foi elaborado por Pedro de Alcântara Bellegarde e Conrado Jacob de Niemeyer em 1863, e dos mapas das províncias do Paraná, Espírito Santo e Santa Catarina, os quais foram impressos pelo Imperial Instituto Artístico, ou, ainda, de diversos outros mapas que comprovam a idéia de que a lucratividade do mercado litográfico permitiu que a iniciativa particular se dedicasse também à impressão e ao comércio de vários tipos de mapas, como, por exemplo, a Planta da Cidade do Rio de Janeiro, produzida por Steinmann em 1831, e o Mapa Geral do Império do Brasil, elaborado por J. H. Leonhart em 1851. Portanto, se relacionarmos esses exemplos com as cifras anteriormente citadas e os problemas acerca da manutenção dos quadros do Arquivo Militar, confirma-se no Brasil tanto a tradição Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto Renato Amado Peixoto européia de descentralização da produção cartográfica de escala, como a constituição de um mercado capaz de suportar uma produção litográfica independente e em contato com o exterior, com a subseqüente necessidade de o processo externo adaptar-se às características do processo interno, o que resultou no esvaziamento das atribuições e funções do Arquivo Militar. 311 A Carta de Niemeyer de 1846 Na década de 1840, a consolidação da discussão do espaço nacional em teatros da narrativa bem definidos em torno do IHGB e da Secretaria dos Negócios Estrangeiros, a descentralização do processo externo e da produção cartográfica em escala e o esvaziamento das funções do Arquivo Militar fizeram com que a primeira Carta Geral do Brasil não nascesse a partir de uma iniciativa do Estado, mas de uma contribuição para o debate da narrativa territorial no IHGB. Construída por Conrado Jacob de Niemeyer durante os anos de 1842 a 1846, a Carta Corográfica do Império do Brasil estabeleceu padrões técnicos e estéticos que seriam endossados pelas Cartas Gerais posteriores e mapas parciais do território, condicionando assim o processo externo às interpretações e limitações do processo interno. Nesse sentido, a composição da Carta de 1846 envolveu um procedimento de escolha do padrão técnico que pode ser caracte- rizado em três níveis de apreensões do processo interno: o primeiro, do geral, relacionado à inserção no universo conhecido das representações cartográficas; o segundo, do particular, relacionado à escolha do repertório das tradições das experimentações do território; o terceiro, do conceitual, relacionado à divulgação e à consolidação das formas percebidas e extraídas da intuição.8 Assim, Niemeyer procurou inicialmente basear sua repre- sentação do território brasileiro sobre o que chamou de Mapa Geral, ou seja, o produto resultante da reunião dos traçados de duas cartas estrangeiras, a Carta da América Meridional, da casa editorial Arrowsmith, e a Carta da Costa Brasileira, do Almirante Roussin. Em seguida, esse Mapa Geral foi modificado e complementado Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... através da consulta a diversos mapas, roteiros, memórias e descrições, sendo que, entre este último tipo de corografias textuais, Niemeyer utilizou especialmente os trabalhos de Cerqueira e Silva (1883), Cunha Mattos (1874) e Aires de Casal (1943), no caso, segundo os trabalhos cartográficos acreditados no debate do IHGB e pela remissão ao cânone ali consagrado. Finalmente, os limites nacionais foram inscritos sobre o produto resultante segundo o Programa Geográfico, de Pinheiro, e a divisão das províncias, de acordo com a Corografia Brasílica, de Aires de Casal. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta de Niemeyer de 1846 312 Já o processo de escolha do padrão estético derivou da decisão de se compor o Mapa Geral a partir da redução e transformação da sua base de dados a uma escala (1:3.000.000) que viabilizasse a composição da Carta Geral em quatro folhas iguais, de acordo com a maior capacidade de impressão da litografia mais bem aparelhada no Brasil naquele momento, a Litografia Rensburg, possibilitando assim que a Carta atingisse o tamanho de 1,50m de altura por 1,50m de largura. A decisão de orientar todo o projeto cartográfico da Carta de 1846 pelo tamanho da maior folha que fosse possível imprimir foi tomada por Niemeyer em função de três objetivos: primeiro, tornar certos detalhes distinguíveis em relação a outros e “dignos de atenção”, especialmente aqueles relativos aos limites com o Paraguai; segundo, diminuir o problema dos erros, através do maior dimensionamento dos elementos geográficos, especialmente da hidrografia; terceiro, equiparar a representação cartográfica do espaço nacional às cartas de grande dimensão impressas na Europa (Instituto..., 1844, 1846; Carta..., 1924). Quanto ao último objetivo, o modelo para Niemeyer eram justamente as grandes cartas gravadas pela casa editorial Arrowsmith, as quais chegavam a medir até dois metros de altura por um metro e quarenta de largura.9 Essas cartas eram também impressas em várias folhas e juntadas para formar o produto final, o qual se destinava a ser exposto emoldurado em grandes paredes, geralmente em órgãos públicos e escolas, diferente dos demais mapas, que simplesmente eram enrolados após a consulta. Portanto, o padrão estético inaugurado por Niemeyer buscava não apenas formatar e inserir o Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto Renato Amado Peixoto Estado brasileiro no espaço, mas ainda construir sua presença, centralidade e monumentalidade através da imposição de sua representação, produzindo um mediador que visava interagir nas relações do indivíduo com o meio social e que lhe seria imposto por um ordenamento das próprias relações entre ele e o Estado.10 As funções específicas desse mediador derivam das transformações culturais e tecnológicas do século XIX, que aumentaram a distinção entre criação e produção11 ao dinamizar os processos de construção e operação da representação, possibilitando a sua constituição enquanto um produto do artifício, ou seja, como uma representação tornada ilimitadamente disponível e que adquiriu novas funções, justamente por essa característica adequar-se às estratégias do processo externo. 313 Finalmente, esse mediador é elaborado sobre as estratégias e táticas desenvolvidas na relação entre o processo externo e o processo interno, quando recebe a expressão de suas tensões através da inscrição ou da negação da inscrição12 de “alegorias” ou “representações simbólicas” como elementos estruturais dos mapas, aos quais também corresponde o condicionamento de sua criação e construção histórica.13 Assim, acreditamos que a interpretação semiológica e iconológica não deva ser utilizada isoladamente, mas entronizada em um método que ultrapasse os aspectos imediatos do mapa e dê conta dos processos de objetivação do ato de representação, sendo ainda capaz de permitir a utilização dos recursos levantados pela História da Cartografia tradicional. Em razão disso, sugerimos que a interpretação semiológica e iconológica dos produtos cartográficos pode se basear nos significados percebidos através do estudo das relações desenvolvidas entre o processo interno e o processo externo, bem como da compreensão de sua inserção no problema geral da forma cartográfica. Como exemplos desse método, apontaremos três aspectos retirados da mesma Carta Niemeyer. Em primeiro lugar, nesse mapa, o meridiano que passa pela cidade do Rio de Janeiro é utilizado como origem de todo o sistema de coordenadas, distinguindo-se dos que eram utilizados usualmente, a saber, os meridianos de Paris Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... ou de Londres. Essa opção pode ser compreendida a partir da inserção da Carta Niemeyer no debate então travado no IHGB sobre a construção da Nação e da Nacionalidade, responsável também por selecionar, disponibilizar e legitimar os textos corográficos a partir dos quais se completou a composição do espaço inscrito na Carta Niemeyer. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Conclusão Acreditamos que a compreensão dos limites e das possibilidades dos produtos cartográficos pode resultar na constituição destes enquanto recursos de grande valia para o ofício do historiador. Portanto, é necessário primeiramente compreender que a transformação dos processos e das práticas cartográficas constitui-se num problema em si mesmo, o qual, muitas vezes, não corresponde às intenções do pesquisador. Em segundo lugar, entendemos que a leitura dos mapas deve ser feita através da investigação das estratégias e táticas inerentes à sua composição, as quais incluem tanto o agenciamento das técnicas e das condições da escrita quanto a distribuição e atribuição de tarefas. Por último, entendemos que é necessário investigar a socialidade dessa composição tendo-se em vista que seu sujeito é também um sistema de relações entre os diversos estratos de sua escritura, e que sua escrita deve ser entendida no mesmo sentido. 315 Niemeyer Chart of 1846 and the reading conditions of cartographic materials Abstract. Niemeyer Chart, printed in 1846, was the first Brazilian General Chart and, through this, we intend to discuss the usage and reading conditions of cartographic products by historians, aiming at the same time, to define its limits and possibilities. Keywords: Niemeyer Chart (1846). Cartographic products. Renato Amado Peixoto 314 314 Em segundo lugar, o destaque dado à divisão provincial pela utilização do colorido quase que a equipara à divisão internacional. Esse destaque pode ser entendido tanto pela ênfase com que o autor que serviu de base à divisão provincial, Manoel Ayres de Casal, trata da questão, quanto pela sobrevivência da questão regional ainda na década de 1840. Em outras cartas da mesma época, era comum que mapas menores ou mesmo desenhos fossem dispostos dentro do mapa principal ou ao redor dele, mas, na Carta Niemeyer, as plantas das capitais das províncias do Rio Grande do Sul, São Paulo, Rio de Janeiro, Minas Gerais, Bahia, Pernambuco, Maranhão e Pará e a planta da Corte verdadeiramente emolduram o Mapa do Brasil, evidenciando, portanto, a sobrevivência da questão regional em meio à construção do nacional. Finalmente, utilizando o mesmo método, podemos com- preender as distintas implicações sociais e políticas da inscrição do espaço através do estudo dos elementos utilizados para descrever o espaço e de sua comparação às representações que se fizeram em torno da construção da Nação, especialmente se considerarmos o binômio civilização x barbárie, conforme idealizado por Ilmar R. de Mattos (1999). Nesse caso, a Carta Niemeyer é prolífica em exemplos dessa representação, como: “Gentio Jacundá tratável e que fala a língua geral”; “Sertão ainda desconhecido e sem cultura”; “Terrenos inteiramente desconhecidos e ocupados por diversas tribos de índios selvagens que embaraçam a navegação fluvial” e “Paritins, Andiras, Araras, Mundrucus e outras nações – Em grande parte domesticados”. Renato Amado Peixoto 1 Ver Guimarães (1988). Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 1 Ver Guimarães (1988). 2 No caso, Saber e Poder implicam-se mutuamente: não existiria relação de poder sem a constituição de um campo correlato de saber, assim como não existiria saber que não pressupusesse e constituísse relações de poder. Ver Harley (2001d, p.87, 2001e, p.37). 3 Em relação à teoria da representação e correlata objetivação da Vontade, ver Schopenhauer (2003, cap. 2). 4 A partir dos conceitos sugeridos por Derrida (2002, p.221-223) em sua leitura da obra de Freud. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... 5 O gênio é entendido, por Schopenhauer, como uma capacidade de conhecimento inata e que se encontra em diversos graus em todos os homens, o que pressupõe serem-lhe inerentes as habilidades da criatividade e do entendimento. Ver Schopenhauer (2003, p. 83-87). 6 A respeito da influência dos estilos e da transmissão de padrões na cartografia manuscrita, ver Marques (1989, p. 87-97). 316 7 Esses dados foram tabulados sobre a descrição pormenorizada do acervo que foi realizada por Cláudio Moreira Bento, pouco antes deste ser confiado ao Arquivo do Exército no Rio de Janeiro. Ver Bento (1985). 7 Esses dados foram tabulados sobre a descrição pormenorizada do acervo que foi realizada por Cláudio Moreira Bento, pouco antes deste ser confiado ao Arquivo do Exército no Rio de Janeiro. Ver Bento (1985). 8 Essa idéia origina-se da relação estabelecida por Schopenhauer entre a música, a realidade e os conceitos abstratos. Ver Schopenhauer (2001, §52). 8 Essa idéia origina-se da relação estabelecida por Schopenhauer entre a música, a realidade e os conceitos abstratos. Ver Schopenhauer (2001, §52). 9 Por exemplo, a carta de 1814 da América do Sul. Ver Arrowsmith (1814). 9 Por exemplo, a carta de 1814 da América do Sul. Ver Arrowsmith (1814). 10 Procuramos aqui adaptar a idéia de mediador de Abraham Moles (1986, p.12- 19), desenvolvida por esse autor para explicar as transformações da representação e de sua operação nas sociedades de consumo. 10 Procuramos aqui adaptar a idéia de mediador de Abraham Moles (1986, p.12- 19), desenvolvida por esse autor para explicar as transformações da representação e de sua operação nas sociedades de consumo. 11 Essa distinção pode ser pensada também a partir da teorização de Abraham Moles (1986, p.15-22), no sentido de que o processo de criação seria correlato à idéia de introdução, invenção e produção do ato de copiar, reproduzir, e que as transformações do século XIX teriam dinamizado a produção, substituindo a criação por uma cadeia operatória, mas entendendo-se esta como um desdobramento da operação da representação em vários níveis visando à reprodução em escala e não necessariamente como parte de um processo de alienação. Notas 1 Ver Guimarães (1988). 2 No caso, Saber e Poder implicam-se mutuamente: não existiria relação de poder sem a constituição de um campo correlato de saber, assim como não existiria saber que não pressupusesse e constituísse relações de poder. Ver Harley (2001d, p.87, 2001e, p.37). 3 Em relação à teoria da representação e correlata objetivação da Vontade, ver Schopenhauer (2003, cap. 2). 4 A partir dos conceitos sugeridos por Derrida (2002, p.221-223) em sua leitura da obra de Freud. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 1 Ver Guimarães (1988). 2 No caso, Saber e Poder implicam-se mutuamente: não existiria relação de poder sem a constituição de um campo correlato de saber, assim como não existiria saber que não pressupusesse e constituísse relações de poder. Ver Harley (2001d, p.87, 2001e, p.37). 3 Em relação à teoria da representação e correlata objetivação da Vontade, ver Schopenhauer (2003, cap. 2). 4 A partir dos conceitos sugeridos por Derrida (2002, p.221-223) em sua leitura da obra de Freud. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 A Carta Niemeyer de 1846 e as condições de leitura... 12 Essa idéia corresponde aproximadamente ao que Harley denomina de “Silêncios” (Silences): para esse autor, o espaço vazio nos mapas estaria ligado a um discurso político e à legitimidade de seu status, enquanto que, em nossa idéia da negação da inscrição, o “Silêncio” não corresponderia a um vazio, mas a um espaço preenchido por uma continuação ou um desdobramento daquele discurso. Ver (2001d, p. 99-100). 13 Arthur Schopenhauer (2001, § 50) identifica a historicidade das “alegorias” e “representações simbólicas” como parte mesmo do problema da compreensão da Representação. 13 Arthur Schopenhauer (2001, § 50) identifica a historicidade das “alegorias” e “representações simbólicas” como parte mesmo do problema da compreensão da Representação. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 ARROWSMITH, Aaron. South America. London: A. Arrowsmith, 1814. BELLEGARDE, Pedro de Alcântara; NIEMEYER, Conrado Jacob de. Carta Corográfica da Província do Rio de Janeiro (Relatório). Rio de Janeiro: Tipografia do Instituto Artístico, 1863. ARROWSMITH, Aaron. South America. London: A. Arrowsmith, 1814. Referências BELLEGARDE, Pedro de Alcântara; NIEMEYER, Conrado Jacob de. Carta Corográfica da Província do Rio de Janeiro (Relatório). Rio de Janeiro: Tipografia do Instituto Artístico, 1863. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto 2004 A Carta Niemeyer de 1846 e as condições de leitura... Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Renato Amado Peixoto BENTO, Cláudio Moreira. Cartografia histórica do Exército. Revista do Instituto Histórico e Geográfico Brasileiro, v. 41, abr./jun. 1985. BRITO, Pedro Torquato Xavier de. História da litografia. Rio de Janeiro: IHGB, s/d. Lata 26, Pasta 1. BRITO, Pedro Torquato Xavier de. Notícia acerca da introdução da arte litográfica e do estado de perfeição em que se acha a cartografia no Império do Brasil. Revista do Instituto Histórico e Geográfico Brasileiro, v. 41, tomo 33, parte II, 1870. 317 317 CARTA de Jacob de Niemeyer para o Visconde de São Leopoldo em 20/9/1843. In: PAUWELS, Geraldo José. Algumas notas sobre a gênese dos números para as áreas do Brasil e seus estados. Porto Alegre: Tipografia do Centro, 1924. p. 7-8. CASAL, Manoel Ayres de. Corografia brasílica ou relação histórico-geográfica do Brasil. São Paulo: E. e H. Laemmert, 1817; Edições Cultural, 1943. II v. DERRIDA, Jacques. Freud e a cena da escritura. In: ______. A escritura e a diferença. São Paulo: Perspectiva, 2002. GUIMARÃES, Manoel Luiz Salgado. Nação e civilização nos trópicos: o Instituto Histórico e Geográfico Brasileiro e o projeto de uma história nacional. Estudos Históricos, Rio de Janeiro, v.1, n.1, p. 5-27, 1988. HARLEY, J. B. Deconstructing the map. In: ______. The new nature of maps: essays in the History of Cartography. Baltimore: The John Hopkins University Press, 2001a. ______. Maps, knowledge and power. In: ______. ______. Baltimore: The John Hopkins University Press, 2001b. ______. Power and legitimation in the English geographical atlases of the eighteenth century In. Baltimore: The John Hopkins University Press, 2001c. ______. Silences and secrecy – The hidden agenda of cartography in early Europe. In. Baltimore: The John Hopkins University Press, 2001d. ______. Text and context in the interpretation in early maps. . In. Baltimore: The John Hopkins University Press, 2001e. INSTITUTO HISTÓRICO E GEOGRÁFICO BRASILEIRO. Carta de Conrado Jacob de Niemeyer ao IHGB oferecendo a Carta Corográfica do Império Brasileiro. Lata 510, Pasta 5, 1846. ______. Nota de Conrado Jacob de Niemeyer, dizendo estar quase pronta a carta corográfica do Império do Brasil. Lata 142, Pasta 49, 1844. MARQUES, Alfredo Pinheiro. The dating of the oldest Portuguese charts. Imago Mundi, 41, p. 87-97, 1989. MARQUES, Alfredo Pinheiro. The dating of the oldest Portuguese charts. Imago Mundi, 41, p. 87-97, 1989. Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. 2004 Anos 90, Porto Alegre, v. 11, n. 19/20, p.299-318, jan./dez. A Carta Niemeyer de 1846 e as condições de leitura... MATOS, Raimundo José da Cunha. Corografia histórica da Província de Goiás. Revista do Instituto Histórico e Geográfico Brasileiro, v. 37, tomo 48, 1874. MATTOS, Ilmar Rohloff. O tempo saquarema: a formação do Estado imperial. 4. ed. Rio de Janeiro: ACCESS, 1999. MOLES, Abraham. O kitsch. São Paulo: Perspectiva, 1986. 318 MUKERJI, Chandra. A new world picture: maps as capital goods for the modern world system. In: ______. From graven images: patterns of modern materialism. New York: Columbia University Press, 1983a. ______. Pictorial prints and the growth of consumerism: class and cosmopolitanism in early modern culture. In. New York: Columbia University Press, 1983b. PANOFSKY, Erwin. Iconografia e iconologia: uma introdução ao estudo da arte da Renascença. In: ______. Significado nas artes visuais. São Paulo: Perspectiva, 1976. PEIXOTO, Renato Amado. A máscara da medusa: a construção do espaço nacional brasileiro através das corografias e da cartografia no século XIX. 2005. Tese (Doutorado) – Universidade Federal do Rio de Janeiro, 2005. ______. Mapeando o vazio: as percepções do espaço nacional nas cartas gerais. In: SIMPÓSIO NACIONAL DE HISTÓRIA, XXII, São Paulo, 2003. Anais Eletrônicos do... São Paulo: ANPUH, 2003. 1 CD-ROM. SCHOPENHAUER, Arthur. A metafísica do belo. São Paulo: Editora Unesp, 2003. ______. O Mundo como vontade e representação. Rio de Janeiro: Contraponto, 2001. SILVA, Ignacio Accioli de Cerqueira e. Corografia paraense ou descrição física, histórica e política da Província do Grão-Pará. Salvador: Tipografia do Diário, 1833 VERNER, Coolie. Copperplate printing. In: WOODWARD, David. Five centuries of map printing. Chicago: University of Chicago Press, 1975.
https://openalex.org/W3203968579	https://talenta.usu.ac.id/jpi/article/download/2687/2040	Indonesian	null	KARAKTERISTIK MORFOLOGI UKURAN TUBUH KERBAU MURRAH DAN KERBAU RAWA DI BPTU SIBORONGBORONG	Jurnal Peternakan Integratif	2,013	cc-by	4,164	Gerli1, Hamdan2 dan Armyn Hakim Daulay2 1. Mahasiswa Program Studi Peternakan Fakultas Pertanian Universitas Sumatera Utara 2. Staf Pengajar Program Studi Peternakan Fakultas Pertanian Universitas Sumatera Utara J. Peternakan Integratif Vol. 1 No. 3 ;276-287 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 ABSTRACT One way to determine the phenotypic diversity of buffaloes is through morphometric observations on each type of buffalo in Indonesia. Morphometric identification can be done by comparing the size and shape of the body. This study aims to obtain information related phenotypic morphometric characteristic of the body Murrah buffalo and swamp buffalo by Principal Component Analysis. The research was conducted in BPTU Siborongborong Kabupaten Tapanuli Utara on Juli up to September 2012. The reseach used 68 swamp buffalo (7 males, 61 females) and 32 Murrah buffaloes (5 males, 27 females) using a survey method. The results statistical showed that the body sizes of Murrah bufallo had higher variances than that Swamp bufallo. Body measurements were showed highly significant differences (P<0,01) in body length , shoulder height, hip height and chest circum, and no significant differences (P>0,05) for chest width, chest depth and hip width. The results principal component analysis showed that the size of the breeds are identifier through shoulder height and the shape is characterized from the chest width. The groups of data between the Murrah bufallo and the Swamp bufallo were significantly different for the shape, while for size has no different. It is concluded that the Murrah bufallo and the Swamp bufallo were significantly different for the shape while has no different for size. Keywords: Murrah Buffalo, Swamp Bufallo, Morphometrics, Principal Component Analysis. Kata Kunci : Kerbau Murrah, Kerbau Rawa, Morfometrik, Analisis Komponen Utama. PENDAHULUAN Kerbau dapat berkembang baik dalam rentang kondisi agroekosistem yang luas dari daerah dengan kondisi yang basah sampai dengan kondisi yang kering (Hardjosubroto, 2006). Di Indonesia sebagian besar terdiri dari kerbau lumpur (swamp bufallo), namun telah muncul berbagai spesifikasi mengikuti anggroekosistem yang membentuknya (Siregar et al., 1997), sementara ada sekitar 5% kerbau sungai seperti Murrah di sekitar Medan (Murti, 2002). Pemanfaatan utama ternak kerbau sampai saat ini selain sumber daging juga merupakan ternak pekerja. Populasi kerbau di Indonesia pada tahun 2005 adalah 2.128.491 ekor, menurun menjadi 2.045.548 ekor pada tahun 2009, dimana sebagian besar kerbau dipelihara oleh peternak kecil dengan tingkat kepemilikan 2-3 ekor. Sementara data pemotongan pada tahun 2005 sekitar 163.848 dan pada tahun 2009 menjadi 166.380 ekor (Ditjennak, 2009). Kualitas kerbau Indonesia pada umumnya mengalami kemunduran, sebagai akibat penurunan mutu genetik dan faktor lain seperti menejemen pemeliharaan yang kurang tepat. Penurunan produktivitas selain dicerminkan dengan penurunan bobot badan sebagai akibat dari penurunan ukuran-ukuran linear permukaan tubuh kerbau, juga disebabkan faktor genetik karena upaya pemuliaan yang belum terarah. Pelestarian keragaman ternak diperlukan dalam upaya mempertahankan sifat-sifat khas yang dapat dimanfaatkan di masa mendatang. Salah satu cara penentuan keragaman fenotipik lokal Indonesia adalah dengan pengamatan morfometrik pada bangsa kerbau lokal Indonesia. Identifikasi morfometrik dilakukan dengan cara menentukan penciri ukuran dan bentuk pada masing-masing kerbau lokal berdasarkan Analisis Komponen Utama (AKU). Bentuk sangat dipengaruhi faktor genetik, sedangkan ukuran lebih dipengaruhi faktor lingkungan. Tujuan pemeliharaan kerbau juga turut mempengaruhi keragaman ukuran pada kerbau-kerbau yang ada di Indonesia. ABSTRAK Salah satu cara untuk menentukan keragaman fenotipik ternak kerbau adalah dengan pengamatan morfometrik pada setiap jenis kerbau di Indonesia. Identifikasi morfometrik dapat dilakukan dengan cara membandingkan ukuran dan bentuk tubuh. Penelitian ini bertujuan untuk memperoleh informasi fenotipik yang berhubungan dengan karakter morfometrik tubuh kerbau murrah dan kerbau rawa berdasarkan Analisis Komponen Utama. Penelitian dilaksanakan di BPTU Siborongborong Kabupaten Tapanuli Utara pada Juli sampai September 2012. Menggunakan 68 kerbau rawa (7 jantan, 61 betina) dan 32 kerbau murrah (5 jantan, 27 betina) dengan metode survei. Hasil analisis statistika menunjukkan ukuran-ukuran tubuh kerbau murrah lebih beragam dibandingkan kerbau rawa. Ukuran menunjukkan perbedaan sangat nyata (P<0,01) pada panjang badan, tinggi pundak, tinggi pingggul dan lingkar dada, dan tidak berbeda nyata (P>0,05) pada lebar dada, dalam dada dan lebar pinggul. Hasil analisis komponenen utama menunjukkan penciri ukuran pada kedua bangsa adalah tinggi pundak dan penciri bentuk adalah lebar dada. Kerumunan data pada kerbau murrah dan kerbau rawa terpisah pada bentuk, sedangkan tidak terpisah pada skor bentuk, sedangkan tidak terpisah pada skor ukuran. Kesimpulan pada penelitian ini adalah kerbau murrah dan kerbau rawa berbeda bentuk sedangkan ukuran sama. 276 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Alat Peralatan yang digunakan adalah tongkat ukur, pita ukur, buku dan alat tulis serta kamera digital. Komputer yang dilengkapi dengan Software statistik MINITAB® 16.2.1.0. sebagai alat bantu olah data. Metode Penelitian ini menggunakan metode survei dengan menggunakan data primer dan data sekunder. Data primer diperoleh dengan dengan pengamatan dan pengukuran langsung terhadap sampel. Pengambilan sampel dilakukan secara purposive sampling, yaitu sampel ditentukan berdasarkan kriteria mencapai umur dewasa tubuh. Data sekunder yang berhubungan dengan penelitian diperoleh dari Dinas Peternakan Sumatara Utara, Balai Pembibitan Ternak Unggul Babi dan Kerbau Sinur Siborong - Borong. Penggolongan umur ditentukan berdasarkan pemunculan tanduk dan penanggalan gigi seri dan diperkirakan sudah mencapai dewasa tubuh serta dari data recording yang ada. Bahan Bahan yang digunakan dalam penelitian ini adalah 100 ekor kerbau yang terdiri atas 68 kerbau rawa (7 jantan, 61 betina) dan 32 kerbau murrah (5 jantan, 27 betina) telah mencapai umur sesuai dengan kriteria yang telah akan dibagi berdasarkan tingkatan umur dan jenis kelamin. Bahan dan Alat Bahan dan Alat Lokasi dan Waktu Penelitian Penelitian dilaksanakan di BPTU Babi dan Kerbau Siborongborong Kabupaten Tapanuli Utara, Sumatera Utara pada Juli sampai September 2012. 277 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Parameter Penelitian Parameter (variabel) yang diamati dalam penelitian ini adalah lingkar dada, lebar dada, dalam dada, tinggi pundak, panjang badan, tinggi pinggul dan lebar pinggul dengan berat badan sebagai tambahan data deskriptif yang diukur mengunakan timbangan. 278 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Gambar 1. Metode pengukuran variabel-variabel linier permukaan tubuh kerbau yang diamati Gambar 1. Metode pengukuran variabel-variabel linier permukaan tubuh kerbau yang diamati Keterangan: Nomor 1-7 berurutan adalah : 1). Lingkar dada. 2). Lebar dada, 3). Dalam dada. 4). Tinggi pundak. 5). Panjang badan. 6). Tinggi pinggul. 7). Lebar pinggul. Bagian bagian tubuh kerbau yang diukur (dinyatakan dalam satuan cm) dan definisinya diuraikan debagai berikut: Bagian bagian tubuh kerbau yang diukur (dinyatakan dalam satuan cm) dan definisinya diuraikan debagai berikut: 1.Lingkar dada (X1) diukur melingkar tepat dibelakang scapula menggunakan pita ukur. 2.Lebar dada (X2) adalah jarak antara penjolan sendi bahu (os scapula) kiri dan kanan, diukur dengan pita ukur. 3.Dalam dada (X3) merupakan jarak antara titik tertinggi pundak dan tulang dada, diukur dengan menggunakan tongkat ukur. 4.Tinggi pundak (X4) jarak tertinggi pundak melalui belakang scapula tegak lurus ke tanah diukur dengan menggunakan tongkat ukur. 5.Panjang badan (X5) adalah garis lurus dari tepi tulang processus spinocus sampai dengan benjolan tulang lapis (os ischium), diukur dengan menggunakan tongkat ukur. 6.Tinggi pinggul (X6) adalah jarak tertingi pinggul secara tegak lurus ke tanah, diukur dengan menggunakan tongkat ukur. 7.Lebar pinggul (X7) diukur dengan tongkat ukur sebagai jarak lebar antara kedua sendi pinggul (Erdiansyah, 2008). 279 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Analisis Data Pengolahan data untuk mendapatkan gambaran dari ukuran tubuh dan bentuk tubuh kedua bangsa kerbau dilakukan dengan menggunakan analisis multivariat yaitu dengan menggunakan Principal Component Analysis atau Analisis Komponen Utama untuk mengetahui hubungan antar variabel dari bangsa kerbau dan digunakan sebagai upaya matematis untuk menyederhanakan variabel menjadi variabel baru, namun variabel baru masih tetap dapat menentukan sebagian besar informasi data asalnya.Karakteristik ukuran tubuh dilakukan dengan menghitung nilai rataan, simpangan baku (S), dan koefisien keragaman (KK) dari setiap sifat yang diamati dengan menggunakan Principal Component Analysis. 1 - n ) x - x( S 2 i   KK% = (100%) x s Keterangan : x = nilai rataan n = jumlah sampel yang diperoleh Xi = ukuran ke-i dari sifat x S = Simpangan baku KK = koefisien keragaman Data ukuran tubuh selanjutnya dianalisis dengan menggunakan Analisis Komponen Utama. Menurut Gaspersz (1992) pengolahan data dengan menggunakan Analisis Komponen Utama dilakukan dengan model matematika sebagai berikut: Keterangan : rangan : Yp = komponen utama ke-p a1p, a2p, ...anp = vektor ciri/vektor Eigen ke-1,...., n pada komponen utama ke-p X1, X2,...,Xn = peubah-peubah yang diamati yaitu: (tinggi pundak, tinggi pinggul, lebar pinggul, panjang badan, lingkar dada, dalam dada, lebar dada) = komponen utama ke-p a1p, a2p, ...anp = vektor ciri/vektor Eigen ke-1,...., n pada komponen utama ke-p 1p, 2p, np g , , p p p X1, X2,...,Xn = peubah-peubah yang diamati yaitu: (tinggi pundak, tinggi pinggul, lebar pinggul, panjang badan, lingkar dada, dalam dada, lebar dada) X1, X2,...,Xn = peubah-peubah yang diamati yaitu: (tinggi pundak, tinggi pinggul, lebar pinggul, panjang badan, lingkar dada, dalam dada, lebar dada) Everitt dan Dunn (1998) menyatakan bahwa komponen utama pertama dinyatakan sebagai vektor ukuran, sedangkan komponen utama kedua sebagai vektor bentuk. Keeratan hubungan antara peubah asal dan komponen utama dapat dilihat melalui besarnya koefisien korelasi antara peubah asal dan komponen utama itu. 280 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Rumus yang digunakan untuk mencari korelasi antara peubah asal dan komponen utama tertentu sebagai berikut: rx,y1= rij = (Gaspersz, 1992). rx,y1= rij = (Gaspersz, 1992). Keterangan: = koefisien korelasi = koefisien korelasi = vektor penciri/vektor Eigen ke-i pada komponen utama ke-j = akar dari nilai penciri/ nilai eigen pada komponen utama ke-j = simpangan baku dari variabel Xi = simpangan baku dari variabel Xi Selanjutnya skor komponen utama yang diperoleh dari persamaan ukuran tubuh disajikan dalam bentuk diagram kerumunan. Analisis Data Diagram kerumunan dibuat berdasarkan skor komponen utama pertama (skor ukuran) sebagai sumbu X dan skor komponen utama kedua (skor bentuk) sebagai sumbu Y; yang diperoleh berdasarkan persamaan ukuran dan bentuk. Perbedaan kerumunan data antara kedua bangsa kerbau yang diamati diperbandingkan pada diagram kerumunan. HASIL DAN PEMBAHASAN Deskriptif Statistik Ukuran Linier Permukaan Tubuh Kerbau Murrah dan Kerbau Rawa Deskriptif Statistik Ukuran Linier Permukaan Tubuh Kerbau Murrah dan Kerbau Rawa Ukuran-ukuran linear peubah ukuran tubuh kerbau murrah dan kerbau rawa yang diukur meliputi tinggi pundak, tinggi pinggul, lebar pinggul, panjang badan, lingkar dada, dalam dada, lebar dada dan penimbangan bobot badan yang dikelompokkan menurut umur dan jenis kelamin berbeda, tabel berikut menyajikan nilai rataan, simpangan baku dan koefisien keragaman pada masing – masing peubah yang diamati disajikan pada Tabel 1 dan Tabel 2. Rataan berat badan kerbau murrah jantan pada kelompok umur 2 – 3,5 tahun 258 kg dan pada kerbau rawa jantan 246 kg. Rawa jantan muda hasil pengamatan didapat tinggi pundak 115,5 cm, tinggi pinggul 117,5 cm, panjang badan 105,5 cm dan lingkar dada 182,5 cm, lebih rendah dibandingkan penelitian (Praharani dan Triwulanningsih, 2007) yaitu tinggi pundak 122,80 cm, tinggi pinggul 125,40 cm, panjang badan 123,20 cm dan lingkar dada 190,22 cm. Kerbau murrah jantan muda memiliki rataan ukuran tubuh tidak jauh berbeda dengan kerbau rawa jantan muda kecuali pada lingkar dada. Dari hasil uji-t terdapat perbedaan yang sangat nyata (P<0,01) pada jantan muda yaitu pada variabel panjang badan. Keragaman ukuran tubuh pada kerbau murrah jantan muda sekitar 1,11 – 8,09 % dan ukuran tubuh pada kerbau rawa jantan muda sekitar 1,77 – 4,47 %. 281 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Ukuran tubuh (tinggi pundak, tinggi pinggul, lebar pinggul, panjang badan, lingkar dada, dalam dada, lebar dada) dipengaruhi oleh umur ternak jenis kelamin. Secara umum, rataan ukuran tubuh kerbau muda lebih rendah dari ternak dewasa. Pada kerbau jantan dewasa ukuran tubuh kerbau rawa tidak berbeda jauh dengan kerbau murrah. Keragaman ukuran tubuh pada kerbau murrah jantan dewasa sekitar 0,76 – 10,60% dan ukuran tubuh pada kerbau rawa jantan dewasa sekitar 1,28 – 6,45%. Tabel 1. Rataan, simpangan baku, dan koefisien keragaman ukuran-ukuran tubuh kerbau murrah dan kerbau rawa jantan Tabel 1. Rataan, simpangan baku, dan koefisien keragaman ukuran-ukuran tubuh murrah dan kerbau rawa jantan Tabel 1. Deskriptif Statistik Ukuran Linier Permukaan Tubuh Kerbau Murrah dan Kerbau Rawa Rataan, simpangan baku, dan koefisien keragaman ukuran-ukuran tubuh kerbau murrah dan kerbau rawa jantan Variabel Bangsa Kelompok umur 2 – 3,5 3,5 – 7 n x ± S KK % n x ± S KK % Bobot badan Murrah 2 258,00±14,71tn 5,70 3 474,20±73,06tn 15,39 Rawa 4 305,17±16,44tn 5,39 3 462,17±43,67tn 9,45 Lingkar dada Murrah 2 162,50±6,36tn 3,92 3 195,00±11,36tn 5,82 Rawa 4 182,50±3,69tn 2,02 3 203,00±6,56tn 3,23 Lebar dada Murrah 2 37,00±1,41tn 3,82 3 49,00±5,19tn 10,60 Rawa 4 39,50±1,29tn 3,63 3 50,33±2,08tn 4,13 Dalam dada Murrah 2 63,50±0,70tn 1,11 3 83,00±6,56tn 7,90 Rawa 4 64,25±2,87tn 4,47 3 82,00±5,29tn 6,45 Tinggi pundak Murrah 2 115,50±4,95tn 4,28 3 138,67±4,04tn 2,91 Rawa 4 115,50±2,08tn 1,80 3 135,00±1,73tn 1,28 Panjang badan Murrah 2 122,00±1,41 1,16 3 148,33±7,64tn 5,15 Rawa 4 105,50±1,70 1,61 3 141,67±4,16 tn 2,94 Tinggi pinggul Murrah 2 111,50±5,65tn 8,09 3 131,00±1tn 0,76 Rawa 4 117,50±2,08tn 1,77 3 132,33±3,78tn 2,86 Lebar pinggul Murrah 2 44,00±2,82tn 6,43 3 57,33±5.5tn 9,61 Rawa 4 44,00±1,41tn 3,21 3 54,67±3,51tn 6,42 Keterangan: Notasi x adalah rataan, n adalah jumlah sampel, S adalah simpangan baku dan KK adalah koefisien keragaman.. Tanda * menunjukkan perbedaan nyata (P<0,05) antar bangsa dalam variabel sedangkan menunjukkan perbedaan sangat nyata (P<0,01) antar bangsa dalam variabel. Tanda (tn) menunjukkan perbedaan tidak nyata (P>0,05) antar bangsa dalam variabel. Rataan tinggi pundak dan panjang badan dari kerbau murrah jantan dewasa pengamatan adalah 138,67 cm dan 148.3 cm, lebih rendah dibandingkan penelitian (Mason, 1974) yaitu 142 dan 151cm, dan menurut Sitorus (2008) yaitu 132 cm dan 132,8 cm. Ukuran lingkar dada kerbau murrah jantan (195 cm) yang diamati juga lebih kecil dari yang didapat Fahimuddin (1975) sebesar 220 cm dan lebih besar dari laporan Sitorus (2008) yaitu 185 cm. Kerbau rawa jantan dewasa hasil pengamatan didapat tinggi pundak 135,5 cm, tinggi pinggul 132,3 cm, panjang badan 141,6 cm dan lingkar dada 203 cm, lebih besar dibandingkan tinggi pundak 127,35 cm, tinggi pinggul 126,82 cm, panjang badan 131 cm dan lingkara dada 196,5 cm (Praharani dan Triwulanningsih, 2007) sedangkan dari hasil 282 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 penelitian Sitorus (2008) tinggi pundak 126,38 cm, tinggi pinggul 125,56 cm, panjang badan 129,5 cm dan lingkar dada 182,16 cm. Tabel 2. Deskriptif Statistik Ukuran Linier Permukaan Tubuh Kerbau Murrah dan Kerbau Rawa 1 No. 3 ;276-287 Kerbau Rawa betina dewasa hasil pengamatan didapat tinggi pundak 125,14 cm, tinggi pinggul 124,49 cm, panjang badan 127,98 cm dan lingkar dada 188,74 cm, lebih besar dibandingkan pada penelitian (Praharani dan Triwulanningsih, 2007) tinggi pundak 122,91 cm, tinggi pinggul 122,72 cm, panjang badan126,96cm, lingkar dada 186,14 cm sedangkan dari hasil penelitian Sitorus (2008) menunjukkan tinggi pundak 122,26 cm, tinggi pinggul 121,38 cm, panjang badan 119,14 cm dan lingkar dada 176,6 cm. Keragaman ukuran tubuh pada kerbau murrah betina dewasa sekitar 3,02 – 9,66% dan ukuran tubuh pada kerbau rawa betina dewasa sekitar 1,89 – 7,51 %. Dari hasil uji-t terdapat perbedaan yang nyata (P<0,05) pada betina induk yaitu pada variabel tinggi pundak sedangkan perbedaan yang sangat nyata (P<0,01) ditunjukkan pada variabel panjang badan dan tinggi pinggul. Deskriptif Statistik Ukuran Linier Permukaan Tubuh Kerbau Murrah dan Kerbau Rawa Rataan, simpangan baku, dan koefisien keragaman ukuran-ukuran tubuh kerbau murrah dan kerbau rawa betina Variabel Bangsa Kelompok umur 2 – 3,5 3,5 – 7 N x ± S KK % n x ± S KK % Bobot badan Murrah 6 241,67±32,85tn 13,59 21 402,98±52,17tn 12,94 Rawa 4 223,65±16,28tn 7,28 57 398,00±38,46tn 9,66 Lingkar dada Murrah 6 155,33±7,17tn 4,62 21 182,71±12,77tn 6,99 Rawa 4 152,00±4,69tn 3,09 57 188,74±7,76tn 4,11 Lebar dada Murrah 6 35,00±1,67tn 4,78 21 40,33±2,56tn 6.34 Rawa 4 36,75±0.96 2,61 57 39,98±3tn 7,51 Dalam dada Murrah 6 65,33±4,80tn 7,35 21 75,43±7,26tn 9,62 Rawa 4 63,50±1,9 tn 3,01 57 75,00±2,9tn 3,89 Tinggi pundak Murrah 6 122,00±5,02 4,11 21 133,00±4,60* 3,46 Rawa 4 112,75±0,5** 0,44 57 125,14±2,44* 1,95 Panjang badan Murrah 6 119,17±7,73tn 6,49 21 137,28±5,57 4,06 Rawa 4 118,25±1,5tn 1,27 57 127,98±2,41 1,89 Tinggi pinggul Murrah 6 115,83±8,23tn 7,11 21 130,28±3,94 3,02 Rawa 4 113,25±1,5tn 1,32 57 124,49±2,46 1,98 Lebar pinggul Murrah 6 41,50±4,13tn 9,96 21 53,80±4.8tn 9,10 Rawa 4 41,75±1,26tn 3,01 57 47,35±3,23tn 6,82 Keterangan: Notasi x adalah rataan, n adalah jumlah sampel, S adalah simpangan baku dan KK adalah koefisien keragaman. Tanda * menunjukkan perbedaan nyata (P<0,05) antar bangsa dalam variabel sedangkan ** menunjukkan perbedaan sangat nyata (P<0,01) antar bangsa dalam variabel. Tanda (tn) menunjukkan perbedaan tidak nyata (P>0,05) antar bangsa dalam variabel. 2. Rataan, simpangan baku, dan koefisien keragaman ukuran-ukuran tubuh kerbau murrah dan kerbau rawa betina Kerbau rawa betina muda hasil pengamatan didapat tinggi pundak 112,75 cm, tinggi pinggul 113,25 cm, panjang badan 118,25 cm dan lingkar dada 152 cm, lebih rendah dibandingkan (Praharani dan Triwulanningsih, 2007) yaitu tinggi pundak 117,29 cm, tinggi pinggul 117,88 cm, panjang badan 118,91 cm, lingkar dada 179,44 cm. Hasil uji-t menunjikkan bahwa terdapat perbedaan yang sangat nyata (P<0,01) pada betina muda yaitu pada variabel tingggi pundak. Kerbau murrah betina muda memiliki rataan ukuran tubuh lebih besar dibanding kerbau rawa betina muda. Keragaman ukuran tubuh pada kerbau murrah betina muda sekitar 4,11 – 9,96% dan ukuran tubuh pada kerbau rawa betina muda sekitar 0.44 – 3.09%. Kerbau murrah betina dewasa hasil pengamatan didapat tinggi pundak 133 cm, tinggi pinggul 130,28 cm, panjang badan 137,28 cm dan lingkar dada 182,71 cm, tidak jauh berbeda dibandingkan laporan Sitorus (2008) yaitu didapati tinggi pundak 133,13 cm, tinggi pinggul 132,5 cm, panjang badan 131,87 cm, kecuali pada lingkar dada 202,59 cm. 283 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 J. Peternakan Integratif Vol. Persamaan Ukuran dan Bentuk Tubuh Kerbau Murrah dan Kerbau Rawa Persamaan skor ukuran dan bentuk tubuh dengan keragaman total dan nilai eigen pada kerbau rawa Persamaan Keragaman total (%) Nilai eigen Ukuran = 0,380X1 + 0,347X2 + 0,370X3 + 0,410X4 + 0,369X5 + 0,391X6 + 0,376X7 73,5 5,114 Bentuk = –0,217X1 – 0,573X2 + 0,169X3 + 0,305X4 + 0,422X5 + 0,304X6 – 0,481X7 13,5 0,944 Keterangan: X1 = Lingkar dada; X2 = Lebar dada; X3 = Dalam dada; X4 = Tinggi pundak; X5 = Panjang badan; X6 = Tinggi Pinggul; X7 = Lebar Pinggul Persamaan Tabel 4 menyajikan persamaan skor ukuran tubuh kerbau rawa yang memiliki keragaman total sebesar 73,5 merupakan proporsi keragaman terbesar diantara komponen – komponen utama yang diperoleh. Nilai eigen yang diperoleh pada persamaan skor ukuran adalah 5,114. Vektor eigen tertinggi pada persamaan ukuran ditemukan pada tinggi pundak (X4) sebesar 0,410 merupakan penciri ukuran kerbau rawa. Korelasi antara skor ukuran dan tinggi pundak ditemukan sebesar +0,190. Persamaan bentuk memiliki keragaman total sebesar 13,5 yang merupakan proporsi keragaman terbesar setelah keragaman total pada persamaan ukuran. Nilai eigen pada persamaan skor bentuk ditemukan sebesar 0,944. Vektor eigen yang tinggi pada persamaan bentuk ditemukan pada lebar dada (X2) sebesar 0,573 merupakan penciri bentuk pada kerbau rawa. Korelasi antara skor bentuk dan lebar dada ditemukan sebesar –0,153. Persamaan Ukuran dan Bentuk Tubuh Kerbau Murrah dan Kerbau Rawa Perasaan ukuran, persamaan bentuk, keragaman total, dan nilai eigen pada kerbau murrah dan kerbau rawa disajikan pada Tabel 3 dan Tabel 4. Tabel 3. Persamaan skor ukuran dan bentuk tubuh dengan keragaman total dan nilai eigen pada kerbau murrah p Persamaan Keragaman total (%) Nilai eigen Ukuran = 0,377X1 + 0,364X2 + 0,368X3 + 0,401X4 + 0,375X5 + 0,386X6 + 0,373X7 78,9 5,526 Bentuk = 0,258X1 + 0,515X2 − 0,378X3 – 0,341X4 + 0,102X5 – 0,498X6 + 0,388X7 6,7 0,468 Keterangan: X1 = Lingkar dada; X2 = Lebar dada; X3 = Dalam dada; X4 = Tinggi pundak; X5 = Panjang badan; X6 = Tinggi Pinggul; X7 = Lebar Pinggul Persamaan Persamaan Persamaan Keragaman total (%) Nilai eigen Ukuran = 0,377X1 + 0,364X2 + 0,368X3 + 0,401X4 + 0,375X5 + 0,386X6 + 0,373X7 78,9 5,526 Bentuk = 0,258X1 + 0,515X2 − 0,378X3 – 0,341X4 + 0,102X5 – 0,498X6 + 0,388X7 6,7 0,468 Keterangan: X1 = Lingkar dada; X2 = Lebar dada; X3 = Dalam dada; X4 = Tinggi pundak; X5 = Panjang badan; X6 = Tinggi Pinggul; X7 = Lebar Pinggul Persamaan skor ukuran tubuh kerbau murrah memiliki keragaman total sebesar 78,9 yang merupakan proporsi keragaman terbesar diantara komponen – komponen utama yang diperoleh. Nilai eigen yang diperoleh pada persamaan skor ukuran adalah 5,526. Vektor eigen tertingi pada persamaan ukuran ditemukan pada tinggi pundak (X4) sebesar 0,401. Korelasi antara skor ukuran dan tinggi pundak ditemukan sebesar +0,123. Tanda positif menunjukkan peningkatan ukuran lingkar dada akan meningkatkan skor ukuran atau sebaliknya. Persamaan bentuk memiliki keragaman total sebesar 6,7 yang merupakan proporsi keragaman terbesar setelah keragaman total pada persamaan ukuran. Nilai eigen pada persamaan skor bentuk ditemukan sebesar 0,468. Vektor eigen tertinggi pada persamaan bentuk ditemukan pada lebar dada (X2) sebesar 0,515 yang merupakan penciri bentuk pada kerbau murrah. Korelasi antara skor bentuk dan lebar dada ditemukan sebesar +0,079. 284 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 4. Persamaan skor ukuran dan bentuk tubuh dengan keragaman total dan nilai eigen pada kerbau rawa Tabel 4. Rekapitulasi Penciri Ukuran dan Bentuk Tubuh pada Kerbau Murrah dan Kerbau Rawa serta Pembentukan Diagram Kerumunan Rekapitulasi penciri ukuran dan bentuk pada kerbau murrah dan kerbau rawa yang diamati berdasarkan persamaan ukuran dan bentuk disajikan pada Tabel 5. Gambar 2 menyajikan diagram kerumunan data kerbau murrah dan kerbau rawa berdasarkan perolehan skor ukuran dan skor bentuk. Tabel 5. Rekapitulasi penciri ukuran dan bentuk tubuh pada kerbau murrah dan kerbau rawa Bangsa Penciri ukuran Penciri bentuk Murrah Tinggi pundak (X4) Lebar dada (X2) Rawa Tinggi pundak (X4) Lebar dada (X2) 285 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 Berdasarkan Tabel 5, Tinggi pundak merupakan penciri ukuran pada semua bangsa kerbau yang diamati. Hal ini divisualisasikan dalam bentuk diagram kerumunan pada Gambar 2. Berdasarkan Tabel 5, Tinggi pundak merupakan penciri ukuran pada semua bangsa kerbau yang diamati. Hal ini divisualisasikan dalam bentuk diagram kerumunan pada Gambar 2. Gambar 2. Diagram kerumunan data skor ukuran dan bentuk tubuh kerbau murrah dan kerbau rawa Gambar 2. Diagram kerumunan data skor ukuran dan bentuk tubuh kerbau murrah dan kerbau rawa Berdasarkan skor ukuran (sumbu-X), kerumunan data kerbau murrah dengan jumlah sampel 32 ekor (5 jantan, 27 betina) berkisar antara 236 – 320; sedangkan dengan jumlah sampel 68 ekor (7 jantan, 61 betina) kerbau rawa menyebar pada rentang 244 – 310. Tampak bahwa kedua bangsa kerbau menempati rentang yang sama, hanya saja pada kerbau rawa kerumunan data lebih terpusat. Hal ini menggambarkan bahwa secara ukuran kerbau murrah dan kerbau rawa adalah sama. Bentuk (fenotipik) dipengaruhi faktor genetik dan lingkungan (Hardjosubroto, 1998). Berdasarkan Tabel 5, penciri bentuk ditemukan pada kerbau murrah dan kerbau rawa adalah lebar dada. Berdasarkan Gambar 2, bentuk kedua kelompok kerbau yang diamati tampak berbeda dilihat dari kerumunan data yang ada. Hal ini dapat dijelaskan dengan tidak adanya komponen yang saling berhimpit antara kerbau murrah dan kerrbau rawa. Berdasarkan skor bentuk (sumbu-Y), kerumunan data kerbau murrah terlihat berada pada posisi bawah yaitu pada -47,5 – -22,5; sedangkan kerbau rawa tampak pada posisi diatasnya yaitu pada 42,5 – 65,1. 286 J. Peternakan Integratif Vol. 1 No. 3 ;276-287 KESIMPULAN Analisis deskriptif menunjukkan ukuran-ukuran tubuh kerbau murrah lebih beragam dibandingkan kerbau rawa. Ukuran-ukuran linier tubuh kerbau murrah berbeda dengan kerbau rawa pada variabel panjang badan pada kerbau jantan umur 2 – 3,5 tahun; variabel tinggi pundak pada kerbau betina umur 2 – 3,5 tahun; variabel tinggi pundak, panjang badan dan tinggi pinggul pada kerbau betina umur 3,5 – 7 tahun. Analisis Komponen Utama menyatakan bahwa tinggi pundak merupakan penciri ukuran dan lebar dada sebagai penciri bentuk pada kerbau murrah dan kerbau rawa. Secara morfologi kerbau murrah dan kerbau rawa sangat berbeda. Hal ini ditunjukkan oleh kerumunan data pada kerbau murrah dan kerbau rawa saling terpisah, walaupun kedua bangsa terlihat sama jika ditinjau dari morfometriknya. DAFTAR PUSTAKA Ditjennak, 2009. Peta potensi wilayah sumber bibit sapi potong lokal dan rencana pengembangannya. www.ditjennak.go.id [7 Januari 2012]. Erdiansyah, E., dan Anneke Anggraeni. 2008. Keagaman Fenotipe dan Pendugaan Jarak Genetic antara Subpopulai Kerbau Rawa Lokal di Kabupaten Dompu, Nusatenggara Barat. Prosiding Seminar dan Lokakarya Nasional Usaha Ternak Kebau Tanah Toraja. 2008. Puslitbang Peternakan, Bogor. Everitt, B. S & G. Dunn. 1998. Applied Multivariate Data Analysis. Jhon Wiley and Sons Inc., Illionois. muddin, M. 1975. Domestic water buffallo. Oxford and IBH publishing co, New Delhi. Gazpersz, V. 1992. Teknik Analisi dalam Penelitian Percobaan. Tarsito, Bandung. Hardjosubroto, W., 1998. Aplikasi Pemuliabiakan di Lapangan. Gramedia widiasarana Indonesia. Jakarta. Hardjosubroto, W. 2006. Kerbau: mutiara yang terlupakan. UGM press. Yogyakarta. Hardjosubroto, W. 2006. Kerbau: mutiara yang terlupakan. UGM press. Yogyakarta. Mason. I.L. 1974. The Husbandry and Health of The Domestic Buffalo. Food and Agriculture Organization of The United Nation, Rome. Mason. I.L. 1974. The Husbandry and Health of The Domestic Buffalo. Food and Agr Organization of The United Nation, Rome. Murti, T. W., 2002. Ilmu Ternak Kerbau. Kanisius. Yogyakarta. Praharani, Lisa., dan E. Triwulanningsih. 2007. Karakterisasi Bibit Kerbau Pada Agroekosistem Dataran Tinggi. Prosiding Seminar dan Lokakarya Nasional Usaha Ternak Kebau Jambi. Puslitbang Peternakan, Bogor. Siregar A. R., P. Situmorang, M. Zulbardi, L. P. Batubara, A Wilson, E. Basuno, S.E. Sinulingga dan C.H. Sirait. 1997. Peningkatan produktivitas kerbau dwiguna. Prosiding Seminar nasional dan veteriner. Bogor. Sitorus, A. J. dan Anneke Anggraeni, 2008. Kkarakterisasi Morfologi dan Estimasi Jarak Genetik Kerbau Rawa, Sungai (Murrah) dan Silangannya di Sumatera Utara. Prosiding Seminar dan Lokakarya Nasional Usaha Ternak Kebau Tanah Toraja. Puslitbang Peternakan, Bogor. 287
https://openalex.org/W4224240778	https://www.researchsquare.com/article/rs-1135954/latest.pdf	English	null	Mapping QTLs for spring green-up, plant vigor, and plant biomass in two lowland switchgrass populations	Molecular breeding	2,022	cc-by	11,307	Mapping QTLs for Spring Green-up, Plant Vigor and Plant Biomass in Two Lowland Switchgrass Populations Dan Chang Mapping QTLs for Spring Green-up, Plant Vigor and Plant Biomass in Two Lowland Switchgrass Populations Dan Chang Mapping QTLs for Spring Green-up, Plant Vigor and Plant Biomass in Two Lowland Switchgrass Populations Introduction Therefore, phenotypes of the overall robustness of plants in early spring could be important indicators of biomass yield potential. Spring green-up and plant vigor are two important developmental traits related to plant biomass yield in switchgrass (Chen et al. 2012; Sadeghpour et al. 2014a). Plant vigor reflects the overall growth capacity and yield in morphology. Spring green-up is a measure of the transition from winter dormancy to active spring regrowth. Early green-up switchgrass takes advantage of the rising temperature and daylength in spring for photosynthesis, and thereby could increases biomass yield with a relative longer growing season than late green-up genotypes (Bhandari et al. 2010). In addition, a long dormancy period over winter and high dormancy rate in early spring could lead to weak plant vigor and slow spring green-up, and potentially reduce biomass yield (Sadeghpour et al. 2014a; Sadeghpour et al. 2014b; Munshaw et al. 2006; Rimi et al. 2011; Curran et al. 2011). Spring regrowth, stem thickness, and biomass yield were studied in lowland switchgrass, demonstrating that variations in these traits were influenced by genetic and environmental factors (Bhandari et al. 2011). However, the genetic bases of spring green-up and plant vigor in switchgrass remain underexplored (Sadeghpour et al. 2014b), and the extent to which these two traits relate to in-season plant biomass is also lacking. Although switchgrass plots are able to maintain productivity for a decade or longer, its establishment is slow (Parrish and Fike 2005; Clifton-Brown et al. 2019). Stands of switchgrass typically produce stable phenotypes in the second year and succeeding years post establishment. Spring green-up was reported to have a high heritability indicating that this trait could be used in phenotypic selection in improving biomass yield (Bhandari et al. 2010). Quantitative trait loci (QTL) mapping is an important technique that connects phenotypic data and genomic loci in plant genetic research and that has the potential to expedite genetic improvement and enhance genetic gain per breeding cycle through MAS, which can be especially valuable for breeding perennial crop such as switchgrass that has multi-year selection cycles. To date, QTL mapping studies on biomass yield and adaptive traits have been conducted in switchgrass (Dong et al. 2015; Lowry et al. 2015; Serba et al. 2015; Chang et al. 2016; Makaju et al. 2018; Tornqvist et al. 2018; Taylor et al. 2018; Ali et al. 2019; Lowry et al. 2019; Taylor et al. 2019). Introduction Switchgrass (Panicum virgatum L.) is a warm-season C4 grass native to North America with a wide geographical distribution range (Bhandari et al. 2010; Wullschleger et al. 2010). The species has been targeted as a predominant bioenergy crop starting in the 1990s due to its high biomass yield potential, wide adaptability to marginal land, fast growth, and low cost in management (Lynd et al. 1991; Sanderson et al. 1996; Bouton et al. 2007; Wang et al. 2010). Substantial genetic diversity exists among and within different ecotypes/cultivars, which provide enormous potential towards improving biomass yield through enhancing its component traits (Bouton 2007; Bhandari et al. 2010). The genome sequence of switchgrass shed lights on genetic mechanisms that have enabled the ancestors of this valuable biomass crop to adapt to cycles of climate warming and cooling (Lovell et al. 2021). Although high biomass yield is the most important breeding objective in developing switchgrass as a biofuel cellulosic crop (Sladden et al. 1991; Walsh et al. 2003; Rose et al. 2008; Casler 2010), biomass yield is a complex trait influenced by many component traits that are controlled by numerous genetic loci (Redfearn et al. 1997; Chang et al. 2016). It is challenging to improve switchgrass biomass yield through traditional breeding methods based on phenotypic or genotypic selection that needs multiple years of breeding and testing cycles. Climate-gene-biomass associations vary considerably among diverged switchgrass gene pools. Lowland ecotype has great biomass yield potential but is winterkill susceptible (Lovell et al, 2021). Perennial grasses such as switchgrass undergo seasonal cycling through winter dormancy and spring regrowth (Sarath et al. 2014). The onset of flowering and seed development before killing frost induces senescence of above ground tissues to avoid adverse environmental conditions. Photosynthates cumulated during a growing season are partly remobilized to below ground organs such as rhizomes, which serve as storage for nutrients to promote regrowth of shoots from rhizome meristems in the following spring. Therefore, the cyclical transition between winter dormancy and spring regrowth is a fundamental developmental trait in the perennial grass. While successful survival from winter dormancy is important, how well the survived plants perform during vegetative growing season is highly relevant to biomass yield. Vigorous plants with a high volume of foliage and fast growth rate are likely to produce a higher biomass yield compared to plants that lack such characteristics. Research Article Keywords: Switchgrass, spring green-up, plant vigor, plant biomass, heritability, QTL DOI: https://doi.org/10.21203/rs.3.rs-1135954/v1 DOI: https://doi.org/10.21203/rs.3.rs-1135954/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Molecular Breeding on April 20th, 2022. See the published version at https://doi.org/10.1007/s11032-022-01296-7. Page 1/14 Abstract Switchgrass ( Panicum virgatum L.) is an important perennial cellulosic bioenergy species. Identification of quantitative trait loci (QTL) controlling important developmental traits is valuable to understanding the genetic basis and breeding high-yielding cultivars. One F 1 hybrid population consisting of 176 individuals derived from NL94 (♀) × SL93 (♂), and one S 1 (first-generation selfed) population of 265 individuals from NL94, were used to study spring green- up, plant vigor and plant biomass in switchgrass. Both populations showed significant variations for genotype, and genotype by environment interactions for the three traits. Plant vigor had strong and positive correlations with plant biomass in both populations. Broad sense heritability estimates of plant vigor ranged from 0.46 to 0.74 and 0.45 to 0.74 in the hybrid and selfed population, respectively. Spring green-up had similar heritability estimates in these two populations (0.42-0.78 in hybrid population, 0.47-0.82 in selfed population). The heritability of plant biomass was above 0.5 (0.54-0.64 in hybrid population, 0.64-0.74 in selfed population). Fifteen and four QTLs were detected for spring green-up in the hybrid and selfed population, respectively. Six and four QTLs were detected for plant vigor in the hybrid and selfed population, respectively. Three QTLs in the hybrid population and one QTL in the selfed population were identified for plant biomass. This study provided new information in understanding the genetic control of biomass components and demonstrated substantial heterotic vigor that could be explored for breeding hybrid cultivars in switchgrass. Introduction Reproductive maturity was a trait closely related to biomass yield. Multiple genomic regions were detected for this trait in two lowland switchgrass populations (Dong et al. 2015). Serba et al. (2015) identified QTLs for biomass yield in a heterozygous pseudo-F1 population and found four stable QTLs across ten environments, which explained 4.9-12.4% of phenotypic variation. Chang et al. (2016) investigated six tillering-related traits in two lowland switchgrass populations (the same populations as in this study), and two stable QTLs for biomass-related traits were detected in multiple environments. Taylor et al. (2019) found significant QTLs for plant height, crown diameter, and plant biomass in different environments in a pseudo-F2 population. However, few studies have been focused on spring green-up and plant vigor in switchgrass. Ali et al. (2019) reported 35 QTLs for spring green-up in a lowland (cv ‘Alamo’) by upland (cv ‘Summer’) switchgrass population, providing valuable knowledge of this developmental trait. A critical issue in the use of QTLs in breeding programs is that they can only be assumed a priori to be relevant to the cross and environment in which they were mapped (Feltus et al. Page 2/14 2006; Yu et al. 2008). Genetic mapping in different populations often results in distinct sets of QTLs due to genetic heterogeneity. For example, in Miscanthus, another well-known bioenergy biomass crop, substantial phenotypic variations of overwintering ability (i.e. transition from winter dormancy to spring regrowth, a trait analogous to spring green-up in this study) have been reported in a large M. sinensis germplasm panel (Dong et al. 2019a), and QTL mapping across multiple populations revealed numerous QTLs that are unique to specific populations (Dong et al. 2018; Dong et al. 2019b). Therefore, more genetic mapping studies are needed to understand the rich genetic diversity underlying agronomic traits in switchgrass, particularly the underexplored yet important traits such as spring green-up and plant vigor. The objectives in this study were to: (1) evaluate phenotypic and genotypic variation for spring green-up, plant vigor, and plant biomass; (2) investigate correlations between the traits; (3) identify QTLs for the traits. 2006; Yu et al. 2008). Genetic mapping in different populations often results in distinct sets of QTLs due to genetic heterogeneity. For example, in Miscanthus another well-known bioenergy biomass crop, substantial phenotypic variations of overwintering ability (i.e. Plant materials and field management One lowland switchgrass hybrid population encompassing 176 progeny from a cross between ‘NL94 LYE 16 × 13’ (NL94) and ‘SL93 7×15’ (SL93) and one lowland selfed population consisting of 265 progeny from NL94, were used in this study (Liu and Wu 2012; Liu et al. 2012; Dong et al. 2015). The parental plants NL94 and SL93 were chosen in 2007 from the Oklahoma State University (OSU) northern lowland (NL) breeding population growing in a low yield environment (LYE) selection nursery and the OSU southern lowland (SL) breeding population, respectively (Liu and Wu 2012). Prior to the initiation of flowering, one potted plant of NL94 and another of SL93 were transferred into a large growth chamber in the OSU Controlled Environmental Research Laboratory. Processed seeds from mature inflorescences from the respective plants were germinated in spring 2009. A total of 456 and 44 progeny were obtained for NL94 and SL93, respectively (Liu and Wu 2012). All of SL93 progeny were removed in subsequent genetic research due to the small number. Finally, 456 progeny from NL94 were divided into two populations based on 12 SSR markers: an NL94 selfed population of 279 progeny and one hybrid population (NL94 x SL93) of 177 progeny (Liu and Wu 2012). The parents and progeny of the two populations were vegetatively propagated in a greenhouse at Oklahoma State University (OSU). Two field trials were established in 2011, one at the OSU Agronomy Farm (36° 12′ N Lat., 97° 08′ W Long), Stillwater, and the other at Cimarron Valley Research Station (35° 59′, 97° 03′), near Perkins, OK. Soil types were Kirkland silt loam in Stillwater and Teller fine sandy loam in Perkins, respectively. A randomized complete block design with three replications was arranged at each of the two locations. Within each block at each field trial, the 441 progeny (265 selfed and 176 hybrid progeny) and two parental genotypes were randomly assigned to 3.21 m ´ 1.07 m plots with three clonal plants per plot. Field management protocols have been described by Dong et al. (2015) and Chang et al. (2016). Briefly, irrigation was provided to two field trials after transplanting to improve establishment. Pre-emergence herbicides including 1.12 kg atrazine (6-chloro-N-ethyl-N-isopropyl-1,3,5-triazine-2,4-diamine) and 1.12 kg Surflan (Oryzalin: 3,5-dinitro-N4N4–dipropylsulfanilamide) active ingredient per hectare were sprayed before spring green-up of switchgrass plants in the following four years (2012-2015). Plant materials and field management Urea was applied with a rate of 67.2 kg/ha N when switchgrass plants started to grow vigorously in each growing season. Hand weeding was implemented as needed. Introduction transition from winter dormancy to spring regrowth a trait analogous to spring green-up in this study) have been reported in a large M. sinensis germplasm panel (Dong et al. 2019a), and QTL mapping across multiple populations revealed numerous QTLs that are unique to specific populations (Dong et al. 2018; Dong et al. 2019b). Therefore, more genetic mapping studies are needed to understand the rich genetic diversity underlying agronomic traits in switchgrass, particularly the underexplored yet important traits such as spring green-up and plant vigor. The objectives in this study were to: (1) evaluate phenotypic and genotypic variation for spring green-up, plant vigor, and plant biomass; (2) investigate correlations between the traits; (3) identify QTLs for the traits. Genotypic data collection and QTL mapping The preexisting genetic maps of these two populations developed by Liu et al. (2012) and Dong et al. (2015) only included 139 and 132 individuals of the selfed and hybrid population, respectively. In order to increase the statistical power in QTL analysis, marker data of previously un-genotyped individuals were obtained as follows. Initially, QTL mapping for plant vigor, spring green-up, and plant biomass were performed on the two preexisting linkage maps (Liu et al. 2012; Dong et al. 2015). Once significant QTLs were identified, SSR markers flanking the QTLs were selected to genotype the remaining 126 individuals of the selfed population and 44 individuals of the hybrid population. Then a second round QTL mapping was conducted by including the marker information of these newly genotyped individuals. We employed this strategy based on two considerations: 1) to assess the stability of identified QTLs, and 2) to expedite genotyping work by focusing on the statistically significant genomic regions. QTL detection was conducted using MapQTL 6.0 (Van Ooijen 2009). Genotype file, map file, and phenotype files were prepared following the software user manual. Interval mapping (IM) was used to do initial genome-wide scan, and then significant markers detected in IM were used as cofactors in a Multiple-QTL model (MQM) to scan additional QTLs (Taylor et al. 2019). QTLs with the LOD threshold above 3.5 were recorded based on the 1000-permutation test (Churchill and Doerge 1994). QTL mapping for spring green-up and plant vigor were both conducted under separate environment (individual combinations of year and location; e.g. 2012STW represents data collected in 2012 at Stillwater field) and across environments (i.e. based on trait least squared means across four years and two locations). Additive allelic effect is of great interest to plant breeding. Given that switchgrass is a tetraploid, up to four distinct alleles could segregate in a bi-parental population such as the hybrid population in this case (i.e., locus heterozygous in both parents). Genotype codes for such scenario would be ab x cd, resulting in four possible genotypes in progeny: ac, ad, bc, and bd (refer to Tables 4, 5 in JoinMap manual: ) ( https://www.kyazma.nl/docs/JM5Manual.pdf). Then QTL additive effects were estimated based on the deviation from the ac genotype (refer to page 41 in MapQTL 6 manual: https://www.kyazma.nl/docs/MQ6Manual.pdf). In contrast, only two alleles were segregating in the selfed population, and QTL additive effects were estimated as the traditional method. Phenotypic variation and broad-sense heritability In most individual environments (location and year combinations), plant vigor in the hybrid population exhibited left skewed distributions whereas the distributions in the selfed population were slightly right skewed (Fig. 1 and Fig. 2). Normally distributed patterns or right skewed distributions were showed for plant biomass in the hybrid population. Large phenotypic variation existed for spring green-up in the two populations. Genotype mean values spanned a wide range in all environments (e.g., 1-100% in 2015PKS; Table 1). Population means also varied considerably across environments, ranging from 35 to 81% and 17 to 44% in the hybrid population and the selfed population, respectively (Fig. 1, Fig. 2 and Table 1). For plant vigor, substantial variations were observed among genotypes within each environment, whereas population means were generally similar across environments (6-8 in hybrid population, 3-4 in selfed population; Table 1). For plant biomass, obvious heterosis and selfing depression for the two populations can be found crossing all individual environments (Table 1). In most individual environments (location and year combinations), plant vigor in the hybrid population exhibited left skewed distributions whereas the d b h lf d l l h l h k d ( d ) ll d b d h k d d b h d f ANOVA indicated that significant effects of genotype, year, and location × year for spring green-up, plant vigor and plant biomass in both populations (Table 2). Significant genotype × location interactions were detected for plant vigor in both populations and plant biomass in the selfed population, but slightly significant genotype × location interactions was showed in the hybrid population. Significant genotype × year interactions were found for spring green-up in both populations and marginally significant (P = 0.0036 in the selfed population) or non-significant (P = 0.0518 in the hybrid population) for plant vigor (Online Resource 1). However, non-significant genotype × year interactions were detected for plant biomass in both populations. Correlations among spring green-up, plant vigor, and plant biomass in the hybrid and selfed populations are presented in Table 2. Only data collected in 2012 and 2013 were used in correlation analysis as plant biomass data were not available in 2014 and 2015. The correlation between spring green-up and plant biomass was marginally positive (0.09) in the selfed population while it was negative (-0.07) in the hybrid population (Table 2). Genotypic data collection and QTL mapping Genetic maps of these two populations used molecular markers developed from multiple sources (Tobias et al. 2008; Okada et al. 2010; Wang et al. 2011). Relationship between the linkage groups and the subgenome designations (N and K) in the switchgrass genome remains unknown. Therefore, primer sequences of mapped markers were retrieved from the respective sources and were compared to switchgrass reference genome (Lovell et al. 2021) using BLASTN 2.6.0+ with an E-value threshold of 0.1. Genotypic data collection and QTL mapping Phenotypic data collection and analysis Phenotypic data were collected in the spring of 2012 (Yr 2) to 2015 (Yr 5). In early April, spring green-up was visually scored for each plant based on a 0-100% scale with 10% increment to evaluate the percentage of green-up tillers covering the whole plant stand area, in which 0 indicated no green tillers and 100％represented green tillers covering the whole plant stand base. Plant vigor data were collected based on visual scoring in May to June of 2012 to 2015. An ordinal scale from 1 to 9 was used to evaluate plant vigor by focusing on plant size (plant width and height), with 1 being the least vigorous (< 10 tillers and short), and 9 being the most vigorous plants. Plant biomass data were collected from December to the following January in 2012 and 2013. Plants were cut at 10 cm above ground, and the three clonal plants within each plot were weighed together, and the average was used for plant biomass of each genotype. Phenotypic data analyses were conducted using R (version 3.6.0) (R Core Team 2019). Pearson correlations between the traits studied were calculated using cor function. To assess the significance of location (STW and PKS), year (2012-2015), genotype, and their interactions, analyses of variance (ANOVAs) were conducted for each trait in each population using the following linear mixed model in lme4 package (Bates et al. 2015). Where Y represents the raw phenotypic data, m is the grand mean, L represents location, A represents year, G represents genotype, B(L) represents block within location, LA represents location by year interaction, LG represents location by genotype interaction, AG represents year by genotype interaction, and e is the random error. Broad-sense heritability (H) was then calculated using the aforementioned model by fitting all model terms as random effects. Variance components were estimated based on the restricted maximum likelihood (REML) method. Equation of H calculation is as follows: Since perennial grass such as switchgrass undergoes an establishment phase, we were also interested in the year-by-year heritability to assess the temporal stability of phenotypes. Therefore, we conducted a second round of ANOVAs for each trait under single-year scenario following model: Where model terms are defined in Eq. 1. Single-year heritability was then calculated following equation 4: Page 3/14 Page 3/14 Plant vigor QTL detection Six significant QTLs for plant vigor with LOD scores from 3.81 to 6.01 and one QTL with a LOD value of 4.7 were discovered in individual environments and the joint-environment in the hybrid population (Table 4). In the selfed population, four QTLs from individual environments were detected with LOD values from 3.54 to 4.77 and one QTL from the joint-environment analysis with a LOD value of 4.39 (Table 5). The phenotypic variance was explained from 7.7 to 12.6％ in the hybrid population, and from 5.5 to 8.3％in the selfed population (Table 4 and Table 5). Among all the significant QTLs in the hybrid population, the QTL between PVGA-1813/1814 and PVGA-1357/1358 on LG 5a was stably expressed in 2014STW, 2015PKS and the joint environment, which accounted for 34.5％ of the total phenotypic variance, and the additive effects of the three genotypes AD, BC and BD for this QTL ranged from -0.65 to 0.93 (Table 4 and Fig. 3). The QTL between PVCA-815/816 and nfsg-50 were also detected in three environments, 2013PKS, 2014STW and the joint environment, accounting for 18.7％ phenotypic variance in the selfed population, with the additive effects ranging from 0.03 to 0.14 (Table 5 and Fig. 3). Correspondence between linkage groups and switchgrass genome High correspondence was found between the nomenclature of genetic linkage groups (a and b) and the subgenome designations (N and K) in the switchgrass genome (Online Resource 3). For both populations, clear correspondences are as follows: LG 1a – Chr 01K, LG 1b – Chr 01N, LG 2a – Chr 02N, LG 2b – Chr 02K, LG 3a – Chr 03K, LG 3b – Chr 03N, LG 4a & 4b – Chr 04N and 04K, LG 5a – Chr 05K, LG 5b – Chr 05N, LG 6a – Chr 06N (the selfed population only), LG 6b – Chr 06K, LG 7a – Chr 07K, LG 8a – Chr 08N, LG 8b – Chr 08K, LG 9a – Chr 09K, LG 9b – Chr 02N. However, LG 6a in the hybrid population largely matched with Chr 07N, indicating the lack of marker coverage on this group. Moreover, LG 7b only contained 3 and 4 markers in the hybrid and selfed population, respectively, and these markers did not correspond to the expected Chr 07N. This is also likely due to the low marker density. Plant biomass QTL detection In the hybrid population, interesting findings showed in individual environments and joint-environments, all of the significant QTLs were focused on LG 5a with the LOD values ranging from 3.75 to 5.69 (Fig. 3). Identical QTLs (e.g., the QTL between PVGA-1971/1972 and PVCAG-2197/2198) and adjacent QTLs (e.g., the QTL between PVCAG-2389/2390 and PVCAG-2167/2168 and the QTL between PVCAG-2167/2168 and PVGA-1971/1972) were found between plant biomass and spring green-up or plant biomass and plant vigor. In the selfed population, only one QTL between sww-1622 and sww-2501 was found on LG 2a with the LOD value 4.15 (Fig. 3). Phenotypic variation and broad-sense heritability Plant vigor was positively correlated with plant biomass in both the hybrid population (0.30) and the selfed (0.26). Between spring green-up and plant vigor, significant positive correlation was found in the selfed population (0.31), while non-significant correlation was found between these two traits in the hybrid population (Table 2). Broad-sense heritabilities of spring green-up and plant vigor were estimated under both single year (i.e., based on single year data across two locations) and joint environments (i.e., based on multiple year data across two locations). For spring green-up, the highest single-year heritabilities were found in 2012, with the value of 0.78 and 0.82 in the hybrid population and selfed population, respectively (Online Resource 2). In subsequent years (2013 to 2015), heritabilities of spring green-up were moderate, ranging from 0.42 to 0.59 in the hybrid population and 0.47 to 0.57 in the selfed population (Online Resource 2). Under joint environments, the heritability of spring green-up was 0.51 in the hybrid population and 0.63 in the selfed population (Table 3). For plant vigor, the highest single-year heritabilities in the two populations were both detected in 2013 with both the value of 0.74 (Online Resource 2). Moreover, the lowest heritabilities of plant vigor in the hybrid population and selfed population were 0.46 in 2015 and 0.45 in 2014, respectively (Online Resource 2). Based on the joint environment analysis, a high heritability (0.80) for plant vigor was observed in the hybrid population, and a moderately high heritability (0.69) was found in the Page 4/14 selfed population (Table 3). As the trait plant biomass, single-year heritability ranged from 0.54 to 0.74 in the hybrid and selfed populations (Online Resource 2). For the joint environments, the heritability of plant biomass was 0.63 in the hybrid population and the same in the selfed population (Table 3). Spring green-up QTLs detection In the hybrid population, a total of 15 QTLs for spring green-up were detected on seven LGs across individual environments (Table 4). Specifically, four QTLs were discovered in 2012STW, three QTLs in each of 2013PKS, 2014PKS and 2015STW. The QTLs of 2012STW, 2013PKS, 2014PKS, and 2015STW cumulatively explained 29.9, 27.4, 27.5 and 24.4％of the phenotypic variance, respectively. Only one QTL was detected in each of 2012PKS, 2014STW and 2015PKS, explaining 9.6, 11.8 and 9.9％ of the phenotypic variance (Table 4). However, only one QTL was identified on LG 2a in the joint environment analysis (Table 4). The significant QTL between markers sww-532 and nfsg-052 on LG 2a was identified in 2014PKS and 2015PKS as well as the joint environment (Table 4 and Fig. 3). The consistent results in multiple environments indicated that this significant QTL was stably expressed. The effects for the genotypes AD, BC and BD were positive for the QTLs on LGs 2b, 9a and 1a in 2015STW while significant QTLs mapped on LGs 1a, 5a and 6b in 2012STW, 2a in 2014PKS, 2015PKS and the joint environments had negative effects (Table 4). In the selfed population, four significant QTLs for spring green-up were identified on three LGs based on individual environments and one significant QTL on LG 6b-1 based on the joint environment (Table 5). Two QTLs in 2012STW, one QTL in each of 2012PKS, 2015PKS and 2015STW explained 18.5, 6.3, 7.1 and 7.1％ phenotypic variance, respectively (Table 5). No significant QTL was detected at the two locations in 2013 and 2014 (Table 5). The QTL between PVAAG- 3017/3018 and PVGA-1115/1116 on LG 6b-1 was detected in 2012PKS, 2012STW and the joint environment analysis (Table 5 and Fig. 3). The additive effects for spring green-up ranged from -5.76 to 5.96 and only one single QTL located on LG 9a in 2015PKS had the positive effect and other QTLs had negative effects (Table 5). Discussion Plant vigor and spring green-up are dynamic traits reflecting the active growth in spring. It has become widely Heritability estimates varied in different environments obviously demonstrated that environments across years and locations are necessary for evaluating heritable traits and providing sufficient information for selection. Broad-sense heritability is statistically higher than narrow-sense heritability because the dominance and epistatic genetic effects are not parsed out from additive effects in calculations. However, it is not easy to obtain accurate genetic effects for the three components. In addition, dominance effects and the epistatic effects are indeed complicated and less readily maneuvered than additive effects in applied breeding programs. Consequently, broad-sense heritability was selected as a method to evaluate the genetic proportion controlling total phenotypic variation. Heritability of the three traits was moderate to high in both the hybrid (Online Resource 2 and Table 3) and selfed populations (Online Resource 2 and Table 3) across years, indicating strong influence of environmental effects as evidenced in the ANOVA analysis (Online Resource 1). A high heritability of 0.82 for spring green-up was reported based on a half-sib family in lowland switchgrass, which could be accounted for more additive effects produced from half-sib family (Bhandari et al. 2010). Plant vigor and spring green-up are dynamic traits reflecting the active growth in spring. It has become widely recognized that the dynamic nature of traits explains the different heritability estimates between environments and populations (Würschum et al. 2014; Pauli et al. 2016). With the advancement in high-throughput phenotyping technologies (HTP), there is a trend to treat heritability as a function across development rather than a fixed time point (Xu et al. 2021). Application of HTP in switchgrass could provide novel insights into the dynamic response to the ever-changing ambient environment in different genotypes, and could enable us to estimate heritability at a finer scale along the temporal dimension. recognized that the dynamic nature of traits explains the different heritability estimates between environments and populations (Würschum et al. 2014; Pauli et al. 2016). With the advancement in high-throughput phenotyping technologies (HTP), there is a trend to treat heritability as a function across development rather than a fixed time point (Xu et al. 2021). Application of HTP in switchgrass could provide novel insights into the dynamic response to the ever-changing ambient environment in different genotypes, and could enable us to estimate heritability at a finer scale along the temporal dimension. Discussion In this study, the QTLs detected for the three traits were distributed unevenly in the linkage groups, and some linkage groups exhibited clusters of QTLs while other linkage groups contained just one QTL or no QTL. In these two populations, the QTLs associated with the three traits were mainly distributed on LGs 1a, 2a, 2b, 5a and 9a. The variability of QTL distribution could be partially explained by the significant genotype by environment effects in the ANOVA analysis (Online Resource 1). The dynamic nature of traits also imposed an inherent factor influencing trait expressions under different environments, and thereby resulted in distinct sets of QTLs. In the QTL analysis, adjacent QTLs were found for spring green-up (the QTLs around nfsg-137, PVAAG-3091/3092, PVCAG- 2517/2518, PVE-1143/1144 and sww-1615), plant vigor (the QTL around PVGA-1513/1514) and plant biomass (the QTL around PVGA-1971/1972 and PVCAG-2197/2198) in the hybrid population (Tables 6, 7) , which suggested that quantitative traits were controlled by multiple closely located genes and (or) influenced by the strong genetic linkage among these QTLs. An in-depth examination on these QTL hotspot regions may reveal more detailed genetic mechanisms controlling agronomic traits in switchgrass, but this requires a sufficiently large population size and high-density genetic maps, as genetic linkage could often yield synthetic associations in genetic mapping studies. From the perspective of applied plant breeding, identifying the exact location of QTLs or candidate genes is perhaps less important than finding the significant genomic regions that can be used to introduce favorable alleles through marker-assisted selection. Moreover, in the hybrid population, the QTL between PVGA-1971/1972 and PVCAG-2197/2198 were detected for spring green-up associated with 2012STW, plant vigor associated with 2012STW and 2014PKS, and plant biomass with 2012STW and 2013PKS. Common genomic regions controlling multiple traits simultaneously in this study suggested that this interval could have a pleiotropic effect or harbor multiple genes, and these three traits may be related in genetic control mechanism despite no significant correlation was detected between spring green-up and plant vigor in the hybrid population (Table 2). Although these two populations share a common parent NL94, the commonality of QTLs was not evident between these two populations, which may be caused by the unique polymorphism in SL93 of the hybrid population. Compared with previous QTL research of switchgrass, we found identical marker intervals and a few markers shared by different traits in the two populations. Discussion Heterosis is one of the primary biological phenomena extensively explored for the success of plant breeding endeavors in many species (East 1936; Gallais 1988; Lippman and Zamir 2007). Both the frequency distributions and population means for the three traits in individual environments indicated heterosis in the hybrid population and inbreeding depression in the selfed population (Fig. 1, Fig. 2 and Table 1). The hybrid progeny means were between or higher than the two parents for these three traits. The hybrid progeny means were higher than the two parents in all individual environments for plant biomass, in the individual environments 2013STW, 2014PKS and 2015PKS for spring green-up and in the individual environments 2012STW, 2013PKS, 2013STW and 2014PKS for plant vigor, indicating substantial transgressive segregation for these two traits. Heterosis in switchgrass was also observed in the study that spring green-up showed transgressive segregation in a hybrid population derived from crossing by AP13 (lowland) and VS16 (upland) (Ali et al., 2019). Different genetic segregations were reported in the full-sib family due to the high heterozygosity between the two parents during meiosis (Lowry et al. 2019). The recombined hybrids surpassed both parents for the traits studied. The transgressive segregation may be derived from the cumulative and complementary effects of favorable genes from both parents. Similarly, the inbred progeny derived from selfing NL 94 parent were segregating. As expected, inbreeding depression for the three traits was observed in the selfed population. Interestingly, some selfed progeny of NL 94 were more vigorous than the parent, Page 5/14 Page 5/14 providing an opportunity to select for better progeny with increased homozygosity. Vigorous progeny have high potential for the development of advanced inbreds. providing an opportunity to select for better progeny with increased homozygosity. Vigorous progeny have high potential for the development of advanced inbreds. Although heterosis is common in crops, the genetic architecture underlying this phenomenon remains elusive due to its complexity. Huang et al. (2016) examined tens of thousands of F2 rice lines of three genetic groups and identified a small number of loci from female parents explaining a large portion of the yield advantage of hybrids. However, no heterosis-associated loci were shared across all lines. Moreover, heterosis is an environmentally modified quantitative phenotype (Williams 1959; Griffing 1990; Schnell and Cockerham 1992). Genetic and genomic analysis such as genetic mapping, genomic sequencing, and transcriptomics will not suffice. Discussion An integrated framework composed of genomic tools, QTL-based phenotyping, and map-based cloning should be employed to understand the role of heterosis in plant breeding and evolution (Lippman and Zamir 2007). As a largely outcrossing species, switchgrass enjoys wide geographical distribution and rich genetic diversity. The highly heterozygous tetraploid genome of switchgrass renders the genetic study of heterosis a challenging task. The availability of rare self-compatible genotype (i.e., NL94 in this study) provided a valuable resource for the development of inbred lines to assist the study of heterosis in switchgrass. Moreover, climate-associated adaptation has resulted in divergence between the northern upland and southern lowland ecotypes that is exemplified by divergent whole-plant morphologies and distinct overwintering ability (Lovell et al. 2021). It is likely that the genetic architectures for heterosis would also be different between ecotypes as that observed in different rice genetic pools (Huang et al. 2016). Therefore, more populations of diverse genetic backgrounds and coordinated long-term study are needed to better understand heterosis in switchgrass. Although heterosis is common in crops, the genetic architecture underlying this phenomenon remains elusive due to its complexity. Huang et al. (2016) examined tens of thousands of F2 rice lines of three genetic groups and identified a small number of loci from female parents explaining a large portion of the yield advantage of hybrids. However, no heterosis-associated loci were shared across all lines. Moreover, heterosis is an environmentally modified quantitative phenotype (Williams 1959; Griffing 1990; Schnell and Cockerham 1992). Genetic and genomic analysis such as genetic mapping, genomic sequencing, and transcriptomics will not suffice. An integrated framework composed of genomic tools, QTL-based phenotyping, and map-based cloning should be employed to understand the role of heterosis in plant breeding and evolution (Lippman and Zamir 2007). As a largely outcrossing species, switchgrass enjoys wide geographical distribution and rich genetic diversity. The highly heterozygous tetraploid genome of switchgrass renders the genetic study of heterosis a challenging task. The availability of rare self-compatible genotype (i.e., NL94 in this study) provided a valuable resource for the development of inbred lines to assist the study of heterosis in switchgrass. Moreover, climate-associated adaptation has resulted in divergence between the northern upland and southern lowland ecotypes that is exemplified by divergent whole-plant morphologies and distinct overwintering ability (Lovell et al. 2021). Discussion It is likely that the genetic architectures for heterosis would also be different between ecotypes as that observed in different rice genetic pools (Huang et al. 2016). Therefore, more populations of diverse genetic backgrounds and coordinated long-term study are needed to better understand heterosis in switchgrass. Marginal correlation coefficients between spring green-up and plant biomass indicated that spring green-up could be a relatively independent trait for plant biomass. A previous study reported that spring green-up showed no correlation with plant biomass resulted from a half-sib family (Bhandari et al. 2010). The correlation analysis between plant vigor and plant biomass indicated that selection for plant vigor during the early stages of growing season may result in a higher plant biomass (Table 2). The consistent results suggested plant vigor could be recommended as an indirect selection criterion for biomass yield. Mature lowland switchgrass plants can grow 2-3 m in height. Phenotyping plant vigor on large mature plants is not readily feasible due to high amounts of foliage and tillers. The results of this study suggest that selection in early stage is a sound alternative. The different correlation results between the spring green-up and plant vigor based on the two populations suggested the genetic background of the populations is an important factor influencing correlations between the traits. Heritability estimates varied in different environments obviously demonstrated that environments across years and locations are necessary for evaluating heritable traits and providing sufficient information for selection. Broad-sense heritability is statistically higher than narrow-sense heritability because the dominance and epistatic genetic effects are not parsed out from additive effects in calculations. However, it is not easy to obtain accurate genetic effects for the three components. In addition, dominance effects and the epistatic effects are indeed complicated and less readily maneuvered than additive effects in applied breeding programs. Consequently, broad-sense heritability was selected as a method to evaluate the genetic proportion controlling total phenotypic variation. Heritability of the three traits was moderate to high in both the hybrid (Online Resource 2 and Table 3) and selfed populations (Online Resource 2 and Table 3) across years, indicating strong influence of environmental effects as evidenced in the ANOVA analysis (Online Resource 1). A high heritability of 0.82 for spring green-up was reported based on a half-sib family in lowland switchgrass, which could be accounted for more additive effects produced from half-sib family (Bhandari et al. 2010). Discussion For example in the hybrid population of this study, the QTL for spring green-up in single environment located in the marker intervals of nfsg-137 and PVAAG- 3091/3092, sww-463 and nfsg-137, PVCAG-2389/2390 and PVCAG-2167/2168, PVAAG-3091/3092 and PVCAG-2517/2518, and sww-1667 and PVCA- Page 6/14 Page 6/14 Page 6/14 Page 6/14 1253/1254 were also detected for biomass-related traits (i.e. plant base size, tillering ability, node numbers and tiller dry weight) in previous research (Chang et al. 2016). The QTL for plant biomass between PVCAG-2197/2198 and PVGA-1649/1650 was the same as the QTL for plant girth in PKS12&13. In the selfed population, the QTLs found in this study between sww-2285 and PVGA-1405/1406, sww-2501 and PVCA-597/598 for spring green-up, as well as PVCA- 815/816 and nfsg-50 for plant vigor just appeared in the previous study referring to those biomass-related traits (Chang et al. 2016). We also found QTLs flanked by common markers in these two studies (i.e. PVCAG-2197/2198, PVCA-179/180, sww-463, nfsg-13, PVGA-1813/1814, and PVCAG-2167/2168) (Chang et al. 2016). These repeated QTLs and common markers could be of great interest in MAS to improve biomass breeding in switchgrass. Moreover, some novel QTLs were found in this study (i.e. QTLs between sww-1615 and sww-333, PVE-413/414 and PVE-1143/1144 and the QTL between sww-532 and nfsg-052 for spring green-up, the QTL between PVE-1361/1362 and PVGA-2107/2108 for plant vigor detected in the hybrid population, QTLs between PVAAG- 3017/3018 and PVGA-1115/1116 for spring green-up, the QTL between sww-2545 and sww-1622 for plant vigor and the QTL between sww-1622 and sww- 2501 for plant biomass detected in the selfed population), which expanded our knowledge in understanding the genetics of biomass and biomass-related traits in switchgrass. QTL mapping studies on yield and yield-related traits in switchgrass are increasing, but still largely rely on single environment and (or) single population. Evaluation of multiple populations under multi-environments is necessary to provide information in plant breeding, particularly in perennial crops such as switchgrass, which is expected to have multiple years of growth for biomass production (Lowry et al. 2019; Lovell et al. 2021). Indeed, maintenance of large- scale multi-year multi-environment field trials are cost prohibitive and labor intensive. Coordinated agricultural projects such as Lovell et al. (2021) and Zhang et al. (2020) laid the cornerstone for a comprehensive understanding of this important bioenergy biomass crop. Conclusion This study characterized phenotypic variations and identified QTLs for spring green-up, plant vigor, and plant biomass in two populations of lowland switchgrass. Both the hybrid and selfed populations showed significant variations for genotype by year or genotype by location interactions. Heterosis in the hybrid population and inbreeding depression in the selfed population indicated the genetic potential in the development of hybrid cultivars in lowland switchgrass. Adjacent and common QTLs detected in the three traits suggested SSR markers flanking the significant QTLs are valuable for marker-assisted selection in lowland switchgrass. The findings in this study will extend the knowledge base of QTLs for biomass components in the species. Declarations Acknowledgements We acknowledge Gary Williams for field wok assistance. Special thanks go to Dr. Carla Goad for her guidance and support in this study. The project was in part sponsored by NSF-EPSCoR, Sun Grant Initiative, Oklahoma Agricultural Experiment Station, and USDA Hatch to Y.Q. Wu. Acknowledgements We acknowledge Gary Williams for field wok assistance. Special thanks go to Dr. Carla Goad for her guidance and support in this study. The project was in part sponsored by NSF-EPSCoR, Sun Grant Initiative, Oklahoma Agricultural Experiment Station, and USDA Hatch to Y.Q. Wu. Funding The project was in part sponsored by NSF-EPSCoR, Sun Grant Initiative, Oklahoma Agricultural Experiment Station, and USDA Hatch to Y.Q. Wu. The project was in part sponsored by NSF-EPSCoR, Sun Grant Initiative, Oklahoma Agricultural Experiment Stat D. Chang carried out the experiment, data analysis and writing of the manuscript, H.X. Dong conducted data analysis and writing of the manuscript, S.Q. Bai designed and supervised the experiment, Y.Q. Wu conceived and supervised the experiment, wrote and finalized the manuscript. All authors approved the submission. All authors approved the submission. All authors approved the submission. Consent to participate References Chen G, Wang Q, Liu YY, et al (2012) Modelling analysis for enhancing seed vigour of switchgrass (Panicum virgatum L.) using an ultrasonic technique. Biomass an-d Bioenergy 47:426–435. https://doi.org/10.1016/j.biombioe.2012.09.015 9. Churchill GA, Doerge RW (1994) Empirical threshold values for quantitative trait m-apping. Genetics. https://do 9. Churchill GA, Doerge RW (1994) Empirical threshold values for quantitative trait m-apping. Genetics. https://doi.org/10.1534/genetics.107.080101 10. Curran WS, Ryan MR, Myers MW, et al (2011) Effectiveness of sulfosulfuron and qu-inclorac for weed control during switchgrass establishment. Weed Technol 25: 5-98–603. https://doi.org/10.1614/wt-d-11-00010.1 11. Clifton-Brown J, Harfouche A, Casler MD, et al (2019) Breeding progress and prepar-edness for mass-scale deployment of perennial lignocellulosic biomass crops sw-itchgrass, miscanthus, willow and poplar. GCB Bioenergy 11:118–151. https://d-oi.org/10.1111/gcbb.12566 11. Clifton-Brown J, Harfouche A, Casler MD, et al (2019) Breeding progress and prepar-edness for mass-scale deployment of perennial lignocellulosic biomass crops sw-itchgrass, miscanthus, willow and poplar. GCB Bioenergy 11:118–151. https://d-oi.org/10.1111/gcbb.12566 11. Clifton Brown J, Harfouche A, Casler MD, et al (2019) Breeding progress and prepar edness for mass scale deployment of perennial lignocell biomass crops sw-itchgrass, miscanthus, willow and poplar. GCB Bioenergy 11:118–151. https://d-oi.org/10.1111/gcbb.12566 12. Dong H, Thames S, Liu L, et al (2015) QTL mapping for reproductive maturity in low-land switchgrass populations. Bioenergy Res 8:1925–1937. https://doi.org/10.1007/s12155-015-9651-9 12. Dong H, Thames S, Liu L, et al (2015) QTL mapping for reproductive maturity in low-land switchgrass populations. Bioenergy Res 8:1925–1937. https://doi.org/10.1007/s12155-015-9651-9 13. Dong H, Liu S, Clark LV, et al (2018) Genetic mapping of biomass yield in three inte-rconnected Miscanthus populations. GCB Bioenergy 10:165–185. https://doi.org/10.1111/gcbb.12472 13. Dong H, Liu S, Clark LV, et al (2018) Genetic mapping of biomass yield in three inte-rconnected Miscanthus populations. GCB Bioenergy 10:165–185. https://doi.org/10.1111/gcbb.12472 14. Dong H, Clark LV, Lipka AE, et al (2019a) Winter hardiness of Miscanthus (III): Ge-nome-wide association and genomic prediction for overwintering abili in Misc-anthus sinensis. GCB Bioenergy 11:930–955. https://doi.org/10.1111/gcbb.12615 14. Dong H, Clark LV, Lipka AE, et al (2019a) Winter hardiness of Miscanthus (III): Ge-nome-wide association and genomic prediction for overwintering ability in Misc-anthus sinensis. GCB Bioenergy 11:930–955. https://doi.org/10.1111/gcbb.12615 15. Dong H, Liu S, Clark LV, et al (2019b) Winter hardiness of Miscanthus (II): Genetic mapping for overwintering ability and adaptation traits in three interconnected Miscanthus populations. GCB Bioenergy 11:706–726. https://doi.org/10.1111/gcbb.12587 15. Dong H, Liu S, Clark LV, et al (2019b) Winter hardiness of Miscanthus (II): Genetic mapping for overwintering ability and adaptation traits in three interconnected Miscanthus populations. GCB Bioenergy 11:706–726. https://doi.org/10.1111/gcbb.12587 16. References East EM (1936) Heterosis. Genetics 21(4): 375–397. https://doi.org/10.1007/BF0298-2541 17. Feltus FA, Hart GE, Schertz KF, et al (2006) Alignment of genetic maps and QTLs be-tween inter- and intra-specific sorghum populations. Theor Appl Genet 112:129-5–1305 17. Feltus FA, Hart GE, Schertz KF, et al (2006) Alignment of genetic maps and QTLs be-tween inter- and intra-specific sorghum populations. Theor Appl Genet 112:129-5–1305 18. Gallais A (1988) Heterosis: its genetic basis and its utilization in plant breeding. Euphytica 39(2):95-104. https://doi.org/10.1007/BF00039859 19 G iffi B (1990) U f t ll d t i t i t t t t h t i h th G ti 126(3) 753 18. Gallais A (1988) Heterosis: its genetic basis and its utilization in plant breeding. Euphytica 39(2):95-104. https://doi.org/10.1007/BF00039859 18. Gallais A (1988) Heterosis: its genetic basis and its utilization in plant breeding. Euphytica 39(2):95-104. https://doi.org/10.1007/BF00039859 18. Gallais A (1988) Heterosis: its genetic basis and its utilization in plant breeding. Euphytica 39(2):95-104. ht 9. Griffing B (1990) Use of a controlled-nutrient experiment to test heterosis hypotheses. Genetics 126(3):753. 0. Huang X, Yang S, Gong J, et al (2016) Genomic architecture of heterosis for yield traits in rice. Nature 537:629– 20. Huang X, Yang S, Gong J, et al (2016) Genomic architecture of heterosis for yield traits in rice. Nature 537:629–633. https://doi.org/10.1038/nature19760 20. Huang X, Yang S, Gong J, et al (2016) Genomic architecture of heterosis for yield traits in rice. Nature 537:629–633. https://doi.org/10.1038/nature19760 1. Lippman ZB, Zamir D (2007) Heterosis: revisiting the magic. Trends in Genetics 23: 60–66. https://doi.org /10. 22. Liu L, Wu Y (2012) Identification of a selfing compatible genotype and mode of inheritance in switchgrass. Bioenergy Res 5:662–668. https://doi.org/10.1007/s12155-011-9173-z 22. Liu L, Wu Y (2012) Identification of a selfing compatible genotype and mode of inheritance in switchgrass. Bioenergy Res 5:662–668. https://doi.org/10.1007/s12155-011-9173-z 23. Liu L, Wu Y, Wang Y, et al (2012) A high-density simple sequence repeat-based gene-tic linkage map of switchgrass. G3 Genes, Genomes, Genet 2:357– 370. https://d-oi.org/10.1534/g3.111.001503 23. Liu L, Wu Y, Wang Y, et al (2012) A high-density simple sequence repeat-based gene-tic linkage map of switchgrass. G3 Genes, Genomes, Genet 2:357– 370. https://d-oi.org/10.1534/g3.111.001503 24. Lovell JT, MacQueen AH, Mamidi S, et al (2021) Genomic mechanisms of climate a-daptation in polyploid bioenergy switchgrass. Nature 590, 438–444. https://doi.o-rg/10.1038/s41586-020-03127-1 24. Lovell JT, MacQueen AH, Mamidi S, et al (2021) Genomic mechanisms of climate a-daptation in polyploid bioenergy switchgrass. Nature 590, 438–444. https://doi.o-rg/10.1038/s41586-020-03127-1 25. References Page 7/14 1. Ali S, Serba DD, Jenkins J, et al (2019) High-density linkage map reveals QTL under-lying growth traits in AP13×VS16 biparental population of switchgrass. GCB Bi-oenergy 11:672–690. https://doi.org/10.1111/gcbb.12592 1. Ali S, Serba DD, Jenkins J, et al (2019) High-density linkage map reveals QTL under-lying growth traits in AP13×VS16 biparental population of switchgrass. GCB Bi-oenergy 11:672–690. https://doi.org/10.1111/gcbb.12592 2. Bates D, Mächler M, Bolker B, Walker SC (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67 2. Bates D, Mächler M, Bolker B, Walker SC (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67:1–48 3. Bhandari HS, Saha MC, Mascia PN, et al (2010) Variation among half-sib families and heritability for biomass yield and other traits in lowland switchgrass (Panicum virgatum L.). Crop Sci 50:2355–2363. https://doi.org/10.2135/cropsci2010.02.0109 3. Bhandari HS, Saha MC, Mascia PN, et al (2010) Variation among half-sib families and heritability for biomass yield and other traits in lowland switchgrass (Panicum virgatum L.). Crop Sci 50:2355–2363. https://doi.org/10.2135/cropsci2010.02.0109 4. Bhandari HS, Saha MC, Fasoula VA, et al (2011) Estimation of genetic parameters f-or biomass yield in lowland switchgrass (Panicum virgatum L.). Sci 51:1525–1533. doi:10.2135/cropsci2010.10.0588. 4. Bhandari HS, Saha MC, Fasoula VA, et al (2011) Estimation of genetic parameters f-or biomass yield in lowland switchgrass (Panicum virgatum L.). Crop Sci 51:1525–1533. doi:10.2135/cropsci2010.10.0588. on JH (2007) Molecular breeding of switchgrass for use as a biofuel crop. Curr. Opin. Genet. Dev. 17(6):553–558 5. Bouton JH (2007) Molecular breeding of switchgrass for use as a biofuel crop. Curr. Opin. Genet. Dev. 17(6 6. Casler MD (2010) Changes in mean and genetic variance during two cycles of within-family selection in switchgrass. Bioenergy Res 3:47–54. https://doi.org/10.1007/s12155-009-9071-9 6. Casler MD (2010) Changes in mean and genetic variance during two cycles of within-family selection in switchgrass. Bioenergy Res 3:47–54. https://doi.org/10.1007/s12155-009-9071-9 7. Chang D, Wu Y, Liu L, et al (2016) Quantitative trait loci mapping for tillering-related traits in two switchgrass populations. Plant Genome 9:1–12. https://doi.org/10.3835/plantgenome2016.01.0010 7. Chang D, Wu Y, Liu L, et al (2016) Quantitative trait loci mapping for tillering-related traits in two switchgrass populations. Plant Genome 9:1–12. https://doi.org/10.3835/plantgenome2016.01.0010 8. Chen G, Wang Q, Liu YY, et al (2012) Modelling analysis for enhancing seed vigour of switchgrass (Panicum virgatum L.) using an ultrasonic technique. Biomass an-d Bioenergy 47:426–435. https://doi.org/10.1016/j.biombioe.2012.09.015 8. References Pauli D, Andrade-Sanchez P, Carmo-Silva AE, et al (2016) Field-based high-through-put plant phenotyping reveals the temporal patterns of quantitative trait loci associated with stress-responsive traits in cotton. G3 6(4): 865-879. https://doi.org/10.1534/g3.115.023515 33. Pauli D, Andrade-Sanchez P, Carmo-Silva AE, et al (2016) Field-based high-through-put plant phenotyping reveals the temporal patterns of quantitative trait loci associated with stress-responsive traits in cotton. G3 6(4): 865-879. https://doi.org/10.1534/g3.115.023515 34. Redfearn DD, Moore KJ, Vogel KP, et al (1997) Canopy architecture and morphology of switchgrass populations differing in forage yield. Agron J 89:262– 269. https://doi.org/10.2134/agronj1997.00021962008900020018x 35. Rimi F, Macolino S, Leinauer B et al (2011) Green-up of seeded bermudagrass cultiv-ars as influenced by spring scalping. Horttechnology 21:230–235. https://doi.or-g/10.21273/horttech.21.2.230 36. Rose IV LW, Das MK, Taliaferro CM (2008) Estimation of genetic variability and he-ritability for biofuel feedstock yield in several populations of switchgrass. Ann Appl Biol 152:11–17. https://doi.org/10.1111/j.1744-7348.2007.00186.x 37. Sadeghpour A, Hashemi M, DaCosta M, et al (2014a) Switchgrass establishment infl-uenced by cover crop, tillage systems, and weed control. Bioenergy Res 7:1402–1410. https://doi.org/10.1007/s12155-014-9485-x 38. Sadeghpour A, Hashemi M, Herbert SJ (2014b) A simple vigor test for adjusting swit-chgrass seeding rate in marginal and fertile soils. Grassl Sci 60:252– 255. https://doi.org/10.1111/grs.12066 39. Sanderson MA, Reed RL, McLaughlin SB, et al (1996) Switchgrass as a sustainable b-ioenergy crop. Bioresource Technology 56: 83–93. https://doi.org/10.1016/0960-8524(95)00176-X 40. Sarath G, Baird LM, Mitchell RB (2014) Senescence, dormancy and tillering in pere-nnial C4 grasses. Plant Sci 217–218(1):140–151. https://doi.org/10.1016/j.plantsci.2013.12.012 41. Schnell FW, Cockerham CC (1992) Multiplicative vs. arbitrary gene action in heteros-is. Genetics 131:461-469. https://doi.org/10.1016/1050- 3862(92)90005-P 42. Serba D, Daverdin G, Bouton JH, Devos KM, et al (2015) Quantitative trait loci(QTL) underlying biomass yield and plant height in switchgrass. BioEnerg Res 8:307–324. doi:10.1007/s12155-014-9523-8. 43. Sladden SE, Bransby DI, Aiken GE (1991) Biomass yield, composition and production costs for eight switchgrass varieties in Alabama. Biomass and Bioenergy 1:1-19–122. https://doi.org/10.1016/0961-9534(91)90034-A 4. Taylor M, Tornqvist CE, Zhao X, et al (2018) Genome-wide association study in pseu-do-F2 populations of switc 44. Taylor M, Tornqvist CE, Zhao X, et al (2018) Genome-wide association study in pseu-do-F2 populations of switchgrass identifies genetic loci affecting heading and an-thesis dates. Front Plant Sci 9:. https://doi.org/10.3389/fpls.2018.01250 44. Taylor M, Tornqvist CE, Zhao X, et al (2018) Genome-wide association study in pseu-do-F2 populations of switchgrass identifies genetic loci affecting heading and an-thesis dates. Front Plant Sci 9:. https://doi.org/10.3389/fpls.2018.01250 heading and an-thesis dates. Front Plant Sci 9:. https://doi.org/10.3389/fpls.2018.01250 45. References Lowry DB, Taylor SH, Bonnette J, et al (2015) QTLs for biomass and developmental traits in switchgrass (Panicum virgatum). Bioenergy Res 8:1856– 1867. https://d-oi.org/10.1007/s12155-015-9629-7 25. Lowry DB, Taylor SH, Bonnette J, et al (2015) QTLs for biomass and developmental traits in switchgrass (Panicum virgatum). Bioenergy Res 8:1856– 1867. https://d-oi.org/10.1007/s12155-015-9629-7 26. Lowry DB, Lovell JT, Zhang L, et al (2019) QTL × environment interactions underlie adaptive divergence in switchgrass across a large latitudinal gradient Proc Natl Acad Sci U S A 116:12933–12941. https://doi.org/10.1073/pnas.1821543116 26. Lowry DB, Lovell JT, Zhang L, et al (2019) QTL × environment interactions underlie adaptive divergence in switchgrass across a large latitudinal gradient. Proc Natl Acad Sci U S A 116:12933–12941. https://doi.org/10.1073/pnas.1821543116 27. Lowry, DB, Lovell JT, Zhang L, et al (2019) QTL x environment interactions underlie adaptive divergence in switchgrass across a large latitudinal gradient. Proceedings of the National Academy of Sciences of the United States of America. https://doi.org/10.1073/pnas.1821543116 27. Lowry, DB, Lovell JT, Zhang L, et al (2019) QTL x environment interactions underlie adaptive divergence in switchgrass across a large latitudinal gradient Proceedings of the National Academy of Sciences of the United States of America. https://doi.org/10.1073/pnas.1821543116 27. Lowry, DB, Lovell JT, Zhang L, et al (2019) QTL x environment interactions underlie adaptive divergence in switchgrass across a large latitudinal gradient. P di f th N ti l A d f S i f th U it d St t f A i htt //d i /10 1073/ 1821543116 28. Lynd LR, Cushman JH, Nichols RJ, et al (1991) Fuel etanol from cellulosic biomass. Science 251:1318–1323. https://doi.org/10.1126/science.251.4999.1318 29. Makaju SO, Wu Y, Anderson MP, et al (2018) Yield-height correlation and QTL local-ization for plant height in two lowland switchgrass populations. Front Agric Sci Eng 5:118–128. https://doi.org/10.15302/J-FASE-2018201 30. Munshaw GC, Ervin EH, Shang C, et al (2006) Influence of late-season iron, nitrogen, and seaweed extract on fall color retention and cold tolerance of four bermudagrass cultivars. Crop Sci 46:272–283. https://doi.org/10.2135/cropsci2005.0078 31. Okada M, Lanzatella C, Saha MC et al (2010) Complete switchgrass genetic maps reveal subgenome collinearity, preferential pairing and multilocus interactions. Genetics 185(3): 745-760. https://doi.org/10.1534/genetics.110.113910 Page 8/14 Page 8/14 32. Parrish DJ, Fike JH (2005) The biology and agronomy of switchgrass for biofuels. CRC Crit Rev Plant Sci 24:423–459. https://doi.org/10.1080/07352680500316433 32. Parrish DJ, Fike JH (2005) The biology and agronomy of switchgrass for biofuels. CRC Crit Rev Plant Sci 24:423–459. https://doi.org/10.1080/07352680500316433 33. References Impacts on the Agricultural Sector . Environ Resource Econ 24:313–333. https://doi.org/10.1023/A:102362 53. Wang D, Lebauer DS, Dietze MC (2010) A quantitative review comparing the yield of switchgrass in monocultures and mixtures in relation to climate and manageme-nt factors. GCB Bioenergy 2:16–25. https://doi.org/10.1111/j.1757-1707.2010.01035.x 53. Wang D, Lebauer DS, Dietze MC (2010) A quantitative review comparing the yield of switchgrass in monocultures and mixtures in relation to climate and manageme-nt factors. GCB Bioenergy 2:16–25. https://doi.org/10.1111/j.1757-1707.2010.01035.x 54. Wang YW, Samuels TD, Wu YQ (2011) Development of 1,030 genomic SSR markers in switchgrass. Theoretical and Applied Genetics 122: 677-686. https://doi.org/10.1007/s00122-010-1477-4 54. Wang YW, Samuels TD, Wu YQ (2011) Development of 1,030 genomic SSR markers in switchgrass. Theoretical and Applied Genetics 122: 677-686. https://doi.org/10.1007/s00122-010-1477-4 55. Williams W (1959) Heterosis and the Genetics of Complex Characters. Nature 184:5-27–530 . https://doi.org/10.1038/184527a0 55. Williams W (1959) Heterosis and the Genetics of Complex Characters. Nature 184:5-27–530 . https://doi.or 55. Williams W (1959) Heterosis and the Genetics of Complex Characters. Nature 184:5-27–530 . https://doi.org/10.1038/184527a0 56. Wullschleger SD, Davis EB, Borsuk ME, et al (2010) Biomass production in switchg-rass across the United States: Database description and determinants of yield. Ag-ron J 102:1158–1168. https://doi.org/10.2134/agronj2010.0087 56. Wullschleger SD, Davis EB, Borsuk ME, et al (2010) Biomass production in switchg-rass across the United States: Database description and determinants of yield. Ag-ron J 102:1158–1168. https://doi.org/10.2134/agronj2010.0087 57. Würschum T, Liu W, Busemeyer L, et al (2014) Mapping dynamic QTL for plant heig-ht in triticale. BMC Genomic Data 15:59. https://doi.org/10.1186/1471-2156-15-59 57. Würschum T, Liu W, Busemeyer L, et al (2014) Mapping dynamic QTL for plant heig-ht in triticale. BMC Genomic Data 15:59. https://doi.org/10.1186/1471-2156-15-59 58. Xu Y, Li Y, Qiu Y (2021) Growth dynamics and heritability for plant high-throughput phenotyping studies using hierarchical functional data analysis. Biometrical Jou-rnal, https://doi.org/10.1002/bimj.202000315 58. Xu Y, Li Y, Qiu Y (2021) Growth dynamics and heritability for plant high-throughput phenotyping studies using hierarchical functional data analysis. Biometrical Jou-rnal, https://doi.org/10.1002/bimj.202000315 59. Yu J, Holland JB, McMullen MD, Buckler ES, et al (2008) Genetic design and statisti-cal power of nested association mapping in maize. Genetics 178:539–551. https://doi.org/10.1534/genetics.107.074245 59. Yu J, Holland JB, McMullen MD, Buckler ES, et al (2008) Genetic design and statisti-cal power of nested association mapping in maize. Genetics 178:539–551. https://doi.org/10.1534/genetics.107.074245 60. Zhang L, Weng X, Behrman K, et al (2020) QTL x environment interactions for panic-le traits in switchgrass (Panicum virgatum). https://doi.org/10.22541/au.160460109.97406017/v1 60. References Taylor M, Tornqvist CE, Zhao X, et al (2019) Identification of quantitative trait loci f-or plant height, crown diameter, and plant biomass in a pseudo-F2 population of 45. Taylor M, Tornqvist CE, Zhao X, et al (2019) Identification of quantitative trait loci f-or plant height, crown diameter, and plant biomass in a pseudo-F2 population of 46. switchgrass. Bioenergy Res 12:267–274. https://doi.org/10.1007/s12155-019-09978-5 46. switchgrass. Bioenergy Res 12:267–274. https://doi.org/10.1007/s12155-019-09978-5 46. switchgrass. Bioenergy Res 12:267–274. https://doi.org/10.1007/s12155-019-09978-5 48. Tobias CM, Sarath G, Twigg P et al (2008) Comparative genomics in switchgrass using 61,585 high-quality expressed sequence tags. The Plant Genom 1(2). https://doi.org/10.3835/plantgenome2008.08.0003 48. Tobias CM, Sarath G, Twigg P et al (2008) Comparative genomics in switchgrass using 61,585 high-quality expressed sequence tags. The Plant Genome 1(2). https://doi.org/10.3835/plantgenome2008.08.0003 49. Tornqvist CE, Taylor M, Jiang Y, et al (2018) Quantitative trait locus mapping for flo-wering time in a lowland × upland switchgrass pseudo-F2 populatio Plant Gen-ome 11:1–9. https://doi.org/10.3835/plantgenome2017.10.0093 49. Tornqvist CE, Taylor M, Jiang Y, et al (2018) Quantitative trait locus mapping for flo-wering time in a lowland × upland switchgrass pseudo-F2 population. Plant Gen-ome 11:1–9. https://doi.org/10.3835/plantgenome2017.10.0093 50. Van Ooijen JW (2009) MapQTL®6. Software for the mapping of quantitative trait lo-ci in experimental populations of diploid species. Kyazma B V 0. Van Ooijen JW (2009) MapQTL®6. Software for the mapping of quantitative trait lo-ci in experimental populat 50. Van Ooijen JW (2009) MapQTL®6. Software for the mapping of quantitative trait lo-ci in experimental populations of diploid species. Kyazma B V 50. Van Ooijen JW (2009) MapQTL®6. Software for the mapping of quantitative trait lo-ci in experimental populations of diploid species. Kyazma B V 51 Walsh ME de la Torre Ugarte DG Shapouri H et al (2003) Bioenergy Crop Producti-on in the United States: Potential Quantities Land Use Changes and 50. Van Ooijen JW (2009) MapQTL®6. Software for the mapping of quantitative trait lo-ci in experimental populations of diploid species. Kyazma B V 51. Walsh ME, de la Torre Ugarte DG, Shapouri H, et al (2003). Bioenergy Crop Producti-on in the United States: Potential Quantities, Land Use Changes, and Economic 51. Walsh ME, de la Torre Ugarte DG, Shapouri H, et al (2003). Bioenergy Crop Producti-on in the United States: Potential Quantities, Land Use Changes, and Economic 51. Walsh ME, de la Torre Ugarte DG, Shapouri H, et al (2003). Bioenergy Crop Producti-on in the United States: Potential Quantities, Land Use Changes, and Economic 52. References Zhang L, Weng X, Behrman K, et al (2020) QTL x environment interactions for panic-le traits in switchgrass (Panicum virgatum). G: genotype, G×A: genotype by year interaction, G×L: genotype by location interaction, E: error Table 4 QTL detection for spring green-up and plant vigor in a hybrid population in lowland switchgrass References https://doi.org/10.22541/au.160460109.97406017/v1 Tables Tables Page 9/14 Table 1 Mean and variation parameters of spring green-up and plant vigor in two switchgrass mapping populations Population Environment Spring green-up Plant vigor Plant biomass (g/plant) NL94 SL93 Mean Min Max SD NL94 SL93 Mean Min Max SD NL94 SL93 Mea Hybrid 2012PKS 69 92 81 10 100 14.36 5 7 7 2 9 1.44 943.67 1377 138 2012STW 67 90 78 12 100 17.32 5 6 7 2 9 1.31 865.33 976.33 140 2013PKS 90 13 56 5 100 20.50 5 7 8 1 9 1.58 843 1460.67 176 2013STW 57 63 67 5 100 21.12 5 7 8 1 9 1.47 1267 1220.33 139 2014PKS 32 30 35 10 90 12.25 5 7 8 1 9 1.55 - - - 2014STW 37 43 37 1 90 15.52 5 7 6 2 9 1.63 - - - 2015PKS 42 35 50 1 100 18.56 5 7 7 2 9 1.45 - - - 2015STW 32 38 36 1 95 16.11 5 7 7 1 9 1.45 - - - Selfed 2012PKS 69 - 44 5 100 20.01 5 - 4 1 8 1.54 943.67 - 356 2012STW 67 - 37 2 100 21.87 5 - 3 1 7 1.11 865.33 - 366 2013PKS 90 - 29 5 90 20.11 5 - 3 1 9 1.33 843 - 529 2013STW 57 - 31 5 100 21.33 5 - 3 1 8 1.11 1267 - 456 2014PKS 32 - 20 1 80 12.26 5 - 3 1 9 1.57 - - - 2014STW 37 - 17 1 70 10.47 5 - 3 1 8 1.11 - - - 2015PKS 42 - 27 1 90 18.92 5 - 4 1 9 1.35 - - - 2015STW 32 - 17 1 70 12.43 5 - 4 1 9 1.23 - - - raits in a hybrid population (above diagonal) and a selfed population (below diagonal) in lowland switchgrass Table 2 Correlation coefficients among traits in a hybrid population (above diagonal) and a selfed population (below diagonal) in lowland switchgrass Trait Spring green-up Plant vigor Biomass yield Spring green-up - 0.05NS† -0.07 Plant vigor 0.31 - 0.30 Biomass yield 0.09 0.26** - Table 2 Correlation coefficients among traits in a hybrid population (above diagonal) and a selfed population (below diagonal) in lowland switchgrass Table 2 Correlation coefficients among traits in a hybrid population (above diagonal) and a selfed population (b nts among traits in a hybrid population (above diagonal) and a selfed population (below diagonal) in lowland sw tability of spring green-up and plant vigor across joint environments in two lowland switchgrass populations. References Table 3 Broad-sense heritability of spring green-up and plant vigor across joint environments in two lowland sw Table 3 Broad-sense heritability of spring green-up and plant vigor across joint environments in two lowland switchgrass populations. Table 5 QTL detection for spring green-up and plant vigor in a selfed population in lowland switchgrass AD, BC, BD represent the additive effects of the deviation between genotype AD, BC or BD and genotype AC; PVE: phenotypic variance explanation; JE: joint environments; Position represents the peak of QTL. Table 5 QTL detection for spring green-up and plant vigor in a selfed population in lowland switchgrass able 5 QTL detection for spring green-up and plant vigor in a selfed population in lowland switchgrass References VE: Phenotypic variance explanation; JE: Joint environments; Position represents the peak of QTL. References Variance & heritability estimation Hybrid Population Selfed Population Spring green-up Plant vigor Plant biomass Spring green-up Plant vigor Plant biomass σ2 G 19.22 0.51 215342 34.02 0.33 55114 σ2 G×A 32.18 0.03 5279 38.72 0.03 1351 σ2 G×L 1.70 0.11 213163 1.19 0.18 54561 σ2 E 231.40 1.56 215342 234.54 1.24 55119 H 0.51 0.80 0.63 0.63 0.69 0.63 G: genotype, G×A: genotype by year interaction, G×L: genotype by location interaction, E: error Table 4 QTL detection for spring green-up and plant vigor in a hybrid population in lowland switchgrass Page 10/14 Page 10/14 Page 10/14 Trait Environment LG Position (cM) LOD Marker interval AD BC BD PVE (％) Spring green-up 2012PKS 9a 21.98 4.90 nfsg-137 PVAAG-3091/3092 11.38 9.84 2.17 9.6 2012STW 1a 78.64 4.19 sww-1615 sww-333 -16.78 -3.84 -4.99 6.5 2012STW 5a 42.44 3.84 PVGA-1971/1972 PVCAG-2197/2198 -7.94 -1.09 -5.87 6.0 2012STW 6b 15.16 4.77 sww-1813 sww-1749 -13.46 -9.36 -6.21 7.5 2012STW 9a 15.98 5.98 sww-463 nfsg-137 3.02 10.20 3.35 9.9 2013PKS 2b 24.05 5.23 PVE-413/414 PVE-1143/1144 4.39 12.77 10.73 9.5 2013PKS 5a 6.00 5.26 PVCAG-2389/2390 PVCAG-2167/2168 2.69 -6.67 7.10 9.6 2013PKS 9a 39.88 4.53 PVAAG-3091/3092 PVCAG-2517/2518 5.04 7.63 12.81 8.3 2014PKS 1a 61.04 4.00 PVCAG-2379/2380 sww-1615 -0.37 3.41 4.73 6.7 2014PKS 2a 39.49 6.28 sww-532 nfsg-052 -7.98 -0.51 -3.28 11.1 2014PKS 2b 33.05 5.64 PVE-1143/1144 sww-2662 -7.42 -0.03 0.20 9.7 2014STW 1a 43.92 5.10 PVGA-2107/2108 PVGA-2119/2120 3.78 -1.89 8.62 11.8 2015PKS 2a 36.49 4.37 sww-532 nfsg-052 -13.40 -9.73 -13.78 9.9 2015STW 1a 13.81 3.64 sww-1667 PVGA-1253/1254 4.53 9.43 0.15 7.0 2015STW 1b 62.21 4.76 PVGA-1401/1402 PVCA-179/180 16.22 -1.34 16.32 9.3 2015STW 2a 73.15 4.13 sww-2545 PVGA-1719/1720 -4.04 1.23 -7.76 8.1 JE 2a 40.49 3.70 sww-532 nfsg-052 -6.24 -2.42 -3.66 7.2 Plant vigor 2012STW 5a 38.44 3.81 PVGA-1971/1972 PVCAG-2197/2198 -0.55 0.21 0.12 8.5 2013STW 9a 40.88 4.08 PVCAG-2517/2518 PVGA-1513/1514 -0.81 1.46 -0.15 8.3 2014PKS 5a 40.44 5.70 PVGA-1971/1972 PVCAG-2197/2198 -0.92 0.06 0.31 12.0 2014STW 1a 0.00 3.92 PVCAG-2537/2538 sww-1667 -0.28 -0.91 0.23 8.7 2014STW 5a 62.31 5.58 PVGA-1813/1814 PVGA-1357/1358 0.28 0.93 0.82 12.1 2015PKS 5a 61.96 6.01 PVGA-1813/1814 PVGA-1357/1358 -0.65 0.40 0.21 12.6 2015PKS 9a 44.53 4.41 PVGA-1513/1514 PVCA-863/864 0.92 0.42 0.15 9.0 2015STW 1a 39.68 3.81 PVE-1361/1362 PVGA-2107/2108 -1.75 -0.26 0.61 7.7 JE 5a 60.96 4.70 PVGA-1813/1814 PVGA-1357/1358 -0.07 0.54 0.45 9.8 Plant biomass 2012PKS 5a 47.37 5.69 PVCAG-2197/2198 PVGA-1649/1650 -333.69 136.75 21.08 13.8 2012STW 5a 38.44 4.21 PVGA-1971/1972 PVCAG-2197/2198 -89.47 191.37 125.27 9.9 2013PKS 5a 41.44 3.75 PVGA-1971/1972 PVCAG-2197/2198 -181.66 49.64 222.22 8.2 2013STW 5a 34.46 3.80 PVCAG-2167/2168 PVGA-1971/1972 38.05 209.82 233.74 7.7 JE 5a 34.46 5.53 PVCAG-2167/2168 PVGA-1971/1972 -117.14 155.53 183.36 13.4 D, BC, BD represent the additive effects of the deviation between genotype AD, BC or BD and genotype AC; PVE: phenotypic variance explanation; JE: joint Page 11/14 Trait Environment LG Position (cM) LOD Marker interval Additive PVE（％） Spring green-up 2012PKS 6b-1 37.44 3.53 PVAAG-3017/3018 PVGA-1115/1116 -5.30 6.3 2012STW 6b-1 38.44 9.16 PVAAG-3017/3018 PVGA-1115/1116 -5.76 12.7 2012STW 9a 64.10 4.24 sww-2285 PVGA-1405/1406 -5.37 5.8 2015PKS 9a 79.39 4.35 PVCAG-2487/2488 PVCA-19/20 5.96 7.1 2015STW 2b 35.18 4.23 sww-2501 PVCA-597/598 -3.06 7.1 JE 6b-1 38.44 5.25 PVAAG-3017/3018 PVGA-1115/1116 -3.79 8.3 Plant vigor 2012PKS 2b 57.75 3.94 sww-83M PVCAG-2647/2648 0.14 7.4 2013PKS 2a 20.00 4.02 PVCA-815/816 nfsg-50 0.08 6.7 2014PKS 2b 25.34 4.77 sww-2545 sww-1622 0.01 8.3 2014STW 2a 17.00 3.54 PVCA-815/816 nfsg-50 0.14 5.5 2015STW 9b 40.96 3.62 PVAAG-2901/2902 sww-166ˍ200 -0.19 6.1 JE 2a 20.00 4.39 PVCA-815/816 nfsg-50 0.03 6.5 Plant biomass 2013PKS 2b 31.63 4.15 sww-1622 sww-2501 10.64 7.9 notypic variance explanation; JE: Joint environments; Position represents the peak of QTL. Figure 3 Significant QTLs for spring green-up, plant vigor, and plant biomass in the hybrid population and selfed population of lowland switchgrass. Supplementary Files Figures Page 12/14 Figure 1 Figure 1 Page 12/14 Frequency distributions of spring green-up and plant vigor in a hybrid population of lowland switchgrass across four years (2012-2015) as well as plant biomass across two years (2012-2013) at two locations [Perkins (PKS) and Stillwater (STW), OK]. Frequency distributions of spring green-up and plant vigor in a hybrid population of lowland switchgrass across four years (2012-2015) as well as plant biomass across two years (2012-2013) at two locations [Perkins (PKS) and Stillwater (STW), OK]. Figure 2 Frequency distributions of spring green-up and plant vigor in a lowland switchgrass selfed population across four years (2012-2015) as well as plant biomass across two years (2012-2013) at two locations [Perkins (PKS) and Stillwater (STW), OK]. Figure 2 Frequency distributions of spring green-up and plant vigor in a lowland switchgrass selfed population across four years (2012-2015) as well as plant biomass across two years (2012-2013) at two locations [Perkins (PKS) and Stillwater (STW), OK]. Page 13/14 Page 13/14 Page 13/14 Figure 3 Significant QTLs for spring green-up, plant vigor, and plant biomass in the hybrid population and selfed population of lowland switchgrass. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. ESM1.docx Figure 3 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. Page 14/14 Page 14/14
https://openalex.org/W4318601660	https://biblio.ugent.be/publication/01GQ78F01E56J3MF15V4S1HKQV/file/01GQ78MHPR39MD51ZYJ4THE969	English	null	Time Is Money in Case of a Button Battery Ingestion	JPGN reports	2,022	cc-by	2,317	CASE REPORT Abstract: Button battery (BB) ingestion is a preventable pediatric health hazard with important morbidity and mortality due to complications. We present 3 pediatric patients with a complicated course after BB ingestion and discuss current guidelines. Urgent endoscopic removal is necessary for every BB impacted in the esophagus. A new strategy before endo- scopic removal is the administration of honey or sucralfate. During endos- copy, rinsing the esophageal mucosae with acetic acid can neutralize the alkalic environment and prevent late complications. Prevention of inges- tion needs to be pursued by increasing awareness and changing legislation of packaging of BB. Case 1 A previously healthy 20 months old girl presented with dys- phagia for 6 months. She was only able to drink or eat mixed foods, but no solids. She suffered from chronic rhonchi starting in the same period. Physical examination showed no respiratory distress. There were stagnating saliva in the throat and a poor weight gain since 6 months. Gastroscopy revealed a BB below the upper esophageal sphincter. After BB removal, a food-containing diverticulum and dis- tal stenosis were visualized. She was tube fed, received antibiotics and a proton pump inhibitor (PPI). The diverticulum was confirmed on an esophagogram (Fig. 1). She was discharged after 2 weeks with tube feeding. It remained necessary until the esophageal diameter allowed liquid and pureed food to pass. Repeated endoscopic dilata- tions and an attempt to endoscopically treat the diverticulum were insufficient to resolve the feeding problems, therefore surgery will be planned. Key Words: foreign body ingestion, pediatrics, dysphagia, esophageal perforation Case 2 An ill-looking 2-year-old boy presented with fever and dys- phagia for solids. He was treated with antibiotics for otitis media. During hospitalization, he deteriorated with pallor and grunting. Laboratory studies revealed increased C-reactive protein (180 mg/L). Thorax radiograph showed a foreign body in the esophagus with a double halo sign on anterior-posterior view and a “step-off” sign on lateral view, that is, a BB (Fig. 2). Computed tomography showed no esophageal perforation but arguments for mediastinitis. Endoscopic removal of the BB was performed and showed necrosis at the esoph- ageal mucosa. Tube feeding was started. Pharmacological therapy consisted of continuing broad-spectrum antibiotics and starting PPI. An esophagogram 2 days later showed a minor perforation. After 1 week tube feeding, oral intake was resumed with pureed and liquid food and discharge after 2 weeks. Endoscopy 6 weeks later showed a non-stenotic esophageal scar. Morbidity and mortality as a result of BB ingestion are mainly the result of esophageal impaction (2). The alkaline esophageal environment increased size and power of the BBs and younger age of the child are associated with an increased esopha- geal impaction risk (2,3). The risk for severe complications further increases with the duration of impaction and remaining voltage of the BB (4). ( ) Based on 3 complicated cases over the past 6 months, presen- tation, diagnosis, and new guidelines will be discussed. INTRODUCTION The frequency of presentation to the emergency room after unintentional foreign body ingestion in children doubled over the past 13 years (1). The ingestion of a button battery (BB) is feared due to associated complications (2). A 7-fold increase of these complica- tions over the past 2 decades, places BB ingestion high on the list of preventable pediatric health hazards (3). Raising awareness among caregivers and general public is essential in the prevention and cor- rect handling of these accidents. Case Report: Gastroenterology Case Report: Gastroenterology Correspondence: Charlotte Bosschaert, MD, Division of Paediatric Gastroenter- ology and Nutrition, Department of Paediatrics, University Hospital Ghent, 3K12D, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: charlotte.boss- chaert@uzgent.be pl Informed patient consent was obtained for publication of the case details. From the Department of Pediatrics, Ghent University Hospital, Ghent Univer- sity, Ghent, Belgium; †Department of Pediatric Surgery, Ghent University Hospital, Ghent University, Ghent, Belgium; and ‡Department of Paedi- atric Intensive Care, Ghent University Hospital, Ghent University, Ghent, Belgium. l Key Words: foreign body ingestion, pediatrics, dysphagia, esophageal perforation @ g Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the North American Society for Pediatric Gastroenterology, Hep- atology, and Nutrition. This is an open access article distributed under the Cre- ative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Received July 7, 2022;accepted August 10, 2022. g The authors report no conflicts of interest. Case 3 Case 1: endoscopic view before and after button battery removal. Result of the esophagogram. FIGURE 2. Case 2: radiograph revealing the presence of a button battery with typical halo sign and posterior step-off in lateral view. tomography scan before removal, to evaluate possible complica- tions (4). In case of symptoms after removal of the battery, an magnetic resonance imaging scan can shed light on the underlying complication (4). As mucosal damage can occur within 2 hours after ingestion, immediate endoscopic removal is necessary for every BB impacted in the esophagus, even if the patient has eaten (4). During endoscopy, good inspection and localization of the negative pole improves the complication risk assessment. Based on small series, neutralization of the remaining hydroxide with acetic acid irrigation (50–150 mL 0.25% acetic acid [= 8 mL acetic acid 3% in 92 mL sterile water]) might reduce late complications. It is only advised in the absence of perforation (10). The majority of batteries located beyond the esoph- agus will pass spontaneously within 7–14 days. Radiograph tracking of the BB progression is advised in asymptomatic patients, unless the battery is detected in the stool. In case of a delayed diagnosis, endoscopic evaluation of the esophagus is advised as it is unknown how long the BB took to transit through the esophagus. Depending on the localization, endoscopic or surgical removal of the BB is rec- ommended if there is no progression or if the patient is experiencing symptoms (4). FIGURE 2. Case 2: radiograph revealing the presence of a button battery with typical halo sign and posterior step-off in lateral view. This episode was followed by repeated endoscopic dilatations with 1 session of steroid injection. This episode was followed by repeated endoscopic dilatations with 1 session of steroid injection. All patients with mucosal damage should be admitted for monitoring. A normal esophagogram after 1–2 days gives the green light for a liquid diet which, if well tolerated, can be expanded to soft food for another 4 weeks. A second look endoscopy after 2–4 days might be considered as it provides prognostic information (11). In case of severe mucosal injury or perforation, broad-spectrum antibi- otics are indicated to treat mediastinitis (4). The use of PPIs has not been studied. DISCUSSION As demonstrated by the clinical cases, timely diagnosis and treatment are essential to avoid complications. Current guidelines include new strategies before BB removal and during endoscopy to prevent complications. The authors provide a flow chart for sus- pected BB ingestion (Fig. 3) (4). During the time-lapse between ingestion and removal, the impact of a recent BB ingestion (<12 hours) in children can be limited using honey (above 12 months of age) or sucralfate (1 g/10 mL) (5). The advised dose for both is 10 mL (2 teaspoons) every 10 minutes with a maximum of 6 doses of honey and 3 doses of sucralfate (4). If no signs of perforation are present, this strategy can be explained to parents when they call the emergency ward. It should never be a reason to delay the endoscopy and the child should remain otherwise nil per os (6). The prevention of these major complications can only be obtained with increased awareness of public and health professionals and changing measures by the government and the industry (10,12). Of all BB ingestions, 70% can be avoided with screw secured com- partments, individual blisters, bitter-tasting BB, and covering one side of a BB (12). Case 3 Received July 7, 2022;accepted August 10, 2022. A 16-month-old girl ingested a foreign body. It was described as a coin on the radiograph. Endoscopic removal 8 hours after ingestion revealed a BB with mucosal injury. One day later, she developed fever with biochemical signs of infection. Com- puted tomography thorax showed a pneumonic infiltrate without signs of pneumomediastinum. Treatment with antibiotics resulted in clinical improvement. One week after BB removal, the patient presented with drooling and food refusal. Endoscopy revealed a tracheoesophageal fistula. Surgical repair of the tracheal perfora- tion with sternocleidomastoid muscle and suture of the esopha- geal perforation was performed. She remained ventilated for 8 days and was treated with PPI and broad-spectrum antibiotics for 3 weeks for mediastinitis. She had paresis of the vocal cords, pos- sibly due to recurrent laryngeal nerve injury. An esophagogram after 3 weeks showed a relapse of tracheoesophageal fistula which closed spontaneously. After 1 month, fluid intake was resumed and discharge home was possible after 5 weeks of hospitalization. From the Department of Pediatrics, Ghent University Hospital, Ghent Univer- sity, Ghent, Belgium; †Department of Pediatric Surgery, Ghent University Hospital, Ghent University, Ghent, Belgium; and ‡Department of Paedi- atric Intensive Care, Ghent University Hospital, Ghent University, Ghent, Belgium. Correspondence: Charlotte Bosschaert, MD, Division of Paediatric Gastroenter- ology and Nutrition, Department of Paediatrics, University Hospital Ghent, 3K12D, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: charlotte.boss- chaert@uzgent.be Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the North American Society for Pediatric Gastroenterology, Hep- atology, and Nutrition. This is an open access article distributed under the Cre- ative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the North American Society for Pediatric Gastroenterology, Hep- atology, and Nutrition. This is an open access article distributed under the Cre- ative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited ISSN: 2691-171X DOI: 10.1097/PG9.0000000000000259 p p y JPGN Reports (2022) 3:4(e259) 1 Case Report FIGURE 1. Case 1: endoscopic view before and after button battery removal. Result of the esophagogram. www.jpgnreports.org 7. Dörterler ME. Clinical profile and outcome of esophageal button battery ingestion in children: an 8-year retrospective case series. Emerg Med Int. 2019;2019:3752645. FIGURE 3. Diagnostic and therapeutic algorithm for button battery ingestions (4). CT, computed tomography; ENT, ear, nose and throat surgeon. FIGURE 3. Diagnostic and therapeutic algorithm for button battery ingestions (4). CT, computed tomography; ENT, ear, nose and throat surgeon. GURE 3. Diagnostic and therapeutic algorithm for button battery ingestions (4). CT, computed tomography; ENT, ear, nose d throat surgeon 7. Dörterler ME. Clinical profile and outcome of esophageal button battery ingestion in children: an 8-year retrospective case series. Emerg Med Int. 2019;2019:3752645. CONCLUSION As symptoms are only present in 30% of patients, they cannot be used to decide whether or not investigations are indicated (7,8). However, severe symptoms (fever, hematemesis, stridor, hoarse- ness, back pain) might be indicative of complications (4). The halo sign (double ring) on radiograph is a well-described item to recognize a BB (8). A lateral radiograph makes it pos- sible to locate the negative pole (the step-off site) and there- fore to anticipate possible complications (4). In case of a delayed diagnosis (>12 hours) (9) or symptoms compatible with complications, it is recommended to perform a computed BB ingestion is a preventable pediatric health hazard with increasing prevalence. Due to fistulization to surrounding tissues, complications are the main cause of morbidity and mortality. BB impaction in the esophagus is an emergency and requires urgent endoscopic removal. New strategies to prevent mucosal damage include administration of honey or sucralfate before endoscopic removal. During endoscopy, acetic acid irrigation of the esopha- geal mucosa might reduce late complications. Prevention by adjusting packaging and secure compartments needs to be pursued by legislation. 2 www.jpgnreports.org Case Report URE 3. Diagnostic and therapeutic algorithm for button battery ingestions (4). CT, computed tomography; ENT, ear, nose throat surgeon. REFERENCES 1. Speidel AJ, Wölfle L, Mayer B, et al. Increase in foreign body and harmful substance ingestion and associated complications in children: a retrospective study of 1199 cases from 2005 to 2017. BMC Pediatr. 2020;20:560. 8. Akilov KA, Asadullaev DR, Yuldashev RZ, et al. Cylindrical and button battery ingestion in children: a single-center experience. Pediatr Surg Int. 2021;37:1461–1466. 2. Varga Á, Kovács T, Saxena AK. Analysis of complications after button battery ingestion in children. Pediatr Emerg Care. 2018;34:443–446. 9. Soto PH, Reid NE, Litovitz TL. Time to perforation for button batteries lodged in the esophagus. Am J Emerg Med. 2019;37:805–809. 3. Litovitz T, Whitaker N, Clark L. Preventing battery ingestions: an analysis of 8648 cases. Pediatrics. 2010;125:1178–1183. 10. Jatana KR, Barron CL, Jacobs IN. Initial clinical application of tissue pH neu- tralization after esophageal button battery removal in children. Laryngoscope. 2019;129:1772–1776. 4. Mubarak A, Benninga MA, Broekaert I, et al. Diagnosis, management, and prevention of button battery ingestion in childhood: a European Society for Paediatric Gastroenterology Hepatology and Nutrition Position Paper. J Pediatr Gastroenterol Nutr. 2021;73:129–136. 11. Ruhl DS, Cable BB, Rieth KK. Emergent treatment of button batteries in the esophagus: evolution of management and need for close second-look esopha- goscopy. Ann Otol Rhinol Laryngol. 2014;123:206–213. 5. Anfang RR, Jatana KR, Linn RL, et al. In response to pH-neutralizing esophageal irrigations as a novel mitigation strategy. Laryngoscope. 2019;129:E163–E164. 12. Lahmar J, Célérier C, Garabédian EN, et al. Esophageal lesions follow- ing button-battery ingestion in children: Analysis of causes and propos- als for preventive measures. Eur Ann Otorhinolaryngol Head Neck Dis. 2018;135:91–94. 6. Jatana KR, Rhoades K, Milkovich S, et al. Basic mechanism of button battery ingestion injuries and novel mitigation strategies after diagnosis and removal. Laryngoscope. 2017;127:1276–1282. 3 www.jpgnreports.org
https://openalex.org/W3025945474	https://mediatum.ub.tum.de/doc/1651454/document.pdf	English	null	Degradation Mechanism of an IrO<sub>2</sub> Anode Co-Catalyst for Cell Voltage Reversal Mitigation Under Transient Operation Conditions of a PEM Fuel Cell	Meeting abstracts/Meeting abstracts (Electrochemical Society. CD-ROM)	2,020	cc-by	24,786	Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** One of the challenges that hinders the widespread commerciali- zation of fuel cell electric vehicles is meeting long-term durability targets.1 Among several conditions that can cause a substantial degradation of membrane electrode assemblies (MEAs), cell (vol- tage) reversal has recently received a great attention.2–4 Cell reversal can occur during the transient operation of proton exchange membrane fuel cells (PEMFCs) on account of a temporary under- supply of H2 to one or several cells in a fuel cell stack.2–5 During cell reversal events, the anode potential rises to ?1 V vs the reversible hydrogen electrode (RHE) potential, causing a transient dissolution of platinum and a continuous oxidation of the carbon support: reversal damages can be substantial even if the cell reversal only occurs over time scales of tens of milliseconds, the control system has to be able to detect cell reversals on a millisecond time scale and to respond very quickly to prevent MEA damage. Therefore, addition of an OER co-catalyst to the anode electrode would seem to be the most simple strategy to mitigate cell reversal damages, and a commonly used anode co-catalyst to facilitate the OER over the COR during cell reversal events is iridium oxide (IrO2).2,4,14,18–20 Numerous studies have investigated the effect of IrO2 co- catalysts on mitigating cell reversal degradation in PEMFC anodes. It is well known that the OER activity of an IrO2 catalyst reliably correlates with the effectiveness of the catalyst for mitigating cell reversal degradation in a PEMFC.20 The low onset potential of the OER on IrO2 prevents the anode potential from reaching high values during cell reversal, and thus signiﬁcantly reduces the extent of the COR at the anode side. Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** 1Chair of Technical Electrochemistry, Department of Chemistry and Catalysis Research Center, Technical University of Munich, 85748 Garching, Germany 2Greenerity GmbH 63457 Hanau Wolfgang Germany IrO2 is the most stable oxygen evolution reaction (OER) catalyst in acidic media and it has been widely used as co-catalyst to mitigate cell reversal damages in the anode of PEM fuel cells (PEMFCs). In this study, a mechanistic understanding of the degradation of an IrO2 anode co-catalyst under transient operation of a PEMFC is provided. Thermogravimetric analysis (TGA) in reductive atmosphere (3.3 vol.% H2/Ar) shows that IrO2 is not stable in H2 containing atmosphere at operational temperatures of PEMFCs. By conducting a series of physical-chemical and electrochemical analyses, it is proven that H2 under the operating conditions in a PEMFC anode can chemically reduce a few outer monolayers of the surface of IrO2 nanoparticles to metallic Ir. The metallic Ir formed on the IrO2 surface can then dissolve during fuel cell start-up/shut-down (SUSD) cycles. At least part of the dissolved Ir species formed in the anode electrode are shown to diffuse through the membrane to the cathode electrode, where they lead to a deterioration of the oxygen reduction reaction (ORR) activity of the Pt cathode catalyst. The consequences of Ir dissolution on the cell reversal tolerance of the anode are also discussed. © 2021 The Author(s). Published on behalf of The Electrochemical Society by IOP Publishing Limited. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited. [DOI: 10.1149/ 1945-7111/ac0d39] Manuscript submitted March 22, 2021; revised manuscript received June 10, 2021. Published June 28, 2021. zE-mail: m.fathi@tum.de Electrochemical Society Student Member. Electrochemical Society Fellow. Journal of The Electrochemical Society, 2021 168 064521 Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1,* Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** Therefore, the focus of the research in this ﬁeld is to attain highly OER-active IrO2 catalysts by different synthesis approaches and to modify the anode catalyst layer design such as to achieve a uniform dispersion of IrO2 in the anode catalyst layer.18–21 Typically, the mitigation performance of an anode OER co-catalyst is evaluated by simulated cell reversal tests (CRTs) in single-cell PEMFCs, in which a constant current (typically 0.2 Acm−2) is applied with air ﬂowing through the cathode compartment and N2 (rather than H2) ﬂowing through the anode compartment.5,22,23 Due to the lack of H2 on the anode electrode during the CRT, the cell potential vs time rapidly drops to large negative values in the beginning of the CRT and then reaches a nearly constant potential plateau whose value gradually decreases over time, until it results in a drastic potential drop that is considered to mark the complete degradation of the anode catalyst layer. The time spent until this ﬁnal potential drop is usually taken as a ﬁgure- of-merit to compare the performance of different anode co-catalysts during cell reversal. The negative cell potential during the CRT corresponds to the difference between the positive cathode potential that is established at the cell current by the ORR on the cathode catalyst and the more positive anode potential at which the cell current can be supplied by the OER and the COR reaction at the anode. Thus, the higher the OER activity of the anode co-catalyst, the less positive will be the anode potential during the CRT, + → + + [ ] + − C 2H O CO 4H 4e 1 2 2 [ ] 1 The carbon oxidation reaction (COR) according to Eq. 1 is thermodynamically favorable at potentials >0.2 VRHE, but due to its slow kinetics, considerable rates are only observed at potentials >0.9 VRHE;6–8 such high anode potentials occur during cell reversal and lead to a collapse of the anode catalyst layer and to cell failure. Degradation Mechanism of an IrO2 Anode Co-Catalyst for Cell Voltage Reversal Mitigation under Transient Operation Conditions of a PEM Fuel Cell Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** Following this, the effect of partial reduction of IrO2 in a PEMFC anode condition when used as an anode co-catalyst is investigated on the durability of PEMFCs upon intermittent opera- tion, namely cell reversal and SUSD (see right panel of Fig. 1). g In addition to the high OER activity of the anode co-catalyst, another requirement a co-catalyst needs to fulﬁl is that it should be stable under the normal operating conditions of a PEMFC anode. While we are not aware of any comprehensive study on the stability of IrO2 in the H2 atmosphere in a PEMFC anode, experiments in a PEM water electrolyzer conﬁguration show that it will undergo surface reduction under these conditions.24 This could ultimately affect the stability of an IrO2 anode co-catalyst, particularly during the repetitive transitions between normal operating conditions, where the anode potential is at ∼0 VRHE, and the high anode potentials (⩾1 VRHE) that occur during cell reversal and system start- up/shut-down (SUSD) transients. An SUSD transient takes place when a H2/airanode-front passes through the anode ﬂow ﬁeld due to the replacement of the H2 gas by air during shut-down and vice versa during start-up.25 The underlying degradation mechanism during SUSD and the effect of various conditions were subject to many studies. It has been shown that SUSD does not only invoke oxidative currents in the cathode electrode, which leads to carbon corrosion and loss of Pt electrochemical surface area (ECSA) at the cathode,26,27 but that it also leads to a signiﬁcant Pt ECSA loss at the anode induced by the anode potential cycling between ∼0 and ∼1 VRHE upon the passage of H2/airanode front.28 These anode voltage cycles enhance the dissolution of an IrO2 catalyst that has been exposed to H2 at elevated temperatures (80 °C), as indicated in a recent publication from our group. Experimental IrO2/TiO2 catalyst speciﬁcation.—A commercial benchmark IrO2 supported on TiO2 OER catalyst (IrO2/TiO2 with 75 wt.% iridium, Elyst Ir75 0480 from Umicore, Germany) is used throughout this study. According to the patent, the IrO2 deposited onto the high surface area TiO2 is heat-treated (Theat-treatment ≈ 400 °C in air atmosphere),30 and it has previously been shown by our group that it is an active and stable OER catalyst for PEMWE applications.31,32 A stoichiometric IrO2 (iridium(IV) oxide) consists of 85.7 wt.% Ir and 14.3 wt.% O. Considering that the here used IrO2/TiO2 catalyst consists of 75 wt.% Ir, its nominal composition can be calculated as: 87.5 wt.% IrO2 (75 wt.% Ir, 12.5 wt.% OIrO2) and 12.5 wt.% TiO2 (7.5 wt.% Ti, 5 wt.% OTiO2). During TGA experiments under H2, the IrO2 phase of this catalyst can be reduced according to the following reaction: [ ] 2 ( ) + ( ) → ( ) + ( ) [ ] IrO s 2H g Ir s 2H O g 2 2 2 2 ( ) + ( ) → ( ) + ( ) IrO s 2H g Ir s 2H O g 2 2 2 Considering that TiO2 cannot be reduced at temperatures where IrO2 will be fully reduced (i.e., at 350 °C, as will be shown later), the total mass loss of the IrO2/TiO2 catalyst upon the complete reduction of IrO2 to Ir would be accompanied by the total loss of OIrO2, which would amount to a mass loss of the IrO2/TiO2 catalyst of 12.5 wt.%. Considering that TiO2 cannot be reduced at temperatures where IrO2 will be fully reduced (i.e., at 350 °C, as will be shown later), the total mass loss of the IrO2/TiO2 catalyst upon the complete reduction of IrO2 to Ir would be accompanied by the total loss of OIrO2, which would amount to a mass loss of the IrO2/TiO2 catalyst of 12.5 wt.%. y Next we will estimate the mass loss upon the reduction of a monolayer of IrO2, assuming a core–shell morphology, with the surface of the TiO2 support covered completely by IrO2 particles. This assumption should be quite reasonable in view of the fact that the IrO2 that is deposited on the TiO2 support has a volume fraction of ∼72% in the ﬁnal IrO2/TiO2 catalyst (based on ρIrO2 = 11.7 g cm−3 and ρTiO2 = 4.23 g cm−3). Experimental Furthermore, since no Ti signal is detected in the XPS Ti2p spectrum of as-received IrO2/TiO2 catalyst (see Fig. A·1), it can be deduced that the TiO2 support particles are completely covered by the IrO2 particles. Based on the patent,30 the pristine TiO2 particles for the preparation of this IrO2/TiO2 catalyst have a speciﬁc surface area of 50 m2g−1 TiO2. This corresponds to an average calculated TiO2 particle size of d ≈28 nm. Considering that the ﬁnal IrO2/TiO2 catalyst contains 87.5 wt.% IrO2 along with the assumption that TiO2 particles are covered by a uniform and compact ﬁlm of crystalline IrO2, an equivalent IrO2 ﬁlm thickness of ∼7.5 nm covering TiO2 support particles can be calculated for the IrO2/TiO2 catalyst. The speciﬁc surface area of this catalyst determined by the Brunauer–Emmett– Teller (BET) method is 27.2 m2g−1 IrO2/TiO2. By assuming a complete coverage of the TiO2 support by IrO2 particles (the schematic in Fig. A·1, inset, shows such a structural assumption), all of the physisorbed N2 determined by the BET measurement would be adsorbed on the outermost IrO2 surface, so that the BET surface area of 27.2 m2g−1 IrO2/TiO2 would represent the area of exposed IrO2 in 1 g of catalyst (SBET(IrO2)). For a zero order estimate of the molar amount of IrO2 in the outermost monolayer of the IrO2 phase, we consider the hypothetical case that all of the IrO2 in the IrO2/TiO2 catalyst would be in the rutile phase, so that the IrO2 surface area can be calculated from the theoretical ionic radii of O2− and Ir4+ in a rutile structure. While a signiﬁcant fraction of the IrO2 phase is actually amorphous, it still has an oxidation state of +4 (see discussion of Fig. 2a), so that the thus estimated surface area should still be reasonable. With this approximation and the above deter- mined BET surface area, one can then estimate the molar amount of IrO2 in the outermost monolayer of the IrO2 phase in 1 g of the In this study, we examine the cell reversal tolerance in the presence of an IrO2 anode co-catalyst as well as its degradation and concomitant poisoning of the cathode catalyst activity upon SUSD cycles. Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** There, we have shown that H2 permeating through the membrane from the hydrogen cathode to the oxygen anode of a PEM water electrolyzer (PEMWE) during open circuit voltage (OCV) conditions leads to a surface reduction of an IrO2 OER catalyst to metallic Ir, which in turn gets oxidized to an amorphous IrOx during the subsequent normal operation period of the PEMWE at the high OER potentials.29 The repetitive cycling between the reducing conditions during OCV periods, where the surface of the IrO2 catalyst is reduced to metallic Ir, and oxidizing conditions during operation, where the IrO2 surface is re-oxidized to an amorphous IrOx, were shown to cause an enhanced dissolution of iridium, concomitant with iridium redeposition in the membrane. Mohammad Fathi Tovini,1,,z Ana Marija Damjanovic,1, Hany A. El-Sayed,1 Jozsef Speder,2 Christian Eickes,2 Jens-Peter Suchsland,2 Alessandro Ghielmi,2 and Hubert A. Gasteiger1,** There are three main strategies to mitigate the damages caused by cell reversal: i) replacing the carbon support by corrosion resistant support materials such as metal oxides;9,10 ii) application of system- mitigation strategies;11,12 and, iii) addition of a co-catalyst to the anode electrode which catalyzes the oxygen evolution reaction (OER), so that the non-destructive OER, rather than the destructive COR, will take place.2,4,5,13,14 The application of corrosion resistant non-carbon support materials has been limited due to their generally lower electrical conductivity, their often insufﬁcient stability parti- cularly at transient fuel cell operating conditions, the risk of cationic contamination and/or degradation of the ionomer in membrane and electrodes, and cost considerations15–17; therefore, graphitized car- bons are the currently used support materials for Pt-based anode catalysts for the hydrogen oxidation reaction (HOR) in PEMFC stacks.5 On the other hand, the implementation of system-mitigation strategies adds complexity and cost to the PEMFC system: as cell non-carbon support materials has been limited due to their generally lower electrical conductivity, their often insufﬁcient stability parti- cularly at transient fuel cell operating conditions, the risk of cationic contamination and/or degradation of the ionomer in membrane and electrodes, and cost considerations15–17; therefore, graphitized car- bons are the currently used support materials for Pt-based anode catalysts for the hydrogen oxidation reaction (HOR) in PEMFC stacks.5 On the other hand, the implementation of system-mitigation strategies adds complexity and cost to the PEMFC system: as cell Journal of The Electrochemical Society, 2021 168 064521 resulting in a lower rate of the COR at the anode and, consequently, in a higher cell reversal tolerance. (ORR) activity of the cathode catalyst, which deteriorates the cell performance. In summary, we will investigate the fundamental aspects of the stability of IrO2 in the presence of H2 using different physical-chemical and electrochemical diagnostics (see left panel of Fig. 1). Following this, the effect of partial reduction of IrO2 in a PEMFC anode condition when used as an anode co-catalyst is investigated on the durability of PEMFCs upon intermittent opera- tion, namely cell reversal and SUSD (see right panel of Fig. 1). (ORR) activity of the cathode catalyst, which deteriorates the cell performance. In summary, we will investigate the fundamental aspects of the stability of IrO2 in the presence of H2 using different physical-chemical and electrochemical diagnostics (see left panel of Fig. 1). Experimental A·2), in order to record the characteristic voltammetry behavior of the as-received or the partially reduced IrO2/TiO2 catalyst powders in this potential range as well as to make sure of the physical stability of the coatings on Au working electrode disk during the experiments. After that, an OER polarization curve was recorded by collecting a CV between 1.2–1.57 VRHE with 20 mVs−1 scan rate where only the very ﬁrst positive-going scan is used for evaluations; during both types of measurements, the electrolyte was purged by bubbling argon in the electrolyte. [ ] 3 where SIrO2 is the cross-sectional area of an IrO2 unit, estimated from the area of two O2−ions and one Ir4+ ion based on their ionic radii (O2−= 126 pm, Ir4+ = 76.5 pm),33 yielding SIrO2 = 1.18 · 10−19 m2 IrO2, and where NA is Avogadro’s number (6.022 × 1023). Considering that the catalyst contains 87.5 wt.% IrO2 with a molecular mass of 224 g mol−1, the total moles of IrO2 in the IrO2/TiO2 catalyst is nIrO2(total) = 3.90 · 10−3 / − mol g . IrO IrO TiO 1 2 2 2 Therefore, the molar fraction of IrO2 that is on the outermost surface of the assumed iridium shell amounts to: = = · · = [ ] ( ) ( ) ( ) − − x n n 3.83 10 mol 3.90 10 mol 0.098 4 IrO ML IrO ML IrO total 4 3 2 2 2 [ ] 4 According to this estimate, one monolayer of IrO2 (MLIrO2) on the IrO2/TiO2 catalyst corresponds to ∼10% of the total moles of IrO2 in the catalyst. Therefore, the reduction of 1 MLIrO2 and, consequently, the formation of 1 monolayer metallic Ir (MLIr) on the surface of the IrO2/TiO2 catalyst via reaction [2], corresponds to the reduction of ∼10% of the total moles of IrO2 in the IrO2/TiO2 catalyst. Referenced to the overall IrO2/TiO2 mass, the reduction of 1 MLIrO2 thus corresponds to an overall mass loss of ∼0.1 · 12.5 wt.% ≈1.25 wt.%, a value which can be used to judge the extent of IrO2 reduction during the later shown TGA experiments with the IrO2/TiO2 catalyst. Experimental Membrane electrode assembly (MEA) preparation and cell assembly.—The stability of IrO2 under the OCV condition of a PEMFC anode was investigated using a 5 cm2 single-cell in a self- designed ﬂow ﬁeld hardware35 and a G60 fuel cell test station (Greenlight Innovation Corp., Canada). These MEAs had an IrO2/TiO2 loading of 2.0 mgIrcm−2 MEA at the anode (no platinum was used at the anode) and platinum supported on Vulcan XC72 carbon (45.8 wt.% Pt/C, TEC10V50E, Tanaka, Japan) with 0.4 mgPtcm−2 MEA loading at the cathode, hot-pressed onto a Naﬁon® 212 membrane (50 μm thick, from Quintech, Germany). The ionomer content of the electrodes was 12 wt.% on the anode and corresponded to an ionomer/carbon mass ratio of 0.65/1 on the cathode, using a Naﬁon ionomer solution (20 wt.% ionomer, D2021 from IonPower, USA). More details on the ink composition and the decal transfer to produce the catalyst coated membranes can be found in our previous study.31 The gas diffusion layer (GDL) used for this experiment was H14C7 (Freudenberg & Co. KG, Germany), and the cell was sealed with PTFE coated ﬁberglass (Fiberﬂon, Fiberﬂon GmbH & Co. KG, Germany) to achieve 20% GDL compression. Rotating disk electrode (RDE) measurements.—Electrochemical measurements were carried out in a three-electrode cell using a static H2 reference electrode (RE) consisted of a Pt wire sealed into one end of a glass tube which was drawn to a capillary and ﬁlled with electrolyte and a bubble of evolved H2 (all voltages of RDE experiments throughout this work are quoted vs the potential of this reversible hydrogen electrode (VRHE)),34 a high surface area Au wire as a counter electrode (CE), and a 5 mm diameter polycrystalline Au disk working electrode (WE) assembled in an interchangeable rotating ring-disk electrode (RRDE) shaft made of polyether ketone (Pine Research Instrumentation, USA). The reference electrode potential was calibrated in a H2 saturated electrolyte prior to each experiment using the Pt ring of the RRDE. A 0.05 M H2SO4 aqueous solution was used as electrolyte, which was prepared by mixing high purity H2SO4 (Ultrapur, 96%, Merck Millipore KGaA, Germany) and ultra-pure water (18.2 MΩ cm at 20 °C, Merck Millipore KGaA, Germany). High purity Ar and H2 (6.0-grade, Westfalen AG) were used to purge the electrolyte. p The SUSD cycling and the CRT measurements were conducted using a 38 cm2 single cell. Experimental Thermogravimetric analysis (TGA) in reductive atmosphere (3.3 vol.% H2/Ar) is used to simulate the reductive environment of PEMFC anode during normal operation, and shows that the near- surface region of IrO2 can be completely reduced to metallic Ir under the operating conditions of a PEMFC anode. The formation of metallic iridium surfaces after TGA experiments to different temperature is proven by X-ray photoelectron spectroscopy (XPS), and we show that the same surface reduction occurs in an MEA upon extended exposure of IrO2 to H2 in a PEMFC anode. Finally, it is shown that anode potential transients, such as those caused by SUSD events, lead to the dissolution of iridium from the IrO2 based anode co-catalyst. Subsequently, the dissolved iridium is shown to diffuse through the membrane and to poison the oxygen reduction reaction Figure 1. A schematic overview of the two main experimentally designed sections in this work and the experiments which are performed in each section. Figure 1. A schematic overview of the two main experimentally designed sections in this work and the experiments which are performed in each section. Journal of The Electrochemical Society, 2021 168 064521 rO2/TiO2 catalyst (nIrO2(ML)): = · = ( · )·( · ) = · [ ] ( ) ( ) − − − S S n N 27.2m 1.18 10 m 6.022 10 3.83 10 mol g 3 IrO2 ML BET IrO2 IrO2 A 2IrO2 19 2IrO2 23 4 IrO2 IrO2 TiO2 1 the polished and cleaned Au working electrode, resulting in a catalyst loading of 0.1 mgIrcm−2 disk. IrO2/TiO2 catalyst (nIrO2(ML)): All the RDE measurements (using the RRDE assembly) were performed using an Autolab potentiostat (PGSTAT302N, Metrohm AG) and a rotator (Pine Research Instrumentation). The electrode rotation rate was ﬁxed at 2500 rounds per minute (RPM) and the electrolyte temperature was maintained at room temperature during the electrochemical measurements. Freshly coated working elec- trodes were dipped in Ar-saturated electrolyte and the uncompen- sated solution resistance between the reference and working electrode was determined by electrochemical impedance spectro- scopy (EIS) from 100 kHz to 100 Hz at open circuit voltage (OCV). Then, cyclic voltammetry (CV) was performed between 0.05–1.35 VRHE with 20 mVs−1 scan rate for 10 cycles (the CVs were started with a positive going scan from OCP to 1.35 VRHE followed by 10 cycles in 0.05–1.35 VRHE, see Fig. Experimental q p p The in situ drying/cleaning step was used in order to desorb all the adsorbed water and to oxidatively remove organic molecules from the surface of the as-received catalyst powder, so that the mass losses during the subsequent reduction step are only due to the reduction of IrO2. The procedure of the in situ drying/cleaning step was as follows: ﬂushing the TGA furnace with Ar (100 mlmin−1) for 5 min at 25 °C, ramping the temperature from 25 to 200 °C (10 Kmin−1) in O2 (100 mlmin−1), holding the temperature at 200 °C for 10 min in O2 (100 mlmin−1), cooling down the furnace from 200 to 25 °C (−10 Kmin−1) in O2 (100 mlmin−1), and ﬁnally purging the TGA furnace with Ar (100 mlmin−1) for 5 min at 25 °C. Since the as-received IrO2/TiO2 catalyst is already heat-treated in air atmosphere at a temperature of ∼400 °C (as stated in the patent),30 the drying/cleaning step at 200 °C in O2 at the beginning of each TGA experiment is not expected to have a signiﬁcant inﬂuence on the as-received catalyst’s physical/chemical properties (surface area, particle size, oxidation state, etc.). p , , ) After the initial drying/cleaning step, the metal oxide reduction step was carried out in two different modes: i) a temperature ramp mode and ii) an isothermal mode. During the temperature ramp experiment, the temperature was ramped from 25 to 500 °C with different heating rates (1, 2.5, 5, and 10 Kmin−1) while supplying 5 vol.%. H2/Ar (40 mlmin−1), which was followed by cooling the furnace from 500 to 25 °C (−20 Kmin−1) in Ar (100 mlmin−1). Since IrO2 was fully reduced at T = 350 °C during the heating step under reductive atmosphere, only the mass proﬁles up to 350 °C are shown in the graphs. The isothermal reduction experiments were carried out by heating the TGA furnace from 25 °C to a given target temperature (80, 100, 120, 160, or 240 °C) with a heating rate of 5 Kmin−1 in Ar (100 mlmin−1), followed by switching the gas supply from Ar to 5 vol.% H2/Ar (40 mlmin−1) and keeping the furnace at the chosen target temperature for 60 min. After this, the furnace was cooled down to 25 °C (−20 Kmin−1) in Ar (100 mlmin−1). Experimental Since IrO2 reduction by H2 occurs only during the 60 min holding temperature period, only the mass proﬁle of this period of the entire experiment is shown in the graphs. For the extended isothermal reduction experiment in Fig. 5a, the experi- mental procedure was identical, except that different longer holding times under reductive conditions at the target temperature of 80 °C were applied. It should be noted that during all the IrO2 reduction steps in TGA experiments, the effective H2 concentration was 3.3 vol.% H2/Ar, since in addition to the 40 mlmin−1 5 vol.% H2/Ar an extra 20 mlmin−1 ﬂow of Ar as cell carrier gas was supplied. Prior to the SUSD cycling and CRT experiments using the 38 cm2 single-cells, the cells were conditioned under H2/air for 8 h at 1 Acm−2 MEA at 80 °C, an outlet pressure of 150 kPaabs, and 100% relative humidity (RH). After conditioning, an H2/air polarization curve was recorded at 80 °C and 100% RH with an outlet pressure of 150 kPaabs and controlled H2/air stoichiometries of 1.5/2. Polarization curves were recorded in galvanostatic mode, with 5 min hold at each current. SUSD cycling was performed according to the US DRIVE Fuel Cell Technical Team Roadmap protocol at 35 °C, ambient cell pressure, and a constant cathode air ﬂow rate corresponding to a stoichiometry of 2 at 1.0 Acm−2 MEA.1 A single SUSD cycle consists of ﬁve steps: i) operating the cell in fuel cell mode at 0.4 Acm−2 MEA and 100% RH for 60 s, with an anode H2 ﬂow corresponding to a stoichiometry of 1.2; ii) setting the anode gas ﬂow and the current to 0, and holding the resulting OCV for 10 s (pre-shutdown step); iii) introducing dry air to the anode compartment at a ﬂow rate that corresponds to a residence time of 0.3 s (shutdown step); iv) setting the air ﬂow rate in the anode to a ﬂow corresponding to a stoichiometry of 1 at 0.1 Acm−2 MEA for 55 s (idle step); and, ﬁnally, v) introducing fully humidiﬁed H2 to the anode compartment at a ﬂow rate corresponding to a stoichiometry of 1.2 at 1.0 Acm−2 MEA (corresponding to a residence time of 0.3 s) and keeping the cell at OCV for 10 s (start-up step). Experimental Finally, 20 μl of the prepared ink was drop-casted on Electrochemical measurements of the MEAs.—The stability of crystalline IrO2 under the OCV condition of a PEMFC anode was Journal of The Electrochemical Society, 2021 168 064521 investigated in a 5 cm2 single-cell using 2000 nccm ﬂow of fully humidiﬁed H2 (anode) and 5000 nccm ﬂow of fully humidiﬁed air (cathode) at 80 °C and 170 kPaabs cell pressure; nccm refers to a volumetric ﬂow rate in units of cm3min−1, referenced to a pressure of 101.3 kPa and a temperature of 0 °C. The cell was kept under these conditions at the OCV for an overall duration of 3 h (pre- conditioning of the MEA was not done prior to this experiment). As soon as H2 is introduced into the anode at 80 °C, metallic Ir sites begin to form on the surface of the catalyst due to the reduction of IrO2 by H2 through Eq. 2. Since the hydrogen oxidation reaction and hydrogen evolution reaction (HOR/HER) activities on iridium are very high,36 the metallic Ir sites on the catalyst surface initiate an open circuit potential of the anode electrode of ∼0 V vs RHE and an open circuit potential of the cathode electrode of ∼0.90–0.95 V vs RHE (corresponding to the OCV of a H2/air cell) throughout the entire 3 h of this OCV experiment. Every half an hour, cyclic voltammograms (CVs) of the anode (serving as working electrode) at ambient pressure and 40 °C were recorded using a Gamry potentiostat (Reference3000, Gamry Instruments, USA), applying a scan rate of 150 mVs−1 in the potential range of 0.07–1.00 V. For this, fully humidiﬁed N2 was fed to the anode (5 nccm) and fully humidiﬁed 5 vol.% H2/Ar to the cathode electrode (500 nccm; serving as the counter and reference electrode); an extended N2 purge of the anode humidiﬁer was conducted before recording the CVs. the TGA furnace. All the gases used in TGA experiments were 5.0-grade and supplied by Westfalen AG. Ar (20 mlmin−1) was used as the cell carrier gas in addition to the reactive gases during all TGA experiment steps. Each TGA experiment was composed of two main steps: i) an initial in situ drying/cleaning step in oxidative atmo- sphere (mass proﬁles of this step are not shown in the ﬁgures) and ii) a subsequent metal oxide reduction step in reductive atmosphere. Experimental The MEAs for these experiments were prepared using platinum supported on Vulcan carbon (50 wt.% Pt/C) with 0.3 mgPtcm−2 MEA loading at the cathode and platinum supported on graphitized Vulcan carbon (20 wt.% Pt/C) with 0.05 mgPtcm−2 MEA loading at the anode. Mitigated anode electrodes were prepared by mixing the as-received IrO2/TiO2 catalyst with the aforementioned Pt/C anode catalyst to achieve a loading of 0.05 mgPtcm−2 MEA + 0.05 mgIrcm−2 MEA (Pt:Ir ratio of 1:1). For simplicity, the MEAs containing the non-mitigated anode (i.e., Pt/C only) and the mitigated anode (Pt/C + IrO2/TiO2) will further on be referred to as “non-mitigated MEA” and “mitigated MEA,” respec- tively. Catalyst layers were prepared with a high equivalent weight ionomer and an ionomer:carbon ratio of 0.65:1. The MEAs were prepared by decal transfer method on 18 μm thick membranes. In this case, the Freudenberg gas diffusion layer H14C10 was used, and the cell was sealed with PTFE-coated ﬁberglass to achieve 10% GDL compression. y Before each measurement, the Au working electrode disk was removed from the RRDE shaft and was polished with 0.3 μm alumina polishing suspension (Buhler AG) and then sonicated in ultrapure water for several times. The catalyst ink suspension was prepared using the as-received or the partially reduced IrO2/TiO2 catalyst powders after the TGA experiments, ultrapure water, and Naﬁon® ionomer solution (5 wt.% ionomer in mixture of lower aliphatic alcohols and water, Sigma Aldrich). The ink composition was adjusted to achieve 0.98 mgIrml−1 ink and 2 wt.% ionomer content in the ﬁnal dried coating consisting of catalyst and ionomer (note: the Ir content of the partially reduced IrO2/TiO2 powders after the TGA experiments was re-calculated based on their mass loss during the TGA experiments). The suspension was sonicated for 30 min in a sonication bath (Elmasonic S 30 H, Elma Schmidbauer GmbH) in order to achieve a homogenous ink. The temperature of the bath was maintained below 35 °C in order to prevent solvent evaporation. Results and Discussion Equation 6 can then be solved based on the FWO method for a given constant α to determine the activation energy: To our knowledge, the reduction of IrO2 in a H2-containing atmosphere has not been considered when using this material as an anode co-catalyst for mitigating cell reversal damages in PEMFCs; recent data, however, showed that IrO2 can be partially reduced to metallic Ir when subjecting an IrO2/TiO2 anode catalyst in a PEM water electrolyzer to cross-over hydrogen at OCV and 80 °C.24,29 In the following, a mechanistic understanding of IrO2 reduction in H2–containing atmospheres is provided. Following this, the long- term stability of IrO2 is evaluated in an isothermal condition at a temperature of 80 °C in a H2-containing atmosphere (simulated PEMFC anode reductive environment). Lastly, the effect of partial reduction of IrO2 in a PEMFC anode condition when used as an anode co-catalyst is investigated on the long-term durability of PEMFCs. ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟ β ( ) = · · − − · [ ] α ( ) Ln ln A E R g 5.523 1.053 E RT 7 a a [ ] 7 where g(α) is the integral form of the kinetic model (f(α)). According to Eq. 7, the activation energy at α = constant can thus be calculated by a linear regression of ln(β) vs 1/T, where T are the temperatures at which the chosen constant α is reached at different heating rates β (in this study, heating rates of 1, 2.5, 5, and 10 Kmin−1 were used). This analysis can be performed for different constant α values, yielding the corresponding activation energies Ea vs α. It has to be noted that Eq. 7 is valid for 20 < · Ea c R T < 60 values, where Tc is the temperature corresponding to the chosen α = constant value obtained for the TGA curve with a heating rate that is closest to the midpoint of the experimental heating rates (i.e., β = 5 Kmin−1 in this study). For · Ea c R T values outside this range, Flynn and Wall suggested corrections to determine accurate Ea values.37 However, all the Ea values in this study fall within the 20 < · Ea c R T <60 range, so that Eq. 7 is valid for calculating the Ea values. Results and Discussion Fundamental aspects of IrO2 reduction in H2-containing atmospheres.—TGA is a well-established technique that can be used to unveil the underlying mechanism of metal oxide reduction even for complex multi-step reactions.41–43 Figure 2a shows the TGA temperature ramp curves of the as-received IrO2/TiO2 catalyst in 3.3 vol.% H2/Ar carried out at four different heating rates (β = 1, 2.5, 5, and 10 Kmin−1). The TGA curves show the onset of a mass loss at temperatures of ∼85 °C–110 °C, depending on the heating rate. The mass loss vs temperature curves are approaching the theoretical mass loss that would be expected for the full reduction of IrO2 in the IrO2/TiO2 catalyst to metallic Ir (i.e., a loss of 12.5 wt.%, see experimental section) at ∼350 °C. Since TiO2 is completely inert to reduction by H2 at this temperature range,44–46 the TGA curves imply that the entire mass loss between 80 °C–350 °C corresponds to the reduction of IrO2 in the IrO2/TiO2 catalyst through Eq. 2. The agreement between the experimental mass loss and that predicted by Eq. 2 indicates that iridium in the here used IrO2/TiO2 catalyst is, at least predominantly, in the form of iridium (IV) oxide. It should be noted that desorption of adsorbed water or any other organic residues on the surface of the catalyst does not contribute to the observed mass loss in the initial stage due to the in situ drying/cleaning step prior to the TGA measurement under reducing conditions (see experimental section). Other analytical methods.—X-ray photoelectron spectroscopy (XPS) was performed on a Kratos Axis Supra spectrometer using monochromatic Al Kα radiation at an energy of 1486.6 eV. The spectra were recorded at a total X-ray source power of 225 W (15 kV and 15 mA current). For XPS measurements, as-received IrO2/TiO2 catalyst powder and catalyst powders after partial reduction in TGA experiments were drop-casted on a copper tape placed on a stainless steel sample holder (in ﬂoating mode, where the sample holder was electrically insulated from the copper tape), and then outgassed overnight in the ultrahigh vacuum (UHV) sample-introduction chamber to remove moisture and contaminants, so that the pressure in the analysis chamber during the XPS data acquisition remained below 1.0 · 10−8 Torr. Experimental H2/air polarization curves at 80 ° C (100% RH, 150 kPaabs, H2/air stoichiometries of 1.5/2.) were recorded after different SUSD cycling intervals, and the potential that corresponds to 1.2 Acm−2 MEA was taken to compare the performance loss of MEAs upon SUSD cycling as shown in Fig. 6a. IrO2 reduction kinetics analysis.—The kinetic analysis of the TGA data was performed based on an isoconversional method to determine the Arrhenius activation energy of the IrO2 reduction step. In this study, we used the Flynn-Wall-Ozawa (FWO) integral method, based on the procedure described in ASTM E1641–16.37,38 This test method is based on the general rate equation derived from Arrhenius equation: CRTs were carried out at 80 °C and 100% RH with a gas outlet pressure of 150 kPaabs. The CRT was initiated by switching the anode gas feed from H2 to N2 (both at 166 nccm) while ﬂowing air in the cathode (333 nccm) and then drawing a constant current of 0.2 Acm−2 MEA. As a result of fuel starvation in the anode, the cell potential rapidly drops to negative values in the beginning of the CRT; the end-of-life (EOL) criterion was deﬁned as the time once the potential reached −1.5 V. ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ α = · · − [ ] α ( ) d dt A f exp E RT 5 a [ ] 5 where α is the reaction extent (fraction of IrO2 reduced to Ir), t is time (min), A is pre-exponential factor, f(α) is the reaction kinetic model, Ea is the activation energy (Jmol−1), R is the gas constant (8.314 Jmol−1K−1), and T is the absolute temperature (in K). By applying a constant heating rate β (in Kmin−1), Eq. 5 can be Thermogravimetric analysis (TGA).—Thermogravimetric ana- lysis (TGA) of the as-received IrO2/TiO2 catalyst powder was performed by a Mettler Toledo TGA/DSC 1 instrument. ∼10 mg IrO2/TiO2 raw powder was weighed into a sapphire crucible (volume 70 μl) closed with a perforated sapphire lid and directly inserted into Journal of The Electrochemical Society, 2021 168 064521 Journal of The Electrochemical Society, 2021 168 064521 were recorded in the 2θ range of 20°–90° with a step size of 0.015° and a hold time of 0.8 s per step. Results and Discussion For XPS measurements on SUSD-cycled and as-prepared MEAs, the MEA samples were cut into small pieces (∼5 × 5 mm) and attached to a self-adhesive copper tape (the electrode to be analyzed facing up) that in turn was attached to a stainless steel sample holder (in ﬂoating mode). Prior to inserting the MEA samples into the UHV sample introduction chamber, they were outgassed for 24 h in a vacuum oven at 80 °C. As only IrO2 can be reduced under these conditions and as the weight loss up to 500 °C (data only shown up to 350 °C in Fig. 2a) corresponds to the complete reduction of IrO2 to Ir, the mass loss plotted on the left y-axis in Fig. 2a can be converted to the fraction of IrO2 reduced to metallic Ir (α), marked by the right y-axis. From this it can be seen that the TGA curves are composed of an initially fast mass loss with increasing temperature for α < 60%, followed by a much more gradual mass loss with temperature for α ranging between 60%–100%. This might suggest that the kinetics of the IrO2 reduction in the IrO2/TiO2 catalyst follows multi-step reaction kinetics, characterized by an initially high rate that gradually slows down as the reduction reaction extent proceeds to α > 60%. In order to get better insights into the IrO2 reduction kinetics for the IrO2/TiO2 catalyst, the Flynn-Wall-Ozawa (FWO) method described in the experimental section is used to perform a kinetic analysis and to calculate the activation energy for the reduction of the IrO2 phase of the IrO2/TiO2 catalyst. The FWO method is a kinetic model- independent analysis method, which can provide the activation energy at each reaction extent α, which is also suitable when several reaction steps are occurring simultaneously and when the activation energy varies with the reaction extent α.47,48 Figure 2b shows the linear regressions of the Ln(β) vs 1/T points which are collected from the TGA curves in Fig. 2a at constant reaction extents α, selected between α = 5%–90%. The horizontal and vertical black High-resolution Ir 4 f and Pt 4 f spectra were collected using a step size of 0.05 eV, 0.6 s/step, and a pass energy of 20 eV; the shown spectra represent the average of 10 individual spectra. All binding energy values are corrected using the adventitious carbon signal (C 1 s = 284.8 eV). Experimental rewritten as: rewritten as: ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ α β = · · − [ ] α ( ) d dT A f exp E RT 6 a [ ] 6 Results and Discussion Interestingly, the clearly lower activation energies up to α = 10% corresponds to the reduction of approximately one monolayer of the IrO2 phase (see above estimates), suggesting that the outer- most monolayer can be reduced more easily than the bulk of the IrO2 phase. This has also already been suggested for the same catalyst based on differential scanning calorimetry data.24 One possible explanation for the apparently more facile reduction of the outermost surface layer(s) of the iridium oxide phase would be that this near- surface region is amorphous, as amorphous iridium oxide was shown to be more readily reducible by H2 (at temperatures of ∼80 °C) compared to crystalline rutile type IrO2.40,49 In summary, the above data suggest that the one or two outermost monolayers of the IrO2 phase of the catalyst consist of an iridium oxide phase that on account of OH surface terminations and/or surface imperfections is amorphous (furtheron referred to A-IrOx), and that has a lower activation energy for reduction to metallic Ir by H2. As the reduction reaction proceeds further into to the sub-surface layers of the IrO2 phase, the reduction of more stable crystalline iridium oxide (C-IrO2) in the bulk of the catalyst starts to take place, indicated by a higher apparent activation energy for mixtures of A-IrOx and C-IrO2. The initially fast mass loss with increasing temperature for α < 60% (see Fig. 2a) suggests that the A-IrOx species are mainly reduced at α < 60%; on the other hand, the more gradual mass loss for α ranging between 60%–100% suggests that the innermost IrO2 phase becomes more and more crystalline, i.e., less reducible. Although TGA temperature ramp experiments are a useful method to compare the temperature range for the reduction of different IrO2 catalysts in an H2-containing atmosphere, they do not provide sufﬁcient information to project the long-term stability of the iridium oxide phase under the isothermal working condition of a fuel cell anode. Therefore, TGA isothermal experiments were designed to study the reduction behavior of the IrO2 phase of the IrO2/TiO2 catalyst at different temperatures of 80 °C–240 °C over a period of 1 h. The mass losses over the course of this isothermal reduction step in 3.3 vol.% H2/Ar (marked here as time = 0) at a given target temperature are plotted in Fig. Results and Discussion The XPS data analysis was performed using the Casa XPS software. A Shirley function was used as background. As it is reported that Ir 4 f spectra exhibit a distinct asymmetric line shape,39,40 a Functional Lorentzian line shape was used for metallic iridium and for iridium oxides, with the parameters set to LF (0.6, 1, 150, 300) and LF (0.3, 1.5, 25, 150), respectively. The ﬁts of the doublets of Ir 4f7/2 and Ir 4f5/2 were ﬁxed to have the same FWHM (full width at half maximum) and to have a (4f7/2)/ (4f5/2) peak area ratio of 4:3. X-ray diffraction was performed using a Stadi P instrument (Stoe & Cie GmbH, Germany) with Cu Kα1 radiation (λ = 1.54059 Å, 50 kV, 30 mA, Ge(111) monochromatized) and a Mythen 1 K areal detector (Dectris Ltd., Switzerland) in transmission mode. About 5 mg of each powder sample was smoothly spread onto a scotch tape and placed in the center of the sample holder hole. The XRD patterns Journal of The Electrochemical Society, 2021 168 064521 Figure 2. (a) TGA temperature ramp experiments with the as-received IrO2/TiO2 catalyst power under 3.3 vol.% H2/Ar at heating rates of 1, 2.5, 5, and 10 Kmin−1 (following the drying/cleaning step; see experimental section). The left y-axis represents the weight% mass loss of the IrO2/TiO2 catalyst vs temperature, while the fraction of the IrO2 phase that is reduced to metallic Ir (α) is given on the right y-axis. The gray horizontal dashed line marks the theoretical mass loss (12.5 wt.%) of the IrO2/TiO2 catalyst upon the complete reduction of the IrO2 phase by H2 to metallic Ir (see Eq. 2). The black horizontal and vertical dotted lines illustrate the data that are used for th l i i b) f ti t t f 10% (b) Li i f Journal of The Electrochemi determined activation energies Ea vs α are plotted in Fig. 2c. It can be seen in Fig. 2c that the activation energy of IrO2/TiO2 reduction is ∼95 and ∼105 kJmol−1 for low reaction extents α of 5% and 10%, respectively; at α ranging between 20%–70% the activation energy is ∼130 ± 5 kJmol−1, then decreasing to ∼115 kJmol−1 as α reaches 90%. Results and Discussion 3a, whereby the mass proﬁles for the initial temperature ramp from 25 °C to the target temperature that is performed in Ar atmosphere are not shown in Fig. 3a. Looking at the entire 1 h time span of the isotherms, it becomes clear that the reduction of the IrO2 phase occurs at temperatures as low as 80 °C, although the reaction is much faster at higher temperatures such as 160 °C or 240 °C, where respectively ∼65% or ∼85% of the iridium oxide phase are reduced within only ∼11 min. An interesting feature is the initial mass gain of ∼0.1–0.2 wt.% when the gas is switched from Ar to 3.3 vol.% H2/Ar at the targeted temperature, as shown in the magniﬁed isotherms in Fig. 3b. This mass gain is likely due to the adsorption of hydrogen on the iridium oxide surface of the IrO2/TiO2 catalyst, as this is reported to be one of the initial steps during the reduction of a metal oxide by H2 gas.40,49,50 Assuming that each of the surface oxygens in the outermost monolayer of the iridium oxide phase were to bond to one H-atom, the molar H-uptake would be two times the moles of IrO2 in the outermost surface layer ( = · ( ) − / − n 3.83 10 mol g , IrO2 ML 4 IrO2 IrO2 TiO2 1 see Eq. 3), corresponding to · − / − 7.66 10 mol g 4 H IrO2 TiO2 1 or ∼0.08 wt.%, which, within the error of measurement, is in the same range as the mass gain observed in Fig. 3b. Figure 2. (a) TGA temperature ramp experiments with the as-received IrO2/TiO2 catalyst power under 3.3 vol.% H2/Ar at heating rates of 1, 2.5, 5, and 10 Kmin−1 (following the drying/cleaning step; see experimental section). The left y-axis represents the weight% mass loss of the IrO2/TiO2 catalyst vs temperature, while the fraction of the IrO2 phase that is reduced to metallic Ir (α) is given on the right y-axis. The gray horizontal dashed line marks the theoretical mass loss (12.5 wt.%) of the IrO2/TiO2 catalyst upon the complete reduction of the IrO2 phase by H2 to metallic Ir (see Eq. 2). The black horizontal and vertical dotted lines illustrate the data that are used for the analysis in b) for a reaction extent of α = 10%. Results and Discussion (b) Linear regression of Ln(β) vs 1/T points collected from a) at constant α values selected between 5%–90%. The squares represent the data points at different constant α values, and the solid lines are the linear regression ﬁts of the data points at each set of α values. (c) Plot of activation energy vs α for the reduction of the IrO2 phase of the IrO2/TiO2 catalyst, calculated based on FWO method (Eq. 7), using the regression line slopes in b). surface oxygens in the outermost monolayer of the iridium oxide phase were to bond to one H-atom, the molar H-uptake would be two times the moles of IrO2 in the outermost surface layer ( = · ( ) − / − n 3.83 10 mol g , IrO2 ML 4 IrO2 IrO2 TiO2 1 see Eq. 3), corresponding to · − / − 7.66 10 mol g 4 H IrO2 TiO2 1 or ∼0.08 wt.%, which, within the error of measurement, is in the same range as the mass gain observed in Fig. 3b. Next we will examine the surface chemistry and the structure of the IrO2/TiO2 catalyst samples after the TGA experiments under 3.3 vol.% H2/Ar in order to identify the nature of reduction products. Figure 4a and b show the XPS Ir4f spectra and XRD patterns, respectively, of the as-received IrO2/TiO2 catalyst and of several (partially) reduced IrO2/TiO2 powders after the TGA experiments. dotted lines in Fig. 2a exemplarily illustrate the data collection at α = 10%. All of the regression lines in Fig. 2b ﬁt to the data points with R2 > 0.99. The activation energy at each α value is calculated from Eq. 7, using the slopes of the ﬁtted lines in Fig. 2b, and the thus Journal of The Electrochemical Society, 2021 168 064521 Figure 3. (a) Isothermal TGA experiments on the reduction of the iridium oxide phase of the IrO2/TiO2 catalyst in 3.3 vol.% H2/Ar at 80, 100, 120, 160, and 240 °C (following the drying/cleaning step; see experimental section). The left y-axis represents the mass loss in wt.% of the IrO2/TiO2 catalyst vs temperature, while the fraction of the IrO2 phase that is reduced to metallic Ir (α) is given on the right y-axis. Results and Discussion The gray horizontal dashed line marks the theoretical mass loss (12.5 wt.%) of the IrO2/TiO2 catalyst upon the complete reduction of the IrO2 phase by H2 to metallic Ir (see Eq. 2). (b) Magniﬁcation of the initial 20 min of a). to the as-received sample (41/59 = 0.69/1). This observation is in line with the reaction mechanism proposed in the context of the discussion of Fig. 2, namely that A-IrOx is being reduced predominantly in the beginning of the reduction reaction. Increasing the isothermal reaction temperature to 240 °C or ramping the temperature from 25 to 500 °C leads to the full reduction of iridium oxide to metallic Ir, which can be seen clearly by an inspection of the XPS data (Fig. 4a) and the XPS peak ﬁtting results (Table I) as well as by the XRD data (Fig. 4b), where all of the features unique to C-IrO2 have disappeared. The sharper diffraction lines for the sample ramped to 500 °C is indicative of more pronounced growth of the metallic Ir domains at this higher ﬁnal reduction temperature. Overall, both TGA temperature ramp and isothermal experiments can in principle be used to study the reduction of IrO2 in H2-containing atmospheres, but the choice of the method depends on the question to be answered. The temperature ramp is a quick experiment for comparing the temperature stability window of different catalysts, while the time consuming isothermal experiment might be more useful when trying to get information at conditions that more closely reﬂect those in real applications. The latter point will be demonstrated in the next section, showing that isothermal TGA experiments can be used to project the behavior of an IrO2/TiO2 catalyst under the conditions of a PEMFC anode. IrO2 reduction in a simulated PEMFC anode reductive envir- onment.—When using IrO2 as an anode co-catalyst in PEMFCs, it is crucial to know the effect of the strongly reducing conditions in the hydrogen anode (∼1 bar H2, 80 °C, and ∼0 V vs RHE) on the chemical stability of IrO2. Results and Discussion The gray horizontal dashed line marks the theoretical mass loss (12.5 wt.%) of the IrO2/TiO2 catalyst upon the complete reduction of the IrO2 phase by H2 to metallic Ir (see Eq. 2). (b) Magniﬁcation of the initial 20 min of a). Table I summarizes the XPS Ir4f peak deconvolution parameters and the calculated composition of each sample based on the ﬁtted spectra. The Ir4f XPS spectrum of the as-received IrO2/TiO2 catalyst (see Fig. 4a) can be ﬁtted to C-IrO2 and A-IrOx components, yielding a composition ratio of A-IrOx:C-IrO2 = 41:59 (for the ﬁtting procedure, see experimental section), with no contributions from metallic iridium being detected. The XRD pattern of the as-received IrO2/TiO2 catalyst (see Fig. 4b) is composed of broad crystallite reﬂections of rutile C-IrO2 from the crystalline portion of the iridium oxide phase (main reﬂections marked by the red dotted lines) and the anatase TiO2 (main reﬂections marked by the green dotted lines) from the TiO2 support of the catalyst. Next, we will examine the XPS and XRD data for the IrO2/TiO2 catalyst after the 1 h isothermal TGA experiment at 120 °C (see blue line in Fig. 3a). The clear detection of metallic Ir in the XPS spectrum of the thus treated IrO2/TiO2 catalyst conﬁrms that the observed mass loss in Fig. 3a corresponds to the partial reduction of IrO2 by H2 through Eq. 2, with metallic Ir being the product of this reaction. The XRD pattern of this sample shows that the metallic Ir forms crystalline domains, indicated by the unique reﬂection at 2θ ≈89° (see right-most gray dotted line in Fig. 4b). In addition, strong diffractions for C-IrO2 and anatase TiO2 are still observed, so that the sample is composed of all three crystalline phases. Based on the XPS analysis (Table I), the surface composition of the sample with respect to metallic iridium and the crystalline and amorphous iridium oxide phases corresponds to Ir:C-IrO2:A-IrOx ≈42:47:11, indicating that the overall fraction of amorphous iridium oxide (∼11%) in the near-surface region has greatly decreased compared to the as-received IrO2/TiO2 sample (∼41%), which is also reﬂected by the much lower A-IrOx:C-IrO2 ratio in the reduced sample (11/47 = 0.23/1) compared The IrO2/TiO2 catalyst mass loss vs time for the long-term isothermal TGA experiment (Fig. Results and Discussion While probing the stability of IrO2 directly in the anode of a PEMFC is possible, this approach has several disadvantages: i) it requires the time-consuming preparation/ optimization of electrodes and their integration in membrane electrode assemblies (MEAs); ii) MEA preparation requires a considerable amount of catalyst (at least a few hundred milligrams), which often is a signiﬁcant hurdle when exploring new catalysts synthesized by different methods; iii) the electrochemical character- ization methods performed in a fuel cell are mainly sensitive to the chemical changes in the near-surface regions of a catalyst, and the analysis of bulk phases (e.g., via XRD) requires cumbersome post- mortem analysis; and, iv) MEA characterization in a fuel cell introduces additional complexity due to multiple reactions occurring in parallel. On the other hand, isothermal TGA experiments can be designed to simulate the reductive environment of PEMFC anodes, whereby no sample preparation is required, only a few milligrams of a sample are needed, and the observed sample mass loss can be directly and quantitatively correlated to the amount of reduced IrO2 (surface and bulk). Therefore, long-term (up to 24 h) isothermal TGA experiments have been performed at 80 °C to investigate the stability of the iridium oxide phase of the IrO2/TiO2 catalyst in the presence of hydrogen, and the results are shown in Fig. 5a. To aid the interpretation of the data, the IrO2/TiO2 catalyst is assumed to have a core–shell morphology, with the surface of the TiO2 support covered completely by IrO2 particles or by an IrO2 ﬁlm (the latter is sketched in Fig. 5c). Nevertheless, Fig. 5c is a simpliﬁed sketch of the IrO2/TiO2 catalyst that is used here. All the assumptions regarding the calculation of the monolayer of IrO2 (MLIrO2) on the IrO2/TiO2 catalyst surface in the experimental section are made while being based on a morphology that TiO2 support is completely covered by IrO2 particles; that is similar to Fig. A·1, inset. Figure 3. (a) Isothermal TGA experiments on the reduction of the iridium oxide phase of the IrO2/TiO2 catalyst in 3.3 vol.% H2/Ar at 80, 100, 120, 160, and 240 °C (following the drying/cleaning step; see experimental section). The left y-axis represents the mass loss in wt.% of the IrO2/TiO2 catalyst vs temperature, while the fraction of the IrO2 phase that is reduced to metallic Ir (α) is given on the right y-axis. Results and Discussion This analysis implies that the reduction of the iridium oxide phase of the IrO2/TiO2 catalyst at 80 °C is not limited to the outermost monolayer(s) of the iridium oxide phase, but that it rather proceeds further towards its bulk; after 24 h in 3.3 vol.% H2/Ar at 80 °C, the data suggest that more than four MLIrO2 equivalents of the IrO2 phase have been reduced, corresponding to α > 40%. capacitive currents are increased by ∼3-fold (green line) compared to those of the as-received IrO2/TiO2 (black line), due to the formation of sub-monolayer amounts of metallic Ir on the catalyst surface. Nevertheless, the CV features still closely resemble that of the as-received catalyst. y Further reduction of the iridium oxide phase of the IrO2/TiO2 catalyst, equating to the reduction of roughly 1 MLIrO2 (i.e., after 3 h, blue asterisk in Fig. 5a), leads to the appearance of voltammetric features for the hydrogen under-potential deposition (Hupd) between 0.05–0.40 VRHE, characteristic of metallic polycrystalline iridium or carbon- supported iridium nanoparticles.52,53 This suggests that the reduction of ∼1 MLIrO2 and thus the formation of ∼1 MLIr on the surface of the IrO2/TiO2 catalyst is sufﬁcient to completely change its voltammetry from that corresponding to heat-treated IrO2 to that corresponding to metallic Ir. After reducing the IrO2/TiO2 catalyst for longer times of 12 or 24 h, where ∼3 or >4 MLIrO2 equivalents are reduced, the CVs reach a steady-state, indicating that the reduction of sub-surface layers of the iridium oxide phase does not further increase the Hupd region. This is due to the fact that H adsorption/desorption occurs only on the outermost surface of the apparently compact and dense metallic Ir phase that is formed during reduction at 80 °C. The reduction of the iridium oxide phase of the IrO2/TiO2 catalyst during the extended isothermal TGA experiment is schematically shown in Fig. 5c. 2 p p g Next, we will show that the above described surface and sub- surface reduction of the IrO2 phase of the IrO2/TiO2 catalyst has a strong impact on its electrochemical behavior. For this, IrO2/TiO2 powder samples were subjected to different isothermal TGA inter- vals of 0.5, 3, 12, and 24 h (marked by the asterisks in Fig. 5a), and their electrochemical behavior was then characterized by RDE measurements and compared with that of the as-received IrO2/TiO2 catalyst. Results and Discussion 5a) reveals that a continuous IrO2 reduction occurs over the entire course of the experiment, not showing any plateau in the mass loss even after 24 h. Based on our estimate (see experimental section) for the mass loss or the reaction extent α which correspond to the reduction of a monolayer of the iridium oxide phase (1 MLIrO2 amounts to ∼1.25 wt.% mass loss or to α ≈10%), the estimated numbers of IrO2 monolayers that are Journal of The Electrochemical Society, 2021 168 064521 Figure 4. Post characterization of the as-received IrO2/TiO2 catalyst and of (partially) reduced IrO2/TiO2 catalyst powders after the TGA experiments in Fig. 3 by: (a) XPS Ir4f spectra and (b) XRD. Sample notations: TGA-120 °C-1h and TGA-240 °C-1h refer to the materials obtained after the 1 h isothermal TGA experiments (see Fig. 3a) and TGA-ramp 25 °C–500 °C refers to the material obtained after the TGA temperature ramp experiment at a heating rate of β = 5 Kmin−1 (see Fig. 2a). Detailed peak deconvolution parameters and the ratios between different Ir species in (a) can be found in Table I. The vertical dotted lines in (b) are the main reference crystalline peak positions for metallic Ir (gray), rutile C-IrO2 (red), and anatase TiO2 (green). Figure 4. Post characterization of the as-received IrO2/TiO2 catalyst and of (partially) reduced IrO2/TiO2 catalyst powders after the TGA experiments in Fig. 3 by: (a) XPS Ir4f spectra and (b) XRD. Sample notations: TGA-120 °C-1h and TGA-240 °C-1h refer to the materials obtained after the 1 h isothermal TGA experiments (see Fig. 3a) and TGA-ramp 25 °C–500 °C refers to the material obtained after the TGA temperature ramp experiment at a heating rate of β = 5 Kmin−1 (see Fig. 2a). Detailed peak deconvolution parameters and the ratios between different Ir species in (a) can be found in Table I. The vertical dotted lines in (b) are the main reference crystalline peak positions for metallic Ir (gray), rutile C-IrO2 (red), and anatase TiO2 (green). reduced to metallic iridium are marked by the orange dotted lines in Fig. 5a. Results and Discussion A·2), whereby the potential that corresponds to the electrode current of 10 mAcm−2 disk is frequently taken as a measure of the OER activity.56 It can be seen in Fig. 5e that the OER activity of the electrodes is increasing with increasing isothermal hold times at 80 °C, whereby the OER activity is improved by 38 mV after a 24 h hold time compared to the as-received IrO2/TiO2 catalyst. Analogous improve- ments of the OER activity (between 20–40 mV) were also observed when subjecting the same IrO2/TiO2 catalyst in the anode catalyst layer of a PEM water electrolyzer to cross-over hydrogen at OCV and 80 °C.24,29 Based on the analysis of Figs. 5a and 5e, such OER activity improvement is directly correlated to the reduction of IrO2 and the formation of metallic Ir in the near-surface layer of the IrO2 phase. It is well known that the product of electrochemical oxidation of metallic Ir (furtheron referred to as E-IrOx, denoting a highly porous, hydrated iridium oxy-hydroxide) has a substantially higher OER activity than heat-treated IrO2,57–59 which explains why the OER activity of the IrO2/TiO2 catalyst increases with increasing reduction time in Fig. 5a. The transformation of metallic Ir to E-IrOx over the course of 10 CVs in the potential window of 0.05–1.35 VRHE prior to the measurement of the OER polarization curves can be seen in Fig. A·2, where the appearance of voltammetric features in the potential range of ∼0.7–1.2 VRHE upon cycling is indicative of the transformation of the near-surface region metallic Ir to E-IrOx. In fact, this feature is more prominent for the samples reduced for 12 and 24 h (Figs. A·2c and A·2d), which showed a nominal reduction of ∼3 and ∼4.5 MLIrO2, respectively. This is due to the fact that upon cycling the electrodes between 0.05–1.35 VRHE, further oxidation of sub-surface metallic Ir leads to the formation of a thicker porous E-IrOx layer on the catalyst surface and to an incorporation of the near-surface active sites to the electrochemical processes.60,61 This phenomenon, so called three-dimensional electrocatalysis,59 explains the higher OER activity of the samples reduced for 12 and 24 h (reduction of ∼3 and ∼4.5 MLIrO2 on IrO2 surface) compared to the sample reduced for 3 h (reduction of ∼1 MLIrO2 on IrO2 surface). IrO2/TiO2 catalyst in a PEMFC anode, its stability was tested in an MEA. Results and Discussion An MEA with an active area of 5 cm2 was prepared, where IrO2/TiO2 was used in the anode electrode and a Pt/C catalyst was used in the cathode electrode. The MEA was held at OCV (Eanode ≈ 0 V vs RHE and Ecathode ≈0.9–0.95 V vs RHE, see experimental section) at 80 °C, with a constant ﬂow of fully-humidiﬁed H2 (2000 nccm) through the anode compartment pressurized at 170 kPaabs (corresponding to a H2 partial pressure of 123 kPa). Every 30 min, anode CVs were recorded at 40 °C, and were compared to the CV of the as-received IrO2/TiO2 catalyst (before ever ﬂowing H2 through the anode), as shown in Fig. 5d. The observed gradual formation of Hupd features of metallic Ir in Fig. 5d suggests that, similar to the TGA experiment, IrO2 reduction upon exposure to H2 also occurs in an MEA in a PEMFC (analogous to what was shown previously for a PEM water electrolyzer conﬁguration24,29). However, the reduc- tion kinetics of the IrO2 phase seems to be faster in the MEA, where it takes ∼2 h for the surface of the IrO2 phase to completely convert to metallic Ir and for the CVs to reach a steady-state. This is most likely due to the higher H2 partial pressure in the MEA experiment (PH2 = 123 kPa) compared to that in the TGA experiment (PH2 = 3.3 kPa). It has to be noted that although the CVs recorded in the MEA reach a steady state after ∼2 h of OCV (see Fig. 5d), it is speculated based on the TGA results (and based on the analogy of the TGA and MEA experiments) that the reduction of the IrO2 phase of the IrO2/TiO2 catalyst also proceeds to subsurface layers of IrO2, even though the formation of bulk metallic Ir is not further traceable by measuring CVs in the MEA. It is also worth mentioning that the high iridium loading of 2.0 mgIrcm−2 MEA used in this section is not relevant for the application of IrO2 as an anode co-catalyst in a PEMFC, and it is only used to show the instability of IrO2 in the reductive atmosphere of a PEMFC anode. The following sections of the paper will show that a more relevant loading of 0.05 mgIrcm−2 MEA will be used in the CRT and SUSD experiments. Results and Discussion Figure 5b shows the CVs (ﬁrst scans out of the 10 measured scans for each sample are shown, the starting positive sweeps from OCP to 1.35 VRHE are removed, see Fig. A·2) of these samples in an Ar saturated 0.05 M H2SO4 electrolyte at a scan rate of 20 mVs−1. Although the surface composition of the as-received IrO2/TiO2 is composed of two different IrO2 species, namely A-IrOx and C-IrO2 (see Table I), the voltammetric features of this sample are resembling those of a highly crystalline and heat-treated IrO2; this is indicative of an electrochemically stable IrO2 phase in the as- received IrO2/TiO2 catalyst.51 It can be seen that after only 0.5 h of the isothermal TGA experiment (green asterisk in Fig. 5a), the In order to validate that the results obtained in the isothermal TGA experiment are representative of what happens to the Journal of The Electrochemical Society, 2021 168 064521 Table I. XPS Ir4f peak deconvolution parameters and the calculated composition of as-received and (partially) reduced IrO2/TiO2 samples based on the ﬁtted XPS Ir4f spectra in Fig. 4a. C-IrO2 refers to crystalline IrO2, A-IrOx refers to amorphous iridium oxide. Details of the ﬁtting procedure are described in the experimental section. Table I. XPS Ir4f peak deconvolution parameters and the calculated composition of as-received and (partially) reduced IrO2/TiO2 samples based on the ﬁtted XPS Ir4f spectra in Fig. 4a. C-IrO2 refers to crystalline IrO2, A-IrOx refers to amorphous iridium oxide. Details of the ﬁtting procedure are described in the experimental section. IrO2/TiO2 catalyst in a PEMFC anode, its stability was tested in an MEA. An MEA with an active area of 5 cm2 was prepared, where IrO2/TiO2 was used in the anode electrode and a Pt/C catalyst was used in the cathode electrode. The MEA was held at OCV (Eanode ≈ 0 V vs RHE and Ecathode ≈0.9–0.95 V vs RHE, see experimental section) at 80 °C, with a constant ﬂow of fully-humidiﬁed H2 (2000 nccm) through the anode compartment pressurized at 170 kPaabs (corresponding to a H2 partial pressure of 123 kPa). Every 30 min, anode CVs were recorded at 40 °C, and were compared to the CV of the as-received IrO2/TiO2 catalyst (before ever ﬂowing H2 through the anode), as shown in Fig. 5d. The observed gradual formation of Hupd features of metallic Ir in Fig. Results and Discussion 5d suggests that, similar to the TGA experiment, IrO2 reduction upon exposure to H2 also occurs in an MEA in a PEMFC (analogous to what was shown previously for a PEM water electrolyzer conﬁguration24,29). However, the reduc- tion kinetics of the IrO2 phase seems to be faster in the MEA, where it takes ∼2 h for the surface of the IrO2 phase to completely convert to metallic Ir and for the CVs to reach a steady-state. This is most likely due to the higher H2 partial pressure in the MEA experiment (PH2 = 123 kPa) compared to that in the TGA experiment (PH2 = 3.3 kPa). It has to be noted that although the CVs recorded in the MEA reach a steady state after ∼2 h of OCV (see Fig. 5d), it is speculated based on the TGA results (and based on the analogy of the TGA and MEA experiments) that the reduction of the IrO2 phase of the IrO2/TiO2 catalyst also proceeds to subsurface layers of IrO2, even though the formation of bulk metallic Ir is not further traceable by measuring CVs in the MEA. It is also worth mentioning that the high iridium loading of 2.0 mgIrcm−2 MEA used in this section is not relevant for the application of IrO2 as an anode co-catalyst in a PEMFC, and it is only used to show the instability of IrO2 in the reductive atmosphere of a PEMFC anode. The following sections of the paper will show that a more relevant loading of 0.05 mgIrcm−2 MEA will be used in the CRT and SUSD experiments. A more active OER catalyst facilitates the OER over the COR during cell reversal in a PEMFC, resulting in longer cell reversal 19 20 IrO2/TiO2 catalyst (black line) and the catalyst after the respective isothermal TGA hold times shown in Fig. 5a. The polarization curves were recorded after 10 CVs in the potential window of 0.05–1.35 VRHE in the Ar-saturated electrolyte (shown in Fig. A·2), whereby the potential that corresponds to the electrode current of 10 mAcm−2 disk is frequently taken as a measure of the OER activity.56 It can be seen in Fig. 5e that the OER activity of the electrodes is increasing with increasing isothermal hold times at 80 °C, whereby the OER activity is improved by 38 mV after a 24 h hold time compared to the as-received IrO2/TiO2 catalyst. Results and Discussion Analogous improve- ments of the OER activity (between 20–40 mV) were also observed when subjecting the same IrO2/TiO2 catalyst in the anode catalyst layer of a PEM water electrolyzer to cross-over hydrogen at OCV and 80 °C.24,29 Based on the analysis of Figs. 5a and 5e, such OER activity improvement is directly correlated to the reduction of IrO2 and the formation of metallic Ir in the near-surface layer of the IrO2 phase. It is well known that the product of electrochemical oxidation of metallic Ir (furtheron referred to as E-IrOx, denoting a highly porous, hydrated iridium oxy-hydroxide) has a substantially higher OER activity than heat-treated IrO2,57–59 which explains why the OER activity of the IrO2/TiO2 catalyst increases with increasing reduction time in Fig. 5a. The transformation of metallic Ir to E-IrOx over the course of 10 CVs in the potential window of 0.05–1.35 VRHE prior to the measurement of the OER polarization curves can be seen in Fig. A·2, where the appearance of voltammetric features in the potential range of ∼0.7–1.2 VRHE upon cycling is indicative of the transformation of the near-surface region metallic Ir to E-IrOx. In fact, this feature is more prominent for the samples reduced for 12 and 24 h (Figs. A·2c and A·2d), which showed a nominal reduction of ∼3 and ∼4.5 MLIrO2, respectively. This is due to the fact that upon cycling the electrodes between 0.05–1.35 VRHE, further oxidation of sub-surface metallic Ir leads to the formation of a thicker porous E-IrOx layer on the catalyst surface and to an incorporation of the near-surface active sites to the electrochemical processes.60,61 This phenomenon, so called three-dimensional 59 Table I. XPS Ir4f peak deconvolution parameters and the calculated composition of as-received and (partially) reduced IrO2/TiO2 samples based on the ﬁtted XPS Ir4f spectra in Fig. 4a. C-IrO2 refers to crystalline IrO2, A-IrOx refers to amorphous iridium oxide. Details of the ﬁtting procedure are described in the experimental section. Sample Peak B.E. Results and Discussion 5e that the OER activity of the electrodes is increasing with increasing isothermal hold times at 80 °C, whereby the OER activity is improved by 38 mV after a 24 h hold time compared to the as-received IrO2/TiO2 catalyst. Analogous improve- ments of the OER activity (between 20–40 mV) were also observed when subjecting the same IrO2/TiO2 catalyst in the anode catalyst layer of a PEM water electrolyzer to cross-over hydrogen at OCV and 80 °C.24,29 Based on the analysis of Figs. 5a and 5e, such OER activity improvement is directly correlated to the reduction of IrO2 and the formation of metallic Ir in the near-surface layer of the IrO2 phase. It is well known that the product of electrochemical oxidation of metallic Ir (furtheron referred to as E-IrOx, denoting a highly porous, hydrated iridium oxy-hydroxide) has a substantially higher OER activity than heat-treated IrO2,57–59 which explains why the OER activity of the IrO2/TiO2 catalyst increases with increasing reduction time in Fig. 5a. The transformation of metallic Ir to E-IrOx over the course of 10 CVs in the potential window of 0.05–1.35 VRHE prior to the measurement of the OER polarization curves can be seen in Fig. A·2, where the appearance of voltammetric features in the potential range of ∼0.7–1.2 VRHE upon cycling is indicative of the transformation of the near-surface region metallic Ir to E-IrOx. In fact, this feature is more prominent for the samples reduced for 12 and 24 h (Figs. A·2c and A·2d), which showed a nominal reduction of ∼3 and ∼4.5 MLIrO2, respectively. This is due to the fact that upon cycling the electrodes between 0.05–1.35 VRHE, further oxidation of sub-surface metallic Ir leads to the formation of a thicker porous E-IrOx layer on the catalyst surface and to an incorporation of the near-surface active sites to the electrochemical processes.60,61 This phenomenon, so called three-dimensional electrocatalysis,59 explains the higher OER activity of the samples reduced for 12 and 24 h (reduction of ∼3 and ∼4.5 MLIrO2 on IrO2 surface) compared to the sample reduced for 3 h (reduction of ∼1 MLIrO2 on IrO2 surface). IrO2/TiO2 catalyst (black line) and the catalyst after the respective isothermal TGA hold times shown in Fig. 5a. The polarization curves were recorded after 10 CVs in the potential window of 0.05–1.35 VRHE in the Ar-saturated electrolyte (shown in Fig. Results and Discussion [eV] FWHM [eV] Area [%] Composition C-IrO2 4f7/2 61.92 0.81 26.24 C-IrO2 4f5/2 64.92 0.81 19.72 IrO2/TiO2 (as-received) C-IrO2 4f7/2 (sat.1) 63.80 2.91 6.83 C-IrO2: 59% C-IrO2 4f5/2 (sat.1) 66.80 2.91 4.94 A-IrOx: 41% C-IrO2 4f5/2 (sat.2) 67.90 1.53 1.32 A-IrOx 4f7/2 62.20 1.79 23.38 A-IrOx 4f5/2 65.20 1.79 17.57 Ir 4f7/2 60.99 0.90 23.84 Ir 4f5/2 63.99 0.90 17.92 C-IrO2 4f7/2 61.71 0.85 20.56 TGA-120 °C-1h C-IrO2 4f5/2 64.71 0.85 15.45 metallic Ir: 42% C-IrO2 4f7/2 (sat.1) 63.5 3.6 5.61 C-IrO2: 47% C-IrO2 4f5/2 (sat.1) 66.5 3.6 4.06 A-IrOx: 11% C-IrO2 4f5/2 (sat.2) 67.71 1.57 1.04 A-IrOx 4f7/2 62.21 1.8 6.63 A-IrOx 4f5/2 65.21 1.8 4.98 TGA-240 °C-1h Ir 4f7/2 60.90 0.87 57.09 metallic Ir: 100% Ir 4f5/2 63.90 0.87 42.91 TGA-ramp 25 °C–500 °C Ir 4f7/2 60.88 0.86 57.09 metallic Ir: 100% Ir 4f5/2 63.88 0.86 42.91 the ﬁtted XPS Ir4f spectra in Fig. 4a. C-IrO2 refers to crystalline IrO2, A-IrOx refers to amorphous iridium oxide. Details of the ﬁtting procedure are described in the experimental section. Sample Peak B.E. [eV] FWHM [eV] Area [%] Composition C-IrO2 4f7/2 61.92 0.81 26.24 C-IrO2 4f5/2 64.92 0.81 19.72 IrO2/TiO2 (as-received) C-IrO2 4f7/2 (sat.1) 63.80 2.91 6.83 C-IrO2: 59% C-IrO2 4f5/2 (sat.1) 66.80 2.91 4.94 A-IrOx: 41% C-IrO2 4f5/2 (sat.2) 67.90 1.53 1.32 A-IrOx 4f7/2 62.20 1.79 23.38 A-IrOx 4f5/2 65.20 1.79 17.57 Ir 4f7/2 60.99 0.90 23.84 Ir 4f5/2 63.99 0.90 17.92 C-IrO2 4f7/2 61.71 0.85 20.56 TGA-120 °C-1h C-IrO2 4f5/2 64.71 0.85 15.45 metallic Ir: 42% C-IrO2 4f7/2 (sat.1) 63.5 3.6 5.61 C-IrO2: 47% C-IrO2 4f5/2 (sat.1) 66.5 3.6 4.06 A-IrOx: 11% C-IrO2 4f5/2 (sat.2) 67.71 1.57 1.04 A-IrOx 4f7/2 62.21 1.8 6.63 A-IrOx 4f5/2 65.21 1.8 4.98 TGA-240 °C-1h Ir 4f7/2 60.90 0.87 57.09 metallic Ir: 100% Ir 4f5/2 63.90 0.87 42.91 TGA-ramp 25 °C–500 °C Ir 4f7/2 60.88 0.86 57.09 metallic Ir: 100% Ir 4f5/2 63.88 0.86 42.91 IrO2/TiO2 catalyst (black line) and the catalyst after the respective isothermal TGA hold times shown in Fig. 5a. The polarization curves were recorded after 10 CVs in the potential window of 0.05–1.35 VRHE in the Ar-saturated electrolyte (shown in Fig. A·2), whereby the potential that corresponds to the electrode current of 10 mAcm−2 disk is frequently taken as a measure of the OER activity.56 It can be seen in Fig. Results and Discussion (e) OER polarization curves of the as-received IrO2/TiO2 catalyst and of the partially reduced IrO2/TiO2 samples taken at 20 mVs−1 and 2500 RPM in Ar-saturated 0.05 M H2SO4 at room temperature (0.1 mgIrcm−2 disk). Figure 5. (a) Extended-time isothermal TGA experiments on the reduction of the iridium oxide phase of the IrO2/TiO2 catalyst in 3.3 vol.% H2/Ar at 80 °C, aiming to simulate the PEMFC hydrogen anode reductive environment. The left y-axis represents the mass loss in wt.% of the IrO2/TiO2 catalyst vs time, while the fraction of the IrO2 phase that is reduced to metallic Ir (α) is given on the right y-axis. The orange dotted lines represent the reduction of 1–4 monolayers of IrO2 (MLIrO2), whereby the reduction of 1 MLIrO2 corresponds to an estimated mass loss of ∼1.25 wt.% or to a reaction extent of α ≈10%; the gray dashed line marks the theoretical mass loss (12.5 wt.%) of the IrO2/TiO2 catalyst upon the full reduction of the IrO2 phase to Ir (see experimental section). The asterisks mark the reduction times for the independently prepared samples for the electrochemical measurements in b) and e). (b) Cyclic voltammograms (CVs) of the as- received IrO2/TiO2 catalyst and of the partially reduced IrO2/TiO2 samples marked in a); they were recorded in an RDE conﬁguration at room temperature between 0.05–1.35 VRHE at a scan rate of 20 mVs−1 in Ar saturated 0.05 M H2SO4 (loading of 0.1 mgIrcm−2 disk) at a rotation rate of 2500 RPM (1st cycle; all 10 cycles are shown in ﬁgure A.2). (c) Schematic representation of the reduction of the IrO2/TiO2 surface to metallic Ir during the extended-time isothermal TGA experiment in a), assuming a core/shell morphology. (d) CVs at 150 mVs−1 of the as-received IrO2/TiO2 catalyst incorporated as anode in a 5 cm2 MEA (2.0 mgIrcm−2 MEA) with a Pt/C cathode (0.4 mgPtcm−2 MEA). The CVs, taken after a series of 30 min holds at OCV (80 °C, 170 kPaabs., 2000 nccm H2 (anode)/ 5000 nccm air (cathode)), were acquired between 0.07–1.00 V at 40 °C and ambient pressure with fully-humidiﬁed N2 (anode) and 5 vol.% H2/Ar (cathode). (e) OER polarization curves of the as-received IrO2/TiO2 catalyst and of the partially reduced IrO2/TiO2 samples taken at 20 mVs−1 and 2500 RPM in Ar-saturated 0.05 M H2SO4 at room temperature (0.1 mgIrcm−2 disk). Results and Discussion A more active OER catalyst facilitates the OER over the COR during cell reversal in a PEMFC, resulting in longer cell reversal tolerance times.19,20 Measuring the OER activity of a catalyst using the RDE technique is a quick and reasonably reliable approach to predict its OER activity in an MEA.54,55 Therefore, Fig. 5e shows the RDE-based OER polarization curves of the as-received Journal of The Electrochemical Society, 2021 168 064521 Figure 5. (a) Extended-time isothermal TGA experiments on the reduction of the iridium oxide phase of the IrO2/TiO2 catalyst in 3.3 vol.% H2/Ar at 80 °C, aiming to simulate the PEMFC hydrogen anode reductive environment. The left y-axis represents the mass loss in wt.% of the IrO2/TiO2 catalyst vs time, while the fraction of the IrO2 phase that is reduced to metallic Ir (α) is given on the right y-axis. The orange dotted lines represent the reduction of 1–4 monolayers of IrO2 (MLIrO2), whereby the reduction of 1 MLIrO2 corresponds to an estimated mass loss of ∼1.25 wt.% or to a reaction extent of α ≈10%; the gray dashed line marks the theoretical mass loss (12.5 wt.%) of the IrO2/TiO2 catalyst upon the full reduction of the IrO2 phase to Ir (see experimental section). The asterisks mark the reduction times for the independently prepared samples for the electrochemical measurements in b) and e). (b) Cyclic voltammograms (CVs) of the as- received IrO2/TiO2 catalyst and of the partially reduced IrO2/TiO2 samples marked in a); they were recorded in an RDE conﬁguration at room temperature between 0.05–1.35 VRHE at a scan rate of 20 mVs−1 in Ar saturated 0.05 M H2SO4 (loading of 0.1 mgIrcm−2 disk) at a rotation rate of 2500 RPM (1st cycle; all 10 cycles are shown in ﬁgure A.2). (c) Schematic representation of the reduction of the IrO2/TiO2 surface to metallic Ir during the extended-time isothermal TGA experiment in a), assuming a core/shell morphology. (d) CVs at 150 mVs−1 of the as-received IrO2/TiO2 catalyst incorporated as anode in a 5 cm2 MEA (2.0 mgIrcm−2 MEA) with a Pt/C cathode (0.4 mgPtcm−2 MEA). The CVs, taken after a series of 30 min holds at OCV (80 °C, 170 kPaabs., 2000 nccm H2 (anode)/ 5000 nccm air (cathode)), were acquired between 0.07–1.00 V at 40 °C and ambient pressure with fully-humidiﬁed N2 (anode) and 5 vol.% H2/Ar (cathode). Results and Discussion Based on the conclusions drawn from the TGA and RDE experiments, and their conﬁrmation by the MEA experiments in a PEMFC, it is clear that at least the near-surface layers of the IrO2 phase of the IrO2/TiO2 catalyst can be reduced to metallic Ir by H2 under the conditions of a PEMFC anode. This formation of metallic Ir at/near the catalyst surface also alters the electrochemical proper- ties of the catalyst, leading to a drastic increase in its OER activity. A consequence of this phenomenon is that OER catalyst testing in an RDE, where the potential is usually kept above 1 V vs RHE, can yield a vastly different OER activity from that of the same catalyst, when it is used as an anode co-catalyst in a PEMFC. For example, the OER activity of the here used IrO2/TiO2 catalyst (corresponding to the black line in Fig. 5e) will be much lower than its activity as a cell reversal mitigation catalyst in a PEMFC anode (more closely corresponding to the yellow line in Fig. 5e). By the same token, IrO2 based catalysts that are made by different synthesis routes could, e. g., be predominantly amorphous (or consisting of hydrous iridium oxide) or highly crystalline, which in turn would lead to a different degree of reducibility under H2 (the higher degree of reducibility of amorphous iridium oxide is suggested by Figs. 2 and 3). As a more easily reducible IrO2 catalyst would be expected to have a higher OER activity, it would also be expected to show a better cell reversal mitigation performance when used as an anode co-catalyst in a PEMFC. This phenomenon might explain the occasional discre- pancy observed between the OER activity of a series of catalysts measured by the RDE technique, and their performance as anode co- catalysts in cell reversal tests (CRTs) in a PEMFC (for example, compare Figs. 3a and 5a in Roh et al.,19 and Figs. 4a and 5a in Moore et al.20). Therefore, the possibly different surface state of an IrO2 based catalyst in conventional RDE based OER activity measurements vs that when used as anode co-catalyst in a PEMFC must be considered. In the next section, further consequences of the near-surface reduction of the IrO2 phase of the IrO2/TiO2 catalyst when used as anode co-catalyst on PEMFC durability will be discussed. Results and Discussion The nearly constant performance over the ﬁrst 100 cycles is likely due to a compensation of the SUSD losses by an activation of the Pt/C cathode catalyst caused by the positive potential excursion during the initial SUSD cycles.62 The overall SUSD induced performance loss with an unmitigated MEA is predominantly due to carbon support corrosion at the cathode, leading to a collapse of the cathode electrode (the so-called cathode thinning) and concomitant O2 transport losses, and to a loss of Pt surface area at the cathode.26 It should be noted that for each SUSD cycle, the anode electrode experiences a potential cycling between ∼0–1.05 VRHE upon the H2/airanode gas front passage, which also causes Pt dissolution and loss of electrochemically active surface area (ECSA) at the anode.28 However, at least for a pure Pt/C anode, the contribution of anode ECSA loss to the overall losses caused by SUSD is negligible, due to the fast hydrogen oxidation reaction (HOR) kinetics on Pt, as long as the anode loadings are not ultra-low (i.e., not =0.05 mgPtcm−2 MEA).28 In order to quantify the degradation rate of the non-mitigated MEA upon SUSD cycling, the H2/air front residence time normal- ized SUSD degradation rate at 35 °C for the non-mitigated MEA was calculated according to Mittermeier et al.26 Considering a potential loss of ∼80 mV over the 500 SUSD cycles (neglecting the initial activation effect of the Pt/C cathode catalyst) and the H2/air front residence time of ∼0.3 s, an average degradation rate of ∼0.5 mVcycle−1 s−1 at 1.2 Acm−2 MEA is obtained. This is in reasonable agreement with the degradation rate of ∼0.3 mVcycle−1 s−1 obtained for SUSDs conducted at 40 °C for cathodes with non-graphitized Vulcan carbon support reported by Mittermeier et al.26 The mitigated MEA was also tested by the same SUSD protocol and the potential proﬁle measured at 1.2 Acm−2 MEA vs SUSD cycling is compared to the non-mitigated MEA in Fig. 6a (dark purple line/symbols). The potential proﬁle of the mitigated MEA at 1.2 Acm−2 is clearly distinct from that of the non- mitigated MEA, as it shows a much larger potential loss in the initial 100 SUSD cycles. This initially rapid degradation is followed by a more gradual voltage decay, so that between 200 and 500 SUSD cycles the residence time normalized degradation rate for the mitigated and the non- mitigated MEA become essentially identical (∼0.4 mVcycle−1 s−1). Results and Discussion 6a), the H2/air performance at 1.2 Acm−2 MEA drops by ∼15 mV over the ﬁrst 10 cycles and then remains roughly constant up to 100 cycles, after which it continuously decreases. The nearly constant performance over the ﬁrst 100 cycles is likely due to a compensation of the SUSD losses by an activation of the Pt/C cathode catalyst caused by the positive potential excursion during the initial SUSD cycles.62 The overall SUSD induced performance loss with an unmitigated MEA is predominantly due to carbon support corrosion at the cathode, leading to a collapse of the cathode electrode (the so-called cathode thinning) and concomitant O2 transport losses, and to a loss of Pt surface area at the cathode.26 It should be noted that for each SUSD cycle, the anode electrode experiences a potential cycling between ∼0–1.05 VRHE upon the H2/airanode gas front passage, which also causes Pt dissolution and loss of electrochemically active surface area (ECSA) at the anode.28 However, at least for a pure Pt/C anode, the contribution of anode ECSA loss to the overall losses caused by SUSD is negligible, due to the fast hydrogen oxidation reaction (HOR) kinetics on Pt, as long as the anode loadings are not ultra-low (i.e., not =0.05 mgPtcm−2 MEA).28 accelerated SUSD experiments are performed at a temperature of 35 °C, according to the US DRIVE Fuel Cell Technical Team Roadmap protocol (see experimental section), so that the strongly temperature-dependent Pt/C catalyst degradation during SUSD events is minimized26 and any degradation that may be caused by the IrO2/TiO2 anode co-catalyst can be more easily determined. Figure 6a shows the cell potential proﬁles at a current density of 1.2 Acm−2 MEA as a function of performed SUSD cycles, determined from H2/air (s = 1.5/2) performance curves at 80 °C, 100% RH, and 150 kPaabs,outlet pressure. For the non-mitigated MEA (turquoise line/symbols in Fig. 6a), the H2/air performance at 1.2 Acm−2 MEA drops by ∼15 mV over the ﬁrst 10 cycles and then remains roughly constant up to 100 cycles, after which it continuously decreases. Results and Discussion While both types of MEAs have the same initial performance (i.e., ∼0.64 V at 1.2 Acm−2), owing to its initially faster SUSD degradation, the mitigated MEA has a ∼40 mV lower performance after 500 SUSD cycles compared to the non-mitigated MEA. This overall ∼40 mV larger voltage loss of the mitigated MEA is apparently due to its initial rapid performance loss and must thus be related to some additional degradation phenomenon originating from IrO2/TiO2 anode co-catalyst in the mitigated MEA. Figure 6. (a) Cell voltages at 1.2 Acm−2 (taken from H2/air performance curves, not shown here) as a function of the performed start-up/shut-down (SUSD) cycles (acc. to the US DRIVE Fuel Cell Technical Team Roadmap protocol at 35 °C, see experimental section) for non-mitigated and mitigated MEAs. H2/air performance curve condition: Tcell = 80 °C, RH = 100%, Pcell = 150 kPaabs,outlet, s = 1.5H2/2air. MEA speciﬁcations: i) cathode with 0.3 mgPtcm−2 MEA loading, ii) anodes with 0.05 mgPtcm−2 MEA (non- mitigated MEA) or with 0.05 mgPtcm−2 MEA + 0.05 mgIrcm−2 MEA (Pt/C + IrO2/TiO2, mitigated MEA) loading. (b) Shirley background-corrected XPS Pt 4 f and Ir 4 f spectra of the anode and cathode electrodes of the pristine and 500-SUSD cycled mitigated MEAs. The vertical dashed line in the Pt 4 f region shows the position of the Pt 4 f7/2 peaks in the spectra of all electrodes. The vertical dashed line in the Ir 4 f region shows the position of the Ir 4f7/2 peaks of the spectra of the anode electrodes (corresponding to the binding energy of A-IrOx, see Table I). For better visualization of iridium crossover into the cathode electrode, the cps values in the Ir 4f regions of the cathode electrodes have been multiplied by 5. Figure 6. (a) Cell voltages at 1.2 Acm−2 (taken from H2/air performance curves, not shown here) as a function of the performed start-up/shut-down (SUSD) cycles (acc. to the US DRIVE Fuel Cell Technical Team Roadmap protocol at 35 °C, see experimental section) for non-mitigated and mitigated MEAs. H2/air performance curve condition: Tcell = 80 °C, RH = 100%, Pcell = 150 kPaabs,outlet, s = 1.5H2/2air. MEA speciﬁcations: i) cathode with 0.3 mgPtcm−2 MEA loading, ii) anodes with 0.05 mgPtcm−2 MEA (non- mitigated MEA) or with 0.05 mgPtcm−2 MEA + 0.05 mgIrcm−2 MEA (Pt/C + IrO2/TiO2, mitigated MEA) loading. Results and Discussion Degradation of an IrO2/TiO2 anode co-catalyst by SUSD cycling.—Based on the results of the previous section it is now clear that during the normal operating conditions of a PEMFC, the IrO2 phase of the IrO2/TiO2 catalyst when used as anode co-catalyst will get (partially) reduced to metallic Ir and, accordingly, its electrochemical behavior will change substantially. This phenom- enon could eventually affect the stability of such an anode co-catalyst during the transient operation of a PEMFC, e.g., during SUSD and cell reversal events. In this regard, an accelerated SUSD test was performed with 38 cm2 active area MEAs with a Pt/C catalyst in the cathode electrode and two different types of anode electrodes, namely either only with Pt/C (furtheron referred to as non-mitigated MEA) or with a mixture of Pt/C and the IrO2/TiO2 as anode co-catalyst (furtheron referred to as mitigated MEA). The Journal of The Electrochemical Society, 2021 168 064521 Figure 6. (a) Cell voltages at 1.2 Acm−2 (taken from H2/air performance curves, not shown here) as a function of the performed start-up/shut-down (SUSD) cycles (acc. to the US DRIVE Fuel Cell Technical Team Roadmap protocol at 35 °C, see experimental section) for non-mitigated and mitigated MEAs. H2/air performance curve condition: Tcell = 80 °C, RH = 100%, Pcell = 150 kPaabs,outlet, s = 1.5H2/2air. MEA speciﬁcations: i) cathode with 0.3 mgPtcm−2 MEA loading, ii) anodes with 0.05 mgPtcm−2 MEA (non- mitigated MEA) or with 0.05 mgPtcm−2 MEA + 0.05 mgIrcm−2 MEA (Pt/C + IrO2/TiO2, mitigated MEA) loading. (b) Shirley background-corrected XPS Pt 4 f and Ir 4 f spectra of the anode and cathode electrodes of the pristine d 500 SUSD l d iti t d MEA Th ti l d h d li i th Pt 4 f accelerated SUSD experiments are performed at a temperature of 35 °C, according to the US DRIVE Fuel Cell Technical Team Roadmap protocol (see experimental section), so that the strongly temperature-dependent Pt/C catalyst degradation during SUSD events is minimized26 and any degradation that may be caused by the IrO2/TiO2 anode co-catalyst can be more easily determined. Figure 6a shows the cell potential proﬁles at a current density of 1.2 Acm−2 MEA as a function of performed SUSD cycles, determined from H2/air (s = 1.5/2) performance curves at 80 °C, 100% RH, and 150 kPaabs,outlet pressure. For the non-mitigated MEA (turquoise line/symbols in Fig. Results and Discussion For the pristine mitigated MEA with an anode co-catalyst (Pt/C + IrO2/TiO2) and a Pt/C cathode these are: i) ORR on the pristine cathode (pink line); ii) OER on the pristine anode (green line); iii) carbon oxidation reaction (COR; black line), whose kinetics are much slower than that of the OER on an iridium based OER catalyst. For the mitigated MEA after SUSD testing, the loss of ORR performance of the cathode electrode (see Fig. 6a) leads to a negative-shift of the ORR (gray line) by a value marked as ①, while a decrease of the OER activity of the anode co-catalyst would lead to a positive-shift of the OER (yellow line) by a value marked as ②. The negative cell voltage during a CRT for a pristine mitigated MEA is marked by ∣Ecell∣-pristine and that of the mitigated MEA after SUSD testing by ∣Ecell∣-post-SUSD; the difference between these two cell voltages corresponds to the loss of OER and ORR performance caused by SUSD testing, i.e., to ①+②. (b) Cell reversal tests (CRTs) of the pristine non-mitigated MEA (turquoise line) as well as of the mitigated MEAs either in pristine state (red line) or after the 500 SUSD cycles shown in Fig. 6a (dark purple line); the MEA speciﬁcations are the same as given in Fig. 6a. CRT condition: i = 0.2 Acm−2 MEA, Tcell = 80◦C, RH = 100%, Pcell = 150 kPaabs,outlet, N2/air ﬂows (166/333 nccm) in anode/cathode. The inset is a magniﬁed view of the ﬁrst 5 min of the CRTs, marking the initial cell potential difference between the two mitigated MEAs, corresponding to the sum of the ORR and OER activity losses (denoted as ①+②in the inset of panel b). according to the patent65 and that its voltammetric features are indicative of highly crystalline IrO2 (see black line in Fig. 5b), one would expect it to have a high stability against transient dissolution during SUSD cycling. However, according to the data shown in Fig. 5d, the near-surface layers of the IrO2/TiO2 anode co-catalyst can be reduced to metallic iridium (from the surface to the core of the catalyst particles, see Fig. 5c) within a few hours when exposed to the H2 environment of a PEMFC anode at ∼0 V vs RHE during the conditioning and recording of a polarization curve (see experi- mental section). Results and Discussion Note that a signiﬁcant fraction of sub-surface IrO2 was also observed in the XPS spectrum of the TGA-1h-120 °C sample in Fig. 4a, even though ∼50% of the IrO2 in this sample was reduced to metallic near-surface Ir during the TGA experiment (see blue line in Fig. 3a). Figure 7. (a) Schematic representation of the current vs potential (refer- enced vs RHE) relationships of the anode and cathode half-cell reactions during a CRT. For the pristine mitigated MEA with an anode co-catalyst (Pt/C + IrO2/TiO2) and a Pt/C cathode these are: i) ORR on the pristine cathode (pink line); ii) OER on the pristine anode (green line); iii) carbon oxidation reaction (COR; black line), whose kinetics are much slower than that of the OER on an iridium based OER catalyst. For the mitigated MEA after SUSD testing, the loss of ORR performance of the cathode electrode (see Fig. 6a) leads to a negative-shift of the ORR (gray line) by a value marked as ①, while a decrease of the OER activity of the anode co-catalyst would lead to a positive-shift of the OER (yellow line) by a value marked as ②. The negative cell voltage during a CRT for a pristine mitigated MEA is marked by ∣Ecell∣-pristine and that of the mitigated MEA after SUSD testing by ∣Ecell∣-post-SUSD; the difference between these two cell voltages corresponds to the loss of OER and ORR performance caused by SUSD testing, i.e., to ①+②. (b) Cell reversal tests (CRTs) of the pristine non-mitigated MEA (turquoise line) as well as of the mitigated MEAs either in pristine state (red line) or after the 500 SUSD cycles shown in Fig. 6a (dark purple line); the MEA speciﬁcations are the same as given in Fig. 6a. CRT condition: i = 0.2 Acm−2 MEA, Tcell = 80◦C, RH = 100%, Pcell = 150 kPaabs,outlet, N2/air ﬂows (166/333 nccm) in anode/cathode. The inset is a magniﬁed view of the ﬁrst 5 min of the CRTs, marking the initial cell potential difference between the two mitigated MEAs, corresponding to the sum of the ORR and OER activity losses (denoted as ①+②in the inset of panel b). With regards to the XPS spectra of the cathode electrodes, no Ir 4 f signal is detected for the pristine cathode, as it only contains the Pt/C catalyst. Results and Discussion This, combined with the fact that metallic iridium surfaces (and their electrochemical oxidation product, E-IrOx) exhibit very high transient dissolution rates, render it very likely that there might be substantial iridium dissolution from the anode co- catalyst during SUSD cycles; if true, this could lead to an iridium loss from the anode electrode and to iridium re-precipitation in the membrane or in the cathode electrode. In order to examine whether iridium dissolution during SUSD cycles leads to a diffusion and re-precipitation of iridium in the oxygen (cathode) electrode, the mitigated MEA was harvested after 500 SUSD cycles and examined by XPS. Figure 6b shows the XPS Pt 4 f and Ir 4 f spectra of the pristine and SUSD cycled anode and cathode electrodes of the mitigated MEA. All the spectra exhibit Pt 4 f doublets with Pt 4f7/2 peaks at ∼71.8 eV (vertical dashed line in Pt 4 f region), indicating that the surface chemistry of the platinum catalyst in both anode and cathode does not change over the 500 SUSD cycles. The Ir 4 f spectra of the pristine and the SUSD cycled anodes are very similar to those of the as-received IrO2/TiO2 catalyst powder (bottom panel in Fig. 4a), with Ir 4f7/2 peaks at ∼62.2 and ∼62.1 eV, respectively. Based on the analysis presented in Table I, the Ir 4 f spectra of the as-received IrO2/TiO2 catalyst are composed of signals from amorphous (A-IrOx) and crystalline (C-IrO2) iridium oxide. Although it is expected that the near-surface layers of the IrO2/TiO2 anode co-catalyst will be partially reduced to metallic Ir during the initial conditioning step and recording of a polarization curve, a metallic Ir signal (∼60.9 eV, see Table I) is not detected in the SUSD cycled anode spectrum. This is most likely due to the electrochemical oxidation of metallic Ir and transition to E-IrOx by potential cycling between ∼0–1.05 V vs RHE during SUSD cycles, where it is reported that E-IrOx is composed of two different iridium oxide species with Ir4f binding energies comparable to the as- received IrO2/TiO2 catalyst in this study.66 Therefore, the Ir4f signal in the SUSD cycled anode spectrum should predominantly reﬂect the contribution of E-IrOx as well as the contribution from the sub- surface IrO2 of the IrO2/TiO2 catalyst that did not get reduced to metallic Ir during the experiments. Results and Discussion (b) Shirley background-corrected XPS Pt 4 f and Ir 4 f spectra of the anode and cathode electrodes of the pristine and 500-SUSD cycled mitigated MEAs. The vertical dashed line in the Pt 4 f region shows the position of the Pt 4 f7/2 peaks in the spectra of all electrodes. The vertical dashed line in the Ir 4 f region shows the position of the Ir 4f7/2 peaks of the spectra of the anode electrodes (corresponding to the binding energy of A-IrOx, see Table I). For better visualization of iridium crossover into the cathode electrode, the cps values in the Ir 4f regions of the cathode electrodes have been multiplied by 5. square-wave like fashion from ∼0 to ∼1.05 and back to ∼0 V vs RHE. These potential cycles, repeated for each SUSD cycle, are well known to lead to the dissolution of iridium (via the formation of Irn+ ions), a mechanism called transient dissolution61,63 (the same process is also responsible for the accelerated dissolution of platinum during the current- or voltage-cycling of fuel cell cathodes).62 The stability of iridium against transient dissolution strongly depends on its chemical and morphological nature. It has been previously shown that transient dissolution rates are ∼2 orders of magnitude higher for metallic Ir compared to highly crystalline IrO2 (usually obtained by heat-treatment),64 and comparable tran- sient dissolution rates have been observed between metallic Ir surfaces and E-IrOx (the product of electrochemical oxidation of To further explore this hypothesis, it is instructive to ﬁrst examine the changes of the anode potential over the course of an SUSD cycle. Under the normal operating conditions of an H2-fed PEMFC anode, the anode potential (and thus the potential of the IrO2/TiO2 anode co-catalyst) is at ∼0 V vs RHE. On the other hand, once the H2 gas in the anode compartment has been replaced by air during an SUSD half-cycle, the anode potential increases to ∼1.05 V vs RHE.28 During a subsequent SUSD half-cycle (i.e., when replacing the air in the anode again by H2), the anode potential will be decreased again to ∼0 V vs RHE. Thus, over the course of a complete SUSD cycle, the anode potential is being moved in a Journal of The Electrochemical Society, 2021 168 064521 Figure 7. (a) Schematic representation of the current vs potential (refer- enced vs RHE) relationships of the anode and cathode half-cell reactions during a CRT. Results and Discussion Here it should be noted that the OER activity of the IrO2/TiO2 catalyst when used as anode co-catalyst is even higher than the OER activity of the as-received material, due to the enhanced OER activity of the (partially) reduced IrO2 phase compared to the initially crystalline IrO2 (compare yellow and black lines in Fig. 5e) that is formed within hours under the conditions of a H2 anode (see Fig. 5d). As a corollary, a non-reducible IrO2 based catalyst would exhibit a lower reversal tolerance due to its lower OER activity. While the SUSD cycling induced iridium dissolution in the anode of the mitigated MEA leads to an ORR activity loss of ∼120 mV at 1.2 Acm−2 (see dark purple line/symbols in Fig. 6a) and of ∼35 mV at 0.2 Acm−2 (data not shown), this would not affect the cell reversal tolerance of the mitigated MEA, as this would not affect the anode potential during cell reversal (green line in Fig. 7a) and thus would not affect the rate of the COR, even though the cell voltage during the CRT would be more negative. Therefore, in this case, one would expect identical cell reversal tolerance for the pristine and for the SUSD cycled mitigated MEA. The cell reversal tolerance would only be affected negatively, if i) the OER activity of the IrO2/TiO2 anode co-catalyst would decrease over the course of SUSD cycling (illustrated by the yellow line in Fig. 7a), and/or if ii) the anode carbon support would be partially corroded by the SUSD cycling due to the H2/air front induced anode potential cycles (see above). The latter is highly unlikely, as only minor anode thinning (∼10%), indicating little carbon support corrosion was observed after 500 SUSD/voltage cycles at 80 °C for a non-graphitized Vulcan carbon support,28 so that for the here conducted 500 SUSD cycles at 35 °C and graphitized Vulcan carbon support, anode carbon support corrosion can be considered negligible (anode thinning of =10%) due to the high apparent activation energy of the COR.75 Whether the SUSD induced iridium dissolution will also affect the OER activity, and thus the cell reversal tolerance of the mitigated MEAs, will be examined in the following. Results and Discussion Hence, the identiﬁcation of the exact nature of iridium in the SUSD cycled cathode electrode is not trivial. However, the ORR activity loss of the mitigated MEA within the ﬁrst ∼100 SUSD cycles that was observed in Fig. 6a could be explained by assuming that iridium dissolved in the anode is deposited on the platinum surface of the Pt/ C cathode catalyst; owing to the ∼100-fold lower ORR activity of iridium compared to platinum, this would lead to a loss of ORR activity of the Pt/C cathode catalyst.68–70 This phenomenon would be analogous to the observations made in the context of (partial) Ru dissolution from PtRu alloy anode catalysts in direct methanol fuel cells or in PEMFCs operated with reformate, where the dissolved Ru species were shown to permeate through the membrane to the cathode and to deteriorate the ORR performance of the Pt/C cathode catalyst.71–74 Based on these considerations, the overall ∼40 mV higher potential loss over 500 SUSD cycles for the mitigated MEA (Fig. 6a) can be assigned to the poisoning of the ORR activity of the Pt/C cathode catalyst by re-deposited crossover iridium. It should be noted that iridium dissolution was also observed when using the same IrO2/TiO2 catalyst as oxygen evolution catalyst in a PEM water electrolyzer that was cycled between OCV and electrolysis mode.29 During the OCV periods, the oxygen electrode (anode) potential dropped to ∼0 V vs RHE due to H2 permeation through the membrane into the anode compartment, while during operation, the anode potential was above 1.5 V vs RHE. In this case, however, the dissolved iridium was precipitated in the H2-saturated membrane phase, which prevented iridium crossover to the hydrogen electrode (cathode). . In contrast to the non-mitigated MEA, the use of IrO2/TiO2 as an anode co-catalyst in the mitigated MEA drastically increases the reversal tolerance time to ∼77 min. This is due to the much higher OER activity of IrO2/TiO2 compared to Pt/C, illustrated in Fig. 7a by the green line for the OER, resulting in a lower anode potential and, consequently, a much lower fraction of the overall current con- tributed by the COR. Results and Discussion h i i b h ll l l i f h Cell reversal for pristine or SUSD cycled MEAs.—Cell reversal tests (CRTs) were performed on pristine and SUSD cycled mitigated MEAs (Pt/C + IrO2/TiO2 anode) to determine the effect of the SUSD induced iridium dissolution on the CRT tolerance of mitigated MEAs; as a reference, a CRT was also performed for the pristine non-mitigated MEA (Pt/C anode). In order to initiate the CRT, the anode gas was switched from H2 to N2, while air was continuously fed to the cathode electrode and a current density of 0.2 Acm−2 MEA was applied. Upon the replacement of H2 by N2, the anodic current initially supplied by the hydrogen oxidation reaction (HOR) will have to be provided by a combination of the oxygen evolution reaction (OER; see green line in Fig. 7a) and the carbon oxidation reaction acc. to Eq. 1 (COR; see black line in Fig. 7a), whose relative contribution to the overall current depends on the OER activity of the anode catalyst(s) and on the nature of the carbon support materials (graphitized carbon supports being more stable than non-graphitized ones).75 The higher the OER/COR activity ratio at a given potential and at a given overall current density, the lower will be the degradation of the anode carbon support over the course of a CRT, i.e., the higher will be the CRT tolerance of an MEA. As the OER and COR currents are only appreciable above ∼1.3–1.4 V vs RHE and as the cathode potential in a PEMFC ranges between ∼0.7–0.9 V vs RHE (see pink line in Fig. 7a), the cell voltage during cell reversal becomes negative (Ecell ≡ Ecathode—Eanode) and is marked by the horizontal arrows in Fig. 7a. As the anode electrode degrades over the course of a CRT (predominantly due to anode carbon support corrosion), the cell voltage becomes more and more negative; in this work, the time to reach a cell voltage of −1.5 V was set as the end-of-life (EOL) criterion and is further on referred to as reversal tolerance time of the MEAs. g As shown in Fig. 7b, the cell reversal tolerance time of the mitigated MEA that was subjected to the 500 SUSD cycles (see Fig. 6a) prior to the CRT is ∼3-fold shorter (∼26 min; see dark purple line in Fig. 7b) than that of the pristine mitigated MEA. Results and Discussion However, a relatively strong Ir 4 f signal could be detected in the XPS spectrum of the cathode electrode after 500 SUSD cycles, indicating iridium dissolution in the anode electrode and subsequent permeation of Irn+ species through the membrane to the cathode electrode (often referred to as crossover). The Ir 4f7/2 peak energy of ∼61.2 eV observed for the SUSD cycled cathode is between the characteristic binding energy of metallic iridium (∼60.9 eV, see Table I) and that observed for E-IrOx (∼61.6 eV for Ir(IV) contribution in E-IrOx) in our previous study.66 The cathode electrode experiences a potential cycling between ∼0.95 to >1.4 V vs RHE during SUSD cycles (more details about potential proﬁle of the cathode electrode during SUSD cycles are provided in the last section). This consequently promotes the oxidation of iridium in cathode during the course of SUSD experiment.61 On the other hand, during the measurement of H2/air polarization curves at different SUSD cycling intervals, the cathode electrode potential metallic Ir by potential cycling between ∼0–1.05 V vs RHE)61,63 rendering metallic Ir and E-IrOx substantially less stable than heat- treated IrO2 when subjected to voltage cycles. Considering that the here used IrO2/TiO2 catalyst was heat-treated above 300 °C metallic Ir by potential cycling between ∼0–1.05 V vs RHE)61,63 rendering metallic Ir and E-IrOx substantially less stable than heat- treated IrO2 when subjected to voltage cycles. Considering that the here used IrO2/TiO2 catalyst was heat-treated above 300 °C Journal of The Electrochemical Society, 2021 168 064521 catalyst, so that its OER kinetics are similar or even slower than the COR of the graphitized carbon support.4,19,75 In the context of the scheme in Fig. 7a, this means that the green line marking the OER kinetics would be shifted positively and would be located at or to the right of the black line marking the COR. In this case, the overall current will be carried to a comparable fraction by both the OER and the COR, leading to a rapid corrosion of the carbon support, which in turn leads to the collapse of the anode catalyst layer and to cell failure. Results and Discussion remains at values below 0.65 V vs RHE for the course of several minutes during each polarization curve, a potential region in which metallic Ir is thermodynamically stable.67 Therefore, the nature of such iridium surface contaminations in the SUSD cycled cathode electrode could be essentially prone to the potential proﬁle experi- enced by the cathode electrode during the experiments. Hence, the identiﬁcation of the exact nature of iridium in the SUSD cycled cathode electrode is not trivial. However, the ORR activity loss of the mitigated MEA within the ﬁrst ∼100 SUSD cycles that was observed in Fig. 6a could be explained by assuming that iridium dissolved in the anode is deposited on the platinum surface of the Pt/ C cathode catalyst; owing to the ∼100-fold lower ORR activity of iridium compared to platinum, this would lead to a loss of ORR activity of the Pt/C cathode catalyst.68–70 This phenomenon would be analogous to the observations made in the context of (partial) Ru dissolution from PtRu alloy anode catalysts in direct methanol fuel cells or in PEMFCs operated with reformate, where the dissolved Ru species were shown to permeate through the membrane to the cathode and to deteriorate the ORR performance of the Pt/C cathode catalyst.71–74 Based on these considerations, the overall ∼40 mV higher potential loss over 500 SUSD cycles for the mitigated MEA (Fig. 6a) can be assigned to the poisoning of the ORR activity of the Pt/C cathode catalyst by re-deposited crossover iridium. It should be noted that iridium dissolution was also observed when using the same IrO2/TiO2 catalyst as oxygen evolution catalyst in a PEM water electrolyzer that was cycled between OCV and electrolysis mode.29 During the OCV periods, the oxygen electrode (anode) potential dropped to ∼0 V vs RHE due to H2 permeation through the membrane into the anode compartment, while during operation, the anode potential was above 1.5 V vs RHE. In this case, however, the dissolved iridium was precipitated in the H2-saturated membrane phase, which prevented iridium crossover to the hydrogen electrode (cathode). remains at values below 0.65 V vs RHE for the course of several minutes during each polarization curve, a potential region in which metallic Ir is thermodynamically stable.67 Therefore, the nature of such iridium surface contaminations in the SUSD cycled cathode electrode could be essentially prone to the potential proﬁle experi- enced by the cathode electrode during the experiments. Results and Discussion Thus, the difference between this value and the ∼88 mV lower initial CRT voltage, equating to ∼53 mV, can only be ascribed to a loss in OER activity of the IrO2/TiO2 anode co-catalyst after the 500 SUSD cycles, thereby corresponding to a positive-shift of the OER potential as compared to the pristine mitigated MEA at the beginning of CRT (≡②). Such loss in OER activity would increase the anode potential and would thus increase the fractional CRT current that is provided by the COR, leading to the observed decrease of the reversal tolerance time. Fig. 7b), respectively. This would signify that the maximum time averaged COR contribution to the overall drawn charge (OER + COR) during CRT would only be ∼0.7% and ∼2.1% for the mitigated MEA (red curve in Fig. 7b) and mitigated MEA after 500 SUSD cycles (dark purple curve in Fig. 7b), respectively. Hence, the contribution of the COR to the overall anodic current can be neglected when using a highly active OER anode co-catalyst. Based on the H2/air performance curves recorded over the course of the SUSD test (data not shown), the increase of the ORR over- potential at 0.2 Acm−2 after 500 SUSD cycles is ∼35 mV (≡①). Thus, the difference between this value and the ∼88 mV lower initial CRT voltage, equating to ∼53 mV, can only be ascribed to a loss in OER activity of the IrO2/TiO2 anode co-catalyst after the 500 SUSD cycles, thereby corresponding to a positive-shift of the OER potential as compared to the pristine mitigated MEA at the beginning of CRT (≡②). Such loss in OER activity would increase the anode potential and would thus increase the fractional CRT current that is provided by the COR, leading to the observed decrease of the reversal tolerance time. and discussion, a schematic representation of the processes occurring in the mitigated MEA with an IrO2/TiO2 anode co-catalyst during the normal operation of a PEMFC and during the shut-down and the start-up half-cycles of an SUSD test are shown in Fig. 8. When using an IrO2 based anode co-catalyst for mitigating cell reversal damages in PEMFCs, it was shown above that the near-surface layer(s) of the IrO2/TiO2 catalyst get chemically reduced to metallic Ir by H2 during the normal operation of the fuel cell, as illustrated in Fig. 8a. Results and Discussion Such chemical alteration of the catalyst surface should improve its reversal tolerance time in a CRT, since metallic iridium that converts to electrochemically oxidized (often referred to as E-IrOx) or hydrous iridium oxide once the potential is increased into the OER region has a higher intrinsic OER activity than the initial heat- treated, highly crystalline IrO2. At the same time, however, metallic Ir is reported to exhibit a ∼2 orders of magnitude higher dissolution rate than crystalline IrO2,64 rendering it more prone to dissolution during the SUSD induced anode potential cycles (∼0–1.05 V vs RHE).28 During the fuel cell shut-down step, described in Fig. 8b, the passage of the air-front pushes the H2 from anode inlet to the anode outlet (from bottom to top in Fig. 8b), whereby the anode potential increases from ∼0 V vs RHE (in the H2-ﬁlled segment) to ∼1.05 V vs RHE (in the air-ﬁlled segment). This leads to platinum and iridium dissolution from the anode catalysts, forming Ptn+ and Irn+ ions that are dissolved in the ionomer phase and can diffuse across the anode, the membrane, and the cathode. As the air-front passes through the anode, the cathode segment adjacent to the air-ﬁlled anode will be at potentials of >1.4 V vs RHE. After the passage of the air-front, the cathode potential will also assume ∼1.05 V vs RHE8 (the cell voltage being ∼0 V); whether re-deposition of Ptn+ and Irn+ (possibly also in form of underpotential deposition on Pt) will occur at such high potential on the Pt/C cathode catalyst is still an open question. In the subsequent start-up step, described in Fig. 8c, a H2-front passes from the anode inlet to the anode outlet, displacing air from the anode compartment. In the H2-ﬁlled anode segment, the potential drops to ∼0 V vs RHE and the dissolved Ptn+ and Irn+ species will deposit on the surface of the anode catalysts. As the nucleation of metallic Pt and Ir is expected to be more facile on metal surfaces compared to nucleation of nanoparticles on the carbon support, one would expect that the dissolved metals deposit preferentially on the platinum and iridium phases. Since the HOR activity of iridium is very high and only ∼3-fold lower than the that of platinum,36 this would not affect the HOR performance of the anode. Results and Discussion On the other hand, since the OER activity of iridium based catalysts is orders of magnitude higher than that of platinum,76 a deposition of platinum There are two possible hypotheses to explain the loss of OER activity of the IrO2/TiO2 anode co-catalyst that is apparently induced by SUSD cycling: i) dissolution of iridium from the anode catalyst during SUSD transients, leading to an overall loss of OER active material in the anode; and/or, ii) dissolution of Pt from the Pt/C anode catalyst due to anode voltage cycling during the SUSD transients and re-deposition of the dissolved Ptn+ species on the Ir/IrO2/TiO2 anode co-catalyst, lowering its OER activity by blocking the OER active iridium surface sites. The former would require a substantial loss of iridium from the anode: at a Tafel slope for the OER of ∼50–60 mV decade−1,31 a loss of 90% of the active iridium sites would lead to an OER activity loss of ∼50–60 mV. Considering a ∼7.5 nm thick layer of IrO2 covering the TiO2 support in the IrO2/TiO2 catalyst (see Fig. A·1, inset), the dissolution of iridium from the surface of the catalyst would constantly expose the underlying iridium active sites in the IrO2 layer, which would consequently participate into OER; therefore, no apparent loss of OER activity is expected until the last few mono-layers of IrO2 covering the TiO2 support particles start to dissolve. As the ratio between the Pt 4 f and the Ir 4 f XPS spectra of the pristine and the SUSD cycled anodes in Fig. 6b do not indicate such high losses of iridium after 500 SUSD cycles, a poisoning of OER active iridium sites by re-deposited platinum seems more likely. Summary of the SUSD induced processes and their effect on the CRT tolerance.—In order to summarize the foregoing analysis Figure 8. Schematic representation of the processes occurring in a mitigated MEA in the presence with an iridium oxide based anode co-catalyst during normal fuel cell operation, fuel cell shut-down, and fuel cell start-up. (a) Normal fuel cell operation: illustrating the chemical reduction of the near-surface layer(s) of the iridium oxide phase of the IrO2/TiO2 anode co-catalyst by H2 to metallic Ir. Results and Discussion In addition, as shown in the inset of Fig. 7b, the initial CRT cell voltage of the SUSD cycled mitigated MEA (dark purple line) is ∼88 mV lower compared to the pristine mitigated MEA (red line). Since the cell voltage during a CRT is the difference between the cathode (ORR) and the anode (OER) half-cell potentials, as shown schema- tically in Fig. 7a, the lower cell voltage of the SUSD cycled mitigated MEA (denoted as ①+②in the inset of Fig. 7a) must originate from a half-cell potential shift of either the ORR (marked by ①in Fig. 7a) and/or the OER (marked by ②). Here it should be noted that with an anode carbon loading of 0.2 mgcm−2 MEA in this study, if all of the carbon support were to corrode at the end of CRT in the anode electrode through Eq. 1 (4e−/CO2), it would correspond to a maximum COR charge of ∼6.4 C cm−2 MEA. The overall charge that is drawn at the end of CRT is ∼924 C cm−2 MEA and ∼312 C cm−2 MEA for the mitigated MEA (red curve in Fig. 7b) and mitigated MEA after 500 SUSD cycles (dark purple curve in Figure 7b shows the CRT for the non-mitigated MEA (turquoise line) as a reference, with a reversal tolerance time of only ∼36 s. This very rapid degradation is due to the poor OER activity of a Pt/C Journal of The Electrochemical Society, 2021 168 064521 Fig. 7b), respectively. This would signify that the maximum time averaged COR contribution to the overall drawn charge (OER + COR) during CRT would only be ∼0.7% and ∼2.1% for the mitigated MEA (red curve in Fig. 7b) and mitigated MEA after 500 SUSD cycles (dark purple curve in Fig. 7b), respectively. Hence, the contribution of the COR to the overall anodic current can be neglected when using a highly active OER anode co-catalyst. Based on the H2/air performance curves recorded over the course of the SUSD test (data not shown), the increase of the ORR over- potential at 0.2 Acm−2 after 500 SUSD cycles is ∼35 mV (≡①). Results and Discussion (b) Fuel cell shut-down step: upon passage of the air-front, the anode potential increases to ∼1.05 V vs RHE,28 leading to platinum and iridium dissolution from the anode catalysts, whereby the resulting Ptn+ and Irn+ ions can diffuse within in the ionomer and membrane phase; after passage of the air-front, the cathode potential will assume ∼1.05 V,8 and re-deposition of crossover iridium on the platinum surface of the Pt/C cathode catalyst could possibly occur. (c) Fuel cell shut-down step: upon passage of the H2-front, the anode potential decreases from ∼1.05 V to ∼0 V vs RHE, and the re-deposition of Ptn+ and Irn+ on the Pt/C and the IrO2/TiO2 catalysts in the anode will occur; after passage of the H2-front, the cathode potential is ∼0.95 V (this lower OCV is due to H2 crossover), and iridium deposition on the Pt/C cathode catalyst becomes more likely. Figure 8. Schematic representation of the processes occurring in a mitigated MEA in the presence with an iridium oxide based anode co-catalyst during normal fuel cell operation, fuel cell shut-down, and fuel cell start-up. (a) Normal fuel cell operation: illustrating the chemical reduction of the near-surface layer(s) of the iridium oxide phase of the IrO2/TiO2 anode co-catalyst by H2 to metallic Ir. (b) Fuel cell shut-down step: upon passage of the air-front, the anode potential increases to ∼1.05 V vs RHE,28 leading to platinum and iridium dissolution from the anode catalysts, whereby the resulting Ptn+ and Irn+ ions can diffuse within in the ionomer and membrane phase; after passage of the air-front, the cathode potential will assume ∼1.05 V,8 and re-deposition of crossover iridium on the platinum surface of the Pt/C cathode catalyst could possibly occur. (c) Fuel cell shut-down step: upon passage of the H2-front, the anode potential decreases from ∼1.05 V to ∼0 V vs RHE, and the re-deposition of Ptn+ and Irn+ on the Pt/C and the IrO2/TiO2 catalysts in the anode will occur; after passage of the H2-front, the cathode potential is ∼0.95 V (this lower OCV is due to H2 crossover), and iridium deposition on the Pt/C cathode catalyst becomes more likely. Figure 8. Schematic representation of the processes occurring in a mitigated MEA in the presence with an iridium oxide based anode co-catalyst during normal fuel cell operation, fuel cell shut-down, and fuel cell start-up. Conclusions In this study, it is shown that when using an IrO2 based anode co- catalyst for mitigating cell reversal damages in PEMFCs, the near- surface layer(s) of the IrO2/TiO2 catalyst get chemically reduced to metallic Ir by H2 during the normal operation of the fuel cell. Such alteration of the near-surface layer(s) of IrO2 drastically affects its stability during the anode potential transients during SUSD cycles, where it was shown that the dissolution of metallic Ir and crossover of the dissolved Irn+ species through the membrane to the cathode electrode cause iridium deposition on the Pt/C cathode catalyst, as evidenced by XPS Ir4f spectra of the cathode electrodes after SUSD cycling. Such iridium-based contamination on the cathode catalyst surface deteriorates the ORR activity of Pt and results in a signiﬁcant performance loss during the normal operation of the fuel cell. At the same time, SUSD transients also cause an OER activity loss of the anode co-catalyst, which was shown to be mainly due to the re- deposition of Pt dissolved from the anode HOR catalyst onto the reduced IrO2 anode co-catalyst, blocking its OER active sites. Figure A·1. XPS Ti2p and Ir4f spectra of as-received IrO2/TiO2 catalyst. The vertically dashed lines in Ti2p region represent the positions of Ti2p3/2 and Ti2p1/2 signals in a TiO2 sample.77 The Ir4f signal is multiplied by 0.5 for a better representation of the data. Inset shows schematic representation of a TiO2 support particle, which is completely covered by IrO2 particles. If the IrO2 layer is assumed as a uniform and compact ﬁlm covering the TiO2 support particle (dTiO2 ≈28 nm), an equivalent IrO2 ﬁlm thickness of ∼7.5 nm can be consequently calculated for this structure. Figure A·1. XPS Ti2p and Ir4f spectra of as-received IrO2/TiO2 catalyst. The vertically dashed lines in Ti2p region represent the positions of Ti2p3/2 and Ti2p1/2 signals in a TiO2 sample.77 The Ir4f signal is multiplied by 0.5 for a better representation of the data. Inset shows schematic representation of a TiO2 support particle, which is completely covered by IrO2 particles. If the IrO2 layer is assumed as a uniform and compact ﬁlm covering the TiO2 support particle (dTiO2 ≈28 nm), an equivalent IrO2 ﬁlm thickness of ∼7.5 nm can be consequently calculated for this structure. Acknowledgments The authors gratefully acknowledge the German Ministry of Education and Research for ﬁnancial support of this work within the innoKA project (BMWi, 03ET6096A). The authors would also like to thank Anna T.S. Freiberg, Philipp J. Rheinländer, and Julien Durst for fruitful scientiﬁc discussions (Chair of Technical Electrochemistry, Technical University of Munich, Germany). Thanks are also extended to Burak Koyuturk and Paulette A. Loichet Torres (Chair of Technical Electrochemistry, Technical University of Munich, Germany) for their help during the XPS measurements. Appendix Appendix Figure A·1. XPS Ti2p and Ir4f spectra of as-received IrO2/TiO2 catalyst. The vertically dashed lines in Ti2p region represent the positions of Ti2p3/2 and Ti2p1/2 signals in a TiO2 sample.77 The Ir4f signal is multiplied by 0.5 for a better representation of the data. Inset shows schematic representation of a TiO2 support particle, which is completely covered by IrO2 particles. If the IrO2 layer is assumed as a uniform and compact ﬁlm covering the TiO2 support particle (dTiO2 ≈28 nm), an equivalent IrO2 ﬁlm thickness of ∼7.5 nm can be consequently calculated for this structure. Results and Discussion As iridium has a much lower ORR activity than Pt,68–70 (underpotential) deposition of iridium on the platinum of the cathode catalyst would lead to the lower ORR activity after SUDS cycles, as deduced from Fig. 6a. In summary, our analysis suggests that the ∼3-fold lower reversal tolerance time observed for the mitigated MEA after 500 SUSD cycles is most likely due to a loss of OER activity of the anode co-catalyst caused by the transient dissolution of platinum from the anode Pt/C catalyst and its partial deposition on the surface of the iridium phase of the IrO2/TiO2 anode co-catalyst. It has to be mentioned that during the start-up process, any dissolved Pt or Ir will immediately re-deposit on the parent particle due to the low anode potential in the presence of H2 (a similar phenomenon was suggested for Pt by Schwämmlein et al.28), so that Irn+ crossover to the cathode can be neglected during the start-up process and should be mainly occurring during the shut-down process. Based on the here proposed mechanisms, the degradation of the ORR activity of the Pt/C cathode catalyst during SUSD cycles could be avoided, if the chemical reduction of the active iridium oxide phase of the IrO2/TiO2 anode co-catalyst could be prevented. Thus, future research towards the development of a more stable anode co-catalyst to mitigate cell reversal degradation would be to focus on stabilization strategies to hinder the chemical reduction of iridium oxide based catalysts by H2 at the operational temperatures of PEMFCs, along with designing high OER active IrO2 catalysts. However, whether Pt would deposit on the surface of a non-reducible iridium oxide based anode co- catalyst and equally degrade its OER activity upon SUSD cycling is still an open question, which requires further investigation in future studies. Last but not least, future research towards the development of new anode co-catalysts should focus on stabilization strategies to hinder the chemical reduction of iridium oxide based catalysts by H2 at the operational temperatures of PEMFCs, along with designing highly OER active IrO2 catalysts. Results and Discussion It has to be mentioned that during the start-up process, any dissolved Pt or Ir will immediately re-deposit on the parent particle due to the low anode potential in the presence of H2 (a similar phenomenon was suggested for Pt by Schwämmlein et al.28), so that Irn+ crossover to the cathode can be neglected during the start-up process and should be mainly occurring during the shut-down process. Based on the here proposed mechanisms, the degradation of the ORR activity of the Pt/C cathode catalyst during SUSD cycles could be avoided, if the chemical reduction of the active iridium oxide phase of the IrO2/TiO2 anode co-catalyst could be prevented. Thus, future research towards the development of a more stable anode co-catalyst to mitigate cell reversal degradation would be to focus on stabilization strategies to hinder the chemical reduction of iridium oxide based catalysts by H2 at the operational temperatures of PEMFCs, along with designing high OER active IrO2 catalysts. However, whether Pt would deposit on the surface of a non-reducible iridium oxide based anode co- catalyst and equally degrade its OER activity upon SUSD cycling is still an open question, which requires further investigation in future studies. atmosphere should be carefully evaluated by a here proposed new methodology prior to using the catalysts in a PEMFC anode. It is based on an extended isothermal TGA experiment at operational temperatures of PEMFCs and under a dilute H2 atmosphere, where the reducibility of the IrO2 catalyst can be reliably and quantitatively examined. After the isothermal TGA experiment, the catalyst powder can be electrochemically investigated by RDE experiments in order to determine the effect of the exposure of the catalyst to a reducing H2 atmosphere on its electrochemical properties. on the surface of the IrO2/TiO2 anode co-catalyst would be expected to drastically reduce its OER activity. Once the H2-front has passed, the open circuit potential of the cathode electrode will be lowered to ∼0.90–0.95 V vs RHE due to the H2 permeation through membrane (corresponding to the OCV of a H2/air cell), at which potential the (underpotential) deposition of iridium ions dissolved in the ionomer phase of the cathode electrode (as well as the deposition of dissolved platinum ions) on the Pt/C cathode catalyst will likely occur. Results and Discussion (a) Normal fuel cell operation: illustrating the chemical reduction of the near-surface layer(s) of the iridium oxide phase of the IrO2/TiO2 anode co-catalyst by H2 to metallic Ir. (b) Fuel cell shut-down step: upon passage of the air-front, the anode potential increases to ∼1.05 V vs RHE,28 leading to platinum and iridium dissolution from the anode catalysts, whereby the resulting Ptn+ and Irn+ ions can diffuse within in the ionomer and membrane phase; after passage of the air-front, the cathode potential will assume ∼1.05 V,8 and re-deposition of crossover iridium on the platinum surface of the Pt/C cathode catalyst could possibly occur. (c) Fuel cell shut-down step: upon passage of the H2-front, the anode potential decreases from ∼1.05 V to ∼0 V vs RHE, and the re-deposition of Ptn+ and Irn+ on the Pt/C and the IrO2/TiO2 catalysts in the anode will occur; after passage of the H2-front, the cathode potential is ∼0.95 V (this lower OCV is due to H2 crossover), and iridium deposition on the Pt/C cathode catalyst becomes more likely. Journal of The Electrochemical Society, 2021 168 064521 on the surface of the IrO2/TiO2 anode co-catalyst would be expected to drastically reduce its OER activity. Once the H2-front has passed, the open circuit potential of the cathode electrode will be lowered to ∼0.90–0.95 V vs RHE due to the H2 permeation through membrane (corresponding to the OCV of a H2/air cell), at which potential the (underpotential) deposition of iridium ions dissolved in the ionomer phase of the cathode electrode (as well as the deposition of dissolved platinum ions) on the Pt/C cathode catalyst will likely occur. As iridium has a much lower ORR activity than Pt,68–70 (underpotential) deposition of iridium on the platinum of the cathode catalyst would lead to the lower ORR activity after SUDS cycles, as deduced from Fig. 6a. In summary, our analysis suggests that the ∼3-fold lower reversal tolerance time observed for the mitigated MEA after 500 SUSD cycles is most likely due to a loss of OER activity of the anode co-catalyst caused by the transient dissolution of platinum from the anode Pt/C catalyst and its partial deposition on the surface of the iridium phase of the IrO2/TiO2 anode co-catalyst. Conclusions In light of the here made observations, it is clear that the reduction of the near-surface layer(s) of the IrO2 anode co-catalyst to metallic Ir compromises its stability during the anode potential transients that occur during SUSD cycles. Therefore, it is suggested that the stability of IrO2-based anode co-catalysts in H2-containing Journal of The Electrochemical Society, 2021 168 064521 Figure A·2. The initial 10 cyclic voltammograms (CVs) of the partially reduced IrO2/TiO2 samples marked in Fig. 5a, namely after (a) 0.5 h, (b) 3 h, (c) 12 h, and (d) 24 h of the TGA isothermal experiment at 80 °C. In order to plot Fig. 5b, the ﬁrst voltammetric cycles out of the measured 10 cycles were taken, whereby the initial anodic going sweeps from OCP (∼0.9 VRHE) to 1.35 VRHE were removed. The arrows show the evolution of the CVs from cycle 1 to 10. The RDE measurements conditions are the same as given in Fig. 5b. Figure A·2. The initial 10 cyclic voltammograms (CVs) of the partially reduced IrO2/TiO2 samples marked in Fig. 5a, namely after (a) 0.5 h, (b) 3 h, (c) 12 h, and (d) 24 h of the TGA isothermal experiment at 80 °C. In order to plot Fig. 5b, the ﬁrst voltammetric cycles out of the measured 10 cycles were taken, whereby the initial anodic going sweeps from OCP (∼0.9 VRHE) to 1.35 VRHE were removed. The arrows show the evolution of the CVs from cycle 1 to 10. The RDE measurements conditions are the same as given in Fig. 5b. ORCID 17. J. Zhang, F. Coms, and S. Kumaraguru, J. Electrochem. Soc., 168, 024520 (2021). 18 I J I H d Y T k El hi A 90 148 (2013) 17. J. Zhang, F. Coms, and S. Kumaraguru, J. Electrochem. Soc., 168, 024520 (2021). 18. I. Jang, I. Hwang, and Y. Tak, Electrochim. Acta, 90, 148 (2013). 18. I. Jang, I. Hwang, and Y. Tak, Electrochim. Acta, 90, 148 (2013). Mohammad Fathi Tovini https://orcid.org/0000-0003-4334-4471 Mohammad Fathi Tovini https://orcid.org/0000-0003-4334-4471 19. C.-W. Roh, H.-E. Kim, J. Choi, J. Lim, and H. Lee, J. Power Sources, 443, 227270 (2019). Hubert A. Gasteiger https://orcid.org/0000-0001-8199-8703 Hubert A. Gasteiger https://orcid.org/0000-0001-8199-8703 20. C. E. Moore, J. Eastcott, M. Cimenti, N. Kremliakova, and E. L. Gyenge, J. Power Sources, 417, 53 (2019). References 21. T. Labi, F. Van Schalkwyk, S. M. Andersen, P. Morgen, S. C. Ray, and J. Chamier, J. Power Sources, 490, 229568 (2021). 1. (https://energy.gov/eere/vehicles/downloads/us-drive-fuel-cell-technical-team- roadmap) (accessed on 03.02.2021) USDRIVE, Fuel Cell Technical Team Roadmap. 1. (https://energy.gov/eere/vehicles/downloads/us-drive-fuel-cell-technical-team- roadmap) (accessed on 03 02 2021) USDRIVE Fuel Cell Technical Team 1. (https://energy.gov/eere/vehicles/downloads/us-drive-fuel-cell-technical-team- roadmap) (accessed on 03.02.2021) USDRIVE, Fuel Cell Technical Team Roadmap. 22. C. Cai, Z. Wan, Y. Rao, W. Chen, J. Zhou, J. Tan, and M. Pan, J. Power Sources, 455, 227952 (2020). 1. (https://energy.gov/eere/vehicles/downloads/us drive fuel cell technical team roadmap) (accessed on 03.02.2021) USDRIVE, Fuel Cell Technical Team Roadmap. ( p gy g roadmap) (accessed on 03.02.2021) USDRIVE, Fuel Cell Technical Team Roadmap. ( ) 23. R. Marić, C. Gebauer, M. Nesselberger, F. Hasché, and P. Strasser, J. Electrochem. Soc., 167, 124520 (2020). p 2. Y. Wang et al., Int. J. Hydrogen Energy, 45, 996 (2020). p 2. Y. Wang et al., Int. J. Hydrogen Energy, 45, 996 (2020). 2. Y. Wang et al., Int. J. Hydrogen Energy, 45, 996 (2020). 3. P. Mandal, B. K. Hong, J.-G. Oh, and S. Litster, J. Power Sourc ( ) 4. P. J. Rheinländer and J. Durst, J. Electrochem. Soc., 168, 024511 B. K. Hong, P. Mandal, J.-G. Oh, and S. Litster, J. Power Sources, 32 25. C. A. Reiser, L. Bregoli, T. W. Patterson, J. S. Yi, J. D. Yang, M. L. Perry, and T. D. Jarvi, Electrochem. Solid-State Lett., 8, A273 (2005). g 5. T. R. Ralph, S. Hudson, and D. P. Wilkinson, ECS Trans., 1, 67 (2006). T. D. Jarvi, Electrochem. Solid-State Lett., 8, A273 (2005) 6. J. P. Meyers and R. M. Darling, J. Electrochem. Soc., 153, A1432 (2006). 26. T. Mittermeier, A. Weiß, F. Hasché, G. Hübner, and H. A. Gasteiger, J. Electrochem. Soc., 164, F127 (2017). 7. R. L. Borup, D. D. Papadias, R. Mukundan, D. Spernjak, D. A. Langlois R. Ahluwalia, K. L. More, and S. Grot, ECS Trans., 69, 1029 (2015). 7. R. L. Borup, D. D. Papadias, R. Mukundan, D. Spernjak, D R. Ahluwalia, K. L. More, and S. Grot, ECS Trans., 69, 1029 (2015 27. T. Mittermeier, A. Weiß, F. Hasché, and H. A. Gasteiger, J. Electrochem. Soc., 165, F1349 (2018). 8. R. Makharia, S. Kocha, P. Yu, M. A. Sweikart, W. Gu, F. Wagner, and H. A. Gasteiger, ECS Trans., 1, 3 (2006). 28. J. N. Schwämmlein, P. J. Rheinländer, Y. References Chen, K. T. Freyer, and H. A. Gasteiger, J. Electrochem. Soc., 165, F1312 (2018). 9. I. C. Halalay, S. Swathirajan, B. Merzougui, M. P. Balogh, 9. I. C. Halalay, S. Swathirajan, B. Merzougui, M. P. Balogh, G. C. Garabedian, and M. K. Carpenter, J. Electrochem. Soc., 158, B313 (2011). M. K. Carpenter, J. Electrochem. Soc., 158, B313 (2011). 29. A. Weiß, A. Siebel, M. Bernt, T. H. Shen, V. Tileli, and H. A. Gasteiger, J. Electrochem. Soc., 166, F487 (2019). 10. C. Gebauer, Z. Jusys, and R. J. Behm, J. Electrochem. Soc., 165, J3342 (2018) 10. C. Gebauer, Z. Jusys, and R. J. Behm, J. Electrochem. Soc 11. J. Wu, X. Z. Yuan, J. J. Martin, H. Wang, J. Zhang, J. Shen, S. Wu, and W. Merida, J P S 184 104 (2008) 11. J. Wu, X. Z. Yuan, J. J. Martin, H. Wang, J. Zhang, J. Shen, S. Wu, and W. Meri J. Power Sources, 184, 104 (2008). 30. B. Klose-Schubert, D. Herein, and H.-G. Reitz, EU Patent EP, 2 608 297 A1 (2013), Awarded June 26. J. Power Sources, 184, 104 (2008). Journal of The Electrochemical Society, 2021 168 064521 36. J. Durst, C. Simon, F. Hasché, and H. A. Gasteiger, J. Electrochem. Soc., 162, F190 (2015). 56. C. C. L. McCrory, S. Jung, I. M. Ferrer, S. M. Chatman, J. C. Peters, and T. F. Jaramillo, JACS, 137, 4347 (2015). T. F. Jaramillo, JACS, 137, 4347 (2015). ( ) 37. J. H. Flynn and L. A. Wall, Journal of Polymer Science Part B: Polymer Letters, 4, 323 (1966). 57. S. Geiger et al., Nat. Catal., 1, 508 (2018). 58. T. Reier, D. Teschner, T. Lunkenbein, A. Bergmann, S. Selve, R. Kraehnert, 58. T. Reier, D. Teschner, T. Lunkenbein, A. Bergmann, S. Selve, R. Kraehnert, R Schlögl and P Strasser J Electrochem Soc 161 F876 (2014) ( ) 38. T. Reier, M. Oezaslan, and P. Strasser, ACS Catal., 2, 1765 (2012). 38. T. Reier, M. Oezaslan, and P. Strasser, ACS Catal., 2, 1765 R. Schlögl, and P. Strasser, J. Electrochem. Soc., 161, F876 (2014 59. L. D. Burke and E. J. M. O’Sullivan, J. Electroanal. Chem. Interfacial Electrochem., 117, 155 (1981). 39. S. J. Freakley, J. Ruiz-Esquius, and D. J. Morgan, Surf. Interface Anal., 49, 794 (2017). ( ) 40. V. Pfeifer et al., Surf. Interface Anal., 48, 261 (2016). 60. E. N. El Sawy and V. I. Birss, J. Mater. Chem., 19, 8244 (2009). 61. S. Cherevko, S. Geiger, O. Kasian, A. Mingers, and K. J. J. Mayrhofer, J. Electroanal. Chem., 774, 102 (2016). 41. D. Yu, M. Zhu, T. A. Utigard, and M. Barati, Miner. Eng., 54, 32 (2013) J. Electroanal. Chem., 774, 102 (2016). 42. K. V. Manukyan, A. G. Avetisyan, C. E. Shuck, H. A. Chatilyan, S. Rouvimov, S. 42. K. V. Manukyan, A. G. Avetisyan, C. E. Shuck, H. A. Chatilyan, S. Rouvimov, S. L. Kharatyan, and A. S. Mukasyan, The Journal of Physical Chemistry C, 119, 16131 (2015). y y y L. Kharatyan, and A. S. Mukasyan, The Journal of Physical Chemistry C, 119, 16131 (2015). 62. G. S. Harzer, J. N. Schwämmlein, A. M. Damjanović, S. Ghosh, and H. A. Gasteiger, J. Electrochem. Soc., 165, F3118 (2018). ( ) 43. J. Dang, G.-H. Zhang, and K.-C. Chou, Int. J. Refract. Met. Hard Mater, 41, 356 (2013). g 63. S. Cherevko, S. Geiger, O. Kasian, A. Mingers, and K. J. J. Mayrhofer, J. Electroanal. Chem., 773, 69 (2016). 44. S. Watanabe, X. Ma, and C. (2013), Awarded June 26. ( ) 12. M. L. Perry, T. Patterson, and C. Reiser, ECS Trans., 3, 783 (2006). ( ) 31. M. Bernt, A. Siebel, and H. A. Gasteiger, J. Electrochem. Soc., 31. M. Bernt, A. Siebel, and H. A. Gasteiger, rnt, A. Siebel, and H. A. Gasteiger, J. Electrochem. Soc., 165, F305 y 13. S. W. Lee, B. H. Lee, T.-Y. Kim, C. Baik, M. S. Kim, G. S. Chai, and C. Pak, Catal. Commun., 130, 105758 (2019). 32. M. Bernt and H. A. Gasteiger, J. Electrochem. Soc., 163, F3179 (2016) Commun., 130, 105758 (2019). g 33. R. D. Shannon, Acta Crystallogr., Sect. A, 32, 751 (1976). 33. R. D. Shannon, Acta Crystallogr., Sect. A, 32, 751 (1976). 14. C. E. Moore, F. Afsahi, A. P. Young, and E. L. Gyenge, The Journal of Physical Chemistry C, 123, 23361 (2019). 34. T. Mittermeier, A. Weiß, H. A. Gasteiger, and F. Hasché, J. Electrochem. Soc., 164, F1081 (2017). 15. H. Schmies et al., Adv. Energy Mater., 8, 1701663 (2018). 35. C. Simon, F. Hasche, D. Muller, and H. A. Gasteiger, ECS Trans., 69, 1293 (2015). 16. E. Hornberger et al., ACS Catal., 8, 9675 (2018). Journal of The Electrochemical Society, 2021 168 064521 Journal of The Electrochemical Society, 2021 168 064521 Song, The Journal of Physical Chemistry C, 113, 14249 (2009). 64. P. Jovanovič et al., JACS, 139, 12837 (2017). 65. M. Lopez, A. Schleunung, and P. Biberbach, EU Patent EP, 1 701 790 B1 (2009), Awarded Sept. 16. 45. J. Li, G. Lu, G. Wu, D. Mao, Y. Guo, Y. Wang, and Y. Guo, Catalysis Science & Technology, 4, 1268 (2014). p 66. A. Hartig-Weiss, M. Miller, H. Beyer, A. Schmitt, A. Siebel, A. T. S. Freiberg, H. A. Gasteiger, and H. A. El-Sayed, ACS Appl. Nano Mater., 3, 2185 (2020). gy, , ( ) 46. H. Zhu, Z. Qin, W. Shan, W. Shen, and J. Wang, J. Catal., 225, 267 (2004). Q g ( ) 47. Z. Chen, J. Dang, X. Hu, and H. Yan, Metals, 8, 751 (2018). 67. M. Pourbaix, Atlas of Electrochemical Equilibria in Aqueous Solutions (National Association of Corrosion Engineers, Houston, Texas) p. 375 (1974). 47. Z. Chen, J. Dang, X. Hu, and H. Yan, Metals, 8, 751 (2018). 48. M. Venkatesh, P. Ravi, and S. P. Tewari, The Journal of Physical Chemistry A, 117, 10162 (2013). 68. D. Yang, B. Li, H. Zhang, and J. Ma, Int. J. Hydrogen Energy, 37, 2447 (2012). g g y g gy ( 69. M. Xiao et al., Angew. Chem. Int. Ed., 58, 9640 (2019). 49. C. Massué, V. Pfeifer, X. Huang, J. Noack, A. Tarasov, S. Cap, and R. Schlögl, ChemSusChem, 10, 1943 (2017). 69. M. Xiao et al., Angew. Chem. Int. Ed., 58, 9640 (2019). 70. E. Antolini, ACS Catal., 4, 1426 (2014). 50. J. Dang, K.-C. Chou, X.-J. Hu, and G.-H. Zhang, Steel Res. Int., 84, 526 (2013). 71. P. Piela, C. Eickes, E. Brosha, F. Garzon, and P. Zelenay, J. Electrochem. Soc., 151, A2053 (2004). 51. S. Geiger, O. Kasian, B. R. Shrestha, A. M. Mingers, K. J. J. Mayrhofer, and 51. S. Geiger, O. Kasian, B. R. Shrestha, A. M. Mingers, K. J. J. Mayrhofer, and S Cherevko J Electrochem Soc 163 F3132 (2016) 72. L. Gancs, B. N. Hult, N. Hakim, and S. Mukerjee, Electrochem. Solid-State Lett., 10, B150 (2007). S. Cherevko, J. Electrochem. Soc., 163, F3132 (2016). 73. E. Antolini, J. Solid State Electrochem., 15, 455 (2011). 52. J. Mozota and B. E. Conway, Electrochim. Acta, 28, 1 (1983). 74. T. T. H. Cheng, N. Jia, and P. He, J. Electrochem. Soc., 157 74. T. T. H. Cheng, N. Journal of The Electrochemical Society, 2021 168 064521 Jia, and P. He, J. Electrochem. Soc., 157, B714 (2010). 53. J. Durst, A. Siebel, C. Simon, F. Hasché, J. Herranz, and H. A. Gasteiger, Energy Environ. Sci., 7, 2255 (2014). 5. W. Gu, P. T. Yu, R. N. Carter, R. Makharia, and H. A. Gaste 75. W. Gu, P. T. Yu, R. N. Carter, R. Makharia, and H. A. Gasteiger, L Starvation in Modern Aspects of Electrochemistry, 49, 45 (2010). 54. M. Bernt, A. Hartig-Weiß, M. F. Tovini, H. A. El-Sayed, C. Sch 54. M. Bernt, A. Hartig-Weiß, M. F. Tovini, H. A. El-Sayed, C. Schramm, J. Schröter, C. Gebauer, and H. A. Gasteiger, Chem. Ing. Tech., 92, 31 (2020). Starvation in Modern Aspects of Electrochemistry, 49, 45 (2010). p f y ( ) 76. T. Reier, M. Oezaslan, and P. Strasser, ACS Catal., 2, 1765 (2012). C. Gebauer, and H. A. Gasteiger, Chem. Ing. Tech., 92, 31 (20 76. T. Reier, M. Oezaslan, and P. Strasser, ACS Catal., 2, 1765 (20 55. M. Fathi Tovini, A. Hartig-Weiß, H. A. Gasteiger, and H. A. El-Sayed, J. Electrochem. Soc., 168, 014512 (2021). 77. C. Fan, C. Chen, J. Wang, X. Fu, Z. Ren, G. Qian, and Z. Wang, Sci. Rep., 5, 11712 (2015).
https://openalex.org/W2589022618	http://ejournal.uinsaizu.ac.id/index.php/komunika/article/download/94/68	Indonesian	null	SejarahNovel Sejarah Indonesia: Komunikasi antara Dunia Sastra dengan Dunia Nyata	Komunika	1,970	cc-by-sa	3,025	JURNAL DAKWAH DAN KOMUNIKASI JURNAL DAKWAH DAN KOMUNIKASI SEJARAH INDONESIA Seperti yang telah tergores dalam catatan yang tebal dan penuh mutiara (bahkan luka, darah, dan air mata), tentu sejarah Indonesia bukan lagi barang baru yang perlu diperdebatkan. Kalau pun ditemukan beberapa hal yang dirasa mengganjal atau menggelinjang, tentu bagian itu tidaklah banyak, dan biasanya cenderung menjadi bahan perbincangan sesaat atau beberapa saat saja. Selebihnya, hampir dapat dikatakan bahwa semua yang terjadi di sini, di negeri ini, relatif telah “menyejarah”. Dari sesuatu yang telah menyejarah itu juga tampak betapa hubungan antara sastra dengan realitas historis demikian dekatnya. Bahkan, dalam beberapa hal, hubungan itu demikian saling mempengaruhi. PENGANTAR: PROBLEMATIKA ISTILAH Ketika kita berbicara mengenai komunikasi, tidak bisa tidak, kita sudah membayangkan adanya korelasi positif atau bahkan interaksi antara dua pihak atau lebih. Demikian pula halnya ketika tulisan kecil ini diberi subjudul Komunikasi ... Sebagai konsekuensinya, perunutan atau pencarian hubungan antara Sastra sebagai dunia imajiner (dunia imajinasi), dunia khayal, dunia angan-angan, dengan Kenyataan (Dunia Nyata) sebagai realitas merupakan kerja tambahan yang harus dilakukan. Meskipun demikian, tentu saja kerja sampingan itu sudah tidak berat lagi karena usaha ke arah itu sudah banyak dilakukan, hasilnya sudah banyak dibukukan, dan pengakuan atasnya pun sudah hampir bisa dikatakan tidak terhitung lagi jumlahnya. Konsep bahwa sastra merupakan salah satu sarana komunikasi antara pengarang dengan pembaca, misalnya, juga telah diakui oleh banyak ahli. Tidak ada masalah. Sementara itu, judul Sejarah Novel Sejarah Indonesia sengaja dipilih karena dari istilahnya saja sebenarnya sudah mengandung problematika. Dari judul itu setidaknya dapat dibicarakan beberapa konsep atau istilah turunan berikut. )Penulis adalah dosen tetap di Jurusan Sastra Indonesia Fakultas Ilmu Budaya UGM Yogyakarta. )Penulis adalah dosen tetap di Jurusan Sastra Indonesia Fakultas Ilmu Budaya UGM Yogyakart Abstract: Literary works bring their spirit of age. Its mean that literary works cant be separated from certain time context. So, there’s communication between literary and reality. Then, can we use “history” on literary works as a source of history? Several people agree that literary can be positioned as mental fact. Therefore, with certain consideration, with certain filter, and comparison and evaluation process, there’s chance that literary, or historical fact inside literary works, being used as one secondary historical source. Keywords: Indonesian historical novel, Literary works, history, reality. Sejarah Novel Sejarah Indonesia: Komunikasi antara Dunia Sastra dengan Dunia Nyata Heru Marwata *) SEJARAH NOVEL INDONESIA Buku sejarah sastra Indonesia pernah ditulis, misalnya oleh Ajip Rosidi. Buku-buku sejenis karya Ajip juga telah dipublikasikan. Sebagai bagian dari karya sastra Indonesia novel tentu saja juga ikut menjadi bahan bahasan buku-buku tersebut. Apakah yang dimaksud dengan “sejarah sastra”? Apakah pustaka yang berisi periodisasi serta daftar pengarang dan karya-karyanya merupakan buku sejarah sastra? Ada ahli yang mengajak (karena merasa perlu) kita menafsir ulang model penulisan sejarah sastra yang demikian itu. Tertarik? JURNAL DAKWAH DAN KOMUNIKASI dibandingkan puisi, cerpen, atau naskah drama. Demikian pula halnya dalam kerangka komunikasi antara dua dunia: sastra dan realita. dibandingkan puisi, cerpen, atau naskah drama. Demikian pula halnya dalam kerangka komunikasi antara dua dunia: sastra dan realita. NOVEL SEJARAH (INDONESIA) Dalam bahan pemicu diskusi tertulis ini novel sejarah diberi batasan sebagai novel yangmemanfaatkan “sejarah” sebagai bahan penulisan, terlepas dari ada tidaknya pengakuan penulis. Adakah novel seperti itu di Indonesia? Jika dikaitkan dengan label resmi, misalnya berupa tulisan Novel Sejarah (di sampul, misalnya), memang sangat sedikit contohnya. Di antara yang sedikit itu trilogi Rara Mandut, Genduk Duku, dan Lusi Lindri karya Mangunwijaya merupakan contoh konkret. Namun, jika dasarnya bukan label, melainkan tanggapan atau pengakuan orang-orang yang berkompeten dalam bidang sastra (saat ini kebanyakan tanggapan/pengakuan seperti itu dimuat/disertakan dalam novel, misalnya di sampul dalam atau belakang, endorsemen), contoh karya yang dapat dimasukkan ke kriteria novel sejarah jumlahnya cukup banyak. Novel-novel karya Pandir Kelana dan Trilogi Gadis Tangsi Suparto Brata dapat disebut sebagai contoh. Juga salah satu di antara karya Pulau Buru Pramoedya. Bagaimana novel-novel itu “memakai” sejarah atau berkomuniksai dengan realitas historis? Apakah dengan demikian berarti “sejarah novel-sejarah” dapat disusun? Atau, jangan-jangan justru sebenarnya semua novel Indonesia adalah novel sejarah. Ini sebuah tantangan bagi para penulis sejarah sastra dan sejarah umum. Jurusan Dakwah STAIN Purwokerto NOVEL INDONESIA Novel adalah salah satu genre sastra yang cukup banyak ditulis dengan menggunakan repertoar atau repertoire (terminologi Wolfgang Iser untuk menyebut “realitas ekstratekstual” dalam istilah Holub) peristiwa historis. Meskipun demikian, tidak berarti bahwa genre sastra lainnya, seperti puisi dan naskah drama, tidak memanfaatkan aspek sejarah. Mengapa novel yang dipilih sebagai bahan diskusi? Dalam kasus ini novel dipandang memiliki lebih banyak keleluasaan karena secara umum hampir semua unsur sastra (dalam istilah Robert Stanton sering disebut tema, fakta, dan sarana sastra) dapat diekspresikan secara rinci dan lebih gamblang dalam novel. Dalam beberapa hal novel juga lebih menyediakan “ruang” KOMUNIKA ISSN: 1978-126 Vol.2 No.2 Jul-Des 2008 pp.131-139 KOMUNIKA ISSN: 1978-126 Vol.2 No.2 Jul-Des 2008 pp.131-139 KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 ISSN: 1978-126 Jurusan Dakwah STAIN Purwokerto JURNAL DAKWAH DAN KOMUNIKASI JURNAL DAKWAH DAN KOMUNIKASI dalam penampang sinkronis dan diakronis. Karya sastra juga berada dalam konteks sejarah khusus dan sejarah umum. Tujuh tesis Jauss sebenarnya dapat dikatakan sebagai tesis komunikasi antara sastra dan kenyataan sejarah, antara dunia imajinasi dengan realitas historis. dalam penampang sinkronis dan diakronis. Karya sastra juga berada dalam konteks sejarah khusus dan sejarah umum. Tujuh tesis Jauss sebenarnya dapat dikatakan sebagai tesis komunikasi antara sastra dan kenyataan sejarah, antara dunia imajinasi dengan realitas historis. Sayang memang bahwa kadang-kadang horison harapan pembaca tidak hanya dipengaruhi oleh pengalaman baca, tetapi juga oleh “kepentingan tertentu”. Akibatnya, karya-karya yang ditengarai berpotensi mempengaruhi “keinginan” dan “keingintahuan” pembaca jusru harus menerima tempelan label tertentu: salah satu label yang paling menakutkan adalah Dilarang. Era berganti, masa berlalu, tanggapan orang pun mengalami perubahan, dan label terhadap karya tertentu juga bisa diklethek seperti stiker. Dari uraian yang terbatas ini ada indikasi kemungkinan karya sastra mampu pula “menyemangati zaman” (bukan hanya membawa semangat zaman). Sebagai ilustrasi, cermati kutipan pendapat Jakob Sumarjo dari halaman akhir roman Anak Semua Bangsa karya Pram berikut. “Kita beruntung menyaksikan novel besar pada dekade ini ... yang ... telah berhasil menggambarkan suasana sosial budaya zamannya.” Secara implisit jelas dalam pernyataan ini ada pengakuan antara sastra sebagai gambaran dunia dengan dunia (nyata) yang dilukiskannya. SEJARAH NOVEL SEJARAH DAN SEJARAH INDONESIA Dalam uraian sebelumnya disajikan semacam penjajaran antara Sejarah Novel dan Sejarah Indonesia. Dengan “semangat” yang sama pendampingan “Sejarah Novel Sejarah” dan “Sejarah Indonesia” pun pasti berterima. Sanusi Pane menulis Surapati dan Robert Anak Surapati serta Sandyakalaning Majapahit pada tahun 30-an. Apakah ada korelasi antara karya-karya Sanusi Pane dengan sejarah bangsa kita? Tampaknya sangat sulit untuk menolak atau menisbikan adanya hubungan tersebut. NOVEL SEJARAH TENTANG INDONESIA Dalam sejarah novel Indonesia dikenal beberapa judul berikut. Rara Mendut (Balai Pustaka), Rara Mendut (Ajip Rosidi), Rara Mendut (Mangunwijaya). Jika dalam ketiga novel itu disebut-sebut nama tempat seperti Kerajaan Mataram dan Pati serta nama-nama tokoh seperti Sultan Agung, Adipati Pragola, dan Tumenggung Wiraguna, adakah artinya bagi sejarah? Burung-burung Manyar karya Mangunwijaya, misalnya, menyebut-nyebut nama UGM {ketika tokoh fiktif Larasati mendapatkan gelar doktor dengan predikat summa cum laude di Balai Senat (pernah terjadikah dalam sejarah UGM?)}, menggunakan tiga latar waktu (prarevolusi, revolusi, dan pascarevolusi) penting di Indonesia, serta memasukkan tokoh Sjahrir yang terkenal. Adakah korelasi positif antara karya itu dengan sejarah Indonesia? Mengapa karya Ki Panji Kusmin Langit Makin Mendung pernah menghebohkan dunia sastra Indonesia pada tahun 60-an? Mengapa Senja di Jakarta Mochtar Lubis dinilai mengritik Soekarno? Mengapa Para Priyayi dan Jalan Menikung serta Sri Sumarahkarya Umar Kayam direkomendasikan untuk dibaca oleh orang (asing) yang ingin mengetahui seluk beluk orang (dan budaya) Jawa? Lasi—yang seperti orang Jepang—dalam Bekisar Merah karya Ahmad Tohari dijadikan istri simpanan oleh seorang pejabat yang merasa perlu—karena gengsi—meniru “gaya kawin” sang pemimpin besar revolusi. Apakah ini sebuah pertanda betapa cukup positifnya hubungan atau betapa terjalin eratnya komunikasi antara sejarah dan sastra atau antara sejarah sastra dan sejarah bangsa? SEJARAH NOVEL DAN SEJARAH INDONESIA Tesis keempat Hans-Robert Jauss yang terkenal adalah Semangat Zaman (lengkapnya 7 tesis itu adalah; (1) pengalaman pembaca, (2) horison harapan, (3) jarak estetik, (4) semangat zaman, (5) rangkaian sastra, (6) sinkronis dan diakronis, serta (7) sejarah khusus dan sejarah umum). Dalam penjelasan konsep semangat zaman ditemukan semacam kesimpulan bahwa karya sastra membawa semangat zamannya. Arti mudahnya, karya sastra membawa “semangat zaman” ketika karya itu ditulis. Hal ini merupakan indikasi pengakuan bahwa karya sastra tidak bisa dilepaskan dari konteks waktu tertentu, lebih mudahnya lagi antara sastra dan realitas terjadi komunikasi. Sejarah novel (Indonesia) tidak dapat dicerabut dari akar sejarah (Indonesia). Jika logikanya di balik, sejarah (Indonesia) telah (bahkan, pasti) “mewarnai” sejarah novel (Indonesia). Dengan logika yang muluk-muluk, tentu tidak tertutup kemungkinan bahwa novel Indonesia juga punya peluang atau potensi untuk mempengaruhi sejarah Indonesia. Pernah-kah kita menyadari bahwa seri Bumi Manusia Pramoedya Ananta Toer (Anak Semua Bangsa, Jejak Langkah, dan Rumah Kaca) pernah“mewarnai” sejarah Indonesia? Semangat zaman adalah bagian dari sejarah. Apa fungsi aspek historis itu bagi novel? Membuat jarak estetik antara karya dan pembaca tidak terlalu renggang. Mengapa? Agar yang ada dalam karya itu tidak menjadi asing dan tidak dikenali pembaca. Bagaimana dengan “kata mutiara” yang mengatakan bahwa sastra selalu berada dalam ketegangan antara tradisi dan inovasi dan bahwa karya sastra dinilai atau dihargai karena kebaruan atau invensinya? Untuk menjawabnya, karena sudah telanjur menyebut secara lengkap, sebaiknya kita membawa kisah ini ke konteks 7 tesis Jauss. Hubungan ketujuh tesis Jauss di atas bersifat dialektis. Secara berurutan, tesis pertama menjadi dasar tesis kedua, demikian seterusnya. Pengalaman baca seseorang akan mempengaruhi horison harapannya. Horison harapan yang dilandasi oleh pengalaman baca itu akan menentukan renggang rekatnya jarak estetik yang terjadi antara karya sastra dengan pembaca, dan seterusnya. Karya sastra membawa semangat zaman. Karya sastra berada dalam rangkaian sastra. Karya sastra berposisi KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 Jurusan Dakwah STAIN Purwokerto KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 ISSN: 1978-126 JURNAL DAKWAH DAN KOMUNIKASI Perhatikan beberapa hal dan “fakta” berikut. Perhatikan beberapa hal dan “fakta” berikut. Suparto menikah dengan Rr. Ariyati, anak seorang petani kaya di Ngombol, Kedu Selatan. Rr. Ariyati lahir di Meurudu 1940, ketika ayahnya jadi serdadu kumpeni. Ketika Perang Dunia II meletus, ayahnya dibubarkan dari kumpeni di Bandung, sedangkan Ariyati bersama tiga orang saudaranya serta ibunya tertinggal bersama istri serdadu kumpeni lainnya di asrama tangsi Medan. Mereka dibawa mengungsi oleh tentara Belanda, tetapi terkejar oleh tentara Jepang di Blankejeren (biografi singkat Suparto yang dimuat di halaman terakhir trilogi novelnya) (realitas historis). Bandingkan biografi di atas dengan “fakta” yang ada dalam novel Suparto ini. Tokoh utama Trilogi Gadis Tangsi adalah Teyi. Adik Teyi, Tumpi, lahir di Meurudu. Tangsi Lorong Belawan berada di Medan. Ayah Teyi seorang serdadu kumpeni. Setelah ayahnya tewas dan Belanda mengalami kekalahan dari tentara Jepang, Teyi, Tumpi, dan ibunya bersama dengan istri-anak para serdadu kumpeni diungsikan oleh tentara Belanda. Rombongan pengungsi ini terkejar tentara Jepang di Blankejeren dan dijadikan tawanan. Akhirnya, keluarga Teyi yang berasal dari Ngombol bisa selamat kembali ke daerah asal. Teyi dan ibu serta adiknya sukses membangun Kerajaan Raminem (nama ibu Teyi) di Ngombol, menjadi tuan tanah dan juragan padi-beras (realitas fiksional). Perhatikan kutipan ini. “Narasumber novel Kerajaan Raminem adalah mertua Suparto Brata” (Kerajaan Raminem, 2006: 470). “Narasumber novel Mahligai di Ufuk Timur ini adalah kesaksian, pengalaman, dan pengamatan Suparto Brata sendiri, asli dan murni” (sampul belakang Mahligai di Ufuk Timur, 2007). Ketika kita mencermati kata “narasumber”, terbayanglah adanya sebuah Isi yang memerlukan Bentuk. Yang disampaikan mertua Suparto adalah Isi, dan yang ditulis Suparto dalam triloginya adalah Bentuk itu, yang lengkap dengan isinya dalam bentuk yang agak lain. Di sini ada dua realitas yang berbeda. Yang disampaikan mertua Suparto (kemungkinan besar) adalah realitas historis, sementara yang di-jlentreh-kan Suparto dalam triloginya adalah realitas fiksional. Demikian pula halnya ketika Suparto sendiri menjadi narasumber bagi novel yang ditulisnya. Dapatkah kita memanfaatkan “sejarah” dalam karya sastra sebagai salah satu sumber sejarah? Ada beberapa orang yang bersepakat bahwa sastra dapat didudukkan sebagai fakta mental. Jadi, dalam beberapa hal, dengan berbagai pertimbangan, dengan filter tertentu, lewat proses komparasi dan evaluasi, sebenarnya tidak tertutup kemungkinan sastra, atau fakta historis dalam sastra, digunakan sebagai salah satu sumber sejarah (sekunder). Dulu, sebut saja novel-novel sebelum tahun 80-an, karya sastra yang menampilkan adegan yang dekat-dekat dengan seksualitas dianggap agak tabu. Bahkan, ada tokoh yang mensinyalir karya sastra dianggap sebagai pose pengarangnya di depan publik. JURNAL DAKWAH DAN KOMUNIKASI Apakah ini berarti bahwa dalam karya fiksi yang bergenre novel, khususnyanovel sejarah, ada muatan sejarahnya? Jika ya, sejauh mana fakta historis fiksional itu bisa dianggap sebagai sebuah kebenaran? Apakah itu berlaku untuk semua karya dari semua penulis? Genduk Duku ... mengungkap suasana tahun-tahun terakhir masa pemerintahan Sultan Agung ... serta suasana yang sendi-sendi historisnya dapat kita lacak dalam laporan-laporan sumber Barat, seperti yang didokumentasikan oleh bekas duta besar VOC di Mataram, ... semua itudiolah dalam ramuan cerita fiktif.... (sampul belakang novel Genduk Dukukarya Mangunwijaya). Jika di sini disebutkan tentang ramuan cerita fiktif, masih layakkah kita mematok realitas imajinatif sebagai sebuah kenyataan? Di mana batas-batasnya? ... Lusi Lindriini memantau, dalam bentuk novel sejarah, secermat mungkin data dan fakta historis Sunan Mangkurat I (abad ke-17), raja kejam Mataram dan zamannya yang penuh peristiwa dramatis. ... wilayah-wilayah (yang sekarang disebut) Bagelen, Magelang, dan Gunung Kidul .... (sampul belakang novel Lusi Lindri Mangunwijaya). Bagaimana kita bisa memilih dan memilah secara tepat yang fakta sejarah dan realitas fiksional? Novel BALADA DARA-DARA MENDUT ini sebentuk dokumentasi, bahkan monumen juga berupa sastra yang “mengabadikan” suatu lembaran sejarahperintisan .... (sampul belakang novel Balada Dara-dara Mendutkarya Mangunwijaya). Novel BALADA DARA-DARA MENDUT ini sebentuk dokumentasi, bahkan monumen juga berupa sastra yang “mengabadikan” suatu lembaran sejarahperintisan .... (sampul belakang novel Balada Dara-dara Mendutkarya Mangunwijaya). Jika dikatakan mengabadikan suatu lembaran sejarah, apakah ini berarti bahwa semua yang dibeberkan adalah nyata adanya? SEJARAH DALAM NOVEL (SEJARAH) Perhatikan beberapa kutipan berikut. Roro Mendut ... dicipta baru dalam bentuk sastra dengan versi khas ... Tanpa meninggalkan pertanggungjawaban segi-segi historisnya yang dilandaskan pada studi tentang Babad Tanah Jawi, dokumen-dokumen .... (sampul belakang novel Roro Mendutkarya Mangunwijaya). Roro Mendut ... dicipta baru dalam bentuk sastra dengan versi khas ... Tanpa meninggalkan pertanggungjawaban segi-segi historisnya yang dilandaskan pada studi tentang Babad Tanah Jawi, dokumen-dokumen .... (sampul belakang novel Roro Mendutkarya Mangunwijaya). KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 ISSN: 1978-126 ISSN: 1978-126 Jurusan Dakwah STAIN Purwokerto JURNAL DAKWAH DAN KOMUNIKASI PENUTUP: APOLOGI Demikian sekadar pemicu diskusi tertulis yang dapat disajikan mengenai komunikasi antara sastra dan realitas, antara dunia angan dengan kenyataan, antara dunia ciptaan pengarang dengan dunia hidup pengarang. Dengan mempertimbangkan beberapa uraian dan sekaligus bukti yang penulis coba beberkan, tampaknya mengakui adanya komunikasi antara dua dunia, sastra dan sejarah, bukanlah pengakuan yang tanpa dasar. Terlalu banyak bukti yang dapat dikemukakan sebagai pendukung atas pengakuan itu. Bagaimana dengan Anda? JURNAL DAKWAH DAN KOMUNIKASI hanya lewat karyanya. Sekarang, sebut saja karya-karya para pengarang wanita seperti Ayu Utami, Djenar Maesa Ayu, Fira Basuki, dan Dewi Lestari, “pose” yang dulunya dianggap agak melanggar ketabuan justru ditengarai sedang ngetren. Sekadar tambahan informasi mengenai pose pengarang ini, beberapa waktu yang lalu UNY nanggap artis Ria Irawan untuk membacakan Vagina Monolog (dan ternyata diikuti oleh seorang peserta reuni FIB UGM dengan membacakan Vagina Monolog di Pendapa Rumah Dinas Bupati Sleman, edan ora?). Masa berganti, rezim bergulir, dan kita mungkin—jika dianugerahi umur panjang—akan menyaksikan wolak-waliking jaman karena yang dianggap baik dalam kriteria sekarang, belum tentu bertahan di era yang telah berubah. Bukankah dalam sejarah juga pernah terjadi fenomena serupa? Bukankah gonta-gantinyabuku teks sejarah merefleksikan adanya peristiwa tersebut? Kita lihat saja. FAKTA DAN FIKSI: REALITA(S) DAN IMAJINASI --> KOMUNIKASI Dalam konteks hubungan antara fakta dan fiksi, di manakah pengarang berposisi? Menurut teori strukturalisme-genetik sosiologi sastra Lucien Goldmann pengarang bukanlah individu,ia transindividu, menulis dengan pandangan dunia tertentu. Sebagai subjek transindividu pengarang “berbicara” (termasuk melalui karya sastra) atas nama kelompok tertentu atau kelompoknya (disadari atau tidak). Konon, pengarang (apalagi pengarang besar/utama) termasuk kelompok orang yang memiliki kepekaan, daya perenungan, dan sekaligus kekuatan berimajinasi yang relatif tinggi. Ketika “berbicara” pengarang tidak hanya menyampaikan informasi atau fakta secara wantah, tetapi telah “membumbuinya” dengan pandangan, pikiran, harapan, serta misi dan visi tertentu. Bagi seorang “koki” fiksi, fakta adalah bahan mentah yang harus diolah agar siap saji, syukur-syukur dengan aroma yang merangsang indera, dengan performa yang mempesona, dengan citarasa yang memanjakan penggemarnya, dan dengan berjuta maksud yang belum tentu dapat kita raba-duga. Makin tinggi kemampuan sang koki fiksi dalam berimajinasi, makin tinggi pula kemungkinan fakta mentah yang diolahnya menjadi demikian mengundang selera (baca), dan dengan demikian, makin mudah pulalah komunikasi antara sastra dan realitas terjalin manis. Dalam konteks ini, semakin besar pulalah probabilitas karya cipta itu “menyejarah” dan mempengaruhi sejarah. Benarkah? JURNAL DAKWAH DAN KOMUNIKASI Perhatikan beberapa hal dan “fakta” berikut. Akibatnya, hampir tidak ada sastrawan yang ingin dicap suka berpose “porno” di hadapan khalayak, meskipun KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 Jurusan Dakwah STAIN Purwokerto ISSN: 1978-126 DAFTAR PUSTAKA Ananta Toer, Pramoedya. 1980. Bumi Manusia.Jakarta: Hasta Mitra. . 1980. Anak Semua Bangsa. Jakarta: Hasta Mitra. . 1980. Rumah Kaca.Jakarta: Hasta Mitra. Brata, Suparto. 2004. Gadis Tangsi. Jakarta: Kompas. . 2006. Kerajaan Raminem. Jakarta: Kompas. . 2007. Mahligai di UfukTimur. Jakarta: Kompas. Barthes, Roland. 1990. S/Z. United Kingdom: Basil Blackwell Ltd. Goldmann, Lucien. 1975. Towards a Sociology of the Novel. London: Tavistock Publication Ltd. Goldmann, Lucien. 1975. Towards a Sociology of the Novel. London: Tavistock Publication Ltd. Goldmann, Lucien. 1975. Towards a Sociology of the Novel. London: Tavistock Publicat Holub, Robert C. 1989. Reception Theory: A Critical Introduction.London:Routledge. Holub, Robert C. 1989. Reception Theory: A Critical Introduction.London:Routledge. Iser, Wolfgang. 1987. The Act of Reading: A Theory of Aesthetic Response.Baltimore & London: The John Hopkins University Press. Jauss, Hans Robert. 1983. Toward an Aesthetic of Reception.Minneapolis: University of Minnesota Press. Iser, Wolfgang. 1987. The Act of Reading: A Theory of Aesthetic Response.Baltimore & London: The John H Jauss, Hans Robert. 1983. Toward an Aesthetic of Reception.Minneapolis: University of Minnesota Press. ISSN: 1978-126 ISSN: 1978-126 JURNAL DAKWAH DAN KOMUNIKASI Mangunwijaya, Y.B. 1993. Balada Dara-dara Mendut.Yogyakarta: Kanisius. Mangunwijaya, Y.B. 1993. Balada Dara-dara Mendut.Yogyakarta: Kanisius. . 1994. Rara Mendut.Jakarta: Gramedia Pustaka Utama. . 1994. Genduk Duku.Jakarta: Gramedia Pustaka Utama. . 1994. Lusi Lindri.Jakarta: Gramedia Pustaka Utama. . 1986. Burung-burung Manyar.?: Djambatan. . 1986. Burung-burung Manyar.?: Djambatan. Stanton, Robert. 1964. An Introduction to Fiction. New York: Holt, Rinehart and Winston, Inc. KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 KOMUNIKA Vol.2 No.2 Jul-Des 2008 pp.131-139 Jurusan Dakwah STAIN Purwokerto ISSN: 1978-126 Jurusan Dakwah STAIN Purwokerto ISSN: 1978-126
https://openalex.org/W3104329483	https://biblio.ugent.be/publication/8680594/file/8680596	English	null	Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History	Tijdschrift voor mediageschiedenis	2,020	cc-by-sa	3,501	Daniel Biltereyst and Philippe Meers Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History So, doing comparative research could be an endeavour that forced us to rethink what we thought we knew best (a film, a screen, a venue, a programme, a screening, …). The comparative mode also forced scholars to rethink, and make clear agreements on, data collection methods and transparent unambiguous protocols for data processing, analysis and interpretation. How do we count the number of venues in a city where mobile cinema or ambulant film screenings are much more common than one where the standard practice of watching a film is sitting in a building with bricks walls? What are the questions we are going to ask people when we want to talk about their past experiences? What are the methodological agreements on the analysis of interviews? Can we just translate interviews in order to compare statements? Some of the problems when doing comparative research are so fundamental that we realise that it might be that we just didn’t think through all of these issues of data collection, processing and analysis before. So, in a nutshell: comparative research on cinema’s history in two or more spatial confines was worthwhile, not only in terms of understanding differences, similarities and cultural specificities (as pointed out by Ercole, Van de Vijver and Treveri Gennari in this collection), but it also forces researchers to think more thoroughly about basic conceptual, theoretical, and methodological issues of the discipline (see the contribution by Klenotic in this special issue). The Alphaville article was a simple attempt to deal with some of those questions. It also thought about a variety of comparative set-ups, where we looked most prominently to varieties of comparative cinema history projects in their choice of methodologies and spatial confines. We came up with four modes of comparing across methodologies and space, with the most common practice being that researchers used different cities and used different research designs and methods. Only few research projects aimed at comparing different cities or other spatial entities (neighbourhoods, countries, …) by using similar methodologies, as was the case with the ECA project (see also articles in this collection by Porubčanská, Meers and Biltereyst and by Çam and Yüksel). Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History In 2016 we published an article in Alphaville on film studies and the comparative mode.1 The piece was part of a special issue on cinema heritage in Europe, and it reflected upon the buzz around doing comparative research within New Cinema History. At that time, several large research projects focused upon comparing issues of film programming, cinemagoing experiences and other aspects related to film exhibition across cities. The issue itself was edited by scholars who were involved in the European Cinema Audiences (ECA) project (see the article by Ercole, Van de Vijver and Treveri Gennari in this issue), which was innovative not only for dealing with an impressive amount of different cities across Europe. The international comparative project was also explorative in dealing with a series of fundamental methodological issues. Collecting data on venues, programming and cinemagoing experiences had already been a burden and a major challenge for film scholars, even if they only concentrated on one venue in one city in one week or even a single day. But when scholars started to compare their findings and ‘data’ across two or more venues, or in more than one city, things got much more complicated. This exercise required something what was never the strongest suit in film studies, i.e. methodological rigor. The latter refers to the strength, solidity and the logic underneath a research design. It deals with making strong agreements on nearly everything. First of all, on definitions and concepts. What is a venue, what is a film, what is a programme? Who is an exhibitor and a distributor? What does watching a film consist of and how different is it from going to a cinema, making a choice, enjoying the picture and talking during and after the show? What is the film experience? And how can we compare that in a reasonable and contextualised way? It was clear then that comparative cinema research forces scholars to rethink most of these concepts because cultural, climatological or historical differences put pressure on our common understanding of these basic constituents of cinema culture. Making a choice for watching a film in an art deco picture palace in Amsterdam, for instance, is quite different from sitting on an open air terrace in the middle of a hot Mexican city where a projector throws some light on a white wall around midnight. Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History At the background of this typology was a social sciences inspired idea that we could also consider revisiting existing data (see the Cinema Memory and the Digital Archive project which revisits Annette Kuhn’s 1930s cinema project), or even replica studies where the same data are re-examined or re-analysed by another scholar. We, however, didn’t want to promote neither one specific mode, nor all of them. All types of comparative research are valuable, and the article just tried to reflect on a meta-methodological level 2 2 TMG 23 (1/2) 2020 Daniel Biltereyst and Philippe Meers on comparative setups within the sub-discipline as a whole. In the article we only focused upon space as a distinctive parameter. The chapter disregarded any other kind of comparative research – like those on comparative modes along time, experiences, films (see contributions by Rabin, and Fuhrmann), and so on. We can compare cinema cultures in this issue across class, gender, sexual orientation, ethnicity, taste cultures, or focus on aspects of the cinema business like comparing film distribution or film flows in two or more cities (e.g. Pafort-Overduin, Lotze, Jernudd and Van Oort on film flows in three cities). We can look at festivals and their respective affective constellations as Petrychyn framed it. It is surprising to see many of the lines or dimensions of comparative research we mentioned – some explicitly, some just in passing – and envisioned – quite optimistically maybe – for a future agenda, here already being realised, just four years later by a range of scholars from across continents. We are thinking about shared (e.g. European port cities) or identical methodologies (see the European Cinema Audiences project); the call for research beyond the US and Europe without an exoticising gaze (rural Turkish cinema (Çam and Yüksel); Jamaica (Martens), The British West Indies (Burns); Brazil (Fuhrmann); South Africa (Maingard)); research going beyond the mere cinema-related context (e.g. tourism, ideology, imperial history in Jamaica, see Martens); or intra-national comparative research (urban and small-town exhibition, for Canada see Whitehead, see also Jernudd and Lundmark), etcetera. Other contributions in this special issue go beyond the comparative towards a multi-dimensional and multi-perspective transnational approach (see Furhmann). New Cinema History’s bandwidth when it comes to periods under study is again illustrated. Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History Every single decade since the start of the medium is in one way or another dealt with in this issue, from the early cinema culture in Canada and the Caribbean, over the classical period in Europe and North-America and the Caribbean, via rural cinema in Turkey in the 1960 and 1970s, to the late 20th and early 21st cinemas in Europe and the US. In some of the articles, analysing cases with different languages proves to be a real challenge, but one that can be overcome (see Ercole, Van de Vijver and Treveri Gennari). At the same time a shared language can be a stimulus for joint international comparative projects, working within a ‘geolinguistic region’ as John Sinclair names it. Here we can refer to the ‘Cultura de la Pantalla’ project, a Spanish language network doing research on cinema cultures (with shared methodologies) in cities across Mexico, Colombia, Spain and the US.2 The invitation from the editors to reflect on this special issue gives us the privileged position not only to comment on the original contributions, but also, to react on some of the constructive 3 critiques authors develop towards comparative New Cinema History as we framed it. Jeff Klenotic’s argument is both interesting and challenging. We truly welcome his highly critical assessment of our comparative approach and his sophisticated call for a ‘splatial framework’, based on the ‘spirit of place’. As one of the prominent voices within New Cinema History, Klenotic stirs up a lively discussion on the possibly reductive strategic character of a certain move towards comparative research. As film studies scholars with a background in media studies and social sciences we are very aware (and wary) of negative tendencies towards mainstreaming a discipline via methods, both in film studies and in communication studies. The countermovement within New Cinema History against this mainstreaming is lively: a focus on multi-level, multi-dimensional approaches, combining political economy inspired structural business analyses with detailed programming studies and adding the missing link of audience studies, illustrate this. More specifically Klenotic challenges our positioning comparative research as ‘an essential part of a discipline’s strategy to become methodologically more mature.’3 In our experience, New Cinema History has never been about designing grand strategies from above, but rather about developing tactics from below in day to day collaborative research practices. Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History This collaborative aspect, of team members working together, and even more so teams from various cultural national contexts, still is one of the original contributions to an otherwise highly individual research field. We are definitely not saying comparative research is ‘better’ than individual micro histories, nor are we reducing comparison to a mechanical or big data limited approach. We do make a point about the importance of a rich variety in comparative, entangled, crossed, parallel, etcetera, histories. Microhistories then are complemented, combined, integrated. By no means did we intend to construct a teleological ‘epistemological ladder’, ‘framing microhistories as methodologically immature and incapable of connecting or analysing macro patterns and forces as they operate in micro places.’4 We firmly believe this is exactly the strength of a New Cinema History take on micro histories: it allows for a dynamic analysis from the micro to the macro, from top down to bottom up, and back. Ercole, Van de Vijver and Treveri Gennari for instance show how a study of micro-histories (in this case, three interviews) allows for a fascinating methodologically reflective comparison. Therefore Klenotic is rightly insisting on the ‘interaction at all scales of experience and historical analysis.’ methodologically reflective comparison. Therefore Klenotic is rightly insisting on the ‘interaction at all scales of experience and historical analysis.’ TMG 23 (1/2) 2020 Daniel Biltereyst and Philippe Meers New Cinema History has never been about a single methodology, let alone that this methodology would build upon a ‘structural heteronormativity’, as Petrychyn claims in his article on queer film festivals.5 He sees the preference for big data as a major cause for the exclusion of these film festivals from the New Cinema History field, but this argument doesn’t stand the test. New Cinema History has always prioritised the micro level and the bottom-up lived experience, as a crucial complement to mainstream quantified data. Marginalised groups and communities are exactly given a voice through the non-text and context oriented approach, where oral history plays a crucial role. Petrychyn rightly points to the fact that film festival studies has developed into a thriving subfield, giving voice and visibility to a wide range of specialised film festivals, and with methods and epistemologies quite similar to New Cinema History. The mutual inspiration with film festival studies is clear. Moreover, this existing interaction has resulted in fruitful exchanges (e.g. panels at NECS conferences, etcetera). Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History The absence of queer film festival research under the discursive umbrella of New Cinema History is probably more due to academic dynamics whereby similar research clusters together, in the sphere of film festival studies. But it is definitely not the result of a hidden agenda of the very open, diverse and anti-dogmatic New Cinema History community. The ‘ephemeral’ to which Petrychyn refers has an important role in New Cinema History. That is exactly one of the main reasons why historical audience research is almost never limited to ‘audience demographics’ or geographically inspired research is neither limited to ‘movie theatres’. Both focus on lived experiences of real people, from all kinds of backgrounds, in a variety of spaces (be it in buildings, drive-ins, mobile or open air cinemas), and on lived cinema cultures as reconstructed within the circulating discourses in a society, through oral history, mental mapping and other, more contextual sources. In this context, Annette Kuhn has been and still is one of the major scholars for this approach within New Cinema History.6 If New Cinema History would really be ‘inattentive to cinema institutions organised by marginalised communities’ it would simply go against its own agenda. Considering pioneers within the approach such as Robert C. Allen, who from very early on focussed on black (and mixed but spatially separate) cinema-going in the rural south of the US; Kate Bowles’ work on rural audiences in New South Wales (Australia), and more recently the efforts of giving a voice to unheard women voices in mid-twentieth century patriarchal societies such as urban and rural Italy, the agenda seems fully in action.7 This does not imply ‘queering New Cinema History’ is not a project worthwhile exploring, we 5 5 can only welcome valuable new perspectives, as the New Cinema History community has always done. But simultaneously bashing the open and non-dogmatic principles the community is built upon seems rather uncalled for. Both Klenotic and Petrychyn to a certain degree seem to assume that a comparative approach within New Cinema History implies big data or a quantitative data-driven research agenda. We can only emphasise the wide methodological possibilities and realities of New Cinema History as a counterargument, as the contributions in this very issue yet again demonstrate. Comparative, Entangled, Parallel and ‘Other’ Cinema Histories. Another Reflection on the Comparative Mode Within New Cinema History And – not to the reader’s surprise we hope – fully rejecting a ‘teleological and centripetal narrative that centres New Cinema History on methods’, we do insist on methodological rigor and transparency, be it comparative or otherwise, combined with an open research spirit whereby basic questions and curiosity guide the researcher towards the most adequate method, rather than leaving it opaque or, more extreme, having the method determine the questions. To conclude these brief reflections, we confirm that comparative research is not only about studying parallel or ‘other’ histories (cf. Hoogland’s article). This special issue strongly illustrates that comparing cinema’s past becomes tremendously interesting when these pasts touched each other at the time, when they had a common ground, or when they were entangled. The histoire croisée approach is much more than a theoretical option because an internationally or even globally oriented medium like cinema has an intensively entangled history. It is entangled in terms of films, of course – film crossing from one city, country or continent to another. Cinema history was entangled in terms of people like filmmakers, stars, camera personnel, businessmen and women being active in different spatial or temporal entities. There was an intensive entanglement in terms of money, strategies, power, public diplomacy (see Fuhrmann). And it was entangled also in cinema reception with audiences consuming foreign pictures, reading non-domestic magazines, dreaming of worlds they could (probably) never (physically) reach. So, entanglement was always at the heart of cinema – in terms of business, flows, content, labour, stories and phantasies. 1 Daniel Biltereyst and Philippe Meers, “New Cinema History and the Comparative Mode: Reflections on Comparing Historical Cinema Cultures,” Alphaville Journal of Film and Screen Media 11 (2016): 13–32. Notes Allen, Going to the Show project, Mapping Moviegoing in North Carolina http://gtts.oasis.unc. edu/; Kate Bowles, “Three Miles of Rough Dirt Road’: Towards an Audience-Centred Approach to Cinema Studies in Australia,” Studies In Australasian Cinema 1(3) (2007): 245–260; Daniela Treveri Gennari, Silvia Dibeltulo, Danielle Hipkins and Catherine O’Rawe, “Analysing Memories Through Video-Interviews: A Case Study of Post-War Italian Cinema-going,” in Routledge Companion to New Cinema History, ed. Daniël Biltereyst, Richard Maltby and Philippe Meers (London: Routledge, 2019): 344–354. Notes 6 TMG 23 (1/2) 2020 Daniel Biltereyst and Philippe Meers 2 J. Sinclair, “Geolinguistic Region as Global Space: The Case of Latin America,” in The New Communications Landscape, ed. G. Wang , J. Servaes and A. Goonasekera (London: Routledge, 2000), 19-32; Philippe Meers, Daniël Biltereyst and José Carlos Lozano, “Cultura de la Pantalla network : writing new cinema histories across Latin America and Europe,” Revista Internacional de Comunicación y Desarollo 2(9) (2018): 158–165. 3 Biltereyst and Meers, “New Cinema History and the Comparative Mode,” 25. 2 J. Sinclair, “Geolinguistic Region as Global Space: The Case of Latin America,” in The New Communications Landscape, ed. G. Wang , J. Servaes and A. Goonasekera (London: Routledge, 2000), 19-32; Philippe Meers, Daniël Biltereyst and José Carlos Lozano, “Cultura de la Pantalla network : writing new cinema histories across Latin America and Europe,” Revista Internacional de Comunicación y Desarollo 2(9) (2018): 158–165. 3 Biltereyst and Meers, “New Cinema History and the Comparative Mode,” 25. 4 Jeffrey Klenotic, “Mapping Flat, Deep, and Slow: On the ‘Spirit of Place’ in New Cinema History,” TMG Journal for Media History 23, no. 1–2 (2020): 12, DOI: 10.18146/2213-0969.2020.TMG202015. 4 Jeffrey Klenotic, “Mapping Flat, Deep, and Slow: On the ‘Spirit of Place’ in New Cinema History,” TMG Journal for Media History 23, no. 1–2 (2020): 12, DOI: 10.18146/2213-0969.2020.TMG202015. 5 Jonathan Petrychyn, “Queering New Cinema History: Affective Methodologies for Comparative Histories,” TMG Journal for Media History 23, no. 1–2 (2020): 1, https://dx.doi.org/10.18146/tmg.588 5 Jonathan Petrychyn, “Queering New Cinema History: Affective Methodologies for Comparative Histories,” TMG Journal for Media History 23, no. 1–2 (2020): 1, https://dx.doi.org/10.18146/tmg.588 E.g. Annette Kuhn, An Everyday Magic: Cinema and Cultural Memory (London; New York: I.B.Tauris, 2002). Annette Kuhn, “Heterotopia, Heterochronia: Place and Time in Cinema Memory,” Screen 45, no.2 (2004): 106–114. Annette Kuhn, Daniel Biltereyst and Philippe Meers, “Memories of Cinemagoing and Film Experience ... An introduction,” Memory Studies, 10, no.1 (2017): 3–16. 7 Robert C. Allen, Going to the Show project, Mapping Moviegoing in North Carolina http://gtts.oasis.unc. edu/; Kate Bowles, “Three Miles of Rough Dirt Road’: Towards an Audience-Centred Approach to Cinema Studies in Australia,” Studies In Australasian Cinema 1(3) (2007): 245–260; Daniela Treveri Gennari, Silvia Dibeltulo, Danielle Hipkins and Catherine O’Rawe, “Analysing Memories Through Video-Interviews: A Case Study of Post-War Italian Cinema-going,” in Routledge Companion to New Cinema History, ed. Daniël Biltereyst, Richard Maltby and Philippe Meers (London: Routledge, 2019): 344–354. 7 Robert C. Biographies Daniel Biltereyst is Professor in Film and Media History and director of the Cinema and Media Studies (CIMS) research centre at Ghent University, Belgium. Besides exploring new approaches to historical media and cinema cultures, he is engaged in work on screen culture as a site of censorship, controversy and public debate. He recently published in the Historical Journal of Film, Radio & Television, Memory Studies and Film Studies. With Richard Maltby and Philippe Meers, he co-edited Explorations in New Cinema History: Approaches and Case Studies (2011), Audiences, Cinema and Modernity: New Perspectives on European Cinema History (2012) and The Routledge Companion to New Cinema History (2019). With Annette Kuhn and Philippe Meers, he co-edited a special issue of Memory Studies (2017) on memories of cinemagoing. He just published Mapping Movie Magazines (Palgrave, with L. Van de Vijver, 2020). 7 Netherlands Institute for Sound and Vision Netherlands Institute for Sound and Vision https://dx.doi.org/10.18146/tmg.795 https://dx.doi.org/10.18146/tmg.795 TMG 23 (1/2) 2020 Daniel Biltereyst and Philippe Meers TMG 23 (1/2) 2020 Daniel Biltereyst and Philippe Meers Philippe Meers is Professor in Film and Media Studies at the University of Antwerp, Belgium, where he is deputy director of the Visual and Digital Cultures Research Center (ViDi). He has published widely on historical and contemporary film cultures and audiences in, e.g. Screen and Media, Culture & Society. With Richard Maltby and Daniel Biltereyst, he co-edited Explorations in New Cinema History: Approaches and Case Studies (2011), Audiences, Cinema and Modernity: New Perspectives on European Cinema History (2012) and The Routledge Companion to New Cinema History (2019). With Annette Kuhn and Daniel Biltereyst, he co-edited a special issue of Memory Studies (2017) on memories of cinemagoing. 8 8 TMG 23 (1/2) 2020 Daniel Biltereyst and Philippe Meers TMG Journal for Media History Volume 23 No (1/2)/2020 TMG Journal for Media History Volume 23 No (1/2)/2020 COPYRIGHT Each article is copyrighted © by its author(s) and is published under license from the author(s). When a paper is accepted for publication, authors will be requested to agree with the Creative Commons Attribution 4.0 International License. 9 9
https://openalex.org/W4226257819	https://link.springer.com/content/pdf/10.1007/JHEP04(2022)181.pdf	English	null	Long-time tails in the SYK chain from the effective field theory with a large number of derivatives	null	2,021	cc-by	16,304	Published for SISSA by Springer Received: December 31, 2021 Revised: March 11, 2022 Accepted: April 8, 2022 Published: April 29, 2022 Received: December 31, 2021 Revised: March 11, 2022 Accepted: April 8, 2022 Published: April 29, 2022 Received: December 31, 2021 Revised: March 11, 2022 Accepted: April 8, 2022 Published: April 29, 2022 Open Access, c⃝The Authors. Article funded by SCOAP3. Keywords: Eﬀective Field Theories, 1/N Expansion, Gauge-Gravity Correspondence Long-time tails in the SYK chain from the eﬀective ﬁeld theory with a large number of derivatives https://doi.org/10.1007/JHEP04(2022)181 Contents 1 Introduction 1 2 Eﬀective ﬁeld theory around thermal equilibrium 3 3 The model 5 3.1 SYK model and hydrodynamics 5 3.2 SYK chain and classical EFT of energy diﬀusion 7 3.3 SYK chain and EFT of nonlinear energy diﬀusion 8 4 Eﬀective action in terms of energy density 9 4.1 Quadratic action and the free propagators 10 4.2 Cubic action and coupling constants 10 5 Loop computations 11 5.1 Self-energy 11 5.2 Long time tails and breakdown of the derivative expansion 12 6 Modiﬁed dispersion relation 13 7 Review, discussion and outlook 16 A Review of Schwinger-Keldysh EFT 18 B KMS conditions 20 B.1 Quadratic action 20 B.2 Cubic action 20 C Energy density in terms of reparametrization mode 22 D Loop computations 23 E Series coeﬃcients and convergence of series 25 Contents 1 Introduction 1 2 Eﬀective ﬁeld theory around thermal equilibrium 3 3 The model 5 3.1 SYK model and hydrodynamics 5 3.2 SYK chain and classical EFT of energy diﬀusion 7 3.3 SYK chain and EFT of nonlinear energy diﬀusion 8 4 Eﬀective action in terms of energy density 9 4.1 Quadratic action and the free propagators 10 4.2 Cubic action and coupling constants 10 5 Loop computations 11 5.1 Self-energy 11 5.2 Long time tails and breakdown of the derivative expansion 12 6 Modiﬁed dispersion relation 13 7 Review, discussion and outlook 16 A Review of Schwinger-Keldysh EFT 18 B KMS conditions 20 B.1 Quadratic action 20 B.2 Cubic action 20 C Energy density in terms of reparametrization mode 22 D Loop computations 23 E Series coeﬃcients and convergence of series 25 Contents 1 Introduction 1 2 Eﬀective ﬁeld theory around thermal equilibrium 3 3 The model 5 3.1 SYK model and hydrodynamics 5 3.2 SYK chain and classical EFT of energy diﬀusion 7 3.3 SYK chain and EFT of nonlinear energy diﬀusion 8 4 Eﬀective action in terms of energy density 9 4.1 Quadratic action and the free propagators 10 4.2 Cubic action and coupling constants 10 5 Loop computations 11 5.1 Self-energy 11 5.2 Long time tails and breakdown of the derivative expansion 12 6 Modiﬁed dispersion relation 13 7 Review, discussion and outlook 16 A Review of Schwinger-Keldysh EFT 18 B KMS conditions 20 B.1 Quadratic action 20 B.2 Cubic action 20 C Energy density in terms of reparametrization mode 22 D Loop computations 23 E Series coeﬃcients and convergence of series 25 1 3 5 5 7 8 9 10 10 11 11 12 13 16 JHEP04(2022)181 C Energy density in terms of reparametrization mode D Loop computations E Series coeﬃcients and convergence of series Long-time tails in the SYK chain from the eﬀective ﬁeld theory with a large number of derivatives JHEP04(2022)181 Navid Abbasi School of Nuclear Science and Technology, Lanzhou University, 222 South Tianshui Road, Lanzhou 730000, China E-mail: abbasi@lzu.edu.cn Abstract: We study nonlinear energy diﬀusion in the SYK chain within the framework of Schwinger-Keldysh eﬀective ﬁeld theory. We analytically construct the corresponding eﬀective action up to 40th order in the derivative expansion. According to this eﬀective action, we calculate the ﬁrst order loop correction of the energy density response function, whose pole is the dispersion relation of energy diﬀusion. As expected, the standard derivative expansion of the classical dispersion relation breaks down due to the long-time tails. However, we ﬁnd that the nonlinear contributions are so that one can still derive the dispersion relation in the power series. In fact, due to the long-time tails, the classical dispersion relation is split into two series distinct from the derivative expansion, and we show they are convergent. The radius of convergence is proportional to the ratio of thermal conductivity to diﬀusion constant. Keywords: Eﬀective Field Theories, 1/N Expansion, Gauge-Gravity Correspondence ArXiv ePrint: 2112.12751 Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction The classical picture of hydrodynamics, as a set of deterministic conservation equations, can only describe the low-energy long-wavelength dissipation processes, without considering ﬂuctuations [1]. In order to include the eﬀect of ﬂuctuations, one method is to place noise terms on the right side of the conservation equations. Then ﬂuctuation-dissipation theorem determines the strength of the noise [2]. However, in this traditional “stochastic” picture, the – 1 – interaction between the noise ﬁelds is ignored. The recently developed eﬀective ﬁeld theory (EFT) of hydrodynamics solves this problem by considering the eﬀects of non-Gaussian noise [3–7].1 In this framework, one can systematically derive the well known stochastic eﬀects, such as long-time tails [15]. The EFT of hydrodynamics has also some new predictions. An example is the discovery of a positive contribution to the magneto-resistance in a U(1) anomalous system [17]. Another interesting example is the prediction of stochastic transport [16]. In two pioneering works, this EFT has also been applied to quantum chaotic systems [18, 19]. The pole- skipping phenomenon as a prediction of such EFT in ref. [18] reveals the hydrodynamic origin of the quantum chaos, at least in maximally chaotic systems [20].2 In another direction, the EFT of hydrodynamics has been recently applied to study the critical ﬂuctuations near the QCD critical point [47, 48]. Much more recently, this EFT has been applied to study the quasicrystals systems [49] and a system in a phase with a spontaneously broken [50]. JHEP04(2022)181 On the other hand, an important problem in classical hydrodynamics is to study the large-order behavior of derivative expansion. In holographic systems, exploring this problem in both the position space and the momentum space shows that the large derivative expansion encodes some information about the lowest non-hydrodynamic modes in the system [51–54]. In this work, inspired by the recent advances in EFT of hydrodynamics mentioned above, we want to investigate the eﬀect of ﬂuctuations on the large-order derivative expansion. As is well known from stochastic [55–58] and EFT [15] studies, the derivative expansion breaks down due to the hydrodynamic ﬂuctuations, and the long-time tails. To see this breakdown, it is suﬃcient to consider the ﬁrst and second order derivatives. Paraphrasing the discussion in the previous paragraph, we then want to determine how much information can be extracted from large-order derivatives in the presence of long-time tails, beyond the breakdown of the derivative expansion. [ ] p pp g y y g p y e also [21–43] and [44, 45] for the holographic and CFT extensions, respectively. 3 2See [22] for the ﬁrst observation of pole-skipping and the relation with hydrodynamics in holography. l [21 43] d [44 45] f th h l hi d CFT t i ti l 3We would like to thank Luca Delacrétaz for pointing this out. 1See [8–12] for earlier works and also [13] for a review. 2See [22] for the ﬁrst observation of pole-skipping and the relation with hydrodynamics in holography. See also [21–43] and [44, 45] for the holographic and CFT extensions, respectively. 3We would like to thank Luca Delacrétaz for pointing this out. 1See [8–12] for earlier works and also [13] for a review. 2 See also [21–43] and [44, 45] for the holographic and CFT extensions, respectively. 3 2See [22] for the ﬁrst observation of pole-skipping and the relation with hydrod d like to thank Luca Delacrétaz for pointing this out. 1 Introduction The eﬀective ﬁeld theory of hydrodynamics is a suitable framework to study the above problem, systematically. To this end, one can construct EFT at large-order in derivatives, and then compute the loop corrections. In a general system, these two expansions, namely the derivative expansion and the loop expansion, should be truncated at the same order. But in a large-N theory, due to suppression of coupling constants in the 1 N expansion, the situation is diﬀerent. One can go through the derivative expansion while dropping out higher-than-one loops [59].3 Therefore, in a theory with a large number of degrees of freedom, we can speciﬁcally study the eﬀect of one-loop interactions on the classical Green’s functions at large-order in the derivative expansion. The system that we choose to study is the low energy SYK chain with a limit of N ≫1. In the continuum limit of the chain, the eﬀective action of the system is found to be a local Schwarzian [60]. This system is not CFT and in contrast to CFT in 1+1 dimension, it has a well-deﬁned hydrodynamic regime [59, 61]. In fact, energy is conserved in the system and its ﬂuctuations diﬀuse in the chain [60, 62]. Thus one can describe the low energy – 2 – dynamics of the system by the EFT of energy diﬀusion [18]. In order to capture the eﬀect of long-time tails in this system, we assume that the diﬀusion constant itself is a function of energy ﬂuctuations. Then by putting two copies of the system on the two legs of closed time path (CTP) contour, we construct the EFT of nonlinear energy diﬀusion in the chain. We analytically construct this EFT up to 40th order in derivatives.4 It is worth noting that since the thermodynamic eﬀective action of the system includes quantum eﬀects, we will use the quantum hydrodynamic EFT, which is valid at ﬁnite ℏand to all order in derivatives [18]. In the next step, we use the above EFT to compute the energy density response function, whose pole is the diﬀusion dispersion relation. In the absence of loop corrections, the “classical” dispersion relation is given in a derivative expansion in momentum space, w = w(q2). Obviously, this series is convergent in the entire range of momenta allowed by the EFT. When including the interactions and loop eﬀects, however, the derivative expansion breaks down. 1 Introduction Nevertheless, we show that one can still derive the diﬀusion pole in the power series, but this time as w = w(\|q\|). Compared to the classical dispersion relation, we ﬁnd two new aspects. First, the classical dispersion relation is split into two modiﬁed dispersion relations, where the number of transport coeﬃcients becomes twice that of the classical dispersion relation. Second, we ﬁnd that the modiﬁed dispersion relation series converge in momentum space. The radius of convergence is proportional to the ratio of the thermal conductivity to the diﬀusion constant. JHEP04(2022)181 In the rest of the paper, we ﬁrst brieﬂy review the EFT of hydrodynamics in section 2. Then in section 3 we discuss the relation between Schwarzian theory and EFT of hydrody- namics. We then explain how to add ﬂuctuations to this picture. In section 3.3 we construct the EFT of ﬂuctuating energy diﬀusion in the SYK chain to 40th order in the derivative expansion. section 5 is devoted to compute the loop corrections of the energy density response function. We then introduce the idea of modiﬁed dispersion relation and calculate the corresponding series coeﬃcients in section 6. Finally, in section 7 we end with review of the results, mentioning possible applications and discussing some followup directions. 4In the previous studies, the EFT of energy diﬀusion was constructed up to ﬁrst [15] or second [16] order in derivatives. 2 Eﬀective ﬁeld theory around thermal equilibrium The main quantity that we want to compute is the energy density response function in a quantum chaotic system at ﬁnite temperature ⟨E(t, x)E(t′, x′)⟩β = Tr ρ0 E(t, x)E(t′, x′) (2.1) (2.1) where ρ0 is the thermal density matrix. Since the energy density is a macroscopic dynamical variable, we would like to compute the above correlator in the framework of eﬀective ﬁeld theory (EFT). We will construct the EFT we want based on the ref. [13]. See appendix A for a review of the method. Considering IEFT = R dxd+1Leﬀ, the most general nonlinear Lagrangian, to second order in a-ﬁelds, i.e. noise ﬁelds, and in the absence of external sources, can be written 4In the previous studies, the EFT of energy diﬀusion was constructed up to ﬁrst [15] or second [16] order in derivatives. – 3 – as [3, 13] (2.2) Leﬀ[σ, Xa] = −H∂tXa −Gi∂iXa + i ∂tXaM1∂tXa + i ∂iXaM2∂iXa + O(X3 a) . (2.2) Leﬀ[σ, Xa] = −H∂tXa −Gi∂iXa + i ∂tXaM1∂tXa + i ∂iXaM2∂iXa + O(X3 a) . In this equation, the coeﬃcient functions H and Gi are (in general nonlinear) functions of ∂tσ(t, x) and its partial derivatives. In fact, it turns out that ∂tσ(t, x) is associated with the energy density E(t, x). Similarly, M1,2 are diﬀerential operators constructed out of ∂tσ(t, x) and diﬀerential operators ∂i and ∂t. Another point with (2.2) is that the classical equation of motion of Xa is simply the equation of conservation of energy JHEP04(2022)181 ∂tH + ∂iGi = 0 . (2.3) (2.3) Thus H and Gi are classical parts of the energy density and energy ﬂux, respectively. By classical here, we mean non-noisy part of the quantities. In any particular system of interest, H and Gi should be used as input data; then by applying KMS conditions to (2.2), one speciﬁes M1 and M2, as well. Before introducing the system we are interested in this work, let us recall that our ﬁnal goal is to compute one-loop corrections to (2.1) from (2.2), to high orders in the derivative expansion. By doing this and obtaining the corresponding energy diﬀusion pole, we will be able to study the large-order behavior of the derivative expansion in the presence of ﬂuctuations. Let us ﬁrst mention our perturbation strategy for constructing Lagrangian functions. We will include three expansions. 1. Expansion around equilibrium. 2 Eﬀective ﬁeld theory around thermal equilibrium The equilibrium state corresponds to σ(t, x) = t and Xa(t, x) = 0. By considering the small deviations as σ(t, x) = t + ϵ(t, x), Xa(t, x) = −ϵa(t, x) , (2.4) (2.4) we construct our EFT to third order in the above out-of-equilibrium ﬁelds. We will see that the cubic Lagrangian is suﬃcient to ﬁnd the ﬁnite one-loop correction to (2.1). 2. Noised ﬁeld expansion. We limit our study to include the eﬀect of ﬂuctuations up to second order in a-ﬁelds. 2. Noised ﬁeld expansion. We limit our study to include the eﬀect of ﬂuctuations up to second order in a-ﬁelds. 2. Noised ﬁeld expansion. We limit our study to include the eﬀect of ﬂuctuations up to second order in a-ﬁelds. 3. Derivative expansion. In order to study the large-order behavior of the hydrodynamic derivative expansion in the presence of ﬂuctuations, we need to construct L to the higher-order derivatives. On the other hand, we would like to ﬁnd all terms in L analytically. By implementing a systematic method, we will construct the Lagrangian to 40th order in the derivative expansion. Although due to the large size of analytic expressions, we only explicitly display the Lagrangian terms to the ﬁfth order, in the appendices. Before concluding this section, let us comment on the size of the spatial derivative in our system. Since our expected EFT should describe a diﬀusion process controlled by (2.3), we are interested in perturbations that behave like ω ∼k2. This together with ∂tσ ∼Xa (see appendix A, below (A.11)) provide our derivative counting scheme in this work. – 4 – 3 The model So far, our discussion about EFT of hydrodynamics has been general, without mentioning any special physical system. When restricting the study to the lower orders in the derivative expansion, it would be feasible to construct Leﬀfor the general system. For example, ref. [15] constructs the energy diﬀusion EFT in a general thermal system up to ﬁrst order in the derivative expansion. But at higher orders in derivatives, the calculation will become more complicated because more possible terms may contribute to H and Gi. Therefore, since our goal is to explore the large-order behavior of the derivative expansion in this paper, we choose to use a speciﬁc system with the well-known H function. JHEP04(2022)181 Our system of interest is the SYK chain. The (0+1) dimensional SYK model [63–65] and also the (1 + 1) dimensional SYK chain [60] have been widely studied in the literature [66]. However, in order to be clear about the problem that we want to address, we need to recall some aspects of the SYK model. In particular, it is well known that the infrared theory in the strong coupling limit is described by hydrodynamics [67]. In section 3.1, we ﬁrst brieﬂy review the model. Then following [18] and by using the language of Schwinger-Keldysh EFT, we revisit the above-mentioned hydrodynamic picture and specify the H function associated with the SYK model. Then in section 3.2, after a quick look at the SYK chain model, we will review its connection with the EFT of hydrodynamics and specify the corresponding H and Gi functions in (2.2) [18]. Everything we will discuss in the following two subsections will be devoted to classical aspects of EFT of hydrodynamics. But our ultimate goal is to couple classical EFT with ﬂuctuations, which is the subject of the next section. 3.1 SYK model and hydrodynamics The SYK model consists of N Majorana fermions χj(τ), j = 1, 2, · · · , N with a random four-fermion interaction. Here τ is the Euclidean time coordinate. At ﬁnite temperature, we have τ ∼τ + β. The random coupling has zero mean Jjklm = 0 and non-zero vari- ance 1 3!N3J2 jklm = J2. The model is non-local in space in the sense that the interaction is all-to-all. This model should be actually considered as a (0 + 1)-dimensional quantum mechanical system. The model is solvable in the large-N limit. Speciﬁcally in the strong coupling limit, N ≫βJ ≫1, the model shows up a conformal symmetry in the infrared limit, that is the invariance under reparameterization τ →f(τ). As a result, the infrared eﬀective action of the model is zero when evaluated on ﬂuctuations that are reparameterizations of Gc. However, it turns out that the corresponding conformal correlators, Gc, are only invariant under SL(2, R) subgroup of this conformal symmetry. In other words, the conformal symmetry is spontaneously broken by the conformal solution Gc. Beyond the conformal limit, the picture above will no longer be the case. In particular, due to explicitly breaking of the conformal symmetry, the eﬀective action becomes non-zero when evaluated on ﬂuctuations δGc, the reparameterizations Gc. For an inﬁnitesimal reparameterization τ →τ + ϵ(τ), one ﬁnds [66] S N = α J Z β 0 dτ 1 2 (ϵ′′)2 −λ2(ϵ′)2 , λ = 2π β , (3.1) (3.1) – 5 – with α being a numerical constant. Now let us take L[ϵ(t)] = C (ϵ′′)2 + λ2(ϵ′)2 with the Lorentzian time coordinate t, and put two copies of it on the two legs of CTP contour. The Schwinger-Keldysh action, with real time coordinate t, then reads with α being a numerical constant. Now let us take L[ϵ(t)] = C (ϵ′′)2 + λ2(ϵ′)2 with the Lorentzian time coordinate t, and put two copies of it on the two legs of CTP contour. The Schwinger-Keldysh action, with real time coordinate t, then reads L[ϵ, ϵa] ⊃L[ϵ1] −L[ϵ2] = C λ2 −∂2 t ∂tϵ ∂tϵa . (3.2) (3.2) Considering (2.4), equation (3.2) is exactly the ﬁrst term in the eﬀective Lagrangian of hydrodynamics (2.2), speciﬁc to a system in (0 + 1) dimension with H(σ) = C λ2 −∂2 t ∂tσ, H0 = C λ2 . 5See [67] for another way of describing the hydrodynamic origin of ϵ. 3.2 SYK chain and classical EFT of energy diﬀusion The SYK chain describes a coupled array of SYK model sites. Each site contains N ≫1 Majorana fermions with four-fermion random coupling Jjklm,x. Here j = 1, 2, · · · , N and x = 1, 2, · · · , M where M is the number of sites. Then there is further coupling between each pair of neighboring sites through another four-fermion random coupling J′ jklm,x. The random couplings Jjklm,x and J′ jklm,x are drawn independently and JHEP04(2022)181 Jjklm,x = J′ jklm,x = 0, 1 3!N3J2 jklm,x = J2 0, N 3J′2 jklm,x = J2 1 . (3.8) (3.8) One can show that the Schwinger-Dyson equations of the model reduce to exactly the same form as those of a (0 + 1)-d SYK model with the coupling constant J = q J2 0 + J2 1 [60]. q As in the SYK model, the eﬀective action of the SYK chain admits an approximate reparametrization symmetry of time in the IR limit. For small deformations of time τ →τ + ϵx(τ), the quadratic eﬀective action is found to be [60] S N = X n,k α 2J ϵn,k ω2 n ω2 n −λ2 + D k2 \|ωn\| ω2 n −λ2 ϵ−n,−k , (3.9) (3.9) where ϵn,k is the Fourier transform of ϵx(τ) and D ∼J2 1/J is the diﬀusion constant. The ﬁrst term in (3.9) is exactly the EFT of SYK model (3.1), if we remove the dependence on k. Recalling (3.6), we ﬁnd that in the limit of an inﬁnite lattice, namely M →∞, this term can be interpreted as a quadratic approximation of the local Schwarzian action ∼ R dt Sch(u(t, x), t). What about the second term in (3.9)? Let us consider two copies of the system placing on the two legs of CTP contour. Then we ﬁnd that L[ϵ, ϵa] ⊃L[ϵ1] −L[ϵ2] = C λ2 −∂2 t ∂tϵ ∂ϵa + D λ2 −∂2 t ∂x∂tϵ ∂xϵa . (3.10) (3.10) The above Lagrangian is actually the quadratic approximation to (2.2) in (1 + 1) dimension with he above Lagrangian is actually the quadratic approximation to (2.2) in (1 + 1) dimension th H = −h λ2 Sch(u(t, x), t) u(t, x) = e−λ σ(t,x) , Gi = −D ∂iH . 3.1 SYK model and hydrodynamics (3.3) (3.3) JHEP04(2022)181 Since H0 represents the energy density in thermal equilibrium σ(t) = t, it is convenient to take C = h λ2 , where h is the thermodynamic energy density. The classical equation of motion is then simply the equation of conservation of energy, i.e. ∂tH = 0. We conclude that the EFT describing dynamics of the reparameterization mode ϵ in the SYK model is actually hydrodynamics.5 We can extend the above-mentioned hydrodynamic action beyond the second order, that is, the regime of nonlinear hydrodynamics. It can be done by generalizing the action (3.1) to a ﬁnite reparameterization τ →τ + f(τ). One ﬁnds [66] S N = −α J Z β 0 dτ Sch(f(τ), τ), Sch(f(τ), τ) = f′′ f′ ′ −1 2 f′′2 f′2 (3.4) (3.4) with f(τ) = tan πτ β . We can have further reparameterization on the Euclidean time circle, τ →σ(τ). Then writing σ(τ) = τ + ϵ(τ) and expanding Sch(f(σ(τ)), τ) in (3.4), we get exactly the two quadratic terms of (3.1). (t) In Lorentzian coordinate, f(τ) transforms to f(t) = tanh πσ(t) β . Inverting this equation, we deﬁne u(t) as 1 2e−λ σ(t) = 1 2 1 −f(t) 1 + f(t) ≡1 2u(t) . (3.5) (3.5) Since −1 2u is SL(2, R) transformed of f, we immediately ﬁnd: Sch(f(t), t) = Sch(−1 2u(t), t). Thus in Lorentzian coordinate, (3.4) takes the following form S = −i Z dt h λ2 Sch(u(t), t), u(t) = e−λ σ(t) , (3.6) (3.6) where we have used 1 2 N α J = C = h λ2 . where we have used 1 2 N α J = C = h λ2 . We can repeat the discussion around (3.2) to ﬁnd the Schwinger-Keldysh analogue of (3.6). Doing so, we ﬁnd that the reparameterization mode in the SYK model, in general, is described by nonlinear hydrodynamics with the corresponding H function given by H = −h λ2 Sch(u(t), t), u(t) = e−λ σ(t) . (3.7) (3.7) Let us recall that equilibrium state corresponds to σ(t) = t. 5See [67] for another way of describing the hydrodynamic origin of ϵ. – 6 – It is worth nothing that the above Schwarzian action goes actually beyond ordinary long distance hydrodynamics. The reason is it includes modes whose time variation rate is comparable to the temperature [68]. 3.1 SYK model and hydrodynamics Correspondingly, the framework of [3] can be used to any large-order in derivatives. Therefore, it can be well applied to study the dynamics of the reparametrization mode in the SYK model. 3.2 SYK chain and classical EFT of energy diﬀusion (3.11) H = −h λ2 Sch(u(t, x), t) u(t, x) = e−λ σ(t,x) , Gi = −D ∂iH . (3.11) (3.11) Thus the second term in (3.10) denotes the diﬀusion of energy in the SYK chain with D being the diﬀusion constant. It should be noted that in our setup, there is only one spatial direction along the Chain. However, we use the subscript i to display the spatial derivative so that we can discuss the generalization of things to higher dimensions later. In summary, given H and Gi by (3.11), the linearized version of equation (2.3) represents the classical linear hydrodynamics of the diﬀusive reparametrization mode in the SYK chain. In next section, we discuss the nonlinear hydrodynamic description of the energy diﬀusion in the SYK chain, taking into account the eﬀects of ﬂuctuations generated by going beyond the quadratic order in (2.2). – 7 – 3.3 SYK chain and EFT of nonlinear energy diﬀusion Let us recall that for the system to be speciﬁed in the framework of EFT of hydro, the H and Gi associated with it need to be determined. As was discussed earlier, our system of interest is the SYK chain with the H and Gi functions given by (3.11). On the other hand, In order to include the interaction between the hydrodynamic ﬁeld ϵ and the ﬂuctuation ﬁeld ϵa, we have to take into account nonlinear terms in the H and Gi. As mentioned before, we aim to construct the cubic interacting Lagrangian. Considering u = e−λ(t+ϵ(t,x)), there are two potential sources of nonlinearity in H and Gi in (3.11): 1. H itself is a nonlinear function of ϵ; in order to construct the cubic Lagrangian, we expand H about its equilibrium value h and keep terms to second order: JHEP04(2022)181 H = −h λ2 Sch(e−λ(t+ϵ(t,x)), t) = h 2 + h ∂tϵ −1 λ2 ∂3 t ϵ + h 1 2(∂tϵ)2 + 3 2λ2 (∂2 t ϵ)2 + 1 λ2 ∂tϵ ∂3 t ϵ + O(ϵ3) . (3.12) (3.12) Notice that although we aim to perform our computations to high orders in the derivative expansion, the above expression is truncated at ∂2 t by construction. Let us remind that as mentioned below (A.11), ∂tδσ ∼∂tϵ ∼O(∂0 t ). 2. The diﬀusion coeﬃcient D can also be a function of energy ﬂuctuations δH = H −h 2. We may write Gi = −D(δH)∂iH = −D0h ∂i∂tϵ−1 λ2 ∂i∂3 t ϵ −D1h ∂i∂tϵ−1 λ2 ∂i∂3 t ϵ ∂tϵ−1 λ2 ∂3 t ϵ +O(ϵ3) (3.13) H)∂iH ∂i∂tϵ−1 λ2 ∂i∂3 t ϵ −D1h ∂i∂tϵ−1 λ2 ∂i∂3 t ϵ ∂tϵ−1 λ2 ∂3 t ϵ +O(ϵ3) (3.13) (3.13) where we have used where we have used D(δH) = D0 + D1 h δH + D2 2h2 (δH)2 + · · · (3.14) (3.14) with Dn = hn ∂nD ∂Hn . Notice that coeﬃcients Dn : n > 1 do not appear in the cubic Lagrangian. It is also worth noting that in general, any of the Dn coeﬃcients can be a diﬀerential operator, constructed out of ∂t and ∂i. However, in this work, we assume that these coeﬃcients are constant. 3.3 SYK chain and EFT of nonlinear energy diﬀusion One important consequence of this assumption is that the classical dispersion relation of energy diﬀusion, namely the relation obtained from linear hydrodynamic equations, will be exact in the derivative expansion and is given by ω = D0k2. In section 6 we will discuss the general case in which D0 itself has a derivative expansion. Finally, one of the problems we will solve in this work is how this simple dispersion relation changes when nonlinear eﬀects are included. Having speciﬁed the ingredients of the model, we should now compute M1 and M2 terms in (2.2). Since each of these terms has two Xa ﬁelds, they are at least of the second order. Therefore in order to construct the cubic Lagrangian, we need to determine M1 and M2 – 8 – operators up to ﬁrst order in ϵ. Then we ﬁnd that the appropriate ansatz in the most general form is as follows operators up to ﬁrst order in ϵ. Then we ﬁnd that the appropriate ansatz in the most general form is as follows M1 = ρ + α1δH α2 + · · · = ρ + α1h ∂tϵ −1 λ2 ∂3 t ϵ α2 + O(ϵ2) , (3.15) M2 = χ + ξ1δH ξ2 + · · · = χ + ξ1h ∂tϵ −1 λ2 ∂3 t ϵ ξ2 + O(ϵ2) . (3.16) (3.15) (3.16) The coeﬃcients in these two equations are all diﬀerential operators, each of which acting on the ﬁrst function on the right. Now the task is to apply the KMS conditions to the eﬀective action and specify these coeﬃcients, perturbatively in the derivative expansion, in terms of D0, D1, β = T −1 and h. See table 1 in appendix B for details. The coeﬃcients in these two equations are all diﬀerential operators, each of which acting on the ﬁrst function on the right. Now the task is to apply the KMS conditions to the eﬀective action and specify these coeﬃcients, perturbatively in the derivative expansion, in terms of D0, D1, β = T −1 and h. See table 1 in appendix B for details. JHEP04(2022)181 Imposing the KMS constraints to the Lagrangian, we have constructed the KMS invariant quadratic and cubic pars of the Lagrangian, i.e. L(2) eﬀ[ϵ, ϵa] and L(3) eﬀ[ϵ, ϵa], to 40th order in the derivative expansion. 3.3 SYK chain and EFT of nonlinear energy diﬀusion However, as discussed in the Introduction, our goal is to compute the energy density correlator deﬁned by (2.1). For this reason, we ﬁnd that it is more appropriate to rewrite the eﬀective Lagrangian in terms of E and ϵa, say Leﬀ[E, ϵa]. To this end, in the next section, we will discuss how to exchange ϵ with E and then explicitly display L(2) eﬀ[E, ϵa] and L(3) eﬀ[E, ϵa]. 4.1 Quadratic action and the free propagators Using the relation between ϵ and E (see appendix C), L(2) eﬀ[ϵ, ϵa] is converted to L(2) eﬀ[E, ϵa] as follows Using the relation between ϵ and E (see appendix C), L(2) eﬀ[ϵ, ϵa] is converted to L(2) eﬀ[E, ϵa] as follows L(2) eﬀ[E, ϵa] = −ϵa ∂tE + D0 ϵa ∂2 i E + iD0 h β ∂iϵa 40 X n=1 χn ∂t λ n−1 ∂iϵa . (4.4) (4.4) From the above quadratic Lagrangian, we ﬁnd the free propagators as G0 EE = 2 D0 T h k2 ω2 + (D0 k2)2 X n=1 χn −i ω λ n−1 , G0 Eϵa = 1 ω + iD0 k2 , G0 ϵaE = −1 ω −iD0 k2 . (4 5) JHEP04(2022)181 (4.5) It should be mentioned that at λ →∞our quadratic Lagrangian simpliﬁes to exactly that of refs. [15, 16]. In this limit, G0 EE is solely given by ﬁrst term of the series. It should be mentioned that at λ →∞our quadratic Lagrangian simpliﬁes to exactly that of refs. [15, 16]. In this limit, G0 EE is solely given by ﬁrst term of the series. In order to compute GR EE, we use the KMS relation [13] In order to compute GR EE, we use the KMS relation [13] G0 EE = coth βω 2 Im GR(0) EE . (4.6) (4.6) We then ﬁnd GR(0) EE = i h D0 k2 1 + ω2 λ2 ω + i D0k2 , (4.7) (4.7) which agrees with [18] and [60].6 We have followed the convention of refs. [3, 13] to deﬁne GR(ω, k) = Gra(ω, k) = −iδ2W δAa0(ω,k)δAr0(−ω,−k). Before ending this section, let us use the above response function to compute the which agrees with [18] and [60].6 We have followed the convention of refs. [3, 13] to deﬁne GR(ω, k) = Gra(ω, k) = −iδ2W δAa0(ω,k)δAr0(−ω,−k). 4 Eﬀective action in terms of energy density The oﬀ-shell hydrodynamic energy density current Jµ E ≡Jµ r = (E, Ji) is deﬁned as Jµ E [13]. Thus the energy density is given by The oﬀ-shell hydrodynamic energy density current Jµ E ≡Jµ r = (E, Ji) is deﬁned as Jµ E [13]. Thus the energy density is given by E = δIEFT[Brµ, Baµ] δAa0 = δIEFT[Brµ, Baµ] δ∂tϵa . (4.1) (4.1) In the second equality we have used Ba0 = Aa0 + ∂tϵa. Calculating (4.1), we ﬁnd In the second equality we have used Ba0 = Aa0 + ∂tϵa. Calculating (4.1), we ﬁnd E = h ∂tϵ −∂3 t ϵ λ2 ! + h 2 (∂tϵ)2 + 3 λ2 (∂2 t ϵ)2 + 2 λ2 ∂3 t ϵ∂tϵ + O(ϵ3) . (4.2) (4.2) Notice that this expression is exact in the derivative expansion. However, ∂tϵ in terms of E can only be found perturbatively in derivatives. We have found ∂tϵ to second order in E and up to 40th order in derivatives. The result related to the ﬁrst ﬁve orders is given in appendix C. The above discussion makes the statement below (A.11) more clear. In the linear regime and to ﬁrst order in derivatives, (4.2) takes the following form: E = h ∂tϵ . (4.3) (4.3) Comparing this with E = c T δT T , we infer that in our EFT, ∂tϵ actually acts as a temperate disturbance in the system. Here, c is the speciﬁc heat. In addition, h is speciﬁed as h = T c. Comparing this with E = c T δT T , we infer that in our EFT, ∂tϵ actually acts as a temperate disturbance in the system. Here, c is the speciﬁc heat. In addition, h is speciﬁed as h = T c. – 9 – 4.1 Quadratic action and the free propagators 4.1 Quadratic action and the free propagators a0( , ) r0( , ) Before ending this section, let us use the above response function to compute the thermal conductivity: a0( , ) r0( , ) Before ending this section, let us use the above response function to compute the thermal conductivity: κ = lim ω→0 lim k→0 ωβ k2 ImGR(0) EE = h β D0 −→ κ = D0 c (4.8) (4.8) where we have used h = T c found below (4.3).7 What we need to do in the following is to compute the loop correction to (4.5) in order to ﬁnd the one-loop corrected version of (4.7). Doing so, we will be able to study the eﬀect of nonlinear ﬂuctuations on the diﬀusion pole. 7Note that Gi in (3.13) can be rewritten in terms of temperature ﬂuctuations: Gi = −κ(δT)∂iT with δT) = κ+κ1 δT T +· · · . In terms of our D0 and D1, one can easily show that κ = D0 c and κ1 = (D0 +D1)c. 6Let us denote that this relation is exact in the sense that it continues to hold to all ord We would like to thank Hong Liu for pointing this out. 7 Let us denote that this relation is exact in the sense We would like to thank Hong Liu for pointing this out. 7 6Let us denote that this relation is exact in the sense that it continues to hold to all orders in derivative. We would like to thank Hong Liu for pointing this out. We would like to thank Hong Liu for pointing this out. 7 5 Loop computations In this section we aim to compute the loop corrections to the retarded Green’s function of the energy density according to the Lagrangian obtained in the previous section. As it is shown in appendix D, the renormalized retarded Green’s function can be formally written as follows GR EE(ω, k) = i (κ + δκ) 1 + ω2 λ2 −κ 2δλ λ ω2 λ2 T k2 ω + i D0 k2 + Σ(ω, k) . (5.1) (5.1) In the expression above, our focus will be on the denominator. Our goal is to compute the self-energy, Σ, perturbatively, in the derivative expansion. Then we will be able to see the inﬂuence of ﬂuctuations on the diﬀusion pole in the derivative expansion. Details of loop computation are given in appendix D. In the next two subsections, we review the results and discuss the consequences. 4.2 Cubic action and coupling constants As in the quadratic Lagrangian, the relation between ϵ and E to convert L(3) eﬀ[ϵ, ϵa] to L(3) eﬀ[E, ϵa]. The result is formally written as L(3) eﬀ= λ1 2 ∂2 i ϵa E2 + i c T 2 λ2 X n=1 n−1 X ℓ=0 g(n) ℓ,n−ℓ−1 λn−1 ∂iϵa∂ℓ t(∂iϵa)∂n−ℓ−1 t E + i c T 2 λ3 X n=3 n−1 X ℓ=1 h(n) ℓ,n−ℓ−1 λn−2 ∂tϵa∂ℓ tϵa∂n−ℓ−1 t E , (4.9) (4.9) 6Let us denote that this relation is exact in the sense that it continues to hold to all orders in derivative. We would like to thank Hong Liu for pointing this out. 7Note that Gi in (3.13) can be rewritten in terms of temperature ﬂuctuations: Gi = −κ(δT)∂iT with κ(δT) = κ+κ1 δT T +· · · . In terms of our D0 and D1, one can easily show that κ = D0 c and κ1 = (D0 +D1)c. – 10 – with the three distinct coupling constants with the three distinct coupling constants λ1 = D1 T c, λ2 = D0 + D1 T c , λ3 = 6 T c λ . (4.10) (4.10) It should be noted that λ1 and λ2 couplings come from M2 term in (2.2), while, λ3 coupling comes from M1 term in that equation. At ﬁrst order in the derivative expansion, the λ1 and λ2 terms are exactly λ and ˜λ terms of equation (B.1) in ref. [15]. Since this Reference does not exceed the ﬁrst order in the derivative expansion, our λ3 term has no analogues there. Note that in (4.9), the λ3 term starts to contribute from the third order in the derivative expansion. ( ) ( ) Due to the complex structure, we do not display g(n) ℓ,n−ℓ−1’s and h(n) ℓ,n−ℓ−1’s in this paper. We refer the reader to the electronic version of the paper on arXiv in which we have explicitly shown these coeﬃcients to 20th order in derivatives. JHEP04(2022)181 9It has recently been discovered that the breakdown of hydrodynamics may also occur in certain condensed matter systems. For instance, the breakdown of diﬀusion at the edge of dirty Quantum Hall systems [70] and breakdown of hydrodynamics in a fracton ﬂuids [71]. 5.1 Self-energy We ﬁnd the cutoﬀindependent part of the self-energy as Σ(ω, k) = cT 2 4D2 0 k2 λ1 k2 −2iω D0 −1/2 (σ1(ω, k) + σ2(ω, k) + σ3(ω, k) + · · · ) , (5.2) (5.2) where σj function denotes the jth order correction in the derivative expansion. We have found that σ1(ω, k) = ω λ1 − ω + iD0k2 λ2 , σ2n(ω, k) = 0 , σ2n+1(ω, k) = ω + iD0k2 F2n ω, iD0k2 λ1 + G2n ω, iD0k2 λ2 , (5.3) (5.3) where F2n and G2n are polynomials of ω and iD0k2 (see table 2 in appendix D). The contribution coming from ﬁrst order, namely σ1, agrees with the result of ref. [15]. where F2n and G2n are polynomials of ω and iD0k2 (see table 2 in appendix D). The contribution coming from ﬁrst order, namely σ1, agrees with the result of ref. [15]. – 11 – We have also deﬁned r = D1/D0. Here, some important points should be mentioned: We have also deﬁned r = D1/D0. Here, some important points should be mentioned: • The corrections at any even order in the derivative expansion vanish. This is due to the Schwarzian’s special form. • The λ3-coupling in (4.9) does not contribute to Σ at any order in the derivative expansion. The reason is that the corresponding Feynman diagrams are fully divergent after the renormalization, without any ﬁnite parts. • The λ3-coupling in (4.9) does not contribute to Σ at any order in the derivative expansion. The reason is that the corresponding Feynman diagrams are fully divergent after the renormalization, without any ﬁnite parts. • Except for the ﬁrst order in derivatives, at any other odd order, the correction vanishes at the classical diﬀusion pole, namely at ω = −iD0k2. • Except for the ﬁrst order in derivatives, at any other odd order, the correction vanishes at the classical diﬀusion pole, namely at ω = −iD0k2. JHEP04(2022)181 JHEP04(2022)181 8See [57] for the discussion about the breakdown of derivative expansion in (2 + 1) and (3 + 1) dime sional cases. 5.2 Long time tails and breakdown of the derivative expansion The standard derivative expansion in our diﬀusion model is based on the assumption that ω ∼k2. On the other hand, our loop computations show that at 1st order in derivative : Σ(ω, k) ∼\|ω\|3/2 ∼(k2)3/2 , at 3rd order in derivative : Σ(ω, k) ∼\|ω\|7/2 ∼(k2)7/2 , · · · t (nodd)th order in derivative : Σ(ω, k) ∼\|ω\|(2nodd+1)/2 ∼(k2)nodd+1/2 . (5.4) at 1st order in derivative : Σ(ω, k) ∼\|ω\|3/2 ∼(k2)3/2 , at 3rd order in derivative : Σ(ω, k) ∼\|ω\|7/2 ∼(k2)7/2 , · · · (5.4) (5.4) at (nodd)th order in derivative : Σ(ω, k) ∼\|ω\|(2nodd+1)/2 ∼(k2)nodd+1/2 . Obviously, these contributions are not consistent with our derivative counting scheme; the self-energy cannot be expressed by the natural powers of ω and k2, but it shows some non-analyticities. The appearance of non-analytic contributions of the form \|ω\|n/2 is the result of nonlinear interactions in LEFT. It is easy to see that the presence of \|ω\|n/2 in GR EE(ω, k →0) leads GR EE(t, x) to show the power-law relaxation ∼t−(n+2)/2. This non-exponential damping of the response function is obviously similar to the long-time tail eﬀect in stochastic hydrodynamics [15, 55]. The nonlinear interactions in our model have also another consequence; the breakdown of derivative expansion. In order to understand why this is the case, we only need to consider (5.4) at ﬁrst order. In the hydrodynamic limit, \|ω\|3/2 is smaller than the ﬁrst order contribution O(ω), but is larger than the second order contribution O(ω2). It simply indicates that the derivative expansion in 1 + 1 dimensional hydrodynamic derivative expansion breaks down beyond the ﬁrst order.8,9 Notice that this statement is independent of the assumptions based on which, we have studied the SYK chain in this work. We expect the same for the non-consonant Dn coeﬃcients in (3.14). 9It has recently been discovered that the breakdown of hydrodynamics may also occur in certain condensed matter systems. For instance, the breakdown of diﬀusion at the edge of dirty Quantum Hall systems [70] and breakdown of hydrodynamics in a fracton ﬂuids [71]. – 12 – 6 Modiﬁed dispersion relation It should be recalled that in our model, the linear hydro dispersion relation is exact for all orders in the derivative expansion, i.e. ω = −iD0k2. This truncation is done just to simplify the calculation. In a more general case, we can consider the following dispersion relation ω = −iD(1)k2 −iD(2)k4 −iD(3)k6 + O(k8) = −i X n=1 D(n)k2n . (6.1) (6.1) By performing tedious calculations, it can be shown that using (6.1) works also leading to (5.4). JHEP04(2022)181 The interesting point is that in any order in derivatives, the corresponding linear and nonlinear contributions, coming from (6.1) and (5.4), are two consecutive powers of (k2)1/2. For instance, at 3rd order, the linear contribution ∼(k2)3 and the nonlinear contribution ∼(k2)7/2. This suggests that: Although the expansion (6.1) fails to work due to nonlinear eﬀects given in (5.4), it is still possible to express ω as a power series of (k2)1/2. it is still possible to express ω as a power series of (k2)1/2. So we ﬁnd it convenient to write the series as follows ω = −i X n=1 k2n D(n,1) ± iD(n,2)(k2)1/2 = −i k2 D(1,1) ± iD(1,2)(k2)1/2 \| {z } 1st order −i k4 D(2,1) ± iD(2,2)(k2)1/2 \| {z } 2nd order +O(k6) . (6.2) (6.2) It is important to note that by “nth order” on the right side of (6.2), we mean the contribution to the dispersion relation that comes from the nonlinear Lagrangian at nth order in the derivative expansion. Let us emphasize that the above expansion is not actually a derivative expansion. The derivative expansion is meaningful only at the Lagrangian level. Our results show that at the level of dispersion relation, the derivative expansion should be replaced by (6.2). From now on we will refer to (6.2) as modiﬁed dispersion relation. The modiﬁed dispersion relation has some properties: 1. The ± in (6.2) indicates that, due to the nonlinear eﬀects, the classical diﬀusion pole is split into two poles with opposite real parts (see ﬁgure 2 below). This is consistent with the same result in the ﬁrst order of the derivative expansion found in ref. [15]. 1. The ± in (6.2) indicates that, due to the nonlinear eﬀects, the classical diﬀusion pole is split into two poles with opposite real parts (see ﬁgure 2 below). This is consistent with the same result in the ﬁrst order of the derivative expansion found in ref. [15]. 6 Modiﬁed dispersion relation Or for practical purposes, is it suﬃcient to use lower-order terms in the modiﬁed series, like what we do in classical hydrodynamics? Answering these questions has to do with the convergence properties of the series. To this end, in the following, we study the convergence of these modiﬁed series. To study the convergence of the series it is convenient to use dimensionless momentum JHEP04(2022)181 To study the convergence of the series, it is convenient to use dimensionless momentum and frequency. To this end, we rearrange the series as w = −i M X n=1 q2n ˜D(n,1)(r, s) ± i ˜D(n,2)(r, s) (q2)1/2 (6.4) (6.4) with w = D0 ω κ2 , q = D0 k κ , ˜D(n,j) = κ2n−3+j D2n−2+j 0 D(n,j) : j = 1, 2 . (6.5) (6.5) The parameters r and s are deﬁned as The parameters r and s are deﬁned as r = D1 D0 , s = κ2 D0 T . (6.6) (6.6) For concreteness, we discuss the convergence of (6.2) for r = 1 2 and s = 250 with M = 40. All 80 coeﬃcients are given in appendix E. For now, let us limit the discussion to real values of momentum. Then (6.4) takes the following simple form For concreteness, we discuss the convergence of (6.2) for r = 1 2 and s = 250 with M = 40. All 80 coeﬃcients are given in appendix E. For now, let us limit the discussion to real values of momentum. Then (6.4) takes the following simple form w = −i M X n=1 q2n ˜D(n,1)(r, s) ± i ˜D(n,2)(r, s) \|q\| ≡−i 2M+1 X n=2 cn\|q\|n . (6.7) (6.7) In ﬁgure 1 we have displayed the logarithm of the absolute value of the coeﬃcients cn. For the mentioned value of the parameters r and s, the slope of the plot at large n tends to 4.724. So the series approximately converges to qc = lim n→∞(ln \|cn+1\| −ln \|cn\|) −1 = lim n→∞ln cn+1 cn −1 ≈0.211 . (6.8) (6.8) Let us denote that the value of qc only depends on the choice of r = D1/D0. In fact, at large n, all coeﬃcients are proportional to s2 resulting in cn+1/cn independent of s. Then the only place where the eﬀect of s may appear is in the low-order terms in ﬁgure 1. 6 Modiﬁed dispersion relation 2. At any order in the derivative expansion, the modiﬁed dispersion relation is determined by twice the transport coeﬃcients of the classical dispersion relation. 2. At any order in the derivative expansion, the modiﬁed dispersion relation is determined by twice the transport coeﬃcients of the classical dispersion relation. 3. Considering (6.1), in our model D(1) = D0 and D(n) = 0, n > 1 (see (3.14) for the deﬁnition of D0). In other words, there is only one nonzero coeﬃcient in the classical dispersion relation. Including the loop corrections, however, we ﬁnd that in the modiﬁed series (6.2), in addition to the coeﬃcient of k2, all other coeﬃcients are also nonzero. For the case of D0 = 2D1, we ﬁnd 3. Considering (6.1), in our model D(1) = D0 and D(n) = 0, n > 1 (see (3.14) for the deﬁnition of D0). In other words, there is only one nonzero coeﬃcient in the classical dispersion relation. Including the loop corrections, however, we ﬁnd that in the modiﬁed series (6.2), in addition to the coeﬃcient of k2, all other coeﬃcients are also nonzero. For the case of D0 = 2D1, we ﬁnd ω = −iD0 k2 ± D2 0 κ ! k3 −i 3 4 D3 0 κ2 ! k4 ± 29 16 D4 0 κ3 ! k5 + O(k6) . (6.3) (6.3) – 13 – As mentioned earlier, κ = D0c and thus in the large-N limit where c is large, κ is also large. This means that all but the ﬁrst term in (6.3) are small corrections in this limit. As expected, in the absence of ﬂuctuations, i.e. in the limit κ →∞, the modiﬁed series returns to the original classical dispersion relation ω = −iD0k2. As mentioned earlier, κ = D0c and thus in the large-N limit where c is large, κ is also large. This means that all but the ﬁrst term in (6.3) are small corrections in this limit. As expected, in the absence of ﬂuctuations, i.e. in the limit κ →∞, the modiﬁed series returns to the original classical dispersion relation ω = −iD0k2. As with the case of derivative expansion in classical hydrodynamics, it is important to understand whether it would be possible to extract any special information from the large order behavior of the modiﬁed series described above. 6 Modiﬁed dispersion relation See appendix E for more details. As a result, we ﬁnd that for a given value of r, the series (6.4) converges to a ﬁnite value. Using (6.5), the dimensionfull radius of convergence is given by kc = qc κ D0 = # κ D0 , (6.9) (6.9) where the numerical factor depends on the value of r. This is the central result of this paper. – 14 – Figure 1. Coeﬃcients of the series (6.7). In our current case, M = 40, so the modiﬁed series is determined by 80 transport coeﬃcients. JHEP04(2022)181 Figure 1. Coeﬃcients of the series (6.7). In our current case, M = 40, so the modiﬁed series is determined by 80 transport coeﬃcients. - - - - - - - - - - - - - - Figure 2. Splitting of the classical diﬀusion mode (represented by large colored dots) into two mode represented by the small dots. Each group of colored path-like points, starting with dark red and ending with purple, shows the change of a particular mode when q discretely increases from 0.1 to 0.2. We have considered 10 regular steps by increments of ∆q = 0.01. Figure 2. Splitting of the classical diﬀusion mode (represented by large colored dots) into two mode represented by the small dots. Each group of colored path-like points, starting with dark red and ending with purple, shows the change of a particular mode when q discretely increases from 0.1 to 0.2. We have considered 10 regular steps by increments of ∆q = 0.01. As mentioned earlier, another eﬀect of nonlinear ﬂuctuations is to split the classical diﬀusion pole into two poles with non-vanishing real parts. It can be seen from ﬁgure 2 that the real parts of these modes are opposite to each other. The appearance of non-vanishing real parts indicates that the diﬀusion process occurs with the propagation of ﬂuctuations in the chain. The two modes illustrated in ﬁgure 2 are similar to the left- and right-moving sound waves in the 1+1 dimensional classical system with conservation of energy and momentum. 10See also [38, 40, 52, 54, 72–86] for recent studies on various aspects of mode collision and convergence radius of hydrodynamics. 7 Review, discussion and outlook In this paper we investigated the eﬀect of hydrodynamic ﬂuctuations in the SYK chain. By constructing the interacting EFT of energy diﬀusion up to 40th order in derivatives, as given by (4.4) and (4.9), we computed the one-loop self-energy to the same order in derivatives (see (5.2)). The special form of derivative corrections in the self-energy led us to the following results: 1. The dispersion relation of the energy diﬀusion does no longer follow the derivative expansion, namely the classical relation w = w(q2). However, the dispersion relation can be still represented as a power series, but in terms of \|q\|, say w = w(\|q\|). We referred to it as the modiﬁed (or quantum) dispersion relation. JHEP04(2022)181 2. The classical dispersion relation is split into two dispersion relations. In addition, up to any order in the derivative expansion, the number of transport coeﬃcients in the modiﬁed dispersion relations is twice that in the classical dispersion relation. 3. We found that the modiﬁed dispersion relation is convergent in the momentum space. The radius of convergent, kc, is proportional to the ratio of the thermal conductivity to the diﬀusion constant (see (6.9)). Since κ = D0 c, one immediately concludes that kc ∼c, that is, it is proportional to the number of degrees of freedom. Therefore, in the large-N limit, kc has a large value. As the ﬁrst generalization of our results, one might think of including the two-loop corrections along with derivative orders larger than 40. Although the latter is a technical problem to obtain better convergence, the former one is physically important; it is actually equivalent to considering 1 N2 corrections to the self-energy. To be more realistic, a larger EFT cut-oﬀvalue should be considered to make room for the inclusion of non-hydrodynamic modes. In the simplest case, only the lowest quasi- normal mode can be included. The corresponding EFT on CTP contour then will have two additional dynamical variables, associated with this quasinormal mode. In this setting, the inﬂuence of nonlinear ﬂuctuations on diﬀusion and non-hydro mode should be studied. Then it would be interesting to see what kind of singularity limits the convergence of the modiﬁed dispersion relation. More realistically, one can think of a 3+1 dimensional model. One way is to consider that the Schwarzian (3.7) depends on three spatial dimensions. 6 Modiﬁed dispersion relation It would be interesting to study these modes at complex momenta to ﬁnd the reason behind the ﬁnite radius of convergence of the modiﬁed series given in (6.9). Following [53], it should be determined whether there are any mode collisions that limit convergence.10 We leave more investigations on this issue for future work. – 15 – 7 Review, discussion and outlook Repeating all the steps taken in the paper, one would arrive at the analogue of (5.2) in 3+1 dimension as follows ˜Σ(ω, k) = cT 2 4D2 0 k2 λ1 k2 −2iω D0 1/2 (˜σ1(ω, k) + ˜σ2(ω, k) + ˜σ3(ω, k) + · · · ) . As it is seen, at the nth order in derivatives, ˜Σ(n) ∼(k2)n+1/2. It can be concluded that for the 3+1 dimensional model, the expansion (6.2) should be changed to allow four transport coeﬃcients at any n; i.e. D(n,j) : j = 1, 2, 3, 4. However, it must be carefully considered; it turns out the coeﬃcients of (k2)n and (k2)n+1/2 will get contributions from two diﬀerent – 16 – orders of n: coeﬃcient of (k2)n = D(n−1,3) + D(n,1) , coeﬃcient of (k2)n+1/2 = D(n−1,4) + D(n,2) . Because of the above pairing between the coeﬃcients D(n,j), one needs to use eﬀectively 2n+4 coeﬃcients. Here, 4 corresponds to D(1,1), D(1,2), D(n,3) and D(n,4); they are not paired with any other coeﬃcients at order n. We leave more investigation on 3+1 dimensional case to future work. Because of the above pairing between the coeﬃcients D(n,j), one needs to use eﬀectively 2n+4 coeﬃcients. Here, 4 corresponds to D(1,1), D(1,2), D(n,3) and D(n,4); they are not paired with any other coeﬃcients at order n. We leave more investigation on 3+1 dimensional case to future work. JHEP04(2022)181 Considering all aforementioned points, one may think of studying the same problem in the case of Quark Gluon Plasma (QGP). The hydrodynamic ﬂuctuations in QGP have been studied previously on the static [56] and also on the evolutionary [87] background. In order to extend the calculations of this paper to the QGP case, two important points should be considered. First, the dynamics of energy density in our model is similar to that of charge density or transverse momentum density in QGP, which is a diﬀusion process. In QGP, however, there are also sound modes. They actually propagate and diﬀuse into the medium. Second, QCD is not a large-N theory. Therefore, to be applicable to QGP, our setting should be extended to include relativistic hydrodynamic ﬂuctuations and also to go beyond one-loop computations. Another direction to explore this problem is holography. In the pioneering work of ref. 12According to the prescription of [89], the “quadratic” eﬀective action for the boundary theory has been computed in several diﬀerent situations [93–95]. 11See [91, 92] for other holographic prescriptions of the Schwinger-Keldysh contour. 11See [91, 92] for other holographic prescriptions of the Schwinger-Keldysh contour. 12According to the prescription of [89], the “quadratic” eﬀective action for the boundary theory has been computed in several diﬀerent situations [93–95]. Acknowledgments We are grateful to Mike Blake, Ali Davody, Luca Delacrétaz, Paolo Glorioso, Sean Hartnoll, Matthias Kaminski, Pavel Kovtun, Hong Liu and Misha Stephanov for valuable discussions and comments. We would also like to thank Armin Ghazi and Omid Tavakol for discussing various aspects of EFT of hydrodynamics. This work was supported by grant number 561119208 “Double First Class” start-up funding of Lanzhou University, China. 7 Review, discussion and outlook [88], the one-loop calculation of Einstein’s gravity in AdS space has been proven to reproduce the long-time tail eﬀect in boundary quantum ﬁeld theory at a ﬁnite temperature. Another interesting calculation can be found in [90], where the leading derivative contribution to the self-energy is directly computed from gravity. But one can do it in yet another way. The idea might be to use the holographic prescription of the Schwinger-Keldysh contour [89]11 and compute the boundary nonlinear eﬀective action from gravity to higher orders in derivatives.12 The next step will be simple, calculating the loop correction of the energy density response function in the boundary. Lastly, it would be interesting to continue the loop computations in this paper and specify all parameters introduced in (5.1). Doing so, the full one-loop corrected response function will be determined. This response function has an important characteristic. It is actually related to a maximally chaotic quantum system [19]. Then it would be very interesting to ﬁnd the pole-skipping phenomenon in this response function and extract the chaos point from it [18]. It is well-known that the chaos point might be located out of the regime validity of hydrodynamics [54, 72]. However, the fact that we have determined Leﬀ to high orders in the derivative expansion allows to probe the chaos point. Then the main question to be answered will be how the Lyapunov exponent and butterﬂy velocity are aﬀected by hydrodynamic ﬂuctuations. – 17 – A Review of Schwinger-Keldysh EFT So, we should replace φ1,2 in (A.2) with A s = 1 2 eW[A1µ,A2µ] = Z Dχ1Dχ2 eiIEFT[χ1,χ2; A1µ,A2µ] . (A.3) (A.3) Then the conservation of Jµ 1,2 translates to gauge invariance of W: Then the conservation of Jµ 1,2 translates to gauge invariance of W: W[A1µ, A2µ] = W[A1µ + ∂µλ1, A2µ + ∂µλ2] . (A.4) (A.4) In addition, one can also show that the generating functional on CPT satisﬁes the following properties: In addition, one can also show that the generating functional on CPT satisﬁes the following properties: W[Aµ, Aµ] = 1 (A.5) reﬂectivity: W ∗[A1µ, A2µ] = W[A2µ, A1µ] (A.6) Cauchy-Schwarz ineq.: Re W[A1µ, A2µ] ≤0 (A.7) KMS condition: W[A1µ(x), A2µ(x)] = W[A1µ(−t, −⃗x), A2µ(−t −iβ, −⃗x)] (A.8) JHEP04(2022)181 (A.7) Now one may ask in what sense χ ﬁeld is associated with ∂µJµ = 0? Let us denote that the above-mentioned IEFT should be such that the equations of motion of χ1,2 are equivalent to conversations of Jµ 1,2. This condition is ﬁxed if χ1,2 always appear with external ﬁelds through the combinations Now one may ask in what sense χ ﬁeld is associated with ∂µJµ = 0? Let us denote that the above-mentioned IEFT should be such that the equations of motion of χ1,2 are equivalent to conversations of Jµ 1,2. This condition is ﬁxed if χ1,2 always appear with external ﬁelds through the combinations B1µ = A1µ + ∂µχ1, B2µ = A2µ + ∂µχ2 . (A.9) (A.9) Therefore the χ1,2 are in fact Stueckelberg ﬁelds associated with the symmetry (A.4). The generating functional (A.3) then can be rewritten as Therefore the χ1,2 are in fact Stueckelberg ﬁelds associated with the symmetry (A.4). The generating functional (A.3) then can be rewritten as eW[A1µ,A2µ] = Z Dχ1Dχ2 eiIEFT[ B1µ,B2µ] . (A.10) (A.10) Now the main question is how to construct IEFT. The separation of scales L ≫ℓmotivates to construct IEFT in a derivative expansion with the expansion parameter ℓ∂µ ∼ℓ L ≪1. However, even writing down the most general local derivative expansion of IEFT[B1µ, B2µ] subject to conditions (A.4)–(A.8) is not enough.13 One further symmetry has to be imposed. To understand it, it is convenient to deﬁne σ = 1 2(χ1 + χ2) , Xa = χ1 −χ2 . A Review of Schwinger-Keldysh EFT JHEP04(2022)181 Below, we start from a microscopic theory and brieﬂy review the steps of ref. [13] to construct EFT around thermal equilibrium. The microscopic analogue of (2.1) can be written as ⟨ψ(t, x)ψ(t′, x′)⟩β where ψ is a microscopic dynamical variable. Such an out of equilibrium observable corresponds to insertion of microscopic ﬁelds ψ1 and ψ2 on the two legs of a closed time path (CTP), namely on the Schwinger-Keldysh contour. Then by coupling the ﬁelds to external sources φ1 and φ2, one can compute the generating functional eW[φ1,φ2] = Z ρ0 Dψ1Dψ2 eiI0[ψ1,φ1]−iI0[ψ2,φ2] , (A.1) (A.1) where I0[ψ1, φ1] is the microscopic action in the presence external source φ. In the language of Wilsonian RG, one may integrate out the UV dynamical variables, the so-called fast modes, in (A.1), to ﬁnd an eﬀective action describing the low energy dynamics. Let us ideally assume that ψ ﬁelds can be separated into UV and IR variables, ϕ and χ, respectively. Integrating out ϕ’s, (A.1) takes the following form: eW[φ1,φ2] = Z ℓmic Dχ1Dχ2 eiIEFT[χ1,χ2; φ1,φ2; ρ0] . (A.2) (A.2) Here, IEFT is the eﬀective action of slow variables χ and is valid at length scales larger than the microscopic scale ℓmic, or equivalently at energy scales much smaller than the UV cutoﬀ1/ℓmic. In the special case that the length (and time) scale of perturbations in the thermal system is much larger than the microscopic length (and time) of relaxation, i.e. L ≫ℓ (and tL ≫τ), non-conserved quantities relax back to equilibrium very fast with the rate of ∼1/τ. However, for conserved quantities, which cannot be locally destroyed, the only way to relax to equilibrium is transport on scales of order L (and tL). One concludes that at such macroscopic scales, which denote the so-called IR limit in (A.2), the only relevant variables χ are those associated with conserved quantities. The EFT of χ modes is indeed hydrodynamics. Eﬀective ﬁeld theory of energy diﬀusion. Let us consider the special case in which the only conserved quantity in the system is energy. In this EFT of energy diﬀusion, the χ ﬁeld in (A.2) is associated with the energy conservation ∂µJµ = 0, with Jµ being the – 18 – energy current coupled to background gauge ﬁeld Aµ. So, we should replace φ1,2 in (A.2) with Asµ, s = 1, 2 energy current coupled to background gauge ﬁeld Aµ. B.1 Quadratic action For a quadratic eﬀective Lagrangian of the form For a quadratic eﬀective Lagrangian of the form For a quadratic eﬀective Lagrangian of the form L(2) eﬀ[B1, B2] = Kµ1µ2 ra Baµ1Brµ2 + i 2 Gµ1µ2 rr Baµ1Baµ2 , (B.2) (B.2) where µ1, µ2 ∈{t, i}, it is found that the KMS constraint in momentum space is given by i Grr(ω) = 1 2 coth βω 2 Kra(ω) −Kar(ω) . (B.3) (B.3) Here, Kar(ω) = Kra(−ω) and for simplicity we have dropped the superscripts µ1 and µ2. In the classical limit ℏ→0, the coth factor on the right side is simply replaced with (βω)−1. However, since quantum eﬀects as well as the higher order derivatives are important in our study, we continue to use the original form (B.3). The corresponding Grr and Kra functions associated with our system, in the absence of external sources, can be written as Kµ1µ2 ra = h 1 + ω2 λ2 ! δµ1 t δµ2 t + (−D0 h) (−iω) 1 + ω2 λ2 ! δµ1 i δµ2 i , (B.4) Gµ1µ2 rr = 2 ρ−ω δµ1 t δµ2 t + 2 χ−ω δµ1 i δµ2 i . (B.5) (B.4) (B.5) Note that we take the Fourier components of Bµ1 and Bµ2 as Bω µ1 and B−ω µ2 , respectively. Plugging above Grr and Kra into (B.3) and expanding it in the derivative expansion, we can specify ρn and χn coeﬃcients, order by order in the derivative expansion. We have computed these coeﬃcients to 40th order in derivatives. The χn coeﬃcients with n ≤5 are found to be: χ1 = 1, χ3 = 1 12 −β2λ2 −12 , χ5 = −1 720β2λ2 β2λ2 −60 . (B.6) (B.6) A Review of Schwinger-Keldysh EFT (A.11) (A.11) Calculating the equations of motion from local IEFT, it turns out that ∂tσ(t, ⃗x) is related to the energy density h + E(t, ⃗x), and Xa(t, ⃗x) corresponds to the noise ﬁeld (h denotes the energy density in thermal equilibrium). This has two direct consequences: Calculating the equations of motion from local IEFT, it turns out that ∂tσ(t, ⃗x) is related to the energy density h + E(t, ⃗x), and Xa(t, ⃗x) corresponds to the noise ﬁeld (h denotes the energy density in thermal equilibrium). This has two direct consequences: 1. The derivative counting scheme should be such that ∂tσ ∼Xa [15]. 1. The derivative counting scheme should be such that ∂tσ ∼Xa [15]. 2. Since only ∂tσ is physical, σ must always appear with at least one time derivative in the eﬀective action. It is equivalent to say that IEFT is required to be invariant under the so-called diagonal shift symmetry [3]: 2. Since only ∂tσ is physical, σ must always appear with at least one time derivative in the eﬀective action. It is equivalent to say that IEFT is required to be invariant under the so-called diagonal shift symmetry [3]: σ(t, ⃗x) →σ(t, ⃗x) + a(⃗x) , X(t, ⃗x) →Xa(t, ⃗x) . (A.12) (A.12) 13Let us suppose Jµ current was related to an internal U(1) symmetry. Then writing down the most general local derivative expansion of IEFT[B1µ, B2µ] would describe a super ﬂuid phase with the spontaneously broken U(1) symmetry [13]. 13Let us suppose Jµ current was related to an internal U(1) symmetry. Then writing down the most general local derivative expansion of IEFT[B1µ, B2µ] would describe a super ﬂuid phase with the spontaneously broken U(1) symmetry [13]. – 19 – B KMS conditions The KMS condition (A.8) is actually a constraint equation on the generating functional in the presence of external sources Aµ. It is shown that a suﬃcient condition for invariance of W[A1µ, A2µ] under (A.8) is that IEFT[B1µ, B2µ] be invariant under The KMS condition (A.8) is actually a constraint equation on the generating functional in the presence of external sources Aµ. It is shown that a suﬃcient condition for invariance of W[A1µ, A2µ] under (A.8) is that IEFT[B1µ, B2µ] be invariant under B1µ(x) →B1µ(−t, −⃗x), B2µ(x) →B2µ(−t −iβ, −⃗x) (B.1) (B.1) which is the same as transformation of background Aµ ﬁeld (see [3, 14] for details). The KMS constraints should be imposed to quadratic and cubic action separately. Here, we do not derive the constraints and just follow ref. [3] and quote their results (see also [69] for an earlier derivation). JHEP04(2022)181 B.2 Cubic action For a cubic eﬀective Lagrangian of the form: L(3) eﬀ[B1, B2] = 1 2 Gµ1µ2µ3 raa Baµ1Brµ2Brµ3 + 1 2 Gµ1µ2µ3 rra Baµ1Baµ2Brµ3 , (B.7) (B.7) – 20 – the KMS constraint in momentum space is given by [3, 15, 69] the KMS constraint in momentum space is given by [3, 15, 69] 2 Grra = − N1 + N2 G∗ aar + N1 Gara + N2 Graa , Ni = coth β ωi 2 . (B.8) (B.8) Notice that any of the above G’s is actually a function of ω1, ω2 and ω3, where ω1+ω2+ω3 = 0. And again, for simplicity, we have dropped the superscripts µ1, µ2 and µ3. The corresponding Graa and Grra functions associated with our system, in the absence of external sources, can be written as Gµ1µ2µ3 raa = −h(−iω2) " D0+D1+3D0 Y 23 +(D0−D1) Y 33 + Y 22 ! +D1 Y 22 Y 33 # δµ1 i δµ2 i δµ3 0 + h 2 " 1+3 Y 23 +2 Y 22 # δµ1 0 δµ2 0 δµ3 0 + 2 ↔3 , (B.9) ! JHEP04(2022)181 (B.9) Gµ1µ2µ3 rra = 1− Y 33 ! αω2,k2 2 αω3,k3 1 δµ1 0 δµ2 0 δµ3 0 +ξω2,k2 2 ξω3,k3 1 δµ1 i δµ2 i δµ3 0 +(1 ↔2). (B.10) Gµ1µ2µ3 rra = 1− Y 33 ! αω2,k2 2 αω3,k3 1 δµ1 0 δµ2 0 δµ3 0 +ξω2,k2 2 ξω3,k3 1 δµ1 i δµ2 i δµ3 0 +(1 ↔2). (B.10) In the above expressions: ω1 + ω2 + ω3 = 0 and we have also deﬁned Y j1j2··· = −i ωj1 λ −i ωj2 λ · · · . (B.11) (B.11) Substituting (B.9) and (B.10) into (B.8) and expanding the two sides in the derivative expansion, we ﬁnd equations between the expansion coeﬃcients α1 and α2, deﬁned in (3.15), and also equations between the expansion coeﬃcients ξ1 and ξ2 (see appendix B). Our goal is to specify the coeﬃcients ξ1, ξ2, α1 and α2 in (B.10). (B.8), perturbatively. It is clear that ξ1 and ξ2 are related to the ﬁrst line of (B.9) while α1 and α2 can be found from the ﬁrst expression in the second line of it. Let us deﬁne α(∂t) = X n=3 α(n) × ∂t λ n−2 , ξ(∂t) = X n=1 ξ(n) × ∂t λ n−1 . B.2 Cubic action n KMS constraints at order n of the derivative expansion 1 D0+D1 2β −T0,0 = 0 2 T0,1−2T1,0 = 0 3 −12β (2T0,0−T0,2+T1,1−2T2,0)+ 1 2β2D0λ2+ 1 2D1 β2λ2+36 = 0 12βT0,2+ 1 2D0 β2λ2−12 + 1 2D1 β2λ2+12 = 0 4 T0,1−T0,3−2T1,0+T1,2−T2,1+2T3,0 = 0 2T1,2−3T0,3 = 0 5 β 1 2βD0λ2 β2λ2−60 −720(T0,2−T0,4−T1,1+T1,3+2T2,0−T2,2+T3,1−2T4,0) +1 2D1 β4λ4−120β2λ2−720 = 0 β 1 2βD0λ2 β2λ2−45 −180(2T0,2−4T0,4+3T1,3−2T2,2) + 1 2D1 β4λ4−90β2λ2−360 = 0 720T0,4+ 1 2βD0λ2 β2λ2+60 + 1 2βD1λ2 β2λ2−60 = 0 Table 1. KMS constraints. n KMS constraints at order n of the derivative expansion 1 D0+D1 2β −T0,0 = 0 2 T0,1−2T1,0 = 0 3 −12β (2T0,0−T0,2+T1,1−2T2,0)+ 1 2β2D0λ2+ 1 2D1 β2λ2+36 = 0 12βT0,2+ 1 2D0 β2λ2−12 + 1 2D1 β2λ2+12 = 0 4 T0,1−T0,3−2T1,0+T1,2−T2,1+2T3,0 = 0 2T1,2−3T0,3 = 0 5 β 1 2βD0λ2 β2λ2−60 −720(T0,2−T0,4−T1,1+T1,3+2T2,0−T2,2+T3,1−2T4,0) +1 2D1 β4λ4−120β2λ2−720 = 0 β 1 2βD0λ2 β2λ2−45 −180(2T0,2−4T0,4+3T1,3−2T2,2) + 1 2D1 β4λ4−90β2λ2−360 = 0 720T0,4+ 1 2βD0λ2 β2λ2+60 + 1 2βD1λ2 β2λ2−60 = 0 Table 1. KMS constraints. KMS constraints at order n of the derivative expansion KMS constraints at order n of the derivative expansion JHEP04(2022)181 Table 1. KMS constraints. Table 1. KMS constraints. Therefore, KMS constraints seem insuﬃcient to specify all the Tj,k coeﬃcients appearing in the Lagrangian. However, after solving the existing equations for an appropriate set of h n−1 2 i + 1 coeﬃcients,14 and applying the solutions to the Lagrangian, it turns out that all n − h n−1 2 i + 1 unspeciﬁed Tj,k coeﬃcients appear as the coeﬃcients of total derivative terms in the cubic Lagrangian L(3) eﬀ. This observation shows that we could actually start with an ansatz which is more constrained than (3.16) and (3.15). However, we continue to use (3.16) and (3.15). We have derived the equations between Tj,k coeﬃcients to 40th order in the derivative expansion. Below, we display the corresponding equations only for the ﬁrst 5 orders. We have also deﬁned Rj,k = αj 1αk 2 and calculated the equations between Rj,k coeﬃcients to the 40th order. The equations can be displayed in a similar way; however, we will not show them in the paper. B.2 Cubic action (B.12) (B.12) In the following, we will ﬁnd equations constraining α(n)’s and ξ(n)’s. For concreteness, let us consider ξ1 and ξ2 at the nth order. In this case the expansion coeﬃcients are ξ(n) 1 and ξ(n) 2 . It is clear that these coeﬃcients always appear in pairs as For concreteness, let us consider ξ1 and ξ2 at the nth order. In this case the expansion coeﬃcients are ξ(n) 1 and ξ(n) 2 . It is clear that these coeﬃcients always appear in pairs as ξ(j) 1 ξ(k) 2 ≡Tj,k . (B.13) (B.13) We ﬁnd that at the nth order in the derivative expansion, there are h n−1 2 i + 1 equations between n independent Tj,k coeﬃcients This can be understood as follows. Equation (B.8) is symmetric with respect to ω1 and ω2. While the symmetry on the right side is obvious, on the left side it is due to (B.10). Thus at any order in the derivative expansion, after imposing ω1 + ω2 + ω3 = 0, equation (B.8) becomes a symmetric polynomial of ω1 and ω2, giving a set of equations. The number of independent equations then is equal to the number of independent coeﬃcients in the expansion of (ω1 + ω2)n, i.e. h n−1 2 i + 1. These equations describe all KMS constraints of order n. On the other hand there are n independent variables: Tn,1, Tn−1,2, · · · , T1,n. – 21 – n KMS constraints at order n of the derivative expansion 1 D0+D1 2β −T0,0 = 0 2 T0,1−2T1,0 = 0 3 −12β (2T0,0−T0,2+T1,1−2T2,0)+ 1 2β2D0λ2+ 1 2D1 β2λ2+36 = 0 12βT0,2+ 1 2D0 β2λ2−12 + 1 2D1 β2λ2+12 = 0 4 T0,1−T0,3−2T1,0+T1,2−T2,1+2T3,0 = 0 2T1,2−3T0,3 = 0 5 β 1 2βD0λ2 β2λ2−60 −720(T0,2−T0,4−T1,1+T1,3+2T2,0−T2,2+T3,1−2T4,0) +1 2D1 β4λ4−120β2λ2−720 = 0 β 1 2βD0λ2 β2λ2−45 −180(2T0,2−4T0,4+3T1,3−2T2,2) + 1 2D1 β4λ4−90β2λ2−360 = 0 720T0,4+ 1 2βD0λ2 β2λ2+60 + 1 2βD1λ2 β2λ2−60 = 0 Table 1. KMS constraints. C Energy density in terms of reparametrization mode In order to ﬁnd the invert equation (4.2), we take an anstaz for ∂tϵ of the form ∂tϵ = X n=0 1 hen ∂n t E λn + X n,m=0 1 h2 en,m ∂n t E∂m t E λn+m . (C.1) (C.1) 14By linearly combining the equations, we have found that the most appropriate set of Tj,k’s at nth order is {Tn,0, Tn−2,2, Tn−1,4, Tn−1,6, · · · , Tn−1,2k−2, · · · }. As it is seen, for k > 2, the kth appropriate Tj,k follows from a general form: Tn−1,2k−2. Finally, in any order n, we choose the ﬁrst n−1 2 + 1 elements of the set as the appropriate coeﬃcients to solve from the KMS equations. – 22 – Substituting this into (4.2), we can then read the coeﬃcients en and en,m, order by order in the derivative expansion. Here is the result related the ﬁrst ﬁve orders: Substituting this into (4.2), we can then read the coeﬃcients en and en,m, order by order in the derivative expansion. Here is the result related the ﬁrst ﬁve orders: ∂2 t E λ2 + ∂4 t E λ4 +O ∂6 t E λ6 !# (C.2) ∂tϵ = 1 h " E+ ∂2 t E λ2 + ∂4 t E λ4 +O ∂6 t E λ6 !# (C.2) −1 h2 " E2 2 + 1 λ2 5 2(∂tE)2+3E∂2 t E + 1 λ4 19 2 (∂2 t E)2+14∂E∂3 t E+5E∂4 t E +O ∂6 t E2 λ6 !# . (C.2) D Loop computations In order to compute the one-loop renormalized Green’s function, we use the standard textbook method: In order to compute the one-loop renormalized Green’s function, we use the standard textbook method: 1 JHEP04(2022)181 ⟨EE⟩= ⟨EE⟩0 + i⟨ESintE⟩−1 2⟨ES2 intE⟩+ · · · . (D.1) (D.1) Then following [15], we parameterize the corrections to the Green’s function as a numerator C(ω, k) and a self-energy Σ(ω, k). The latter corresponds to diagrams that resum, shown by the gray blobs below, while the former corresponds to those that do not so, shown by shaded blobs in the following. Diagrammatically, we write GEϵa(p)(−C(p))GϵaE(p) = ; GEϵa(p)(−Σ(p))GEE(p) = = . (D.2) (D.2) Then the Green’s functions can be written as GEE(p) = 1 − = G0 Eϵa(p)(−C(p))G0 ϵaE(p) 1 + G0 EϵaΣ(p) −Σ∗(p)G0 ϵaE(p) . (D.3) (D.3) By using the free propagators given in (4.5), we simply ﬁnd GEE(ω, k) = C(ω, k) ω2 + D2 0k4 + 2 ω Re Σ(ω, k) + 2 D0 k2 Im Σ(ω, k) (D.4) (D.4) where where C(ω, k) = 2 T 2 κ k2 40 X n=1 χn −i ω λ n−1 + loop corrections . (D.5) (D.5) Then by using KMS relations, we can parameterize the one-loop retarded Green’s function as (5.1). In the following, we explain in detail how to compute C(p) and Σ(p). The numerator C(p), up to ﬁrst order in loop corrections, is given by the following diagram with p = (ω, k) GEϵa(p)(−C(p))GϵaE(p) = GEϵa(p)( C(p))GϵaE(p) . – 23 – Since we are not interested in computing C(p) we do not write the corresponding loop integrals. The self-energy Σ(p) is what we want to compute. Diagrammatically, and up to ﬁrst order in loop corrections, we have: Since we are not interested in computing C(p) we do not write the corresponding loop integrals. The self-energy Σ(p) is what we want to compute. Diagrammatically, and up to ﬁrst order in loop corrections, we have: GEϵa(p)(−Σ(p))GEE(p) = which leads to JHEP04(2022)181 p) = quartic+λ2 1k2 Z p′ k′2GϵaE(p′)GEE(p+p′)−icT 2λ1k2 Z p′ ˜λ2 (k′2+kk′)GϵaE(p′)GEϵa(p+p′) Σ(p) = quartic+λ2 1k2 Z p′ k′2GϵaE(p′)GEE(p+p′)−icT 2λ1k2 Z p′ ˜λ2 (k′2+kk′)GϵaE(p′)GEϵa(p+p′) −iT 3λ1k2 Z p′ ˜λ3 (ω′2+ωω′)GϵaE(p′)GEϵa(p+p′). D Loop computations (D.6) (D.6) In the above expression In the above expression In the above expression ˜λ2 = 1 2λ2 X n=1 n−1 X ℓ=0 g(n) ℓ,n−ℓ−1 λn−1 (−i)n−1 h ω′ℓ+ (ω + ω′)ℓi ωn−ℓ−1 , ˜λ3 = 1 2λ3 X n=3 n−1 X ℓ=1 h(n) ℓ,n−ℓ−1 λn−2 (−i)n−1 h ω′ℓ+ (ω + ω′)ℓi ωn−ℓ−1 , (D.7) (D.7) with λ1, λ2 and λ3 given in (4.10). Let us recall that h and g coeﬃcients have been given in appendix. In eq. (D.6), “quartic” denotes the contribution coming from the quartic Lagrangian. We have found that this term does not have any cutoﬀindependent part. From (D.7), we see that at ﬁrst order in derivatives, namely n = 1, ˜λ2 = λ2 and ˜λ3 = 0. This is exactly the case considered in [15]. In this case, the frequency integrals are UV ﬁnite and one can easily perform the momentum integrals by considering a hard cutoﬀmomentum. Beyond n = 1, however, the frequency integrals are UV divergent, as can be seen by substituting (D.7) into (D.6). In order to regularize them, we follow the ideas developed in [14] and use the following regularization for the three integrals in (D.6), respectively: GEE(ω′, k′) → Λ Λ + iω n−1 GEE(ω′, k′), ˜λ2,3 → Λ Λ −iω n−1 ˜λ2,3 (D.8) (D.8) where Λ is a UV energy cutoﬀand n denotes the order of derivative, which is actually 40 in our case. Note that to perform the integral in the λ2 1 term, we close the contour along the semicircle in the lower half of the complex ω-plane, while for the next two integrals in (D.6), we close the contour along the upper semicircle in the complex plane. These choices are consistent with the regularizations introduced in (D.8). After performing the frequency integrals, we consider a hard cutoﬀmomentum Λ′ and evaluate the momentum integrals. Another point that should be noted is the inclusion of ghosts. As mentioned in [3], some anti-commuting ghosts variables and BRST symmetry are necessary to protect the unitarity condition (A.5) at the loop level. However, it was shown that they do not play any role in low energy dynamics [13, 14]. D Loop computations – 24 – ( ) (2n+1) F2n and G2n functions deﬁned in (5.3) (3) F2 = ω(β2λ2+12)(ω+iD0k2) 48λ2 G2 = − D2 0k4(β2λ2+r(β2λ2+12)−12)−2iD0k2ω(β2λ2+r(β2λ2+12)−12)+ω2(β2λ2+r(β2λ2+12)−36) 48λ2(r+1) (5) F4 = − β2ω(β2λ2−60)(ω+iD0k2)(3D2 0k4−6iD0k2ω+ω2) 11520λ2 G4 = 720ω4(β2λ2−8)−120ω2(5β2λ2+24)(D2 0k4−2iD0k2ω+ω2)+β2λ2(D4 0k8−4iD3 0k6ω+2D2 0k4ω2−12iD0k2ω3+ω4)(β2λ2+r(β2λ2−60)+60) 11520λ4(r+1) Table 2. Corrections to the self energy. n ˜D(n,1) ˜D(n,2) n ˜D(n,1) ˜D(n,2) 1 1 1 21 2.0216×1020s2 −9.47945×1020s2 2 0.75 −1.8125 22 −4.44677×1021s2 2.08675×1022s2 3 −5.35938 1.38778×10−17s2 23 9.79602×1022s2 −4.6002×1023s2 4 −0.03125s2 0.0427909s2 24 −2.16094×1024s2 1.01541×1025s2 5 0.15915s2 −0.642968s2 25 4.77276×1025s2 −2.24397×1026s2 6 −2.69663s2 11.5966s2 26 −1.05531×1027s2 4.96422×1027s2 7 50.7156s2 −224.481s2 27 2.33576×1028s2 −1.09928×1029s2 8 −1002.63s2 4509.76s2 28 −5.17466×1029s2 2.4364×1030s2 9 20398.8s2 −92694.3s2 29 1.14737×1031s2 −5.40439×1031s2 10 −422827.0s2 1.93498×106s2 30 −2.54607×1032s2 1.1997×1033s2 11 8.87966×106s2 −4.08468×107s2 31 5.65396×1033s2 −2.66505×1034s2 12 −1.88292×108s2 8.69585×108s2 32 −1.2564×1035s2 5.92405×1035s2 13 4.02259×109s2 −1.86355×1010s2 33 2.79367×1036s2 −1.31764×1037s2 14 −8.64469×1010s2 4.01494×1011s2 34 −6.21553×1037s2 2.93238×1038s2 15 1.86673×1012s2 −8.68793×1012s2 35 1.38363×1039s2 −6.52941×1039s2 16 −4.04713×1013s2 1.88687×1014s2 36 −3.08165×1040s2 1.4546×1041s2 17 8.80382×1014s2 −4.11067×1015s2 37 6.86682×1041s2 −3.24202×1042s2 18 −1.92062×1016s2 8.97928×1016s2 38 −1.53082×1043s2 7.22897×1043s2 19 4.2004×1017s2 −1.96596×1018s2 39 3.41408×1044s2 −1.61255×1045s2 20 −9.20617×1018s2 4.3131×1019s2 40 −7.61714×1045s2 3.5984×1046s2 Table 3. Coeﬃcients of series (6.7) for r = 1 2. (2n+1) F2n and G2n functions deﬁned in (5.3) (3) F2 = ω(β2λ2+12)(ω+iD0k2) 48λ2 G2 = − D2 0k4(β2λ2+r(β2λ2+12)−12)−2iD0k2ω(β2λ2+r(β2λ2+12)−12)+ω2(β2λ2+r(β2λ2+12)−36) 48λ2(r+1) (5) F4 = − β2ω(β2λ2−60)(ω+iD0k2)(3D2 0k4−6iD0k2ω+ω2) 11520λ2 G4 = 720ω4(β2λ2−8)−120ω2(5β2λ2+24)(D2 0k4−2iD0k2ω+ω2)+β2λ2(D4 0k8−4iD3 0k6ω+2D2 0k4ω2−12iD0k2ω3+ω4)(β2λ2+r(β2λ2−60)+60) 11520λ4(r+1) F2n and G2n functions deﬁned in (5.3) JHEP04(2022)181 Table 3. Coeﬃcients of series (6.7) for r = 1 2. Table 3. Coeﬃcients of series (6.7) for r = 1 2. E Series coeﬃcients and convergence of series All 80 coeﬃcients of series (6.4) for r = 1 2 are displayed in table 3. To explain why the ﬁrst few coeﬃcients are independent of s2, let us recall from (6.3) that the coeﬃcient of kn seem to be of order Dn−1 0 /κn−2. But it turns out that this only works for n < 7; the leading contribution to the coeﬃcient of kn for n ≥7 will be something like Dn−3 0 /(T 2κn−6). This is simply due to the special form of the Schwarzian; i.e. the 1/λ2 ∼1/T 2 term in (3.12) contributes at least from order ∂3 t ∼ω3 ∼k6. This change of behavior in the coeﬃcients is the same as what we see in the coeﬃcients of rescaled series (6.4) in table 3. – 25 – Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] L.D. Landau and E.M. Lifshitz, Fluid Mechanics, Course of Theoretical Physics, vol. 6, Pergamon Press, Oxford, U.K. (1987) [DOI]. [2] L.D. Landau and E.M. Lifshitz, Statistical Physics Part 2, Course of Theoretical Physics, vol. 9, Butterworth-Heinemann, Oxford, U.K. (1980) [DOI]. JHEP04(2022)181 [3] M. Crossley, P. Glorioso and H. Liu, Eﬀective ﬁeld theory of dissipative ﬂuids, JHEP 09 (2017) 095 [arXiv:1511.03646] [INSPIRE]. [4] F.M. Haehl, R. Loganayagam and M. Rangamani, Adiabatic hydrodynamics: The eightfold way to dissipation, JHEP 05 (2015) 060 [arXiv:1502.00636] [INSPIRE]. [5] K. Jensen, N. Pinzani-Fokeeva and A. Yarom, Dissipative hydrodynamics in superspace, JHEP 09 (2018) 127 [arXiv:1701.07436] [INSPIRE]. [6] F.M. Haehl, R. Loganayagam and M. Rangamani, Eﬀective Action for Relativistic Hydrodynamics: Fluctuations, Dissipation, and Entropy Inﬂow, JHEP 10 (2018) 194 [arXiv:1803.11155] [INSPIRE]. [7] K. Jensen, R. Marjieh, N. Pinzani-Fokeeva and A. Yarom, A panoply of Schwinger-Keldysh transport, SciPost Phys. 5 (2018) 053 [arXiv:1804.04654] [INSPIRE]. [8] S. Dubovsky, L. Hui, A. Nicolis and D.T. Son, Eﬀective ﬁeld theory for hydrodynamics: thermodynamics, and the derivative expansion, Phys. Rev. D 85 (2012) 085029 [arXiv:1107.0731] [INSPIRE]. [9] S. Endlich, A. Nicolis, R.A. Porto and J. Wang, Dissipation in the eﬀective ﬁeld theory for hydrodynamics: First order eﬀects, Phys. Rev. D 88 (2013) 105001 [arXiv:1211.6461] [INSPIRE]. [10] S. Grozdanov and J. Polonyi, Viscosity and dissipative hydrodynamics from eﬀective ﬁeld theory, Phys. Rev. D 91 (2015) 105031 [arXiv:1305.3670] [INSPIRE]. [11] P. Kovtun, G.D. Moore and P. Romatschke, Towards an eﬀective action for relativistic dissipative hydrodynamics, JHEP 07 (2014) 123 [arXiv:1405.3967] [INSPIRE]. [12] M. Harder, P. Kovtun and A. Ritz, On thermal ﬂuctuations and the generating functional in relativistic hydrodynamics, JHEP 07 (2015) 025 [arXiv:1502.03076] [INSPIRE]. [13] H. Liu and P. Glorioso, Lectures on non-equilibrium eﬀective ﬁeld theories and ﬂuctuating hydrodynamics, PoS TASI2017 (2018) 008 [arXiv:1805.09331] [INSPIRE]. [14] P. Gao, P. Glorioso and H. Liu, Ghostbusters: Unitarity and Causality of Non-equilibrium Eﬀective Field Theories, JHEP 03 (2020) 040 [arXiv:1803.10778] [INSPIRE]. [15] X. Chen-Lin, L.V. Delacrétaz and S.A. Hartnoll, Theory of diﬀusive ﬂuctuations, Phys. Rev. Lett. 122 (2019) 091602 [arXiv:1811.12540] [INSPIRE]. [16] A. Jain and P. Kovtun, Late Time Correlations in Hydrodynamics: Beyond Constitutive Relations, Phys. Rev. Lett. 128 (2022) 071601 [arXiv:2009.01356] [INSPIRE]. – 26 – [17] N. Sogabe, N. Yamamoto and Y. Yin, Positive magnetoresistance induced by hydrodynamic ﬂuctuations in chiral media, JHEP 09 (2021) 131 [arXiv:2105.10271] [INSPIRE]. [18] M. Blake, H. Lee and H. References Liu, A quantum hydrodynamical description for scrambling and many-body chaos, JHEP 10 (2018) 127 [arXiv:1801.00010] [INSPIRE]. [19] M. Blake and H. Liu, On systems of maximal quantum chaos, JHEP 05 (2021) 229 [arXiv:2102.11294] [INSPIRE]. [20] J. Maldacena, S.H. Shenker and D. Stanford, A bound on chaos, JHEP 08 (2016) 106 [arXiv:1503.01409] [INSPIRE]. [21] M. Blake, R.A. Davison and D. Vegh, Horizon constraints on holographic Green’s functions, JHEP 01 (2020) 077 [arXiv:1904.12883] [INSPIRE]. JHEP04(2022)181 [22] S. Grozdanov, On the connection between hydrodynamics and quantum chaos in holographic theories with stringy corrections, JHEP 01 (2019) 048 [arXiv:1811.09641] [INSPIRE]. [23] M. Natsuume and T. Okamura, Holographic chaos, pole-skipping, and regularity, PTEP 2020 (2020) 013B07 [arXiv:1905.12014] [INSPIRE]. [24] M. Natsuume and T. Okamura, Nonuniqueness of Green’s functions at special points, JHEP 12 (2019) 139 [arXiv:1905.12015] [INSPIRE]. [25] M. Natsuume and T. Okamura, Pole-skipping with ﬁnite-coupling corrections, Phys. Rev. D 100 (2019) 126012 [arXiv:1909.09168] [INSPIRE]. [26] X. Wu, Higher curvature corrections to pole-skipping, JHEP 12 (2019) 140 [arXiv:1909.10223] [INSPIRE]. [27] Y. Ahn, V. Jahnke, H.-S. Jeong and K.-Y. Kim, Scrambling in Hyperbolic Black Holes: shock waves and pole-skipping, JHEP 10 (2019) 257 [arXiv:1907.08030] [INSPIRE]. [28] W. Li, S. Lin and J. Mei, Thermal diﬀusion and quantum chaos in neutral magnetized plasma, Phys. Rev. D 100 (2019) 046012 [arXiv:1905.07684] [INSPIRE]. [29] N. Ceplak, K. Ramdial and D. Vegh, Fermionic pole-skipping in holography, JHEP 07 (2020) 203 [arXiv:1910.02975] [INSPIRE]. [30] S. Das, B. Ezhuthachan and A. Kundu, Real time dynamics from low point correlators in 2d BCFT, JHEP 12 (2019) 141 [arXiv:1907.08763] [INSPIRE]. [31] N. Abbasi and J. Tabatabaei, Quantum chaos, pole-skipping and hydrodynamics in a holographic system with chiral anomaly, JHEP 03 (2020) 050 [arXiv:1910.13696] [INSPIRE]. [32] Y. Liu and A. Raju, Quantum Chaos in Topologically Massive Gravity, JHEP 12 (2020) 027 [arXiv:2005.08508] [INSPIRE]. [33] Y. Ahn, V. Jahnke, H.-S. Jeong, K.-Y. Kim, K.-S. Lee and M. Nishida, Pole-skipping of scalar and vector ﬁelds in hyperbolic space: conformal blocks and holography, JHEP 09 (2020) 111 [arXiv:2006.00974] [INSPIRE]. [34] Y. Ahn, V. Jahnke, H.-S. Jeong, K.-S. Lee, M. Nishida and K.-Y. Kim, Classifying pole-skipping points, JHEP 03 (2021) 175 [arXiv:2010.16166] [INSPIRE]. [35] K.-Y. Kim, K.-S. Lee and M. Nishida, Holographic scalar and vector exchange in OTOCs and pole-skipping phenomena, JHEP 04 (2021) 092 [Erratum ibid. 04 (2021) 229] [arXiv:2011.13716] [INSPIRE]. – 27 – [36] K. References Sil, Pole skipping and chaos in anisotropic plasma: a holographic study, JHEP 03 (2021) 232 [arXiv:2012.07710] [INSPIRE]. [37] H. Yuan and X.-H. Ge, Pole-skipping and hydrodynamic analysis in Lifshitz, AdS2 and Rindler geometries, JHEP 06 (2021) 165 [arXiv:2012.15396] [INSPIRE]. [38] N. Abbasi and M. Kaminski, Constraints on quasinormal modes and bounds for critical points from pole-skipping, JHEP 03 (2021) 265 [arXiv:2012.15820] [INSPIRE]. [39] N. Ceplak and D. Vegh, Pole-skipping and Rarita-Schwinger ﬁelds, Phys. Rev. D 103 (2021) 106009 [arXiv:2101.01490] [INSPIRE]. [40] H.-S. Jeong, K.-Y. Kim and Y.-W. Sun, Bound of diﬀusion constants from pole-skipping points: spontaneous symmetry breaking and magnetic ﬁeld, JHEP 07 (2021) 105 [arXiv:2104.13084] [INSPIRE]. JHEP04(2022)181 [41] H. Yuan and X.-H. Ge, Analogue of the pole-skipping phenomenon in acoustic black holes, Eur. Phys. J. C 82 (2022) 167 [arXiv:2110.08074] [INSPIRE]. [42] M. Blake and R.A. Davison, Chaos and pole-skipping in rotating black holes, JHEP 01 (2022) 013 [arXiv:2111.11093] [INSPIRE]. [43] K.-Y. Kim, K.-S. Lee and M. Nishida, Construction of bulk solutions for towers of pole-skipping points, arXiv:2112.11662 [INSPIRE]. [44] F.M. Haehl and M. Rozali, Eﬀective Field Theory for Chaotic CFTs, JHEP 10 (2018) 118 [arXiv:1808.02898] [INSPIRE]. [45] D.M. Ramirez, Chaos and pole skipping in CFT2, JHEP 12 (2021) 006 [arXiv:2009.00500] [INSPIRE]. [46] A. Jain, P. Kovtun, A. Ritz and A. Shukla, Hydrodynamic eﬀective ﬁeld theory and the analyticity of hydrostatic correlators, JHEP 02 (2021) 200 [arXiv:2011.03691] [INSPIRE]. [47] J. Chao and T. Schaefer, Multiplicative noise and the diﬀusion of conserved densities, JHEP 01 (2021) 071 [arXiv:2008.01269] [INSPIRE]. [48] N. Sogabe and Y. Yin, Oﬀ-equilibrium non-Gaussian ﬂuctuations near the QCD critical point: an eﬀective ﬁeld theory perspective, JHEP 03 (2022) 124 [arXiv:2111.14667] [INSPIRE]. [49] M. Baggioli and M. Landry, Eﬀective Field Theory for Quasicrystals and Phasons Dynamics, SciPost Phys. 9 (2020) 062 [arXiv:2008.05339] [INSPIRE]. [50] L.V. Delacrétaz, B. Goutéraux and V. Ziogas, Damping of Pseudo-Goldstone Fields, Phys. Rev. Lett. 128 (2022) 141601 [arXiv:2111.13459] [INSPIRE]. [51] M.P. Heller, R.A. Janik and P. Witaszczyk, Hydrodynamic Gradient Expansion in Gauge Theory Plasmas, Phys. Rev. Lett. 110 (2013) 211602 [arXiv:1302.0697] [INSPIRE]. [52] B. Withers, Short-lived modes from hydrodynamic dispersion relations, JHEP 06 (2018) 059 [arXiv:1803.08058] [INSPIRE]. [53] S. Grozdanov, P.K. Kovtun, A.O. Starinets and P. Tadić, Convergence of the Gradient Expansion in Hydrodynamics, Phys. Rev. Lett. 122 (2019) 251601 [arXiv:1904.01018] [INSPIRE]. [54] S. Grozdanov, P.K. Kovtun, A.O. Starinets and P. Tadić, The complex life of hydrodynamic modes, JHEP 11 (2019) 097 [arXiv:1904.12862] [INSPIRE]. References – 28 – [55] P. Kovtun and L.G. Yaﬀe, Hydrodynamic ﬂuctuations, long time tails, and supersymmetry, Phys. Rev. D 68 (2003) 025007 [hep-th/0303010] [INSPIRE]. [56] P. Kovtun, G.D. Moore and P. Romatschke, The stickiness of sound: An absolute lower limit on viscosity and the breakdown of second order relativistic hydrodynamics, Phys. Rev. D 84 (2011) 025006 [arXiv:1104.1586] [INSPIRE]. [57] P. Kovtun, Lectures on hydrodynamic ﬂuctuations in relativistic theories, J. Phys. A 45 (2012) 473001 [arXiv:1205.5040] [INSPIRE]. [58] A. Shukla, Hydrodynamic ﬂuctuations and long-time tails in a ﬂuid on an anisotropic background, Nucl. Phys. B 968 (2021) 115442 [arXiv:2101.10000] [INSPIRE]. JHEP04(2022)181 JHEP04(2022)181 [59] L.V. Delacretaz, Heavy Operators and Hydrodynamic Tails, SciPost Phys. 9 (2020) 034 [arXiv:2006.01139] [INSPIRE]. [60] Y. Gu, X.-L. Qi and D. Stanford, Local criticality, diﬀusion and chaos in generalized Sachdev-Ye-Kitaev models, JHEP 05 (2017) 125 [arXiv:1609.07832] [INSPIRE]. [61] C.P. Herzog, P. Kovtun, S. Sachdev and D.T. Son, Quantum critical transport, duality, and M-theory, Phys. Rev. D 75 (2007) 085020 [hep-th/0701036] [INSPIRE]. [62] C. Choi, M. Mezei and G. Sárosi, Pole skipping away from maximal chaos, JHEP 02 (2021) 207 [arXiv:2010.08558] [INSPIRE]. [63] A. Kitaev, A simple model of quantum holography (part 1), talk at KITP, April 7, 2015, http://online.kitp.ucsb.edu/online/entangled15/kitaev/. [64] A. Kitaev, A simple model of quantum holography (part 2), talk at KITP, May 27, 2015, http://online.kitp.ucsb.edu/online/entangled15/kitaev2/. [65] S. Sachdev and J. Ye, Gapless spin ﬂuid ground state in a random, quantum Heisenberg magnet, Phys. Rev. Lett. 70 (1993) 3339 [cond-mat/9212030] [INSPIRE]. [66] J. Maldacena and D. Stanford, Remarks on the Sachdev-Ye-Kitaev model, Phys. Rev. D 94 (2016) 106002 [arXiv:1604.07818] [INSPIRE]. [67] K. Jensen, Chaos in AdS2 Holography, Phys. Rev. Lett. 117 (2016) 111601 [arXiv:1605.06098] [INSPIRE]. [68] J. Maldacena, D. Stanford and Z. Yang, Conformal symmetry and its breaking in two dimensional Nearly Anti-de-Sitter space, PTEP 2016 (2016) 12C104 [arXiv:1606.01857] [INSPIRE]. [69] E. Wang and U.W. Heinz, A Generalized ﬂuctuation dissipation theorem for nonlinear response functions, Phys. Rev. D 66 (2002) 025008 [hep-th/9809016] [INSPIRE]. [70] L.V. Delacretaz and P. Glorioso, Breakdown of Diﬀusion on Chiral Edges, Phys. Rev. Lett. 124 (2020) 236802 [arXiv:2002.08365] [INSPIRE]. [71] P. Glorioso, J. Guo, J.F. Rodriguez-Nieva and A. Lucas, Breakdown of hydrodynamics below four dimensions in a fracton ﬂuid, arXiv:2105.13365 [INSPIRE]. [72] N. Abbasi and S. Tahery, Complexiﬁed quasinormal modes and the pole-skipping in a holographic system at ﬁnite chemical potential, JHEP 10 (2020) 076 [arXiv:2007.10024] [INSPIRE]. [73] A. Jansen and C. References Pantelidou, Quasinormal modes in charged ﬂuids at complex momentum, JHEP 10 (2020) 121 [arXiv:2007.14418] [INSPIRE]. – 29 – [74] M. Baggioli, How small hydrodynamics can go, Phys. Rev. D 103 (2021) 086001 [arXiv:2010.05916] [INSPIRE]. [75] D. Arean, R.A. Davison, B. Goutéraux and K. Suzuki, Hydrodynamic Diﬀusion and Its Breakdown near AdS2 Quantum Critical Points, Phys. Rev. X 11 (2021) 031024 [arXiv:2011.12301] [INSPIRE]. [76] M.P. Heller, A. Serantes, M. Spaliński, V. Svensson and B. Withers, Hydrodynamic gradient expansion in linear response theory, Phys. Rev. D 104 (2021) 066002 [arXiv:2007.05524] [INSPIRE]. [77] M. Asadi, H. Soltanpanahi and F. Taghinavaz, Critical behaviour of hydrodynamic series, JHEP 05 (2021) 287 [arXiv:2102.03584] [INSPIRE]. JHEP04(2022)181 [78] M. Baggioli, U. Gran and M. Tornsö, Collective modes of polarizable holographic media in magnetic ﬁelds, JHEP 06 (2021) 014 [arXiv:2102.09969] [INSPIRE]. [79] N. Wu, M. Baggioli and W.-J. Li, On the universality of AdS2 diﬀusion bounds and the breakdown of linearized hydrodynamics, JHEP 05 (2021) 014 [arXiv:2102.05810] [INSPIRE]. [80] S. Grozdanov, A.O. Starinets and P. Tadić, Hydrodynamic dispersion relations at ﬁnite coupling, JHEP 06 (2021) 180 [arXiv:2104.11035] [INSPIRE]. [81] M.P. Heller, A. Serantes, M. Spaliński, V. Svensson and B. Withers, Hydrodynamic Gradient Expansion Diverges beyond Bjorken Flow, Phys. Rev. Lett. 128 (2022) 122302 [arXiv:2110.07621] [INSPIRE]. [82] H.-S. Jeong, K.-Y. Kim and Y.-W. Sun, The breakdown of magneto-hydrodynamics near AdS2 ﬁxed point and energy diﬀusion bound, JHEP 02 (2022) 006 [arXiv:2105.03882] [INSPIRE]. [83] K.-B. Huh, H.-S. Jeong, K.-Y. Kim and Y.-W. Sun, Upper bound of the charge diﬀusion constant in holography, arXiv:2111.07515 [INSPIRE]. 4] Y. Liu and X.-M. Wu, Breakdown of hydrodynamics from holographic pole collision, JHEP 01 (2022) 155 [arXiv:2111.07770] [INSPIRE]. [85] C. Cartwright, M.G. Amano, M. Kaminski, J. Noronha and E. Speranza, Convergence of hydrodynamics in rapidly spinning strongly coupled plasma, arXiv:2112.10781 [INSPIRE]. [86] I. Amado, C. Hoyos-Badajoz, K. Landsteiner and S. Montero, Hydrodynamics and beyond in the strongly coupled N = 4 plasma, JHEP 07 (2008) 133 [arXiv:0805.2570] [INSPIRE]. [87] Y. Akamatsu, A. Mazeliauskas and D. Teaney, A kinetic regime of hydrodynamic ﬂuctuations and long time tails for a Bjorken expansion, Phys. Rev. C 95 (2017) 014909 [arXiv:1606.07742] [INSPIRE]. [88] S. Caron-Huot and O. Saremi, Hydrodynamic Long-Time tails From Anti de Sitter Space, JHEP 11 (2010) 013 [arXiv:0909.4525] [INSPIRE]. [89] P. Glorioso, M. Crossley and H. Liu, A prescription for holographic Schwinger-Keldysh contour in non-equilibrium systems, arXiv:1812.08785 [INSPIRE]. [90] G. Cheng and B. [94] Y. Bu, M. Fujita and S. Lin, Ginzburg-Landau eﬀective action for a ﬂuctuating holographic superconductor, JHEP 09 (2021) 168 [arXiv:2106.00556] [INSPIRE]. [95] Y. Bu, T. Demircik and M. Lublinsky, All order eﬀective action for charge diﬀusion from Schwinger-Keldysh holography, JHEP 05 (2021) 187 [arXiv:2012.08362] [INSPIRE]. References Swingle, Scrambling with conservation laws, JHEP 11 (2021) 174 [arXiv:2103.07624] [INSPIRE]. [91] J. de Boer, M.P. Heller and N. Pinzani-Fokeeva, Holographic Schwinger-Keldysh eﬀective ﬁeld theories, JHEP 05 (2019) 188 [arXiv:1812.06093] [INSPIRE]. [92] K. Skenderis and B.C. van Rees, Real-time gauge/gravity duality: Prescription, Renormalization and Examples, JHEP 05 (2009) 085 [arXiv:0812.2909] [INSPIRE]. – 30 – [93] J.K. Ghosh, R. Loganayagam, S.G. Prabhu, M. Rangamani, A. Sivakumar and V. Vishal, Eﬀective ﬁeld theory of stochastic diﬀusion from gravity, JHEP 05 (2021) 130 [arXiv:2012.03999] [INSPIRE]. [94] Y. Bu, M. Fujita and S. Lin, Ginzburg-Landau eﬀective action for a ﬂuctuating holographic superconductor, JHEP 09 (2021) 168 [arXiv:2106.00556] [INSPIRE]. [95] Y. Bu, T. Demircik and M. Lublinsky, All order eﬀective action for charge diﬀusion from Schwinger-Keldysh holography, JHEP 05 (2021) 187 [arXiv:2012.08362] [INSPIRE]. JHEP04(2022)181 – 31 –
https://openalex.org/W3082097521	https://www.mdpi.com/2075-4701/10/9/1160/pdf?version=1598526917	English	null	Numerical Analysis of Effect of Initial Bubble Size on Captured Bubble Distribution in Steel Continuous Casting Using Euler-Lagrange Approach Considering Bubble Coalescence and Breakup	Metals	2,020	cc-by	9,291	Received: 23 July 2020; Accepted: 25 August 2020; Published: 27 August 2020 Abstract: A mathematic model considering the bubble coalescence and breakup using the Euler-Lagrange approach has been developed to study the eﬀect of the initial bubble size on the distribution of bubbles captured by the solidiﬁcation shell. A hard sphere model was applied for dealing with the bubble collision. Advanced bubble coalescence and breakup models suitable for the continuous casting system and an advanced bubble captured criteria have been identiﬁed established with the help of user-deﬁned functions of FLUENT. The predictions of bubble behavior and captured bubble distribution agree with the water model and plant measurements well respectively. The results show that the number of small bubbles captured by solidiﬁcation shell is much higher than that of large bubbles. What is more, the number of captured bubbles at the sidewalls decreases with the distance from the meniscus. For the case of large gas ﬂow rate (gas ﬂow fraction of 8.2%), the initial size of bubbles has little eﬀect on bubble captured distribution under various casting speeds. When the gas ﬂow rate is small (gas ﬂow fraction of 4.1%), the number density of captured bubbles increases as the initial bubble size increases, and the eﬀect of initial bubbles size on captured bubble number density is ampliﬁed when the casting speed decreases. The average captured bubble diameter is about 0.12–0.14 mm. Additionally, for all cases, the initial bubble size hardly aﬀects the average size of captured bubbles. ywords: continuous casting mold; bubble interaction; initial bubble size; captured bubble distribution Numerical Analysis of Eﬀect of Initial Bubble Size on Captured Bubble Distribution in Steel Continuous Casting Using Euler-Lagrange Approach Considering Bubble Coalescence and Breakup Weidong Yang 1, Zhiguo Luo 1,2,, Nannan Zhao 1 and Zongshu Zou 1,2, Weidong Yang 1, Zhiguo Luo 1,2,, Nannan Zhao 1 and Zongshu Zou 1,2, 1 School of Metallurgy, Northeastern University, Shenyang 110819, China; 1710503@stu.neu.edu.cn (W.Y.); 1901564@stu.neu.edu.cn (N.Z.) 2 State Key Laboratory of Rolling and Automation, Northeastern University, Shenyang 110819, China * Correspondence: luozg@smm.neu.edu.cn (Z.L.); zouzs@mail.neu.edu.cn (Z.Z.); Tel.: +86-13889212571 (Z.L.); +86-13609878273 (Z.Z.) Weidong Yang 1, Zhiguo Luo 1,2,, Nannan Zhao 1 and Zongshu Zou 1,2, 1 School of Metallurgy, Northeastern University, Shenyang 110819, China; 1710503@stu.neu.edu.cn (W.Y.); 1901564@stu.neu.edu.cn (N.Z.) 2 State Key Laboratory of Rolling and Automation, Northeastern University, Shenyang 110819, China * Correspondence: luozg@smm.neu.edu.cn (Z.L.); zouzs@mail.neu.edu.cn (Z.Z.); Tel.: +86-13889212571 (Z.L.); +86-13609878273 (Z.Z.) metals metals www.mdpi.com/journal/metals 1. Introduction The bubbles captured by the solidifying steel shell during the continuous casting process are the main cause of defects of blisters and slivers in the slab. Argon gas is usually injected from the submerged entry nozzle (SEN) to withstand nozzle clogging [1,2]. The transport of argon bubbles has a great eﬀect on the product quality. The bubbles escaping at the slag layer, while not casting the slag entrapment, are helpful for removing the inclusions by attaching them to the slag, which is good for the slab quality. However, the bubbles captured near the meniscus cause surface defects, and those captured deep in the mold result internal defects, which is harmful for the slab quality [3]. Argon gas is injected through the porous refractory at the upper part of the SEN wall. The initial size of argon bubble is aﬀected by the refractory properties, gas ﬂow rate and ﬂuid velocity [4]. After entering the mold, both the evolution and redistribution of bubbles have great impact on the ﬂuid ﬂow and product quality. Due to the high temperature and invisibility, it is diﬃcult to study the Metals 2020, 10, 1160; doi:10.3390/met10091160 www.mdpi.com/journal/metals 2 of 15 Metals 2020, 10, 1160 bubble motion in the actual continuous casting process. Many water model experiments have been conducted to investigate the bubble behaviors in the mold [5–9]. Mathematical simulation is another choice, which was extensively adopted [10–14]. However, there are still challenges in the numerical simulation due to the complexity and lack of reliable theory of the bubble behaviors. Early mathematical simulations of bubble motion in the mold ignored the bubble coalescence and breakup, which results in the diﬀerence between the simulation results and reality. With the development of population balance model (PBM), much research has considered the bubble interaction using the Euler-Euler method [7,15–17]. This method needs lower computational demand, but can not get the information of individual information [18]. There is still little research considering the bubble interaction in the mold with a Euler-Lagrange method [19–21]. Recent years, some papers simulate the bubble transport in the mold considering the bubble interaction in the mold using the Euler-Lagrange method [22,23]. However, the coalescence and breakup models have too many assumptions. Most research gets the well known conclusion that large bubbles tend to escape at the top surface, while small bubbles tend to arrive at the side wall and depth of the mold. 1. Introduction Next, the small bubbles approaching the solidiﬁcation front may be captured or pushed back to the ﬂow [24]. To simulate the bubble capture by the solidifying shell, a reasonable capture criterion is needed. Recently, an advanced captured criterion based on the local force balance between the bubble and the solidiﬁcation front was proposed [25,26]. For analyzing the information of entrapped bubbles, such as size, location and number density, the Euler-Lagrange method is necessary. Many papers have studied the motion of particles or bubbles transport in the mold and the capture by a solidiﬁcation shell [20,21,24,26–28]. However, in those research, the change of bubbles size due to the bubble collision and breakup in the molten steel ﬂow was neglected, so it can be only analyzed qualitatively the removal rate of each size bubble with a ﬁxed particle number. However, the number of each bubble size will change after coalescence and breakup and is aﬀected with diﬀerent operation conditions. For example, various casting speeds (usually ranging from 1 to 1.7 m/min) cause various dissipation rates in the system, which in turn aﬀects the bubble breakup, and diﬀerent gas ﬂow rates (usually gas ﬂow fraction of 3–10%) cause diﬀerent bubble sizes and a bubble number aﬀecting the bubble coalescence and breakup in the continuous casting process. So, for the quantitative analysis of bubble entrapment in the actual continuous casting process, the bubble coalescence and breakup must be considered. What is more, the initial bubble size is an important parameter to determine the bubble secondary distribution in the mold and aﬀects the bubble captured by the solidiﬁcation shell. Lee et al. [4] studied the eﬀect of operation conditions on initial bubble formation by the water model, and the results showed that the initial bubble size ranges from 1 to 2.5 mm. Some papers investigated the eﬀect of initial bubble size on the bubble transport by mathematical simulation. However, the bubble coalescence and breakup were ignored in those papers, and it was only qualitative analyzed the particle motion and the remove fraction of each ﬁxed size [26–28]. Few researchers have investigated the eﬀect of initial bubble size on the bubble captured by the solidiﬁcation shell considering bubble coalescence and breakup. The purpose of the work is to establish a mathematic model considering bubble coalescence and breakup using the Euler-Lagrange approach to analyze the bubble motion in the mold and bubble captured by the solidiﬁcation shell. 1. Introduction The location, size and number density of entrapment bubbles were considered in the present paper. In addition, the eﬀect of the initial bubble size under diﬀerent operation conditions on captured bubble distribution was studied. 2.1. Governing Equations Governing Equations List of Symbols Governing equations for continuous phase Continuity: ∂ ∂t(αlρl) + ∇· (αlρlul) = 0 Momentum: ∂ ∂t(αlρlul) + ul · ∇(αlρlul) = −∇p + ∇· [αl(µl + µt)]∇ul + FI αl—volume fraction of liquid phase ρl, ul, µl—density, average velocity, viscosity of liquid phase µt—turbulent viscosity FI—interfacial forces between two phases Governing equations for bubble mb dub dt = FDra + FPre + FBuo + FVir + FGra + FLif Drag force FDra = CDra ρl\|u−ub\|(u−ub) 2 πd2 b 4 Pressure gradient force FPre = 1 6πd3 bρl du dt Buoyancy force Fbuo = −1 6πd3 bρl · g Virtual mass force Fvir = 1 6πd3 bCVirρl d dt(u −ub) Gravity force FGra = 1 6πd3 bρb · g Lift force FLif = −1 6πd3 bCLifρl(ub −u)(∇× u) ub, mb, ρb—velocity, mass, density of bubble CDra—drag coeﬃcient CVir—virtual mass coeﬃcient (0.5) Pa Table 1. Governing equations for the model. ub, mb, ρb—velocity, mass, density of bubble CDra—drag coeﬃcient CVir—virtual mass coeﬃcient (0.5) Pa Governing equations for turbulence model (standard k-ε model) Governing equations for turbulence model (standard k ε model) αlρl ∂k ∂t + u·∇k = −∇ αl µt σk ∇k + αlGk −αlρlε αlρl ∂ε ∂t + u·∇ε = −∇ αl µt σε ∇ε + αlCl ε k Gk −αlC2ρl ε2 k Gk = µt ∂ui,j ∂xj + ∂ui,j ∂xi ∂ui,j ∂xj Cµ(0.99), σk(1.0), C1(1.44), C2(1.92)—standard model constants .2. Bubble Coalescence Model 2.1. Governing Equations A Euler-Lagrange model is necessary to get the information of bubbles captured by the solidiﬁcation shell. The model of bubble motion and size history, written in C language, is treated in a Lagrangian way, according to the Newton’s Second Law. The eﬀect of the discrete phase volume on the continuous 3 of 15 Metals 2020, 10, 1160 phase is considered in the model, which is neglected in the DPM (Discrete Phase Model). The collision of bubbles is modeled by means of the hard sphere model. The liquid phase is solved with the commercial package FLUENT 18.1 (ANSYS Inc., Canonsburg, PA, USA, www.ansys.com). The coupling between two phases is realized by computing the liquid phase volume of each grid and adding interfacial forces as a source term, with the help of extensive user-deﬁned functions of FLUENT. The governing equations of two-phase are summarized in Table 1. Table 1. Governing equations for the model. Governing Equations List of Symbols Governing equations for continuous phase Continuity: ∂ ∂t(αlρl) + ∇· (αlρlul) = 0 Momentum: ∂ ∂t(αlρlul) + ul · ∇(αlρlul) = −∇p + ∇· [αl(µl + µt)]∇ul + FI αl—volume fraction of liquid phase ρl, ul, µl—density, average velocity, viscosity of liquid phase µt—turbulent viscosity FI—interfacial forces between two phases Governing equations for bubble mb dub dt = FDra + FPre + FBuo + FVir + FGra + FLif Drag force FDra = CDra ρl\|u−ub\|(u−ub) 2 πd2 b 4 Pressure gradient force FPre = 1 6πd3 bρl du dt Buoyancy force Fbuo = −1 6πd3 bρl · g Virtual mass force Fvir = 1 6πd3 bCVirρl d dt(u −ub) Gravity force FGra = 1 6πd3 bρb · g Lift force FLif = −1 6πd3 bCLifρl(ub −u)(∇× u) ub, mb, ρb—velocity, mass, density of bubble CDra—drag coeﬃcient CVir—virtual mass coeﬃcient (0.5) Pa Table 1. Governing equations for the model. 2.2. Bubble Coalescence Model In the present work, the bubble collision was resolved by the hard sphere model. By integrating the works of Yang et al. [29,30], a bubble coalescence model considering the bubbles size and oﬀ-center degree was used in the present work to determine if the bubble collision will result in coalescence or not. With the equivalent diameter of two collision bubbles smaller than 2.3 mm, bubbles coalesce when We 1 −B2 < 0.16 + B/24 We = rev2 rρl σ (1) (1) where We is the relative Weber number; re = 2r1r2/(r1 + r2) is the equivalent radius of two interacting bubbles and vr is the relative velocity of bubbles. B = b r1 + r2 (2) (2) where B is the dimensionless number characterizing the oﬀ-center degree. Where r1 and r2 are the radii of the two bubbles and b is the distance from the center of the one bubble to the relative velocity vector originating from the center of the other bubble at contact. where B is the dimensionless number characterizing the oﬀ-center degree. Where r1 and r2 are the radii of the two bubbles and b is the distance from the center of the one bubble to the relative velocity vector originating from the center of the other bubble at contact. With the equivalent diameter of two collision bubbles larger than 2.3 mm, two collision bubbles result in coalescence if the relative approach velocity exceeds 0.11 m/s [31,32]. Metals 2020, 10, 1160 4 of 15 2.3. Bubble Breakup Model 2.3. Bubble Breakup Model The bubble breakup model used in the present work is the model proposed by Yang et al. [30]. In the turbulent two-phase ﬂow, a bubble breaks up into two daughter bubbles if the uneven pressure acting on the bubble surface larger than the surface force [33]. The breakup of the bubble occurs if the bubbles exceed a maximum stable size, dmax. dmax = We3/5 crit σ3/5 ρ3/5 l ε−2/5 (3) (3) where Wecrit is the critical Weber number for bubble break-up with a value of 0.53; ε is the turbulent kinetic energy dissipation rate and σ is the gas–liquid surface tension. Besides the bubble breakup criteria, the volume fraction of the daughter bubble is the other essential parameter. According to the work of Yang et al. [30], the volume fraction of daughter bubble suit for the tanh function, following the U-shape of daughter size distribution, in the steel continuous casting system. fbv = 1 2[1 + tanh(14.4x −7.2)] (4) (4) where fbv is the volume fraction of daughter bubble and x is a random variable from 0 to 1. 2.4. Bubble Capture Model and Predict Sample Observations The bubble capture model as proposed by Yuan and Thomas [25,26,28] was used in the present work. This model considers the eﬀect of PDAS (primary dendrite arm spacing), bubble size and local ﬂow ﬁeld. If the diameters of bubbles are smaller than the PDAS, the bubbles will be captured when touching the solidiﬁcation shell. When the bubbles are larger than PDAS, the force balance is considered to determine if bubbles are captured by the solidiﬁcation shell when the local ﬂow velocity changes. Besides the forces introduced in the previous section, three additional forces, including the lubrication force Flub, surface tension gradient force FGrad and Van der Waals force FIV, were exerted on the captured result. 2 2 Flub = 6πusol R2 b ht rd rd + Rb !2 (5) (5) where Rb is the bubble radius; usol is the solidiﬁcation front moving velocity and the details about it can be found elsewhere; ht is the thickness between the bubble and dendrite tip and rd is the radius of dendrite tip. FGrad = −mβπRb ξ2  ξ2 −R2 b β ln "(ξ + Rb)[α(ξ −Rb) + β] (ξ −Rb)[α(ξ + Rb) + β] # + 2Rb α −β α2 ln "α(ξ + Rb) + β α(ξ −Rb) + β #) (6) (6) where α = 1 + nC0, β = (C∗−C0)nrd, ξ = Rb+ + rd + ho and m, n, C0 and C∗are the model parameters, which are given in the previous work. FIV = 2π(σsb −σsl −σbl) rdRb rd + Rb a2 o h2 t (7) (7) ao is the steel atomic diameter, σsb, σsl and σbl represent surface tensions for solid–bubble, solid–liquid and bubble–liquid respectively. For comparing with plant measurement, it is essential to determine which layer of product steel the bubbles appear on. Whether the captured bubble q with radius Rq at the distance Dq beneath the casting product surface can be found in the examined layer of the sample w at the distance Dw beneath the surface of test sample is shown in Figure 1. The solidiﬁcation shell thickness is determined by s = 3 √ t [28]. The solidiﬁcation time t is calculated as t = L/vc = 40L, where L is the length beneath 5 of 15 kness is Metals 2020, 10, 1160 g p beneath the sur the meniscus and vc is the casting speed. 2.4. Bubble Capture Model and Predict Sample Observations So, Dq = S\| Lq= 3 p 40Lq, where Lq is the distance between the entrapped bubble q and the meniscus. c length beneath the meniscus and vc is the casting speed. So, =S =3 40 q q q L D L , where q L is the distance between the entrapped bubble q and the meniscus the meniscus and vc is the casting speed. So, Dq = S\| Lq= 3 p 40Lq, where Lq is the distance between the entrapped bubble q and the meniscus. length beneath the meniscus and vc is the casting speed. So, =S =3 40 q q q L D L , where q L is the distance between the entrapped bubble q and the meniscus. Figure 1. Schematic of the model features for the bubble capture description. Figure 1. Schematic of the model features for the bubble capture description. Figure 1 Schematic of the model features for the bubble capture description Figure 1. Schematic of the model features for the bubble capture description. g 2 5 N i l I l i 2.5. Numerical Implementation g 2 5 N i l I l i 2.5. Numerical Implementation 2.5. Numerical Implementation To quantitatively validate the mathematic model, the benchmark case in the paper of Jin et al. [28] was built. The computational domain and boundary conditions are shown in Figure 2. Table 2 presents the geometry parameters and operation conditions. The liquid phase flow field was solved with the commercial package FLUENT. The model of bubble was written by C. The couple between two phases was realized by computing the liquid phase volume of each grid and adding interfacial forces as a source term, with the help of extensive user-defined functions of FLUENT. The time step was set as 0.001 s, and the total simulation time was 60 s. The bubbles were assumed as spheres with th i iti l di t i f 1 t 2 t thi ti diti b d L t l [4] To quantitatively validate the mathematic model, the benchmark case in the paper of Jin et al. [28] was built. The computational domain and boundary conditions are shown in Figure 2. Table 2 presents the geometry parameters and operation conditions. The liquid phase ﬂow ﬁeld was solved with the commercial package FLUENT. The model of bubble was written by C. The couple between two phases was realized by computing the liquid phase volume of each grid and adding interfacial forces as a source term, with the help of extensive user-deﬁned functions of FLUENT. The time step was set as 0.001 s, and the total simulation time was 60 s. The bubbles were assumed as spheres with the initial diameter ranging from 1 to 2 mm at this operation condition, based on Lee et al. [4]. Metals 2020, 10, x FOR PEER REVIEW 6 of 15 Figure 2. Schematic diagram of the mold with boundary conditions. Figure 2. Schematic diagram of the mold with boundary conditions. Figure 2. Schematic diagram of the mold with boundary conditions. Figure 2. Schematic diagram of the mold with boundary conditions. 6 of 15 Metals 2020, 10, 1160 Table 2. Geometry parameters and operation conditions. Table 2. Geometry parameters and operation conditions. g 2 5 N i l I l i 2.5. Numerical Implementation Parameter Value SEN port width × height 65 × 83 mm2 Nozzle port angle 15◦down Submergence depth of SEN 160 mm Slab width × thickness 1300 × 230 mm2 Slab length 2000 mm Casting speed 1.5 m/min Argon volume fraction 8.2 vol pct Density of liquid 7000 kg/mm3 Viscosity of liquid 0.0063 kg/(m·s) Density of gas 0.5 kg/mm3 Viscosity of gas 0.0000212 kg/(m·s) Initial bubble diameter 1 (30%), 1.5 (45%), 2 (25%) g p 3 2 C i f B bbl C t d i S l 3.2. Comparison of Bubbles Captured in Samples 3.2. Comparison of Bubbles Captured in Samples For validating the computation result, the measurement of samples with six layers examined on each by Jin et al. [4], shown in Figure 4a, were compared with the computation results. For the plant measurements, the part above the examining layer was milled away, and then an optical microscope was used to record the bubble diameter and number of the examining layer [4]. Figure 4b,c shows comparison of bubble number density and mean diameter between the simulation and measurement at the wide face. It can be seen that the computation simulation predicted 0.1–0.2 bubble per cm2 on the first four layers at the wide face, which matched the measurements of the sample well. On the fifth and sixth layers, there were less than 0.05 bubble per cm2. The bubbles in these two layers were entrapped at the downward recirculation region, where only a few small bubbles could reach. Figure 4c shows the average bubble diameter at the wide face. The simulation result shows that the average bubble diameter slightly decreased and the distance beneath the surface was 0.1–0.15 mm, which also matched the measurement. Figure 4d,e shows the bubble number density and the bubble diameter of simulation and measurement at the narrow face. The size and number of bubbles captured at the narrow was similar with the wide face, and the For validating the computation result, the measurement of samples with six layers examined on each by Jin et al. [4], shown in Figure 4a, were compared with the computation results. For the plant measurements, the part above the examining layer was milled away, and then an optical microscope was used to record the bubble diameter and number of the examining layer [4]. Figure 4b,c shows comparison of bubble number density and mean diameter between the simulation and measurement at the wide face. It can be seen that the computation simulation predicted 0.1–0.2 bubble per cm2 on the ﬁrst four layers at the wide face, which matched the measurements of the sample well. On the ﬁfth and sixth layers, there were less than 0.05 bubble per cm2. The bubbles in these two layers were entrapped at the downward recirculation region, where only a few small bubbles could reach. Figure 4c shows the average bubble diameter at the wide face. 3.1. Bubble Distribution The bubble distribution in the mold from the computation model was compared with a simple water model results in Figure 3. From Figure 3, it can be noticed that the entire bubble distribution from the computation model agreed qualitatively well with the water model. Most of the bubbles ﬂoated up near the SEN due to the buoyancy, while smaller bubbles got to the further location following with the liquid ﬂow. Due to the introduction of bubble interaction, the bubble size distribution was controlled by the bubble coalescence and breakup. In the submerged entry nozzle, the bubble was small at the initial size and the number of bubble was large, so a lot of bubbles coalesced to larger ones, as shown in Figure 3. At the location near the SEN ports, due to the high turbulent stress, and the large bubble size causing by amounts of coalescence of bubbles in the SEN, a lot of bubbles broke up, as shown in Figure 3. It can be noticed that the bubble distribution and bubble interaction agreed well in the mold with the experiments results. However, there were more bubbles at the top surface in the water model than the simulation results. The bubbles that got to the gas–liquid interface would stay for a while in the water model resulting in the aggregation of bubbles, but the bubbles that get to the interface will be removed immediately by the program setting in the mathematic simulation. 7 of 15 7 of 15 Metals 2020, 10, 1160 Metals 2020, 10, x FO Figure 3. Comparison of bubble behavior between the simulation and water model. Figure 3. Comparison of bubble behavior between the simulation and water model. Figure 3 Comparison of bubble behavior between the simulation and water model Figure 3. Comparison of bubble behavior between the simulation and water model. g p 3 2 C i f B bbl C t d i S l 3.2. Comparison of Bubbles Captured in Samples 4. Results and Discussion into the narrow face, and th the top surface after out of t 4.1. Two-Phase Flow in the Mold Figure 6 shows the whole afte 25 I Fi u e 6 the bubb Metals 2020, 10, x FOR PEER REVIEW Figure 5 shows the ﬂuid velocity counter and vector in the mold at the center cross-sectional plane. It can be seen that two jet ﬂows were in the upper recirculation region. One jet ﬂow impacted into the narrow face, and then it branched into an upper and a down ﬂows. The other jet ﬂow got to the top surface after out of the port, which was driven by the bubble buoyancy. after 25 s. In Figure 6, the bubble size is presented with color for a better visualization of the small bubbles. It can be noticed that the large bubbles (green color) floated up to the top surface close to the SEN. Additionally, only the bubbles (blue color) smaller than 1 mm could get to the narrow face and the deep of the mold, with the reason that the effect of drag force on the small bubbles was greater than that of buoyancy. Most of the bubbles captured by the solidification shell were these 4.1. Two-Phase Flow in the Mold Figure 5 shows the fluid velocity counter and vector in the mold at the center cross-sectional plane. It can be seen that two jet flows were in the upper recirculation region. One jet flow impacted into the narrow face, and then it branched into an upper and a down flows. The other jet flow got to Figure 5. Fluid velocity and streamlines in the mold. Figure 5. Fluid velocity and streamlines in the mold. of the port, which was driven by the bubble buoyanc e whole instantaneous bubble distribution in the co he bubble size is presented with color for a better vis ed that the large bubbles (green color) floated up to t only the bubbles (blue color) smaller than 1 mm could ld, with the reason that the effect of drag force on oyancy. Most of the bubbles captured by the solidific Figure 5. Fluid velocity and streamlines in the mold. Figure 5. Fluid velocity and streamlines in the mold. Figure 6 shows the whole instantaneous bubble distribution in the continuous casting mold after 25 s. In Figure 6, the bubble size is presented with color for a better visualization of the small bubbles. g p 3 2 C i f B bbl C t d i S l 3.2. Comparison of Bubbles Captured in Samples The simulation result shows that the average bubble diameter slightly decreased and the distance beneath the surface was 0.1–0.15 mm, which also matched the measurement. Figure 4d,e shows the bubble number density and the bubble diameter of simulation and measurement at the narrow face. The size and number of bubbles captured at the narrow was similar with the wide face, and the simulation results matched the measurements. 8 of 15 8 of 15 8 of 15 8 of 15 Metals 2020, 10, 1160 Metals 2020 10 x FOR Figure 4. (a) Locations of samples and examined layers. Comparison between simulation and plant examination: (b) bubble number density on a wide face; (c) average bubble diameter on a wide face; (d) bubble number density on a narrow face and (e) average bubble diameter on a narrow face. 4 Results and Discussion Figure 4. (a) Locations of samples and examined layers. Comparison between simulation and plant examination: (b) bubble number density on a wide face; (c) average bubble diameter on a wide face; (d) bubble number density on a narrow face and (e) average bubble diameter on a narrow face. Figure 4. (a) Locations of samples and examined layers. Comparison between simulation and plant examination: (b) bubble number density on a wide face; (c) average bubble diameter on a wide face; (d) bubble number density on a narrow face and (e) average bubble diameter on a narrow face. Figure 4. (a) Locations of samples and examined layers. Comparison between simulation and plant examination: (b) bubble number density on a wide face; (c) average bubble diameter on a wide face; (d) bubble number density on a narrow face and (e) average bubble diameter on a narrow face. 9 of 15 onal Metals 2020, 10, 1160 Figure 5 sho 4.2. Distribution of Bubble Captured Location The bubble captured locations at the narrow and wide faces are discussed in this section. Diﬀerent from the previous papers with a constant bubble size, the number of small bubbles captured by the solidiﬁcation shell was determined by the actual ﬂow ﬁeld and the bubble interaction. The distribution of the bubble entrapped location under 1.5 m/min casting speed and a bubble fraction of 8.2% is shown in Figure 7. For a better analysis of the bubble entrapped location, the bubble was divided into three levels: small bubble (diameter smaller than 0.2 mm), medium-size bubble (diameter ranges from 0.2 to 1 mm) and large bubble (diameter larger than 1 mm). From Figure 7, it can be seen that many small bubbles were captured by the solidifying shell, and the number of bubbles decreased with the distance from the top surface. In the narrow face, a number peak of captured bubbles appeared at the region about 0.5 mm below the top surface, where the mainstream impacts. In the deep part of the wide face, more bubbles were captured near the narrow face. From Figure 7, it can be noticed that the number of entrapment bubbles with medium-size bubbles and large size was far less than the small bubbles. There was a small amount of medium-size bubbles captured in the deep, but almost no large bubbles were captured in the deep of the mold. Additionally, the most of the large bubbles were entrapped close to the top surface. Metals 2020, 10, x FOR PEER REVIEW 10 of 15 The bubble captured locations at the narrow and wide faces are discussed in this section. Different from the previous papers with a constant bubble size, the number of small bubbles captured by the solidification shell was determined by the actual flow field and the bubble interaction. The distribution of the bubble entrapped location under 1.5 m/min casting speed and a bubble fraction of 8.2% is shown in Figure 7. For a better analysis of the bubble entrapped location, the bubble was divided into three levels: small bubble (diameter smaller than 0.2 mm), medium-size bubble (diameter ranges from 0.2 to 1 mm) and large bubble (diameter larger than 1 mm). From Figure 7, it can be seen that many small bubbles were captured by the solidifying shell, and the number of bubbles decreased with the distance from the top surface. 4. Results and Discussion into the narrow face, and th the top surface after out of t It can be noticed that the large bubbles (green color) ﬂoated up to the top surface close to the SEN. Additionally, only the bubbles (blue color) smaller than 1 mm could get to the narrow face and the deep of the mold, with the reason that the eﬀect of drag force on the small bubbles was greater than that of buoyancy. Most of the bubbles captured by the solidiﬁcation shell were these small bubbles. Figure 5. Fluid velocity and streamlines in the mold. Figure 6. The whole bubble distribution at the time of 20 s in the mold. of Bubble Captured Location Figure 6. The whole bubble distribution at the time of 20 s in the mold. Figure 6. The whole bubble distribution at the time of 20 s in the mold. Figure 6. The whole bubble distribution at the time of 20 s in the mold. Figure 6. The whole bubble distribution at the time of 20 s in the mold. Metals 2020, 10, 1160 10 of 15 10 of 15 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubble 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubbles 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubbles The initial size of bubbles may affect the bubble secondary size distribution after the interaction of bubbles and the complex effect by the flow field, which in turn affects the capture of bubbles by the solidification shell. The effect of the initial size of bubbles on the distribution of captured bubbles may vary with different casting speeds and gas flow rates. For analyzing the distribution of captured bubbles, number density and average size of captured bubbles are discussed in this section. The number density of captured bubbles was calculated as the ratio of the number of captured bubbles to the area during the casting time. The area during the casting time was calculated as: Sk = vc × tc × Lk, where Sk is the area during the casting time, vc is the casting speed, k is the wide or narrow face, tc is the casting time and Lk is the length of wide face or narrow face. From the work of Lee et al. [4], few initial bubbles generated from the porous refractory are smaller than 1 mm. So, to investigate the effect of initial bubble size on the captured bubble distribution, three different initial bubble sizes of 1 mm, 1.5 mm and 2 mm were compared in this section. p Table 3 shows the distribution of captured bubbles at the wide and narrow faces with three diﬀerent initial bubble sizes under the operation of the casting speed of 1.5 m/min and gas ﬂow rate of 8.2% gas volume fraction. The number density of captured bubbles on the wide face and narrow face was about 3.6 and 5.2 per cm2, which clearly indicates that there were more bubbles per unit area on the narrow face. Since the main ﬂuid stream rushed to the narrow face, more bubbles were carried to the narrow face. The average size of captured bubbles was about 0.130 and 0.126 mm respectively. Due to the greater ﬂuid velocity towards the narrow face, a larger drag force acted on the bubbles facing the direction to the narrow face, resulting in larger bubbles brought to the narrow face. With the diﬀerent initial bubble sizes, there were no obvious diﬀerences in the number density and size of captured bubbles. 4.2. Distribution of Bubble Captured Location In the narrow face, a number peak of captured bubbles appeared at the region about 0.5 mm below the top surface, where the mainstream impacts. In the deep part of the wide face, more bubbles were captured near the narrow face. From Figure 7, it can be noticed that the number of entrapment bubbles with medium-size bubbles and large size was far less than the small bubbles. There was a small amount of medium-size bubbles captured in the deep, but almost no large bubbles were captured in the deep of the mold. Additionally, the most of the large bubbles were entrapped close to the top surface. Figure 7. Locations of bubbles captured by the solidification shell. (a) Small bubble; (b) medium-size bubble and (c) large bubble. Figure 7. Locations of bubbles captured by the solidiﬁcation shell. (a) Small bubble; (b) medium-size bubble and (c) large bubble. Additionally, the most of the large bubbles were entrapped close to the top surface. A i io a y, e o o e a ge pp p Figure 7. Locations of bubbles captured by the solidification shell. (a) Small bubble; (b) medium-size bubble and (c) large bubble. Figure 7. Locations of bubbles captured by the solidiﬁcation shell. (a) Small bubble; (b) medium-size bubble and (c) large bubble. Metals 2020, 10, 1160 11 of 15 11 of 15 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubble Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 2.177 0.1357 3.113 0.1379 1.5 2.185 0.1336 3.221 0.1388 2 2.294 0.1322 3.412 0.1370 Table 5. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.7 m/min and gas ﬂow rate of 8.2% gas volume fraction. Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 6.272 0.1181 8.976 0.1221 1.5 6.277 0.1179 8.968 0.1229 2 6.265 0.1175 8.971 0.1226 Table 5. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.7 m/min and gas ﬂow rate of 8.2% gas volume fraction. Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 6.272 0.1181 8.976 0.1221 1.5 6.277 0.1179 8.968 0.1229 2 6.265 0.1175 8.971 0.1226 Table 5. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.7 m/min and gas ﬂow rate of 8.2% gas volume fraction. Table 6 shows the number density and average size of captured bubbles with diﬀerent initial bubble sizes. For all cases, the gas ﬂow rate was 4.1% argon volume fraction and the casting speed was 1.5 m/min. The results indicate that the bubble number density decreased comparing with the gas ﬂow rate of 8.2% argon volume fraction. For diﬀerent initial bubble sizes, it can be found that the captured bubble number density decreased with the decrease of initial bubble size. The captured bubble number density with the initial bubble size of 2 mm was about 1.21 times of that of 1 mm. For the small gas ﬂow rate (argon volume fraction of 4.1%), the probability of collision between bubbles was lower than that of a large gas volume fraction. 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubble In the operation of casting speed of 1.5 m/min and gas ﬂow rate of 8.2% gas volume fraction, the gas volume was large enough and the ﬂow ﬁeld was suﬃciently turbulent, so the bubbles in the SEN with diﬀerent initial bubble sizes could fully collide. The number and size of large bubbles, which could break up to the small bubbles that could be captured by the solidiﬁed shell, had little diﬀerence under diﬀerent initial bubble sizes. So, there were no obvious diﬀerences at the number density and size of captured bubbles with diﬀerent initial bubble sizes. The number density and size of captured bubbles with the casting speed of 1.7 m/min and 1.3 m/min and gas ﬂow rate of 8.2% gas volume fraction are present in Tables 4 and 5. It can be noticed that more bubbles were captured and the average bubble size decreased with increasing casting speed. It also can be found that when the gas ﬂow rate was 8.2%, the initial bubble size had no obvious eﬀect on the captured bubble distribution at the operation of the casting speed of 1.7 and 1.3 m/min. The reason was same as the casting speed of 1.5 m/min. Table 3. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.5 m/min and gas ﬂow rate of 8.2% gas volume fraction. Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 3.681 0.1271 5.281 0.1314 1.5 3.522 0.1256 5.113 0.1288 2 3.581 0.1261 5.334 0.1305 Table 3. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.5 m/min and gas ﬂow rate of 8.2% gas volume fraction. 12 of 15 Metals 2020, 10, 1160 Table 4. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.3 m/min and gas ﬂow rate of 8.2% gas volume fraction. 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubble Therefore, in the SEN, the bubbles with a large initial size could coalesce into more large bubbles, which could break up into the small bubbles that were captured by the solidiﬁed shell, than small initial size bubbles. So, the captured bubble number density with the large initial bubble size was larger than that with a small initial bubble size. The eﬀect of the initial bubbles size on captured bubble distribution under the condition of casting speed of 1.7 m/min and gas ﬂow rate of 4.1% gas volume fraction is presented in Table 7. It can be found that the captured bubble number density with the initial bubble size of 2 mm was about 1.05 times of that of 1 mm. For the case of a gas ﬂow rate of 4.1% argon volume fraction, the eﬀect of the initial bubble size on the captured bubble number density decreased when the casting speed increased. By increasing the casting speed, the maximum velocity that bubbles can get will increase, and the bubble motion will be more chaotic. Therefore, the probability of collision between bubbles increases with increasing casting speed, and the diﬀerence in the number of large bubbles under diﬀerent initial bubble sizes will be reduced. This is why the eﬀect of the initial bubbles size on captured bubble distribution decreased with the increase of casting speed. From Table 8, for the case of casting speed of 1.3 m/min, it can be found that the captured bubble number density with the initial bubble size of 2 mm was about 1.55 times of that with an initial bubble size of 1 mm. Similar to the previous explanation, for the case of gas ﬂow rate of 4.1% argon volume fraction and casting speed of 1.3 m/min, the gas volume fraction was small, and the bubbles motion was more stable with a lower casting speed. Therefore, the probability of collision between bubbles was lower with a lower casting speed. So, the eﬀect of initial bubble size on the captured bubble number density was higher. From the simulation results, it can be noticed that the initial bubble size had no signiﬁcant eﬀect on the captured bubble size. The reason is that the average size of bubbles that can reach the advancing solidiﬁed shell was mainly determined by the casting speed. 13 of 15 Metals 2020, 10, 1160 Table 6. 4.3. Eﬀect of the Initial Bubble Size on the Distribution of Captured Bubble Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.5 m/min and a gas ﬂow rate of 4.1% gas volume fraction. Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 1.583 0.1267 2.313 0.1313 1.5 1.634 0.1262 2.433 0.1305 2 1.882 0.1268 2.803 0.1311 Table 7. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.7 m/min and a gas ﬂow rate of 4.1% gas volume fraction. Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 0.712 0.1336 1.084 0.1388 1.5 0.883 0.1324 1.343 0.1365 2 1.112 0.1329 1.653 0.1343 Table 8. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.3 m/min and a gas ﬂow rate of 4.1% gas volume fraction. Diameter of Initial Bubble (mm) Number Density of Captured Bubble on the Wide Face (per cm2) Average Size of Captured Bubble on the Wide Face (mm) Number Density of Captured Bubble on the Narrow Face (per cm2) Average Size of Captured Bubble on the Narrow Face (mm) 1 2.663 0.1177 4.265 0.1225 1.5 2.756 0.1173 4.336 0.1233 2 2.831 0.1176 4.453 0.1218 Table 7. Captured bubble distribution with diﬀerent initial bubble sizes at the operation casting speed of 1.7 m/min and a gas ﬂow rate of 4.1% gas volume fraction. Metals 2020, 10, 1160 Metals 2020, 10, 1160 14 of 15 Author Contributions: Z.Z., Z.L. and W.Y. conceived and designed the study; W.Y. and N.Z. did the numerical simulation; all the authors discussed the results; W.Y. wrote the paper; Z.Z. and Z.L. edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: No funding has supported this work. Funding: No funding has supported this work. Acknowledgments: The authors wish to express thanks to Yang You and Weiqiang Liu for the academic communication. Acknowledgments: The authors wish to express thanks to Yang You and Weiqiang Liu for cademic communication. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References . Bai, H.; Thomas, B.G. Turbulent ﬂow of liquid steel and argon bubbles in slide-gate tundish nozzles: P model development and validation. Metall. Mater. Trans. B 2001, 32B, 253–267. [CrossRef] model development and validation. Metall. Mater. Trans. B 2001, 32B, 253–267. [CrossRef] 2. Thomas, B.G.; Huang, X.; Sussman, R.C. Simulation of argon gas ﬂow eﬀects in a continuous slab caster. Metall. Mater. Trans. B 1994, 25, 527–547. [CrossRef] . Thomas, B.G.; Huang, X.; Sussman, R.C. Simulation of argon gas ﬂow eﬀects in a continuous slab ca Metall. Mater. Trans. B 1994, 25, 527–547. [CrossRef] 3. Bai, H.; Thomas, B.G. Eﬀects of clogging argon injection and continuous casting-conditions on ﬂow and air aspiration in submerged-entry nozzles. Metall. Mater. Trans. B 2001, 32B, 707–722. [CrossRef] 3. Bai, H.; Thomas, B.G. Eﬀects of clogging argon injection and continuous casting-conditions on ﬂow and air aspiration in submerged-entry nozzles. Metall. Mater. Trans. B 2001, 32B, 707–722. [CrossRef] 4. Lee, G.G.; Thomas, B.G.; Kim, S.H. Eﬀect of refractory properties on initial bubble formation in continuous-casting nozzles. Met. Mater. Int. 2010, 16, 501–506. [CrossRef] 4. Lee, G.G.; Thomas, B.G.; Kim, S.H. Eﬀect of refractory properties on initial bubble formation in continuous-casting nozzles. Met. Mater. Int. 2010, 16, 501–506. [CrossRef] g 5. Sing, V.; Dash, S.K.; Sunitha, J.S.; Ajmani, S.K.; Das, A.K. Experimental simulation and mathematical modeling of air bubble movement in slab caster mold. ISIJ Int. 2006, 46, 210–218. [CrossRef] g 5. Sing, V.; Dash, S.K.; Sunitha, J.S.; Ajmani, S.K.; Das, A.K. Experimental simulation and mathematical modeling of air bubble movement in slab caster mold. ISIJ Int. 2006, 46, 210–218. [CrossRef] 6. Timmel, K.; Shevchenko, N.; Röder, M.; Anderhuber, M.; Gardin, P.; Eckert, S.; Gerbeth, G. Visualization of liquid metal two-phase ﬂows in a physical model of the continuous casting process of steel. Metall. Mater. Trans. B 2014, 46, 700–710. [CrossRef] 6. Timmel, K.; Shevchenko, N.; Röder, M.; Anderhuber, M.; Gardin, P.; Eckert, S.; Gerbeth, G. Visualization of liquid metal two-phase ﬂows in a physical model of the continuous casting process of steel. Metall. Mater. Trans. B 2014, 46, 700–710. [CrossRef] 7. Liu, Z.; Li, L.; Qi, F.; Li, B.; Jiang, M.; Tsukihashi, F. Population balance modeling of polydispersed bubbly Flow in continuous-casting using multiple-size-group approach. Metall. Mater. Trans. B 2014, 46, 406–420. [CrossRef] 8. Zhang, L.; Yang, S.; Cai, K.; Li, J.; Wan, X.; Thomas, B.G. References Investigation of ﬂuid ﬂow and steel cleanliness in the continuous casting strand. Metall. Mater. Trans. B 2007, 38, 63–83. [CrossRef] 9. Iguchi, M.; Kasai, N. Water model study of horizontal molten steel-air two-phase jet in a continuous casting mold. Metall. Mater. Trans. B 2000, 31, 453–460. [CrossRef] 10. Zhang, W.; Luo, S.; Chen, Y.; Wang, W.; Zhu, M. Numerical simulation of ﬂuid ﬂow, heat transfer, species transfer, and solidiﬁcation in billet continuous casting mold with M-EMS. Metals 2019, 9, 66. [CrossRef] 11. Ni, P.; Ersson, M.; Jonsson, L.; Zhang, T.A.; Jönsson, P. Eﬀect of immersion depth of a swirling ﬂow tundish SEN on multiphase ﬂow and heat transfer in mold. Metals 2018, 8, 910. [CrossRef] 12. Yang, H.; Vanka, S.P.; Thomas, B.G. Modeling argon gas behavior in continuous casting of steel. JOM 2018, 70, 2148–2156. [CrossRef] 13. Lai, Q.; Luo, Z.; Hou, Q.; Zhang, T.; Wang, X.; Zou, Z. Numerical study of inclusion removal in steel continuous casting mold considering interactions between bubbles and inclusions. ISIJ Int. 2018, 58, 2062–2070. [CrossRef] 14. Kubo, N.; Ishii, T.; Kubota, J.; Aramaki, N. Two-phase ﬂow numerical simulation of molten steel and argon gas in a continuous casting cold. ISIJ Int. 2002, 42, 1251–1258. [CrossRef] 15. Liu, Z.Q.; Qi, F.S.; Li, B.K.; Cheung, S.C.P. Modeling of bubble behaviors and size distribution in a slab continuous casting mold. Int. J. Multiph. Flow 2016, 79, 190–201. [CrossRef] 16. Liu, Z.; Qi, F.; Li, B.; Jiang, M. Multiple size group modeling of polydispersed bubbly ﬂow in the mold: An analysis of turbulence and interfacial force models. Metall. Mater. Trans. B 2014, 46, 933–952. [CrossRef] 16. Liu, Z.; Qi, F.; Li, B.; Jiang, M. Multiple size group modeling of polydispersed bubbly ﬂow in the mold: An analysis of turbulence and interfacial force models. Metall. Mater. Trans. B 2014, 46, 933–952. [CrossRef] 17. Liu, Z.; Li, B. Large-eddy simulation of transient horizontal gas-liquid ﬂow in continuous casting using dynamic subgrid-scale model. Metall. Mater. Trans. B 2017, 48, 1833–1849. [CrossRef] 18 Deen N G Solberg T Hjertager B H Large edd simulation of the gas liquid ﬂow in a square cross sectioned 16. Liu, Z.; Qi, F.; Li, B.; Jiang, M. Multiple size group modeling of polydispersed bubbly ﬂow in the mold: An analysis of turbulence and interfacial force models. Metall. Mater. Trans. B 2014, 46, 933–952. [CrossRef] 17. Liu, Z.; Li, B. 5. Conclusions The Euler-Lagrange model was used to simulate the bubble transport in the continuous casting mold and bubble captured by the solidiﬁcation shell. The bubble coalescence and breakup were considered in the model, and an advanced bubble captured model was established. The location, number density and size of captured bubbles were analyzed in the present work. Besides, the eﬀect of the initial bubble size on the captured bubble distribution under diﬀerent operation conditions was discussed. The conclusions are the following: 1. Most of large bubbles ﬂoated up to the top surface close to the SEN, and only some small bubbles could get to the narrow face and the deep of the mold. 2. The number of small bubbles (diameter smaller than 0.2 mm) captured by the solidiﬁcation shell was far more than medium-size (diameter ranges from 0.2 to 1 mm) and large bubble (diameter larger than 1 mm). The number of small and medium-size bubbles captured by the solidiﬁcation shell decreased with the distance from the top surface. The large bubbles were captured only near the top surface. 3. For the case of a large gas ﬂow rate, the initial bubble size had no obvious eﬀect on the number density and average size of captured bubbles under diﬀerent casting speeds. When the gas ﬂow rate was small, the number density of captured bubbles by the solidiﬁcation shell decreased with the decrease of the initial bubble size, and the eﬀect of initial bubble size on captured bubble number density increased when the casting speed decreased. For all cases, the initial bubble size hardly aﬀected the average size of captured bubbles. References Large-eddy simulation of transient horizontal gas-liquid ﬂow in continuous casting using 17. Liu, Z.; Li, B. Large-eddy simulation of transient horizontal gas-liquid ﬂow in continuous casting using dynamic subgrid-scale model. Metall. Mater. Trans. B 2017, 48, 1833–1849. [CrossRef] 8. Deen, N.G.; Solberg, T.; Hjertager, B.H. Large eddy simulation of the gas-liquid ﬂow in a square cross-sectio bubble column. Chem. Eng. Sci. 2001, 56, 6341–6349. [CrossRef] 18. Deen, N.G.; Solberg, T.; Hjertager, B.H. Large eddy simulation of the bubble column. Chem. Eng. Sci. 2001, 56, 6341–6349. [CrossRef] 19. Liu, C.; Luo, Z.; Wang, N.; Zhang, T.; Zou, Z. Mathematical modeling of multi-sized argon gas bubbles motion and its impact on melt ﬂow in continuous casting mold of steel. J. Iron Steel Res. Int. 2014, 21, 403–407. [CrossRef] Metals 2020, 10, 1160 15 of 15 15 of 15 20. Liu, Z.; Li, B.; Jiang, M. Transient asymmetric ﬂow and bubble transport inside a slab continuous-casting mold. Metall. Mater. Trans. B 2013, 45, 675–697. [CrossRef] 21. Liu, Z.Q.; Li, B.K.; Jiang, M.F.; Tsukihashi, F. Euler-Euler-Lagrangian modeling for two-phase ﬂow and particle transport in continuous casting mold. ISIJ Int. 2014, 54, 1314–1323. [CrossRef] 22. Zhang, T.; Luo, Z.G.; Liu, C.L.; Zhou, H.; Zou, Z.S. A mathematical model considering the interaction of bubbles in continuous casting mold of steel. Powder Technol. 2015, 273, 154–164. [CrossRef] 22. Zhang, T.; Luo, Z.G.; Liu, C.L.; Zhou, H.; Zou, Z.S. A mathematical model considering the interaction of bubbles in continuous casting mold of steel. Powder Technol. 2015, 273, 154–164. [CrossRef] 23. Zhang, T.; Luo, Z.; Zhou, H.; Ni, B.; Zou, Z. Analysis of two-phase ﬂow and bubbles behavior in a continuous casting mold using a mathematical model considering the interaction of bubbles. ISIJ Int. 2016, 56, 116–125. [CrossRef] 24. Zhang, L.; Wang, Y. Modeling the entrapment of nonmetallic inclusions in steel continuous-casting billets. JOM 2012, 64, 1063–1074. [CrossRef] 25. Yuan, Q. Transient Study of Turbulent Flow and Particle Transport During Continuous Casting of Steel Slabs. Ph D Thesis University of Illinois at Urbana Champaign Urbana Champaign IL USA 2004 25. Yuan, Q. Transient Study of Turbulent Flow and Particle Transport During Continuous Casting of Steel Slabs. Ph.D. Thesis, University of Illinois at Urbana-Champaign, Urbana-Champaign, IL, USA, 2004. 26. Thomas, B.G.; Yuan, Q.; Mahmood, S.; Liu, R.; Chaudhary, R. Transport and entrapment of particles in steel continuous casting. Metall. Mater. Trans. B 2013, 45, 22–35. [CrossRef] 27. References Liu, Z.; Li, B. Transient motion of inclusion cluster in vertical-bending continuous casting caster considering heat transfer and solidiﬁcation. Powder Technol. 2016, 287, 315–329. [CrossRef] 28. Jin, K.; Thomas, B.G.; Ruan, X. Modeling and measurements of multiphase ﬂow and bubble entrapment in steel continuous casting. Metall. Mater. Trans. B 2015, 47, 548–565. [CrossRef] 29. Yang, W.; Luo, Z.; Lai, Q.; Zou, Z. Study on bubble coalescence and bouncing behaviors upon oﬀ-center collision in quiescent water. Exp. Therm. Fluid Sci. 2019, 104, 199–208. [CrossRef] 30. Yang, W.; Luo, Z.; Gu, Y.; Liu, Z.; Zou, Z. Simulation of bubbles behavior in steel continuous casting mold using an Euler-Lagrange framework with modiﬁed bubble coalescence and breakup models. Powder Technol. 2020, 361, 769–781. [CrossRef] 31. Ribeiro, C.P.; Mewes, D. The eﬀect of electrolytes on the critical velocity for bubble coalescence. Chem. Eng. J. 2007, 126, 23–33. [CrossRef] 32. Ribeiro, C.P.; Mewes, D. On the eﬀect of liquid temperature upon bubble coalescence. Chem. Eng. Sci. 2006, 61, 5704–5716. [CrossRef] 33. Sevik, M.; Park, S.H. The Splitting of drops and bubbles by turbulent ﬂuid ﬂow. J. Fluid Eng. 1973, 95, 53–60. [CrossRef] 33. Sevik, M.; Park, S.H. The Splitting of drops and bubbles by turbulent ﬂuid ﬂow. J. Fluid Eng. 1973, 95, 53–60. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2018828520	https://europepmc.org/articles/pmc4370580?pdf=render	English	null	Electrographic Waveform Structure Predicts Laminar Focus Location in a Model of Temporal Lobe Seizures In Vitro	PloS one	2,015	cc-by	8,316	Electrographic Waveform Structure Predicts Laminar Focus Location in a Model of Temporal Lobe Seizures In Vitro Christopher Adams1, Natalie E. Adams2, Roger D. Traub3,4, Miles A. Whittington2* 1 Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, United Kingdom, 2 Hull York Medical School, The University of York, York, United Kingdom, 3 Dept. Physical Sciences, IBM TJ Watson Research Center, New York, New York, United States of America, 4 Department of Neurology, Columbia University, New York, New York, United States of America * Miles.whittington@hyms.ac.uk OPEN ACCESS OPEN ACCESS Citation: Adams C, Adams NE, Traub RD, Whittington MA (2015) Electrographic Waveform Structure Predicts Laminar Focus Location in a Model of Temporal Lobe Seizures In Vitro. PLoS ONE 10(3): e0121676. doi:10.1371/journal.pone.0121676 Academic Editor: Liset Menendez de la Prida, Consejo Superior de Investigaciones Cientificas— Instituto Cajal, SPAIN Received: November 10, 2014 Accepted: February 3, 2015 Published: March 23, 2015 Copyright: © 2015 Adams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Adams C, Adams NE, Traub RD, Whittington MA (2015) Electrographic Waveform Structure Predicts Laminar Focus Location in a Model of Temporal Lobe Seizures In Vitro. PLoS ONE 10(3): e0121676. doi:10.1371/journal.pone.0121676 Academic Editor: Liset Menendez de la Prida, Consejo Superior de Investigaciones Cientificas— Instituto Cajal, SPAIN Received: November 10, 2014 Accepted: February 3, 2015 Published: March 23, 2015 Copyright: © 2015 Adams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Adams C, Adams NE, Traub RD, Whittington MA (2015) Electrographic Waveform Structure Predicts Laminar Focus Location in a Model of Temporal Lobe Seizures In Vitro. PLoS ONE 10(3): e0121676. doi:10.1371/journal.pone.0121676 Academic Editor: Liset Menendez de la Prida, Consejo Superior de Investigaciones Cientificas— Instituto Cajal, SPAIN Copyright: © 2015 Adams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are available at www.carmen.org.uk for anyone who registers as a user with CARMEN e-science resource. Funding: This study was supported by the Wellcome Trust, The Wolfson Foundation, NSF (DMS- 1042134). IBM TJ Watson Research Center provided support in the form of salaries for author RT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section. RESEARCH ARTICLE Abstract Temporal lobe epilepsy is the most common form of partial-onset epilepsy and accounts for the majority of adult epilepsy cases in most countries. A critical role for the hippocampus (and to some extent amygdala) in the pathology of these epilepsies is clear, with selective removal of these regions almost as effective as temporal lobectomy in reducing subsequent seizure risk. However, there is debate about whether hippocampus is ‘victim’ or ‘perpetra- tor’: The structure is ideally placed to ‘broadcast’ epileptiform activity to a great many other brain regions, but removal often leaves epileptiform events still occurring in cortex, particu- larly in adjacent areas, and recruitment of the hippocampus into seizure-like activity has been shown to be difficult in clinically-relevant models. Using a very simple model of acute epileptiform activity with known, single primary pathology (GABAA Receptor partial block- ade), we track the onset and propagation of epileptiform events in hippocampus, parahippo- campal areas and neocortex. In this model the hippocampus acts as a potential seizure focus for the majority of observed events. Events with hippocampal focus were far more readily propagated throughout parahippocampal areas and into neocortex than vice versa. The electrographic signature of events of hippocampal origin was significantly different to those of primary neocortical origin – a consequence of differential laminar activation. These data confirm the critical role of the hippocampus in epileptiform activity generation in the temporal lobe and suggest the morphology of non-invasive electrical recording of neocorti- cal interictal events may be useful in confirming this role. Propagation of Epileptiform Activity in Temporal Cortex Once initiated such discharges spread through area CA1 and can then be widely ‘broadcast’ to a multitude of cortical and sub-cortical regions [4]. It is not surprising then that, in patients with unitemporal seizure origin almost 90% are seizure-free following surgical hippocampal re- moval [5]—a rate as good, if not better than temporal lobectomy but far less iatrogenic damage. However, this remaining ca. 10% of patients in whom epileptiform activity persists after hippo- campectomy fuel the debate about whether the hippocampal formation is victim or perpetrator in epilepsy. Hippocampal recruitment de novo following sclerosis is difficult, with very severe damage predicted to be required before the hippocampus itself acts as a source (rather than just a distributor) of epileptiform activity [6]. Propagation paths for aberrant activity out of hippocampus are not clear—following status epilepticus further seizure-like events have multi- ple apparent focal origins with variable and discontiguous patterns of propagation from them [7]. Competing Interests: The status of RTas an employee of IBM does not in any way alter the authors' adherence to PLOS ONE policies on sharing data and materials. Further complicating this picture is the suggestion that hippocampus proper and parahip- pocampal areas both have equal epileptogenic potential [7]. In addition, models of epileptiform activity in these brain regions reveal different patterns of seizure focus and spread apparently depending on the nature of the pathology introduced [8,9]. For example, the low magnesium model of epileptiform activity involves both boosting NMDA receptor-mediated excitatory neurotransmission and also reducing GABAA-receptor mediated inhibition [10]. Similarly the 4-AP model involves blockade of neuronal repolarisation via Kv1 potassium channels [11], but also—at least in hippocampus—the formation of depolarising, excitatory GABAergic network activity [12]. This suggests a need to quantify seizure origin and spread within these areas, using models that introduce specific and well-documented primary pathology are needed. Here we use a very basic acute seizure model (selective GABAA receptor-mediated disinhi- bition alone) combined with voltage sensitive dye imaging to investigate further the origin of epileptiform activity and its spread along the hippocampal-neocortical axis. Introduction A role for the hippocampus in temporal lobe epilepsies is clear. Local circuit properties—par- ticularly recurrent excitatory synaptic connection density in area CA3 [1] and critical depen- dence on strong GABA-ergic inhibition in areas CA2/CA1 [2] allow local circuits to generate intense, hypersynchronous discharges under the influence of epileptogenic pathology [3]. PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 1 / 13 NE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Methods Data were obtained from horizontal slices, 0.45 mm thick, from normal (non-epileptic) adult male Wistar rats obtained from B&K Universal, housed in pairs and given free access to food and water prior to terminal anaesthesia (Isoflurane followed by ketamine/xylazine). Slice prep- aration was briefly as follows: Terminally anaesthetised rats were intracardially perfused with buffered, ice-cold sucrose solution. The brain was removed and transferred to a vibratome. Re- sulting slices were maintained at 34°C at the interface between warm, wet 95% O2/ 5%CO2 and a perfusate of artificial cerebrospinal fluid (aCSF) containing (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 1 MgSO4, 1.2 CaCl2, 24 NaHCO3 and 10 glucose. Spontaneous epileptiform events were induced by bath application of bicuculline (0.1–0.2 mM). All tissue preparation was per- formed in accordance with the UK Animals (Scientific Procedures) act 1986 and with consent from the University of York Animal Ethics Committee. Electrographic data were recorded as local field potentials using glass micropipettes filled with aCSF (resistance 0.1–0.5 MO), digitised at 2 kHz and bandpass filtered at 0.5–100 Hz). Spatiotemporal patterns of activity were studied by loading slices with the voltage-sensitive dye (VSD) di-8-ANEPPS in 0.5% ethanol/DMSO containing aCSF fluid for 1–3h. Slices were then illuminated through a 10x objective by 532 nm (Coherent) laser and changes in output spectra recorded in the red range at 200 Hz using a Micam Ultima CCD camera (100 x 100 pixels, 25 mm square). Only a maximum of 6 x 10s epochs of data were taken from each slice to pre- vent result bias from bleaching under laser light. Fluorescence data were converted to 16 bit, grayscale TIFF stacks and exported to Matlab for analysis: Spatially, each frame was back- ground-subtracted, detrended and ‘post’-filtered at 2x2 pixels. Each pixel was then temporally PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 2 / 13 Propagation of Epileptiform Activity in Temporal Cortex manipulated using a Savitzky-Golay differentiation filter. Seizure wavefronts were detected as positive crossings of a threshold set at 2SD above baseline (non-epileptiform) activity. Velocity of propagation measurement were estimated (no compensation for curvature of structures was used) from the spread of these wavefronts across the slices. Wavefront data was rendered in 3D-space (x-, y- slice coordinates and time) using the Iso2mesh toolbox [13]. All raw data and bespoke analysis routines are available on the ‘CARMEN’ website (Carmen.org.uk). doi:10.1371/journal.pone.0121676.g001 Results 57 spontaneous interictal events lasting 0.2–2.8s were captured from 25 slices from 12 rats. Events were not stereotyped in terms of amplitude or origin and were divided here into 4 main types depending on region of initiation and pattern of propagation (Fig. 1). These were as fol- lows: Type 1, Non-propagating events originating in hippocampus (14/57). Type 2, Non-prop- agating events originating in primary auditory cortex (Au1, 11/57). Type 3a, propagating events originating in hippocampus, projecting through subiculum, medial and lateral entorhi- nal cortices and perirhinal cortex to neocortex (30/57). Type 3b, a subset of type 3a events Fig 1. Example VSD recordings of transient, spontaneous epileptiform discharges at three sites along the hippocampal—neocortical axis. A. Cartoon illustrating the 3 sites the example fluorescence changes illustrated were taken from: 1—stratum radiatum of area CA1 of the hippocampus, 2—layer 5/6 of medial entorhinal cortex (mEC), 3—layer 5/6 of auditory cortex (Au1). Box shows the coverage of the CCD chip used to record the data. Scale bar 1 mm. B/C. Examples of the 4 subtypes of spatiotemporal, interictal activity seen, each example is 2 x 2 pixel binned and temporally filtered (see methods). Bi. Non-propagating events originating in hippocampus. Bii. Non-propagating events originating in Au1 (note amplitude-degraded event visible in mEC but not hippocampus). Ci. Propagating, reverberating events originating in hippocampus, projecting to neocortex and returning to hippocampus. Cii. Propagating, non-reverberating events originating inhippocampus. Scale bars B/C 0.1% Δf, 2 sec. Fig 1. Example VSD recordings of transient, spontaneous epileptiform discharges at three sites along the hippocampal—neocortical axis. A. Cartoon illustrating the 3 sites the example fluorescence changes illustrated were taken from: 1—stratum radiatum of area CA1 of the hippocampus, 2—layer 5/6 of medial entorhinal cortex (mEC), 3—layer 5/6 of auditory cortex (Au1). Box shows the coverage of the CCD chip used to record the data. Scale bar 1 mm. B/C. Examples of the 4 subtypes of spatiotemporal, interictal activity seen, each example is 2 x 2 pixel binned and temporally filtered (see methods). Bi. Non-propagating events originating in hippocampus. Bii. Non-propagating events originating in Au1 (note amplitude-degraded event visible in mEC but not hippocampus). Ci. Propagating, reverberating events originating in hippocampus, projecting to neocortex and returning to hippocampus. Cii. Propagating, non-reverberating events originating inhippocampus. Scale bars B/C 0.1% Δf, 2 sec. Results 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Propagation of Epileptiform Activity in Temporal Cortex originating in hippocampus, also projecting to neocortex but then returning to hippocampus through each intermediate structure along the hippocampal-neocortical axis (7/30). In only two cases were events captured that originated in intermediate structures—both arising in deep layers of medial entorhinal cortex. In no cases were events captured that originated in neocor- tex and projected to hippocampus. In order to understand the signatures of each event that cor- responded to each type of dynamic behaviour we first considered differences within hippocampus. Hippocampal spatiotemporal profiles of propagating and non- propagating interictal-like events Type 1 and type3a&b events both always began at the CA1/CA2 border before rapidly spread- ing in both directions along the cornu ammonis to area CA3 and along the CA1 subregion (Fig. 2). The majority (72%) of captured interictal-like events began like this. Each subtype ex- hibited maximum percentage fluorescence change at the origin (0.14 ± 0.22 (type 1), 0.16 ± 0.15 (Type 3a), 0.12 ± 0.24 (Type 3b), P>0.1, n = 14, 23 & 7 respectively, Fig. 2A). In ad- dition, no significant difference was found in the extent and speed of spread through CA3. Mid-CA3 percentage fluorescence changes were degraded slightly from those at the focus (0.10 ± 0.03 (type 1), 0.08 ± 0.02 (Type 3a), 0.08 ± 0.03 (Type 3b), P>0.1, n = 14, 23 & 7 respec- tively, Fig. 2A). Mean rate of spread of the wavefront was between 0.03 and 0.05 m.s-1 at 34°C in each case (Fig. 2C). However, quantifiable differences in the pattern of spread along area CA1 were observed when comparing events that remained in hippocampus (type 1) and those that propagated to neocortex (types 3a/b). Non-propagating type 1 events projected slowly (0.17 ± 0.03 m.s-1) for only ca. 0.5 mm along CA1 whereas type 3a/b events propagated along the entire CA1 axis to subiculum at an initial, rapid rate (0.37 ± 0.07 m.s-1, P<0.05 cf non prop- agating events along CA1) before slowing at the CA1-subiculum border (Fig. 2C). Type 3 events fell into two subcategories: those that projected to neocortex and terminated, and those that projected to neocortex and then back to area CA1. We therefore next compare the spatiotemporal dynamics of these forward (hippocampus—neocortex) and back (neocortex to hippocampus) propagations. PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Pattern of propagation through periallocortex Propagation of interictal-like events from hippocampus through periallocortex was rapid and exhibited saltatory properties—discrete ‘jumps’ of activity from one locus in the general propa- gation direction to a more distal locus, followed by both forward and back propagation (Fig. 3, left panel). It involved recruitment of deep layers exclusively. From hippocampus activity jumped to the distal end of medial entorhinal cortex (mEC) before jumping further to the distal end of lateral entorhinal cortex (lEC). After each jump, activity propagated rapidly back to- wards subiculum (0.47 ± 0.11 ms-1, Fig. 3B). From lEC an apparent boundary to propagation was suggested by the considerable slowing of further propagation through peririnal cortex (0.02 ± 0.11 m.s-1, see discussion). Overall propagation time from CA1 focus to deep layers of primary auditory neocortex (Au1) was 25 ± 8 ms (n = 30) for all type 3 events. In complete contrast, interictal-like events propagating back to hippocampus from previ- ously invaded neocortex showed near monotonic, considerably slower conduction speeds and no evidence for saltatory conduction despite also exclusively utilising connectivity within deep cortical layers (Fig. 3A, B). Overall return conduction time from Au1 to area CA1 was 74 ± 15 ms (P<0.05 cf propagation times from hippocampal events described above, n = 7). These reverberatory events were initiated in Au1 on the decay of the fluorescence change (membrane potential depolarisation) caused by the initial invasion of neocortex by activity originating PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 4 / 13 Propagation of Epileptiform Activity in Temporal Cortex Fig 2. Spatiotemporal properties of non-propagating vs. propagating events originating in hippocampus. A. Example interictal fluorescence chang along the CA axis for non-propagating and propagating events. Not both types of event originate in CA1 (towards the CA2 border), both types propagate retrogradely into CA3, but only propagating events successfully invade subiculum. Scale bar 0.1% Δf, 1 sec. B. Spatiotemporal maps of the two event types in hippocampus. Only areas demonstrating activity over threshold are represented on the colormap, overlaid on the transmitted light (non-fluorescent) slice image. Main areas are labelled DG (dentate gyrus), CA2,3 (cornu ammonis subdivisdion 2,3), Sub (subiculum). Note the activity is concentrated in mid stratum radiatum. Colormap represents time from event onset (cool-hot). C. Mean event onset and spread (n = 6 slices) viewed along the CA1 horizontal ax (as illustrated by the cartoon insert). Propagation of Epileptiform Activity in Temporal Cortex Fig 3. Comparison of propagation dynamics for events of hippocampal origin propagating to neocortex, and the reverberative return wave. A. Spatiotemporal maps of activity spread through subiculum (top left of the field of view), medial & lateral entorhinal cortex and perirhinal cortex towards Au1. Only areas demonstrating activity over threshold are represented on the colormap, overlaid on the transmitted light (non-fluorescent) slice image. Note the activity is concentrated in deep cortical layers only. Colormap represents time from event onset (cool-hot). Scale bar 1mm. B. Mean event spread (n = 6 slices) viewed along the periallocortical horizontal axis (as illustrated by the cartoon insert). ‘Zero’ distance is set for the CA1/subiculum border. Note this is different from Fig. 2C as the majority of area CA1 was not present in the view field for these experiments. An individual example of an interictal wavefront (1st threshold crossing) is shown in gray, blue lines show mean ± s.e.mean. Note the rapid, saltatory-like spread of activity from hippocampus through periallocortex (the ‘jump’ in activity position with time followed by both forward and back (shown as dotted lines) propagation instead of just monotonic forward propagation) and the very slow conduction from lateral entorhinal to perirhinal cortices (asterisk in A and B). In contrast, the reverberative return wave from Au1 to hippocampus was relatively monotonic. d i 10 1371/j l 0121676 003 Fig 3. Comparison of propagation dynamics for events of hippocampal origin propagating to neocortex S i l f i i d h h bi l ( l f f h fi ld f i ) di l & l l hi Fig 3. Comparison of propagation dynamics for events of hippocampal origin propagating to neocortex, and the reverberative return wave. A. Spatiotemporal maps of activity spread through subiculum (top left of the field of view), medial & lateral entorhinal cortex and perirhinal cortex towards Au1. Only areas demonstrating activity over threshold are represented on the colormap, overlaid on the transmitted light (non-fluorescent) slice image. Note the activity is concentrated in deep cortical layers only. Colormap represents time from event onset (cool-hot). Scale bar 1mm. B. Mean event spread (n = 6 slices) viewed along the periallocortical horizontal axis (as illustrated by the cartoon insert). ‘Zero’ distance is set for the CA1/subiculum border. Note this is different from Fig. 2C as the majority of area CA1 was not present in the view field for these experiments. An individual example of an interictal wavefront (1st threshold crossing) is shown in gray, blue lines show mean ± s.e.mean. Note the rapid, saltatory-like spread of activity from hippocampus through periallocortex (the ‘jump’ in activity position with time followed by both forward and back (shown as dotted lines) propagation instead of just monotonic forward propagation) and the very slow conduction from lateral entorhinal to perirhinal cortices (asterisk in A and B). In contrast, the reverberative return wave from Au1 to hippocampus was relatively monotonic. doi:10.1371/journal.pone.0121676.g003 doi:10.1371/journal.pone.0121676.g003 from area CA1 (Fig. 1C). The 7 events captured began 0.42–0.78 seconds after the initial inva- sion, at a time when the fluorescence signal had still not decreased to half its initial maximum (see discussion). Pattern of propagation through periallocortex An individual example of an interictal wavefront (1st threshold crossing) is shown in gray, blue lines show mean ± s.e. mean. Note the mean initial propagation velocity is very rapid along CA1 (slope of wavefront, dotted lines) in opposite directions for the two events, but in each case, velocity slows markedly on reaching CA3 and subiculum (see results for quantification). doi:10.1371/journal.pone.0121676.g002 Propagation of Epileptiform Activity in Temporal Cort Fig 2. Spatiotemporal properties of non-propagating vs. propagating events originating in hippocampus. A. Example interictal fluorescence changes along the CA axis for non-propagating and propagating events. Not both types of event originate in CA1 (towards the CA2 border), both types propagate retrogradely into CA3, but only propagating events successfully invade subiculum. Scale bar 0.1% Δf, 1 sec. B. Spatiotemporal maps of the two event types in hippocampus. Only areas demonstrating activity over threshold are represented on the colormap, overlaid on the transmitted light (non-fluorescent) slice image. Main areas are labelled DG (dentate gyrus), CA2,3 (cornu ammonis subdivisdion 2,3), Sub (subiculum). Note the activity is concentrated in mid stratum radiatum. Colormap represents time from event onset (cool-hot). C. Mean event onset and spread (n = 6 slices) viewed along the CA1 horizontal axis (as illustrated by the cartoon insert). An individual example of an interictal wavefront (1st threshold crossing) is shown in gray, blue lines show mean ± s.e. mean. Note the mean initial propagation velocity is very rapid along CA1 (slope of wavefront, dotted lines) in opposite directions for the two events, but in each case, velocity slows markedly on reaching CA3 and subiculum (see results for quantification). doi:10.1371/journal.pone.0121676.g002 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 5 / 13 Laminar structure of events of neocortical or hippocampal origin In addition to the majority events with a hippocampal focus a number of interictal-like events were seen to originate in Au1. Of the 11 type 2 events captured none of them resulted in activi- ty propagating to hippocampus. This was in contrast to the reverberatory events described 6 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Propagation of Epileptiform Activity in Temporal Cortex above, arising from the initial activation of neocortex by hippocampus. In an attempt to under- stand why events with Au1 foci did not propagate outside neocortex we studied in more detail their local dynamics. Initial observation revealed that the two types of cortical activity were ini- tiated in completely different layers within Au1 (Fig. 4A): 11/11 events with Au1 focus initiated in layer 2/3 whereas all 30 events projected from hippocampus began in layers 5/6. Interesting- ly, of these 30 events, only the 7 that resulted in reverberatory activity back to hippocampus also generated fluorescence changes above threshold in superficial layers. Interictal-like events with origin in Au1 rapidly projected from layer 2/3 to layer 5 with a conduction time of 8 ± 2 ms (n = 11, Fig. 4B). Projection to deeper layers became progressively slower and unreliable. The events remained horizontally confined to a spread of 1.5 ± 0.4 mm (layers 2/3) and 1.6 ± 0.6 mm (layers 5/6) (n = 11, P>0.1) around the focus with a distinct bias to propagation towards periallocortex rather than more rostral associational and Fig 4. Comparison of propagation dynamics within neocortex. A. Spatiotemporal maps of activity spread through neocortical laminae for events originating in cortex (Type 2 events, left panel) and those spreading to cortex from hippocampus (Type 3 events with (3b) or without (3a) backpropagation, right panel). Only areas demonstrating activity over threshold are represented on the colormap, overlaid on the transmitted light (non-fluorescent) slice image. Colormap represents time from event onset (cool-hot). B. Mean event spread (n = 6 slices, left panel, n-5 slices right panel) viewed along the radial cortical axis from pia to subcortical while matter (as illustrated by the cartoon insert). An individual example of an interictal wavefront (1st threshold crossing) is shown in gray, blue lines show mean ± s.e.mean. Note the superficial focus for events of neocortical origin and the deep focus for events propagating from hippocampus. doi:10.1371/journal.pone.0121676.g004 Laminar structure of events of neocortical or hippocampal origin d i 10 1371/j l 0121676 004 Fig 4. Comparison of propagation dynamics within neocortex. A. Spatiotemporal maps of activity spread through neocortical laminae for events originating in cortex (Type 2 events, left panel) and those spreading to cortex from hippocampus (Type 3 events with (3b) or without (3a) backpropagation, right panel). Only areas demonstrating activity over threshold are represented on the colormap, overlaid on the transmitted light (non-fluorescent) slice image. Colormap represents time from event onset (cool-hot). B. Mean event spread (n = 6 slices, left panel, n-5 slices right panel) viewed along the radial cortical axis from pia to subcortical while matter (as illustrated by the cartoon insert). An individual example of an interictal wavefront (1st threshold crossing) is shown in gray, blue lines show mean ± s.e.mean. Note the superficial focus for events of neocortical origin and the deep focus for events propagating from hippocampus. doi:10.1371/journal.pone.0121676.g004 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 7 / 13 Propagation of Epileptiform Activity in Temporal Cortex somatosensory areas (see Fig. 4A, left panel). The 7 events captured that originated in hippo- campus and recruited superficial Au1 had opposite laminar profiles to those described above. Origin was always in layers 5/6 with a slower conduction time to layers 2/3 of 17 ± 3 ms (n = 7, P<0.05 cf conduction in the opposite direction for events of Au1 origin). Two additional fea- tures were of note: Firstly, once activated, horizontal propagation within layers 2/3 was near- identical to that seen for events of Au1 origin. Spread was 1.3 ± 0.3 mm with, again, a distinct bias to propagation towards periallocortex. Secondly, activity in deep layers was ‘en passant’— propagating to Au1 through the deep layers and continuing out of Au1 during and after activa- tion of layers 2/3 (Fig. 4B, right panel). These contrasting local dynamic signatures of interic- tal-like events—particularly their laminar differences—suggested that they should be distinguishable with conventional electrophysiological measures of local field potentials. Consequences for electrographic recordings There were always 4 threshold cross- ings when threshold was set to +2SD of the traces. Mean line length from first to last threshold crossing was 1.62 ± 0.19 s (n = 30, P<0.05 cf. line lengths for type 2 events in auditory cortex above). Mean peak-peak amplitudes were not significantly different from the biphasic events with primary Au1 origin (46 ± 12 μV, n = 5, P>0.1). Again, paired LFP recordings from layers 2/3 and 5/6 confirmed the deep to superficial propagation of this type of event within neocor- tex (cf. Fig. 4B). In addition to the different laminar origin of the type 2 and type 3a/b auditory cortical events the type 3 events were associated with a longer duration of fluorescence change (depolarisation) in layers 5/6; Type 2, 0.18 ± 0.09 s vs. type 3a/b, 0.70 ± 0.15 s (P<0.05). In contrast type 3a/b events, with hippocampal origin, were associated with pial LFPs show- ing a triphasic form and initial positive-going deflection. There were always 4 threshold cross- ings when threshold was set to +2SD of the traces. Mean line length from first to last threshold crossing was 1.62 ± 0.19 s (n = 30, P<0.05 cf. line lengths for type 2 events in auditory cortex above). Mean peak-peak amplitudes were not significantly different from the biphasic events with primary Au1 origin (46 ± 12 μV, n = 5, P>0.1). Again, paired LFP recordings from layers 2/3 and 5/6 confirmed the deep to superficial propagation of this type of event within neocor- tex (cf. Fig. 4B). In addition to the different laminar origin of the type 2 and type 3a/b auditory cortical events the type 3 events were associated with a longer duration of fluorescence change (depolarisation) in layers 5/6; Type 2, 0.18 ± 0.09 s vs. type 3a/b, 0.70 ± 0.15 s (P<0.05). PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Consequences for electrographic recordings Comparison of fluorescence changes concurrently with local field potential (LFP) recordings from the pial surface of Au1 were used to relate the spatiotemporal pattern of interictal-like events to a surrogate for intra- or extra-cranial recordings in vivo (Fig. 5). When comparing type 2 events of primary neocortical origin with type 3b events of hippocampal origin we found a distinct difference in the shape of the superficial LFP: Type 2 events that originated in superficial layers of Au1 were accompanied by biphasic LFPs with initial negative-going deflec- tions. With a threshold set at +2 standard deviations (SD) from the trace mean there were al- ways 2 threshold crossings for this event type. Mean line length from first to last crossing (filtered data) was 1.17 ± 0.12 s (n = 14 events). Mean peak-peak amplitude deflection of the pial signal was 42 ± 10 μV (n = 5, Fig. 5A, upper panel). Paired LFP recordings from layers 2/3 and 5/6 during these events of auditory origin confirmed the propagation from superficial to deep neocortex as seen with the fluorescence recordings (Fig. 5A, mid and lower panels). Comparison of fluorescence changes concurrently with local field potential (LFP) recordings from the pial surface of Au1 were used to relate the spatiotemporal pattern of interictal-like events to a surrogate for intra- or extra-cranial recordings in vivo (Fig. 5). When comparing type 2 events of primary neocortical origin with type 3b events of hippocampal origin we found a distinct difference in the shape of the superficial LFP: Type 2 events that originated in superficial layers of Au1 were accompanied by biphasic LFPs with initial negative-going deflec- tions. With a threshold set at +2 standard deviations (SD) from the trace mean there were al- ways 2 threshold crossings for this event type. Mean line length from first to last crossing (filtered data) was 1.17 ± 0.12 s (n = 14 events). Mean peak-peak amplitude deflection of the pial signal was 42 ± 10 μV (n = 5, Fig. 5A, upper panel). Paired LFP recordings from layers 2/3 and 5/6 during these events of auditory origin confirmed the propagation from superficial to deep neocortex as seen with the fluorescence recordings (Fig. 5A, mid and lower panels). In contrast type 3a/b events, with hippocampal origin, were associated with pial LFPs show- ing a triphasic form and initial positive-going deflection. PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Discussion These data confirm the dominant role for hippocampus in most temporal lobe epilepsies. While the model used here is simplistic at best it clearly showed that, within the hippocampal- neocortical axis, most epileptiform events (over 70%) had a focus along the CA3/CA1 bound- ary [2]. As with clinical observations following hippocampectomy, a small number of events of primary neocortical origin were also present, however, these were seen to be highly localised and of extremely low risk of spread and recruitment of other brain regions. In contrast, two thirds of the events demonstrating a hippocampal focus effectively recruited all the periallocor- tical areas present in the slice as well as proximal neocortical regions. In addition, there was a risk of repetitive activation of hippocampus via reverberation of these projected events. Pairing superficial layer local field potential traces with the VSD data showed than events of neocortical origin had biphasic voltage waveforms whereas events of hippocampal origin had triphasic 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Transverse in PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 9 / 13 Propagation of Epileptiform Activity in Temporal Cortex vitro hippocampal slice work (slices in this study were horizontal) has shown that epileptiform events can occur at either end of area CA3 [16, 17] with GABAergic disinhibition alone favour- ing area CA2/CA3a [18]. In each case area CA1 activation had an absolute requirement of CA3 involvement. As these and the present study all used disinhibition models this may reflect the starkly different patterns of network inhibition seen transversely compared to horizontally in the hippocampus proper [19]. For example, a distinct role for area CA2 in shaping propagatory events from DG/CA3 has been demonstrated with inhibition intact [20]. This was difficult to explore further here as we used a disinhibition model and also did not routinely record from area CA3c and the hilus. With intact hippocampal circuitry disinhibition-induced epileptiform activity has been shown to originate further along area CA1 towards subiculum, further com- plicating the issue of precise origin of epileptiform activity within the hippocampal circuit [21]. The incidence of both hippocampal origin and probability of event propagation out of hip- pocampus were highly consistent with seizures seen in medically refractory temporal lobe epi- lepsies. In the clinical case approximately 80% of seizures had a hippocampal focus and 60– 80% of these spread to neocortex [22,23]. The reasons for this lack of stereotypy in event prop- agation may stem from the nature of the primary pathology underlying the seizures and the way this is modelled. In particular, potassium conductances appear to play a role. In the present study only GABAA receptors were partially blocked, leaving GABAB receptors intact. While this form of inhibition has not been shown to affect seizure induction and spread in neocortex [24], it has been shown to be involved in termination of seizures within the hippocampal for- mation [25]. vitro hippocampal slice work (slices in this study were horizontal) has shown that epileptiform events can occur at either end of area CA3 [16, 17] with GABAergic disinhibition alone favour- ing area CA2/CA3a [18]. In each case area CA1 activation had an absolute requirement of CA3 involvement. As these and the present study all used disinhibition models this may reflect the starkly different patterns of network inhibition seen transversely compared to horizontally in the hippocampus proper [19]. For example, a distinct role for area CA2 in shaping propagatory events from DG/CA3 has been demonstrated with inhibition intact [20]. This was difficult to explore further here as we used a disinhibition model and also did not routinely record from area CA3c and the hilus. With intact hippocampal circuitry disinhibition-induced epileptiform activity has been shown to originate further along area CA1 towards subiculum, further com- plicating the issue of precise origin of epileptiform activity within the hippocampal circuit [21]. The incidence of both hippocampal origin and probability of event propagation out of hip- pocampus were highly consistent with seizures seen in medically refractory temporal lobe epi- lepsies. In the clinical case approximately 80% of seizures had a hippocampal focus and 60– 80% of these spread to neocortex [22,23]. The reasons for this lack of stereotypy in event prop- agation may stem from the nature of the primary pathology underlying the seizures and the way this is modelled. In particular, potassium conductances appear to play a role. In the present study only GABAA receptors were partially blocked, leaving GABAB receptors intact. While this form of inhibition has not been shown to affect seizure induction and spread in neocortex [24], it has been shown to be involved in termination of seizures within the hippocampal for- mation [25]. Differences in propagation trajectories seen in the present study compared with similar in vitro studies also suggest the role for potassium conductances involved in afterhyperpolarisa- tions in principal cells and their resting membrane potentials. In the present study only deep layers of periallocortical regions were recruited and acted as channels for hippocampal event spread (and return, Fig. 3). Models using disinhibition combined with lower magnesium ion concentration (to boost NMDA receptor-dependent synaptic excitation), or 4-aminopyridine to block Kv1-subtype of potassium channels [11] have shown recruitment of more superficial entorhinal cortex and dentate gyrus [9,10]. In addition, region-specific differences in the bal- ance of synaptic inhibition and excitation has also been suggested to account for both propaga- tion trajectory patterns and dynamics and whether focal seizures spread at all [26]. Changes in microcircuit connectivity may also account for the saltatory nature of propagation of hippo- campal events to neocortex as previously suggested [27], but do not appear to provide insight into the directionality of this phenomenon seen here: Saltatory propagation trajectories were only seen in the hippocampal-to-neocortical direction and not vice versa (Fig. 3). Propagation of Epileptiform Activity in Temporal Cortex Fig 5. Laminar origin of local and hippocampally-projected neocortical discharges is reflected in the electrographic waveform structure. A. Upper traces. Average of 12 events measured with an extracellular electrode at the pia to model non-invasive cortical recordings (pia). Shaded region represents +/- 2 SD of the mean trace shown. Note the overt biphasic vs. triphasic shape of the electrographic events when comparing interictal-like discharges of neocortical vs. hippocampal origin respectively. Middle traces. Concurrent superficial (L2/3) and deep (L5/6) cortical local field potential (LFP) recordings showing different laminar onsets. Lower traces. Example 2x2-binned fluorescence changes for the two event types. Scale bars 20μV (upper), 0.2 mV (middle), 0.1% ΔF (lower), 0.5 sec. B. Selected frames from the events in A showing the sequence of spatiotemporal activation for the two interictal-like events. Note the more overt, diffuse activation of deep layers for events propagated from hippocampus. d i 10 1371/j l 0121676 005 Fig 5. Laminar origin of local and hippocampally-projected neocortical discharges is reflected in the electrographic waveform structure. A. Upper traces. Average of 12 events measured with an extracellular electrode at the pia to model non-invasive cortical recordings (pia). Shaded region represents +/- 2 SD of the mean trace shown. Note the overt biphasic vs. triphasic shape of the electrographic events when comparing interictal-like discharges of neocortical vs. hippocampal origin respectively. Middle traces. Concurrent superficial (L2/3) and deep (L5/6) cortical local field potential (LFP) recordings showing different laminar onsets. Lower traces. Example 2x2-binned fluorescence changes for the two event types. Scale bars 20μV (upper), 0.2 mV (middle), 0.1% ΔF (lower), 0.5 sec. B. Selected frames from the events in A showing the sequence of spatiotemporal activation for the two interictal-like events. Note the more overt, diffuse activation of deep layers for events propagated from hippocampus. doi:10.1371/journal.pone.0121676.g005 (spike and wave-like) waveforms (Fig. 5). Given the difficulty in directly recording hippocam- pal events with conventional, non-invasive EEG methods [14], these data suggest the form of interictal cortical surface electrographic activity may be of use in determining causal nature of hippocampal pathology in temporal lobe epilepsies. The origin and spread within hippocampus seen here is largely consistent with previous re- ports on epileptiform events in acutely disinhinited hippocampus [2,15]. However, different tissue preparations have been shown to yield different foci within hippocampus. PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Propagation of Epileptiform Activity in Temporal Cortex extreme does the hippocampus act as a channel for repetitive epileptiform activity [6]. Howev- er, this may not invalidate the model used in the present study. In approximately 30% of cases of drug intractable temporal lobe epilepsy no lesions were seen but hippocampectomy was to a large extent successful [31], suggesting sclerosis is not a prerequisite for a key role for hippo- campus in seizure generation. The difference in propagation trajectories for hippocampofugal compared to hippocampo- petal events suggested different constraints for seizure spread to and from this region. The very rapid, saltatory spread from CA1 through entorhinal cortices may reflect the recruitment of physiologically relevant pathways for hippocampal communication with neocortex. However, the apparent, at least partial, barrier formed by the perirhinal cortex suggests a critical role in seizure spread from hippocampus. In general perirhinal cortex is spared in mesial temporal lobe epilepsy. However, if epileptiform activity is present here it can be severe [32]. It has been shown to be a highly sensitive area for intervention for seizure propagation and resistance to kindling [33,34] but is rarely removed in epilepsy surgery [35]. Within neocortex the fate of interictal epileptiform events was seen to be very different de- pending on whether the events were of neocortical origin or projected from hippocampus. In the former case (type 2 events here) events always started in superficial layers as seen for a number of acute epileptiform activity models (eg. Ref [36], but see also ref [37]). Projection to deep layers was rapid and seen for all events. In contrast, type 3 events invading neocortex from a hippocampal focus began in deep layers, as has been shown to occur occasionally in zero magnesium models [37]. Projection to superficial layers was seen in less than 50% of cases and, when present, occurred relatively slowly. This may reflect the ca. 5-fold less excitatory connectivity from deep to superficial cf. superficial to deep layers seen in primary sensory neo- cortex [38]. This imbalance between descending and ascending interlaminar connections may also, in part, explain the occurrence of reverberative epileptiform events: These were only seen in cases where superficial layers were activated. The slow conduction times to superficial layers may ensure that subsequent reactivation of deep layers occurs at an ideal time while neurons in layers 5/6 are no longer refractory, but are still relatively depolarised. Conclusions The propagation analyses presented here suggest that the small number of events of neocortical origin seen following hippocampectomy may not be of clinical significance owing to their high- ly localised nature and absence of overt spread to other brain regions. They also reinforce the suggestion that the perirhinal cortex may be an effective, putative target region for surgical in- tervention in temporal lobe epilepsy. In addition, these preliminary data suggest that it may be possible to identify whether seizure activity has a hippocampal focus by detailed analysis of neocortical activity alone. In addition, this re-en- trant activation of deep layers appeared also to underlie the different electrographic shapes of neocortically recorded events of local or hippocampal origin (Fig. 5). This dependence on sei- zure origin for seizure discharge shape has been noted in other models [39], where deep (ven- triculocisternal) origins had more ‘spike and wave—like’ morphologies compared to those with origins towards the surface of cortex. Of particular importance for reverberative activity is the dentate gyrus. This region is acti- vated by layer 2 entorhinal cortical neurons via the perforant path, rather than the direct tem- poroammonic pathway to area CA1 originating in layer 3 and deep layers as seen here [28]. The dentate gyrus is remarkably resistant to seizure spread, not because of GABAA receptor- mediated inhibition (as reduced in this study), but because of a highly hyperpolarised, potassi- um channel-dependent resting membrane potential [29]. With acute disinhibition alone, as used here, the seizure-resisting effects of intrinsic potassium conductances in superficial perial- locortical and dentate gyral neurons would be intact. In the pilocarpine model of epilepsy this is manifest as a preservation of the filtering properties of the dentate gyrus for neocortical input to hippocampus via the performat path [30]. Interestingly this study also showed a huge (ca. 10-fold) increase in excitatory effects of temporoammonic inputs directly to area CA1— the only active trajectory seen in the present study for re-entrant activity into hippocampus. However, in models of chronic hyperexcitability the bombardment of dentate gyrus with epi- leptiform events of cortical origin gradually induces cell death. Only when this becomes 10 / 13 References 1. Wong RK, Traub RD, Miles R. Cellular basis of neuronal synchrony in epilepsy. Adv Neurol. 1986; 44: 583–92. PMID: 3706021 2. Traub RD, Knowles WD, Miles R, Wong RK. Models of the cellular mechanism underlying propagation of epileptiform activity in the CA2-CA3 region of the hippocampal slice. Neuroscience 1987; 21: 457– 70. PMID: 3039403 3. Kibler AB, Durand DM. Orthogonal wave propagation of epileptiform activity in the planar mouse hippo- campus in vitro. Epilepsia 2011; 52: 1590–600. doi: 10.1111/j.1528-1167.2011.03125.x PMID: 21668440 4. Catenoix H, Magnin M, Mauguière F, Ryvlin P. Evoked potential study of hippocampal efferent projec- tions in the human brain. Clin Neurophysiol. 2011; 122: 2488–97. doi: 10.1016/j.clinph.2011.05.007 PMID: 21669549 5. Tsai ML, Chatrian GE, Holubkov AL, Temkin NR, Shaw CM, Ojemann GA. Electrocorticography in pa- tients with medically intractable temporal lobe seizures. II. Quantification of epileptiform discharges fol- lowing successive stages of resective surgery. Electroencephalogr Clin Neurophysiol.1993; 87: 25–37. PMID: 7687951 6. Dyhrfjeld-Johnsen J, Santhakumar V, Morgan RJ, Huerta R, Tsimring L, Soltesz I. Topological determi- nants of epileptogenesis in large-scale structural and functional models of the dentate gyrus derived from experimental data. J Neurophysiol. 2007; 97: 1566–87. PMID: 17093119 7. Holtkamp M, Buchheim K, Elsner M, Matzen J, Weissinger F, Meierkord H. Status epilepticus induces increasing neuronal excitability and hypersynchrony as revealed by optical imaging. Neurobiol Dis. 2011; 43: 220–7. doi: 10.1016/j.nbd.2011.03.014 PMID: 21440625 8. Ferrea E, Maccione A, Medrihan L, Nieus T, Ghezzi D, Baldelli P et al. Large-scale, high-resolution electrophysiological imaging of field potentials in brain slices with microelectronic multielectrode arrays. Front. Neural Circuits 2012; 6:80. doi: 10.3389/fncir.2012.00080 PMID: 23162432 9. Barbarosie M, Avoli M. CA3-driven hippocampal-entorhinal loop controls rather than sustains in vitro limbic seizures. J Neurosci. 1997; 17: 9308–14. PMID: 9364076 10. Traub RD, Jefferys JG, Whittington MA. Enhanced NMDA conductance can account for epileptiform ac- tivity induced by low Mg2+ in the rat hippocampal slice. J Physiol.1994; 478: 379–93. PMID: 7965853 11. Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA et al. Pharmacological charac- terization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably express- ed in mammalian cell lines. Mol Pharmacol. 1994; 45: 1227–34. PMID: 7517498 12. Avoli M, Perreault P, Olivier A, Villemure JG. 4-Aminopyridine induces a long-lasting depolarizing GABA-ergic potential in human neocortical and hippocampal neurons maintained in vitro. Neurosci Lett. 1988; 94: 327–32. PMID: 2849735 13. Acknowledgments The authors wish to dedicate this work to the late Prof Colin Ingram, without whom none of this would have been possible. 11 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 Propagation of Epileptiform Activity in Temporal Cortex Author Contributions Conceived and designed the experiments: CA NA MW. Performed the experiments: CA NA. Analyzed the data: NA RT. Contributed reagents/materials/analysis tools: NA. Wrote the paper: RT MW. PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 References Fang Q, Boas D. Tetrahedral mesh generation from volumetric binary and gray-scale images. Proc IEEE Intl. symposium on Biomedical Imaging. 2009;1142–1145. 14. Smith SJM. EEG in the diagnosis, classification, and management of patients with epilepsy. J. Neurol. Neurosurg Psychiatry 2005; 76: ii2–ii7. PMID: 15961864 15. Knowles WD, Traub RD, Strowbridge BW. The initiation and spread of epileptiform bursts in the in vitro hippocampal slice. Neuroscience 1987; 21:441–55. PMID: 3039402 16. Gjerstad L, Andersen P, Langmoen IA, Lundervold A, Hablitz J. Synaptic triggering of epileptiform dis- charges in CA2 pyramidal cells in vitro. Acta Physiol Scand. 1981; 113: 245–52. PMID: 7315453 17. Traub RD, Jefferys JGR, Miles R. Analysis of the propagation of disinhibition-induced afterdischarges along the guinea-pig hippocampal slice in vitro. J Physiol. 1993; 472: 267–87. PMID: 8145144 18. Colom LV, Saggau P. Spontaneous interictal-like activity originates in multiple areas of the CA2-CA3 region of hippocampal slices. J Neurophysiol. 1994; 71: 1574–85. PMID: 8035236 19. Gloveli T, Dugladze T, Rotstein H, Traub RD, Monyer H, Heinemann U et al. Orthogonal arrangement of rhythm generating microcircuits in the hippocampus. Proc Natl Acad Sci USA 2005; 102: 13295– 300. PMID: 16141320 PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 12 / 13 Propagation of Epileptiform Activity in Temporal Cortex 20. Sekino Y, Obata K, Tanifuji M, Mizuno M, Murayama J. Delayed signal propagation via CA2 in rat hip- pocampal slices revealed by optical recording. J Neurophysiol. 1997; 78: 1662–8. PMID: 9310451 21. Biodo D, Jesuthasan N, DeCurtis M, Uva L. Network dynamics during the progression of seizure-like events in the hippocampal-parahippocampal regions. Cerebral Cortex 2014; 24: 163–73. doi: 10.1093/ cercor/bhs298 PMID: 23048021 22. Spencer SS, Williamson PD, Spencer DD, Mattson RH. Human hippocampal seizure spread studied by depth and subdural recording: The hippocampal commissure. Epilepsia 1987; 28: 479–89. PMID: 3653050 23. Spencer SS, Spencer DD, Williamson PD, Mattson R. Combined depth and subdural electrode investi- gation in uncontrolled epilepsy. Neurology 1990; 40: 74–9. PMID: 2296386 24. Federico P, MacVicar BA. Imaging the induction and spread of seizure activity in the isolated brain of the guinea pig: the roles of GABA and glutamate receptors. J Neurophysiol. 1996; 76: 3471–92. PMID: 8930287 25. Stringer JL, Lothman EW. Pharmacological evidence indicating a role of GABAergic systems in termi- nation of limbic seizures. Epilepsy Res. 1990; 7: 197–204. PMID: 1705225 26. Menendez de la Prida L, Pozo MA. PLOS ONE \| DOI:10.1371/journal.pone.0121676 March 23, 2015 References Excitatory and inhibitory control of epileptiform discharges in com- bined hippocampal/entorhinal cortical slices. Brain Res. 2002; 940:27–35. PMID: 12020871 27. Wadman WJ, Gutnick MJ. Non-uniform propagation of epileptiform discharge in brain slices of rat neo- cortex. Neuroscience 1993; 52: 255–62. PMID: 8450945 28. van Groen T, Miettinen P, Kadish I. The entorhinal cortex of the mouse: organization of the projection to the hippocampal formation. Hippocampus 2003; 13: 133–49. PMID: 12625464 29. Lambert JD, Jones RS. A re-evaluation of excitatory amino acid-mediated synaptic transmission in rat dentate gyrus. J Neurophysiol. 1990; 64: 119–32. PMID: 1974917 30. Ang CW, Carlson GC, Coulter DA. Massive and specific dysregulation of direct cortical input to the hip- pocampus in temporal lobe epilepsy. J Neurosci. 2006; 26: 11850–6. PMID: 17108158 31. Wieser HG. Selective amygdalo-hippocampectomy for temporal lobe epilepsy. Epilepsia 1988; 29 suppl 2: S100–13. PMID: 3168957 32. Biagini G, D'Antuono M, Benini R, de Guzman P, Longo D, Avoli M. Perirhinal cortex and temporal lobe epilepsy. Front Cell Neurosci. 2013; 7: 1–10. doi: 10.3389/fncel.2013.00001 PMID: 23355802 33. Fukumoto S, Tanaka S, Tojo H, Akaike K, Takigawa M. Perirhinal cortical lesion suppresses the sec- ondary generalization in kainic acid-induced limbic seizure. Psychiatry Clin Neurosci. 2002; 56: 561–7. PMID: 12193248 34. Akman O, Karson A, Aker RG, Ates N, Onat FY. Perirhinal cortical kindling in rats with genetic absence epilepsy. Neurosci Lett. 2010; 479: 74–8. PMID: 20560164 35. Bonilha L, Martz GU, Glazier SS, Edwards JC. Subtypes of medial temporal lobe epilepsy: influence on temporal lobectomy outcomes? Epilepsia 2012; 53: 1–6. doi: 10.1111/epi.12029 PMID: 23216573 36. Borbély S, Halasy K, Somogyvári Z, Détári L, Világi I. Laminar analysis of initiation and spread of epilep- tiform discharges in three in vitro models. Brain Res Bull. 2006; 69: 161–7. PMID: 16533665 37. Tsau Y, Guan L, Wu JY. Epileptiform activity can be initiated in various neocortical layers: an optical im- aging study. J Neurophysiol. 1999; 82: 1965:73. PMID: 10515986 38. Lefort S, Tomm C, Floyd Sarria JC, Petersen CC. The excitatory neuronal network of the C2 barrel col- umn in mouse primary somatosensory cortex. Neuron 2009; 61: 301–16. doi: 10.1016/j.neuron.2008. 12.020 PMID: 19186171 39. Fisher RS, Prince DA. Spike-wave rhythms in cat cortex induced by parenteral penicillin. I. Electroen- cephalographic features. Electroencephalogr Clin Neurophysiol. 1977; 42: 608–24. PMID: 67022 13 / 13
https://openalex.org/W3123709896	https://pub.epsilon.slu.se/22630/1/zhang_s_et_al_210222.pdf	English	null	Remote Sensing Supported Sea Surface pCO2 Estimation and Variable Analysis in the Baltic Sea	Remote sensing	2,021	cc-by	15,551	Citation: Zhang, S.; Rutgersson, A.; Philipson, P.; Wallin, M.B. Remote Sensing Supported Sea Surface pCO2 Estimation and Variable Analysis in the Baltic Sea. Remote Sens. 2021, 13, 259. https://doi.org/10.3390/ rs13020259 Received: 19 November 2020 Accepted: 12 January 2021 Published: 13 January 2021 Received: 19 November 2020 Accepted: 12 January 2021 Published: 13 January 2021 Keywords: pCO2; remote sensing; random forest; variable importance; the Baltic Sea Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- ms in published maps and institutio- nal afﬁliations. Remote Sensing Supported Sea Surface pCO2 Estimation and Variable Analysis in the Baltic Sea Shuping Zhang 1,, Anna Rutgersson 1 , Petra Philipson 2 and Marcus B. Wallin 1,3 1 Department of Earth Sciences, Uppsala University, SE-752 36 Uppsala, Sweden; Anna.Rutgersson@met.uu.se (A.R.); Marcus.Wallin@slu.se (M.B.W.) 1 Department of Earth Sciences, Uppsala University, SE-752 36 Uppsala, Sweden; Anna.Rutgersson@met.uu.se (A.R.); Marcus.Wallin@slu.se (M.B.W.) Correspondence: shuping.zhang@geo.uu.se Abstract: Marginal seas are a dynamic and still to large extent uncertain component of the global carbon cycle. The large temporal and spatial variations of sea-surface partial pressure of car- bon dioxide (pCO2) in these areas are driven by multiple complex mechanisms. In this study, we analyzed the variable importance for the sea surface pCO2 estimation in the Baltic Sea and de- rived monthly pCO2 maps for the marginal sea during the period of July 2002–October 2011. We used variables obtained from remote sensing images and numerical models. The random forest algorithm was employed to construct regression models for pCO2 estimation and produce the importance of different input variables. The study found that photosynthetically available radiation (PAR) was the most important variable for the pCO2 estimation across the entire Baltic Sea, followed by sea surface temperature (SST), absorption of colored dissolved organic matter (aCDOM), and mixed layer depth (MLD). Interestingly, Chlorophyll-a concentration (Chl-a) and the diffuse attenuation coefﬁcient for downwelling irradiance at 490 nm (Kd_490nm) showed relatively low importance for the pCO2 estimation. This was mainly attributed to the high correlation of Chl-a and Kd_490nm to other pCO2-relevant variables (e.g., aCDOM), particularly in the summer months. In addition, the variables’ importance for pCO2 estimation varied between seasons and sub-basins. For example, the importance of aCDOM were large in the Gulf of Finland but marginal in other sub-basins. The model for pCO2 estimate in the entire Baltic Sea explained 63% of the variation and had a root of mean squared error (RMSE) of 47.8 µatm. The pCO2 maps derived with this model displayed realistic seasonal variations and spatial features of sea surface pCO2 in the Baltic Sea. The spatially and seasonally varying variables’ importance for the pCO2 estimation shed light on the heterogeneities in the biogeochemical and physical processes driving the carbon cycling in the Baltic Sea and can serve as an important basis for future pCO2 estimation in marginal seas using remote sensing techniques. The pCO2 maps derived in this study provided a robust benchmark for understanding the spatiotemporal patterns of CO2 air-sea exchange in the Baltic Sea. remote sensing Citation: Zhang, S.; Rutgersson, A.; Philipson, P.; Wallin, M.B. Remote Sensing Supported Sea Surface pCO2 Estimation and Variable Analysis in the Baltic Sea. Remote Sens. 2021, 13, 259. https://doi.org/10.3390/ rs13020259 1. Introduction Global oceans are an important sink of atmospheric CO2 and uptake approximately 30% of the global anthropogenic CO2 emissions [1]. As the global ocean uptake of CO2 in- creases by a rate proportional to the atmospheric CO2, substantial differences exist between oceans and marginal seas [1,2]. The changing air-sea exchange of CO2 in marginal seas, particularly those at high-latitude, is found to be the major source of uncertainties in the estimate of ocean CO2 uptake [3,4]. As the atmospheric CO2 is as rather globally homoge- nous, sea surface partial pressure of carbon dioxide (pCO2) in the marginal sea is the key component for precisely determining the direction of the air-sea exchange of CO2. Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/remotesensing Remote Sens. 2021, 13, 259. https://doi.org/10.3390/rs13020259 Remote Sens. 2021, 13, 259 2 of 22 Therefore, deriving maps of the changing pCO2 for marginal seas over time is critical for precise estimate of global air-sea exchange and ocean uptake of CO2 [2,3,5]. Therefore, deriving maps of the changing pCO2 for marginal seas over time is critical for precise estimate of global air-sea exchange and ocean uptake of CO2 [2,3,5]. Generally, sea surface pCO2 is jointly determined by biogeochemical processes, vertical and horizontal mixing of sea water, and the air-sea exchange of CO2 [6,7]. Many sea surface variables related to these processes are can be retrieved from remote sensing images. Given their vast spatial coverages, remotely sensed sea surface variables have increasingly been used in sea surface pCO2 estimation. Remotely sensed Chlorophyll-a concentration (Chl-a) is commonly used as an indicator of biological activities in water [8]. Sea surface temperature (SST) largely determines the solubility of CO2 in sea water and has been frequently used to estimate pCO2 from remote sensing [9–13]. In addition, bacteria respira- tion produces CO2 by decomposing dissolved organic matter (DOM) [14,15]. Therefore, absorption of colored dissolved organic matter (aCDOM) retrieved from remote sensing images was used in sea surface pCO2 estimation [16,17]. Furthermore, after [18] found from in-situ measurements that sea surface salinity (SSS) was highly related to sea surface pCO2, SSS derived directly from remote sensing images or remotely sensed aCDOM were adopted to support sea surface pCO2 estimate [16,19]. 1. Introduction Kd_490nm, a proxy of water trans- parency, was derived from remote sensing and included in sea surface pCO2 estimation to indicate the effect of biological activities [16]. Mixed layer depth (MLD) determines thermal stratiﬁcation between different water masses and, however, is not retrievable with remote sensing approaches. Therefore, some studies used the MLD obtained from ocean models to support the derivation of sea surface pCO2 maps [9,12]. Similarly, model-yield gross pri- mary production (GPP) and net primary production (NPP) were also included to support pCO2 estimation by approximating the biological control on pCO2 in sea water [9,12]. p y pp g g p Sea surface pCO2 in many global marginal seas have been estimated with various remote sensing supported approaches [9,12,16,17,20–23]. Most of the studies chose the variables based on empirical knowledge and focused on deriving pCO2 maps with small estimate errors (e.g., RMSE). However, few studies have investigated the spatiotemporal variabilities of the variable’s relevance to sea surface pCO2 in marginal seas. Considering the high spatial variabilities in the controlling forces of sea surface pCO2 in marginal seas, some studies divided the targeted seas into sub-basins/subsets and separately constructed models for pCO2 retrieval in each of the sub-basins/subset [12,22,24]. Though this strategy produced maps of good quality in the sub-basins/subsets, it provided little knowledge on the variables’ relevance to pCO2 distribution. Furthermore, Reference [25] regarded the sea surface pCO2 in the targeted area as a mixture of the pCO2 controlled by different processes (e.g., vertical mixing and biological uptake) and determined each of the processes separately from different sets of variables. Despite the successfully applications in multiple marginal seas [10,25,26], their method was often limited to pCO2 estimation in summer time and thus fails to provide information for other seasons. Overall, large space remains for investigation on variables’ relevance (importance) in sea surface pCO2 estimate across different time and space. The Baltic Sea is a semi-enclosed marginal sea located in northern Europe. The car- bon budget of the Baltic Sea displays considerable seasonal and interannual variabilities. To date, the few studies attempting to estimate sea surface pCO2 in the Baltic Sea using remote sensing approaches, e.g., [12]., have barely provided information on the variables’ relevance/importance to the pCO2 estimate for this marginal sea. 1. Introduction In this study, we aimed to analyze the importance of different variables for pCO2 estimation and derive improved monthly pCO2 maps for the Baltic Sea from 2002 to 2011. We conducted the following: (1) ﬁltering the in-situ pCO2 data for the model training and validation; (2) assessing the relative importance of the input variables for the pCO2 estimation on different spatial and seasonal scales; and (3) deriving pCO2 maps for the Baltic Sea. 3.1. Remote Sensing Products The Moderate Resolution Imaging Spectroradiometer (MODIS) on board Aqua satellite was designed for ocean surface investigations. The sensor maps the earth every two days from July 2002 on. A MODIS image consists of 36 spectral bands covering the spectrum of wavelength from 0.63 to 14.38 µm. Images from MODIS Aqua have been successfully used to detect coastal water clarity [36], survey red tides [37], map lake suspended matter [38], and retrieve coastal dissolved organic carbon [39]. Variables, like Chl-a and SST retrieved from MODIS-Aqua images with already mature algorithms, have been widely used to estimate sea surface pCO2 or simulate sea surface CO2 ﬂux in different oceans and marginal seas [11,16,17,40,41]. From the National Aeronautics and Space Administration (NASA) Goddard Space Flight Center (https://oceancolor.gsfc.nasa.gov/), we obtained the level-3 monthly mean MODIS products of PAR, Kd_490 nm and SST covering the period of August 2002–October 2020. All data have a spatial resolution equivalent to 4×4 km at the equator (Table 1). Table 1. Variables used to estimate partial pressure of carbon dioxide (pCO2) in the study. Data Variables Platform Type Spatial Resolution Time Span Provider MODIS Aqua PAR, SST, Kd_490nm Space-borne satellite 4 km August 2002–November 2011 Ocean Color Web MERIS Chl-a, aCDOM Space-borne satellite 300 m August 2002–November 2011 NEMO-NORDIC SSS, MLD Model 4 km August 2002–November 2011 CMEMS The Medium Resolution Imaging Spectrometer (MERIS) on board Envisat satel- lite was designed for ocean color observation. During its life span from 2002 to 2011, MERIS mapped the earth every 1–3 days and measured water surface radiances in 15 spec- tral bands from visible to infrared spectrum. Up to now, MERIS data have been frequently Table 1. Variables used to estimate partial pressure of carbon dioxide (pCO2) in the study. Data Variables Platform Type Spatial Resolution Time Span Provider MODIS Aqua PAR, SST, Kd_490nm Space-borne satellite 4 km August 2002–November 2011 Ocean Color Web MERIS Chl-a, aCDOM Space-borne satellite 300 m August 2002–November 2011 NEMO-NORDIC SSS, MLD Model 4 km August 2002–November 2011 CMEMS Table 1. Variables used to estimate partial pressure of carbon dioxide (pCO2) in the study. The Medium Resolution Imaging Spectrometer (MERIS) on board Envisat satel- lite was designed for ocean color observation. During its life span from 2002 to 2011, MERIS mapped the earth every 1–3 days and measured water surface radiances in 15 spec- tral bands from visible to infrared spectrum. 2. Study Area The Baltic Sea is located at high latitudes (55–60◦N) in Europe. As the sun illumination and temperature there exhibit signiﬁcant seasonal changes, the Baltic Sea and adjacent Remote Sens. 2021, 13, 259 3 of 22 3 of 22 terrestrial ecosystems also undergo high seasonality. In addition, the wide span of the Baltic Sea in latitude forms a large spatial gradient in sun illumination and the corresponding environment condition, like SST. The Baltic Sea has restricted water exchange with the open North Atlantic Ocean via the Danish straits and is a semi-enclosed marginal sea. More than 600 rivers drain the catchment of total 1.7 million km2 and export to the Baltic Sea substan- tial freshwater and terrigenous substances, including organic carbon [27–30]. Therefore, the Baltic Sea is characterized with a high concentration of CDOM, and most part of the sea presents as “brown water”. With varying inputs from different rivers, the sub-basins of the Baltic Sea create highly heterogeneous biogeochemical conditions in this marginal sea. Consequently, the pCO2 distribution in the Baltic Sea displays evident seasonality and spatial heterogeneity [31]. Upwelling characterized with evident seasonality and spatiality occurs frequently in the Baltic Sea and brings up deep water of high pCO2 up to 2000 µatm to the sea surface [32,33]. The high concentration of nutrients brought up together with the deep water leads to cyanobacteria and phytoplankton blooms after the upwelling event, which further complicates the pCO2 distribution in the Baltic Sea [34]. p p Till now, nearly all the pCO2 related studies in the Baltic Sea were based on in-situ measurements from ship and/or buoys, and the ﬁndings are often valid for limited sites of the sea. Therefore, analyzing variables’ relevance and obtaining reliable pCO2 maps is critical for better understanding the carbon cycle and the air-sea exchange in the Baltic Sea [35]. 3. Data We chose the variables for pCO2 estimation based on previous studies and the charac- teristic of the Baltic Sea. The variables SST, photosynthetically available radiation (PAR), Chl-a, Kd_490 nm, and aCDOM were remotely sensed. SSS and MLD were produced by the numerical model NEMO-NORDIC together with data assimilation. In-situ pCO2 mea- surements from three different sources were used to train and validate the model for pCO2 estimation. 3.1. Remote Sensing Products Up to now, MERIS data have been frequently Remote Sens. 2021, 13, 259 4 of 22 used to investigate water related issues in global ocean and marginal seas, including mapping sea algae coverage [42], detecting phytoplankton bloom [43] and cyanobacterial bloom [44], and estimating Chl-a, aCDOM, and suspended matter [45–49]. Most of these studies targeted at European lakes and seas and demonstrated the great potential of MERIS data for investigating these waters. Speciﬁcally, [45] found that Chl-a retrieved from MERIS for the Baltic Sea had similar distributions to that of in-situ measurements. The MERIS data from the MERCI data base (https://merisrr-merci-ds.eo.esa.int/ merci) were used to retrieve Chl-a and aCDOM for the Baltic Sea with the Free University of Berlin (FUB) processor which were especially developed for European coastal waters. Invalid pixels (i.e., land, mixture of land and water, various cloud types, and cloud shadow) were masked out from MERIS images before the Chl-a and aCDOM retrieval. The perfor- mance of Chl-a and aCDOM retrieved from MERIS with FUB processor in the Baltic Sea were assessed to be excellent [49,50]. In this study, the daily Chl-a and aCDOM derived from MERIS images were aggregated monthly and resampled to 4×4 km. The Chl-a and aCDOM derived from the full MERIS archive spans from July 2002 to December 2011. Comparison of the contributions of the Chl-a products from MODIS and MERIS to pCO2 estimation in the method employed here did not show signiﬁcant differences (Figure S2). 3.2. Modeled Data MLD and SSS are important variables for pCO2 estimates. However, remotely sensed SSS have much coarser resolutions than other variables, such as Chl-a, and MLD is not yet obtainable from remote sensing. Alternatively, modeled MLD and SSS have been applied in many studies on sea surface pCO2 estimation [9,12,20,51,52]. Therefore, we employed the monthly MLD and SSS produced by the NEMO-NORDIC model which is a a Baltic and North Sea model based on the NEMO ocean engine and a local singular evolutive interpolated Kalman (LSEIK)ﬁlter data assimilation with a spatial resolution of 4 × 4 km [53] (Table 1). Validation of the modeled SSS against the station observation demonstrated a bias smaller than 0.5 ppt and a RMSE of 0.5 ppt [53]. 3.3. In-Situ Data We used all the in-situ sea surface pCO2 measurements available in the Baltic sea during August 2002–November 2011 (Table 2 and Figure 1). They included the data from the Surface Ocean CO2 Atlas (SOCAT) (2nd Version) [54], the measurements from a moored buoy at Östergarnsholm site [55], and data from [56]. Table 2. The in-situ measurements used in this study. Table 2. The in-situ measurements used in this study. Data Source Acquisition Platform Time Period Location No. Measurement No. Measurements after Aggregation & Filtering SOCAT Ship June 2002–October 2011 Baltic Sea 194,565 194,565 Östergarnsholm SEMI at a bouy May 2005–December 2011 Central Baltic Sea 6631 23 [56] Station & ships June 2000–September 2009 Gulf of Bothnia 6328 1060 Table 2. The in-situ measurements used in this study. All the data in SOCAT have undergone quality control and were of error < 10 µatm [54,57]. We used pCO2 measurements acquired from 2002 to 2011 to match the remotely sensed variables. The data from SOCAT for this period were obtained from the Finnpartner vessels which travelled between Lübeck and Helsinki every second day [58]. The pCO2 measurements are available every 1–2 min and appear as a series of points distributed along the ship tracks (Figure 1A). g p g At the Östergarnsholm site, the sea surface pCO2 is measured by a submersible au- tonomous moored instrument (SAMI) mounted on a buoy mooring one kilometer east off the island Östergarnsholm in the central Baltic Sea (Figure 1A). The SAMI sensor was installed four meters below the water surface and has recorded the pCO2 there every 30 or 60 min from 2005 May to the present [55]. The pCO2 measurement from Östergar- nsholm site also fulﬁlls the accuracy criterion of <10 µatm. Remote Sens. 2021, 13, 259 5 of 22 The pCO2 data used by [56] ﬁlled the data gap left by the previous two data sources in the Gulf of Bothnia. The data set consisted of both manual bottle measurements from discrete stations and continuous ferry box measurements obtained with the same method as the vessel data in SOCAT (Figure 1A). The measurements were mainly from the year of 2006, 2009, and 2010. More details about the data are available from [56]). Figure 1. The location where the in-situ pCO2 were measured in the Baltic Sea from August 2002 to November 2011 (A) and the density distribution of the in-situ pCO2 measurements (B). 3.3. In-Situ Data The numbers in parentheses indicate the sub-basins where the variables’ importance was analyzed. (1) Gulf of Bothnia, including Bothnia Bay (north) and Bothnia Sea (south); (2) Gulf of Finland (north) and Gulf of Riga (south); (3) Baltic Proper; (4) Arkona Basin. Figure 1. The location where the in-situ pCO2 were measured in the Baltic Sea from August 2002 to November 2011 (A) and the density distribution of the in-situ pCO2 measurements (B). The numbers in parentheses indicate the sub-basins where the variables’ importance was analyzed. (1) Gulf of Bothnia, including Bothnia Bay (north) and Bothnia Sea (south); (2) Gulf of Finland (north) and Gulf of Riga (south); (3) Baltic Proper; (4) Arkona Basin. 4.1. Random Forest Random forest is a tree-assembled model where the trees are constructed based on a set training samples [59]. Random forest has shown excellent performance in classiﬁ- cation and regression [60,61]. Therefore, it has been used in various ﬁelds. For example, it has been used to estimate gross primary production of vegetation from remote sensing images [62], downscaling the soil moisture data and chlorophyll ﬂuorescence of coarse resolutions etc. [63,64]. With respect to pCO2 estimation from remote sensing data [17] derived pCO2 maps for the Gulf of Mexico with an RMSE of 31.7 µatm using a similar tree-based algorithm. In addition, [16] compared random forest with other commonly used approaches (e.g., multiple linear regression) and proved that random forest was a robust algorithm for sea surface pCO2 estimation from remote sensing data in the Gulf of Mexico [16]. In this study, random forest models were trained to express the relationship between the in-situ pCO2 measurements and spatially and temporally co-located variables (i.e., Chl-a, aCDOM, SST, PAR, Kd490nm, SSS, and MLD). Each random forest model contained a number of tree (known as Ntree) with each node splitting to a number of leaves (known as Mtry). At each node, a bootstrapped subset of randomly selected training samples was used to construct the relationship between the Mtry variables (e.g., Chl- and SST) and the dependent variable (i.e., pCO2) in the form of split leaves [65]. The tree grew as the nodes were produced and connected in a cascade manner. Each decision tree was independently produced. The forest construction was ﬁnished as the trees grew to Ntree, a user-deﬁned number of trees [59]. The ﬁnal random forest is a set of trees with best Remote Sens. 2021, 13, 259 6 of 22 performance in expressing the relationship between variables in the training samples. Further details on the random forest model are to be found in Breiman (2001). Each random forest model contained 500 trees (N tree = 500) of the leaf size of three (Mtry = 3). We used the random forest algorithm implemented in the package randomForest [66] for the open access software R [67]. Subsequently, the importance of each variable in the random forest model was also extracted and analyzed. The importance of a variable Xm was determined by the mean decrease accuracy (MDA) of the random forest model when the variable Xm is randomly permuted in the training samples [59]. 4.1. Random Forest Therefore, the importance of variable Xm in a random forest model indicates its contributions/relevance to the model and the response of corresponding variable to the pCO2 variation in the training data set. For each variable, the importance was derived independently. The variables are not complementary to each other in pCO2 estimate, Therefore, the sum of the variables’ importance cannot stay as constant value, like 100%, across different time and spatial scale. 4.2. Filtering In-Situ Data The diurnal differences of sea surface pCO2 in the Baltic Sea can reach up to 40 µatm [68], and using only the data from day time or night time would introduce 8% to 36% error on monthly air–sea CO2 ﬂuxes [69]. Pre-analysis also found that using in-situ pCO2 measurements from 24 h for sea surface pCO2 estimation would increase the uncertainty of results by 30–60 µatm (Supplementary Materials Figure S2). Therefore, we only used the in-situ pCO2 measurements obtained during the exact period of the two satellites (i.e., MODIS Aqua and MERIS) passes over the Baltic Sea, i.e., 9:00–14:00 UTC 00. Subsequently, the in-situ data were aggregated monthly to match the frequency of the remotely sensed and modelled variables. The variables exactly co-located to the in-situ pCO2 measurements were extracted and used for random forest model construction and validation. Using the variables (e.g., SST) derived for the months characterized with frequent upwelling occurrences can signiﬁcantly affect the monthly pCO2 estimates by introducing large biases (Figure S3). Therefore, the upwelling effect should be eliminated to the largest possible extent. To achieve this, we constructed a random forest model using in-situ data from each month as validation data and the rest as training data. All the models with the alternative absence of in-situ data from each month were constructed with identi- cal settings. Inspection on the mean absolute errors (MAE) and RMSE of these models showed that the following monthly data were dominated by upwelling (i.e., large bias): 2003–09, 2006–09, 2006–08, 2009–07, 2009–09, 2009–10, 2011–04, 2011–08, 2011–09, 2011–10 (Figure S4). Nearly all of them were in fall when upwelling prevails in the Baltic Sea [32]. In-situ pCO2 measurements from these months were eliminated from training and vali- dating the model. Sea surface pCO2 maps in these months were not predicted as it would produce misestimation for these months. p After narrowing the time window of in-situ pCO2 measurements down to 9:00–14:00, aggregating these in-situ pCO2 measurements monthly, and ﬁltering out the data from the upwelling dominated months, 10,769 in-situ pCO2 measurements with matching variables remained, as shown in Figure S1. 4.4. Constructing the Fnal Model for pCO2 Estimation in the Baltic Sea We constructed a ﬁnal random forest model for pCO2 estimation in the entire Baltic Sea. This model was trained with the in-situ pCO2 measurements in odd months of even years (e.g., March 2002) and even months of odd years (e.g., April 2003) and validated with the remaining data. By doing this, both the training and validation data covered each of the 12 months in a year and the pCO2 relevant processes from each month. Exchanging the training data and validation data yielded models with nearly the same performance (Figure S7). The monthly mean pCO2 distribution in the entire Baltic Sea were predicted with this model. The Pearson correlations of the pCO2 estimated with above model to each of the variables were analyzed. In order to speed up the processing, the correlation was analyzed on a 0.5◦× 0.5◦grid form. In each month, the mean of pCO2 and the means of each targeted variables (e.g., Chl-a) in the same grid cell was derived. The Pearson correlations between pCO2 and each of the variables in each grid cell were obtained across the study period of 2002–2011. 4.5. Comparing the Random Forest to Self-Organized Map (SOM) and Multiple Linear Regression (MLR) for pCO2 Estimation in the Baltic Sea 4.5. Comparing the Random Forest to Self-Organized Map (SOM) and Multiple Linear Regression (MLR) for pCO2 Estimation in the Baltic Sea SOM is an artiﬁcial neuronal network algorithm which classiﬁes the input samples into a number of classes, based on their Euclidian distance from each other in the space determined by the variables of the input data [20,70]. Often, the number of classes (neuron) are given a priori in a grid format (e.g., 2 × 5). Each class corresponds to a neuron which contains the coefﬁcients determining the relationship between the variables and the dependent variable in the same class, which is also called labelling the class with the dependent variable (output). In the case of sea surface pCO2 estimation with SOM, the remotely sensed variables, like Chl-a and SST, in the training data, are used to calculate the distance between the input samples for classiﬁcation. In the pCO2 prediction with such a SOM model, the samples will be attributed with the pCO2 of a class to whom the sample show the closest distance to. Detailed description of a SOM application for sea surface pCO2 estimation by remote sensing data is available in Telszewski et al. 4.3. Analyzing Variables’ Importance for pCO2 Estimation We derived the variables’ importance to the pCO2 estimation on two scales: spatially and temporally. On the spatial scale, the random forest models were constructed both for the overall Baltic Sea and its sub-basins indicated in Figure 1B. In each sub-basin, a random forest model was trained with the in-situ in the sub-basin from 2/3 of the months from random selection. Each model was then validated with the in-situ data in the sub-basin from the rest 1/3 months. We constructed 50 random forest models in each sub-basin with the training and validation data selected in such way. In the temporal analysis of the variables’ importance to the pCO2 estimates, the in-situ measurements were divided Remote Sens. 2021, 13, 259 7 of 22 into different seasons. Speciﬁcally, February–April was spring, May–July was summer, and August–October was fall. The limited availability of satellite data due to frequent and extensive cloud coverage in November, December, and January did not allow for such analysis during these months. Like the spatial analysis, in-situ data from 2/3 of the months from random selection were used for training and the rest 1/3 for validation. Fifty random forest models were constructed in each season with the training data selected in the same manner and validated with the corresponding complementary data. 5. Results 5.1. Spatiotemporal Characteristics of Variable Importance to pCO2 Estimation On the entire Baltic Sea scale, PAR was the most important variable (mean importance of 66%) for the sea surface pCO2 estimate during 2002–2011. It meant that the errors of the random forest model constructed without PAR would be by 66% higher than that constructed with PAR. PAR was followed by SST, MLD, aCDOM, and SSS with mean importance of 21%, 20%, 15%, and 14%, respectively. Chl-a and Kd_490nm showed the lowest importance of 12% and 10% (Figure 2A). Figure 2. Variables’ importance for pCO2 estimate in the Baltic Sea and its sub-basins. (A) Variables’ importance in the 50 models trained with in-situ data in the entire Baltic Sea from 2/3 months of random selection; (B–D) Variables’ importance in the 50 models trained with in-situ data from each sub-basin from 2/3 months of random selection. (E) The RMSEs of the 50 models trained with in-situ data from the four regions, respectively. CDM in the sub-ﬁgures donates the aCDOM derived from Medium Resolution Imaging Spectrometer (MERIS) images, KED stands for Kd_490nm. Figure 2. Variables’ importance for pCO2 estimate in the Baltic Sea and its sub-basins. (A) Variables’ importance in the 50 models trained with in-situ data in the entire Baltic Sea from 2/3 months of random selection; (B–D) Variables’ importance in the 50 models trained with in-situ data from each sub-basin from 2/3 months of random selection. (E) The RMSEs of the 50 models trained with in-situ data from the four regions, respectively. CDM in the sub-ﬁgures donates the aCDOM derived from Medium Resolution Imaging Spectrometer (MERIS) images, KED stands for Kd_490nm. The variables importance differed among the sub-basins of the Baltic Sea. Compare to the pCO2 estimate in the entire Baltic Sea (Figure 2A), the importance of PAR, SST, aCDOM, SSS, and MLD for pCO2 estimation in the Gulf of Finland (i.e., sub-basin No.2) increased by 26%, 13 %, 15%, 5%, and 1% (Figure 2B). For pCO2 estimation in this sub-basin, PAR was still the most importance variable. With the mean importance of 25%, aCDOM and SST are the next most importance variables, followed by SSS and MLD with respective importance of 18% and 16% (Figure 2B). The importance of Chl-a and aCDOM to the pCO2 estimation in The variables importance differed among the sub-basins of the Baltic Sea. 4.4. Constructing the Fnal Model for pCO2 Estimation in the Baltic Sea (2009). SOM and its variants have been widely used to estimate sea surface pCO2 with support of remote sensing products [11,12,20,71–74]. In this study, we used the SOM algorithm implemented in the R packages of kohonen [75]. We set the size neurons (class) grid to be 25 × 20, in order to have the total number of classes same to the number of trees in the random forest models constructed in this study. Furthermore, multiple linear regression (MLR) has been used in many studies for estimating sea surface pCO2 in marginal seas and performed good results [9,16]. Therefore, we compared the performance of SOM, MLR, and random forest in the sea surface pCO2 estimation in the Baltic Sea. During the comparison, the same variables were used in the three algorithms without any preselection. Random forest, SOM, and MLR models were trained with the identical data and validated likewise. Two schemes of training data selection were adopted, one with in-situ pCO2 mea- surements from 2/3 of the months from random selection (scheme Number 1, same as in Section 4.3) and the other one using 2/3 of in-situ pCO2 measurements from random selection as training data (scheme Number 2). Scheme Number 2 was similar to the training data selection by [12]. In both schemes, the validation data were the complementary of the training data. Remote Sens. 2021, 13, 259 8 of 22 5. Results Compare to the pCO2 estimate in the entire Baltic Sea (Figure 2A), the importance of PAR, SST, aCDOM, SSS, and MLD for pCO2 estimation in the Gulf of Finland (i.e., sub-basin No.2) increased by 26%, 13 %, 15%, 5%, and 1% (Figure 2B). For pCO2 estimation in this sub-basin, PAR was still the most importance variable. With the mean importance of 25%, aCDOM and SST are the next most importance variables, followed by SSS and MLD with respective importance of 18% and 16% (Figure 2B). The importance of Chl-a and aCDOM to the pCO2 estimation in Remote Sens. 2021, 13, 259 9 of 22 the southern Baltic Sea (i.e., sub-basins No.3–4) were similar to that for the overall Baltic Sea, with slightly lower importance of SSS in sub-basin No.3 (Figure 2A). The ﬁltering and time window narrowing down left the Gulf of Bothnia (i.e., sub-basin No.1, Figure 1) with the in-situ data from March 2006 and September 2009. It hampered the construction of random forest model for pCO2 estimate in this sub-basin, due to the strategy of data from 2/3 months for model training. The 50 random forest models constructed in the Baltic Sea, sub-basin No.2, sub-basin No.3, and sub-basin No.4 had the means of 49 µatm, 72 µatm, 50 µatm, and 43 µatm, respectively. p y The variables’ importance for pCO2 estimation also varied on seasonal scales. For the sea surface pCO2 estimate in the entire Baltic Sea during February–April, PAR was the most important variable with mean importance of 56%, followed by MLD (20%), SSS (15%), SST (15%), and aCDOM (10%). Chl-a and Kd_490nm showed mean importance of 8% (Figure 3B). From May to July, all the variables displayed a similar importance (12–14%), with Kd_490nm (7%) and MLD (5%) (Figure 3C). The low importance of all the variables in May-July means that during this period the alternative absence of the variables in the models constructed did not signiﬁcantly change the accuracies of the respective models. In another word, during May-July, the combination of any six out of the seven variables used in the study can well cover the variations of pCO2 in the Baltic Sea. 5.2. pCO2 Maps from Final Random Forest Model The ﬁnal random forest model for sea surface pCO2 estimation for the entire Baltic Sea engaged all the variables, namely, PAR, Chl-a, aCDOM, SST, Kd_490nm, SSS, and MLD. Its RMSE was 47.8 µatm and its coefﬁcient of determination (i.e., R2) was 0.63 (Figure 4A). The mean absolute error (MAE) of the model was -3.26 µatm, implying a slight overall underestimate of pCO2. The pCO2 predicted with this model exhibited minor overesti- mates for pCO2 larger than 450 µatm and slight overestimates for pCO2 around 200 µatm (Figure 4A). Both the estimated and observed pCO2 values were mainly in the range of 100–500 µatm, with a few pCO2 observations between 500 µatm and 600 µatm (Figure 4A). Figure 4. The ﬁnal random forest model for the pCO2 estimate. (A) Quality performance of the model, where the red dashed line is the regression line between the pCO2 observation and the estimate and black dashed line is 1:1 line; (B) the variables’ importance in the model. Figure 4. The ﬁnal random forest model for the pCO2 estimate. (A) Quality performance of the model, where the red dashed line is the regression line between the pCO2 observation and the estimate and black dashed line is 1:1 line; (B) the variables’ importance in the model. The variable importance in the ﬁnal model was similar to that in Figure 2A. Speciﬁcally, PAR was the most important variable, followed by SST, MLD, and aCDOM. Ch-a and Kd_490nm showed the lowest importance (Figure 4B). p g For the period of August 2002–October 2011, pCO2 maps covering the entire Baltic Sea were retrieved for each month except November, December, January, and February, when the remotely sensed variables were not available due to frequent cloud coverage. Taking the year of 2005 as example (Figure 5), the sea surface pCO2 in the Baltic Sea were in the range of 100–500 µatm. On the spatial scale, the pCO2 maps exhibited rea- sonable transitions in the Baltic Sea (Figure 5). In addition, detailed features of the pCO2 variation were also displayed in those maps. For example, in April 2005, much lower pCO2 was present at the river mouths in the southern Baltic Sea compared to other areas. In May 2005, a strip of low pCO2 was present in the central Baltic Proper. 5. Results For pCO2 estimation in the entire Baltic Sea in the period of August-October, PAR and SST were the ﬁrst two most important variables with respective importance of 38% and 31% (Figure 3D), followed by MLD (16%) and SSS (12%) and the rest variables with importance of 10%. Chl-a and Kd_490nm showed overall low importance for the pCO2 estimate across Baltic Sea, regardless of the season. From November to the following January, the dense cloud cover over the Baltic Sea region barely allowed any optical images qualiﬁed for the retrieval of remote sensed variables. The RMSEs of the 50 models were in the range of 30–80 µatm. The models trained with data from May–July showed the smaller RMSEs (41 µatm) than those trained with in-situ data from February–April and August–October (52 µatm and 55 µatm) (Figure 3D). Figure 3. Variables’ importance for pCO2 estimate in the entire Baltic Sea in different seasons (A–C) and the RMSEs of the corresponding 50 models (D). Figure 3. Variables’ importance for pCO2 estimate in the entire Baltic Sea in different seasons (A–C) and the RMSEs of the corresponding 50 models (D). Remote Sens. 2021, 13, 259 10 of 22 Overall, PAR showed the highest importance for pCO2 estimate in the Baltic Sea across different seasons and locations. SST was the second most important variable. aCDOM is important for pCO2 estimate in the Gulf of Finland. MLD is important for pCO2 estimate in all the sub-basins of the Baltic Sea but varied seasonally. SSS is important for pCO2 estimation in the Baltic Sea both spatially and temporally. Chl-a, which has been commonly considered as the determining variable for pCO2, showed low importance to the pCO2 estimate over the entire Baltic Sea and its sub-basins. Kd_490nm showed low importance for pCO2 estimation in the Baltic Sea across different seasons and sub-basins. 5.2. pCO2 Maps from Final Random Forest Model In September 2005, an area of pCO2 higher than both August and October was displayed in the southern Baltic Sea (Figure 5). 11 of 22 Remote Sens. 2021, 13, 259 Figure 5. Seasonal distribution of pCO2 in the Baltic Sea in the year of 2005 and the large rivers draining to the Baltic Sea. nal distribution of pCO2 in the Baltic Sea in the year of 2005 and the large rivers draining to the Baltic Sea. Figure 5. Seasonal distribution of pCO2 in the Baltic Sea in the year of 2005 and the large rivers draini The sea surface pCO2 in the Baltic Sea exhibited significant seasonal variations (Figure 5). Generally, low (undersaturated) pCO2 conditions of 100–300 µatm prevailed during sum- mer months (e.g., July) and the winter months (e.g., October) were characterized by over- saturated pCO2 conditions of up to 500 µatm (Figure 5). The pCO2 variation at different sites in the Baltic Sea also exhibited these characteristics (Figure 6). ( g ) The sea surface pCO2 in the Baltic Sea also showed signiﬁcant spatial gradient and variation along the months, particularly between April and September (Figure 5). In April, July, and August, the southern central Baltic Sea (excluding the sub-basin No.4 in Figure 1B) often displayed pCO2 approximately 100–150 µatm lower than the northern sub-basins (Figure 5). In May, the Gulf of Finland and the Gulf of Riga (Sub-basin No.2 in Figure 1B) showed the lowest pCO2 of 100 µatm in the Baltic Sea. In June, sea surface pCO2 in the two narrow gulfs increased slightly, while the Gulf of Bothnia exhibits its lowest seas surface pCO2 in a year. In September, the sea surface pCO2 in the southern Baltic Sea increased rapidly and displayed a reversed the gradient to that in August. In October, the pCO2 in the entire Baltic Sea was in the range of 380–420 µatm, rather homogenous in comparison to other months (Figures 5 and 6). On the other hand, different areas in the Baltic Sea showed their minimum pCO2 at different time. While the Gulf of Finland (No.42 in Figure 6A) and the Baltic Proper (i.e., No.61 in Figure 6A) had two seasonal minima in May and July, respectively, the Bothnia Sea (i.e., No.8 in Figure 6A) and the Bothnia Bay (No.28 in Figure 6A) showed their only seasonal minima of 180–250 µatm in June. 5.2. pCO2 Maps from Final Random Forest Model Thirdly, the seasonal change points of pCO2 int the Baltic Sea varied spatially. The pCO2 in the Bothnia Bay and Bothnia Sea started decreasing in May (Figure 6B,C), but the pCO2 in the Baltic Proper and Gulf of Finland in the south showed this change already in April, one month earlier (Figure 6D,E). The pCO2 in the Gulf of Bothnia (i.e., No.8 and 28 in Figure 6A) increased already in July, but such changes in the pCO2 in the southern Baltic Sea were delayed by one month to August. Consequently, in August, when pCO2 in the northern Baltic displayed are almost equal to the values in winter months (Figure 6B,C), pCO2 in the Baltic Proper and Gulf of Finland remained on the level of its summer value (Figure 6D,E). Furthermore, in the Gulf of Finland (i.e., No.42 in Figure 6A), signiﬁcant inter-annual pCO2 differences were present in April and August (Figure 6D), but, in the Baltic Proper (i.e., No.62, Figure 6A), this occurred in May, July, and August (Figure 6E). mote Sens. 2021, 13, 259 12 o Figure 6. Seasonal cycle of monthly pCO2 at different sites in the Baltic Sea. The pCO2 estimate for the months deter- mined with signiﬁcant upwelling effect in Section 4.2 were excluded from this analysis. (A): the location of the sites, (B–E): the seasonal cycle of monthly pCO2 at sites in sub-ﬁgure A. 12 of 22 Remote Sens. 2021, 13, 259 Figure 6. Seasonal cycle of monthly pCO2 at different sites in the Baltic Sea. The pCO2 estimate for the months deter- mined with signiﬁcant upwelling effect in Section 4.2 were excluded from this analysis. (A): the location of the sites, (B–E): the seasonal cycle of monthly pCO2 at sites in sub-ﬁgure A. Across the period of 2002–2011, the estimated pCO2 were correlated to the variables in the Baltic Sea to different degrees in different directions, varying spatially (Figure 7). The Chl-a-pCO2 correlation varied between −0.5 and 0.5, with general positive correlation in the northern Baltic Sea and negative correlation in the south. The estimated pCO2 were generally negatively correlated to the co-located aCDOM in the Baltic Sea with correlation coefﬁcients ranging from −1 to 0, and the correlation exhibited larger absolute coefﬁcients than Chl-a-pCO2 correlation, particularly in the southern Baltic Sea. 5.2. pCO2 Maps from Final Random Forest Model SST-pCO2 correlation mostly exhibited negative coefﬁcients (i.e., from −0.5 to 0) in the Baltic Sea, with larger absolute values in the south than in the north. Exceptionally high positive SST-pCO2 Remote Sens. 2021, 13, 259 13 of 22 13 of 22 correlation, up to 0.8, was present in the very west part of the Baltic Sea. The PAR-pCO2 correlation in the Baltic presented the largest absolute coefﬁcients and pCO2 was mostly negatively correlated to PAR in the entire Baltic Sea (i.e., from −1 to −0.6), showing the same pattern to the SST-pCO2 correlation. Kd_490nm-pCO2 correlation showed the similar pattern as Chl-a-pCO2, with slightly higher absolute coefﬁcients in the southeastern coasts. SSS exhibited high positive correlation to the co-located pCO2 at the coastal waters with values ranging from 0 to 0.8, mostly at 0. MLD was positively correlated to pCO2 in the entire Baltic Sea with large absolute coefﬁcients (0.5–1), except in the very north and west part of the sea. Figure 7. The spatial correlation between the estimate pCO2 and the variables the study period of 2002–2011. The size of grid cells is 0.5◦× 0.5◦. Minus value the variable was negatively correlated to the pCO2 there and the positive values mean positive correlations. Figure 7. The spatial correlation between the estimate pCO2 and the variables the study period of 2002–2011. The size of grid cells is 0.5◦× 0.5◦. Minus value the variable was negatively correlated to the pCO2 there and the positive values mean positive correlations. 5.3. Comparison of Random Forest and SOM arison of random forest, SOM, and multiple linear regression (MLR) in pCO2 estimation in the Baltic Sea. Figure 8. Comparison of random forest, SOM, and multiple linear regression (MLR) in pCO2 estim g p p g p (A,B) Comparison of a random forest model (i.e., RF in the ﬁgures) to the SOM model and MLR model trained with exactly the same in-situ data from 2/3 of months selected randomly; (C) Histograms of RMSE of 50 models trained in the same manner as in A and B; (D,E) Comparison of a random forest model to the SOM model and MLR model trained with exactly the same 2/3 of the in-situ data selected randomly; (F) Histograms of RMSE of 50 models constructed the same manner as in (D,E). 5.3. Comparison of Random Forest and SOM In the both schemes of training and validation data selection described in Section 4.5, majority of validation data were in the range of 100–500 µatm. The pCO2 estimated with random forest were in the same range as the validation data (Figure 8A,C). In con- trast, the SOM model constrained the pCO2 estimate into the range of 230–430 µatm (Figure 8A,C), particularly in the scheme No.2 where the training data were the randomly selected pCO2 measurements (Figure 8C). In addition, often one pCO2 value estimated from SOM responded to a large range of observed pCO2, forming evident horizontal features in the cross-validation (Figure 8A,D), particularly when the prediction covers multiple months. However, such patterns were not notable in the pCO2 estimated with random forest or MLR (Figure 8B,E). g In an example of 50 experiments where the training data were selected with scheme No.1 (Figure 8A,B), the coefﬁcient of determination of the random forest model prediction was 0.68, much larger than 0.58 and 0.6, the coefﬁcient of determination of the prediction with the SOM and MLR trained with the identical pCO2 measurements. The mean RMSE of the 50 random forest models trained with training data selected with scheme No.1 was 49 µatm, while the mean RMSE of their SOM and MLR counterparts were 55 and 62 µatm (Figure 8C). In the case of training data selected with scheme No.2, the mean RMSE of the 50 random forest models was 24 µatm, signiﬁcantly lower than 30 and 48 µatm, the respective means of RMSEs of the 50 SOM models and MLR models trained with the same sets of training data (Figure 8F). This indicated random forest outperformed SOM in the pCO2 estimation in the Baltic Sea. 14 of 22 Remote Sens. 2021, 13, 259 Figure 8. Comparison of random forest, SOM, and multiple linear regression (MLR) in pCO2 estimation in the Baltic Sea. (A,B) Comparison of a random forest model (i.e., RF in the ﬁgures) to the SOM model and MLR model trained with exactly the same in-situ data from 2/3 of months selected randomly; (C) Histograms of RMSE of 50 models trained in the same manner as in A and B; (D,E) Comparison of a random forest model to the SOM model and MLR model trained with exactly the same 2/3 of the in-situ data selected randomly; (F) Histograms of RMSE of 50 models constructed the same manner as in (D,E). 6.1. Characteristics of Variable Contribution to the pCO2 Estimate The high importance of PAR for pCO2 in the Baltic Sea and its sub-basins and the high correlation of this variable to sea surface pCO2 are attributed to the high seasonality of the sun illumination. Located at the high latitude of the Baltic Sea 54–66◦N (Figure 1), the sun illumination in the central Baltic Sea, for example, varies from 6 h in winter to 18 h in summer. As phytoplankton photosynthesis is largely determined by the available sun illumination, it is reasonable that seasonality of pCO2 aligns with that of PAR. In addition, river discharge loaded with CDOM, etc. is also characterized with high seasonality and, to large extent, synchronized to PAR [30], so is the bacteria respiration dependent on the available organic matter. Therefore, it is reasonable that PAR exhibited high importance for sea surface pCO2 estimation in the Baltic Sea and its sub-basins. The importance of PAR in the pCO2 estimate in the Baltic Sea in different seasons can be attributed to the wide span of the Baltic Sea (12◦) in latitude (Figure 1) and the resultant large gradient in sun illumination. On a day in spring, the sun illumination in the southern Baltic Sea is 2–3 h longer than that in the north, same for fall. The gradients in PAR largely impose differences in the intensities of phytoplankton photosynthesis, SST distribution, and ultimately to CO2 uptake of sea water via primary production. As for in summer when PAR and other variables displayed similar but low importance, sun illumination in the northern Baltic Sea is up to 6 h longer than in the southern Baltic Sea, displaying an even larger spatial gradient across the Baltic Sea than in other seasons. However, owing to snowmelt, the co-current freshwater discharge and the nutrients it loads are all very high in the Baltic Sea in late spring and early summer [30], create a high spatiality in the nutrient and DOM etc. Yet, the spatial pattern of cDOM etc. are likely different from that of PAR, depending on the sizes of catchment and land cover types. When all the processes determining pCO2 take place with similarly high intensities, none of the variables exhibit prominent importance, but all of them jointly determined the pCO2 in the Baltic Sea in summertime with similar degree (importance). 6.1. Characteristics of Variable Contribution to the pCO2 Estimate Likewise, the other variables also exhibited low importance for pCO2 estimate in May–July (Figure 3B). Yet, this was the case for the Baltic Sea, as for its applicability in other marginal seas, and the situation should be treated carefully. pp y g , y Overall, PAR exhibited the highest importance for the pCO2 estimation in the Baltic Sea across different sub-basin and nearly in every season, except summer. In addition, the PAR-pCO2 correlation coefﬁcients were of the largest absolute values among all the variable-pCO2 correlations (Figure 7). The high importance of PAR for pCO2 in the Baltic Sea and its sub-basins and the high correlation of this variable to sea surface pCO2 are attributed to the high seasonality of the sun illumination. Located at the high latitude of the Baltic Sea 54–66◦N (Figure 1), the sun illumination in the central Baltic Sea, for example, varies from 6 h in winter to 18 h in summer. As phytoplankton photosynthesis is largely determined by the available sun illumination, it is reasonable that seasonality of pCO2 aligns with that of PAR. In addition, river discharge loaded with CDOM, etc. is also characterized with high seasonality and, to large extent, synchronized to PAR [30], so is the bacteria respiration dependent on the available organic matter. Therefore, it is reasonable that PAR exhibited high importance for sea surface pCO2 estimation in the Baltic Sea and its sub-basins. The importance of PAR in the pCO2 estimate in the Baltic Sea in different seasons can be attributed to the wide span of the Baltic Sea (12◦) in latitude (Figure 1) and the resultant large gradient in sun illumination. On a day in spring, the sun illumination in the southern Baltic Sea is 2–3 h longer than that in the north, same for fall. The gradients in PAR largely impose differences in the intensities of phytoplankton photosynthesis, SST distribution, and ultimately to CO2 uptake of sea water via primary production. As for in summer when PAR and other variables displayed similar but low importance, Overall, PAR exhibited the highest importance for the pCO2 estimation in the Baltic Sea across different sub-basin and nearly in every season, except summer. In addition, the PAR-pCO2 correlation coefﬁcients were of the largest absolute values among all the variable-pCO2 correlations (Figure 7). 6.1. Characteristics of Variable Contribution to the pCO2 Estimate We analyzed the importance of different variables to the pCO2 estimation in the Baltic Sea using random forest on different spatial and temporal scales. It was evident that the spatiotemporal variability in the variable’s importance was high, but some general patterns were visible. Chl-a displayed overall low importance (small contribution) to the pCO2 estimate across different spatial and temporal scales in the Baltic Sea (Figures 2 and 3). The Chl-a- pCO2 correlation in the Baltic Sea was also relatively low, compared to the other variables’ correlation to pCO2 (Figure 7). This was in contrast to previous ﬁndings that Chl-a was closely related to pCO2 in global oceans [13] and marginal seas, like the Gulf of Mexico [10]. The limited importance of Chl-a is probably due to: (1) In addition to Chl-a, PAR, and SST are also fundamental factors for the photosynthesis induced biological ﬁxation of carbon; (2) The studies that established or conﬁrmed correlations between Chl-a and pCO2 did not include aCDOM [13,76]. But high correlation (r > 0.9) was found between remotely sensed Chl-a and aCDOM in the Gulf of Mexico [17] and West Florida Shelf [41]. Chl-a and aCDOM also displayed similar spatiotemporal patterns in the Baltic Sea (Figure S8). In the analysis of variables’ importance, aCDOM exhibited a more pronounced response to pCO2 variation than Chl-a (Figure 2A), as it showed higher correlation to pCO2 than Chl-a did (Figure 7). Remote Sens. 2021, 13, 259 15 of 22 15 of 22 Similarly, sea surface pCO2 in the Gulf of Mexico is more closely related to aCDOM than to Chl-a [41]. However, despite its low importance for sea sur face pCO2 estimate in the Baltic Sea at all the spatial and temporal scales and its general low correlation to pCO2 (Figures 2, 3 and 7), we still regarded Chl-a as an important variable for the pCO2 estimation in the Baltic Sea. This is particularly the case during summer (i.e., May–July), when the cyanobacteria and phytoplankton blooms takes place often, uptakes CO2 and reduces the sea surface pCO2 in the Baltic Sea [58]. The low importance of Chl-a in May–Jul (summer in this study) (Figure 3B) is very likely that, during this time, the effect of absent Chl-a in the model was compensated by variables highly correlated to Chl- a during in this time (e.g., CDOM and SST). 6.1. Characteristics of Variable Contribution to the pCO2 Estimate g p Concerning the determination of the seasonality in sea surface pCO2, the Julian day of the year (DOY) has been frequently in previous studies [12,16]. However, in this study, PAR holds two advantages over DOY. Firstly, PAR is a direct measure of sun radiation available for photosynthesis, and it has physical meaning, while DOY is a proxy of the seasonality. Secondly, a trigonometric conversion is often applied on DOY to correctly proximate the seasonality. Speciﬁcally, the minus cosine of DOY was used for pCO2 estimate in waters in the northern hemisphere and cosine of DOY for waters in the southern hemisphere [16,18]. Consequently, a trigonometric conversion of DOY attributes a spatially constant value in the entire hemisphere and overlook the effect spatial gradient of sun illumination. In contrast, PAR captures well the spatial gradient of sun illumination along the longitude and express its effect on photosynthesis in the water. Therefore, we suggest that future sea surface pCO2 estimation consider the participation of PAR instead of DOY (Figure 1). Remote Sens. 2021, 13, 259 16 of 22 16 of 22 The SST holds the same position in the pattern of variables’ importance for pCO2 estimate in the Baltic Sea and its sub-basin (Figure 2). This was probably because the seasonality magnitudes of SST in each sub-basin are on the same order, particularly when the sub-basins are relatively small and well mixed horizontally. In many cases, despite its correlation to pCO2 being on the same order as the Chl-a-pCO2 and Kd_490nm-pCO2 correlations, SST showed a larger importance than Chl-a, which aligned with the prediction error produced by alternatively omitting the variables by [17]. In the pCO2 estimates for the Baltic Sea in different seasons, SST was more important in August–October than in other seasons (Figure 3). This was probably because, in fall, the large spatial gradient in SST in the Baltic Sea responded more to the pCO2 distribution at a similar degree as the PAR does, but more than other variables. For example, the sea surface in the Gulf of Bothnia starts freezing already in October and lower down the primary production, whereas the southern Baltic Sea remains open water at time and allow the biological CO2 uptake [77]. 6.1. Characteristics of Variable Contribution to the pCO2 Estimate p Despite its low importance for the pCO2 estimate for the entire Baltic Sea, aCDOM ex- hibited more important for the pCO2 estimate in the Gulf of Finland than in other sub- basins (Figure 2B). The aCDOM -pCO2 correlation in the Baltic Sea is also relatively large, particularly at the coast and in the Gulf of Finland (Figure 7). As mentioned previously, bacteria respiration produces CO2 by decomposing organic carbons, like DOM [14,15]. The relatively narrow waters of the Gulf of Finland receive a large terrestrial input of DOM from the rivers, including the Neva, which drains the largest sub-catchment of the Baltic Sea, approximately 1/6 of the total Baltic Sea catchment [30]. The changes of sea surface pCO2 in the Gulf of Finland largely responded to the changes in CDOM there. Therefore, aCDOM is important for pCO2 estimation in the Gulf of Finland (Figure 2B) and thus in the Baltic Sea, as well. Similar mechanism very likely applies at coastal waters re- ceiving river discharges. Moreover, this study used the aCDOM derived from MERIS images. The MERIS sensor was succeeded by the Ocean and Land Color Instrument (OLCI) sensors on Sentinel-3 satellites in 2016. Therefore, aCDOM derived from OLCI images will likely play an equivalent role in the pCO2 estimate in the Baltic Sea and other similar waters. Though less than PAR and sometimes slightly less than SST, MLD was important for the pCO2 estimation in the Baltic Sea and all its sub-basins (Figure 2B). pCO2 in the Baltic Sea is largely and positively correlated to MLD (Figure 7). This is probably resulted from the seasonally varying amount of fresh water discharged by the many rivers and lay above the relatively saline and heavy water [78]. In addition, seasonal winds in the Baltic Sea might have jointly determined the high variation of MLD [32] and, consequently, the vertical mixing of sea water and pCO2, as well. In this study, Kd_490 nm showed low importance to the pCO2 estimation in the Baltic Sea, regardless of season or sub-basin (Figures 2 and 3) and a relatively weaker correlations to pCO2 (i.e., from −0.7 to 0), compared to variables, like PAR and aCDOM. This aligns with the previously found negatively correlation between Kd_490 nm and pCO2 in the Gulf of Mexico [16]. 6.1. Characteristics of Variable Contribution to the pCO2 Estimate Here, we argue that the reasons behind the low contribution of Chl-a to pCO2 estimation very likely also applied to Kd_490nm. This argument is well supported by previous studies. It is found that Kd_490nm in the Baltic Sea was a function of inherent optical properties, i.e., absorption and scattering of phytoplankton, and effects of illumination and viewing angle [79,80]. Furthermore, [81] observed a strong positive correlation between Kd_490nm and river discharge into the Baltic Sea and the latter is rich of CDOM. In addition, a positive correlation of Kd_490nm to Chl-a and aCDOM were noticed in the Baltic Sea (S9), and the Kd_490nm-pCO2 and aCDOM-pCO2 correlations also exhibited similar patterns (Figure 7). 6.2. Impact of Unbalanced In-Situ Measurements Distribution on the Model for pCO2 Estimate The processes controlling pCO2 across the Baltic sea (e.g., phytoplankton photosynthe- sis, bacteria respiration and runoff) vary spatially and temporally [30,82] and thus increase the difﬁculties in mapping pCO2 in the Baltic Sea with high accuracy; (4) Upwelling take places in the Baltic Sea with varying frequencies among years and months [83] and com- plicates the pCO2 process in multiple manners [34,84]. Even though we eliminated the months dominated by upwelling, few upwelling might have remained in the rest of the months and increased the RMSE of the model; (5) Most importantly, the random forest model covered the processes that took places in the entire Baltic Sea in all the seasons in the period of 2002–2011. This task itself is a challenging one due to the above factors. All these factors rendered deriving sea surface pCO2 in the Baltic Sea more challenging than in other marginal seas. g The random forest algorithm outperformed SOM and MLR in the sea surface pCO2 estimation (Figure 8). We attributed this to how the three algorithms treated the variables. In random forest, a series of forests were constructed, and the most effective one was chosen for prediction [59,65]. While the variables and training samples were randomly selected for the tree construction, the best model was the one with little participation of the unimportant variables. In contrast, when the mode was constructed with SOM, all the input variables had the same weights [70]. This very likely ampliﬁed the contribution of the unimportant or correlated variables and suppresses the important ones at the corresponding temporal and spatial scale, thus caused misestimates (Figure 8A,C). The variants of SOM, such as SOMLO, probably also inherit such effects. MLR attributed weights to the input variables by determining their correlation coefﬁcients to the dependent variables. The effect of the coefﬁcients is very evident in the case when the training samples were chosen across months and cover a large variation. For example, in the experiments in Figure 8A–C, the samples covered 2/3 of the months and performed RMSE similar to that of random forest and better than SOM. In contrast, in the experiment where the samples were 2/3 of the entire in-situ data set from random selection, samples from the same season/months of high similarity were likely used. 6.2. Impact of Unbalanced In-Situ Measurements Distribution on the Model for pCO2 Estimate 6.2. Impact of Unbalanced In-Situ Measurements Distribution on the Model for pCO2 Estimate The in-situ pCO2 measurements available in the Baltic Sea during 2002–2011 were unevenly distributed, namely, relatively sparse measurements in the north and dense measurements in the south (Figure 1). In order to ensure the participation of the in- Remote Sens. 2021, 13, 259 17 of 22 17 of 22 situ data from the northern Baltic Sea, we selected in-situ data month-wise to train and validate the model for pCO2 estimation, instead of randomly selecting from the in-situ measurements. However, this measure led to the missing determination of variables’ importance for the Gulf of Bothnia due to the few months of in-situ measurements in this basin (i.e., March 2006 and September 2009). In the future, including additional in-situ pCO2 measurements from the Gulf of Bothnia can help analyze the variables’ importance for the pCO2 estimate in that region and understand the processes controlling pCO2 there. These additional in-situ pCO2 measurements are also expected to improve the RMSE of pCO2 estimate for the entire Baltic Sea. Despite the unbalanced distribution of in-situ data in the Baltic Sea, the monthly pCO2 maps were retrieved for the Baltic Sea for the period of August–October 2011 (Figure 5). The RMSE of the model for pCO2 estimation was 47.8 µatm (Figure 4), slightly larger than 25 µatm and 31.7 µatm, the RMSEs of the models constructed by [16] and [17], respectively, for pCO2 estimation in the Gulf of Mexico using similar tree-based regression algorithms. Still, the RMSE of 47.8 µatm is relatively small for pCO2 estimation in the Baltic Sea, considering the following factors: (1) the pCO2 estimation was undertaken on the monthly frequency, where the in-situ data from entire month was integrated to the few days with remote sensing images; (2) The magnitudes of the seasonal changes in pCO2 in the Baltic Sea are much larger than that in middle or low latitude marginal seas. For example, the pCO2 in the Baltic Sea was in the range of 100–600 µatm (Figure 8), while, in the Gulf of Mexico, it was 200–450 µatm [16], and, in the South China Sea, it was 250–450 µatm [11]; (3). 6.3. pCO2 Maps for the Baltic Sea and Its Spatiotemporal Characteristics In this study, we produced the monthly pCO2 maps for the entire Baltic Sea over the period of August 2002–October 2020. These maps showed that pCO2 across the Baltic Sea was characterized by strong seasonality, generally, high pCO2 in winter and low pCO2 in summer (Figures 5 and 6). The trend aligned well with that derived from in- situ data in the Baltic Sea [85]. The seasonality of pCO2 in the Baltic Sea was similar to that in the marginal sea of Gulf of Maine but different from the one observed in Gulf of Mexico by [16]. In addition, the range of seasonal pCO2 variation in the Baltic Sea (i.e., 100–500 µatm) was larger than that observed for the two marginal seas (i.e., 300–500 µatm) (Figures 5 and 6) [16]. These different seasonal variations trends and variables’ importance (e.g., Kd_490nm) suggest that the processes determining the pCO2 in the Baltic Sea are likely different from that observed in other seas, or same processes work on different intensity, for example, the gradient in PAR. In addition to the similar seasonal trend, minor differences exist in the seasonal trends of pCO2 in the Baltic Sea. For example, Baltic Proper and the Gulf of Finland showed pCO2 minima both in May and July, while, in the Bothnia Bay and Bothnia Sea, it was only shown on minima in June (Figure 6). May is the time when most rivers pass their annual peak of water levels [30], and, in July, the daytime is the longest in a year in Baltic Sea, with the most sunny days. In addition, different areas in the Baltic Sea showed interannual variations in different months (Figure 6). For example, the waters in the Gulf of Finland exhibited large interannual variation in April (Figure 6D), when the large river input take place in the sub-basin [27]. The Baltic Proper showed such variations during May–July (Figure 6E), when the primary production is high in this sub-basin and upwelling also occurs very often there [58,68]. This indicates that the dominantly driver of pCO2 are spatially variable across the Baltic Sea. The pCO2 maps derived from this model exhibited continuous transitions between the sub-basins of the Baltic Sea (Figure 5). Therefore, these maps are a signiﬁcant improvement from those produced in previous studied by dividing the Baltic sea into different sub-basins [12]. 6.2. Impact of Unbalanced In-Situ Measurements Distribution on the Model for pCO2 Estimate Given that the time window of in-situ data was narrowed down to 9:00–14:00, and the in-situ data from the months dominated by upwelling were also removed, we did not consider the effect of outlier on the modeling and the errors produced by the models were regarded to be from the misestimate of the models. Overall, random forest performs better than MLR and SOM regardless of the variation range of the training data. MLR performs better than SOM when the training data cover a large Remote Sens. 2021, 13, 259 18 of 22 18 of 22 variation, and SOM performs better than MLR when the training data cover a relatively small variation. 6.3. pCO2 Maps for the Baltic Sea and Its Spatiotemporal Characteristics 7. Conclusions This study analyzed the variables’ importance in the pCO2 estimation for the Baltic Sea across different time and sub-basins with the support of remote sensing and derived pCO2 maps for the Baltic Sea from August 2002 to October 2011. We found that the contributions of the variables to pCO2 retrieval for the Baltic Sea vary both spatially and temporally and likely replicated the spatiotemporal characteristics of the driving forces. Among all the variables, PAR was the most important, followed by SST and MLD. Chl-a contributed surprisingly little to the pCO2 estimate. aCDOM was important for the pCO2 estimation for the Gulf of Finland and the Gulf of Riga. The random forest model used for the pCO2 estimate for the entire Baltic Sea had the RMSE of 47.8 µatm, MAE of −3.26 µatm, and coefﬁcient of determination of 0.63. These pCO2 maps derived in this study are one of the most reliable pCO2 ﬁelds in the Baltic Sea and can potentially support determining the role of the Baltic Sea as sink/source of the atmospheric CO2. Moreover, the variables importance/relevance from this study can provide a benchmark for understanding the different drivers of pCO2 in the Baltic Sea and how they vary in different time and space. In the Baltic Sea region, frequent clouds in November, December, and January lead to the absence of pCO2 maps during those three months. This is an inevitable situation considering the high-latitude location of the Baltic Sea. Derivation of sea surface pCO2 for the Baltic Sea in the wintertime needs to be achieved by combining the remote sensing supported results with additional sources information, e.g., modeling. 19 of 22 19 of 22 Remote Sens. 2021, 13, 259 Supplementary Materials: The following are available online at https://www.mdpi.com/2072-429 2/13/2/259/s1, Figure S1: Spatial and temporal distributions of the in-situ data used for training and validating the pCO2 estimate. Figure S2: Diurnal effect on the pCO2 estimate. Figure S3: Scenarios where the upwelling affects the pCO2 estimate from remote sensing images. Figure S4: The effect of upwelling in the pCO2 estimate with remote sensing image. Figure S5: The monthly mean product of Chl-a derived from MODIS and MERIS images in May, July and September 2011 mapping the Baltic Sea. Figure S6: aCDOM from MODIS and MERIS in the Baltic Sea. Figure S7: The performance differences between of Chl-a from MODIS and Chl-a from MERIS in the pCO2 estimate. 7. Conclusions Figure S8: Alternative of the ﬁnal model for pCO2 estimate in the entire Baltic Sea. Figure S9: Relationship between variables in the Baltic Sea. Author Contributions: S.Z., A.R. and P.P. designed the study. S.Z. did the data collection, analysis and manuscript preparation. Writing—review & editing, S.Z., A.R., P.P. and M.B.W. Investigation, S.Z., P.P. and M.B.W. All authors have read and agreed to the published version of the manuscript. Funding: Swedish National Space Board (Project No: 174/17) and the BONUS Blue Baltic (Call No. 2015-101: Integrated carboN and TracE Gas monitoRing for the bALtic sea) funded this study. Funding: Swedish National Space Board (Project No: 174/17) and the BONUS Blue Baltic (Call No. 2015-101: Integrated carboN and TracE Gas monitoRing for the bALtic sea) funded this study. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The computations were performed on Swedish National Infrastructure for Computing (SNIC) through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) (Project No. SNIC 2019-8-223 & SNIC2019-30-7). The MODIS data were provided by Physical Oceanography Distributed Active Archive Center (PODAAC: https://podaac.jpl.nasa. gov/). Copernicus Marine Environment Monitoring Service (CMEMS: https://marine.copernicus. eu/) provided SSS and MLD data. The ICOS (Integrated Carbon Observation System) station Östergarnsholm is funded by Swedish Research Council and Uppsala University. Bernd Schneider and Matti Perttilä coordinated in the provision of original data used by Löfﬂer et al. (2012). We are thankful to the constructive comments from three anonymous reviewers. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Gruber, N.; Clement, D.; Carter, B.R.; Feely, R.A.; Van Heuven, S.; Hoppema, M.; Ishii, M.; Key, R.M.; Kozyr, A.; Lauvset, S.K.; et al. The oceanic sink for anthropogenic CO2 from 1994 to 2007. Science 2019, 363, 1193–1199. [CrossRef] 1. Gruber, N.; Clement, D.; Carter, B.R.; Feely, R.A.; Van Heuven, S.; Hoppema, M.; Ishii, M.; Key, R.M.; Kozyr, A.; Lauvset, S.K.; et al. The oceanic sink for anthropogenic CO2 from 1994 to 2007. Science 2019, 363, 1193–1199. [CrossRef] p g 2. Laruelle, G.G.; Cai, W.; Hu, X.; Gruber, N.; MacKenzie, F.T.; Regnier, P. Continental shelves as a variable but increasing global sink for atmospheric carbon dioxide. Nat. Commun. 2018, 9, 1–11. [CrossRef] b b h l f ll i l [ f] 2. Laruelle, G.G.; Cai, W.; Hu, X.; Gruber, N.; MacKenzie, F.T.; Regnier, P. Continental shelves as a sink for atmospheric carbon dioxide. Nat. Commun. 2018, 9, 1–11. [CrossRef] 3. Gruber, N. Carbon at the coastal interface. Nat. Cell Biol. 2015, 517, 148–149. [CrossRef] 4. Laruelle, G.G.; Lauerwald, R.; Pfeil, B.; Regnier, P. Regionalized global budget of the CO2exchangeat the air-water interface in continental shelf seas. Glob. Biogeochem. Cycles 2014, 28, 1199–1214. [CrossRef] g y 5. Hofmann, E.E.; Cahill, B.; Fennel, K.; Friedrichs, M.A.; Hyde, K.; Lee, C.; Mannino, A.; Najjar, R.G.; O’Reilly, J.E.; Wilkin, J.; et al. Modeling the Dynamics of Continental Shelf Carbon. Annu. Rev. Mar. Sci. 2011, 3, 93–122. [CrossRef] 6. Fennel, K.; Wilkin, J.; Previdi, M.; Najjar, R. Denitriﬁcation effects on air-sea CO2 ﬂux in the coastal ocean.pdf. Geophys. Res. Lett. 2008, 35, 1–5. [CrossRef] 7. Xue, L.; Cai, W.-J.; Hu, X.; Sabine, C.L.; Jones, S.M.; Sutton, A.J.; Jiang, L.-Q.; Reimer, J.J. Sea surface carbon dioxide at the Georgia time series site (2006–2007): Air–sea ﬂux and controlling processes. Prog. Oceanogr. 2016, 140, 14–26. [CrossRef] 7. Xue, L.; Cai, W.-J.; Hu, X.; Sabine, C.L.; Jones, S.M.; Sutton, A.J.; Jiang, L.-Q.; Reimer, J.J. Sea surface carbon dioxide at the Georgia time series site (2006–2007): Air–sea ﬂux and controlling processes. Prog. Oceanogr. 2016, 140, 14–26. [CrossRef] 8. Schneider, B.; Müller, J.D. Biogeochemical Transformations in the Baltic Sea–Observations through Carbon Dioxide Glasses; Springer: l / d lb G S 9. Chierici, M.; Signorini, S.R.; Mattsdotter-Björk, M.; Fransson, A.; Olsen, A. Surface water fCO2 algorithms for the high-latitude Paciﬁc sector of the Southern Ocean. Remote Sens. Environ. 2012, 119, 184–196. [CrossRef] 10. Chen, S.; Hu, C.; Cai, W.; Yang, B. References Estimating surface pCO2 in the northern Gulf of Mexico: Which remote sensing model to use? Cont. Shelf Res. 2017, 151, 94–110. [CrossRef] f 11. Jo, Y.-H.; Dai, M.; Zhai, W.; Yan, X.-H.; Shang, S. On the variations of sea surfacepCO2in the northern South China Sea: A remote sensing based neural network approach. J. Geophys. Res. Space Phys. 2012, 117, 1–13. [CrossRef] 12. Parard, G.; Charantonis, A.A.; Rutgersson, A. Remote sensing the sea surface CO2 of the Baltic Sea using the SOMLO methodology. Biogeosciences 2015, 12, 3369–3384. [CrossRef] 13. Fay, A.R.; McKinley, G. Correlations of surface ocean pCO2 to satellite chlorophyll on monthly to interannual timescales. Glob. Biogeochem. Cycles 2017, 31, 436–455. [CrossRef] Remote Sens. 2021, 13, 259 20 of 22 14. Gustafsson, E.; Omstedt, A.; Gustafsson, B.G. The air-water CO2 exchange of a coastal sea—A sensitivity study on factors that inﬂuence the absorption and outgassing of CO2 in the Baltic Sea. J. Geophys. Res. Oceans 2015, 120, 5342–5357. [CrossRef] 15. Gustafsson, E.; Deutsch, B.; Gustafsson, B.; Humborg, C.; Mörth, C.-M. Carbon cycling in the Baltic Sea—The fate of allochthonous organic carbon and its impact on air–sea CO2 exchange. J. Mar. Syst. 2014, 129, 289–302. [CrossRef] 16. Chen, S.; Hu, C.; Barnes, B.B.; Wanninkhof, R.; Cai, W.-J.; Barbero, L.; Pierrot, D. A machine learning approach to estimate surface ocean pCO2 from satellite measurements. Remote Sens. Environ. 2019, 228, 203–226. [CrossRef] 17. Lohrenz, S.E.; Cai, W.; Chakraborty, S.; Huang, W.-J.; Guo, X.; He, R.; Xue, Z.G.; Fennel, K.; Howden, S.; Tian, H. Satellite estimation of coastal pCO2 and air-sea ﬂux of carbon dioxide in the northern Gulf of Mexico. Remote Sens. Environ. 2018, 207, 71–83. [CrossRef] 18. Ikawa, H.; Faloona, I.; Kochendorfer, J.; Paw, K.T.; Oechel, W. Air–sea exchange of CO2 at a Northern California coastal site along the California Current upwelling system. Biogeosciences 2013, 10, 4419–4432. [CrossRef] p g y g 19. Joshi, I.D.; Ward, N.D.; D’Sa, E.J.; Osburn, C.L.; Bianchi, T.S.; Oviedo-Vargas, D. Seasonal Trends i CO2 Fluxes in Apalachicola Bay, Florida, From VIIRS Ocean Color. J. Geophys. Res. Biogeosci. 2018, p g y g Joshi, I.D.; Ward, N.D.; D’Sa, E.J.; Osburn, C.L.; Bianchi, T.S.; Oviedo-Vargas, D. Seasonal Trends in Surfa CO2 Fluxes in Apalachicola Bay, Florida, From VIIRS Ocean Color. J. Geophys. Res. Biogeosci. 2018, 123, 2466–2484. [CrossRef] 20. References Telszewski, M.; Chazottes, A.; Schuster, U.; Watson, A.J.; Moulin, C.; Bakker, D.C.E.; González-Dávila, M.; Johannessen, T.; Körtzinger, A.; Lüger, H.; et al. Estimating the monthly pCO2 distribution in the North Atlantic using a self-organizing neural network. Biogeosciences 2009, 6, 1405–1421. [CrossRef] 21. Friedrich, T.; Oschlies, A. Neural network-based estimates of North Atlantic surface pCO2 from satellit study. J. Geophys. Res. Space Phys. 2009, 114, 1–12. [CrossRef] y p y p y 22. Hales, B.; Strutton, P.G.; Saraceno, M.; Letelier, R.; Takahashi, T.; Feely, R.; Sabine, C.; Chavez, F. Satellite-based prediction of pCO2 in coastal waters of the eastern North Paciﬁc. Prog. Oceanogr. 2012, 103, 1–15. [CrossRef] 23. Salisbury, J.; VanDeMark, D.; Hunt, C.W.; Campbell, J.W.; McGillis, W.R.; McDowell, W.H. Seasonal observations of surface waters in two Gulf of Maine estuary-plume systems: Relationships between watershed attributes, optical measurements and surface pCO2. Estuar. Coast. Shelf Sci. 2008, 77, 245–252. [CrossRef] p 24. Signorini, S.R.; Mannino, A.; Najjar, R.G., Jr.; Friedrichs, M.A.M.; Cai, W.-J.; Salisbury, J.; Wang, Z.A.; Thomas, H.; Shadwick, E.H. Surface ocean p CO2 seasonality and sea-air CO2 ﬂux estimates for the North American east coast. J. Geophys. Res. Oceans 2013, 118, 5439–5460. [CrossRef] 25. Bai, Y.; Cai, W.-J.; He, X.; Zhai, W.; Pan, D.; Dai, M.; Yu, P. A mechanistic semi-analytical method for remotely sensing sea surfacepCO2 in river-dominated coastal oceans: A case study from the East China Sea. J. Geophys. Res. Oceans 2015, 120, 2331–2349. [CrossRef] [ ] 26. Song, X.; Bai, Y.; Cai, W.; Chen, C.-T.A.; Pan, D.; He, X.; Zhu, Q. Remote Sensing of Sea Surface pCO2 in the Bering Sea in Summer Based on a Mechanistic Semi-Analytical Algorithm (MeSAA). Remote Sens. 2016, 8, 558. [CrossRef] 27 Bergström S ; Carlsson B River runoff to the Baltic Sea Ambio 1994 23 280 287 g 28. Omstedt, A.; Elken, J.; Lehmann, A.D.; Piechura, J. Knowledge of the Baltic Sea physics gained during the BALTEX and related programmes. Prog. Oceanogr. 2004, 63, 1–28. [CrossRef] 29. Meier, H.E.M.; Rutgersson, A.; Reckermann, M. An Earth System Science Program for the Baltic Sea Region. Eos 2014, 95, 109–110. [CrossRef] 30. Käyhkö, J.; Apsite, E.; Bolek, A.; Filatov, N.; Kondratyev, S.; Korhonen, J.; Kriauˇci¯unien˙e, J.; Lindström, G.; Nazarova, L.; Pyrh, A.; et al. Recent Change—River Run-off and Ice Cover. In Second Assessment of Climate Change for the Baltic Sea Basin; l l d l / d lb 30. References Käyhkö, J.; Apsite, E.; Bolek, A.; Filatov, N.; Kondratyev, S.; Korhonen, J.; Kriauˇci¯unien˙e, J.; Lindström, G.; Nazarova, L.; Pyrh, A.; et al. Recent Change—River Run-off and Ice Cover. In Second Assessment of Climate Change for the Baltic Sea Basin; Regional Climate Studies Series; Springer: Berlin/Heidelberg, Germany, 2015; pp. 99–115. ISBN 9783319160054. 31. Schneider, B.; Dellwig, O.; Kuli´nski, K.; Omstedt, A.; Pollehne, F.; Rehder, G.; Savchuk, O. Ecological processes in the Baltic Sea. In Biological Oceanography of the Baltic Sea; Snoeijs-Leijonmalm, P., Schubert, H., Radziejewska, T., Eds.; Springer Nature: Berlin/Heidelberg, Germany, 2017; Volume 30, pp. 87–278. ISBN 9789400706675. g y pp 32. Lehmann, A.; Myrberg, K. Upwelling in the Baltic Sea—A review. J. Mar. Syst. 2008, 74, S3–S12. [CrossRef] g y pp ehmann, A.; Myrberg, K. Upwelling in the Baltic Sea—A review. J. Mar. Syst. 2008, 74, S3–S12. [CrossRef] orman M ; Parampil S R ; Rutgersson A ; Sahlée E Inﬂuence of coastal upwelling on the air–sea gas exc y g p g y 33. Norman, M.; Parampil, S.R.; Rutgersson, A.; Sahlée, E. Inﬂuence of coastal upwelling on the air Baltic Sea Basin. Tellus B Chem. Phys. Meteorol. 2013, 65, 1–16. [CrossRef] 33. Norman, M.; Parampil, S.R.; Rutgersson, A.; Sahlée, E. Inﬂuence of coastal upwelling on the air–sea gas exchange of CO2 in a Baltic Sea Basin. Tellus B Chem. Phys. Meteorol. 2013, 65, 1–16. [CrossRef] mund, N.; Nausch, G.; Voss, M. Upwelling events may cause cyanobacteria blooms in the Baltic Sea. J. M –76. [CrossRef] 35. Wesslander, K.; Hall, P.; Hjalmarsson, S.; Lefèvre, M.; Omstedt, A.; Rutgersson, A.; Sahlée, E.; Tengberg, A. Observed carbon dioxide and oxygen dynamics in a Baltic Sea coastal region. J. Mar. Syst. 2011, 86, 1–9. [CrossRef] 36. Barnes, B.B.; Hu, C.; Schaeffer, B.A.; Lee, Z.; Palandro, D.; Lehrter, J.C. MODIS-derived spatiotemporal water clarity patterns in optically shallow Florida Keys waters: A new approach to remove bottom contamination. Remote Sens. Environ. 2013, 134, 377–391. [CrossRef] 37. Hu, C.; Muller-Karger, F.E.; Taylor, C.J.; Carder, K.L.; Kelble, C.; Johns, E.; Heil, C.A. Red tide detection and tracing using MODIS ﬂuorescence data: A regional example in SW Florida coastal waters. Remote Sens. Environ. 2005, 97, 311–321. [CrossRef] 38. Shi, K.; Zhang, Y.; Zhu, G.; Liu, X.; Zhou, Y.; Xu, H.; Qin, B.; Liu, G.; Li, Y. Long-term remote monitoring of total suspended matter concentration in Lake Taihu using 250 m MODIS-Aqua data. Remote Sens. Environ. 2015, 164, 43–56. References [CrossRef] 39. Liu, B.; D’Sa, E.J.; Joshi, I.D. Multi-decadal trends and inﬂuences on dissolved organic carbon distribution in the Barataria Basin, f d d / b [ f] 37. Hu, C.; Muller-Karger, F.E.; Taylor, C.J.; Carder, K.L.; Kelble, C.; Johns, E.; Heil, C.A. Red tide detection and tracing using MODIS ﬂuorescence data: A regional example in SW Florida coastal waters. Remote Sens. Environ. 2005, 97, 311–321. [CrossRef] g p , , [ ] 38. Shi, K.; Zhang, Y.; Zhu, G.; Liu, X.; Zhou, Y.; Xu, H.; Qin, B.; Liu, G.; Li, Y. Long-term remote monitoring of total suspended matter concentration in Lake Taihu using 250 m MODIS-Aqua data. Remote Sens. Environ. 2015, 164, 43–56. [CrossRef] 39. Liu, B.; D’Sa, E.J.; Joshi, I.D. Multi-decadal trends and inﬂuences on dissolved organic carbon distribution in the Barataria Basin, Louisiana from in-situ and Landsat/MODIS observations. Remote Sens. Environ. 2019, 228, 183–202. [CrossRef] 21 of 22 21 of 22 Remote Sens. 2021, 13, 259 40. Parard, G.; Charantonis, A.; Rutgersson, A. Using satellite data to estimate partial pressure of CO2 in the Baltic Sea. J. Geophys. Res. Biogeosci. 2016, 121, 1002–1015. [CrossRef] g 41. Chen, S.; Hu, C.; Byrne, R.H.; Robbins, L.L.; Yang, B. Remote estimation of surface pCO2 on the West Florida Shelf. Cont. Shelf Res. 2016, 128, 10–25. [CrossRef] 42. Gower, J.; King, S. Satellite Images Show the Movement of Floating Sargassum in the Gulf of Mexico and Atlantic Ocean. Nat. Preced. 2008, 1–13. [CrossRef] Borstad, G.; Brown, L. Detection of intense plankton blooms using the 709 nm band of the MERIS imaging Remote Sens. 2005, 26, 2005–2012. [CrossRef] 43. Gower, J.; King, S.; Borstad, G.; Brown, L. Detection of intense plankton blooms using the 709 nm ban spectrometer. Int. J. Remote Sens. 2005, 26, 2005–2012. [CrossRef] 44. Matthews, M.W. Eutrophication and cyanobacterial blooms in South African inland waters: 10 years of MERIS observations. Remote Sens. Environ. 2014, 155, 161–177. [CrossRef] 45. Attila, J.; Kauppila, P.; Kallio, K.Y.; Alasalmi, H.; Keto, V.; Bruun, E.; Koponen, S. Applicability of Earth Observation chlorophyll-a data in assessment of water status via MERIS—With implications for the use of OLCI sensors. Remote Sens. Environ. 2018, 212, 273–287. [CrossRef] , [ ] 46. Vilas, L.G.; Spyrakos, E.; Palenzuela, J.M.T. Neural network estimation of chlorophyll a from MERIS full resolution data for the coastal waters of Galician rias (NW Spain). Remote Sens. Environ. 2011, 115, 524–535. References Pfeil, B.; Olsen, A.; Bakker, D.C.E.; Hankin, S.; Koyuk, H.; Kozyr, A.; Malczyk, J.; Manke, A.; Metzl, N.; Sabine, C.L.; et al. A uniform, quality controlled Surface Ocean CO2 Atlas (SOCAT). Earth Syst. Sci. Data 2013, 5, 125–143. [CrossRef] 58 Schneider B ; Kaitala S ; Maunula P Identiﬁcation and quantiﬁcation of plankton bloom events in the Baltic Sea by continuous A uniform, quality controlled Surface Ocean CO2 Atlas (SOCAT). Earth Syst. Sci. Data 2013, 5, 125–143. [CrossRef] 58. Schneider, B.; Kaitala, S.; Maunula, P. Identiﬁcation and quantiﬁcation of plankton bloom events in the Baltic Sea by continuous pCO and chloroph ll a measurements on a cargo ship J Mar Syst 2006 59 238 248 [CrossRef] , q y 2 ( ) y , , [ ] 58. Schneider, B.; Kaitala, S.; Maunula, P. Identiﬁcation and quantiﬁcation of plankton bloom events in the Baltic Sea by continuous pCO2 and chlorophyll a measurements on a cargo ship. J. Mar. Syst. 2006, 59, 238–248. [CrossRef] 58. Schneider, B.; Kaitala, S.; Maunula, P. Identiﬁcation and quantiﬁcation of plankton bloom events in the pCO2 and chlorophyll a measurements on a cargo ship. J. Mar. Syst. 2006, 59, 238–248. [CrossRef] p p y g p y 59. Breiman, L. Random Forest; University of California Berkeley: Berkeley, CA, USA, 2001. 59. Breiman, L. Random Forest; University of California Berkeley: Berkeley, CA, USA, 2001. 60. Belgiu, M.; Drăgut, L. Random forest in remote sensing: A review of applications and future directions. ISPRS J. Photogramm. Remote Sens. 2016, 114, 24–31. [CrossRef] A.; Van Der Linden, S. Classifying Multilevel Imagery from SAR and Optical Sensors by Decision Fusion Remote Sens. 2008, 46, 1457–1466. [CrossRef] 61. Waske, B.; Member, A.; Van Der Linden, S. Classifying Multilevel Imagery from SAR and Optical IEEE Trans. Geosci. Remote Sens. 2008, 46, 1457–1466. [CrossRef] 62. Wolanin, A.; Camps-Valls, G.; Gómez-Chova, L.; Mateo-García, G.; Van Der Tol, C.; Zhang, Y.; Guanter, L. Estimating crop primary productivity with Sentinel-2 and Landsat 8 using machine learning methods trained with radiative transfer simulations. Remote Sens. Environ. 2019, 225, 441–457. [CrossRef] 63. Wei, Z.; Meng, Y.; Zhang, W.; Peng, J.; Meng, L. Downscaling SMAP soil moisture estimation with gr regression over the Tibetan Plateau. Remote Sens. Environ. 2019, 225, 30–44. [CrossRef] 64. Liu, X.; Guanter, L.; Liu, L.; Damm, A.; Malenovský, Z.; Rascher, U.; Peng, D.; Du, S.; Gastellu-Etchegorry, J.-P. References [CrossRef] l ll h ll f b é h 47. Loisel, H.; Mangin, A.; Vantrepotte, V.; Dessailly, D.; Dinh, D.N.; Garnesson, P.; Ouillon, S.; Lefebvre, J.-P.; Mériaux, X.; Phan, T.M. Variability of suspended particulate matter concentration in coastal waters under the Mekong’s inﬂuence from ocean color (MERIS) remote sensing over the last decade. Remote Sens. Environ. 2014, 150, 218–230. [CrossRef] g 48. Kutser, T.; Verpoorter, C.; Paavel, B.; Tranvik, L.J. Estimating lake carbon fractions from remote sensing data. Remote Sens. Environ. 2015, 157, 138–146. [CrossRef] 49. Kratzer, S.; Brockmann, C.; Moore, G. Using MERIS full resolution data to monitor coastal waters—A case study from Himmer- fjärden, a fjord-like bay in the northwestern Baltic Sea. Remote Sens. Environ. 2008, 112, 2284–2300. [CrossRef] j j y 50. Schroeder, T.; Schaale, M.; Fischer, J. Retrieval of atmospheric and oceanic properties from MERIS measurements: A new Case-2 water processor for BEAM. Int. J. Remote Sens. 2007, 28, 5627–5632. [CrossRef] p 51. Olsen, A.; Brown, K.R.; Chierici, M.; Johannessen, T.; Neill, C.J. Sea-surface CO2 fugacity in the subpolar North Atlantic. Biogeosciences 2008, 5, 535–547. [CrossRef] 52. Chierici, M.; Olsen, A.; Johannesen, T.; Trinañes, J.; Wanninkhof, R. Algorithms to estimate the carbon dioxide uptake in the northern North Atlantic using shipboard observations, satellite and ocean analysis data. Deep Sea Res. Part II Top. Stud. Oceanogr. 2009, 56, 630–639. [CrossRef] [ ] 53. Axell, L. CMEMS Baltic Sea Physical Reanalysis Product BALTICSEA_REANALYSIS_PHY_003_011; EU Copernicus Marine Service: Toulouse, France, 2019. 54. Bakker, D.C.E.; Pfeil, B.; Landa, C.S.; Metzl, N.; O’Brien, K.M.; Olsen, A.; Smith, K.; Cosca, C.; Harasawa, S.; Jones, S.D.; et al. A multi-decade record of high-quality fCO2 data in version 3 of the Surface Ocean CO2 Atlas (SOCAT). Earth Syst. Sci. Data 2016, 8, 383–413. [CrossRef] , [ ] 55. Rutgersson, A.; Pettersson, H.; Nilsson, E.; Bergström, H.; Wallin, M.B.; Sahlée, E.; Wu, L.E.; Mårtensson, E.M. Using land-based stations for air–sea interaction studies. Tellus A Dyn. Meteorol. Oceanogr. 2019, 72, 1–23. [CrossRef] 56. Löfﬂer, A.; Schneider, B.; Perttilä, M.; Rehder, G. Air–sea CO2 exchange in the Gulf of Bothnia, Baltic Sea. Cont. Shelf Res. 2012, 37, 46–56. [CrossRef] 57. Pfeil, B.; Olsen, A.; Bakker, D.C.E.; Hankin, S.; Koyuk, H.; Kozyr, A.; Malczyk, J.; Manke, A.; Metz A uniform, quality controlled Surface Ocean CO2 Atlas (SOCAT). Earth Syst. Sci. Data 2013, 5, 125–143 57. References Downscaling of solar-induced chlorophyll ﬂuorescence from canopy level to photosystem level using a random forest model. Remote Sens. Environ. 2019, 231, 110772. [CrossRef] 65. Breiman, L. Bagging predictions. Mach. Learn. 1996, 24, 123–140. [CrossRef] 65. Breiman, L. Bagging predictions. Mach. Learn. 1996, 24, 123 140. [CrossRef] 66. Liaw, A.; Wiener, M. Breiman and Cutler’s Random Forests for Classiﬁcation and Regression; R Package Version: 4.6–14. 2018. Available online: https://www.stat.berkeley.edu/~{}breiman/RandomForests/ (accessed on 10 November 2020). 22 of 22 22 of 22 Remote Sens. 2021, 13, 259 67. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2018. Available online: https://www.R-project.org/ (accessed on 10 November 2020). p p j g 68. Wesslander, K. The Carbon Dioxide System in the Baltic Sea Surface Waters; University of Gotenburg: G 68. Wesslander, K. The Carbon Dioxide System in the Baltic Sea Surface Waters; University of Gotenburg: Gothenburg, Sweden, 2011. 69. Bozec, Y.; Merlivat, L.; Baudoux, A.-C.; De Beaumont, L.; Blain, S.; Bucciarelli, E.; Danguy, T.; Grossteffan, E.; Guillot, A.; Guillou, J.; et al. Diurnal to inter-annual dynamics of pCO2 recorded by a CARIOCA sensor in a temperate coastal ecosystem (2003–2009). Mar. Chem. 2011, 126, 13–26. [CrossRef] 70. Kohonen, T. Self-Organization and Associative Memory, 3rd ed.; Huang, T.S., Kohonen, T., Schroeder, M.R., Eds.; Springer: Berlin/Heidelberg, Germany; New York, NY, USA; London, UK; Paris, France; Tokyo, Japan; Hong Kong, China, 2001; ISBN 9783540513872. 71. Landschützer, P.; Gruber, N.; Bakker, D.C.E.; Schuster, U.; Nakaoka, S.; Payne, M.R.; Sasse, T.P.; Zeng, J. A neural network-based estimate of the seasonal to inter-annual variability of the Atlantic Ocean carbon sink. Biogeosciences 2013, 10, 7793–7815. [CrossRef] 72. Le Quéré, C.; Moriarty, R.; Andrew, R.M.; Canadell, J.G.; Sitch, S.; Korsbakken, J.I.; Friedlingstein, P.; Peters, G.P.; Andres, R.J.; Boden, T.A.; et al. Global Carbon Budget. Earth Syst. Sci. Data 2015, 7, 349–396. [CrossRef] 71. Landschützer, P.; Gruber, N.; Bakker, D.C.E.; Schuster, U.; Nakaoka, S.; Payne, M.R.; Sasse, T.P.; Zeng, J. A neural network-based estimate of the seasonal to inter-annual variability of the Atlantic Ocean carbon sink. Biogeosciences 2013, 10, 7793–7815. [CrossRef] y g 72. Le Quéré, C.; Moriarty, R.; Andrew, R.M.; Canadell, J.G.; Sitch, S.; Korsbakken, J.I.; Friedlingstein, Boden, T.A.; et al. Global Carbon Budget. Earth Syst. Sci. Data 2015, 7, 349–396. [CrossRef] 73. Landschützer, P.; Laruelle, G.G.; Roobaert, A.; Regnier, P. A uniform pCO2 climatology combining open and coastal oceans. Earth Syst. Sci. References Data 2020, 12, 2537–2553. [CrossRef] y 74. Yasunaka, S.; Siswanto, E.; Olsen, A.; Hoppema, M.; Watanabe, E.; Fransson, A.; Chierici, M.; Murata, A.; Lauvset, S.K.; Wanninkhof, R.; et al. Arctic Ocean CO2 uptake: An improved multiyear estimate of the air–sea CO2 ﬂux incorporating chlorophyll a concentrations. Biogeosciences 2018, 15, 1643–1661. [CrossRef] p y g 75. Wehrens, R.; Kruisselbrink, J. Flexible Self-Organizing Maps in kohonen 3.0. J. Stat. Softw. 2018, 87, 75. Wehrens, R.; Kruisselbrink, J. Flexible Self-Organizing Maps in kohonen 3.0. J. Stat. Softw. 2018, 87, 1–18. [CrossRef] 76. Croft, H.; Chen, J.; Wang, R.; Mo, G.; Luo, S.; Luo, X.; He, L.; Gonsamo, A.; Arabian, J.; Zhang, Y.; et al. The global distribution of leaf chlorophyll content. Remote Sens. Environ. 2020, 236, 111479. [CrossRef] 76. Croft, H.; Chen, J.; Wang, R.; Mo, G.; Luo, S.; Luo, X.; He, L.; Gonsamo, A.; Arabian, J.; Zhang, Y.; et leaf chlorophyll content. Remote Sens. Environ. 2020, 236, 111479. [CrossRef] 77. Haapala, J.J.; Ronkainen, I.; Schmelzer, N.; Sztobryn, M. Recent Change—Sea Ice. In Second Assessment of Climate Change for the Baltic Sea Basin; Springer: Berlin/Heidelberg, Germany, 2015; pp. 145–153. ev, O. Main upwelling regions in the Baltic Sea—A statistical analysis based on three-dimensional modelling 2003, 8, 97–112. 78. Myrberg, K.; Andrejev, O. Main upwelling regions in the Baltic Sea—A statistical analysis based on thre Boreal Environ. Res. 2003, 8, 97–112. 79. Pierson, D.C.; Kratzer, S.; Strömbeck, N.; Håkansson, B. Relationship between the attenuation of downwelling irradiance at 490 nm with the attenuation of PAR (400–700 nm) in the Baltic Sea. Remote Sens. Environ. 2008, 112, 668–680. [CrossRef] 80. Stramska, M.; Stramski, D.; Mitchell, B.G.; Mobley, C.D. Estimation of the absorption and backscattering coefﬁcients from in water radiometric measurements. Limnol. Oceanogr. 2000, 45, 628–641. [CrossRef] 81. Stramska, M.; ´Swirgo´n, M. Inﬂuence of atmospheric forcing and freshwater discharge on interannual variability of the vertical diffuse attenuation coefﬁcient at 490 nm in the Baltic Sea. Remote Sens. Environ. 2014, 140, 155–164. [CrossRef] 82. Algesten, G.; Wikner, J.; Sobek, S.; Tranvik, L.J.; Jansson, M. Seasonal variation of CO2 saturat of marine net heterotrophy. Glob. Biogeochem. Cycles 2004, 18, 1–7. [CrossRef] 82. Algesten, G.; Wikner, J.; Sobek, S.; Tranvik, L.J.; Jansson, M. Seasonal variation of CO2 saturation in the Gulf of Bothnia: Indications of marine net heterotrophy. Glob. Biogeochem. Cycles 2004, 18, 1–7. [CrossRef] 83. Lehmann, A.D.; Myrberg, K.; Höﬂich, K. References A statistical approach to coastal upwelling in the Baltic Sea satellite data for 1990–2009. Oceanologia 2012, 54, 369–393. [CrossRef] g 84. Lips, I.; Lips, U.; Liblik, T. Consequences of coastal upwelling events Finland (Baltic Sea). Cont. Shelf Res. 2009, 29, 1836–1847. [CrossRef] g 84. Lips, I.; Lips, U.; Liblik, T. Consequences of coastal upwelling events on physical and chemical patterns in the central Gulf of Finland (Baltic Sea). Cont. Shelf Res. 2009, 29, 1836–1847. [CrossRef] 84. Lips, I.; Lips, U.; Liblik, T. Consequences of coastal upwelling events on physical and chemical patterns in the central Gulf of Finland (Baltic Sea). Cont. Shelf Res. 2009, 29, 1836–1847. [CrossRef] 85. Wesslander, K.; Omstedt, A.; Schneider, B. Inter-annual and seasonal variations in the air–sea CO2 balance in the central Baltic Sea and the Kattegat. Cont. Shelf Res. 2010, 30, 1511–1521. [CrossRef] ( ) f [ ] 85. Wesslander, K.; Omstedt, A.; Schneider, B. Inter-annual and seasonal variations in the air–sea CO2 balance in the central Baltic Sea and the Kattegat. Cont. Shelf Res. 2010, 30, 1511–1521. [CrossRef] f 85. Wesslander, K.; Omstedt, A.; Schneider, B. Inter-annual and seasonal Sea and the Kattegat. Cont. Shelf Res. 2010, 30, 1511–1521. [CrossRef]
W2981928918.txt	https://www.mdpi.com/1422-0067/20/21/5305/pdf?version=1572437767	en		Which Low-Abundance Proteins are Present in the Human Milieu of Gamete/Embryo Maternal Interaction?	International journal of molecular sciences	2,019	cc-by	12,650	International Journal of Molecular Sciences Article Which Low-Abundance Proteins are Present in the Human Milieu of Gamete/Embryo Maternal Interaction? Analuce Canha-Gouveia 1 , A. Paradela 2 , António Ramos-Fernández 2 , Maria Teresa Prieto-Sánchez 3 , Maria Luisa Sánchez-Ferrer 3 , Fernando Corrales 2 and Pilar Coy 1, * 1 2 3 * Department of Physiology, Faculty of Veterinary, University of Murcia, Campus Mare Nostrum, IMIB-Arrixaca, 30100 Murcia, Spain; analuce.canha@um.es Proteomics Laboratory, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), 28049 Madrid, Spain; proteobotics@gmail.com (A.R.-F.); alberto.paradela@cnb.csic.es (A.P.); fcorrales@cnb.csic.es (F.C.) Department of Obstetrics & Gynecology, “Virgen de la Arrixaca” University Clinical Hospital, IMIB-Arrixaca, 30100 Murcia, Spain; mt.prieto@um.es (M.T.P.-S.); marisasanchez@um.es (M.L.S.-F.) Correspondence: pcoy@um.es Received: 2 October 2019; Accepted: 23 October 2019; Published: 24 October 2019 Abstract: The improvement of the embryo culture media is of high relevance due to its influence on successful implantation rates, pregnancy, neonatal outcomes, and potential effects in adult life. The ideal conditions for embryo development are those naturally occurring in the female reproductive tract, i.e., the oviductal and uterine fluids. To shed light on the differences between chemical and natural media, we performed the first comparative study of the low abundance proteins in plasma, uterine, and oviductal fluid collected, simultaneously, from healthy and fertile women that underwent a salpingectomy. The rationale for this design derives from the fact that high-abundant proteins in these fluids are usually those coming from blood serum and frequently mask the detection of low abundant proteins with a potentially significant role in specific processes related to the embryo–maternal interaction. The proteomic analysis by 1D-nano LC ESI-MSMS detected several proteins in higher amounts in oviductal fluid when compared to uterine and plasma samples (RL3, GSTA1, EZRI, DPYSL3, GARS, HSP90A). Such oviductal fluid proteins could be a target to improve fertilization rates and early embryo development if used in the culture media. In conclusion, this study presents a high-throughput analysis of female reproductive tract fluids and contributes to the knowledge of oviductal and uterine secretome. Keywords: low abundance proteins; human reproductive fluids; salpingectomy 1. Introduction Assisted reproductive technologies (ART) have grown worldwide to assist the increasing number of patients who request these methods to conceive [1]. This successful development of ART has been achieved by continuous critical analysis of all methods performed and materials used [2]. Recently, culture media has received special attention [3]. During intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF), the development of human-preimplantation embryos takes place in an artificial environment (i.e., a petri dish with chemically defined or semi defined culture media). Nowadays, it is possible to find culture media on the market composed of different formulations that vary from simple salt solutions to more complex compositions that include synthetically derived proteins and growth factors [4]. The presence of proteins, other than albumin in these chemically Int. J. Mol. Sci. 2019, 20, 5305; doi:10.3390/ijms20215305 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5305 2 of 20 defined solutions, is usually avoided [5]. The batch-to-batch variability in protein composition has been linked to fluctuating pregnancy rates in clinics [6]. To overcome this variability, to minimize the risk of disease transmission and to achieve stable high success rates, protein supplement has evolved from initial donor or patient plasma/serum formulas to recombinant albumin [7]. Although current culture media are showing consistency and promote higher pregnancy rates than the ones used before, there is a growing concern of a possible link between the composition of the culture media and the phenotype of the offspring [8]. The developmental origins of health and disease (DOHaD) hypothesis proposed that in utero stress is associated with an increased risk of disorders in adulthood. This hypothesis has been extended to include the putative effect of the oviductal and periconceptional environment to future progeny health [9]. In animal models, extensive data revealed the presence of genetic and epigenetic alterations, especially in imprinted genes, derived from the use of ART [10–12]. In recent years, the long-term side effect of ART on further development in humans has also been studied. Several works have suggested that the composition of the media in which embryos are cultured may have an impact on the quality of embryos generated in IVF/ICSI cycles, thereby influencing implantation and pregnancy rates [8,13]. In 2016, a single-center cohort study did not find significant effects on birthweight, malformation risk (minor and major), offspring growth, and frequency of medical concerns according to culture media [14]. However, a contemporary study found a correlation between the IVF media and perinatal outcomes in a randomized controlled trial. The outcomes considered in this study were the number of viable embryos grown, the rate of successful implantations, the pregnancy rates, and the birthweights of the newborns [15]. Due to this uncertainty, it is imperative to invest more effort in the improvement of embryo culture conditions, trying to mimic the in vivo environment where embryos develop. Oviductal and uterine fluids (OF, UF) contain essential factors of different origin and composition that are pivotal for the development of gametes, zygotes, and later, embryos. These include nutrients, hormonal and non-hormonal factors, electrolytes, and other macromolecules [16]. Additionally, the volume, pH, and osmolality of these fluids are precisely regulated. If we compare reproductive fluids with artificial culture media, we observe that most of the lipids, hormones, glycosaminoglycans, proteins, and exosomes found in vivo are missed [16,17]. Even though embryos show some flexibility and are able to adapt to changes in growth-media, as in the case of protein-free media [18], it is clear that the multiple components of reproductive fluids play an active role in the whole process [19]. As aforementioned, human recombinant albumin is present in most IVF culture media. This protein has been recognized for its important role in embryo culture and is the most abundant macromolecule in the human oviduct [20]. This high proportion is a limiting factor to effective detection of low abundance proteins, which could play an important role for the embryo [21] and could be of interest in the design and development of culture media similar to physiological fluids. While the use of natural reproductive fluids in culture media may seem impractical at present, the inclusion of some of these proteins produced in the laboratory, such as recombinant albumin, may help to shorten the enormous distance between synthetic media and natural fluids, reducing the stress for the embryo. To bridge the discrepancy between chemical and biological media, we have performed the first comparative study of the proteomes obtained from three different fluids collected simultaneously from the same donor, namely uterine fluid (UF), oviductal fluid (OF), and plasma (P), aiming to find low abundance proteins of special significance for embryo development. Samples were collected from healthy and fertile women who underwent a salpingectomy. The amount and quality of the samples allowed us to perform immuno-depletion of the high-abundance serum proteins to detect specific lower-abundance proteins in each fluid [22]. The comparison between the three types of fluids and between individuals constitutes the first quantitative comparison of human oviductal and uterine proteins and could help to improve embryo culture media. Int. J. Mol. Sci. 2019, 20, 5305 3 of 20 2. Results Int.Sample J. Mol. Sci. 2019, 20, 5305 2.1. Collection and Immunoaffinity Depletion 3 of 20 OF, UF, and P were collected from three healthy women (BRA52, BRA54, and BRA57) to compare OF, UF, and P were collected from three healthy women (BRA52, BRA54, and BRA57) to their proteomic profile. The volumes of reproductive fluids obtained from each donor are shown in compare their proteomic profile. The volumes of reproductive fluids obtained from each donor are Table 1. shown in Table 1. Table 1. Volume of reproductive fluids collected from each donor. Table 1. Volume of reproductive fluids collected from each donor. Collected Fluids BRA-52 BRA-54 BRA-57 Collected Fluids BRA-52 BRA-54 BRA-57 70.9 49 57 70.9 49 100 59 UF - uterine fluid (µL) 260 100 59 As for plasma, 500 µL of the sample was obtained in the three cases. Immunoaffinity As for plasma, 500 depletion µL of theofsample wasabundant obtainedproteins in the was threeperformed cases. Immunoaffinity chromatography-based the most (Supplementary chromatography-based depletion of the most abundant proteins performed Table S1). The unbound fraction (Mr < 3000 Da), containing the low-abundancewas protein fraction of (Supplementary Table S1). The unbound fraction (Mr < 3000 Da), containing the low-abundance each sample, was used for the analysis. The MS and MSMS spectra obtained were used for protein fractionand of each sample, was used for the samples, analysis. using The MS MSMS spectra obtainedby identification label-free quantification of the the and statistical design developed were used for identification and label-free quantification of the samples, using the statistical designof Proteobotics SL. Identified peptides and proteins and their corresponding statistical estimates developed by are Proteobotics Identified peptides and their corresponding statistical significance describedSL. in Supplementary Tableand S1. proteins The parameters used for the search, as well as estimates of significance are described in Supplementary Table S1. The parameters used for the search, the codes for the four search engines used, are summarized in Supplementary Table S2. as well as the codes for the four search engines used, are summarized in Supplementary Table S2. OF—oviductal fluid (µL) 57 OF - oviductal fluid (µL) UF—uterine fluid (µL) 260 2.2. Data Analysis and Quantification 2.2. Data Analysis and Quantification 2.2.1. Hierarchical Clustering 2.2.1. Hierarchical Clustering Label-free quantification (LFQ) values were analyzed hierarchical clustering. Samples were Label-free quantification (LFQ) values were analyzed byby hierarchical clustering. Samples were grouped into two main clusters: plasma samples and reproductive fluids. The cluster corresponding grouped into two main clusters: plasma samples and reproductive fluids. The cluster corresponding to reproductive fluids was divided into two subpopulations corresponding to oviductal and uterine to reproductive fluids was divided into two subpopulations corresponding to oviductal and uterine samples, respectively, where the samples from patients BRA54 and BRA52 were closer (Figure 1). samples, respectively, where the samples from patients BRA54 and BRA52 were closer (Figure 1). Figure 1. Hierarchical clustering of the samples based on label-free quantification (LFQ) values of Figure 1. Hierarchical clustering of the samples based on label-free quantification (LFQ) values of protein identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of proteolytic protein identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of proteolytic peptides from the plasma (P), oviductal fluid (OF), and uterine fluid (UF) samples of the three patients peptides from the plasma (P), oviductal fluid (OF), and uterine fluid (UF) samples of the three patients (52, 54, and 57 years old). (52, 54, and 57 years old). 2.2.2. Principal Component Analysis (PCA) 2.2.2. Principal Component Analysis (PCA) PCA showed an increased dispersion of the samples compared to the previous hierarchical PCAHowever, showed an increased dispersion of the samples compared the previous hierarchical clustering. it was still possible to distinguish the three groups to of samples: OF, UF, and P. clustering. However, it was still possible to distinguish the three groups of samples: OF, UF, and P. The population most closely grouped were oviductal samples, while uterine samples showed the highest dispersion (Figure 2). Int. J. Mol. Sci. 2019, 20, 5305 4 of 20 The population most closely grouped were oviductal samples, while uterine samples showed the highest 2). Int. J. Mol.dispersion Sci. 2019, 20,(Figure 5305 4 of 20 Figure Figure 2. 2. Principal Principal component component analysis analysis (PCA) (PCA) of of all all identified identified proteins proteins from fromlabel-free label-freeexperiments. experiments. 2.2.3. Comparative Analysis 2.2.3. Comparative Analysis Quantitative data obtained for the three groups of samples were normalized and compared: Quantitative data obtained for the three groups of samples were normalized and compared: OF OF versus UF (Supplementary Table S3), OF versus P (Supplementary Table S4), and UF versus P versus UF (Supplementary Table S3), OF versus P (Supplementary Table S4), and UF versus P (Supplementary Table S5). Regulated proteins for each paired comparison were color-coded according (Supplementary Table S5). Regulated proteins for each paired comparison were color-coded to the statistical confidence (Table 2). according to the statistical confidence (Table 2). Table 2. Summary of data analysis and quantification of 1D-nano LC ESI-MSMS for each pairwise Table 2. Summary of data analysis and quantification of 1D-nano LC ESI-MSMS for each pairwise comparison (oviductal fluid/uterine fluid (OF/UF), plasma/oviductal fluid (P/OF), and P/UF), indicating comparison (oviductal fluid/uterine fluid (OF/UF), plasma/oviductal fluid (P/OF), and P/UF), the number of abundance proteins for each of the contrasts with different degrees of confidence. indicating the number of abundance proteins for each of the contrasts with different degrees of The protein abundance for each paired comparison were color-coded according to the statistical confidence. The protein abundance for each paired comparison were color-coded according to the confidence (green or red for high grade, yellow or orange for medium grade, yellow or light orange for statistical confidence (green or red for high grade, yellow or orange for medium grade, yellow or light low degree of confidence and white for no significantly different expression). orange for low degree of confidence and white for no significantly different expression). Abundance Color-Code According to the OF/P UF/P OF/U OF/UF Statistical Confidence OF/P UF/P Abundance Color-Code According to the Statistical Confidence F truly null fraction (π0, proportion of contrasts under 77.90 77.90% 60.40 77.90 60.40% 77.90%(π0, proportion of contrasts under the null hypothesis) truly null fraction the null hypothesis) % % % Confident high abundant (q-value < 0.01, positive 15 49 Confident high 13abundant (q-value < 0.01, positive log fold change) 15 49 13 log fold change) Likely high positive abundant (q-value < 0.05, positive log 25 107 52 Likely high abundant (q-value < 0.05, log fold change) 25 107 52 fold change) Putative high abundant (q-value > 0.05 but p-value < 1−π0, positive log fold 308 601 398 Putative high abundant (q-value > 0.05 but p-value < 308 601 change) 398 1−π0, positive log fold change) 1016 1016 552 958 552 no differentially 958 abundant no differentially abundant Putative low abundant (q-value > 0.05negative but p-value Putative low abundant (q-value > 0.05 but p-value < 1−π0, log <fold 232 127 232 216 127 216 1−π0, negative log fold change) change) Likely low negative abundantlog (q-value < 0.05, negative log 16 27 (q-value < 0.05, fold change) 1633 33 Likely low abundant 27 fold change) 14 67 27 Confident low abundant Confident (q-value < low 0.01,abundant negative (q-value log fold < change) 0.01, negative log 14 67 27 fold change) 1626 1625 1602 # hypotheses tested 1626 1625 1602 # hypotheses tested 70 119 256 # hypotheses 119 significant with q <#0.05 70256 hypotheses significant with q < 0.05 The comparison between UF and P identified 1602 proteins with at least two unique peptides (false discovery rate FDR <1%) and, thus, was selected for further comparative analysis. Of these, 119 proteins met the criteria established for considering a protein as differentially expressed (q < 0.05). In addition, 1625 proteins were identified and quantified when OF was compared to P showing a higher number of either up- or downregulated proteins (n = 256) with q < 0.05. The lowest number of proteins (n = 70, q < 0.05) differentially expressed was found in the comparison between reproductive fluids, in which 1626 proteins were identified and quantified. Int. J. Mol. Sci. 2019, 20, 5305 5 of 20 The comparison between UF and P identified 1602 proteins with at least two unique peptides (false discovery rate FDR <1%) and, thus, was selected for further comparative analysis. Of these, 119 proteins met the criteria established for considering a protein as differentially expressed (q < 0.05). Int. J. Mol. Sci. 2019, 20, 5305 5 of 20 In addition, 1625 proteins were identified and quantified when OF was compared to P showing a higher number of either up- or downregulated proteins (nof=proteins 256) with q < 0.05. Theexpressed lowest number of The pairwise comparison with a higher percentage differentially (q < 0.05) proteins 70, < 5305 0.05) differentially expressed was found in OF-P the comparison between reproductive Int.relation J. Mol.(n Sci.= 20 in to2019, theq20, total number of proteins identified was the (15.8%), and the lowest was5 of OFfluids, in which 1626 proteins were identified and quantified. UF (4.3%) (Figure 3). The Thepairwise pairwisecomparison comparisonwith withaahigher higherpercentage percentageof ofproteins proteinsdifferentially differentiallyexpressed expressed(q(q<<0.05) 0.05) in ofof proteins identified was thethe OF-P (15.8%), andand the the lowest waswas OF-UF inrelation relationtotothe thetotal totalnumber number proteins identified was OF-P (15.8%), lowest OFUF (4.3%) (Figure (4.3%) (Figure 3). 3). Figure 3. Percentage of proteins differentially abundant after analysis and quantification of 1D-nano LC ESI-MSMS data for three pairwise comparisons: oviductal fluid versus uterine fluid (OF/UF), oviductal fluid versus (OF/P) and uterine fluid versus plasma and (UF/P) (q < 0.05). of 1D-nano Figure 3. Percentage ofplasma proteins differentially abundant after analysis quantification Figure 3. Percentage of three proteins differentially abundant after analysis and quantification 1D-nano LC ESI-MSMS data for pairwise comparisons: oviductal fluid versus uterine fluidof(OF/UF), LC ESI-MSMS data for three pairwise comparisons: oviductal fluid versus uterine fluid (OF/UF), In the UF/P comparison, 7.4% of proteins were differentially abundant. This trend remained oviductal fluid versus plasma (OF/P) and uterine fluid versus plasma (UF/P) (q < 0.05). oviductal fluid versus plasma (OF/P) and uterine fluid versus plasma (UF/P) (q < 0.05). when low and high abundant proteins were compared. These results confirm both hierarchical and In thecomponent UF/P comparison, 7.4% of proteins were abundant. This trend when principal analyses, which revealed thedifferentially highest differences between OF remained and P samples. In the UF/P comparison, 7.4% of proteins were differentially abundant. This trend remained low and high abundant proteins were compared. confirm both hierarchical Conversely, both reproductive fluids (OF andThese UF),results showed the lowest percentageand of principal proteins when low and high abundant proteins were compared. These results confirm both hierarchical and component analyses, which revealed the highest differences between OF and P samples. Conversely, differentially abundant, confirming the highest degree of similarity of these reproductive fluids principal component analyses, which revealed the highest differences between OF and P samples. both reproductive fluids (OF and UF), showed the lowest percentage of proteins differentially abundant, (Figure 3). Conversely, both reproductive fluids considering (OF and UF), the lowest confirming the highest degree of similarity of these reproductive (Figurepercentage 3). A Venn analysis was performed all showed proteinsfluids differentially abundantof(qproteins < 0.05) differentially abundant, confirming the highest degree of similarity of these reproductive fluids A Venn analysis performed all proteins differentially abundant (q < high 0.05) (Figure 4), (Figure 4), with thewas main aim of considering detecting specific differences between low and abundant (Figure 3). with the main aim of detectingfluids specific differences proteins both in reproductive and plasma. between low and high abundant proteins both in A Vennfluids analysis performed considering all proteins differentially abundant (q < 0.05) reproductive and was plasma. (Figure 4), with the main aim of detecting specific differences between low and high abundant proteins both in reproductive fluids and plasma. Figure Figure 4.4. Venn Venn diagram diagram of of proteins proteins that that showed showed aa significant significant (q (q<< 0.05) 0.05) low low abundance abundance (A) (A) or or high high abundance abundance(B). (B). Figure only 4. Venn of proteins that showed significant (q < 0.05) 4A), low abundance (A) or high When thediagram low abundant proteins were aconsidered (Figure it was observed that the abundance (B). highest number of proteins were found when OF was compared to P (96). Additionally, 11 of these low abundant proteins were also found when we compared UF versus P and UF versus OF (Figure When only theTable low abundant proteins were considered (Figure it was observed the 5, Supplementary S6A5). STRING network analysis of these 4A), 11 proteins showed that several Int. J. Mol. Sci. 2019, 20, 5305 6 of 20 When only the low abundant proteins were considered (Figure 4A), it was observed that the highest number of proteins were found when OF was compared to P (96). Additionally, 11 of these low abundant proteins were also found when we compared UF versus P and UF versus OF (Figure 5,Sci. Supplementary Int. J. Mol. Sci. 2019, 5305 Table S6A5). STRING network analysis of these 11 proteins showed several Int. J. Mol. 2019, 20,20, 5305 6 6ofof2020 biological processes involved in the regulation of protein processing: the protein activation cascade and complement activation. The reactome pathways were mainly from the complement cascade and and complement activation. The reactome pathways were mainly from the complement cascade and and complement activation. The reactome pathways were mainly from the complement cascade and the innate immune system. the innate immune system. the innate immune system. Figure 5.5.5.Venn diagram of proteins that showed aa significant (q(q(q << 0.05) low abundance and the Figure Venn diagram proteins that showed asignificant significant <0.05) 0.05) low abundance and the Figure Venn diagram ofof proteins that showed low abundance and the corresponding interactome for the 11 low abundant proteins detected in the three comparison pairs corresponding interactome for the low abundant proteins detected the three comparison pairs corresponding interactome for the 1111 low abundant proteins detected inin the three comparison pairs (UF/P, OF/P, and OF/UF). (UF/P, OF/P, and OF/UF). (UF/P, OF/P, and OF/UF). However, there were 3939 abundant proteins in in compared to to 6, 6, However, there were low abundant proteins OF compared P(Figure (Figure Supplementary However, there were 39low low abundant proteins inOF OF compared toPP(Figure 6,Supplementary Supplementary Table S6A3). STRING analysis of these proteins also showed that they were involved in the regulation TableS6A3). S6A3).STRING STRINGanalysis analysisofofthese theseproteins proteinsalso alsoshowed showedthat thatthey theywere wereinvolved involved the regulation Table inin the regulation of protein processing, protein activation cascade, and complement activation. Reactome pathways were of protein processing, protein activation cascade, and complement activation. Reactome pathways of protein processing, protein activation cascade, and complement activation. Reactome pathways mostly related to the complement cascade, the innate immune system, and the and regulation of insulin weremostly mostlyrelated related thecomplement complement cascade, the innateimmune immunesystem, system, andthe theregulation regulation were totothe cascade, the innate ofof growth factor. In the UF/P comparison, 53 proteins were downregulated, 3 of which were exclusively insulingrowth growthfactor. factor.InInthe theUF/P UF/Pcomparison, comparison,5353proteins proteinswere weredownregulated, downregulated,3 3ofofwhich whichwere were insulin low abundant in this fluid (retinol-binding protein 4, fibronectin, and (Supplementary exclusively low abundant thisfluid fluid(retinol-binding (retinol-binding protein fibronectin,and andflasminogen flasminogen exclusively low abundant ininthis protein 4,4,flasminogen fibronectin, Table S6A7). Both reproductive fluids (OF and UF) showed only 11 out of 29 proteins significantly low (SupplementaryTable TableS6A7). S6A7).Both Bothreproductive reproductivefluids fluids(OF (OFand andUF) UF)showed showedonly only1111out outofof2929proteins proteins (Supplementary abundant, including lactotransferrin, neutrophil gelatinase-associated lipocalin, and coactosin-like significantlylow lowabundant, abundant,including includinglactotransferrin, lactotransferrin,neutrophil neutrophilgelatinase-associated gelatinase-associatedlipocalin, lipocalin, significantly protein, among others (Supplementary Table S6A1). STRING analysis (Figure 6) of these 11 proteins andcoactosin-like coactosin-likeprotein, protein,among amongothers others(Supplementary (SupplementaryTable TableS6A1). S6A1).STRING STRINGanalysis analysis(Figure (Figure6)6) and linked reactome pathways from the immune system and by interleukins between most of the these proteins linked reactome pathways from thesignaling immunesystem system andsignaling signaling interleukins ofofthese 1111proteins linked reactome pathways from the immune and bybyinterleukins proteins, besides Coatomer subunit beta (COPB2), adapter molecule crk isoform Crk-II (CRK), Thy-1 betweenmost mostofofthe theproteins, proteins,besides besidesCoatomer Coatomersubunit subunitbeta beta(COPB2), (COPB2),adapter adaptermolecule moleculecrk crkisoform isoform between membrane glycoprotein (THY1) and secreted frizzled-related protein 4 (SFRP4). Crk-II(CRK), (CRK),Thy-1 Thy-1membrane membraneglycoprotein glycoprotein(THY1) (THY1)and andsecreted secretedfrizzled-related frizzled-relatedprotein protein4 4(SFRP4). (SFRP4). Crk-II Figure 6.6.Venn diagram of proteins showing a significant (q < 0.05) low and the corresponding Figure 6.Venn Venn diagram proteins showing significant <0.05) 0.05)low low abundance andthe the Figure diagram ofofproteins showing a asignificant (q(q<abundance abundance and interactome for the 11 low abundant proteins in OF compared to UF. correspondinginteractome interactomefor forthe the1111low lowabundant abundantproteins proteinsininOF OFcompared comparedtotoUF. UF. corresponding Both reproductive fluids showed 39 low abundant proteins in comparison to P Bothreproductive reproductivefluids fluidsshowed showed3939low lowabundant abundantproteins proteinsinincomparison comparisontotoP P(Supplementary (Supplementary Both (Supplementary Table S6A6), mainly linked to regulation of protein processing, protein activation TableS6A6), S6A6),mainly mainlylinked linkedtotoregulation regulationofofprotein proteinprocessing, processing,protein proteinactivation activationcascade, cascade, Table cascade, complement activation, and formation of fibrin clots. The highest number of high abundant complement activation, and formation of fibrin clots. The highest number of high abundant proteins complement activation, and formation of fibrin clots. The highest number of high abundant proteins proteins was found when OF was compared to P (154), with 92 of these exclusively found in the wasfound foundwhen whenOF OFwas wascompared comparedtotoP P(154), (154),with with9292ofofthese theseexclusively exclusivelyfound foundininthe thecomparison comparison was OF/P(Supplementary (SupplementaryTable TableS7A3). S7A3).The TheUF UFshowed showed6565high highabundant abundantproteins proteinscompared comparedtotoP,P,with with OF/P 23 of them exclusive to this comparison (Supplementary Table S7A7). The comparison between both 23 of them exclusive to this comparison (Supplementary Table S7A7). The comparison between both reproductivefluids fluidsshowed showedthat thatonly only1818out outofof4040proteins proteinswere wereexclusively exclusivelyhigh highabundant abundant reproductive (SupplementaryTable TableS7A1). S7A1).Together, Together,the thereproductive reproductivefluids fluidsshowed showed4040high highabundant abundantproteins proteins (Supplementary compared to P (Supplementary Table S7A6) and 2 high abundant proteins (40S ribosomal protein S19 compared to P (Supplementary Table S7A6) and 2 high abundant proteins (40S ribosomal protein S19 Int. J. Mol. Sci. 2019, 20, 5305 7 of 20 comparison OF/P (Supplementary Table S7A3). The UF showed 65 high abundant proteins compared to P, with 23 of them exclusive to this comparison (Supplementary Table S7A7). The comparison between Int. J. Mol. Sci. 2019, 20, 5305 7 of 20 both reproductive fluids showed that only 18 out of 40 proteins were exclusively high abundant (Supplementary Table chain) S7A1). were Together, fluids showed abundant proteins and Tubulin alpha-1C foundthe in reproductive the three comparative pairs: 40 UFhigh versus P, OF versus P, compared to P (Supplementary Table S7A6) and 2 high abundant proteins (40S ribosomal protein S19 and UF versus OF (Supplementary Table S7A5). and Tubulin alpha-1C chain) were found in the three comparative pairs: UF versus P, OF versus P, and versus OF (Supplementary Table S7A5). 2.2.4.UF Targeted Mass Spectrometry (MRM/SRM) = 9; 3Spectrometry OF, 3 UF, and(MRM/SRM) 3 P) were reanalyzed by target mass spectrometry to validate 23 2.2.4.Samples Targeted(nMass proteins selected on the basis of differences found in the quantitative analysis (some were classified Samples (n = 9; 3 OF, 3 UF, and 3 P) were reanalyzed by target mass spectrometry to validate as confident high abundant or confident low abundant, others as putative or likely high or low 23 proteins selected on the basis of differences found in the quantitative analysis (some were classified as abundant) and based on their previously described roles in fertilization or embryo–maternal confident high abundant or confident low abundant, others as putative or likely high or low abundant) communication (e.g., OVGP1. PLMN, HS90A, or PDIA3) [23–26] (Supplementary Table S8). and based on their previously described roles in fertilization or embryo–maternal communication We confirmed that most of the low abundant proteins (RL3, GSTA1, EZRI, DPYL3, GARS, (e.g., OVGP1. PLMN, HS90A, or PDIA3) [23–26] (Supplementary Table S8). HSP90A) were significantly present in the three types of fluids according to this sequence: OF > UF > We confirmed that most of the low abundant proteins (RL3, GSTA1, EZRI, DPYL3, GARS, HSP90A) P or OF > UF and absent in P (TSTD1, OVGP1, NNRE, EF2) (Supplementary Table S9, Supplementary were significantly present in the three types of fluids according to this sequence: OF > UF > P or OF table S10, Figure 7). However, some proteins (EF1D, PDIA3, CLH1, ENPL) were present in similar > UF and absent in P (TSTD1, OVGP1, NNRE, EF2) (Supplementary Tables S9 and S10, Figure 7). amounts in reproductive fluids: OF = UF > P or OF = UF and absent in P (PTGR2, THY1, IPO9, NGAL) However, some proteins (EF1D, PDIA3, CLH1, ENPL) were present in similar amounts in reproductive (Supplementary Table S9, Supplementary Table S10, Figure 7). For these proteins (TSTD1, OVGP1, fluids: OF = UF > P or OF = UF and absent in P (PTGR2, THY1, IPO9, NGAL) (Supplementary NNRE, EF2, PTGR2, THY1, IPO9, NGAL), we were unable to confirm their presence in plasma Tables S9 and S10, Figure 7). For these proteins (TSTD1, OVGP1, NNRE, EF2, PTGR2, THY1, IPO9, samples as we could not detect any of the proteotypic peptides designed as protein-specific targets NGAL), we were unable to confirm their presence in plasma samples as we could not detect any of the (Figure 7). proteotypic peptides designed as protein-specific targets (Figure 7). Figure Summary of of the the low low abundance abundance proteins proteins detected detected by by target target mass mass spectrometry, spectrometry, from from the Figure 7. 7. Summary the 23 23 selected candidates, in in the Several proteins proteins were were detected in high selected candidates, the female female reproductive reproductive tract tract fluids. fluids. Several detected in high amounts in comparison to plasma samples (EFD1,(EFD1, PDIA3,PDIA3, GARS, GARS, ENPL, PTGR2, amounts in inreproductive reproductivefluids fluids in comparison to plasma samples ENPL, EF2), while others were low abundant (PLMN, TETN), as expected. Several proteins were detected in PTGR2, EF2), while others were low abundant (PLMN, TETN), as expected. Several proteins were high amounts in OF when compared to UF and P samples (RL3, EZRI, DYL3, HS90A, NNRE, OVGP1). detected in high amounts in OF when compared to UF and P samples (RL3, EZRI, DYL3, HS90A, On the other hand, THY1, IPO9, COTL1, NGAL, found in higher NNRE, OVGP1). Onthe theproteins other hand, theCLH1, proteins THY1, CLH1, IPO9, SFRP4 COTL1,were NGAL, SFRP4 were amounts uterine fluid. GSTA1 and TSD1 detected as exclusively high abundant proteins in found in in higher amounts in uterine fluid. were GSTA1 and TSD1 were detected as exclusively high oviductal fluid, in comparison to plasma, while TRFL was exclusively high abundant in uterine fluid. abundant proteins in oviductal fluid, in comparison to plasma, while TRFL was exclusively high abundant in uterine fluid. In addition, there were some samples where we detected only one of the proteotypic peptides (e.g., TRFL). On the other hand, SFRP4, COTL1, and TRFL seemed to be more abundant in UF than Int. J. Mol. Sci. 2019, 20, 5305 8 of 20 In addition, there were some samples where we detected only one of the proteotypic peptides (e.g., TRFL). On the other hand, SFRP4, COTL1, and TRFL seemed to be more abundant in UF than in the other samples. Finally, some proteins (e.g., TETN, PLMN) appeared to be more abundant in P than in the reproductive fluids. 3. Discussion Different IVF culture media influence the rates of successful implantation, pregnancy, and neonatal outcomes [15]. Recently, it has been shown that culture media supplemented with natural female reproductive fluids has improved IVF efficiency, morphological embryo quality, and epigenetic reprogramming profiles in pig blastocysts, compared to culture media without these supplements [27]. This finding has encouraged the study of the reproductive fluids aimed at the detection of elements that are lacking in embryo culture media [28]. 3.1. How to Analyze the Reproductive Fluids? 3.1.1. The Most Adequate Method to Collect and Perform A Proteomic Characterization Despite the oviduct being necessary in nature for optimal gamete maturation, capacitation, selection, and embryo development, detailed information about oviduct secretions and function is still scarce, mainly in humans, due to the difficulty in obtaining appropriate samples [21]. The proteomic characterization of uterine fluid [29] has been recognized for essential components that could be added to the culture media. However, the different collection methods (e.g., aspiration or uterine flushes) have not yet revealed a consistent proteomic pattern of this fluid [30–32]. This study shows a proteomic characterization of the low abundance proteins in reproductive fluids (oviductal and uterine) that are significantly detected relative to the plasma of healthy, young, and fertile women during the secretory phase of the menstrual cycle. The method to collect human reproductive fluids is one of the challenges for the proteomic study of these fluids, mainly due to the small volume of the samples and the difficulty of accessing samples from healthy women without damaging the endometrial cavity or the oviduct. Such damage could result in subsequent bleeding, mostly in the labile endometrium, which could alter the original composition of the collected fluids. In this study, the reproductive fluids were collected with the Mucat device (CDD Laboratoire) adapted with a thinner and smoother distal tip, which reduces the risk of endometrial damage and blood contamination of the samples. This collection method overcomes the limitations to those described previously, such as the reduced volume obtained by aspiration with embryo transfer devices and washing of soluble factors from the glycocalyx with uterine flushes [33]. Our collection method provided enough volume to efficiently perform the protein analysis and quantification by 1D-nano LC ESI-MSMS. The study of the transcriptome and secretome of reproductive fluids achieved through cutting-edge and robust technology used in our work is important to elucidate the role of the female tract and the identification of potentially crucial oviductal factors contributing to the success of fertilization and early embryonic development [16,34,35]. Previous proteomic studies have characterized the greater presence of serum proteins (e.g., serum albumin and immunoglobulins) in these fluids [36], but their abundance can mask the detection of less abundant proteins, which could be of interest in the future development of culture media similar to physiological fluids. Therefore, we decided to perform an efficient depletion of these major proteins to study the reproductive fluids secretome. The MS and MSMS spectra obtained were used for the identification and quantification of the samples, free labeling or label-free format, using the powerful statistical design developed by Proteobotics SL [37], that eliminates likely false positives in peptide and protein identification. Subsequently, a considerable number of proteins connoted as differentially abundant with statistical significance (q < 0.05) were identified, some of them which have not been previously identified in other studies. One example of the robust nature of this statistical design is the fact that changes in protein expression identified the oviduct-specific glycoprotein (Q12889) only as a protein Int. J. Mol. Sci. 2019, 20, 5305 9 of 20 putatively high abundant in OF, even though its abundance in this fluid is much higher than in UF or P [38,39]. These data confirm that the proteins detected as differentially abundant under our conditions were accurate. Indeed, with the MSM approach, OVGP1 was detected with higher abundance in OF than in UF and was not detected in plasma, confirming what was expected. 3.1.2. The Most Suitable Study Population Our set of samples of only three individuals was highly valuable compared to other studies because it was possible to test the reproductive fluids and plasma of the same women, avoiding bias due to individual variability. Another factor that improved efficiency was the homogeneity of the samples, ensured by the strict inclusion criteria that we established: less than 40 years old, healthy, no use of birth control pills, fertile (at least one healthy child born) and collection at the same phase of the menstrual cycle (secretory phase). This phase of menstrual cycle was selected since our goal was to identify low abundant proteins present in these fluids after ovulation and until implantation, since it is the phase corresponding to the time point when human embryos develop under in vitro conditions (from ICSI or IVF until embryo transfer at day 3 or 5) and embryo–maternal communication is highly necessary. This phase occurs after ovulation, when the endometrium experiences several changes, including the transformation of glands and slowing of stromal proliferation [40]. Additionally, it is characterized by the abundant presence of endoplasmic reticulum in the glandular epithelial cells, displacement of nuclei centrally, and accumulation of glycogen-rich vacuoles, which are lost 6 days after ovulation and corresponds to maximal glandular secretory activity [41]. 3.2. Common Differences in Protein abundance of Reproductive Fluids Versus Plasma The proteomic analysis performed in this study validates that OF, UF, and P are different from each other (Figures 1 and 2), but the reproductive fluids share most of the identified proteins. In humans, the intramural portion of the uterine tube does not allow a real physical separation between the oviductal and uterine environments. Therefore, it is reasonable to think that there is smooth communication between these anatomical regions. This fact is corroborated by the ability of human embryos, such as other primate embryos, to develop if exposed to the uterine environment prematurely or to implant in the oviduct (ectopic pregnancy), which does not happen in other non-primate mammals. Furthermore, in cases of hydrosalpinx, bilateral salpingectomy is recommended for women before undergoing IVF to improve birth success rates, since hydrosalpinx fluid may alter endometrial receptivity [42–44]. Although the importance of prostaglandins in the oviduct has been previously highlighted [45], our study showed for the first time that prostaglandin reductase 2, which forms the stable prostaglandin PGD2, PGE2, or PGF2α, is high abundant in OF in comparison to P, by shotgun and MRM analysis. This analysis also detected Prostaglandin reductase 2 in uterine fluid samples (Supplementary Table S8_MRM Results). Elongation factors 2 and 1 were mainly high abundant in OF compared to plasma by the shotgun approach, but MRM analysis also detected significant differences between OF and UF. The reduced expression of ELF1 was proposed as a candidate marker for early diagnosis of cervical cancer [46]. Protein disulfide-isomerase A3 was also detected in the reproductive fluids of these healthy women compared to P by shotgun and MRM analysis, although these proteins were detected before in the endometrium from early-secretory (LH + 2) to mid-secretory phase (LH + 7) in women with unexplained infertility [47]. Although endoplasmin (ENPL) has been linked mainly to oviductal fluid in other species, our study also detected it in uterine fluid [48]. The low abundant proteins were mainly related to complement cascade, regulation of the inflammatory response, and the protein activation cascade. Plasminogen (PLMN) that was detected and quantified for the first time in OF some years ago [49,50] was low abundant in all comparison pairs FO/P, FO/FU, FU/P by shotgun analysis, but MRM analysis did not detect any significant differences. It has been previously shown that the plasminogen/plasmin system is activated during gamete interaction and regulates sperm entry into the oocyte [23,49]. Additionally, it is regulated by progesterone at the transcription level [51]. Therefore, we expected high abundance at the proliferative Int. J. Mol. Sci. 2019, 20, 5305 10 of 20 phase and not at the secretory phase when our samples were collected, as indeed was observed. In the same pathway, tetranectin (CLEC3B), a plasminogen-binding protein belonging to the family of C-type lectins [52], was also detected as a low abundant protein in OF and UF when compared to P in our data. Previous studies have found specific biomarkers of the secretory phase and window of implantation in human endometria, such as osteopontin (OPN), epidermal growth factor (EGF), Fibronectin, Vitronectin, Secreted Phosphoprotein 1 (SPP1), Laminin, Insulin Like Growth Factor Binding Protein 1 (IGFBP1), Transformin Growth Factor (TFG), Homeobox-leucine zipper protein (Hox10), Interleukin-6 (Il6), Leukemia inhibitory factor (LIF), Bone morphogenetic protein 2 (BMP2), Left-right determination factor 2 (LEFTY2,) Cytosolic phospholipase A2 (CPLA2), Prostaglandin G/H synthase 2 (COX2), and Prostaglandin E2 (PGE) [53,54]. Interestingly, in our study, these biomarkers were not detected since they were not differentially expressed in the reproductive fluids. For example, fibronectin in UF was detected as a putative low abundant protein. However, in OF, it was detected as confident low abundant. On the other hand, laminin (Q16363) was detected as putative upregulated in UF relatively to P, while OF, when compared to P, did not show any significant difference in amount detected but was spotted as putative low abundant compared to UF. The transforming growth factor-beta-induced protein (Q15582) was also detected as putative high abundant in UF and putative low abundant in OF. These discrepancies among these array studies have already been described in previous publications for several genes (De) [55], which might be explained by differences in the study design or comparison of mid-secretory phase endometrium to either proliferative or early secretory endometrium. 3.3. Differentially abundance Proteins in Oviductal Fluid The changes in protein abundance observed between reproductive fluids and plasma have allowed us to identify a greater number of differentially abundant proteins in the OF than in the UF compared to P. From these, the high abundant proteins identified were predominantly involved in cellular catabolic process, biosynthesis of amino acids and organic substances, organic and aromatic compounds, and catabolic acid signaling. Some of these proteins have not been detected before in the OF, such as CCT2—T-complex protein 1 subunit beta. However, this protein is associated with capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae, so it is reasonable its detection in the fluid where fertilization takes place [56]. Another protein detected was ACTR3—Actin-related protein 3, whose specific function has not been yet determined. However, this protein is a major constituent of the ARP2/3 complex, known to be involved in the maintenance of the asymmetric (MII) spindle position in mouse oocytes [57]. Arpc1 protein, which is related to spermiation under the regulation of estrogen [58], was also detected in our study. Thiosulfate glutathione sulfurtransferase (TST), which provides the link between the first step in mammalian H2S metabolism performed by the sulfide:quinone oxidoreductase [59], was detected as a highly expressed protein for the first time in this fluid when compared to uterine fluid and plasma samples. These results were also confirmed by MRM analysis. However, our study also corroborated the presence of interesting proteins previously described in other species [16,21], such as oviduct-specific glycoprotein (OVGP1), which was detected in oviductal fluid in higher amount relatively to uterine fluid. By shotgun analysis, heat shock protein HSP 90-alpha was high abundant exclusively in OF compared to P, but by MRM analysis, it was detected in UF (although in small quantities). This protein acts like a chaperone of the progesterone receptor (PR) and is essential for the maintenance of its functional activity [60]. Therefore, HSP 90-alpha should contribute to the functionality of progesterone, which is crucial for the early-stage embryo–maternal communication and maintenance of pregnancy [61] and could represent a strong candidate to be present in the culture media, apart from OVGP1. Ezrin, detected in our study, was overexpressed in OF compared to P and UF by shotgun and MRM analysis; likewise, Ezrin was previously detected as a candidate with a role in the final process of oocyte maturation, which occurs in the oviduct and involves zona pellucida hardening [26]. Int. J. Mol. Sci. 2019, 20, 5305 11 of 20 Uba3 was also detected as an high abundant protein in the OF compared to P, which is supported by previous studies, where the corresponding gene expression of Uba3 mRNA in the uterus, ovary, skeletal muscle, and neural tissues was detected, with lower abundance in kidney, intestine, stomach, and liver [62]. The high abundance of some proteins involved in apoptotic processes, such as E3 ubiquitin-protein ligase (HUWE1) and DNA fragmentation factor subunit alpha (DFFA), was also evident in our data. This is not surprising since previous studies have shown the high abundance of several apoptotic proteins in the female reproductive tract [63] and even caspases, which are involved in the development of preimplantation human embryos [64]. 3.4. Differentially abundant Proteins in Uterine Fluid As the aforementioned stated, UF showed fewer significantly abundant proteins than OF when compared to P. The detected high abundant proteins are involved in the immune response and granulocyte activation. These results corroborate previous studies that have described the complex interaction between stromal cells and immune cells at the secretory phase of the superficial endometrium, namely the CD56-/CD16+ uterine natural killer (uNK) cells, which plays a role in maternal allorecognition of fetal trophoblasts rather than a cytotoxic role. This “cycling” cell population, which is regulated by regional steroid hormones, local chemokines, and interleukins, increases after ovulation and disappears if there is no pregnancy [40]. One of the proteins that was high abundant in our UF samples was the Thy-1 membrane glycoprotein (THY1). The Thy-1 membrane glycoprotein was high abundant exclusively in UF in relation to P by shotgun, but with MRM analysis, it was not possible to detect the peptides in all samples. This protein has been previously linked to the mechanisms that allow trophoblast cells to fuse with maternal host cells and is imperceptible for maternal immune effectors due to the maternal Thy-I supply to trophoblast somatic hybrids [65], therefore, may play an important role in implantation. The shotgun and MRM analysis showed that the secreted frizzled-related protein 4 (SFRP4) was also detected largely in uterine fluid, as previously described [66]. The lactoferrin estrogen-responsive protein, previously detected in the uterus of mice and rats, was also spotted in uterine fluids by the proteomic techniques performed in this study [67]. The clathrin heavy chain 1 was high abundant exclusively in UF related to P by the shotgun approach, but MRM analysis also detected significant differences between OF and P. Clathrin heavy chain has been shown to be important for viability, embryogenesis, and RNA interference (RNAi) in arthropods, such as Drosophila melanogaster and Metaseiulus occidentalis [68]. The 60S ribosomal protein L3 is a component of the large subunit of cytoplasmic ribosomes [69]. L ribosomal proteins were detected in reproductive fluids compared to P by shotgun and MRM analysis. The same detection was achieved for the first time by Importin-9, although it was previously linked to Type-I interferons IFN-ε [70], which is constitutively expressed by cells of the reproductive tract [71]. Finally, coactosin-like protein was detected by shotgun and MRM analysis for the first time and was exclusively high abundant in UF in relation to P and OF. The present data corroborate that reproductive fluids represent an important source of biomarkers with potential interest in the development of better embryo culture media and that each one show particular proteomic profiles according to their roles in the different stages at the beginning of the life cycle (i.e., fertilization, first cleavages, blastocyst implantation). Therefore, it would make sense the design of specific culture media be adapted to the needs of the gametes, zygotes, or embryos at every in vitro culture step in the assisted reproduction laboratories. 4. Materials and Methods 4.1. Study Population This research study (internal code 2016_3_6_HCUVA) was approved on 25th of April of 2016 by CEIC (Comité etico de Investigación Clinica) Virgen de la Arrixaca. The sampling was carried out at Int. J. Mol. Sci. 2019, 20, 5305 12 of 20 the Service of Obstetrics and Gynaecology of the University Clinical Hospital Virgen de la Arrixaca in Murcia, Spain. Patients who underwent a planned bilateral salpingectomy by laparoscopy, from June 2016 until June 2018 and fulfilled the inclusion criteria, were invited to participate in the study. Inclusion criteria were the following: premenopausal women with no hormonal treatment during the three months before surgery, normal menstrual cycles, and absence of uterine or tubal pathology as established by transvaginal ultrasound analysis. To select a homogenous population, besides the general inclusion criteria, 3 women (out of 62 initially recruited) were selected based on their similar age (31, 33, and 39 years old), proof of healthy progeny, and because surgery was performed in the secretory phase of the menstrual cycle. Samples (UF and OV, and blood) were stored at the reproductive fluid collection of Biobank-Mur (Biobanco en Red de la Región de Murcia, PT13/0010/0018; PT17/0015/0038, integrated in the Spanish National Biobanks Network, B.000859), until proteomic analysis, following standard operating procedures with approval of the Ethical and Scientific Committees of the hospital, and in accordance with Directive 2004/ 23 /EC of the European Parliament and of the Council of March 31, 2004 concerning human blood and its components, Law 14/2007, of July 3, of Biomedical Research, and Royal Decree of Biobanks 1716/2011, of November 18. Informed consent was obtained from all participants. The patients were identified with specific study codes to protect their confidentiality: BRA52, BRA54, BRA57 (Table 3). Table 3. Demographic data of the recruited patients. Demographic Data BRA-52 BRA-54 BRA-57 Age (years) Menarche (years) Parity Menstrual cycle duration Sample collection day (Menstrual Cycle phase) 33 11 G4C4 30 days Day 18: early secretory phase 39 14 G2P2 30 days Day 17: early secretory phase 31 10 G2C1P1 29/30 days Day 22: secretory phase 4.2. Sample Collection One fasting blood sample was collected, from the median cubital vein before surgery, to an EDTA-containing tube and centrifuged immediately at 1200× g for 15 min at 4 ◦ C to separate the plasma fraction from blood cells. Plasma was aliquoted and frozen at −80 ◦ C until analysis. Uterine fluid was obtained with an adapted Mucat device (Laboratoire CCD, Paris, France) in the surgery room before the procedure. This class I medical device, complying with Directive 93/42/EEC, indicated for direct exocervical or endocervical aspiration and Hühner test, was adapted to be easily introduced into the uterus. Once introduced, aspiration of the fluid was performed with the integrated plunger, which slides up and down when pushed by a flexible acetal resin shaft, without a syringe. Aspirated volumes varied from 59 to 260 µL. Upon laparoscopic intervention, fallopian tubes were removed, transferred to ice-cold Petri dishes, and dissected. Once dissected, fallopian tubes were clamped in both extremities: A—the one closest to the isthmus (the narrowest part of the tube) and B—the one contiguous to the infundibulum (the widest part of the tube). Afterward, with an ascendant manual mechanical pressure (from the extremity A to B), the oviductal fluid accumulated at the B portion of the ampulla. Then, the clamping scissor that closed this end was opened, and the fluid was aspirated with the Mucat device. Aspirated volumes varied from 49 to 70.9 µL. Once the fluids were collected in EDTA K2 (1.8 mg/mL) tubes, they were immediately centrifuged at 7000× g for 15 min at 4 ◦ C to remove cell debris, and the supernatant was aliquoted and frozen at −80 ◦ C until analysis. Int. J. Mol. Sci. 2019, 20, 5305 13 of 20 4.3. Immunoaffinity Depletion All samples were diluted 5-fold with 1X dilution buffer (Tris-Buffered Saline (TBS); 10 mM Tris-HCl with 150 mM NaCl, pH 7.4), filtered using a 0.45 µm pore-size spin filter to remove particulate materials, and centrifuged at 9000× g for 1 min. To deplete Albumin, IgG, α1-Antitrypsin, IgA, IgM, Transferrin, Haptoglobin, α2-Macroglobulin, Fibrinogen, Complement C3, α1-Acid Glycoprotein (Orosomucoid), HDL (Apolipoproteins A-I and A-II), and LDL (mainly Apolipoprotein B)), an AKTAprime plus FPLC (General Electric, Seoul, South Korea) was used to inject 100–200 µL per sample in a Seppro IgY 14 LC5 column (Sigma–Aldrich, St.Louis, MO, USA) with a constant flow rate of 0.5 mL/min for 20 min, followed by a washing step at a flow rate of 2 mL/min for 3 min. Non bound proteins (depleted fraction) were collected in the flow-through fraction. Due to the large volume of collected fractions, depleted samples were concentrated using Amicon™ Ultra-15 Centrifugal Filter Units (Millipore, MA, USA). Concentrated samples were reconstituted in a chaotropic buffer containing 8 M urea, 2 M thiourea, and 100 mM triethylammonium bicarbonate (TEAB) pH 8.5. Concentrated fractions were stored at −80 ◦ C. 4.4. Tryptic Digestion Samples of 10 µg of each depleted protein were dissolved in 8 M urea, 25 mM ammonium bicarbonate, reduced, and alkylated with iodoacetamide, according to a method previously described (López-Ferrer et al., 2004) [72]. Urea concentration was reduced to 2 M with 25 mM ammonium bicarbonate (final volume 40 µL), and the samples digested overnight at 37 ◦C with trypsin (Sigma-Aldrich), with a sample/enzyme ratio of 25:1. After digestion, samples were desalted using ZipTip C18 (Merck, MA, USA) [72]. 4.5. Liquid Chromatography and Mass Spectrometer Analysis A 2 µg aliquot of each digested sample was subjected to 1D-nano LC ESI-MSMS analysis using a nano liquid chromatography system (Eksigent Technologies nanoLC Ultra 1D plus, SCIEX, Foster City, CA, USA) coupled via a Nanospray III source to a high-speed Triple TOF 5600 mass spectrometer (SCIEX, Foster City, CA, USA). The analytical column used was a silica-based reversed-phase Acquity UPLC M-Class Peptide BEH C18 Column, 75 µm I.D. × 150 mm length, 1.7 µm particle size, and 130 Å pore size (Waters, MA, USA). The trap column was a C18 Acclaim PepMapTM 100 (Thermo Scientific, Waltham, MA, USA), 100 µm × 2 cm, 5 µm particle diameter, 100 Å pore size, switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 2 µL/min. The nano-pump provided a flowrate of 250 nL/min and was operated under gradient elution conditions. Peptides were separated using a 250 min gradient ranging from 2% to 90% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid). The injection volume was 5 µL. Data acquisition was performed with a TripleTOF 5600 System (SCIEX, Foster City, CA). Data were acquired using an ionspray voltage floating (ISVF) 2300 V, curtain gas (CUR) 35, interface heater temperature (IHT) 150 ◦ C, ion source gas 1 (GS1) 25, declustering potential (DP) 100 V. All data were acquired using information-dependent acquisition (IDA) mode with Analyst TF 1.7 software (SCIEX, Framingham, MA, USA). For IDA parameters, 0.25 s MS survey scans (mass range 350–1250 Da) were followed by 35 MS/MS scans of 100ms (mass range 100–1800, total cycle time was 4 s). Switching criteria were ion m/z greater than 350 and smaller than 1250, with a charge state of 2 to 5 and an abundance threshold of more than 90 counts (cps). Former target ions were excluded for 15 s. IDA rolling collision energy (CE) parameters script was used for automatically controlling the CE. 4.6. Targeted Mass Spectrometry (MRM/SRM) To validate the results obtained by 1D-nano LC ESI-MSMS, a total of 2 µg per sample was used for SRM / MRM-directed proteomics using an Eksigent 1D Plus liquid nanocromatograph coupled to a SCIEX 55000 QTRAP quadrupole triple mass spectrometer and using a 60-min gradient. A blank was inserted between samples. A total of 291 transitions were monitored, corresponding to 81 specific peptides of 23 different proteins (Table 4). Int. J. Mol. Sci. 2019, 20, 5305 14 of 20 Table 4. Selected proteins for MRM validation. Gene Name Gene Description EF1D Elongation factor 1-delta RL3 60S ribosomal protein L3 PDIA3 Protein disulfide-isomerase A3 GSTA1 Glutathione S-transferase A1 EZRI Ezrin DPYL3 Isoform LCRMP-4 of Dihydropyrimidinase-related protein 3 GARS Glycine–tRNA ligase TSTD1 Thiosulfate:glutathione sulfurtransferase THY1 Thy-1 membrane glycoprotein CLH1 Clathrin heavy chain 1 IPO9 Importin-9 ENPL Endoplasmin HS90A Heat shock protein HSP 90-alpha NNRE NAD(P)H-hydrate epimerase PTGR2 Prostaglandin reductase 2 EF2 Elongation factor 2 COTL1 Coactosin-like protein NGAL Neutrophil gelatinase-associated lipocalin SFRP4 Secreted frizzled-related protein 4 Protein Class Plasma proteins Predicted intracellular proteins FDA approved drug targets Plasma proteins Predicted intracellular proteins Predicted secreted proteins Ribosomal proteins Enzymes Plasma proteins Predicted secreted proteins Enzymes Plasma proteins Predicted intracellular proteins Cancer-related genes Plasma proteins Predicted intracellular proteins Predicted intracellular proteins Disease-related genes Plasma proteins Potential drug targets Predicted secreted proteins Predicted intracellular proteins CD markers Plasma proteins Predicted membrane proteins Predicted secreted proteins Cancer-related genes Plasma proteins Predicted intracellular proteins Predicted intracellular proteins Predicted secreted proteins Transporters Predicted localization Intracellular, Secreted Cancer-related genes Plasma proteins Predicted intracellular proteins Predicted secreted proteins Cancer-related genes Plasma proteins Predicted intracellular proteins Disease related genes Enzymes Potential drug targets Predicted secreted proteins Enzymes Predicted intracellular proteins Cancer-related genes Disease-related genes Plasma proteins Predicted intracellular proteins Plasma proteins Predicted intracellular proteins Candidate cardiovascular disease genes Plasma proteins Predicted secreted proteins Candidate cardiovascular disease genes Plasma proteins Predicted secreted proteins Int. J. Mol. Sci. 2019, 20, 5305 15 of 20 Table 4. Cont. Gene Name Gene Description TRFL Lactotransferrin PLMN Plasminogen TETN Tetranectin OVGP1 Oviduct-specific glycoprotein Protein Class Cancer-related genes Plasma proteins Predicted intracellular proteins Predicted secreted proteins Cancer-related genes Candidate cardiovascular disease genes Disease-related genes Enzymes FDA approved drug targets Plasma proteins Predicted secreted proteins Cancer-related genes Plasma proteins Predicted intracellular proteins Predicted secreted proteins Plasma proteins Predicted secreted proteins As the number of transitions was too high to analyze, using a single method, the method of analysis was converted into two sub-methods (1 and 2) where 12 proteins and their corresponding peptides and transitions were monitored in each. Nine total samples (3 OF, 3 UF, and 3 P) were monitored using both sub-methods, and the raw data files in wiff format were analyzed using the Skyline 4.2 program. The analysis determined the areas corresponding to each transition and peptide. In cases where peptides could not be detected in samples, quantification results are not shown. In cases in which it was possible to monitor more than one peptide per protein, the areas corresponding to the transitions of each peptide and the areas of the peptides of each protein were added to obtain a total summed-area for each protein. 4.7. Data Analysis and Quantification MS/MS spectra in the form of raw data files were processed to mgf format using PeakView® 2.2 Software (SCIEX, Foster City, CA, USA) and mgf files searched using Mascot Server 2.5.1, (London, UK) OMSSA 2.1.9, X!TANDEM 2013.02.01.1, and Myrimatch 2.2.140 against a composite target/decoy database built from the 71,785 sequences of the Homo sapiens reference proteome found at Uniprot (January 2018), together with commonly occurring laboratory contaminants. An initial 35 ppm X!TANDEM search was used to recalibrate the precursor ion mass measurements in all MS/MS spectra. Search engines were then configured to match potential peptide candidates with mass error tolerance of 10 ppm and fragment ion tolerance of 0.02 Da, allowing for up to two missed tryptic cleavage sites and a maximum isotope error (13C) of 1, considering fixed carbamidomethylation of cysteine and variable oxidation of methionine, pyroglutamic acid from glutamine or glutamic acid at the peptide N-terminus, and acetylation of the protein N-terminus. Score distribution models were used to compute peptide-spectrum match p-values1, and spectra recovered by a false discovery rate (FDR) ≤ 0.01 (peptide-level) filter were selected for label-free quantitative analysis using parent ion intensities. The label-free quantification (LFQ) values of protein identified in nine samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were analyzed by hierarchical clustering, principal component analysis (PCA) and Venn Diagram using R Core Team (2013) (R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria), was used to observe if the different values would be grouped in a set of clusters corresponding to each type of sample or patient, in which each cluster would be distinct from each other, and the objects within each cluster would be broadly similar to each other. Protein–protein interaction networks were estimated by STRING (Szklarczyk et al. Nucleic Acids Res. 2015 43(Database issue): D447-52). Differential abundance was quantified using linear models, and statistical significance was measured using q-values (FDR). All analyses were conducted using software from Proteobotics (Madrid, Spain) [37,73]. Int. J. Mol. Sci. 2019, 20, 5305 16 of 20 The targeted mass spectrometry (MRM/SRM) data, namely the areas corresponding to each transition and peptide, were transformed to log 2 scale and a paired sample t-test was performed to determine whether the mean difference between two sets (OF/P, OF/UF, UF/P) was zero. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD015980. 5. Conclusions This study presents a high-throughput analysis of female reproductive tract fluids during the secretory phase of the menstrual cycle, which constitutes a novel contribution to the knowledge of the oviductal and uterine secretomes. The present data corroborate that reproductive fluids represent an important source of biomarkers with potential interest in the development of improved embryo culture media. Currently, recombinant albumin is added to the media because it is considered an essential supplement for embryo development and has been shown to be endocytosed by the embryo from the medium [5], as was also demonstrated for the oviductal OVGP1 [74]. Therefore, it is plausible that the reproductive fluids contain many other proteins with important roles in embryo development itself and can also act as carriers for other embryonic growth factors. Previous studies have focused on UF, but our study demonstrates that OF is also a rich fluid with essential proteins that could be a target to improve fertilization rates and early embryo development if used in the culture media, namely EZRIN, HSP90, or OVGP1. More studies with similar designs and with established standard operating procedures are needed to corroborate our results/hypothesis and find consistent markers in secretions of the female reproductive tract. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/21/5305/s1. Author Contributions: Conceptualization, F.C. and P.C.; Data curation, A.C.-G., A.P. and A.R.-F.; Formal analysis, A.C.-G., A.P., A.R.-F. and P.C.; Funding acquisition, P.C.; Investigation, A.C.-G., M.T.P.-S., M.L.S.-F. and F.C.; Methodology, A.C.-G and A.P.; Project administration, P.C.; Resources, M.T.P.-S. and M.L.S.-F.; Software, A.R.-F.; Validation, A.P. and F.C.; Writing—original draft, A.C.-G. and P.C.; Writing—review and editing, A.P., A.R.-F., M.T.P.-S., M.L.S.-F. and F.C. Funding: This study was funded by MINECO-FEDER (AGL 2015-66341-R), 20040/GERM/16, and the European Union, Horizon 2020 Marie Sklodowska-Curie Action, REPBIOTECH 675526. Acknowledgments: We thank the Service of Obstetrics and Gynecology of the University Clinical Hospital “Virgen de la Arrixaca” in Murcia, Spain, for the collaboration in sample collection. We want to particularly acknowledge the patients and the BioBank “Biobanco en Red de la Región de Murcia” (PT13/0010/0018; PT17/0015/0038) integrated into the Spanish National Biobanks Network (B.000859) for its collaboration. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations OF UF P Oviductal fluids Uterine fluids Plasma References 1. 2. 3. Adamson, G.D.; de Mouzon, J.; Chambers, G.M.; Zegers-Hochschild, F.; Mansour, R.; Ishihara, O.; Banker, M.; Dyer, S. International Committee for Monitoring Assisted Reproductive Technology: World report on assisted reproductive technology, 2011. Fertil. Steril. 2011, 110, 1067–1080. [CrossRef] [PubMed] Chronopoulou, E.; Harper, J.C. IVF culture media: Past, present and future. Hum. Reprod. Update 2015, 21, 39–55. [CrossRef] [PubMed] Sunde, A.; Brison, D.; Dumoulin, J.; Harper, J.; Lundin, K.; Magli, M.C.; Van Den Abbeel, E.; Veiga, A. Time to take human embryo culture seriously. Hum. Reprod. 2016, 31, 2174–2182. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 5305 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 17 of 20 Morbeck, D.E.; Paczkowski, M.; Fredrickson, J.R.; Krisher, R.L.; Hoff, H.S.; Baumann, N.A.; Moyer, T.; Matern, D. Composition of protein supplements used for human embryo culture. J. Assist. Reprod. Genet. 2014, 31, 1703–1711. [CrossRef] Blake, D.; Svalander, P.; Jin, M.; Silversand, C.; Hamberger, L. Protein supplementation of human IVF culture media. J. Assist. Reprod. Genet. 2002, 19, 137–143. [CrossRef] [PubMed] Bavister, B.D. Culture of preimplantation embryos: Facts and artifacts . Hum. Reprod. Update 1995, 1, 91–148. [CrossRef] Bungum, M.; Humaidan, P.; Bungum, L. Recombinant human albumin as protein source in culture media used for IVF: A prospective randomized study. Reprod. Biomed. Online 2002, 4, 233–236. [CrossRef] Youssef, M.M.; Mantikou, E.; van Wely, M.; Van der Veen, F.; Al-Inany, H.G.; Repping, S.; Mastenbroek, S. Culture media for human pre-implantation embryos in assisted reproductive technology cycles. Cochrane Database Syst. Rev. 2015, 8e, 291–295. [CrossRef] Sinclair, K.D.; Rutherford, K.M.D.; Wallace, J.M.; Brameld, J.M.; Stöger, R.; Alberio, R.; Sweetman, D.; Gardner, D.S.; Perry, V.E.A.; Adam, C.L.; et al. Epigenetics and developmental programming of welfare and production traits in farm animals. Reprod. Fertil. Dev. 2016, 28, 1443–1478. [CrossRef] Fernández-Gonzalez, R.; Moreira, P.; Bilbao, A.; Jiménez, A.; Pérez-Crespo, M.; Ramírez, M.A.; De Fonseca, F.R.; Pintado, B.; Gutiérrez-Adán, A. Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proc. Natl. Acad. Sci. USA 2004, 101, 5880–5885. [CrossRef] El Hajj, N.; Haaf, T. Epigenetic disturbances in in vitro cultured gametes and embryos: Implications for human assisted reproduction. Fertil. Steril. 2013, 99, 632–641. [CrossRef] [PubMed] Canovas, S.; Campos, R.; Aguilar, E.; Cibelli, J.B. Progress towards human primordial germ cell specification in vitro. Mol. Hum. Reprod. 2017, 23, 4–15. [CrossRef] [PubMed] Mantikou, E.; Youssef, M.A.F.M.; van Wely, M.; van der Veen, F.; Al-Inany, H.G.; Repping, S.; Mastenbroek, S. Embryo culture media and IVF/ICSI success rates: A systematic review. Hum. Reprod. Update 2013, 19, 210–220. [CrossRef] [PubMed] Bouillon, C.; Léandri, R.; Desch, L.; Ernst, A.; Bruno, C.; Cerf, C.; Chiron, A.; Souchay, C.; Burguet, A.; Jimenez, C.; et al. Does Embryo Culture Medium Influence the Health and Development of Children Born after In Vitro Fertilization? PLoS ONE 2016, 11, e0150857. [CrossRef] Kleijkers, S.H.M.; Mantikou, E.; Slappendel, E.; Consten, D.; Van Echten-Arends, J.; Wetzels, A.M.; Van Wely, M.; Smits, L.J.M.; Van Montfoort, A.P.A.; Repping, S.; et al. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: A multicenter RCT. Hum. Reprod. 2016, 31, 2219–2230. [CrossRef] [PubMed] Avilés, M.; Gutiérrez-Adán, A.; Coy, P. Oviductal secretions: Will they be key factors for the future ARTs? MHR Basic Sci. Reprod. Med. 2010, 16, 896–906. [CrossRef] [PubMed] Galileo, D.S.; Bathala, P.; Fereshteh, Z.; Martin-DeLeon, P.A.; Li, K.; Al-Dossary, A.A. Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: Murine OVS play a pivotal role in sperm capacitation and fertility. MHR Basic Sci. Reprod. Med. 2018, 24, 143–157. Schramm, R.D.; Al-Sharhan, M. Fertilization and early embryology: Development of in-vitro-fertilized primate embryos into blastocysts in a chemically defined, protein-free culture medium. Hum. Reprod. 1996, 11, 1690–1697. [CrossRef] Tarahomi, M.; Vaz, F.; Zafardoust, S.; Fatemi, F.; VanWely, M.; Mohammadzadeh, A.; Repping, S.; Hamer, G.; Mastenbroek, S. Human uterine fluid composition is distinct from clinically used preimplantation embryo culture media. Fertil. Steril. 2018, 110, e364. [CrossRef] Leese, H.J. The formation and function of oviduct fluid. J. Reprod. Fert 1988, 82, 843–856. [CrossRef] Coy, P.; Yanagimachi, R. The Common and Species-Specific Roles of Oviductal Proteins in Mammalian Fertilization and Embryo Development. Bioscience 2015, 65, 973–984. [CrossRef] Hannan, N.J.; Stoikos, C.J.; Stephens, A.N.; Salamonsen, L.A. Depletion of high-abundance serum proteins from human uterine lavages enhances detection of lower-abundance proteins. J. Proteome Res. 2009, 8, 1099–1103. [CrossRef] [PubMed] Mondéjar, I.; Grullón, L.A.; García-Vázquez, F.A.; Romar, R.; Coy, P. Fertilization outcome could be regulated by binding of oviductal plasminogen to oocytes and by releasing of plasminogen activators during interplay between gametes. Fertil. Steril. 2012, 97, 453–461. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 5305 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 18 of 20 Coy, P.; Cánovas, S.; Mondéjar, I.; Saavedra, M.D.; Romar, R.; Grullón, L.; Matás, C.; Avilés, M. Oviduct-specific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy. Proc. Natl. Acad. Sci. USA 2008, 105, 15809–15814. [CrossRef] Coy, P.; García-Vázquez, F.A.; Visconti, P.E.; Avilés, M. Roles of the oviduct in mammalian fertilization. Reproduction 2012, 144, 649–660. [CrossRef] Mondéjar, I.; Martínez-Martínez, I.; Avilés, M.; Coy, P. Identification of potential oviductal factors responsible for zona pellucida hardening and monospermy during fertilization in mammals. Biol. Reprod. 2013, 89, 67. [CrossRef] Canovas, S.; Ivanova, E.; Romar, R.; García-Martínez, S.; Soriano-Úbeda, C.; García-Vázquez, F.A.; Saadeh, H.; Andrews, S.; Kelsey, G.; Coy, P. DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids. Elife 2017, 6, e23670. [CrossRef] Berlanga, O.; Bradshaw, H.B.; Vilella-Mitjana, F.; Garrido-Gómez, T.; Simón, C. How endometrial secretomics can help in predicting implantation. Placenta 2011, 32, S271–S275. [CrossRef] Zhang, Y.; Wang, Q.; Wang, H.; Duan, E. Uterine Fluid in Pregnancy: A Biological and Clinical Outlook. Trends Mol. Med. 2017, 23, 604–614. [CrossRef] Salamonsen, L.A.; Edgell, T.; Rombauts, L.J.F.; Stephens, A.N.; Robertson, D.M.; Rainczuk, A.; Nie, G.; Hannan, N.J. Proteomics of the human endometrium and uterine fluid: A pathway to biomarker discovery. Fertil. Steril. 2013, 99, 1086–1092. [CrossRef] Di Quinzio, M.K.W.; Oliva, K.; Holdsworth, S.J.; Ayhan, M.; Walker, S.P.; Rice, G.E.; Georgiou, H.M.; Permezel, M. Proteomic analysis and characterisation of human cervico-vaginal fluid proteins. Aust. New Zeal. J. Obstet. Gynaecol. 2007. [CrossRef] [PubMed] Ametzazurra, A.; Matorras, R.; García-Velasco, J.A.; Prieto, B.; Simón, L.; Martínez, A.; Nagore, D. Endometrial fluid is a specific and non-invasive biological sample for protein biomarker identification in endometriosis. Hum. Reprod. 2009, 24, 954–965. [CrossRef] [PubMed] Hannan, N.J.; Nie, G.; Rainzcuk, A.; Rombauts, L.J.F.; Salamonsen, L.A. Uterine Lavage or Aspirate: Which View of the Intrauterine Environment? Reprod. Sci. 2012, 19, 1125–1132. [CrossRef] [PubMed] Mateos, J.; Carneiro, I.; Corrales, F.; Elortza, F.; Paradela, A.; del Pino, M.S.; Iloro, I.; Marcilla, M.; Mora, M.I.; Valero, L.; et al. Multicentric study of the effect of pre-analytical variables in the quality of plasma samples stored in biobanks using different complementary proteomic methods. J. Proteom. 2017, 150, 109–120. [CrossRef] [PubMed] Casado-Vela, J.; Rodriguez-Suarez, E.; Iloro, I.; Ametzazurra, A.; Alkorta, N.; García-Velasco, J.A.; Matorras, R.; Prieto, B.; González, S.; Nagore, D.; et al. Comprehensive proteomic analysis of human endometrial fluid aspirate. J. Proteome Res. 2009, 10, 4622–4632. [CrossRef] [PubMed] Tjokronegoro, A.; Sirisinha, S. Quantitative Analysis of Immunoglobulins and Albumin in Secretion of Female Reproductive Tract*Supported by Research Grants GA BMS 7209 and GA BMS 7309 from The Rockefeller Foundation and by the National Research Council of Thailand. Fertil. Steril. 1975, 26, 413–417. [CrossRef] Ramos-Fernández, A.; Paradela, A.; Navajas, R.; Albar, J.P. Generalized method for probability-based peptide and protein identification from tandem mass spectrometry data and sequence database searching. Mol. Cell. Proteom. 2008, 7, 1748–1754. [CrossRef] Xu, H.; Zhu, Q.; Ji, X.; Wang, Y.; He, Y.; Chen, Z.; Sun, Y.; Hu, S.; Yao, G. Transcriptomic Changes During the Pre-Receptive to Receptive Transition in Human Endometrium Detected by RNA-Seq. J. Clin. Endocrinol. Metab. 2014, 99, E2744–E2753. Woo, M.M.M.; Alkushi, A.; Verhage, H.G.; Magliocco, A.M.; Leung, P.C.K.; Gilks, C.B.; Auersperg, N. Gain of OGP, an estrogen-regulated oviduct-specific glycoprotein, is associated with the development of endometrial hyperplasia and endometrial cancer. Clin. Cancer Res. 2004, 10, 7958–7964. [CrossRef] Brosens, J.J.; Gellersen, B. Cyclic Decidualization of the Human Endometrium in Reproductive Health and Failure. Endocr. Rev. 2014, 35, 851–905. Bazer, F.W.; Spencer, T.E.; Johnson, G.A.; Burghardt, R.C.; Wu, G. Comparative aspects of implantation. Reproduction 2009, 138, 195–209. [CrossRef] [PubMed] Strandell, A.; Lindhard, A. Why does hydrosalpinx reduce fertility? The importance of hydrosalpinx fluid. Hum. Reprod. 2002, 17, 1141–1145. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 5305 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 19 of 20 Ng, E.H.; Ajonuma, L.C.; Lau, E.Y.; Yeung, W.S.; Ho, P.C. Adverse effects of hydrosalpinx fluid on sperm motility and survival. Hum. Reprod. 2000, 15, 772–777. [CrossRef] [PubMed] Meyer, W.R.; Castelbaum, A.J.; Somkuti, S.; Sagoskin, A.W.; Doyle, M.; Harris, J.E.; Lessey, B.A. Hydrosalpinges adversely affect markers of endometrial receptivity. Hum. Reprod. 1997, 12, 1393–1398. [CrossRef] [PubMed] Kodithuwakku, S.P.; Miyamoto, A.; Wijayagunawardane, M.P.B. Spermatozoa stimulate prostaglandin synthesis and secretion in bovine oviductal epithelial cells. Reproduction 2007, 133, 1087–1094. [CrossRef] [PubMed] Zhao, Q.; He, Y.; Wang, X.-L.; Zhang, Y.-X.; Wu, Y.-M. Differentially expressed proteins among normal cervix, cervical intraepithelial neoplasia and cervical squamous cell carcinoma. Clin. Transl. Oncol. 2015, 17, 620–631. [CrossRef] [PubMed] Manohar, M.; Khan, H.; Sirohi, V.K.; Das, V.; Agarwal, A.; Pandey, A.; Siddiqui, W.A.; Dwivedi, A. Alteration in endometrial proteins during early- and mid-secretory phases of the cycle in women with unexplained infertility. PLoS ONE 2014, 9, e111687. [CrossRef] Papp, S.M.; Fröhlich, T.; Radefeld, K.; Havlicek, V.; Kösters, M.; Yu, H.; Mayrhofer, C.; Brem, G.; Arnold, G.J.; Besenfelder, U. A novel approach to study the bovine oviductal fluid proteome using transvaginal endoscopy. Theriogenology 2019, 132, 53–61. [CrossRef] Coy, P.; Jimenez-Movilla, M.; Garcia-Vazquez, F.A.; Mondejar, I.; Grullon, L.; Romar, R. Oocytes use the plasminogen-plasmin system to remove supernumerary spermatozoa. Hum. Reprod. 2012, 7, 1985–1993. [CrossRef] Grullón, L.A.; Gadea, J.; Mondéjar, I.; Matás, C.; Romar, R.; Coy, P. How Is Plasminogen/Plasmin System Contributing to Regulate Sperm Entry into the Oocyte? Reprod. Sci. 2013, 20, 1075–1082. Roldan-Olarte, M.; Garcia, D.C.; Jimenez-Diaz, M.; Valdecantos, P.A.; Miceli, D.C. In vivo and in vitro expression of the plasminogen activators and urokinase type plasminogen activator receptor (u-PAR) in the pig oviduct. Anim. Reprod. Sci. 2012, 136, 90–99. [CrossRef] [PubMed] Holtet, T.L.; Graversen, J.H.; Clemmensen, I.; Thogersen, H.C.; Etzerodt, M. Tetranectin, a trimeric plasminogen-binding C-type lectin. Protein Sci. 1997, 6, 1511–1515. [CrossRef] [PubMed] Bhusane, K.; Bhutada, S.; Chaudhari, U.; Savardekar, L.; Katkam, R.; Sachdeva, G. Secrets of Endometrial Receptivity: Some Are Hidden in Uterine Secretome. Am. J. Reprod. Immunol. 2016, 75, 226–236. [CrossRef] [PubMed] Revel, A.; Achache, H. Endometrial receptivity markers, the journey to successful embryo implantation. Hum. Reprod. Update 2006, 12, 731–746. Aghajanova, L.; Hamilton, A.E.; Giudice, L.C. Uterine receptivity to human embryonic implantation: Histology, biomarkers, and transcriptomics. Semin. Cell Dev. Biol. 2008, 19, 204–211. [CrossRef] [PubMed] Redgrove, K.A.; Anderson, A.L.; Dun, M.D.; McLaughlin, E.A.; O’Bryan, M.K.; Aitken, R.J.; Nixon, B. Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae. Dev. Biol. 2011, 356, 460–474. [CrossRef] [PubMed] Yi, K.; Unruh, J.R.; Deng, M.; Slaughter, B.D.; Rubinstein, B.; Li, R. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes. Nat. Cell Biol. 2011, 13, 1252–1258. [CrossRef] Kumar, A.; Dumasia, K.; Gaonkar, R.; Sonawane, S.; Kadam, L.; Balasinor, N.H. Estrogen and androgen regulate actin-remodeling and endocytosis-related genes during rat spermiation. Mol. Cell. Endocrinol. 2015, 404, 91–101. [CrossRef] Melideo, S.L.; Jackson, M.R.; Jorns, M.S. Biosynthesis of a central intermediate in hydrogen sulfide metabolism by a novel human sulfurtransferase and its yeast ortholog. Biochemistry 2014, 53, 4739–4753. [CrossRef] Haverinen, M.; Passinen, S.; Syvälä, H.; Pasanen, S.; Manninen, T.; Tuohimaa, P.; Ylikomi, T. Heat shock protein 90 and the nuclear transport of progesterone receptor. Cell Stress Chaperones 2001, 6, 256–262. [CrossRef] Wetendorf, M.; DeMayo, F.J. The progesterone receptor regulates implantation, decidualization, and glandular development via a complex paracrine signaling network. Mol. Cell. Endocrinol. 2012, 357, 108–118. [CrossRef] [PubMed] Reed, C.A.; Osborne, E.; Kirk, E.A.; Bailey, J.A.; Fan, M.; Nephew, K.P.; Long, X.; Gize, E.A.; Bigsby, R.M. The Activating Enzyme of NEDD8 Inhibits Steroid Receptor Function. Mol. Endocrinol. 2002, 16, 315–330. Int. J. Mol. Sci. 2019, 20, 5305 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 20 of 20 Boeddeker, S.J.; Hess, A.P. The role of apoptosis in human embryo implantation. J. Reprod. Immunol. 2015, 108, 114–122. [CrossRef] [PubMed] Mendoza, C.; Greco, E.; Ubaldi, F.; Martinez, F.; Tesarik, J.; Rienzi, L.; Iacobelli, M. Caspase activity in preimplantation human embryos is not associated with apoptosis. Hum. Reprod. 2002, 17, 1584–1590. Bukovský, A.; Presl, J.; Zidovský, J. Association of some cell surface antigens of lymphoid cells and cell surface differentiation antigens with early rat pregnancy. Immunology 1984, 52, 631–640. Fagerberg, L.; Hallström, B.M.; Oksvold, P.; Kampf, C.; Djureinovic, D.; Odeberg, J.; Habuka, M.; Tahmasebpoor, S.; Danielsson, A.; Edlund, K.; et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol. Cell. Proteom. 2014, 13, 397–406. [CrossRef] Jefferson, W.N.; Padilla–Banks, E.; Newbold, R.R. Lactoferrin is an estrogen responsive protein in the uterus of mice and rats. Reprod. Toxicol. 2000, 14, 103–110. [CrossRef] Wu, K.; Hoy, M.A. Clathrin heavy chain is important for viability, oviposition, embryogenesis and, possibly, systemic RNAi response in the predatory mite Metaseiulus occidentalis. PLoS ONE 2014, 9, e110874. [CrossRef] Biedka, S.; Micic, J.; Wilson, D.; Brown, H.; Diorio-Toth, L.; Woolford, J.L.J. Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes. J. Cell Biol. 2018, 217, 2503–2518. [CrossRef] Matsumiya, T.; Xing, F.; Ebina, M.; Hayakari, R.; Imaizumi, T.; Yoshida, H.; Kikuchi, H.; Topham, M.K.; Satoh, K.; Stafforini, D.M. Novel role for molecular transporter importin 9 in posttranscriptional regulation of IFN-epsilon expression. J. Immunol. 2013, 191, 1907–1915. [CrossRef] Hermant, P.; Francius, C.; Clotman, F.; Michiels, T. IFN-epsilon is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines. PLoS ONE 2013, 8, e71320. [CrossRef] [PubMed] López-Ferrer, D.; Martínez-Bartolomé, S.; Villar, M.; Campillos, M.; Martín-Maroto, F.; Vázquez, J. Statistical Model for Large-Scale Peptide Identification in Databases from Tandem Mass Spectra Using SEQUEST. Anal. Chem. 2004, 76, 6853–6860. [CrossRef] [PubMed] Lopez-Serra, P.; Marcilla, M.; Villanueva, A.; Ramos-Fernandez, A.; Palau, A.; Leal, L.; Wahi, J.E.; Setien-Baranda, F.; Szczesna, K.; Moutinho, C.; et al. A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect. Nat. Commun. 2014, 5, 3608. [CrossRef] [PubMed] Algarra, B.; Maillo, V.; Avilés, M.; Gutiérrez-Adán, A.; Rizos, D.; Jiménez-Movilla, M. Effects of recombinant OVGP1 protein on in vitro bovine embryo development. J. Reprod. Dev. 2018, 64, 433–443. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4307407398	https://www.researchsquare.com/article/rs-1984059/latest.pdf	English	null	Flexible Uretero-Renoscopy Versus Mini-Percutaneous Nephrolithotomy for Treatment of Renal Stones 2 Cm or Less; A Randomised, Comparative Study	Research Square (Research Square)	2,022	cc-by	137	Keywords: Posted Date: October 27th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1984059/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. EDITORIAL NOTE: The full text of this preprint has been withdrawn by the authors while they make corrections to the work. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Page 1/2 Page 1/2 Abstract The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Full Text The authors have withdrawn this preprint from Research Square. Page 2/2
https://openalex.org/W2074923652	https://jmedicalcasereports.biomedcentral.com/counter/pdf/10.4076/1752-1947-3-7300	English	null	Postnatal onset of severe growth retardation after in utero exposure to carbamazepine and phenobarbital: a case report	Journal of medical case reports	2,009	cc-by	1,687	Introduction Epilepsy is common, affecting 0.5% to 1% of the population. Of these, a third are women in reproductive age, and approximately 1 in 250 pregnancies are exposed to antiepileptic drugs. In utero exposure to antiepileptic drugs can result in several different teratogenic effects including major malformations, dysmorphic facial fea- tures, intrauterine growth retardation, learning and behavioral problems. We report on a child exposed in utero to both carbamazepine and phenobarbital. He Received: 19 February 2008 Accepted: 23 January 2009 Published: 12 June 2009 Journal of Medical Case Reports 2009, 3:7300 doi: 10.4076/1752-1947-3-7300 Received: 19 February 2008 Accepted: 23 January 2009 Published: 12 June 2009 Journal of Medical Case Reports 2009, 3:7300 doi: 10.4076/1752-1947-3-7300 This article is available from: http://jmedicalcasereports.com/jmedicalcasereports/article/view/7300 © 2009 Liguori and Cianfarani; licensee Cases Network Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction: Anticonvulsant drugs taken by pregnant women to prevent seizures are among the most common causes of potential harm to the fetus. While the immediate harmful effects manifesting as congenital abnormalities are well known, the long-term effects on growth of children exposed in utero to antiepileptic drugs are still uncertain. Case presentation: A 7-year-old boy presented to our clinic with severe short stature. His height was 110.4 cm (−2.4 standard deviation score), with a target height of 177 cm (+0.35 standard deviation score). Height corrected for target height was −2.75 standard deviation score. He presented with mild dysmorphic facial features, hypospadias and postnatal onset of severe growth retardation. Biochemical and endocrine tests were in the normal range. The child was exposed in utero to both carbamazepine and phenobarbital. Conclusion: This case report shows for the first time that prenatal exposure to antiepileptic drugs may induce postnatal onset of severe growth retardation, suggesting the need for growth and endocrine monitoring of offspring exposed in utero to anticonvulsant drugs. presented with mild dysmorphic facial features, hypospa- dias and postnatal onset of growth retardation. Open Access Case report Postnatal onset of severe growth retardation after in utero exposure to carbamazepine and phenobarbital: a case report Alice Liguori and Stefano Cianfarani* Address: ‘Rina Balducci’ Center of Pediatric Endocrinology, Department of Public Health and Cell Biology, Tor Vergata University, 00133 Rome Italy Address: ‘Rina Balducci’ Center of Pediatric Endocrinology, Department of Public Health and Cell Biology, Tor Vergata University, 00133-Rome, Italy Email: AL - aliceliguori@tiscali.it; SC* - stefano.cianfarani@uniroma2.it * Corresponding author Case presentation A 7-year-old boy presented to our clinic with short stature. His height was 110.4 cm (−2.4 standard deviation score (SDS)), with a target height of 177 cm (+0.35 SDS). Height corrected for target height was −2.75 SDS. His weight was 16.5 kg. Body mass index (BMI) was 13.5 (−1.8 SDS). He was born at term after an uneventful pregnancy. Birth weight Page 1 of 3 (page number not for citation purposes) Journal of Medical Case Reports 2009, 3:7300 http://jmedicalcasereports.com/jmedicalcasereports/article/view/7300 Figure 2. Patient’s phenotype. was 4260 g (+1.3 SDS), birth length was 53 cm (+1.1 SDS) and birth head circumference was 37 cm (+1.3 SDS). He was born with hypospadias and underwent surgery at the age of two years. During pregnancy, his mother had undergone antiepileptic therapy with carbamazepine (200 mg bid) and phenobarbital (100 mg bid). His growth was normal during the first 12 months of age, thereafter it slowed down progressively (Figure 1). On physical examination, ocular hypertelorism, arched eyebrows, epicanthal folds, broad nasal bridge, low-set ears, and shortness of the thumb were noted (Figure 2). Bone age was six years. Neurocognitive function was normal. Liver and renal function test results, electrolytes, calcium, phosphorus, and celiac disease mar- kers were within the normal range. Urine examination was normal and thyroid function tests were normal. Arginine and growth hormone releasing hormone (GHRH) + arginine testing showed normal growth hormone (GH) responses (GH peaks 25 mg/L and 25.3 mg/L, respectively; normal values ≥10 mg/L and 20 mg/L, respectively). Insulin- like growth factor-I (IGF-I) concentrations were in the low normal range (90 mg/L, −1.8 SDS), whereas IGFBP-3 levels were within the normal range (2.9 mg/L, +0.2 SDS). Renal and cardiac ultrasound scans were normal. Skeletal X-rays Figure 1. Postnatal growth of the child exposed in utero to carbamazepine and phenobarbital. Figure 2. Patient’s phenotype. Figure 2. Patient’s phenotype. showed a short first metacarpal bone but no sign of skeletal dysplasias. Dysmorphologic evaluation did not reveal any particular syndrome. Chromosome analysis disclosed a normal 46,XY, karyotype. References 1. Kini U, Adab N, Vinten J, Fryer A, Clayton-Smith J: Dysmorphic features: an important clue to the diagnosis and severity of fetal anticonvulsivant syndromes. Arch Dis Child Fetal Neonatal Ed 2006, 91:90-95. , 2. Meador KJ, Baker GA, Finnel RH, Kalayjian LA, Liporace JD, Loring DW, Mawer G, Pennell PB, Smith JC, Wolff MC for the NEAD Study Group: In utero antiepileptic drug exposure: fetal death and malformations. Neurology 2006, 67:407-412. 2. Meador KJ, Baker GA, Finnel RH, Kalayjian LA, Liporace JD, Loring DW, Mawer G, Pennell PB, Smith JC, Wolff MC for the NEAD Study Group: In utero antiepileptic drug exposure: fetal death and malformations. Neurology 2006, 67:407-412. 3. Artama M, Auvinen A, Raudaskoski T, Isojärvi I, Isojärvi J: Anti- epileptic drug use of women with epilepsy and congenital malformations in offspring. Neurology 2005, 64:1874-1878. p g gy 4. Manent JB, Jorquera I, Mazzucchelli I, Depaulis A, Perucca E, Ben- Ari Y, Represa A: Fetal exposure to GABA-acting antiepileptic drugs generates hippocampal and cortical dysplasias. Epilepsia 2007, 48:684-693. 5. Wide K, Winbladh B, Tomson T, Kallen B: Body dimensions of infants exposed to antiepileptic drugs in utero: observations spanning 25 years. Epilepsia 2000, 41:854-861. 5. Wide K, Winbladh B, Tomson T, Kallen B: Body dimensions of infants exposed to antiepileptic drugs in utero: observations spanning 25 years. Epilepsia 2000, 41:854-861. 6. Perucca E: Birth defects after prenatal exposure to antiepilep- tic drugs Lancet Neurol 2005 4:781-786 p g y p p 6. Perucca E: Birth defects after prenatal exposure to antiepilep- tic drugs. Lancet Neurol 2005, 4:781-786. g 7. Arulmozhi T, Dhanaraj M, Rangaraj R, Vengatesan A: Physical growth and psychomotor development of infants exposed to antiepileptic drugs in utero. Neurol India 2006, 54:42-47. Our case is consistent with two previous reports showing either impaired physical growth in infants exposed to anticonvulsant drugs in utero in spite of normal birth size [7] or increased frequency of major malformations, microcephaly, and growth retardation in infants exposed to carbamazepine compared with control infants [8]. However, the severity of growth retardation and the full investigation of GH-IGF-I axis make our case unique. The finding of reduced IGF-I levels despite normal GH peak responses to stimulation tests raises the issue of a potential disrupting effect of the in utero antiepileptic exposure on postnatal GH-IGF-I axis function. 8. p p g 8. Consent Written informed consent was obtained from both parents of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in Chief of this journal. Competing interests The authors declare that they have no competing interests. Conclusion This case report shows for the first time that prenatal exposure to antiepileptic drugs may induce postnatal onset of severe growth retardation, thus suggesting the need for growth and endocrine monitoring of offspring exposed in utero to anticonvulsant drugs. Discussion Data on the effects of prenatal exposure to carbamazepine and/or phenobarbital are conflicting. In a retrospective study of 375 children aged from six months to 16 years born to 219 mothers with epilepsy, Kini et al. [1] reported short stature in 6.5% of children exposed to Figure 1. Postnatal growth of the child exposed in utero to carbamazepine and phenobarbital. Page 2 of 3 (page number not for citation purposes) Page 2 of 3 (page number not for citation purposes) Journal of Medical Case Reports 2009, 3:7300 Journal of Medical Case Reports 2009, 3:7300 http://jmedicalcasereports.com/jmedicalcasereports/article/view/7300 carbamazepine and 6.4% of children exposed to poly- therapy. In a prospective observational study across 25 epilepsy centers in the USA and UK, Meador et al. [2] observed that more adverse outcomes were observed in pregnancies with in utero valproate exposure. In children exposed in utero to carbamazepine, the following con- genital malformations have been reported: absent kidney, duplicate renal pelvis, hypospadias, and inguinal hernia. In a cohort of female patients with epilepsy, Artama et al. [3] reported that the risk for congenital malformations was not elevated in offspring of mothers using carbamazepine, oxcarbazepine, or phenytoin (as monotherapy or poly- therapy without valproate). In rats, Manent et al. [4] reported that prenatal exposure to vigabatrin and valpro- ate, which act on GABA signaling, induces hippocampal and cortical dysplasias, which are likely to result from a neuronal migration defect and neuronal death. In contrast, offspring of rats exposed to carbamazepine showed no clear-cut evidence of dysplasias. Wide et al. [5] found a significant reduction in weight, head circumference and length, which tended to improve toward the first year and was marked in babies exposed to polytherapy and also in babies exposed to carbamazepine monotherapy. However, it has to be pointed out that nearly all studies on the adverse fetal effects of antiepileptic drugs have methodo- logical shortcomings, including retrospective or inade- quately prospective design, insufficient sample size, recruitment and assessment bias, limited length of follow-up, questionable choice of controls, and failure to account for potential confounders [6]. Authors’ contributions AL and SC followed up the patient in the clinics, performed the literature review, drafted the manuscript, and read and approved the final version of the manuscript. References Holmes LB, Harvey EA, Coull BA, Huntington KB, Khoshbin S, Hayes AM, Ryan LM: The teratogenicity of anticonvulsant drugs. N Engl J Med 2001, 344:1132-1138. Do you have a case to share? Submit your case report today • Rapid peer review • Fast publication • PubMed indexing • Inclusion in Cases Database Any patient, any case, can teach us something www.casesnetwork.com Abbreviations GH, growth hormone; GHRH, growth hormone releasing hormone; IGF-I, insulin-like growth factor-I; IGFBP-3, insulin-like growth factor binding protein-3.
https://openalex.org/W4388569872	https://ijmscr.org/index.php/ijmscrs/article/download/1237/1034	English	null	Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration	International journal of medical science and clinical research studies	2,023	cc-by	3,600	Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration Paola Lizbeth Zamudio Naranjo1, Elsa María Roselli Gutiérrez2, Mildred Andrea Zúñiga Onofre3, Monica Tamayo Garza 4, José Manuel Montero de Anda5, Sergio Emmanuel Vargas López 6 1,2,3,4,5,6 Universidad Autónoma de Guadalajara, Guadalajara. México. ABSTRACT Urolithiasis, the formation of urinary calculi, remains a prevalent and clinically significant urological disorder worldwide. Despite extensive research, the etiology of urolithiasis remains multifactorial and not entirely understood. Emerging evidence suggests that the urinary microbiome, previously considered sterile, plays a pivotal role in the pathogenesis and progression of urolithiasis. This article delves into the intricate relationship between urolithiasis and the urinary microbiome, elucidating the key microbial players, their potential mechanisms of action, and the clinical implications of this association. We explore recent advancements in metagenomics, metabolomics, and microbial profiling techniques that have revolutionized our understanding of the urinary microbiome in urolithiasis. A comprehensive understanding of the interplay between urolithiasis and the urinary microbiome holds promise for novel diagnostic and therapeutic strategies, offering new avenues for the prevention and management of this debilitating condition. Published On: 06 November 2023 Available on: https://ijmscr.org/ KEYWORDS: urolithiasis, calculi, microbiome INTRODUCTION the urinary tract, their dynamic interactions, and their potential role in stone formation. Furthermore, we will examine how dysbiosis of the urinary microbiome may contribute to stone recurrence and investigate the mechanisms underlying these phenomena. By shedding light on these intricate connections, we aim to pave the way for innovative diagnostic and therapeutic strategies that harness the power of the urinary microbiome to mitigate the burden of urolithiasis.2,3 Urolithiasis, the formation of calculi within the urinary tract, represents a significant medical challenge affecting millions of individuals globally. Despite advances in diagnostic and therapeutic modalities, the exact pathogenesis of urolithiasis remains elusive, and its recurrence rates remain high. Conventionally, urolithiasis has been attributed primarily to physicochemical factors, including urinary supersaturation with stone-forming salts and inadequate urine volume. However, emerging research has unveiled a previously underappreciated player in the intricate urolithiasis puzzle— the urinary microbiome.1 International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 11 November 2023 Page No: 2627-2630 International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 11 November 2023 Page No: 2627-2630 International Journal of Medical Science and Clinical Research Studies ISSN(print): 2767-8326, ISSN(online): 2767-8342 Volume 03 Issue 11 November 2023 Page No: 2627-2630 Volume 03 Issue 11 November 2023 Page No: 2627-2630 2627 Volume 03 Issue 11 November 2023 Corresponding Author: Paola Lizbeth Zamudio Naranjo Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration Chronic inflammation in the urinary tract can also promote stone development.5 The Urinary Microbiome Connection: Recent advancements in microbiome research have unveiled the urinary microbiome as a novel player in urolithiasis epidemiology. Shifts in microbial composition, often referred to as dysbiosis, have been associated with stone formation. Dysbiosis may result from factors such as antibiotic use, recurrent urinary tract infections, and dietary habits, all of which can influence stone risk.4 8. Dietary Factors: High dietary intake of oxalate-rich foods, purines, sodium, and inadequate fluid consumption can exacerbate stone formation by altering urinary composition and increasing the risk of supersaturation.5 Immunocompromised Individuals: Certain populations, such as those with immunosuppression due to conditions like HIV or organ transplantation, are at an increased risk of urolithiasis, possibly due to altered urinary microbiome profiles and immune responses.4 9. Genetic Predisposition: Genetic mutations affecting various transporters and enzymes involved in calcium and oxalate metabolism can heighten the susceptibility to urolithiasis. These genetic factors can disrupt normal urinary homeostasis.5 10. Urinary Microbiome Dysbiosis: Recent research has uncovered that shifts in the composition and function of the urinary microbiome can influence the pathophysiology of urolithiasis. Dysbiosis may contribute to alterations in urinary pH, the production of biofilms on stone surfaces, and the modulation of immune responses, all of which can affect stone formation, retention, and recurrence.5 Corresponding Author: Paola Lizbeth Zamudio Naranjo Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration Crystals can adhere to renal papillary surfaces, leading to stone growth over time.5 Recurrence Rates: Urolithiasis is often characterized by a recurrent nature. Approximately 50% of individuals who experience an initial stone episode will develop additional stones within five years. This high recurrence rate underscores the importance of preventative measures.4 3. Crystal Growth and Aggregation: Once crystals form, they can grow and aggregate to form macroscopic stones. Growth is influenced by urinary pH, ionic strength, and the presence of inhibitors and promoters. Crystals can adhere to renal papillary surfaces, leading to stone growth over time.5 4. Stone Adhesion: Adherence of crystals to renal tubular cells and organic matrix components in the urinary tract is facilitated by various adhesive molecules. This adherence promotes crystal retention and further contributes to stone growth.5 4. Stone Adhesion: Adherence of crystals to renal tubular cells and organic matrix components in the urinary tract is facilitated by various adhesive molecules. This adherence promotes crystal retention and further contributes to stone growth.5 Geographic Variation: Urolithiasis exhibits notable geographic variation, with a higher prevalence in certain regions. Factors contributing to these disparities include dietary habits, climate, genetics, and socioeconomic factors. Geographic Variation: Urolithiasis exhibits notable geographic variation, with a higher prevalence in certain regions. Factors contributing to these disparities include dietary habits, climate, genetics, and socioeconomic factors. Ethnic and Racial Factors: Epidemiological studies have identified variations in urolithiasis rates among different ethnic and racial groups. For instance, individuals of South Asian descent appear to have a higher risk of developing kidney stones.4 5. Anatomical Factors: Anatomical anomalies, such as ureteral strictures, kidney malformations, and renal cysts, can lead to urinary stasis and create ideal conditions for stone formation. Obstructions can also hinder stone passage.5 6. Metabolic Factors: Metabolic abnormalities, such as hypercalciuria, hyperuricosuria, and hyperoxaluria, can result from genetic predisposition or dietary factors. These metabolic disturbances contribute to an increased concentration of stone-forming substances in the urine 5 Obesity and Metabolic Syndrome: The global rise in obesity and metabolic syndrome has coincided with an increased prevalence of urolithiasis. These conditions are often associated with metabolic abnormalities, including hypercalciuria and insulin resistance, which predispose individuals to stone formation.4 concentration of stone-forming substances in the urine.5 7. Infections and Inflammation: Infection-related urolithiasis is primarily associated with struvite stones. Urease-producing bacteria, such as Proteus and Klebsiella, hydrolyze urea to ammonia, leading to an increase in urinary pH and struvite crystal formation. EPIDEMIOLOGY Urolithiasis, a common urological condition characterized by the formation of urinary stones, has a significant global epidemiological footprint. The elucidation of its epidemiological intricacies, coupled with the burgeoning understanding of the urinary microbiome, promises to shed light on potential risk factors, patterns of occurrence, and novel preventive strategies.3 Historically, the urinary tract was considered a sterile environment. Still, recent investigations utilizing advanced molecular techniques, such as high-throughput sequencing and metagenomics, have revealed a diverse microbial community residing within the urinary system. This revelation has ignited a paradigm shift, underscoring the potential influence of the urinary microbiome on urolithiasis formation and recurrence.1,2 Prevalence: Urolithiasis represents a widespread medical concern, with prevalence rates varying geographically. Studies have shown that the prevalence is highest in regions with hot climates and elevated temperatures, potentially due This article embarks on an in-depth exploration of the intriguing relationship between urolithiasis and the urinary microbiome. We will delve into the microbial composition of Corresponding Author: Paola Lizbeth Zamudio Naranjo 2627 Volume 03 Issue 11 November 2023 2627 Volume 03 Issue 11 November 2023 Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration to increased dehydration and a greater propensity for urinary supersaturation 3 4 urinary microbiome is param comprehension of this condition Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration to increased dehydration and a greater propensity for urinary supersaturation.3,4 urinary microbiome is param comprehension of this condition. Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration urinary microbiome is paramount to advancing our comprehension of this condition.4,5 1. Urinary Supersaturation: The cornerstone of urolithiasis pathogenesis is the urinary supersaturation of stone-forming salts, including calcium oxalate, calcium phosphate, uric acid, and struvite. Supersaturation occurs when the concentration of these solutes exceeds their saturation point, triggering crystallization.5 Gender Disparities: Epidemiological data consistently reveal a higher prevalence of urolithiasis in males compared to females, with a male-to-female ratio typically ranging from 2:1 to 3:1. This gender disparity is partially attributed to hormonal and anatomical differences affecting urinary calcium excretion and urinary pH.4 2. Nucleation: Nucleation, the initial formation of microscopic crystals within the urinary system, is a pivotal step in stone development. Factors such as urinary stasis, high concentrations of stone-forming ions, and the presence of heterogeneous nucleating sites can facilitate crystal nucleation.5 Age Distribution: Urolithiasis exhibits a bimodal age distribution. The first peak occurs in the third and fourth decades of life, predominantly affecting men. The second peak occurs in the sixth to seventh decades, with a more even distribution between genders. Pediatric stone disease is also an emerging concern.4 Recurrence Rates: Urolithiasis is often characterized by a recurrent nature. Approximately 50% of individuals who experience an initial stone episode will develop additional stones within five years. This high recurrence rate underscores the importance of preventative measures.4 Geographic Variation: Urolithiasis exhibits notable geographic variation, with a higher prevalence in certain regions. Factors contributing to these disparities include dietary habits, climate, genetics, and socioeconomic factors. Ethnic and Racial Factors: Epidemiological studies have identified variations in urolithiasis rates among different ethnic and racial groups. For instance, individuals of South Asian descent appear to have a higher risk of developing kidney stones.4 3. Crystal Growth and Aggregation: Once crystals form, they can grow and aggregate to form macroscopic stones. Growth is influenced by urinary pH, ionic strength, and the presence of inhibitors and promoters. Crystals can adhere to renal papillary surfaces, leading to stone growth over time.5 4. Stone Adhesion: Adherence of crystals to renal tubular cells and organic matrix components in the urinary tract is facilitated by various adhesive molecules. This adherence promotes crystal retention and further contributes to stone growth.5 3. Crystal Growth and Aggregation: Once crystals form, they can grow and aggregate to form macroscopic stones. Growth is influenced by urinary pH, ionic strength, and the presence of inhibitors and promoters. 2628 Volume 03 Issue 11 November 2023 ETIOLOGIES Clinical Implications and Diagnostic Potential: Our examination of the clinical implications highlighted the potential for microbiome-based diagnostic tools in urolithiasis. The urinary microbiome may offer valuable information for risk stratification, guiding preventive measures, and predicting stone recurrence. Developing these diagnostic modalities could revolutionize urological practice. 5. Therapeutic Prospects: The discussion delved into the promising therapeutic prospects that emerge from acknowledging the microbial dimension in urolithiasis. Targeting microbial dysbiosis may offer a novel therapeutic approach to stone management. Modulation of the urinary microbiome through probiotics, prebiotics, or other interventions could become an integral part of stone prevention and treatment strategies. 4. Staphylococcus: Staphylococcus species, typically associated with skin and mucosal surfaces, have also been detected in the urinary microbiome. Their role in urolithiasis is not entirely clear, but they may contribute to biofilm formation on stone surfaces. 6,7 5. Enterococcus: Enterococcus species are found in the urinary microbiome and can influence urinary pH. Their presence may have implications for stone formation, although the specific mechanisms are still being elucidated. 8 5. Enterococcus: Enterococcus species are found in the urinary microbiome and can influence urinary pH. Their presence may have implications for stone formation, although the specific mechanisms are still being elucidated. 8 the specific mechanisms are still being elucidated. 8 6. Bacterial Biofilms: Biofilm formation by various bacteria, including Proteus mirabilis and Escherichia coli, on the surface of urinary stones is a key mechanism in urolithiasis. Biofilms provide a protective environment for bacteria, allowing them to adhere to stone surfaces, multiply, and promote stone growth. 8 6. Bacterial Biofilms: Biofilm formation by various bacteria, including Proteus mirabilis and Escherichia coli, on the surface of urinary stones is a key mechanism in urolithiasis. Biofilms provide a protective environment for bacteria, allowing them to adhere to stone surfaces, multiply, and promote stone growth. 8 6. Research Directions: Our discussion also pointed towards key avenues for future research. Further investigations are needed to elucidate the intricate mechanisms by which bacteria contribute to stone formation and to better understand the dynamics of microbial dysbiosis. Prospective studies examining the effectiveness of microbiome-targeted interventions in stone prevention and management are warranted. 7. Microbial Dysbiosis: Shifts in the composition and diversity of the urinary microbiome, often referred to as dysbiosis, can disrupt the delicate balance of urinary tract ecology. ETIOLOGIES 1. Struvite-Forming Bacteria: Struvite stones, composed of magnesium ammonium phosphate, are often linked to urinary tract infections (UTIs) caused by urease-producing bacteria. These bacteria, which include Proteus mirabilis, Klebsiella pneumoniae, and some strains of Escherichia coli, hydrolyze urea to ammonia, leading to an increase in urinary pH and the precipitation of struvite crystals. 6 2. Microbial Influence on Urolithiasis: The discussion has illuminated how specific bacteria, such as urease-producing organisms like Proteus mirabilis and Klebsiella pneumoniae, can directly impact stone formation through the hydrolysis of urea, leading to an increase in urinary pH and the subsequent precipitation of struvite crystals. Furthermore, the presence of bacteria within urinary stone biofilms has been linked to stone growth and recurrences. 2. Corynebacterium: Corynebacterium species have been identified in the urinary microbiome and are known to influence stone formation by modulating urinary pH. Some Corynebacterium strains can elevate urinary pH, contributing to the formation of alkaline stones like struvite or calcium phosphate. 6 2. Corynebacterium: Corynebacterium species have been identified in the urinary microbiome and are known to influence stone formation by modulating urinary pH. Some Corynebacterium strains can elevate urinary pH, contributing to the formation of alkaline stones like struvite or calcium phosphate. 6 3. Microbiome Dysbiosis: The concept of dysbiosis, involving shifts in microbial composition and function, emerged as a central theme in our discussion. Dysbiosis within the urinary microbiome has been associated with various factors, including antibiotic use, recurrent urinary tract infections (UTIs), and metabolic disorders. These shifts in microbial equilibrium may be predisposing factors for stone formation. 3. Microbiome Dysbiosis: The concept of dysbiosis, involving shifts in microbial composition and function, emerged as a central theme in our discussion. Dysbiosis within the urinary microbiome has been associated with various factors, including antibiotic use, recurrent urinary tract infections (UTIs), and metabolic disorders. These shifts in microbial equilibrium may be predisposing factors for stone formation. 3. Lactobacillus: Lactobacillus is a common member of the urinary microbiome. Certain species of Lactobacillus are acid-producing bacteria that can lower urinary pH, potentially mitigating the risk of stone formation, particularly in individuals prone to calcium oxalate stones. 6,7 4. Clinical Implications and Diagnostic Potential: Our examination of the clinical implications highlighted the potential for microbiome-based diagnostic tools in urolithiasis. The urinary microbiome may offer valuable information for risk stratification, guiding preventive measures, and predicting stone recurrence. Developing these diagnostic modalities could revolutionize urological practice. 4. Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration Understanding the complex pathophysiology of urolithiasis and its intricate relationship with the urinary microbiome is pivotal for developing targeted therapeutic interventions and preventive strategies. Further research into the urinary microbiome's role in stone formation holds promise for innovative approaches to managing this recurrent and debilitating condition.5 discussions and insights that shape our understanding of urolithiasis pathogenesis and potential avenues for future research and clinical application. 1. The Paradigm Shift: Sterile No More: One of the pivotal revelations arising from this study is the transformation of the traditional view of the urinary tract as a sterile environment. The recognition of a dynamic urinary microbiome, characterized by diverse bacterial communities, challenges the conventional understanding of urolithiasis pathophysiology. 1. The Paradigm Shift: Sterile No More: One of the pivotal revelations arising from this study is the transformation of the traditional view of the urinary tract as a sterile environment. 1. The Paradigm Shift: Sterile No More: One of the pivotal revelations arising from this study is the transformation of the traditional view of the urinary tract as a sterile environment. The recognition of a dynamic urinary microbiome, characterized by diverse bacterial communities, challenges the conventional understanding of urolithiasis pathophysiology. ETIOLOGIES Dysbiosis may result from factors such as antibiotic use, recurrent UTIs, or metabolic disorders, potentially predisposing individuals to stone formation. 9 7. Holistic Stone Management: Finally, our findings underscore the importance of a holistic approach to stone management. Combining traditional physicochemical strategies with microbiome-focused interventions may provide the most effective means of preventing stone PATHOPHYSIOLOGY Urolithiasis, a multifaceted disorder characterized by the formation of urinary calculi, involves a complex interplay of physiological, biochemical, and microbial factors within the urinary tract. Understanding the intricate pathophysiological mechanisms behind urolithiasis and its association with the 2628 Volume 03 Issue 11 November 2023 2629 Volume 03 Issue 11 November 2023 Corresponding Author: Paola Lizbeth Zamudio Naranjo REFERENCES I. Price TK, Dune T, Hilt EE, Thomas-White KJ, Kliethermes S, Brincat C, et al. The clinical urine culture: Enhanced techniques improve detection of clinically relevant microorganisms. J Clin Microbiol. 2016;54(5):1216-1222. doi:10.1128/JCM.00044-16 The Urinary Microbiome's Emerging Significance: The once- held notion of the urinary tract as a sterile environment has given way to the recognition of a complex and dynamic urinary microbiome. This microbial community, composed of various bacterial species, plays a role in the urolithiasis landscape that cannot be ignored. II. Wolfe AJ, Toh E, Shibata N, Rong R, Kenton K, Fitzgerald M, et al. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol. 2012;50(4):1376-83. doi:10.1128/JCM.05852-11 Bacterial Diversity and Stone Formation: We have explored the specific bacteria implicated in urolithiasis, including urease-producing organisms such as Proteus mirabilis and Klebsiella pneumoniae, as well as other members of the urinary microbiome like Corynebacterium, Lactobacillus, Staphylococcus, and Enterococcus. These bacteria influence urinary pH, crystal nucleation, and the formation of stone- promoting biofilms. III. Pearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, Fok C, et al. The female urinary microbiome: A comparison of women with and without urgency urinary incontinence. MBio. 2014; 5(4):e01283-14. doi:10.1128/mBio.01283-14. IV. IV. Siddiqui H, Nederbragt AJ, Lagesen K, Jeansson SL, Jakobsen KS. Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons. BMC Microbiol. 2011; 11:244. doi:10.1186/1471-2180-11-244 22047020 Dysbiosis as a Precursor to Stone Formation: Dysbiosis within the urinary microbiome, characterized by shifts in microbial composition and function, appears to be a key factor in stone pathogenesis. Factors such as antibiotic use, recurrent urinary tract infections, and metabolic disorders can disrupt the balance of the urinary microbiota, potentially predisposing individuals to stone development. V. Hilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, Mueller ER, et al. Urine is not sterile: Use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. J Clin Microbiol. 2014;52(3):871-876. doi:10.1128/JCM.02876-13 Clinical Implications: Understanding the urinary microbiome's role in urolithiasis has significant clinical implications. It opens doors for the development of microbiome-based diagnostic tools that may aid in risk stratification and personalized prevention strategies. Furthermore, targeting microbial dysbiosis may represent a novel therapeutic approach in stone management. VI. Karstens L, Asquith M, Davin S, Stauffer P, Fair D, Gregory WT, et al. Does the urinary microbiome play a role in urgency urinary incontinence and its severity?. Urolithiasis and its Interplay with the Urinary Microbiome: A Comprehensive Exploration formation, reducing recurrence rates, and improving patient outcomes. microbiological insights into u exciting prospects for enhanced di i d h i l microbiological insights into urolithiasis research offers exciting prospects for enhanced patient care, personalized medicine, and the potential to alleviate the burden of stone disease. As we venture further into this realm, we embark on a path toward transformative advancements in urological practice. CONCLUSIONS In this comprehensive exploration of the intricate relationship between urolithiasis and the urinary microbiome, we have unveiled a fascinating interplay between microbial dynamics and stone formation within the urinary tract. Our findings contribute valuable insights to the evolving understanding of urolithiasis pathogenesis and open promising avenues for novel diagnostic and therapeutic strategies. DISCUSSION The exploration of the intricate interplay between urolithiasis and the urinary microbiome has unveiled a complex and evolving landscape within the realm of urology. Our in-depth investigation into this relationship has led to several critical 2629 Volume 03 Issue 11 November 2023 2630 Volume 03 Issue 11 November 2023 REFERENCES Front Cell Infect Microbiol. 2016; 6:78. doi:10.3389/fcimb.2016.00078. Preventive Strategies: Acknowledging the microbial component in urolithiasis pathophysiology underscores the importance of comprehensive preventive strategies. These strategies should encompass not only traditional measures to address physicochemical risk factors but also interventions that promote urinary microbiome health and diversity. VII. VII. Thomas-White KJ, Hilt EE, Fok C, Pearce MM, Mueller ER, Kliethermes S, et al. Incontinence medication response relates to the female urinary microbiota. Int Urogynecol J. 2016; 27(5):723-33. doi:10.1007/s00192-015-2847-x VIII. VIII. Kramer H, Soyibo A, Forrester T, Boyne M, Markossian T, Durazo-Arvizu R, et al. The burden of chronic kidney disease and its major risk factors in Jamaica. Kidney Int. 2018;94(5):840-42. doi:10.1016/j.kint.2018.07.025. Future Directions: The dynamic nature of the urinary microbiome and its influence on urolithiasis demand continued research efforts. Future investigations should focus on elucidating the precise mechanisms by which bacteria contribute to stone formation, refining our understanding of microbial dysbiosis, and developing microbiome-targeted interventions. IX. Wolfe AJ, Brubaker L. Sterile urine and the presence of bacteria. Eur Urol. 2015; 68(2):173-4. doi:10.1016/j.eururo.2015.02.041. In conclusion, our exploration of urolithiasis and its intricate association with the urinary microbiome has illuminated new facets of this age-old condition. The integration of Corresponding Author: Paola Lizbeth Zamudio Naranjo
https://openalex.org/W2110812199	https://europepmc.org/articles/pmc3657628?pdf=render	English	null	Brain dysfunction as one cause of CFS symptoms including difficulty with attention and concentration	Frontiers in physiology	2,013	cc-by	5,004	Correspondence: p Benjamin H. Natelson, Department of Pain Medicine and Palliative Care, Suite 4K, PACC, 10 Union Square East, New York, NY 10003, USA. e-mail: bnatelson@chpnet.org Keywords: fatigue, pathophysiology, oxidative stress, causative hypotheses, syndrome noting similarities between CFS and FM, suggested an explana- tory model based on the concept of symptom ampliﬁcation—that is, a common psychological tendency to somatize or miscon- strue the signiﬁcance of normal physical sensation. Moving from the question as to whether CFS and FM are the same or differ- ent illnesses is the conclusion that many patients with CFS have their illness based on somatization. We did explore this hypoth- esis early on and learned that the probability of any one patient receiving the diagnosis of Somatization Disorder depended on whether or not his/her symptoms were coded as psychiatric or physical. If the coder decided the infectious, rheumatological, and neuropsychiatric symptoms of CFS were psychiatric, then nearly every patient would be classiﬁed as having Somatization Disorder, but if the symptoms were coded as physical, then nearly none would be so classiﬁed. Based on this assessment, we eschewed the idea of “lumping” patients into one explana- tory rubric and have continued our efforts to “split” patients into discrete subgroups. Chronic fatigue syndrome (CFS) is a medically unexplained ill- ness characterized by new onset of fatigue, severe enough to produce a substantial decrease in activity, and accompanied by infectious, rheumatological, and neuropsychiatric symptoms (Fukuda et al., 1994). One of the most common and disabling symptoms is the subjective complaint of “brain fog” or inability to concentrate and do multiple tasks at the same time. Our earli- est study provided objective evidence of a problem with attention and concentration (DeLuca et al., 1993). Chronic fatigue syndrome (CFS) is a medically unexplained ill- ness characterized by new onset of fatigue, severe enough to produce a substantial decrease in activity, and accompanied by infectious, rheumatological, and neuropsychiatric symptoms (Fukuda et al., 1994). One of the most common and disabling symptoms is the subjective complaint of “brain fog” or inability to concentrate and do multiple tasks at the same time. Our earli- est study provided objective evidence of a problem with attention and concentration (DeLuca et al., 1993). Because CFS is deﬁned using a clinical case deﬁnition, patients with this diagnosis must constitute a heterogeneous group, prob- ably with multiple causative agents—much as was the case of the eighteenth century diagnosis of dropsy. published: 20 May 2013 doi: 10.3389/fphys.2013.00109 Benjamin H. Natelson1,2 1 Director, Pain and Fatigue Study Center, Department of Pain Medicine and Palliative Care, Beth Israel Medical Center, Manhattan, New York, NY, USA 2 Professor of Neurology, Albert Einstein College of Medicine, Bronx, New York, NY, USA We have been able to reduce substantially patient pool heterogeneity by identifying phenotypic markers that allow the researcher to stratify chronic fatigue syndrome (CFS) patients into subgroups. To date, we have shown that stratifying based on the presence or absence of comorbid psychiatric diagnosis leads to a group with evidence of neurological dysfunction across a number of spheres. We have also found that stratifying based on the presence or absence of comorbid ﬁbromyalgia leads to information that would not have been found on analyzing the entire, unstratiﬁed patient group. Objective evidence of orthostatic intolerance (OI) may be another important variable for stratiﬁcation and may deﬁne a group with episodic cerebral hypoxia leading to symptoms. We hope that this review will encourage other researchers to collect data on discrete phenotypes in CFS to allow this work to continue more broadly. Finding subgroups of CFS suggests different underlying pathophysiological processes responsible for the symptoms seen. Understanding those processes is the ﬁrst step toward developing discrete treatments for each. Reviewed by: y J. Thomas Cunningham, University of North Texas Health Science Center, USA Abdu Adem, United Arab Emirates University, United Arab Emirates REVIEW ARTICLE published: 20 May 2013 doi: 10.3389/fphys.2013.00109 REVIEW ARTICLE published: 20 May 2013 doi: 10.3389/fphys.2013.00109 Edited by: Edited by: Julian M. Stewart, New York Medical College, USA Reviewed by: J. Thomas Cunningham, University of North Texas Health Science Center, USA Abdu Adem, United Arab Emirates University, United Arab Emirates Correspondence: Benjamin H. Natelson, Department of Pain Medicine and Palliative Care, Suite 4K, PACC, 10 Union Square East, New York, NY 10003, USA. e-mail: bnatelson@chpnet.org Julian M. Stewart, New York Medical College, USA Correspondence: Our ﬁndings of increased numbers of abnormalities in the NO-PSYCH patients are of interest when compared to a subset analysis of an ear- lier study (Greco et al., 1997): while no signiﬁcant difference in rate of abnormalities was found between a group of unstratifed patients and controls, they did report a difference signiﬁcant at the 0.06 level between a subgroup of patients without depression compared to healthy controls; thus our data do replicate this ﬁnd- ing. The sum of these data suggests that some CFS patients may have a brain marker of their disease in the form of small nonspe- ciﬁc MRI white matter abnormalities, occurring predominantly in the frontal lobes. These studies led to our current working hypothesis—that a subgroup of patients with CFS has an under- lying neurological disease which leads to the symptoms of fatigue and cognitive dysfunction. may have an underlying encephalopathy producing the symptoms of CFS. The other way we have stratiﬁed our CFS patient sample is on the presence or absence of ﬁbromyalgia—characterized by the patient reporting widespread pain and having tenderness in at least 11 of 18 tender points subjected to palpation (Wolfe et al., 1990). When comparing CFS patients with and without comorbid ﬁbromyalgia, we found that only patients in the CFS only group and not those with both CFS plus ﬁbromyalgia had abnormal neuropsychological test results (Cook et al., 2005). A recent paper from another group corroborated our ﬁnding of normal cogni- tive function in ﬁbromyalgia (Mohs et al., 2012). Unfortunately, we do not have data as to psychiatric status for these subjects; however other studies have revealed that patients with CFS only have approximately half the psychiatric comorbidity than those with comorbid FM (Ciccone and Natelson, 2003); these data support our working hypothesis that the group of CFS patients with no co-existing psychiatric problems has an underlying brain disease. These results support our hypothesis that stratifying CFS patients into subgroups can lead to improved understanding of the cognitive complaints of all CFS patients. We have two major hypotheses for how the changes in blood ﬂow affect neural func- tioning adversely, leading to increased ventricular lactate and/or small lesions in frontal white matter, and then to symptoms. The ﬁrst of these, spearheaded by my colleague Dr. Correspondence: Dikoma Shungu of Weil-Cornell, is an outgrowth of Martin Pall’s hypoth- esis (Pall, 2000) that some immunological or infectious trigger activates one or more pro-inﬂammatory cytokines which induce a synthesis chain leading to the production of peroxynitrite, a potent pro-apoptotic, and pro-inﬂammatory reactive oxygen or nitrogen species (Hoffman et al., 1997; Basu, 2008). This pro- cess is inhibited by adequate antioxidant capacity/reserves, of which glutathione (GSH) is the most abundant and important component. In the face of inadequate levels of antioxidants, per- oxynitrite will react with arachidonic acid in cell membrane phospholipids, to form isoprostanes, compromising membrane and cell function. A ﬁnal metabolic step frees the isoprostanes from the cell membrane phospholipids, allowing them to move into body ﬂuids, where they can be measured to provide an objective and reliable measure for the presence of oxidative stress. Using the same stratiﬁcation strategy, we moved on to other studies. First, we collected spinal ﬂuid from 45 CFS patients and 13 healthy controls. As expected, the healthy controls had white blood counts per high power ﬁeld and protein concentrations that were all below the high value for laboratory normals. In con- trast, we found that nearly 30% of the patients had elevations in one or both of these variables. While there was no difference in lifetime rates of Axis I pathology, the group with the abnor- mal spinal ﬂuids had signiﬁcantly fewer cases with a diagnosis of current depression than the group with the normal spinal ﬂuids (Natelson et al., 2005). Our next study looked at cere- bral blood ﬂow in CFS (Yoshiuchi et al., 2006). We found that CFS patients as a group had signiﬁcant reductions in cerebral blood ﬂow across a number of brain in regions. However, those patients with no comorbid psychiatric diagnosis had more areas of reduced blood ﬂow compared to controls than patients who did have comorbid psychiatric diagnoses. Finally, we re-evaluated data on ventricular lactate which had shown increases for CFS relative to controls (Mathew et al., 2009). Nine of the 16 CFS patients fell into a “high” group (i.e., displaying higher ventric- ular lactate levels that were one SD above the CFS group mean) which was higher than every healthy volunteer and was responsi- ble for the overall group effect. Correspondence: The condition of dropsy evolved to a host of more speciﬁc diagnoses includ- ing congestive heart failure and other organ failures when the pathological causes of the speciﬁc organ dysfunction leading to dropsy were discovered. Our approach has been to try to strat- ify along certain phenotypic criteria to try to develop more homogeneous subgroups. In contrast, some researchers have been impressed by the large amount of comorbidity occurring among several of these syndromes—namely, CFS and ﬁbromyalgia (FM). Conﬁrming this with data from our Center, we found ∼37% of patients with CFS also fulﬁlled case criteria for FM (Ciccone and Natelson, 2003), and one study of patients with FM reported that about 20% also fulﬁlled criteria for CFS (Buchwald and Garrity, 1994). Regarding this overlap between syndromes, Wessely et al. (1999), for example, have suggested that the “similarities between them outweigh the differences” and Barsky and Borus (1999), Over the years, we have used two major strategies for stratify- ing patients. The ﬁrst used the presence or absence of comorbid psychiatric diagnosis—usually major depressive disorder. Our ﬁrst target was the neuropsychological dysfunction we had orig- inally reported (DeLuca et al., 1993). In a follow up study, we stratiﬁed our patients into those with or without comorbid Axis I diagnoses: we found that the group with no comorbid psychi- atric diagnoses was the group with the worst neuropsychological test results (DeLuca et al., 1997). After doing an initial study which showed increased numbers of anatomic abnormalities on May 2013 \| Volume 4 \| Article 109 \| 1 www.frontiersin.org www.frontiersin.org Genesis of symptoms in CFS Natelson brain magnetic resonance imaging (Natelson et al., 1993), we fol- lowed that study up after stratifying our patients into PSYCH or NO-PSYCH groups. Again, we found that the patients without psychiatric comorbidity were the ones with the highest number of abnormalities—usually small, circumscribed areas of T2 signal in frontal lobes (Lange et al., 1999). To start determining whether these were insigniﬁcant epiphenomena or perhaps functioned to affect physical function, we asked if physical function as assessed by the SF-36 correlated with MRI ﬁndings and in fact found that those with the lesions had worse physical function than those with normal brain MRIs (Cook et al., 2001). Frontiers in Physiology \| Integrative Physiology Correspondence: In exploratory post-hoc analyses, these 9 CFS patients were compared with the remaining 6 CFS patients with normal lactate levels (“normal”), which revealed that the only differences between the two CFS subgroups was greater overall functional disability on the SF-36 (P = 0.039), and a trend toward greater lifetime Axis I mood disorder comorbid- ity (4 of 7 vs. 1 of 9, Fisher Exact probability test, P = 0.07) in the CFS group with “low” lactate levels compared to CFS patients with higher ventricular lactate (Mathew et al., unpublished data). Although preliminary, these data suggest that “no-psych” patients This model is pertinent to CFS for the following reasons: ﬁrst, two studies have reported that isoprostanes are elevated in CFS (Kennedy et al., 2005; Robinson et al., 2009). And sec- ond, we have found that cortical GSH is reduced by 36% in CFS (Shungu et al., 2012). These data lead to the inference that brain oxidative stress molecules are increased in CFS; these molecules have potent vasoconstrictor effects on cerebral arteri- oles (Hoffman et al., 1997) and thus could explain the decreases in regional cerebral blood ﬂow that we have now found in a number of studies (Yoshiuchi et al., 2006; Biswal et al., 2011; Shungu et al., 2012). Finally the impaired cellular metabolism produced by these molecules could, via co-occurring increases in anaerobic glycolytic activity, lead to the increased ventricular CSF lactate in CFS shown to occur by Shungu and colleagues in three separate studies (Mathew et al., 2009; Murrough et al., May 2013 \| Volume 4 \| Article 109 \| 2 Frontiers in Physiology \| Integrative Physiology Genesis of symptoms in CFS Natelson on a visual analog scale (Natelson et al., 2007). While patients both with and without OI had higher anxiety levels while supine than controls that difference did not increase in the OI group during orthostatic challenge. In fact, there were no differences in groups apart from the initial shift noted at base- line. Based on that result, we hypothesized that orthostatically- induced hyperventilation represented a physiological response to intra-thoracic hypovolemia related to blood pooling distally. Novak et al. have come to a similar conclusion in their studies of cerebral blood ﬂow during orthostatic challenge in syncope patients. Correspondence: Only one study in CFS patients exists to date on the effects of orthostatic challenge on some measure of brain blood ﬂow—i.e., middle cerebral arte- rial blood velocity assessed by transcranial Doppler (Razumovsky et al., 2003). Using this technique, no signiﬁcant difference was found in resting middle cerebral artery blood velocity between patients and controls in the supine posture, and no difference was found during orthostatic stress. However, only 10% of patients in this study had OI, and this rate was the same as that in controls. Not ﬁnding differences in either supine or challenged postures may have two explanations: ﬁrst, cerebral blood veloc- ity may not reﬂect the same measure as absolute cerebral blood ﬂow and second, it may be important to have a larger group of patients with OI and then to compare blood ﬂow in that group to those without OI. If that experiment were done, we might expect that the patients with OI but not those without OI will have dramatic decreases in cerebral blood ﬂow with com- mensurate increases in resistance due to orthostatically-induced hyperventilation. Subsequently attention has turned away from delayed hypotension to orthostatic tachycardia—deﬁned as an increase in heart rate from those in the supine position of ≥30 beats per minute or rates of ≥130 beats per minute while standing. Later studies suggested an important age factor in determining the physiological form of OI. Almost every adolescent with CFS had orthostatic tachycardia (Stewart et al., 1999), but this was not the case with adults (Natelson et al., 2007) where rates appear to be lower ranging from 10% (Naschitz et al., 2006) to 27% (Hoad et al., 2008) with the higher rate found in UK patients who reported having the diagnosis of CFS rather than being directly diagnosed. In our own study of adult patients (Natelson et al., 2007), orthostatic tachycardia was not common, occurring in only 11% of patients, not signiﬁcantly different from rates in healthy controls. While orthostatic tachycardia is not common in adults, ortho- static hyperventilation—end tidal CO2 values lowered to below 30 mmHg during orthostatic challenge—seems to be substan- tially more common in that we found it occurred in 21% of adult CFS patients compared to 3% in healthy controls (Natelson et al., 2007). Correspondence: They postulated that patients hyperventilate for several reasons: ﬁrst, to help pull blood into the chest by increasing pre-load mechanically and next to combat decreases in blood pressure by tachypnea-induced vasoconstriction (Novak et al., 1998). 2010; Shungu et al., 2012). One possibility that our studies have ruled out is mitochondrial dysfunction from the accumu- lation of free radicals: we found brain ATP and associated high energy phosphates to be normal in CFS (Shungu et al., 2012). Thus, we are focusing on the oxidative stress hypothesis in this model of CFS. The second explanatory hypothesis is directed to the ∼25% of CFS patients with physiological evidence of orthostatic intoler- ance (OI). Early work by David Streeten (Streeten and Anderson, 1992) tied chronic symptoms including fatigue to a form of OI characterized by delayed hypotension induced by orthostatic stress. Subsequent work made the link with CFS directly. The researchers did tilt testing with pharmacological potentiation in young adults with CFS and reported that most patients developed delayed hypotension accompanied by increased fatigue com- pared to healthy controls (Bou-Holaigah et al., 1995). While several groups have replicated this ﬁnding in older patient pop- ulations (DeLorenzo et al., 1997; Freeman and Komaroff, 1997; Schondorf et al., 1999), even more groups have not, including the original research team reporting the ﬁnding (Razumovsky et al., 2003), another group studying twins (Poole et al., 2000), yet another studying a community sample of CFS patients (Jones et al., 2005), and our own carefully controlled study of unmedicated and uninstrumented patients (LaManca et al., 1999). Finding orthostatic tachycardia or hyperventilation is a phys- iological conﬁrmation of the complaint of OI. While we do not know whether physiological manifestation of OI correlates with plasma isoprostanes or, inversely, with brain GSH (we are currently doing studies to answer this question), another possi- bility exists. Using two methods for determining absolute cerebral blood ﬂow, we have demonstrated reductions in CFS patients as a group in the resting state (Yoshiuchi et al., 2006; Biswal et al., 2011). We reported average blood ﬂow and thus some patients had blood ﬂow in the normal range while others had ﬂow that was quite reduced relative to controls. We hypothesize that the OI group may be the ones with the reduced brain blood ﬂow at rest and that this group of patients may show further decreases in brain blood ﬂow during orthostatic challenge. www.frontiersin.org ACKNOWLEDGMENTS Dr. Natelson’s effort on this review was supported in time by NIH grant #NS-075653. REFERENCES patients devoid of psychiatric disease. J. Neurol. Neurosurg. Psychiatry 62, 151–155. patients devoid of psychiatric disease. J. Neurol. Neurosurg. Psychiatry 62, 151–155. Natelson, B. H., Tseng, C.-L., and Ottenweller, J. E. (2005). Spinal ﬂuid abnormalities in patients with chronic fatigue syndrome. Clin. Diagn. Lab. Immunol. 12, 53–55. chronic fatigue syndrome. Clin. Physiol. 19, 111–120. Barsky, A. J., and Borus, J. F. (1999). Functional somatic syndromes. Ann. Intern. Med. 130, 910–921. Lange, G., DeLuca, J., Maldjian, J. A., Lee, H., Tiersky, L. A., and Natelson, B. H. (1999). Brain MRI abnormali- Lange, G., DeLuca, J., Maldjian, J. A., Lee, H., Tiersky, L. A., and Natelson, B. H. (1999). Brain MRI abnormali- ties exist in a subset of patients with Lange, G., DeLuca, J., Maldjian, J. A., Lee, H., Tiersky, L. A., and Natelson, B. H. (1999). Brain MRI abnormali- ties exist in a subset of patients with chronic fatigue syndrome. J. Neurol. Sci. 171, 3–7. DeLuca, J., Johnson, S. K., and Natelson, B. H. (1993). Information processing efﬁciency in chronic fatigue syndrome and multiple sclerosis. Arch. Neurol. 50, 301–304. Basu, S. (2008). F2-Isoprostanes in human health and diseases: from molecular mechanisms to clini- cal implications. Antioxid. Redox Signal. 10, 1405–1434. Novak, V., Spies, J. M., Novak, P., McPhee, B. R., Rummans, T. A., and Low, P. A. (1998). Hypocapnia and cerebral hypoperfusion in orthostatic intolerance. Stroke 29, 1876–1881. chronic fatigue syndrome. J. Neurol. Sci. 171, 3–7. Mathew, S. J., Mao, X., Keegan, K. A., Levine, S. M., Smith, E. L., Heier, L. A., et al. (2009). Ventricular cere- brospinal ﬂuid lactate is increased in chronic fatigue syndrome compared with generalized anxiety disorder: an in vivo 3.0 T (1)H MRS imaging study. NMR Biomed. 22, 251–258. Freeman, R., and Komaroff, A. L. (1997). Does the chronic fatigue syndrome involve the autonomic nervous system? Am. J. Med. 102, 357–364. Biswal, B., Kunwar, P., and Natelson, B. H. (2011). Cerebral blood ﬂow is reduced in patients with chronic fatigue syndrome as assessed by arterial spin labeling. J. Neurol. Sci. 301, 9–11. Pall, M. L. (2000). Elevated, sustained peroxynitrite levels as the cause of chronic fatigue syndrome. Med. Hypotheses 54, 115–125. Fukuda, K., Straus, S. E., Hickie, I., Sharpe, M. C., Dobbins, J. G., and Komaroff, A. (1994). ACKNOWLEDGMENTS The chronic fatigue syndrome: a comprehen- sive approach to its deﬁnition and study. Ann. Intern. Med. 121, 953–959. Bou-Holaigah, I., Rowe, P. C., Kan, J., and Calkins, H. (1995). The rela- tionship between neurally mediated hypotension and the chronic fatigue syndrome. JAMA 274, 961–967. Poole, J., Herrell, R., Ashton, S., Goldberg, J., and Buchwald, D. (2000). Results of isoproterenol tilt table testing in monozygotic twinds discordant for chronic fatigue syndrome. Arch. Intern. Med. 160, 3461–3468. Mohs, R., Mease, P., Arnold, L. M., Wang, F., Ahl, J., Gaynor, P. J., et al. (2012). The effect of duloxetine treatment on cognition in patients with ﬁbromyalgia. Psychosom. Med. 74, 628–634. Buchwald, D., and Garrity, D. (1994). Comparison of patients with chronic fatigue syndrome, ﬁbromyalgia, and multiple chemical sensitivities. Arch. Intern. Med. 154, 2049–2053. Greco, A., Tannock, C., Brostoff, J., and Costa, D. C. (1997). Brain MR in chronic fatigue syndrome. AJNR 18, 1265–1269. Murrough, J. W., Mao, X., Collins, K. A., Kelly, C., Andrade, G., Nestadt, P., et al. (2010). Increased ven- tricular lactate in chronic fatigue syndrome measured by 1H MRS imaging at 3T. II: comparison with major depressive disorder. NMR Biomed. 23, 643–650. Razumovsky, A. Y., DeBusk, K., Calkins, H., Snader, S., Lucas, K. E., Vyas, P., et al. (2003). Cerebral and systemic hemodynamics changes during upright tilt in chronic fatigue syndrome. J. Neuroimaging 13, 57–67. Hoad, A., Spickett, G., Elliott, J., and Newton, J. (2008). Postural orthostatic tachycardia syndrome is an under-recognized condition in chronic fatigue syndrome. QJM 101, 961–965. Ciccone, D. S., and Natelson, B. H. (2003). Comorbid illness in the chronic fatigue syndrome: a test of the single syndrome hypothesis. Psychosom. Med. 62, 268–275. Robinson, M., Gray, S. R., Watson, M. S., Kennedy, G., Hill, A., Belch, J. J., et al. (2009). Plasma IL-6, its solu- ble receptors and F-isoprostanes at rest and during exercise in chronic fatigue syndrome. Scand. J. Med. Sci. Sports 13, 1–9. Naschitz, J. E., Mussaﬁa-Priselac, R., Kovalev, Y., Zaigraykin, N., Slobodin, G., Elias, N., et al. (2006). Patterns of hypocapnia on tilt in patients with ﬁbromyalgia, chronic fatigue syndrome, nonspeciﬁc dizziness, and neurally mediated syncope. Am. J. Med. Sci. 331, 295–303. Hoffman, S. W., Moore, S., and Ellis, E. F. (1997). Isoprostanes: free radical- generated prostaglandins with con- strictor effects on cerebral arterioles. Stroke 28, 844–849. Cook, D. B., Lange, G., DeLuca, J., and Natelson, B. H. (2001). Correspondence: When we broke out the data by gender, we found ortho- static hyperventilation to occur in 35% of the female patients—a very substantial proportion (Natelson et al., 2007). The patients with orthostatic hyperventilation showed a progressive fall in end tidal CO2 during orthostatic challenge—a trend also seen, albeit much milder, in CFS patients without OI or in healthy controls. In a parallel study, a group of Israeli investigators (Naschitz et al., 2006) also monitored end tidal CO2 in CFS patients and found a rate of 31% showing hypocapnia (gender not speciﬁed). If this is the case, then we would expect that it is the patients with OI who are at increased risk of developing the increased T2- signal lesions we have reported to occur via a process leading to sporadic cerebral hypoxia. As a corollary, we would expect that it is these patients who have the most difﬁculty with cognitive pro- cessing. Unfortunately, we had not been assessing CFS patients for OI at the time of these earlier studies—a deﬁciency we are now remedying in current work. We expect that OI will be more common in patients with no history of psychiatric diagnosis than in those with such diagnoses. The common explanation for hyperventilation is anxiety. We speciﬁcally asked volunteers in our study to rate their anxiety May 2013 \| Volume 4 \| Article 109 \| 3 www.frontiersin.org www.frontiersin.org Genesis of symptoms in CFS Natelson In conclusion, we have been able to reduce substantially patient pool heterogeneity by identifying phenotypic markers that allow the researcher to stratify patients into subgroups. To date, we have shown that stratifying based on the presence or absence of comorbid psychiatric diagnosis leads to a group with evidence of neurological dysfunction across a number of spheres. We have also found that stratifying based on the presence or absence of comorbid ﬁbromyalgia leads to information that would not have been found on analyzing the entire, unstratiﬁed patient group. Objective evidence of OI may be another important variable for stratiﬁcation and may produce some of the symptoms of CFS via intermittent cerebral hypoxia. We hope that this review will encourage other researchers to collect data on discrete pheno- types in CFS to allow this work to continue more broadly. Finding subgroups of CFS suggests different underlying pathophysiologi- cal processes responsible for the symptoms seen. *Correspondence: Understanding those processes is the ﬁrst step toward developing discrete treat- ments for each. Conﬂict of Interest Statement: The author declares that the research ACKNOWLEDGMENTS Relationship of brain MRI abnormalities and physical functional status in CFS. Int. J. Neurosci. 107, 1–6. Jones, J. F., Nicholson, A., Nisenbaum, R., Papanicolaou, D. A., Solomon, L., Boneva, R., et al. (2005). Orthostatic instability in a population-based study of chronic fatigue syndrome. Am. J. Med. 118, 1415.e19–1415.e28. Cook, D. B., Nagelkirk, P. R., Peckerman, A., Poluri, A., Mores, J., and Natelson, B. H. (2005). Exercise and cognitive performance in Chronic Fatigue Syndrome. Med. Sci. Sports Exerc. 37, 1460–1467. Schondorf, R., Benoit, J., Wein, T., and Phaneuf, D. (1999). Orthostatic intolerance in the chronic fatigue syndrome. J. Auton. Nerv. Syst. 75, 192–201. Natelson, B. H., Cohen, J. M., Brassloff, I., and Lee, H.-J. (1993). A con- trolled study of brain magnetic res- onance imaging in patients with the chronic fatigue syndrome. J. Neurol. Sci.120, 213–217. Shungu, D. C., Weiduschat, N., Murrough, J. W., Mao, X., Pillemer, S., Dyke, J. P., et al. (2012). Increased ventricular lactate in chronic fatigue syndrome. III. Relationships to cortical glutathione and clinical symptoms implicate oxidative stress in disorder pathophysiology. NMR Biomed. 25, 1073–1087. Kennedy, G., Spence, V. A., McLaren, M., Hill, A., Underwood, C., and Belch, J. J. (2005). Oxidative stress levels are raised in chronic fatigue syndrome and are associated with clinical symptoms. Free Radic. Biol. Med. 39, 584–589. DeLorenzo, F., Hargreaves, J., and Kakkar, V. V. (1997). Pathogenesis and management of delayed ortho- static hypotension in patients with chronic fatigue syndrome. Clin. Auton. Res. 7, 185–190. Natelson, B. H., Intriligator, R., Cherniack, N. S., Chandler, H. K., and Stewart, J. M. (2007). Hypocapnia is a biological marker for orthostatic intolerance in some patients with chronic fatigue syndrome. Dyn. Med. 6:2. doi: 10.1186/1476-5918-6-2 LaManca, J. J., Peckerman, A., Walker, J., Kesil, W., Cook, S., Taylor, A., et al. (1999). Cardiovascular response during head-up tilt in DeLuca, J., Johnson, S. K., Ellis, S. P., and Natelson, B. H. (1997). Cognitive functioning is impaired in chronic fatigue syndrome Stewart, J. M., Gewitz, M. H., Weldon, A., Arlievsky, N., Li, K., and Munoz, Stewart, J. M., Gewitz, M. H., Weldon, A., Arlievsky, N., Li, K., and Munoz, J. (1999). Orthostatic intolerance J. (1999). Orthostatic intolerance May 2013 \| Volume 4 \| Article 109 \| 4 Frontiers in Physiology \| Integrative Physiology Genesis of symptoms in CFS Natelson in adolescent chronic fatigue syn- drome. Pediatrics 103, 116–121. concentration. Front. Physiol. 4:109. doi: 10.3389/fphys.2013.00109 This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology. Copyright © 2013 Natelson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third- party graphics etc. May 2013 \| Volume 4 \| Article 109 \| 5 ACKNOWLEDGMENTS Rheumatology 1990 criteria for the classiﬁcation of ﬁbromyalgia: report of the Multicenter Criteria Committee. Arthritis Rheum. 33, 160–172. Rheumatology 1990 criteria for the classiﬁcation of ﬁbromyalgia: report of the Multicenter Criteria Committee. Arthritis Rheum. 33, 160–172. Rheumatology 1990 criteria for the classiﬁcation of ﬁbromyalgia: report of the Multicenter Criteria Committee. Arthritis Rheum. 33, 160–172. was conducted in the absence of any commercial or ﬁnancial relationships that could be con- strued as a potential conﬂict of interest. in adolescent chronic fatigue syn- drome. Pediatrics 103, 116–121. concentration. Front. Physiol. 4:109. doi: 10.3389/fphys.2013.00109 concentration. Front. Physiol. 4:109. doi: 10.3389/fphys.2013.00109 This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology. This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology. This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology. Streeten, D. H. P., and Anderson, G. H. (1992). Delayed orthostatic intolerance. Arch. Intern. Med. 152, 1066–1072. Yoshiuchi, K., Farkas, J., and Natelson, B. H. (2006). Patients with chronic fatigue syndrome have reduced absolute cortical blood ﬂow. Clin. Physiol. Funct. Imaging 26, 83–86. Copyright © 2013 Natelson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third- party graphics etc. Wessely, S., Nimnuan, C., and Sharpe, M. (1999). Functional somatic syn- dromes: one or many? Lancet 354, 936–939. Received: 06 December 2012; paper pending published: 07 March 2013; accepted: 28 April 2013; published online: 20 May 2013. Wolfe, F., Smythe, H. A., Yunus, M. B., Bennett, R. M., Bombardier, C., Goldenberg, D. L., et al. (1990). The American College of Citation: Natelson BH (2013) Brain dys- function as one cause of CFS symptoms including difﬁculty with attention and Conﬂict of Interest Statement: The author declares that the research Conﬂict of Interest Statement: The author declares that the research May 2013 \| Volume 4 \| Article 109 \| 5 www.frontiersin.org www.frontiersin.org
https://openalex.org/W2738131613	https://europepmc.org/articles/pmc5526870?pdf=render	English	null	Fragment optimization for GPCRs by molecular dynamics free energy calculations: Probing druggable subpockets of the A 2A adenosine receptor binding site	Scientific reports	2,017	cc-by	11,619	Fragment optimization for GPCRs by molecular dynamics free energy calculations: Probing druggable subpockets of the A2A adenosine receptor binding site Received: 24 November 2016 Accepted: 23 May 2017 Published: xx xx xxxx Received: 24 November 2016 Accepted: 23 May 2017 Published: xx xx xxxx Pierre Matricon 1, Anirudh Ranganathan2, Eugene Warnick3, Zhan-Guo Gao3, Axel Rudling2, Catia Lambertucci4, Gabriella Marucci4, Aitakin Ezzati2, Mariama Jaiteh1, Diego Dal Ben 4, Kenneth A. Jacobson 3 & Jens Carlsson1 Fragment-based lead discovery is becoming an increasingly popular strategy for drug discovery. Fragment screening identifies weakly binding compounds that require optimization to become high- affinity leads. As design of leads from fragments is challenging, reliable computational methods to guide optimization would be invaluable. We evaluated using molecular dynamics simulations and the free energy perturbation method (MD/FEP) in fragment optimization for the A2A adenosine receptor, a pharmaceutically relevant G protein-coupled receptor. Optimization of fragments exploring two binding site subpockets was probed by calculating relative binding affinities for 23 adenine derivatives, resulting in strong agreement with experimental data (R2 = 0.78). The predictive power of MD/FEP was significantly better than that of an empirical scoring function. We also demonstrated the potential of the MD/FEP to assess multiple binding modes and to tailor the thermodynamic profile of ligands during optimization. Finally, MD/FEP was applied prospectively to optimize three nonpurine fragments, and predictions for 12 compounds were evaluated experimentally. The direction of the change in binding affinity was correctly predicted in a majority of the cases, and agreement with experiment could be improved with rigorous parameter derivation. The results suggest that MD/FEP will become a powerful tool in structure-driven optimization of fragments to lead candidates. Fragment-based lead discovery (FBLD) has rapidly become a well-established technique in early drug develop- ment1. Several lead candidates developed using FBLD have already reached clinical trials, resulting in two FDA approved drugs2. In contrast to high-throughput screening (HTS), where large numbers (~105–106) of drug-sized molecules are tested experimentally, FBLD focuses on smaller libraries (typically 1000–5000 compounds) with molecules of low molecular weight (<300 Da)2, 3. By limiting the size of the molecules in the library, fragment screening achieves a much broader coverage of chemical space than HTS due to the astronomical number of possible drug-like molecules. The low molecular complexity of fragments also reduces the probability for steric mismatches with the receptor, leading to the discovery of ligands that optimally complement subpockets of the binding site4, 5. Consequently, screening of fragment libraries often delivers high hit-rates and diverse starting points for lead development2, 3. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 24 November 2016 Accepted: 23 May 2017 Published: xx xx xxxx Results M i Mapping binding site subpockets using free energy calculations for fragment ligands. Analysis of available A2AAR crystal structures in complex with agonists18 and antagonists17, 24 revealed that the orthosteric site, i.e. the binding site of the native agonist, has several subpockets that could accommodate fragment-like ligands (Fig. 1). Hydrogen bonding to Asn253 has been identified as a key interaction for ligand recognition and this part of the binding site has also been demonstrated to be a hot-spot for fragment binding21, 24. Fragment-sized ligands occupying this region could be further optimized by extension into two additional buried subpockets of the orthosteric site (Fig. 1). The first of these is the ribose-recognizing site (pocket A) and the second is a pocket located below the adenine moiety of adenosine (pocket B). To explore if MD/FEP could guide fragment growth into the two different subpockets, relative binding free energies (ΔΔGbind) for 20 pairs of adenine-derived com- pounds (Table 1) were calculated using the thermodynamic cycle shown in Fig. 2. The relative binding affinity for a compound pair was calculated from alchemical transformations of one ligand into another in complex with the receptor and in aqueous solution (Fig. 2)13. Experimental binding affinities from radioligand binding assays were available for 20 adenine derivatives (1–17, 19, 22–23)22, 23 and were determined in this work for three additional adenine-based compounds (18, 20, and 21, Supplementary Table 1). The compound pairs differed by one to five heavy atoms and spanned up to >500-fold changes in binding affinity. Adenine-based ligands devoid of a ribose- like group are typically antagonists of the A2AAR, which was also confirmed for four selected compounds (5, 19, 22, 23) by measuring inhibition of agonist-induced cAMP production (Supplementary Figure 1). Based on these results, a high-resolution structure of the A2AAR in an inactive conformation (PDB code 4EIY)17 was used in the simulations, and initial ligand binding modes were generated by aligning the adenine moiety to the adenine-like core of the co-crystallized antagonist. All MD simulations were performed in a spherical system centered on the binding site with explicit representation of protein, solvent, membrane, and ligand. Each MD/FEP calculation was divided into several steps, corresponding to transformations for electrostatics, Lennard-Jones, and relevant bonded force field energy terms. Fragment optimization for GPCRs by molecular dynamics free energy calculations: Probing druggable subpockets of the A2A adenosine receptor binding site However, the ligands that emerge from fragment screening typically have low affinities and, in the second step of FBLD, these compounds need to be optimized to yield potent and selective lead candidates. 1Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE- 75124, Uppsala, Sweden. 2Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Stockholm, Sweden. 3Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892, United States. 4Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, Via S. Agostino 1, 62032, Camerino (MC), Italy. Pierre Matricon and Anirudh Ranganathan contributed equally to this work. Correspondence and requests for materials should be addressed to J.C. (email: jens.carlsson@icm.uu.se) Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 1 www.nature.com/scientificreports/ Fragment-to-lead optimization has proved to be a very challenging step in FBLD2. Prioritization of fragments for optimization is often guided by ligand efficiency (defined as the free energy of binding divided by the num- ber of heavy atoms of the compounds6) and access to atomic resolution information regarding binding modes7. Recently, more intricate criteria, e.g. based on the thermodynamic binding profiles of the fragments, have also been suggested to be an important factor in the selection of starting points for optimization8. Two main strat- egies for fragment-to-lead optimization, “linking” and “growing”, have been proposed2. Although “linking” of compounds occupying different subpockets of a binding site has been successful in some cases, “growing” of the fragment by iterative additions of smaller chemical groups has become more widely used2, 3. In either case, access to high-resolution crystal structures of fragments bound to the target often makes crucial contributions to the optimization process7. Whereas computational methods for structure-based ligand design are routinely used for drug-sized molecules9, applications of such approaches to fragment optimization have been more scarce10. The fact that fragments are weak ligands, only occupy a small fraction of the binding site, and may have multiple binding modes adds extra levels of complexity that are challenging to predict with simplified models such as empirical scoring functions. Furthermore, scoring functions developed for computer-aided ligand design have been parameterized based on drug-like compounds, and it has been suggested that these may not be suitable for fragment ligands11, 12. Fragment optimization for GPCRs by molecular dynamics free energy calculations: Probing druggable subpockets of the A2A adenosine receptor binding site Molecular dynamics (MD) simulations in combination with alchemical free energy methods, which explicitly consider contributions to binding from conformational flexibility and interactions with water molecules, could provide a rigorous approach to guide fragment optimization13, but this technique has only recently been applied to FBLD10. Accurate predictions of relative binding affinities for analogs to ligands identified by fragment screening could improve the efficiency of FBLD, further establishing this approach as a groundbreaking strategy for early drug development. g g gy y g p In this work, the utility of MD combined with alchemical free energy methods in fragment optimization was explored for the human A2A adenosine receptor (A2AAR), a G protein-coupled receptor (GPCR) relevant for drug development14 against Parkinson’s disease15 and cancer16. Multiple high-resolution crystal structures of the A2AAR have recently been determined17, 18 and numerous fragment-sized ligands have been identified to this target19–21, making it an ideal test case for evaluating a computational approach. Calculation of relative binding affinities using MD simulations in combination with the free energy perturbation (MD/FEP) method was first benchmarked retrospectively for 23 fragment-sized compounds22, 23. The MD/FEP technique was also used to assess multiple binding modes and predict the thermodynamic signatures governing changes in binding affin- ity, which are both factors of major interest in the optimization process. In a second step, MD/FEP was applied prospectively to predict relative affinities for 12 fragment-sized compounds with unknown binding affinities, fol- lowed by experimental evaluation of these in pharmacological assays. In light of the results, the feasibility of using MD simulations in combination with alchemical free energy methods as a tool in fragment-to-lead optimization will be discussed. Results M i The number of steps and simulation length of each MD/FEP calculation were optimized to achieve convergence and the uncertainty of each step was <0.4 kcal/mol in all cases, with an average of 0.1 kcal/mol for all transformations. Three independent sets of simulations with an average total length of close to 100 ns were used to calculate the relative binding free energy for each compound pair (a total of 1.9 µs for the 20 compound pairs).hi The first set of 10 pairs of adenine derivatives (Table 1) probed opportunities for growing fragments into the ribose-recognizing site (pocket A, Fig. 1) and mainly involved substitutions in the N9-position of the adenine scaffold (Table 1). The average unsigned error for this set of relative binding free energies was 0.66 kcal/mol, resulting in strong correlation with experimental binding data (Fig. 3, R2 = 0.75). Relative free energies involving compounds 1 and 11 were not included in the analysis of correlation with experimental data as reliable Ki values could not be determined for these compounds due to their lack of binding at the highest tested concentration Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 2 www.nature.com/scientificreports/ Figure 1. (A) Orthosteric binding site of the A2AAR shown as white cartoon with Asn253 in sticks. The adenine group is shown in sticks with carbon atoms in gold and hydrogen bonds indicated with black dashed lines. Two adjacent subpockets are shown as spheres with yellow (pocket A, ribose group of endogenous agonist adenosine from the crystal structure with PDB code 2YDO)18 and cyan (pocket B, furan group of antagonist ZM241385 from the crystal structure with PDB code 4EIY)17 carbon atoms. (B) Two adenine-based and three fragment-sized ligands of the A2AAR. Ki values are provided for the fragment ligands21, 22, 24. (C) 2D structures of compounds 1–23. The R-groups are shown in Table 1. Figure 1. (A) Orthosteric binding site of the A2AAR shown as white cartoon with Asn253 in sticks. The adenine group is shown in sticks with carbon atoms in gold and hydrogen bonds indicated with black dashed lines. Two adjacent subpockets are shown as spheres with yellow (pocket A, ribose group of endogenous agonist adenosine from the crystal structure with PDB code 2YDO)18 and cyan (pocket B, furan group of antagonist ZM241385 from the crystal structure with PDB code 4EIY)17 carbon atoms. (B) Two adenine-based and three fragment-sized ligands of the A2AAR. Results M i Ki values are provided for the fragment ligands21, 22, 24. (C) 2D structures of compounds 1–23. The R-groups are shown in Table 1. Figure 2. Thermodynamic cycle used to calculate relative free energies of binding (ΔΔGbind) from MD simulations. Alchemical transformations of the ligands L and L’ were performed in aqueous solution (ΔGaq, left panel) and bound to the receptor (ΔGprot, right panel). The protein is shown as green cartoon and the ligand is depicted in sticks. Water molecules are shown as red spheres and membrane carbon atoms are represented by grey spheres. Figure 2. Thermodynamic cycle used to calculate relative free energies of binding (ΔΔGbind) from MD simulations. Alchemical transformations of the ligands L and L’ were performed in aqueous solution (ΔGaq, left panel) and bound to the receptor (ΔGprot, right panel). The protein is shown as green cartoon and the ligand is depicted in sticks. Water molecules are shown as red spheres and membrane carbon atoms are represented by grey spheres. (Ki > 100 μM). However, it should be noted that MD/FEP correctly predicted the direction of the shift in binding free energies in both cases. One example of successful fragment growth into pocket A was observed for addi- tion of a methyl group in the N9-position of the adenine scaffold (compounds 3 and 4). This resulted in a large improvement of the binding affinity (2.0 kcal/mol), which was also reproduced by the calculated free energy change of 2.4 kcal/mol. Interestingly, substituents larger than two heavy atoms in pocket A typically reduced binding affinities. For example, replacement of a 9-ethyl substituent by 2-hydroxyethyl, isobutyl or propyl moie- ties led to losses of binding affinity, which were also captured the MD/FEP calculations.hff (Ki > 100 μM). However, it should be noted that MD/FEP correctly predicted the direction of the shift in binding free energies in both cases. One example of successful fragment growth into pocket A was observed for addi- tion of a methyl group in the N9-position of the adenine scaffold (compounds 3 and 4). This resulted in a large improvement of the binding affinity (2.0 kcal/mol), which was also reproduced by the calculated free energy change of 2.4 kcal/mol. Interestingly, substituents larger than two heavy atoms in pocket A typically reduced binding affinities. Results M i The experimentally determined values23 indicated a >1000-fold loss of binding affinity, as compound 23 showed no significant binding (reported Ki value > 100 μM), whereas the calculated value suggested that the two compounds had similar affinity. As this was a major outlier among the considered compound pairs, compound 23 was retested in a radioligand binding assay at the A2AAR. The Ki value was determined to be 95 nM for compound 23 (Supplementary Table S1), lead- ing to a relative free energy of −0.8 kcal/mol, which was in better agreement with the prediction and resulted in a strong correlation with experiment for the second compound set (R2 = 0.75, Fig. 3).hfi were dependent on the substituent in the N9-position. This interdependency of the two substituents was also captured by the calculated binding free energies for this subset of four compound pairs (2–3, 5, 9, and 14–17). Another series of compounds with an ethyl group in the N9-position and varying substituents in C8-position (9 and 18–23) was also considered. A 10-fold increase of binding affinity compared to compound 9 was obtained for the C8-methyl substituent (compound 18) and addition of a hydroxyl group in the same position (compound 20) also resulted in improved binding. The relative binding free energies for a majority of the considered pairs exploring pocket B were within 1 kcal/mol of the experimental value (Fig. 3). However, for the pair consisting of 8-alkoxy-9-methyladenine derivatives 23 and 21, there was a large discrepancy between the published experi- mental affinity and relative binding free energy calculated from MD simulations. The experimentally determined values23 indicated a >1000-fold loss of binding affinity, as compound 23 showed no significant binding (reported Ki value > 100 μM), whereas the calculated value suggested that the two compounds had similar affinity. As this was a major outlier among the considered compound pairs, compound 23 was retested in a radioligand binding assay at the A2AAR. The Ki value was determined to be 95 nM for compound 23 (Supplementary Table S1), lead- ing to a relative free energy of −0.8 kcal/mol, which was in better agreement with the prediction and resulted in a strong correlation with experiment for the second compound set (R2 = 0.75, Fig. Results M i 3).hfi g p p ( , g ) The ability of MD/FEP to predict changes in affinity was further highlighted by the strong correlation between experimental and predicted binding free energies for the full set of 18 compound pairs (R2 = 0.78, Fig. 3). To assess the influence of experimental uncertainties on this result, the correlation was calculated for 1000 random selections of either the maximal or minimal Ki value obtained from the 95% confidence interval, which resulted in R2 = 0.75 with a standard deviation of 0.1. It should also be noted that the correlations between the exper- imentally determined relative binding affinities and trivial size-descriptors, e.g. the difference in the number of heavy atoms (R2 = 0.10) or predicted 1-octanol/water partition coefficients (AlogP, R2 = 0.44), were low. In order to compare our results to an empirical scoring function, the adenine derivatives were also docked to the orthosteric site using the GLIDE docking program25 and binding free energies were calculated with the standard precision (SP) scoring function for the 18 compound pairs. All of the docked compounds reproduced the binding mode expected from crystal structures of the A2AAR in complex with adenine-based ligands. The correlation with experimentally determined relative binding free energies for GLIDE-SP (R2 = 0.42, Supplementary Figure 2) was similar to that obtained for ALogP and significantly lower than for MD/FEP. Assessment of alternative binding modes and thermodynamic signatures of fragment bind- ing. The use of MD/FEP in fragment optimization could be limited by the uncertainty associated with ligand binding modes if a crystal structure of the complex is not available. As fragments are small, such compounds can bind in a large number of orientations and it may be challenging to rank these with more simplified models, e.g. molecular docking scoring functions12, 26. The calculations for 9-methyl adenine derivatives 2 and 3 were extended to explore two alternative binding modes identified by the molecular docking study carried out by Lambertucci et al.22 (Fig. 4A and B). Both proposed binding modes predicted a hydrogen bond between the exo- cyclic nitrogen of the adenine moiety and the side chain oxygen of Asn253. The first binding mode involved an additional hydrogen bond between the N7 of the adenine-moiety and the side chain nitrogen donor of Asn253, leading to an orientation that was essentially identical to that observed in the crystal structure of the A2AAR in complex with adenosine (Fig. Results M i For example, replacement of a 9-ethyl substituent by 2-hydroxyethyl, isobutyl or propyl moie- ties led to losses of binding affinity, which were also captured the MD/FEP calculations.hff gfi y p The second compound set explored the effects of substituents in the C8-position of the adenine scaffold, which extended into pocket B. A bromine in this position (compound 3) led to a 58-fold increase in affinity compared to the unsubstituted compound 2. This effect was also captured by the free energy calculations, which predicted an improvement in binding corresponding to 2.3 kcal/mol between compounds 2 and 3, in close agreement with the experimental value (2.4 kcal/mol). Interestingly, changes in affinity from addition of a bromine in the C8-position Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 3 www.nature.com/scientificreports/ Figure 3. Comparison of calculated and experimental relative binding free energies (ΔΔGbind) for 18 compound pairs. The solid line represents prefect agreement between calculated and experimental data whereas the dotted lines represent an absolute deviation of 1 kcal/mol. Experimental and computational error bars correspond to the data reported in Table 1. Figure 3. Comparison of calculated and experimental relative binding free energies (ΔΔGbind) for 18 compound pairs. The solid line represents prefect agreement between calculated and experimental data whereas the dotted lines represent an absolute deviation of 1 kcal/mol. Experimental and computational error bars correspond to the data reported in Table 1. were dependent on the substituent in the N9-position. This interdependency of the two substituents was also captured by the calculated binding free energies for this subset of four compound pairs (2–3, 5, 9, and 14–17). Another series of compounds with an ethyl group in the N9-position and varying substituents in C8-position (9 and 18–23) was also considered. A 10-fold increase of binding affinity compared to compound 9 was obtained for the C8-methyl substituent (compound 18) and addition of a hydroxyl group in the same position (compound 20) also resulted in improved binding. The relative binding free energies for a majority of the considered pairs exploring pocket B were within 1 kcal/mol of the experimental value (Fig. 3). However, for the pair consisting of 8-alkoxy-9-methyladenine derivatives 23 and 21, there was a large discrepancy between the published experi- mental affinity and relative binding free energy calculated from MD simulations. Results M i The calculated free energies suggested that the pose derived from the binding mode of adenosine in A2AAR crystal structures, which was also used in the MD/FEP calculations, was favored by 6.5 ± 0.1 kcal/mol. The population of the alternative binding mode was thus predicted to be very low and would not influence the experimentally measured binding affinity significantly.h i The enthalpic and entropic components of the binding free energy are increasingly attracting interest in drug discovery as these can provide more information on the driving forces of ligand binding27. Although the exper- imental binding free energy differences for compound 3 relative to compounds 2 and 4 were accurately repro- duced by the MD/FEP calculations, it was not clear from visual inspection why the addition of a single heavy atom resulted in such a large change in binding affinity in both cases. To further quantify the change in binding free energy, it was decomposed into enthalpy and entropy components using a relationship analogous to the van’t Hoff equation. MD/FEP calculations were carried out for the two compound pairs at 13 different temperatures between 270 and 330 K. The enthalpy and entropy components could then be derived from the slope and inter- cept of the relation between ΔΔGbind/T and 1/T (Fig. 4C and D)28. These calculations demonstrated that the pre- dicted affinity increase for compound 3 relative to compound 2 was driven by entropy (−TΔΔSbind = −7.1 kcal/ mol), which was counteracted by an unfavorable enthalpy contribution (ΔΔHbind) of +4.8 kcal/mol. In contrast, the gain in affinity for compound 3 relative to compound 4 was enthalpy driven (ΔΔHbind = −5.6 kcal/mol and −TΔΔSbind = + 3.4 kcal/mol). Overall, there were only small differences in receptor structure between the three complexes based on the MD simulations, suggesting that changes in the solvent network could be responsible for the distinct thermodynamic profiles. MD snapshots of the water molecules in the binding site were clustered to identify hydration sites in the vicinity of the ligands using the algorithm developed by Young et al.29. Comparison of the solvent networks revealed that introduction of the 9-methyl group (compound 2) displaced an ordered water molecule in pocket A (Fig. 4E) whereas the 8-bromine (compound 3) replaced a different ordered water in pocket B (Fig. 4F). Results M i 1)18. In the alternative orientation, a hydrogen bond with the N1 atom of the Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 4 www.nature.com/scientificreports/ Figure 4. (A) Binding mode of compound 3 based on an A2AAR crystal structure in complex with a related ligand (PDB code 4EIY). (B) Alternative binding mode for compound 3. (C,D) Determination of entropy and enthalpy components of the relative binding free energy from MD/FEP calculations at different temperatures for compounds 3 and 4 (C), and compounds 2 and 3 (D). (E,F) Maps of binding site solvent structure from clustering of snapshots from a simulation of compound 4 (E) and 2 (F) in complex with the A2AAR. The corresponding bromine and methyl substituents in compound 3 are represented as transparent grey sticks. In both cases, the water molecule displaced by compound 3 is shown as a red sphere. The orthosteric binding site of the A2AAR is shown as a white cartoon with a key residue in sticks. The binding modes of the ligands are shown in sticks with carbon atoms in gold and hydrogen bonds indicated with black dashed lines. Figure 4. (A) Binding mode of compound 3 based on an A2AAR crystal structure in complex with a related ligand (PDB code 4EIY). (B) Alternative binding mode for compound 3. (C,D) Determination of entropy and enthalpy components of the relative binding free energy from MD/FEP calculations at different temperatures for compounds 3 and 4 (C), and compounds 2 and 3 (D). (E,F) Maps of binding site solvent structure from clustering of snapshots from a simulation of compound 4 (E) and 2 (F) in complex with the A2AAR. The corresponding bromine and methyl substituents in compound 3 are represented as transparent grey sticks. In both cases, the water molecule displaced by compound 3 is shown as a red sphere. The orthosteric binding site of the A2AAR is shown as a white cartoon with a key residue in sticks. The binding modes of the ligands are shown in sticks with carbon atoms in gold and hydrogen bonds indicated with black dashed lines. adenine moiety was instead obtained, resulting in a second distinct binding mode. The relative binding affinity of the two poses (Fig. 4A and B) was calculated by alchemically transforming one binding mode into the other via an intermediate compound (24) to assess their probability (Supplementary Figure 3). Results M i The large increases in binding affinity hence appeared to be connected to changes in binding site solvation in both cases, but were the result of different thermodynamic profiles. Prospective predictions for three nonpurine fragment series. To further challenge the MD/FEP method, calculations were extended to 12 fragment-sized nonpurine heterocycles of unknown affinity, which Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 5 www.nature.com/scientificreports/ represented three series with varying levels of modeling difficulty (Fig. 5). The first two compound series were adenine-like and had substituents that explored pocket B. Compound 25, [1,2,4]triazolo[1,5-a][1,3,5] triazin-7-amine, was a substructure of the A2AAR antagonist ZM241385 (Fig. 1). Similar to the adenine deriv- atives described in previous sections, this scaffold represented a case with high confidence regarding the frag- ment binding mode and predictions were made for three compounds in this series (25–27). The second scaffold, derivatives of [1,2,4]triazolo[1,5-a]pyridin-8-amine 28, was less similar to adenine, but was assumed to maintain hydrogen bond interactions with Asn253 (Fig. 5), and relative affinities were predicted for three 2-alkyl analogs (29–31) that probed pocket B. The third series of five variously substituted derivatives of N-(benzo[d]thiazol-2-yl) acetamide 32 was unrelated to adenine. This represented the most challenging series, as the binding mode of this scaffold was unknown. After the MD/FEP predictions had been completed, the 12 compounds were evaluated experimentally using radioligand binding assays, and Ki values were determined for the ligands that showed >50% displacement at 300 μM (Supplementary Table S2). The computational and experimental results for the three series of fragments are summarized in Fig. 5.hfi g g The relative binding affinities for substituted triazolo-triazin-amine derivatives 26 and 27 compared to unsub- stituted 25 were calculated using the same protocol as for the series of adenine derivatives. The MD/FEP calcula- tions predicted that both compound 26 (0.6 kcal/mol) and 27 (4.3 kcal/mol) had higher affinity than compound 25, which was also confirmed experimentally. Compounds 26 and 27 had Ki values of 78.5 and 20 μM, respec- tively, which were large improvements over compound 25 that only displayed 34% radioligand displacement at 300 μM. For the second series of analogs, the calculated relative binding free energy (ΔΔGcalc = 0.4 kcal/mol) correctly predicted that the 2-methyl substituted triazolopyridine 29 (31% at 300 μM) had higher affinity than compound 28, which showed close to negligible radioligand displacement at 300 μM (17%). Results M i However, the 2-ethyl and 2-isopropyl substituted compounds 30 (49% at 300 μM) and 31 (223 μM) were incorrectly ranked relative to compound 29 (31% at 300 μM). Both compounds 30 and 31 were predicted to be weaker than compound 29 by 0.9 and 2.2 kcal/mol, respectively. The discrepancies for triazolopyridines 30 and 31 were intriguing considering the close agreement with experiment for the adenine-like ligands. To understand the origin of these erroneous predictions, the MD simulation trajectories for the ethyl-substituted compound 30 and methoxy-substituted compound 21 were inspected visually. The main difference between compounds 30 and 21 was found to be the torsional angles of the substituents protruding into pocket B. For compound 21, the methoxy group primarily sampled angles that were within the plane of the adenine moiety, whereas the ethyl group did not align with the plane of the aromatic ring. Torsion angle scans for compound 30 using density functional theory (DFT) revealed large errors in the force field parameters both in the location of the minimum and the energy barrier height of the potential energy curve (Fig. 5E), whereas there was reasonable agreement between OPLSAA_2005 and DFT for compound 21 (Supplementary Figure 4). MD/FEP calculations were then repeated for compounds 30 and 31 using a force field term for the torsion that reproduced the DFT calculations. The calculated relative binding free energies (to compound 29) changed from −0.9 to + 0.1 kcal/mol for compound 30 and from −2.2 to + 0.7 kcal/ mol for compound 31. These shifts in calculated values resulted in accurate ranking of the two ligands relative to 29 (Supplementary Table S2).f pp y For the third series of compounds, 2-acetamido-benzothiazole (compound 32) was the core scaffold and had a Ki value of 79 μM. 4-Hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine- 1-carboxamide (tozadenant), a compound that has been in clinical trials for the treatment of Parkinson’s disease30, could essentially be considered as a superstructure of this fragment. However, tozadenant could not be accom- modated in the crystal structure used for the adenine derivatives in a manner that allowed for hydrogen bonding with residue Asn253. Hence, prior to experimental evaluation of this fragment series, an alternative binding site conformation based on a different A2AAR crystal structure24 was used. In this conformation, alternative side chain rotamers for His264 and Glu169 lead to a more open binding pocket, which could accommodate tozadenant and compound 32. Results M i This binding pose for compound 32 was found to be stable in MD simulations, and a representa- tive snapshot was used as starting point for the FEP calculations (Fig. 5C). The effects of adding a methyl group at three different positions (4, 5 and 6) of the benzothiazole ring of compound 32 were evaluated computationally (Fig. 5C, compounds 33–35). Improvements of affinity corresponding to 0.3 and 0.4 kcal/mol were predicted by MD/FEP for compounds 33 and 34 respectively, whereas a large loss of binding was obtained for compound 35. The predictions for compounds 33 and 34 agreed reasonably well with the subsequently determined 7-fold increases of affinity. Compound 35 was the weakest ligand of the three analogs with only a two-fold increase of affinity, but the MD/FEP calculations had predicted a loss of binding free energy in this case (2.7 kcal/mol). To further optimize compound 33, MD/FEP calculations were carried out for the 4-methoxy-substituted com- pound 36. The experimentally determined 44-fold increase of affinity (corresponding to 2.3 kcal/mol, Fig. 5F) compared to compound 32 was partially captured by the MD/FEP calculations, which predicted a 0.8 kcal/mol improvement in binding free energy. Hence, whereas the direction of the change in binding affinity was correct, the magnitude of the improvement in affinity was underestimated. To investigate if prediction accuracy could be improved by increasing sampling, we retrospectively extended the simulations by doubling the production time for the transformation between compounds 36 and 32, which resulted in improved agreement with experimental data (ΔΔGbind = 1.3 kcal/mol). Discussionh The focus of this work was to evaluate using MD simulations in combination with free energy calculations as a tool for fragment optimization. Three key results emerged from calculations of relative binding affinities for frag- ments ligands of the A2AAR, a GPCR that has been intensively studied as a drug target14. First, there was a strong correlation between calculated and experimental relative binding free energies for ligands based on an adenine scaffold. Remarkably, the direction of the shift in binding free energy was correctly predicted for all of the adenine 6 Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 www.nature.com/scientificreports/ Figure 5. (A–C) Binding modes and summary of MD/FEP predictions for three nonpurine compound series. The orthosteric binding site of the A2AAR is shown as a white cartoon with key residues in sticks. The predicted binding modes of the ligands are shown in sticks with carbon atoms in gold and hydrogen bonds indicated with black dashed lines. The experimental result for each fragment is shown as its Ki (μM) or % displacement of radioligand binding at 300 μM. The performed MD/FEP calculations are represented with arrows in red and green, which corresponds to accurate and erroneous predictions, respectively. (D) Summary of agreement of MD/FEP calculations with experimental data. The percentage of accurate predictions of the direction of the binding free energy change is shown. (E) Potential energy curve for the indicated torsion calculated from OPLSAA_2005, DFT (QM), and a molecular mechanics potential fitted to the DFT results (QM-Derived). (F) Concentration-effect curves for displacement of radiolabeled A2AAR antagonist [3H]ZM241385 by compounds 27 and 36. Figure 5. (A–C) Binding modes and summary of MD/FEP predictions for three nonpurine compound series. The orthosteric binding site of the A2AAR is shown as a white cartoon with key residues in sticks. The predicted binding modes of the ligands are shown in sticks with carbon atoms in gold and hydrogen bonds indicated with black dashed lines. The experimental result for each fragment is shown as its Ki (μM) or % displacement of radioligand binding at 300 μM. The performed MD/FEP calculations are represented with arrows in red and green, which corresponds to accurate and erroneous predictions, respectively. (D) Summary of agreement of MD/FEP calculations with experimental data. The percentage of accurate predictions of the direction of the binding free energy change is shown. Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 Discussionh (E) Potential energy curve for the indicated torsion calculated from OPLSAA_2005, DFT (QM), and a molecular mechanics potential fitted to the DFT results (QM-Derived). (F) Concentration-effect curves for displacement of radiolabeled A2AAR antagonist [3H]ZM241385 by compounds 27 and 36. derivatives that had an absolute experimental free energy change >0.5 kcal/mol. Second, the potential of MD/ FEP calculations to assess alternative binding modes and predict thermodynamic signatures of fragment binding was demonstrated, which could be used to tailor ligand properties during optimization. Finally, prospective pre- dictions for three compound series and evaluation of these in pharmacological assays highlighted opportunities and challenges for the use of MD/FEP calculations in FBLD.h The potential of MD/FEP to guide fragment optimization was clearly demonstrated by the excellent results obtained for the series of adenine derivatives22, 23. Substantial changes in affinity could be achieved by intro- duction of substituents in the C8- and N9-positions of this scaffold. These effects were not obvious by visual inspection of the complexes and changes in affinity did not correlate with trivial descriptors such as heavy atom count. The fact that addition of diverse substituents to the same subpocket improved binding affinities likely reflects a complex interaction network involving structural water molecules, polar and non-polar side chains. These effects were accurately captured by the MD/FEP calculations, but not by docking in combination with an empirical scoring function. As previously demonstrated by Warren et al., empirical scoring functions are more suitable for screening of large chemical databases to prioritize compounds for experimental testing rather than ranking closely related ligands by affinity31. The improved accuracy for MD/FEP may be due to explicitly tak- ing into account water molecules, induced fit effects and associated entropic contributions to the binding9. Discussionh 2D structures of the compounds are shown in Fig. 1C. aUncertainties are calculated as the standard error of the mean based on the maximal and minimal affinities values obtained from the 95% confidence intervals of the experimentally determined Ki values. Experimental Ki values can be found in Supplementary Table 1. bAverage relative binding free energy from three independent trajectories with uncertainties estimated as the standard error of the mean. cKi value from reference 23/Remeasured Ki value in this work (Supplementary Table 1). performance of docking scoring functions can also be further improved for specific targets by considering the effects of specific water molecules22, 32, 33, but such protocols may not be readily transferred to other ligand series or targets. Consideration of the enthalpic and entropic components of the binding free energy has recently been suggested to be an important metric to guide fragment optimization8. In addition to improved predictions of relative affinities, MD/FEP calculations make it possible to characterize the driving forces behind a change in free energy, which has previously been applied successfully to study ion hydration28 and enzyme catalysis34. In this work, we used the same approach to investigate the large differences in binding observed for two compound pairs from the series of adenine-based ligands. The affinity gains obtained for a substituent in the C8-position of ade- nine were found to be associated with a large increase of entropy and displacement of a binding site water mole- cule in pocket B, which appeared to be a classic example of the hydrophobic effect. Interestingly, the same hotspot has previously been identified based on MD-derived maps of the solvent network in the A2AAR binding site35, 36. In contrast, the addition of methyl substituent in the N9-position, which involved displacement of a water mole- cule from pocket B, led to a decrease of entropy and the improvement of the binding affinity was instead driven by enthalpy. Large improvements of affinity due to the addition of a single heavy atom, which has been referred to as the “magic methyl” effect, may hence have completely different thermodynamic origins. Although the predictions of the entropy and enthalpy contributions to the relative binding free energies will need to be further tested by comparison to experimental data, our results suggest that MD/FEP is not limited to guiding affinity optimization, but can also be used to tailor the thermodynamic profile of ligands. Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 Discussionh The Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 7 www.nature.com/scientificreports/ Ligand pair (A → B) cmpd A cmpd B ΔΔGbind (kcal/mol) R1 R2 R1 R2 Experimentala Calculatedb 1 → 2 pocket A — — H CH3 <−1.6 −3.9 ± 0.2 3 → 4 Br CH3 Br H 2.0 ± 0.3 2.4 ± 0.0 5 → 3 Br CH3CH2 Br CH3 0.5 ± 0.3 −1.2 ± 0.0 6 → 5 Br HOCH2CH2 Br CH3CH2 −1.5 ± 0.2 −2.5 ± 0.1 7 → 5 Br CH3CH2CH2 Br CH3CH2 −1.1 ± 0.2 −0.5 ± 0.1 8 → 5 Br (CH3)2CHCH2 Br CH3CH2 −2.9 ± 0.3 −2.3 ± 0.1 9 → 2 H CH3CH2 H CH3 0.7 ± 0.2 0.2 ± 0.0 10 → 9 H HOCH2CH2 H CH3CH2 −1.0 ± 0.2 −1.4 ± 0.1 11→ 9 H (CH3)2CHCH2 H CH3CH2 <−2.3 −3.2 ± 0.0 12 → 13 H HOCH2CH2CH2 H CH3CH2CH2 0.5 ± 0.2 0.6 ± 0.4 3 →2 Pocket B Br CH3 H CH3 2.4 ± 0.2 2.3 ± 0.2 5 → 9 Br CH3CH2 H CH3CH2 2.2 ± 0.3 1.5 ± 0.0 14 → 15 Br cC5H9 H cC5H9 0.0 ± 0.3 −2.2 ± 0.2 16 → 17 Br CH2CHCH2CH2 H CH2CHCH2CH2 1.0 ± 0.2 0.3 ± 0.2 18 → 9 CH3 CH3CH2 H CH3CH2 1.4 ± 0.2 1.4 ± 0.0 19 → 9 furyl CH3CH2 H CH3CH2 3.8 ± 0.2 5.4 ± 0.2 20 → 9 OH CH3CH2 H CH3CH2 0.4 ± 0.2 0.1 ± 0.1 21 → 18 CH3O CH3CH2 CH3 CH3CH2 1.3 ± 0.1 2.7 ± 0.1 22 → 21 CH3CH2O CH3CH2 CH3O CH3CH2 −0.3 ± 0.2 −0.4 ± 0.0 23 → 21 (CH3)2CHO CH3CH2 CH3O CH3CH2 <−4.9/−0.8c 0.0 ± 0.1 Table 1. Calculated and experimental relative binding free energies for 20 compound pairs based on adenine. 2D structures of the compounds are shown in Fig. 1C. aUncertainties are calculated as the standard error of the mean based on the maximal and minimal affinities values obtained from the 95% confidence intervals of the experimentally determined Ki values. Experimental Ki values can be found in Supplementary Table 1. bAverage relative binding free energy from three independent trajectories with uncertainties estimated as the standard error of the mean. cKi value from reference 23/Remeasured Ki value in this work (Supplementary Table 1). Table 1. Calculated and experimental relative binding free energies for 20 compound pairs based on adenine. MD/FEP calculations MD/FEP calculations. The MD simulations were performed using a high-resolution crystal structure of the A2AAR (PDB accession code: 4EIY, 1.8 Å)17. In a first step, a hydrated 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membrane bilayer was first equilibrated around the A2AAR structure with periodic boundary conditions using the 4.5.5 version of GROMACS47. These simulations were setup using the GPCR-ModSim protocol48 and the OPLS all atom (OPLSAA) force field37, TIP3P waters49, and Berger lipid parameters50. All protein atoms were tightly restrained to their initial coordinates and the hydrated membrane was equilibrated for a total of 40 ns at 300 K. All MD/FEP calculations were carried out starting from the membrane equilibrated A2AAR system using the program Q51 with the same force field. Ligand parameters were obtained using the OPLSAA_2005 version implemented in the program hetgrp_ffgen (Schrödinger, LLC, New York, NY, 2017). The simulations were car- ried out at 310 K in a sphere of 18 Å radius centered on the ligand. All protein, water, and ligand atoms within 18 Å of the center of the sphere were explicitly included in the simulations. Atoms close to the sphere edge were restrained to their initial coordinates and atoms beyond the sphere edge were excluded from nonbonded interac- tions. Asp, Glu, Lys, and Arg residues within 15 Å of the sphere center were protonated according to their most probable states at pH 7 and ionizable residues closer to the sphere edge were set to their neutral state. The proto- nation states of the histidines in the binding site were set by manual inspection. His278, His250, and His264 were protonated at Nδ, Nε, and both nitrogen positions, respectively. The series of 2-acetamido-benzothiazole deriva- tives (compounds 32–36) were simulated with conformations of His264 and Glu169 obtained from an alternate crystal structure of the A2AAR24. In these simulations His264 was protonated at the Nδ position. The SHAKE52 algorithm was applied to constrain all solvent bonds and angles and the water molecules at the sphere surface were subjected to radial and polarization restraints according to the SCAAS model51, 53. A nonbonded cutoff of 10 Å was used for all atoms except the ligand, for which no cutoff was applied. Long-range electrostatic interactions were treated with the local reaction field method54. The time step was set to 1 fs and nonbonded pair lists were updated every 25 steps. Discussionh In the case of the A2AAR, fragment screening against stabilized receptor constructs by biophysical methods19, 20 and computationally using molecular docking21 have led to the discovery of diverse starting points for development of lead compounds. The determination of multiple high-resolution crystal structures of GPCRs in complex with fragments24, 46 provides exciting opportunities to apply computational meth- ods in FBLD for GPCRs. Our results demonstrate that the MD/FEP approach can contribute to efficient optimiza- tion of fragment hits, which is key for successful use of FBLD in drug development. The combination of molecular docking screening for fragment identification and efficient ligand optimization via MD/FEP has the potential to become a powerful addition to the toolbox of methods used in fragment-based drug discovery. Discussionh i Application of the MD/FEP technique to fragment-sized molecules has several advantages from a methodo- logical standpoint. Molecular mechanics force fields are likely more accurate for fragment- than for drug-like com- pounds as parameters are typically developed based on fragment-sized molecules37. Furthermore, it should be more feasible to reach convergence of the free energy calculations for fragments as such molecules typically have fewer degrees of freedom than drugs. Comparison of our results to a recent study that applied the MD/FEP technique to two series of lead-like A2AAR ligands supports this idea38. The lower correlation with experiment obtained for two series of adenine-derived ligands may reflect that these were of lead-like size and interacted with the flexible extra- cellular loops whereas the fragments considered in this work had limited conformational flexibility and extended into a relatively rigid TM region. Interestingly, two recent benchmarks of binding free energy calculations for a large number of soluble targets showed a similar trend10, 39. It should be noted that access to information regarding the binding mode for a representative ligand was likely a key contributor to the accuracy of the MD/FEP calculations in all cases. Hence, if a high-resolution structure of a representative complex is available, MD/FEP calculations can be a valuable technique for ligand optimization and the approach appears to be particularly suited for fragments.hf q g p pp pp p y g The prospective predictions carried out for three different fragment series revealed potential pitfalls for the use of MD/FEP to predict ligand binding affinities. In this case, the compounds had affinities in the high micromo- lar to millimolar range, which closely mimicked the scenario encountered in fragment-to-lead optimization3. In Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 8 www.nature.com/scientificreports/ agreement with the results obtained for the adenine derivatives, the predictions were excellent for the fragments derived from a ligand co-crystallized with the A2AAR. The second fragment series illustrated the importance of high quality torsional force field parameters for small molecule ligands. The MD/FEP calculations based on OPLSAA_2005 parameters were not in agreement with experimental data, whereas a DFT-derived torsional poten- tial resulted in correct ranking of the compounds. This result suggests that force field parameters should be used with caution even for fragment-sized molecules. As we focused only on congeneric series of compounds with mainly non-polar substituents, the performance of the partial atomic charges from OPLSAA_2005 was not assessed in this work. Discussionh For example, consideration of different heterocyclic compounds could involve large changes in charge distri- bution, which may not be accurately represented by empirical partial charges. Fortunately, as fragments will have a small number of atoms and rotatable bonds, torsions and partial charges could in principle be derived using ab initio methods prior to the MD/FEP calculations to further enhance modeling accuracy. In line with these ideas, particular focus was put on improvements of torsional potentials and partial atomic charges in the recently released OPLS3 force field for organic molecules40. The last series of fragments illustrated challenges associated with lack of crystal structure information regarding fragment binding modes. Modeling of the binding mode involved consideration of several binding site conformations and required expert knowledge regarding ligand recognition by the target. Encouragingly, the direction of the change in binding free energy was correctly predicted in three out of four cases, but the magnitude of the shift in affinity was not always captured. Considering the many uncertainties involved in modeling of fragment binding modes, fragment optimization in the absence of a crystal structure should be con- sidered to be very challenging. In these instances, the use of metadynamics41 and MD/FEP in combination with mutagenesis studies42, 43 to identify ligand binding modes hold promise. Binding modes that reproduce the initial structure-activity relationships could be used to guide compound selection in the following rounds of optimization. The major advances made in molecular and structural biology for GPCRs44, 45 make it possible to apply FBLD to numerous targets of therapeutic interest. In the case of the A2AAR, fragment screening against stabilized receptor constructs by biophysical methods19, 20 and computationally using molecular docking21 have led to the discovery of diverse starting points for development of lead compounds. The determination of multiple high-resolution crystal structures of GPCRs in complex with fragments24, 46 provides exciting opportunities to apply computational meth- ods in FBLD for GPCRs. Our results demonstrate that the MD/FEP approach can contribute to efficient optimiza- tion of fragment hits, which is key for successful use of FBLD in drug development. The combination of molecular docking screening for fragment identification and efficient ligand optimization via MD/FEP has the potential to become a powerful addition to the toolbox of methods used in fragment-based drug discovery. The major advances made in molecular and structural biology for GPCRs44, 45 make it possible to apply FBLD to numerous targets of therapeutic interest. MD/FEP calculations In the simulations of the ligands in aqueous solution, the compound was positioned in the center of the sphere and a weak harmonic restraint was applied to a central atom (e.g. C5 of the adenine scaffold) to prevent it from approaching the sphere edge. Clustering of the water network in the binding site was carried out based on a simulation of 8 ns with the receptor and ligand restrained to their starting coordinates, from which 8000 snapshots were extracted and processed using the algorithm of Young et al.29.h p p g g g The relative binding free energy for a pair of compounds was calculated in multiple steps using MD/FEP: (i) The transformation of partial charges and (ii) combined transformation of Lennard-Jones (LJ) and parameters involving covalent bonds in several MD/FEP calculations. If multiple heavy atoms were annihilated, a separate MD/FEP calculation was carried out to remove these in a step-wise manner. A soft-core potential was intro- duced for the atom in a first step, followed by removal of the resulting van der Waals potential42, 55. The force field parameters describing angles, bonds, and improper torsions were retained for annihilated atoms whereas the torsional potential was removed in some cases to improve convergence. The total free energy was calculated as the sum of the results obtained in each step. Each MD/FEP calculation was divided into n intermediate states that Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 9 www.nature.com/scientificreports/ were equilibrated separately. The potential (Um) defining each state was a linear combination of energy functions describing the start- (A) and endpoint (B) of the transformation were equilibrated separately. The potential (Um) defining each state was a linear combination of energy functions describing the start- (A) and endpoint (B) of the transformation λ λ = − + U U U (1 ) m m A m B λ λ = − + U U U (1 ) (1) m m A m B (1) where λm varies from 0 to 1. The FEP calculations involving partial charges were performed using 11 states whereas the number of λ values used to transform LJ and bonded parameters varied from 40 to 122 steps. Each receptor-ligand complex was equilibrated for 475 ps at each λ value. In this simulation, harmonic restraints on the protein and ligand atoms were released in several steps and the temperature was gradually increased to 310 K. MD/FEP calculations The equilibration step was followed by 250 ps of unrestrained simulation, from which potential energies were extracted. The same transformation was carried out in a spherical water droplet. In this case, the system was equil- ibrated for 350 ps, followed by 100 ps of unrestrained simulation. The free energy difference between states A and B was calculated by summing up the free energy differences of the n intermediate states using ∑ Δ = − → = − − − + G kT e ln (2) A B FEP m n U U kT m 1 1 ( )/ m m 1 (2) where 〈…〉m represents an ensemble average on the potential Um, which is calculated from the MD simulations13. Three replicates, which were initiated from different starting velocities for the atoms in the system, were per- formed for each state and these were exponentially averaged in calculations of the free energy. The uncertainty of a transformation was quantified as the difference in free energy obtained by applying the FEP formula in the forward and reverse direction and was optimized by increasing the number of λ values or simulation length until convergence was obtained. The uncertainty of a calculated relative binding free energy was estimated as the stand- ard error of the mean of three independent trajectories.h The enthalpy (ΔΔHbind) and entropy (ΔΔSbind) contribution to the relative binding free energy were calcu- lated from a relationship analogous to the van’t Hoff equation28. Each FEP transformation was carried out at 5 K intervals between 270 and 330 K to calculate the temperature dependence of the relative binding free energy (ΔΔGbind). The entropy and enthalpy components were then calculated from the slope and intercept of the rela- tion between ΔΔGbind/T and 1/T: ΔΔ = ΔΔ −ΔΔ G T T H S 1 (3) bind bind bind (3) Molecular docking and ALogP calculations. The docking calculations were carried out with GLIDE25 (version 6.0, Schrödinger, LLC, New York, NY, 2017) using the standard precision (SP) protocol and default set- tings. The same A2AAR crystal structure (PDB code 4EIY17) and protonation states for the ionizable residues as in the MD simulations were used. Prior to docking, all non-protein atoms (e.g. water molecules) were removed and the binding site was defined based on the co-crystallized ligand. ALogP values were calculated using Maestro (Release 2017–1: Maestro, Schrödinger, LLC, New York, NY, 2017). Torsion scans. Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 MD/FEP calculations Potential energy profiles for the ethyl and methoxy substituents of compounds 21 and 30 were calculated using Gaussian0956. The torsional scans were performed using B3LYP/DFT with the 6-311++G(d,p) basis set57–60 and a full geometry optimization was carried out at each point. The corresponding force field poten- tial energies were calculated based on the geometries obtained from the DFT optimization. Radioligand binding and functional assays. Radioligand binding assays for compounds 25–36 were performed as previously described61 using membrane preparations from Chinese hamster ovary (CHO) or human embryonic kidney (HEK)293 cells stably expressing the human A1, A2A or A3AR. The following radioli- gands: antagonist [3H]DPCPX (0.5 nM); antagonist [3H]ZM241385 (1.0 nM); and agonist [125I]AB-MECA (0.2 nM) were used for A1, A2A, and A3ARs, respectively. Binding parameters were calculated using Prism 6 software (GraphPAD, San Diego, CA, USA). IC50 values obtained from competition curves were converted to Ki values using the Cheng-Prusoff equation. Data were expressed as mean ± standard error. The radioligand binding assays for compounds 18, 20, 21, and 23 were performed as previously described23. Each Ki value was determined from at least three independent experiments. p p Four adenine derivatives (compounds 5, 19, 22, and 23) were evaluated in functional assays. CHO cells, stably transfected with the human A2AAR and transiently with firefly luciferase biosensor, were grown adherently and maintained in Dulbecco’s Modified Eagles Medium with nutrient mixture F12 (DMEM/F12 with phenol red), supplemented with 10% FBS (Fetal Bovine Serum), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5µg/mL amphotericin, 1 mM Sodium pyruvate, and 0.1 mg/mL Geneticin (G418) at 37 °C, and aerated with 5% CO2: 95% O2. Cells were harvested in CO2 independent media and counted in a Neobauer chamber. The desiderate cell number was incubated in equilibration medium containing a 3% v/v GloSensor cAMP reagent stock solution, 10% FBS and 87% CO2 independent medium. After 2 hours of incubation cells were dispensed in wells of 384 well plate and when a steady-state basal signal was obtained, different concentrations of antagonists were added. After 10 min, 1 µM of 5′-N-ethylcarboxamidoadenosine (NECA), the reference agonist, was injected. Responses were expressed as percentage of the Maximal Relative Luminescence Units (RLU). Inhibition-response curves were fitted by a non-linear regression using a Prism 4.0 program (GraphPad Software, San Diego, CA, USA). Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 10 www.nature.com/scientificreports/ References 1. Johnson, C. N., Erlanson, D. A., Murray, C. W. & Rees, D. C. Fragment-to-lead medicinal chemistry publications in 2015. J. Med. Chem. 60, 89–99 (2017). 2. Erlanson, D. A., Fesik, S. W., Hubbard, R. E., Jahnke, W. & Jhoti, H. Twenty years on: the impact of fragments on drug discovery. Nat Rev. Drug Discov. 15, 605–619 (2016). g 3. Erlanson, D. A. Introduction to fragment-based drug discovery. Top. Curr. Chem. 317, 1–32 (2012). 4. Congreve, M., Chessari, G., Tisi, D. & Woodhead, A. J. Recent developments in fragment-based drug discovery. J. Med. Chem. 51 3661–3680 (2008). 5. Leach, A. R. & Hann, M. M. Molecular complexity and fragment-based drug discovery: ten years on. Curr. Opin. Chem. Biol. 15 489–496 (2011).hfi 6. Hopkins, A. L., Keseru, G. M., Leeson, P. D., Rees, D. C. & Reynolds, C. H. The role of ligand efficiency metrics in drug discovery Nat. Rev. Drug Discov. 13, 105–121 (2014). g Murray, C. W. & Blundell, T. L. Structural biology in fragment-base 8. Ferenczy, G. G. & Keseru, G. M. Enthalpic efficiency of ligand binding. J. Chem. Inf. Model. 50, 1536–1541 (2010). fi 9. Kitchen, D. B., Decornez, H., Furr, J. R. & Bajorath, J. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat. Rev. Drug Discov. 3, 935–949 (2004). pp g 0. Steinbrecher, T. B. et al. Accurate binding free energy predictions in fragment optimization. J. Chem. Inf. Model. 55, 2411–2420 (2015) d l f d k b l d l d d f l 10. Steinbrecher, T. B. et al. Accurate binding free energy pre 10. Steinbrecher, T. B. et al. Accurate binding free energy predictions in fragment optimization. J. Chem. Inf. Model. 55, 2411–2420 (2015). 11. Sándor, M., Kiss, R. & Keserű, G. M. Virtual fragment docking by Glide: a validation study on 190 protein−fragment complexes. J. Chem. Inf. Model. 50, 1165–1172 (2010). 1. Sándor, M., Kiss, R. & Keserű, G. M. Virtual fragment docking by Glide: a validation study on 190 protein−fragment complexes. J Chem. Inf. Model. 50, 1165–1172 (2010). f 12. Verdonk, M. L. et al. Docking performance of fragments and druglike compounds. J. Med. Chem. 54, 5422–5431 (2011). 13. Kollman, P. Free-energy calculations - Applications to chemical and biochemical phenomena. Chem. Rev. 93, 2395–2417 (1993) 4. Chen, J. F., Eltzschig, H. K. & Fredholm, B. B. Adenosine receptors as drug targets–what are the challenges? Nat. Rev. References Drug Discov 12, 265–286 (2013). 5. Armentero, M. T. et al. Past, present and future of A2A adenosine receptor antagonists in the therapy of Parkinson’s disease Pharmacol. Therapeut. 132, 280–299 (2011).i h p ( ) 16. Hatfield, S. M. & Sitkovsky, M. A2A adenosine receptor antagonists to weaken the hypoxia-HIF-1alpha driven immunosuppression and improve immunotherapies of cancer. Curr. Opin. Pharmacol. 29, 90–96 (2016). p 17. Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012). g y 18. Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474, 521–525 (2011).i 9. Chen, D. et al. Fragment screening of GPCRs using biophysical methods: identification of ligands of the adenosine A2A receptor with novel biological activity. ACS Chem. Biol. 7, 2064–2073 (2012). g y 20. Congreve, M. et al. Fragment screening of stabilized G-protein-coupled receptors using biophysical methods. Methods Enzymol. 493, 115–136 (2011). 21. Chen, D., Ranganathan, A., IJzerman, A. P., Siegal, G. & Carlsson, J. Complementarity between in silico and biophysical screening approaches in fragment-based lead discovery against the A2A adenosine receptor. J. Chem. Inf. Model. 53, 2701–2714 (2013). 22. Lambertucci, C. et al. 8-Bromo-9-alkyl adenine derivatives as tools for developing new adenosine A2A and A2B receptors lig Bioorg. Med. Chem. 17, 2812–2822 (2009). g 3. Volpini, R. et al. Adenosine A2A receptor antagonists: New 8-substituted 9-ethyladenines as tools for in vivo rat models of Parkinson’ disease. ChemMedChem 4, 1010–1019 (2009).f 24. Dore, A. S. et al. Structure of the adenosine A2A receptor in complex with ZM241385 and the xanthines XAC and caffeine. Structure 19, 1283–1293 (2011). 5. Halgren, T. A. et al. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med. Chem. 47, 1750–1759 (2004). 26. Nervall, M., Hanspers, P., Carlsson, J., Boukharta, L. & Åqvist, J. Predicting binding modes from free energy calculations. J. Med. Chem. 51, 2657–2667 (2008). 27. Klebe, G. Applying thermodynamic profiling in lead finding and optimization. Nat. Rev. Drug Discov. 14, 95–110 (2015). l Å b l h d f lk l l f l l d l h 27. Klebe, G. Applying thermodynamic profiling in lead finding an Å ii 28. Carlsson, J. & Åqvist, J. Absolute hydration entropies of alkali metal ions from molecular dynamics simulations. J. Phys. Chem. B 113, 10255–10260 (2009). 29. References Young, T., Abel, R., Kim, B., Berne, B. J. & Friesner, R. A. Motifs for molecular recognition exploiting hydrophobic enclos protein-ligand binding. P. Natl. Acad. Sci. USA 104, 808–813 (2007).l g g 30. Hauser, R. A. et al. Tozadenant (SYN115) in patients with Parkinson’s disease who have motor fluctuations on levodopa: a ph double-blind, randomised trial. Lancet Neurol. 13, 767–776 (2014). ( ) 31. Warren, G. L. et al. A critical assessment of docking programs and scoring functions. J. Med. Chem. 49, 5912–5931 (2006). 31. Warren, G. L. et al. A critical assessment of docking programs and scoring functions. J. Med. Chem. 49, 5912–5931 (2006). 32 Hi C B i T & Sh W H d ti it th d i l i SAR f t i l l i l bi di t th 31. Warren, G. L. et al. A critical assessment of docking programs and scoring functions. J. Med. Chem. 49, 5912–5931 (2006). 32 Higgs, C , Beuming, T & Sherman, W Hydration site thermodynamics explain SARs for triazolylpurines analogues binding to th 32. Higgs, C., Beuming, T. & Sherman, W. Hydration site thermodynamics explain SARs for triazolylpurines analogues bindin A2A Receptor. ACS Med. Chem. Lett. 1, 160–164 (2010). 2A p 33. Lenselink, E. B., Beuming, T., Sherman, W., van Vlijmen, H. W. & IJzerman, A. P. Selecting an optimal number of binding site to improve virtual screening enrichments against the adenosine A2A receptor. J. Chem. Inf. Model. 54, 1737–1746 (2014). Å p g g vist, J., Kazemi, M., Isaksen, G. V. & Brandsdal, B. O. Entropy and e d ll b q py y y 35. Andrews, S. P., Mason, J. S., Hurrell, E. & Congreve, M. Structure-based drug design of chromone antagonists of the adenosi receptor. MedChemComm 5, 571–575 (2014).t p 6. Mason, J. S. et al. High end GPCR design: crafted ligand design and druggability analysis using protein structure, lipophilic hotspot and explicit water networks. In Silico Pharmacol. 1, 1–12 (2013).i p ( ) 37. Jorgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. Development and testing of the OPLS all-atom force field on conformat i d i f i li id J A Ch S 118 11225 11236 (1996) p 37. Jorgensen, W. L., Maxwell, D. S. & Tirado-Rives, J. Development and testing of the OPLS all-atom force field on conformational energetics and properties of organic liquids. J. Am. Chem. Soc. Author Contributions P.M., A.R., K.A.J., and J.C. designed the research project. P.M., A.R., A.E., M.J., A.Ru., and J.C. performed and analyzed the MD simulations. C.L., G.M., D.D.B., E.W., Z.G., and K.A.J. carried out and analyzed the radioligand binding and functional assays. All authors contributed to the writing of the manuscript. P.M., A.R., K.A.J., and J.C. designed the research project. P.M., A.R., A.E., M.J., A.Ru., and J.C. performed and analyzed the MD simulations. C.L., G.M., D.D.B., E.W., Z.G., and K.A.J. carried out and analyzed the radioligand binding and functional assays. All authors contributed to the writing of the manuscript. www.nature.com/scientificreports/ Chem Phys. 72, 5639–5648 (1980).fif y 59. Clark, T., Chandrasekhar, J., Spitznagel, G. W. & Schleyer, P. V. Efficient diffuse function-augmented basis sets for anion calculations. Iii. The 3-21 + G basis set for first-row elements, Li-F. J. Comput. Chem. 4, 294–301 (1983). hi 60. Frisch, M. J., Pople, J. A. & Binkley, J. S. Self-consistent molecular-orbital methods. 25. Supplementary functions for gaussian- sets. J. Chem. Phys. 80, 3265–3269 (1984). y 61. Carlsson, J. et al. Structure-based discovery of A2A adenosine receptor ligands. J. Med. Chem. 53, 3748–3755 (2010). y 61. Carlsson, J. et al. Structure-based discovery of A2A adenosine receptor ligands. J. Med. Chem. 53, 3748–3755 (2010). y 61. Carlsson, J. et al. Structure-based discovery of A2A adenosine receptor ligands. J. Med. Chem. 53, 3748–3755 (2010). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 46. Christopher, J. A. et al. Biophysical fragment screening of the beta1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design. J. Med. Chem. 56, 3446–3455 (2013). 47. Pronk, S. et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatic 845–854 (2013). ( ) 48. Rodríguez, D., Bello, X. & Gutiérrez-de-Terán, H. Molecular modelling of G protein-coupled receptors through the web. Mol. Inform. 31, 334–341 (2012).i f ( ) 9. Mahoney, M. W. & Jorgensen, W. L. A five-site model for liquid water and the reproduction of the density anomaly by rigid nonpolarizable potential functions. J. Chem. Phys. 112, 8910–8922 (2000).l p p y ( ) 50. Berger, O., Edholm, O. & Jähnig, F. Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophys. J. 72, 2002–2013 (1997). hydration, constant pressure, and constant temperature. Biophys. J. Å y p p p y 51. Marelius, J., Kolmodin, K., Feierberg, I. & Åqvist, J. Q. A molecular dynamics program for free energy calculations and empirical valence bond simulations in biomolecular systems. J. Mol. Graph. Model. 16, 213–225 (1998). y p 2. Ryckaert, J. P., Ciccotti, G. & Berendsen, H. J. C. Numerical-integration of cartesian equations of motion of a system with constraints Molecular-dynamics of N-alkanes. J. Comput. Phys. 23, 327–341 (1977).f 53. King, G. & Warshel, A. A surface constrained all-atom solvent model for effective simulations of polar solutions. J. Chem. Phys. 91, 3647–3661 (1989).i ( ) 54. Lee, F. S. & Warshel, A. A local reaction field method for fast evaluation of long-range electrostatic interactions in molecular simulations. J. Chem. Phys. 97, 3100–3107 (1992). y ( ) 5. Beutler, T. C., Mark, A. E., Vanschaik, R. C. & Gerber, P. R. & Vangunsteren, W. F. Avoiding singularities and numerical instabilitie in free-energy calculations based on molecular simulations. Chem. Phys. Lett. 222, 529–539 (1994). gy y ( ) 56. Gaussian 09, Frisch, M. J. et al., Gaussian, Inc., Wallingford CT, 2016. . Gaussian 09, Frisch, M. J. et al., Gaussian, Inc., Wallingford CT, 2 g 7. Raghavachari, K., Binkley, J. S., Seeger, R. & Pople, J. A. Self-consistent molecular-orbital methods. 20. Basis set for correlated wave functions. J. Chem. Phys. 72, 650–654 (1980). 8. Mclean, A. D. & Chandler, G. S. Contracted gaussian-basis sets for molecular calculations. 1. 2nd Row Atoms, Z = 11–18. J. Acknowledgementsh g This work was supported by grants from the Swedish Research Council (2013-5708) and the Science for Life Laboratory to J.C. and by funding from the NIDDK Intramural Research Program to K.A.J. Computational resources were provided by the Swedish National Infrastructure for Computing (SNIC). J.C., P.M., A.R., A.Ru., and M.J. participate in the European COST Action CM1207 (GLISTEN). References 118, 11225–11236 (1996). nd properties of organic liquids. J. Am. Chem. Soc. 118, 11225–112 g g 8. Lenselink, E. B. et al. Predicting binding affinities for GPCR ligands using free-energy perturbation. ACS Omega 1, 293–304 (2016) l d l bl d f l l d b d d d b f d 38. Lenselink, E. B. et al. Predicting binding affinities for GPCR ligands using free-energy perturbation 38. Lenselink, E. B. et al. Predicting binding affinities for GPCR ligands using free energy perturbation. ACS Omega 1, 293 304 (2016). 39. Wang, L. et al. Accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field. J. Am. Chem. Soc. 137, 2695–2703 (2015).ih gy pi 0. Harder, E. et al. OPLS3: A force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput 12, 281–296 (2016).i 1. Clark, A. J. et al. Prediction of protein-ligand binding poses via a combination of induced fit docking and metadynamics simulations J. Chem. Theory Comput. 12, 2990–2998 (2016). h y p 42. Boukharta, L., Gutierrez-de-Teran, H. & Åqvist, J. Computational prediction of alanine scanning and ligand binding energetics in G-protein coupled receptors. PLoS Comput. Biol. 10, e100358 (2014). Åf 43. Keranen, H., Gutierrez-de-Teran, H. & Åqvist, J. Structural and energetic effects of A2A adenosine receptor mutations on agonist and antagonist binding. PloS One 9, e108492 (2014). g g 4. Katritch, V., Cherezov, V. & Stevens, R. C. Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol Toxicol. 53, 531–556 (2013). 45. Rodriguez, D., Ranganathan, A. & Carlsson, J. Discovery of GPCR ligands by molecular docking screening: Novel opportunities provided by crystal structures. Curr. Top. Med. Chem. 15, 2484–2503 (2015). Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 11 Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-04905-0 Supplementary information accompanies this paper at doi:10.1038/s41598-017-04905-0 Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Reports \| 7: 6398 \| DOI:10.1038/s41598-017-04905-0 12
https://openalex.org/W3045536489	https://repub.eur.nl/pub/129358/Repub_129358_O-A.pdf	English	null	Prevalence and abundance of selected genes conferring macrolide resistance genes in COPD patients during maintenance treatment with azithromycin	Antimicrobial resistance and infection control	2,020	cc-by	6,115	RESEARCH Open Access Prevalence and abundance of selected genes conferring macrolide resistance genes in COPD patients during maintenance treatment with azithromycin Remco S. Djamin1†, Sander Talman1† , Eefje J. A. Schrauwen2,3†, Christian J. H. von Wintersdorff4, Petra F. Wolffs4, Paul H. M. Savelkoul4, Sevim Uzun1, René Kerstens5, Menno M. van der Eerden6 and Jan A. J. W. Kluytmans2,7 Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 https://doi.org/10.1186/s13756-020-00783-w Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 https://doi.org/10.1186/s13756-020-00783-w Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 https://doi.org/10.1186/s13756-020-00783-w Abstract Objectives: Maintenance treatment with macrolide antibiotics has shown to be effective in reducing exacerbations in COPD patients. A major concern with prolonged treatment with antibiotics is the development of bacterial resistance. In this study we determined the effect of azithromycin on the development and acquisition of resistance to macrolides in the nasopharyngeal flora in COPD patients. Methods: This study was part of the COLUMBUS trial, a randomised, double-blind, placebo-controlled trial to measure the effect of maintenance treatment with azithromycin in 92 COPD patients on the exacerbation rates during a 12-month period. In order to determine resistance to macrolides, we used a targeted metagenomic approach to measure the presence and relative abundance of specific macrolide resistance genes ermB, ermF and mefA in throat samples collected at different time-points during this 12-month period. Results: There was no increased risk for acquisition of macrolide resistance genes in the azithromycin group compared to the placebo group in COPD patients. However, loss of the macrolide resistance gene ermB was increased overtime in the placebo treated group compared to the azithromycin group (n = 5 for the placebo group versus n = 0 for the azithromycin group at 12 months; p = 0.012). The change in relative abundance of the three macrolide-resistance genes showed that all but one (ermF) increased during treatment with azithromycin. Conclusions: The acquisition rate of macrolide resistance genes in COPD patients treated with azithromycin maintenance therapy was limited, but the relative abundance of macrolide resistance genes increased significantly over time compared to placebo. p p This study was part of the COLUMBUS trial (Clinicaltrials.gov, NCT00985244). Study design and participants This study was part of the COLUMBUS trial (Clinical- trials.gov, NCT00985244), a randomised, double blind, placebo-controlled trial to measure the effect of main- tenance treatment with azithromycin in COPD patients on the exacerbation rates during a 12-month period. The study protocol and primary results have been pub- lished earlier [9, 17]. Adult patients (≥18 years) with a diagnosis of COPD who had received treatment for three or more exacerbations in the previous year were ran- domly assigned to receive 500 mg azithromycin or pla- cebo three times a week for 12 months (total of 92 patients). © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 8 Page 2 of 8 Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Sample collection During the treatment period, throat samples (e-swabs) were collected at baseline, 6 months and 12 months, as well as during each exacerbation that required admission to the hospital. E-swabs were stored at −80 °C until mo- lecular analysis was performed. Bacteria may acquire resistance by antibiotic efflux or poor drug penetration, resulting in reduced concentra- tions of the intracellular antibiotics. Antibiotics render ineffective due to drug target site modification, due to genetic mutation of the target, posttranslational target modification or antibiotics are inactivated by modifica- tion or hydrolysis [17–20]. Introduction [24]. In addition, the mefA gene is responsible for a macrolide efflux pump system [25, 26]. Some of these genes are known to persist on mobile genetic elements, which easily facilitate the spread of these resistance genes. Chronic obstructive pulmonary disease (COPD) is an important cause of morbidity and mortality [1]. Exacer- bations in COPD patients impose a large burden on health care costs and are important events in disease progression [2, 3]. In the present study the randomized control trial by Uzun et al. was futher explored in order to determine the effect of azithromycin maintenance therapy on the dynamics of macrolide resistance genes in the pharyngeal microbiota of COPD patients [9]. We used a targeted (PCR-based) metagenomic approach to deter- mine the presence and relative abundance of specific macrolide resistance genes; ermB, ermF and mefA. COPD exacerbations are mainly caused by bacterial and viral infections, leading to airway inflammation [4, 5]. Macrolides have antimicrobial, anti-inflammatory and anti-viral effects, which make them potentially useful in reducing COPD exacerbations [6]. Hence, maintenance treatment with macrolide antibiotics has shown to be ef- fective in reducing exacerbations in COPD patients [7–9]. fective in reducing exacerbations in COPD patients [7 9]. A major concern with prolonged treatment with antibi- otics is the development of bacterial resistance [10–12]. Seven major classes of antibiotics have been described, β- lactams and glycopeptides (inhibit cell wall synthesis); macrolides, aminoglyclosides and tetracyclines (protein synthesis); daptomycin (cell membrane function); platen- symycin (fatty acid biosynthesis). Bacteria use two mecha- nisms to achieve resistance: the first is intrinsic resistance and the second is acquired resistance [13]. Intrinsic resist- ance is the ability to resist the action of specific antibiotics due to inherent structural or functional properties. Pseudomonas aeruginosa, for example is resistant for cer- tain classes of antibiotics due to the absence of susceptible target sites for particular antibiotics [14]. Furthermore, Pseudomonas aeruginosa, S. aureus and e.coli possesses several genes, associated with intrinsic resistance to sev- eral classes of antibiotics like B lactams, aminoglyclosides and fluoroqinolones [15, 16]. Molecular methods The extraction of DNA was performed from the collected e-swabs™(COPAN BV), using the EasyMAG (Biomér- ieux). Real-time PCR was performed to detect and quan- tify genes responsible for resistance to macrolides; ermB, ermF and mefA. These three specific genes were chosen since these are the most common mobile antibiotic resist- ance genes that confer macrolide resistance. Amplification of ermB was performed as described earlier [27]. Primers to target mefA and the forward primer for ermF were adapted from earlier described studies [28, 29]. A reverse primer for ermF was designed by performing an nBLAST in GenBank for the ermF gene sequence (NG_047826.1) and aligning all resulting sequences with > 75% query coverage (identity: 94–100%) using MAFFT (http://mafft. cbrc.jp/alignment/software/), after which a primer hom- ologous to all sequences was chosen. Gyrase and topoisomerase IV are the two type II topoi- somerases utilized in bacteria. Inhibition of those topoisome- rases by quinolone based antibiotics prevents uncoiling of DNA strands, thereby preventing replication of bacteria. Ac- quired resistances against quinolone antibiotics is achieved by mutations in the quinolone binding site of gyrase and topoisomerase IV (gyrA or parC gene) [21], emerging the need of non-quinolone based chemical compounds [12]. The use of macrolides has been associated with the devel- opment of macrolide resistance in oral commensal strepto- coccal microbiota [22]. However, the effect of maintenance treatment with macrolides on resistance in patients with COPD has given controversial results [7–9, 23]. Macrolide resistance can be caused by several mecha- nisms. Target modification is mediated by one or more rRNA erm methylases, which change a site in 23S rRNA Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Page 3 of 8 Page 3 of 8 Page 3 of 8 The 16S ribosomal DNA was amplified as a reference gene to normalize for the amount of bacterial DNA in the samples, using previously described primers [30]. All targets were amplified by using a MyiQ Single-Color Real-Time PCR Detection System (BioRad, Hercules, CA, USA) in 25-μL reactions containing 12.5 μL iQ SYBR Green Supermix (BioRad), 300 nM of both the re- spective targets forward and reverse primer and 5-μL template DNA. Primer sequences, amplicon sizes and PCR cycling conditions are displayed in Table 1. Statistical methods The prevalence of macrolide genes between the treatment groups was compared using a χ2 test. In addition, acquisi- tion and loss of different resistance genes between differ- ent treatment groups were compared using a χ2 test. Study population The COLUMBUS trial was a single centre study that took place at the Amphia Hospital (Breda, the Netherlands) between May 19, 2010 and June 18, 2013. The placebo group consisted of 47 patients and the azithromycin group of 45 patients. The baseline charac- teristics of these 92 patients are described in Table 2. The COLUMBUS trial was a single centre study that took place at the Amphia Hospital (Breda, the Netherlands) between May 19, 2010 and June 18, 2013. The placebo group consisted of 47 patients and the azithromycin group of 45 patients. The baseline charac- teristics of these 92 patients are described in Table 2. Molecular methods For all antibiotic resistance gene targets, specificity of the assay was investigated by melting curve analysis of all samples and amplicon sequencing of 10 random positive samples using the PCR primers and an ABI BigDye Terminator v1.1 Cycle Sequencing Kit. Sequencing data were ob- tained on an ABI 3730 DNA Analyzer (Applied Biosys- tems, Foster City, CA, USA). All used PCR assays were specifically designed or evaluated for use in metage- nomics analyses. During evaluation of the assay, results were confirmed using sequencing. During current ana- lysis each positive signal was manually inspected to be a specific amplification signal by comparing the melting curve to that of the positive control. Samples with a non-identical melting curve were not considered as posi- tive. Efficiencies of the assays were determined to be 103.1% (16S rDNA), 99.7% (ermF) and 105.1% (mefA). described earlier [31]. This method is standard to meas- ure the relative change in mRNA expression levels by using real-time PCR. Here, we measure the relative amount of target DNA present rather than measuring mRNA expression. The 16S rDNA was used as the refer- ence gene. In order to perform paired-analysis, multiple throat samples from one patient have to be available in which the presence of the gene of interest was detected. If the gene of interest was not present, this sample was excluded from the paired sample analysis. Ratio’s log- transformed, in order to create a more homogenous population, were compared between treatment arms using the Wilcoxon rank sum test. In addition, descrip- tive statistics (n, mean, median, SD) and graphical pre- sentations were provided for both time points. Changes from baseline in relative resistance gene abundances (ratio) were evaluated between samples of month 6 (and month 12) and samples of baseline using the same ΔΔCT method with a Pfaffl modification to correct for PCR efficiency as described earlier (1). Prevalence of macrolide resistance genes present in pharyngeal microbiota A comparison of the resistance gene abundances be- tween treatment groups was performed based on the samples of both month 6 and month 12. These compari- sons were based on the change from baseline, relative to the amount of 16S DNA present, using real-time PCR. These ratios – or fold changes – were calculated for ermB, ermF and mefA using the ΔΔCT method with a Pfaffl modification to correct for PCR efficiency as At baseline throat samples were taken in 36 (n = 36/47; 77%) patients in the placebo group and in 44 (n = 44/45; 98%) patients in the azithromycin group. At month 6 and 12 the available samples were, 30 and 27 in placebo group and 34 and 32 in azithromycin group, respectively. The 11 patients in the placebo group and one patient in azithromycin group without a throat sample granted no permission to take an extra throat swab. Table 1 PCR conditions and primer sequences Primer Sequence 5′ - 3’ Amplicon size (bp) Cycling conditions 16SrDNA_F CCTACGGGNGGCWGCAG 465 1 × 95 °C, 3’ 16SrDNA_R GACTACHVGGGTATCTAATCC 35 × 95 °C, 15″; 55 °C, 20″; 72 °C, 30″ ermB_F AAGGGCATTTAACGACGAAACTG 438 1 × 95 °C 3’ ermB_R ATTTATCTGGAACATCTGTGGTATG 40 × 95 °C 15″, 60 °C 20″, 72 °C 30″ ermF_F CGACACAGCTTTGGTTGAAC 120 1 × 95 °C 3′ ermF_R TTTGACACCACTTTGAAAGGAAA 40 × 95 °C 15″, 58 °C 20″, 72 °C 30″ mefA CCTGCAAATGGCGATTATTT 199 1 × 95 °C 3′ mefA AATAGCAAGCACTGCACCAG 40 × 95 °C 15″, 58 °C 20″, 72 °C 30″ Table 1 PCR conditions and primer sequences Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Page 4 of 8 Table 2 Baseline characteristics Azithromycin group (n = 47) Placebo group (n = 45) Male 22 (46·8%) 18 (40%) Age (years) 64·7 (10·2) 64·9 (10·2) Current smoker 20 (43%) 9 (20%) AECOPD in past year 4·0 (1·2) 4·0 (1·1) Hospitalisation due to AECOPD 1·0 (1·1) 0·7 (0·8) Spirometry after bronchodilation FEV1 (L) 1·1 (0·47) 1·1 (0·43) FEV1 (% of predicted) 44·2 (19·3) 45·0 (19·5) FVC (L) 2·9 (0·8) 2·7 (0·92) FVC (% of predicted) 92·5 (22·2) 88·9 (20·3) FEV1/FVC (%) 38·0 (11·7) 40·3 (12·4) GOLD stages I 2 (4·3%) 3 (6·7%) II 14 (29·8%) 12 (26·7%) III 18 (38·3%) 20 (44·4%) IV 13 (27·7%) 10 (22·2%) Data are in n (%) or mean (SD), unless otherwise stated. AECOPD = acute exacerbations of COPD. Prevalence of macrolide resistance genes present in pharyngeal microbiota FEV1 Forced expiratory volume in 1 s. FVC Forced vital capacity. GOLD Global Initiative for Chronic Obstructive Pulmonary Disease Regarding the azithromycin group, the prevalence of the ermF and ermB genes at 6 months was 67.7% (n = 23/34) and 97.1% (n = 33/34) versus 68.8% (n = 22/32) and 100% (n = 32/32) at 12 months (p = n.s.). Compari- son of the ermF prevalence between the placebo and azi- thromycin groups showed no significant differences at 6 and 12 months (p = 0.05 and p = 0.109). The difference in prevalence of ermB increased significantly over time in the azithromycin group compared to the placebo treated group (p = 0.029 6 months, p = 0.002; 12 months). Of the 80 patients with a baseline sample taken, 58 (72%) also had a sample at ‘month 6’ and 53 (66%) at ‘month 12’. A total of 43 patients (54%) had samples at both ‘month 6’ and ‘month 12’. This percentage was comparable between treatment arms: 20 (56%) in pla- cebo and 23 (52%) in azithromycin. The macrolide resistance gene mefA was present in all available throat samples at all time points. Before treatment, prevalence of the macrolide resistance genes ermF and ermB were respectively 44.4% (n = 16/36) and 86.1% (n = 31/36) in the placebo group (n = 36), and respectively 59.1% (n = 26/44) and 97.7% (n = 43/44) in the azithromycin group (n = 44) (p = 0.261 ermF, p = 0.085 ermB) (Table 3). Loss and acquisition of macrolide-resistance in Loss and acquisition of macrolide-resistance in pharyngeal microbiota during and after treatment with placebo or azithromycin After 6 and 12 months of placebo treatment, the ermF and ermB genes were detected in 43.3% (n = 13/30), 80% (n = 24/30) at 6 months, and 48.1% (n = 13/27) and 74.1% (20/27) at 12 months of the throat samples tested, corres- pondingly, with no statistical differences regarding the pres- ence of resistance genes between the treatment groups. In the placebo group, 27 patients had throat swabs avail- able from visits at baseline and 6 months while 26-paired samples were available from baseline and 12 months. For the azithromycin group, there were 34 paired samples (from baseline and 6 months) and 30 pairs (from base- line and 12 months). Table 3 Prevalence of ermF and ermB macrolide resistance genes over time ermF % (pos/all samples) ermB % (pos/all samples) Prevalence Placebo Azithromycin P value Placebo Azithromycin P value Baseline 44,4 (16/36) 59,1 (26/44) 0.261 86,1 (31/36) 97,7 (43/44) 0.085 M6 43,3 (13/30) 67,6 (23/34) 0.050 80,0 (24/30) 97,1 (33/34) 0.029 M12 48,1 (13/27) 68,8 (22/32) 0.109 74,1 (20/27) 100,0 (32/32) 0.002* Prevalence of ermB is statistically significant in the Azithromycin group at M6 and M12 compared to the Placebo group (Chi-square, Pearson corrected) Table 3 Prevalence of ermF and ermB macrolide resistance genes over time Page 5 of 8 Page 5 of 8 Page 5 of 8 Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 The loss and acquisition of macrolide resistance genes (mefA, ermF and ermB) in pharyngeal microbiota before and after treatment of the paired samples is shown in Table 3. During the trial, no differences were detected in the pres- ence of the mefA gene in the pharyngeal microbiota. were detected in the acquisition rates. However, the high prevalence of all resistance genes at baseline, with mefA being present in 100% of cases should be taken into con- sideration. Looking at the relative abundance of the macrolide-resistance genes over-time, a statistical increase of all tested genes in the azithromycin group compared to the placebo group was observed. For the patients without the macrolide genes ermF and ermB present in their pharyngeal microbiota at baseline (nermF = 15 and nermB = 4 in placebo, nermF = 16 and nermB = 1 in azithromycin), no statistical differences were observed in the acquisition rates between the pla- cebo and azithromycin treated groups. Loss and acquisition of macrolide-resistance in pharyngeal microbiota during and after treatment with placebo or azithromycin Long-term treatment with macrolides might influence the microbiological profile and antibiotic resistance in air- ways. The acquisition of respiratory pathogens and macro- lide resistant microorganisms as a result of maintenance treatment with macrolides in COPD patients has been ad- dressed in three studies [7–9]. It is important to note that these studies did not have the ability to measure quantita- tive differences over-time. Seemungal and colleagues found no difference in colonization rates with macrolide- resistant organisms between the macrolide and placebo group during1 year of treatment [7]. In contrast with these findings, earlier analysis of our COLUMBUS study found fewer patients in the azithromycin group with macrolide- resistant bacteria in sputum samples compared to those in the placebo group [9]. Albert et al. however, observed an increase in the incidence of colonization with macrolide- resistant organisms in the azithromycin group compared to the placebo group [8]. In summary, it can be stated that there is conflicting evidence about the influence of main- tenance treatment with macrolides on the acquisition of macrolide resistant respiratory pathogens in COPD pa- tients. In the current study, only a small difference in ac- quisition rate of macrolide resistance genes between patients treated with azithromycin or placebo could be demonstrated, nevertheless, a statistical increase in the relative abundance of the tested genes was found. This lat- ter finding suggests that maintenance therapy with azi- thromycin does influence the presence of macrolide resistance genes, which indicates towards changes in microbiological profile. However, from the patients with the macrolide genes ermF and ermB present (nermF = 12 and nermB = 23in pla- cebo, nermF = 18 and nermB = 33 in azithromycin) none of the patients treated with azithromycin lost the ermF and ermB gene over time, while for the placebo group, 1 and 3 patients lost the ermF and ermB gene after 6 months, respectively. Moreover, in 5 patients in the placebo group, the ermB gene was lost after 12 months, there- fore, the number of patients that lost the gene was statis- tically significant higher in the placebo group compared to the azithromycin group (p = 0.012). Relative gene abundances of the macrolide resistant genes during and after treatment with placebo or azithromycin A large part of the patients in both groups already had de- tectable levels of macrolide genes at baseline. This enabled us to compare the relative abundance of the genes in throat samples to determine the effect of the treatment on the abundance of these genes. Figure 1 depicts the overall abundance change of a resistance gene (log-transformed). The relative gene abundance of mefA after 6 months of treatment was substantially higher in the azithromycin ver- sus placebo group −0.39, n = 26; p = 0.002) (Fig. 1, Table 4). Determining the overall increase or decrease of the abun- dance of the ermF gene showed that this gene increased over time after treatment with azithromycin (0.86, n = 18 M6 and 1.04, n = 17 M12) compared to the placebo group (−0.14, n = 11 M6 and 0.15, n = 12 M12) as well, which was only significant at 12 months p = 0.0124 (Fig. 1, Table 4). With regard to the macrolide gene ermB, the relative gene abundance was significantly increased over time in the azi- thromycin group (0.69, n = 33 M6 and 0.89, n = 30 M12) compared to the placebo group (−0.32, n = 20 M6 and − 0.42, n = 18 M12) after 6 and 12 months of treatment (p = 0.01 and p = 0.001, respectively) (Fig. 1, Table 4). To our knowledge, this is the first randomised con- trolled double blind study in a COPD population, in which the effect of long-term treatment with macrolides on the acquisition and relative abundance of macrolide resistance genes using a targeted metagenomic approach has been evaluated. However, this study has some limitations. Un- fortunately, throat samples were not obtained from all pa- tients at regular visits. Furthermore, throat samples were not cultured in order to assess the changes in the micro- biological profile and resistance patterns. One additional option would be to assess the microbiota based on the 16SrDNA amplified in the samples. Finally, in this study we focused on three genes, which are involved in macro- lide resistance. It is known that more genes and targeted mutations are involved in this process [32–34]. Discussion During the study, only for the ermB gene, a significant dif- ference in prevalence between the azithromycin group and the placebo group was measured over time which was attributed to a loss of this resistance gene within the pla- cebo group. For the ermF and mefA gene, no differences The consequences of this study for daily practice are unclear. The clinical benefit of macrolide maintenance Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Page 6 of 8 therapy in COPD patients with frequent exacerbations has been demonstrated repeatedly [7 9] In the most re recommendation is accompanied by the advice that the possibility of developing resistant organisms should be Fig. 1 Relative changes in gene abundance at 6 and 12 months after treatment with placebo or azithromycin. Changes related to baseline gene abundance of mefA (a, b), ermF (c, d) and ermB (e, f) at 6 and 12 months are shown. Results are visualized in box-plots with median and 10th and 90th percentiles (dots show outliers). The dotted line shows the zero line. Mean logs ratio and statistics are shown in Table 4 Fig. 1 Relative changes in gene abundance at 6 and 12 months after treatment with placebo or azithromycin. Changes related to baseline gene abundance of mefA (a, b), ermF (c, d) and ermB (e, f) at 6 and 12 months are shown. Results are visualized in box-plots with median and 10th and 90th percentiles (dots show outliers). The dotted line shows the zero line. Mean logs ratio and statistics are shown in Table 4 therapy in COPD patients with frequent exacerbations has been demonstrated repeatedly [7–9]. In the most re- cent update of the GOLD guidelines it is recommended to consider the addition of a macrolide in COPD pa- tients treated with long-acting beta2 agonists/long-acting muscarinic antagonists/inhalation corticosteroids com- bination, who still have exacerbations [35]. This recommendation is accompanied by the advice that the possibility of developing resistant organisms should be taken into consideration in the decision making. As indicated, at the start of the study the prevalence of macrolide-resistance genes were already high in throat samples. This may be the result of historical exposure to (macrolide) antibiotics in this specific study population, Djamin et al. Received: 8 May 2020 Accepted: 14 July 2020 Received: 8 May 2020 Accepted: 14 July 2020 Funding Th 10. Serisier DJ. Risks of population antimicrobial resistance associated with chronic macrolide use for inflammatory airway diseases. Lancet Respir Med. 2013;1:262–74. https://doi.org/10.1016/S2213-2600(13)70038-9. 10. Serisier DJ. Risks of population antimicrobial resistance associated with chronic macrolide use for inflammatory airway diseases. Lancet Respir Med. 2013;1:262–74. https://doi.org/10.1016/S2213-2600(13)70038-9. This investigator-initiated study was funded by a trust called SoLong, which is associated with the department of respiratory medicine of the Amphia hospital in the Netherlands. The trust had no role in the design of the study, nor in analysis, interpretation of data or in writing the manuscript. 11. Li H, Liu DH, Chen LL, et al. Meta-Analysis of the adverse effects of long- term azithromycin use in patients with chronic lung diseases. Antimicrob Agents Chemother. 2014;58:511–7. https://doi.org/10.1128/AAC.02067-13. Discussion Antimicrobial Resistance and Infection Control (2020) 9:116 Page 7 of 8 Page 7 of 8 Page 7 of 8 Table 4 Comparison of mean logs ratios of the different macrolide–resistant genes before, during and after treatment Placebo Azithromycin Mean SD Mean SD P value** mefA M6 −0.22 (n = 27) 1.00 0.51 (n = 34) 0.47 0.0001 M12 −0.39 (n = 26) 1.21 0.33 (n = 30) 0.68 0.002 ermF M6 −0.14 (n = 11) 1.35 0.86 (n = 18) 0.99 0.0687 M12 0.15 (n = 12) 0.94 1.04 (n = 17) 0.80 0.0124 ermB M6 −0.32 (n = 20) 1.48 0.69 (n = 33) 0.93 0.0116 M12 −0.42 (n = 18) 1.33 0.89 (n = 30) 1.05 0.0013 mean of the log gene abundance ratio compared to baseline *p values for comparison of mean abundance at either 6 or 12 months and baseline, by Wilcoxon ranked sum test Table 4 Comparison of mean logs ratios of the different macrolide–resistant genes before, during and after treatment (NL29500.101.09). All patients agreed and signed an informed consent before entering this study. Consent for publication Not applicable. References 1. Suissa S, Dell’Aniello S, Ernst P. Long-term natural history of chronic obstructive pulmonary disease: Severe exacerbations and mortality. Thorax. 2012;67:957–63. https://doi.org/10.1136/thoraxjnl-2011-201518. 2. Miravitlles M, Murio C, Guerrero T, et al. Pharmacoeconomic evaluation of acute exacerbations of chronic bronchitis and COPD. Chest. 2002;121:1449– 55. https://doi.org/10.1378/chest.121.5.1449. In conclusion, this study showed that the acquisition rate of macrolide resistance genes in COPD patients treated with azithromycin maintenance therapy was lim- ited, but the relative abundance of macrolide resistance genes increased significantly over time compared to pla- cebo. The clinical implications of these findings are un- clear and at this time we consider the observed clinical benefits for this specific group of patients to outweigh the risks of antimicrobial resistance. It is recommended to monitor development of resistance carefully when treating patients for prolonged periods with antibiotics. 3. Donaldson GC, Seemungal TAR, Bhowmik A, et al. Relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease. Thorax. 2002;57:847–52. https://doi.org/10.1136/thorax. 57.10.847. 4. Rohde G, Borg I, Wiethege A, et al. Inflammatory response in acute viral exacerbations of COPD. Infection. 2008;36:427–33. https://doi.org/10.1007/ s15010-008-7327-5. 4. Rohde G, Borg I, Wiethege A, et al. Inflammatory response in acute viral exacerbations of COPD. Infection. 2008;36:427–33. https://doi.org/10.1007/ s15010-008-7327-5. 5. Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med. 2008;359:2355. https://doi.org/ 10.1056/NEJMra0800353. 6. Yamaya M, Azuma A, Takizawa H, et al. Macrolide effects on the prevention of COPD exacerbations. Eur Respir J. 2012;40:485–94. https://doi.org/10. 1183/09031936.00208011. 7. Seemungal TAR, Wilkinson TMA, Hurst JR, et al. Long-term Erythromycin Therapy Is Associated with Decreased Chronic Obstructive Pulmonary Disease Exacerbations. Am J Respir Crit Care Med. 2008;178:1139–47. https:// doi.org/10.1164/rccm.200801-145OC. Authors’ contributions RSD SU JAJWK d MME Authors’ contributions RSD, SU, JAJWK, and MME contributed to the study design. RSD, ST, EJAS, CJHW, PFW, PHMS, SU, RK, MME, JAJWK participated in data interpretation. RSD, ST, EJAS, CJHW, PFW, PHMS, SU, RK, MME, JAJWK edited the manuscript. RSD, ST, EJAS, JAJWK and MME wrote the manuscript. RK did the statistical analysis. CJHW, PFW, PHMS, EJAS, JAJWK were responsible for microbiologic analysis. RSD, SU contributed to sample collection. RSD, ST, JAJWK and MME did the literature search. SU, RSD enrolled the study participants. RSD, ST, EJAS contributed equally to this manuscript. The author(s) read and approved the final manuscript. 8. Albert RK, Connett J, Bailey WC, et al. Azithromycin for Prevention of Exacerbations of COPD. N Engl J Med. 2011;365:689–98. https://doi.org/10. 1056/NEJMoa1104623. 9. Uzun S, Djamin RS, Kluytmans JAJW, et al. Azithromycin maintenance treatment in patients with frequent exacerbations of chronic obstructive pulmonary disease (COLUMBUS): A randomised, double-blind, placebo- controlled trial. Lancet Respir Med. 2014;2:361–8. https://doi.org/10.1016/ S2213-2600(14)70019-0. Availability of data and materials 12. Badshah SL, Ullah A. New developments in non-quinolone-based antibiotics for the inhibiton of bacterial gyrase and topoisomerase IV. Eur J Med Chem. 2018;152:393–400. https://doi.org/10.1016/j.ejmech.2018.04.059. The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request. The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request. 13. Lynch JP, Clark NM, Zhanel GG. Evolution of antimicrobial resistance among Enterobacteriaceae (focus on extended spectrum β-lactamases and carbapenemases). Expert Opin Pharmacother. 2013;14:199–210. https://doi. org/10.1517/14656566.2013.763030. 13. Lynch JP, Clark NM, Zhanel GG. Evolution of antimicrobial resistance among Enterobacteriaceae (focus on extended spectrum β-lactamases and carbapenemases). Expert Opin Pharmacother. 2013;14:199–210. https://doi. org/10.1517/14656566.2013.763030. Ethics approval and consent to participate Authors’ contributions RSD, SU, JAJWK, and MME contributed to the study design. RSD, ST, EJAS, CJHW, PFW, PHMS, SU, RK, MME, JAJWK participated in data interpretation. RSD, ST, EJAS, CJHW, PFW, PHMS, SU, RK, MME, JAJWK edited the manuscript. RSD, ST, EJAS, JAJWK and MME wrote the manuscript. RK did the statistical analysis. CJHW, PFW, PHMS, EJAS, JAJWK were responsible for microbiologic analysis. RSD, SU contributed to sample collection. RSD, ST, JAJWK and MME did the literature search. SU, RSD enrolled the study participants. RSD, ST, EJAS contributed equally to this manuscript. The author(s) read and approved the final manuscript. Author details 1 Author details 1Department t of Respiratory Medicine, Amphia Hospital, Molengracht 21, 4818 CK Breda, The Netherlands. 2Laboratory for Microbiology and Infection Control, Amphia Hospital, Breda, The Netherlands. 3Academy for Technology and Environmental Health, Avans University of Applied Sciences, Breda, the Netherlands. 4Department of Medical Microbiology, Maastricht University Medical Center+, Maastricht, The Netherlands. 5Orion Statistical Consulting BV, Hilvarenbeek, The Netherlands. 6Department of Respiratory Medicine, Erasmus Medical Center, Rotterdam, The Netherlands. 7Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands. since only COPD patients with a minimum of three exacerbations in the previous year, have been in- cluded in this study. This could be an argument to consider macrolide maintenance treatment only in this specific category of COPD patients. However, this high prevalence has also been observed in a healthy travel population, as shown in the study of von Win- tersdorff et al., with an ermB gene presence in 99.2% in fecal samples [27]. since only COPD patients with a minimum of three exacerbations in the previous year, have been in- cluded in this study. This could be an argument to consider macrolide maintenance treatment only in this specific category of COPD patients. However, this high prevalence has also been observed in a healthy travel population, as shown in the study of von Win- tersdorff et al., with an ermB gene presence in 99.2% in fecal samples [27]. Ethics approval and consent to participate The independent Ethics Committee “Toetsingscommissie Wetenschappelijk Onderzoek Rotterdam” (TWOR) granted approval for his study The independent Ethics Committee “Toetsingscommissie Wetenschappelijk Onderzoek Rotterdam” (TWOR) granted approval for his study Djamin et al. Antimicrobial Resistance and Infection Control (2020) 9:116 Page 8 of 8 Page 8 of 8 34. Garza-Ramos G, Xiong L, Zhong P, et al. Binding site of macrolide antibiotics on the ribosome: New resistance mutation identifies a specific interaction of ketolides with rRNA. J Bacteriol. 2001;183:6898–907. https://doi.org/10. 1128/JB.183.23.6898-6907.2001. 14. Aslam B, Wang W, Arshad MI, et al. Antibiotic resistance: a rundown of a global crisis. Infect Drug Resist. 2018;11:1645–58. https://doi.org/10.2147/IDR. S173867. 15. Blake KL, O’Neill AJ. Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents. J Antimicrob Chemother. 2013;68:12–6. https://doi.org/10.1093/jac/dks373. 35. Global Initiative for Chronic Obstructive Lung Disease - Global Initiative for Chronic Obstructive Lung Disease - GOLD. 2020;89. https://goldcopd.org/. Accessed 14 Jan 2020. 35. Global Initiative for Chronic Obstructive Lung Disease - Global Initiative for Chronic Obstructive Lung Disease - GOLD. 2020;89. https://goldcopd.org/. Accessed 14 Jan 2020. 16. Liu A, Tran L, Becket E, et al. Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: Generating an antibiotic bar code. Antimicrob Agents Chemother. 2010;54:1393–403. https://doi.org/10. 1128/AAC.00906-09. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. Floyd JL, Smith KP, Kumar SH, et al. LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus. Antimicrob Agents Chemother. 2010;54:5406–12. https://doi.org/10.1128/AAC.00580-10. 18. Hu RM, Liao ST, Huang CC, et al. An Inducible Fusaric Acid Tripartite Efflux Pump Contributes to the Fusaric Acid Resistance in Stenotrophomonas maltophilia. PLoS One. 2012;7. https://doi.org/10.1371/journal.pone.0051053. 19. Kim C, Mwangi M, Chung M, et al. The mechanism of heterogeneous beta- lactam resistance in MRSA: Key role of the stringent stress response. PLoS One. 2013;8. https://doi.org/10.1371/journal.pone.0082814. 20. Ogawa W, Onishi M, Ni R, et al. Functional study of the novel multidrug efflux pump KexD from Klebsiella pneumoniae. Gene. 2012;498:177–82. https://doi.org/10.1016/j.gene.2012.02.008. 21. Hooper DC, Jacoby GA. Topoisomerase Inhibitors: Fluoroquinolone Mechanisms of Action and Resistance. Cold Spring Harb Perspect Med. 2016;6:a025320. https://doi.org/10.1101/cshperspect.a025320. 22. Malhotra-Kumar S, Lammens C, Coenen S, et al. Effect of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a randomised, double-blind, placebo- controlled study. Lancet. 2007;369:482–90. https://doi.org/10.1016/S0140- 6736(07)60235-9. 23. D H. Azithromycin for Prevention of Exacerbations of COPD. N Engl J Med. 2011;365:2234–7. https://doi.org/10.1056/NEJMc1111248. 24. Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrob Agents Chemother. 1991;35:1267–72. https://doi.org/10.1128/AAC.35.7.1267. 25. Clancy J, Petitpas J, Dib‐Hajj F, et al. Molecular cloning and functional analysis of a novel macrolide‐resistance determinant, mefA , from Streptococcus pyogenes. Mol Microbiol. 1996;22:867–79. https://doi.org/10. 1046/j.1365-2958.1996.01521.x. 26. Tait-Kamradt A, Clancy J, Cronan M, et al. mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrob Agents Chemother. 1997;41:2251–5. https://doi.org/10.1128/aac. 41.10.2251. 27. von Wintersdorff CJH, Penders J, Stobberingh EE, et al. High rates of antimicrobial drug resistance gene acquisition after international travel, the Netherlands. Emerg Infect Dis. 2014;20:649–57. https://doi.org/10.3201/ eid2004.131718. 28. Szczepanowski R, Linke B, Krahn I, et al. Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiology. 2009;155:2306–19. https://doi.org/10.1099/mic.0. 028233-0. 29. Chen J, Yu Z, Michel FC, et al. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems. Appl Environ Microbiol. 2007;73:4407–16. https://doi. org/10.1128/AEM.02799-06. 30. Klindworth A, Pruesse E, Schweer T, et al. Publisher’s Note Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencingbased diversity studies. Nucleic Acids Res. 2013;41. https://doi. org/10.1093/nar/gks808. 30. Klindworth A, Pruesse E, Schweer T, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencingbased diversity studies. Nucleic Acids Res. 2013;41. https://doi. org/10.1093/nar/gks808. 31. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:45e–45. https://doi.org/10.1093/nar/29.9. e45. 31. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:45e–45. https://doi.org/10.1093/nar/29.9. e45. 32. Leclercq R. Mechanisms of Resistance to Macrolides and Lincosamides: Nature of the Resistance Elements and Their Clinical Implications. Clin Infect Dis. 2002;34:482–92. https://doi.org/10.1086/324626. 32. Leclercq R. Mechanisms of Resistance to Macrolides and Lincosamides: Nature of the Resistance Elements and Their Clinical Implications. Clin Infect Dis. 2002;34:482–92. https://doi.org/10.1086/324626. 33. Varaldo PE, Montanari MP, Giovanetti E. Genetic elements responsible for erythromycin resistance in streptococci. Antimicrob Agents Chemother. 2009;53:343–53. https://doi.org/10.1128/AAC.00781-08. 33. Varaldo PE, Montanari MP, Giovanetti E. Genetic elements responsible for erythromycin resistance in streptococci. Antimicrob Agents Chemother. 2009;53:343–53. https://doi.org/10.1128/AAC.00781-08.
https://openalex.org/W3036507153	https://europepmc.org/articles/pmc7309994?pdf=render	English	null	Clinical features of aseptic meningitis with varicella zoster virus infection diagnosed by next-generation sequencing: case reports	BMC infectious diseases	2,020	cc-by	5,787	Clinical features of aseptic meningitis with varicella zoster virus infection diagnosed by next-generation sequencing: case reports Lanlan Chen1†, Yao Xu1†, Chunfeng Liu2, Hong Huang3, Xingxing Zhong1, Cancan Ma1, Haina Zhao1 and Yingzhu Chen1* Abstract Background: The aseptic meningitis caused by varicella zoster virus (VZV) reactivation was less described in the literature, most of which were detected by means of polymerase chain reaction. The authors presented 4 adult immunocompetent patients with acute aseptic meningitis with VZV infection diagnosed by next-generation sequencing (NGS). Case presentation: Four patients were admitted to the hospital with headache and fever between March 2018 and August 2019. The median ages were 37 years (range 22–52 years). The median symptoms onset to clinic time was 3.5 days (range 3–6 days). Two patients had signs of meningeal irritation. Rash occurred after the meningitis symptoms in 1 patient (time from meningitis symptoms to rash, 2 days). No other sign or symptom was reported. The brain Magnetic resonance imaging and electroencephalography were normal in all patients. Cerebrospinal fluid (CSF) samples were obtained at a median of 4 days (range 3–7 days) from the meningitis symptoms onset. Opening pressure of lumbar puncture after admission were high in these cases (median 256 mm H2O; range 165–400 mm H2O). White blood cell counts and protein levels were significantly elevated in CSF samples (median 317 × 10^6/L, range 147–478 × 10^6/L; median 1.41 g/L, range 0.57–1.79 g/L). The cytology of CSF demonstrated a lymphocytic pleocytosis, and most multinuclear cells. The culture of CSF was negative for all 4 cases, while T-cell spot test was positive for 2 cases, who were administrated with anti-tuberculosis treatment for suspicious tuberculous meningitis. NGS of CSF (the Vision Medical Research Institute) detected specific sequences of VZV in the 4 cases within 72 h after admission. The inappropriate treatment were stopped while acyclovir were continued intravenously for 10–14 days. All patients recovered completely. Conclusions: VZV is an infectious agent that causes aseptic meningitis in immunocompetent adults and could not be accompanied by skin manifestations. The NGS of CSF is a rapid detection for the identification and differentiation of meningitis in patients, which is of great importance for providing the rapid and accurate diagnosis and the targeted antimicrobial therapy for central nervous system infection. Keywords: Next-generation sequencing, Varicella zoster virus, Cerebrospinal fluid, Aseptic meningitis, Case report * Correspondence: yzchendr@163.com †Lan Lan Chen and Yao Xu contributed equally to this work. Abstract 1Department of Neurology, Northern Jiangsu People’s Hospital, Medical College of Yangzhou University, Yangzhou 225001, China Full list of author information is available at the end of the article * Correspondence: yzchendr@163.com †Lan Lan Chen and Yao Xu contributed equally to this work. 1Department of Neurology, Northern Jiangsu People’s Hospital, Medical College of Yangzhou University, Yangzhou 225001, China Full list of author information is available at the end of the article Chen et al. BMC Infectious Diseases (2020) 20:435 https://doi.org/10.1186/s12879-020-05155-8 Chen et al. BMC Infectious Diseases (2020) 20:435 https://doi.org/10.1186/s12879-020-05155-8 Open Access Case no. 2 O N On November 2018, a 22-year-old man was admitted to Northern Jiangsu People’s Hospital because of fever (37.7 °C), severe headache, and nausea for 3 days. One day before admission, he noticed vescicles in a very lim- ited region of the left dorsal skin. No other sign or symptom was reported (Table 1). He was diagnosed of varicella when he was 4 years old. Upon admission, his vital signs were unremarkable, and the skin lesions lo- cated in a small part of the area supplied with level of 10 thoracic were considered consistent with a diagnosis with herpes zoster. Neurological examination was sig- nificant for neck stiffness and positive for Kernig signs. The white blood cell count was 11.23 × 10^9/L with 79% neutrophils. The serum electrolytes, hemoglobin, erythrocyte sedimentation rate, procalcitonin, and C-reactive protein were normal. The T-cell spot test was positive. The cerebro- spinal fluid (CSF) contained 147 × 10^6/L white blood cells (WBCs) (reference range, <8 × 10^6/L); CSF protein was 1.72 g/L (reference range, 0.2–0.4 g/L) (Table 2). Brain Mag- netic resonance imaging (MRI) revealed pre-existing brain trauma lesions. The patient was started on intravenous acyclovir for possible herpes simplex virus infection, ceftriax- one for possible bacterial disease, as well as rifampin, isonia- zid, pyrazinamide, and ethambutol for the possible tuberculosis infection. His CSF sample was immediately sent The white blood cell count was 12.18 × 10^9/L, while 81% neutrophils. The inflammatory biomarkers, same with those tested in case 1, were within normal ranges. The opening pressure of lumbar puncture was 235 mm H2O (reference range, 90–180 mm H2O). The cytology of CSF demonstrated WBCs with 200 × 10^6/L, and an increased protein concentration (0.57 g/L) (Table 2). The CSF was positive for VZV by NGS (Vision Medical Re- search Institute), with unique reads of sequences of 13, 190, the genome coverage of 98.83%, and read depth of 11.87 X (Table 3, Fig. 1, Additional file 3). Table 1 Demographic characteristics and clinical findings for cases Case No. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. BMC Infectious Diseases (2020) 20:435 Page 2 of 7 Page 2 of 7 Chen et al. BMC Infectious Diseases (2020) 20:435 Case presentation Case no. 1 On March 2018, a 52-year-old man was admitted to Northern Jiangsu People’s Hospital because of headache for 4 days and fever (37.6 °C) for 2 days. He had no other symptoms. The constant and progressive headache did not relieved after the routine treatment such as oral nonsteroidal analgesics. He had a history of chicken pox when he was 7 years old. He had symptomatic epilepsy since 8 years ago after a brain trauma, which occurred every 6 to 12 months. He had no sign of meningeal irri- tation, and vital signs were normal. No rash was seen at any stage (Table 1). Background for pathogen detection by NGS at Vision Medical Research Institute, and it was sequenced on Illumina NextSeq500 plat- form using a 75-cycle single-end (see Additional file 1 for de- tailed detection process) [5]. The identified number of unique reads mapped on the VZV genome sequence was 17, 137, with genome coverage of 99.73%, and read depth of 17.9 X (Table 3, Fig. 1). After filtering out low-complexity and shorter reads, the NGS analyses of other viruses, bacter- ial, mycoplasma, and tubercle bacillus were negative (Add- itional file 2). Aseptic meningitis is an inflammation of the meninges associated with acute onset of headache, fever and neck stiffness, with pleocytosis of the cerebrospinal fluid, and no growth on routine bacterial culture [1]. The leading recognizable causes of aseptic meningitis include non- polio human enteroviruses, mumps virus, lymphocytic choriomeningitis virus and herpesviruses [2]. Varicella zoster virus (VZV) reactivation is recognized as one of the most common neurological infectious diseases and VZV the second most frequent virus causing encephal- itis [3]. VZV meningitis were less described in the litera- ture, most of which involved adolescent or elderly patients and meanwhile the viruses were detected by means of polymerase chain reaction (PCR) [4]. After the diagnosis with VZV meningitis within 48 h after admission, the antibiotic and anti-tuberculosis treatment were stopped immediately. The acyclovir (10 mg/kg t.i.d) were continued intravenously for 10 days. The patient’s condition improved quickly. A repeat lum- bar puncture on the ninth day after admission revealed improved WBCs (39 × 10^6/L) and protein (0.88 g/L) (Table 2). Thus he was discharged, and a month later on follow-up in the outpatient clinic, he had recovered completely. We here described cases of 4 immunocompetent adults with aseptic meningitis due to VZV reactivation diagnosed by next-generation sequencing (NGS). Case no. 2 O N Gender Age (y) Duration of symptome before admittion (d) Duration of hospitalization (d) Headache Fever Cutaneous zoster Neck stiffness Kernig signs EEG MRI History of chicken pox 1 male 52 4 10 + + – – – – – + 2 male 22 3 10 + + + + + – – + 3 male 45 6 14 + + – – – – – uncertain 4 male 29 3 10 + + – + – – – + Table 1 Demographic characteristics and clinical findings for cases Chen et al. BMC Infectious Diseases (2020) 20:435 Page 3 of 7 Table 2 Routine laboratory evalutions of CSF of the four cases Case No. Days from symptom onset to CSF collection (d) Days from CSF collection to diagnosis (d) Total time to diagnosis (d) Pressure (mm H2O) WBC (×106/ L) Multinuclear (%) Protein (g/L) Glucose (mmol/L) Serum glucose (mmol/L) 1 4 2 2 165 147 1.4 1.72 3.82 6.80 1 13 / / 120 39 1.0 0.88 4.97 6.95 2 3 2 2 235 200 1.0 0.57 3.88 4.74 2 11 / / 70 130 0.8 0.40 4.97 5.93 3 7 2 3 400 478 0.2 1.79 7.03 8.37 3 16 / / 240 91 0.4 0.65 4.06 5.79 4 4 2 3 275 434 0.7 1.10 3.72 4.77 4 12 / / 175 175 0.9 0.37 3.42 4.64 Note: Total time to diagnosis = days to CSF collection+ days from CSF collection to NGS result Table 2 Routine laboratory evalutions of CSF of the four cases as well as rifampin, isoniazid, pyrazinamide, and etham- butol for the possible tuberculosis infection. His CSF sample was immediately tested by NGS at Vision Med- ical Research Institute. The sequencing detection identi- fied 1330 sequence reads uniquely corresponding to the VZV, with the genome coverage of 34.77% (Table 3, Fig. 1, Additional file 4). The patient was treated with acyclovir and ceftriaxone after admission. After VZV was detected, ceftriaxone was stopped and acyclovir (10 mg/kg t.i.d) was continued intravenously for 10 days. The therapy was highly effect- ive and the patient’s clinical condition rapidly improved. The CSF on the eighth day after the admission revealed WBCs with 130 × 10^6/L, and protein (0.40 g/L) (Table 2). The patient was discharged after 10-day treatment. An examination carried out about 4 weeks later did not find any sign or symptom of disease. Case no. 2 O N After etiological diagnosis was identified, antibiotic and anti-tuberculosis treatment were stopped immedi- ately. The intravenously acyclovir was continued for total 14 days, while mannitol was for the total 11 days with a gradually decreased dose. The disease made rapid progress. A repeat lumbar puncture on the eleventh day after admission revealed improved WBCs (91 × 10^6/L) and protein (0.65 g/L) (Table 2). Thus he was discharged from hospital. A month later on follow-up in the out- patient clinic, he had recovered completely. Case no. 3 On August 2019, a 45-year-old man with headache for 6 days was admitted to our hospital. He felt fatigued and headache, never measured his temperature. No other sign or symptom was reported. No positive sign of neurological examination was detected (Table 1). A low fever of less than 38.0 °C (37.8 °C on the first day, 37.6 °C on the second day, 37.7 °C on the third day) was found every afternoon since the second day after admission. The white blood cell count was 13.45 × 10^9/L, while 79% neutrophils. The inflammatory biomarkers, same with those tested in case 1 and case 2, were within nor- mal ranges. The lumbar puncture on the second day after admission revealed significantly increased opening pressure of CSF (400 mm H2O). The CSF contained 478 × 10^6/L WBCs; CSF protein was 1.79 g/L (Table 2). The T-cell spot test was positive. The patient was treated with intravenous mannitol, acyclovir, ceftriaxone, Case no. 4 On August 2019, a 29-year-old man with a fever (38.0 °C) and a progressive headache was admitted to Northern Jiangsu People’s Hospital. His disease began with headache and nausea 3 days earlier. No other symp- tom was developed. Neurological examination was mod- erate for neck stiffness and negative for Kernig signs (Table 1). The white blood cell count was 13.73 × 10^9/ L, while 82% neutrophils. The results of routine inflam- matory biomarkers, same with those tested in other cases, were normal. The second day after the admission, Table 3 Number, percentage, genome coverage, and read depth of unique reads for the sequences of varicella zoster virus in the rcentage, genome coverage, and read depth of unique reads for the sequences of varicella zoster virus in the Table 3 Number, percentage, genome coverage, and read depth of unique reads for the sequences of varicella zoster virus in the CSF samples Case No. Pathogen Unique reads Percentage, % Genome Coverage, % Read Depth (X) 1 varicella zoster virus 17,137 100 99.73 17.90 2 varicella zoster virus 13,190 100 98.83 11.87 3 varicella zoster virus 1330 100 34.77 1.25 4 varicella zoster virus 102 96.23 4.32 1.03 Note: In case 1, 2, and 3, only VZV has been detected by NGS. So, the percentage is 100% for each of them. In case 4, there were 102 reads of VZV and 4 reads of Human parvovirus B19 detected by NGS (Additional file 5). So, the percentage is 96.23% (102/106) for case 4 Note: In case 1, 2, and 3, only VZV has been detected by NGS. So, the percentage is 100% for each of them. In case 4, there were 102 reads of VZV and 4 reads of Human parvovirus B19 detected by NGS (Additional file 5). So, the percentage is 96.23% (102/106) for case 4 Chen et al. Discussion and conclusions the opening pressure of lumbar puncture showed 275 mm H2O, and the CSF contained 434 × 10^6/L WBCs (Table 2). The patient was started on intravenous acyclovir of 10 mg/ kg t.i.d and mannitol of 250 ml b.i.d. DNA of VZV was iden- tified in the CSF using NGS (Vision Medical Research Insti- tute). The sequencing detection identified 102 sequence reads uniquely corresponding to the VZV, with genome coverage of 4.32% (Table 3, Fig. 1, Additional file 5). The intravenous acyclovir were administered for 10 days, and the patient’s condition improved quickly. A repeat lumbar punc- ture on the eighth day after the admission revealed normal pressure of 175 mm H2O, improved WBCs (129 × 10^6/L), and normal protein (0.37 g/L) (Table 2). Thus he was dis- charged from hospital. A month later on follow-up in the outpatient clinic, he had recovered completely. the opening pressure of lumbar puncture showed 275 mm H2O, and the CSF contained 434 × 10^6/L WBCs (Table 2). The patient was started on intravenous acyclovir of 10 mg/ kg t.i.d and mannitol of 250 ml b.i.d. DNA of VZV was iden- tified in the CSF using NGS (Vision Medical Research Insti- tute). The sequencing detection identified 102 sequence reads uniquely corresponding to the VZV, with genome coverage of 4.32% (Table 3, Fig. 1, Additional file 5). The intravenous acyclovir were administered for 10 days, and the patient’s condition improved quickly. A repeat lumbar punc- ture on the eighth day after the admission revealed normal pressure of 175 mm H2O, improved WBCs (129 × 10^6/L), and normal protein (0.37 g/L) (Table 2). Thus he was dis- charged from hospital. A month later on follow-up in the outpatient clinic, he had recovered completely. VZV, belonging to the group of alpha-herpes viruses, causes varicella (chickenpox) and herpes zoster. Varicella usually results in mild to moderate illness in mainly childhood or immunocompetent patients with dissemi- nated vesicular rash. After primary infection, VZV re- mains latent in sensory cranial nerve ganglia or dorsal root ganglia, when reactivated, replicates along the course of the nerve and appears as a localized vesicular skin rash. Viral reactivation can cause a wide range of neurologic disease, most frequently manifesting as her- pes zoster and post-herpetic neuralgia [3]. Older age, immunocompromised state, bone marrow transplant re- cipients and possibly pregnancy are risk factors associ- ated with higher severity of VZV. Case no. 4 BMC Infectious Diseases (2020) 20:435 Page 4 of 7 0 20 40 60 80 100 120 0 50 100 150 200 r e b m u n s d a e R Nucleotide Position along VZV Genome a 0 20 40 60 80 100 120 0 100 200 300 400 Reads number Nucleotide Position along VZV Genome b 0 20 40 60 80 100 120 0 5 10 15 20 r e b m u n s d a e R Nucleotide Position along VZV Genome c 0 2 40 60 80 100 120 0 2 4 6 8 Reads number Nucleotide Position along VZV Genome d Fig. 1 Reads mapping to VZV genome, generated with NGS from CSF. a In case No. 1, the viral reads (17,137 reads) corresponded to varicella zoster virus, with a genome coverage of 99.73%. b In case No. 2, the viral reads (13,190 reads) corresponded to varicella zoster virus, with a genome coverage of 99.83%. c In case No. 3, the viral reads (1330 reads) corresponded to varicella zoster virus, with a genome coverage of 34.77%. d In case No. 4, the viral reads (102 reads) corresponded to varicella zoster virus with a genome coverage of 4.32% 0 20 40 60 80 100 120 0 100 200 300 400 Reads number Nucleotide Position along VZV Genome b 0 20 40 60 80 100 120 0 50 100 150 200 r e b m u n s d a e R Nucleotide Position along VZV Genome a a 0 2 40 60 80 100 120 0 2 4 6 8 Reads number Nucleotide Position along VZV Genome d 0 20 40 60 80 100 120 0 5 10 15 20 r e b m u n s d a e R Nucleotide Position along VZV Genome c d c Nucleotide Position along VZV Genome Nucleotide Position along VZV Genome Fig. 1 Reads mapping to VZV genome, generated with NGS from CSF. a In case No. 1, the viral reads (17,137 reads) corresponded to varicella zoster virus, with a genome coverage of 99.73%. b In case No. 2, the viral reads (13,190 reads) corresponded to varicella zoster virus, with a genome coverage of 99.83%. c In case No. 3, the viral reads (1330 reads) corresponded to varicella zoster virus, with a genome coverage of 34.77%. d In case No. Case no. 4 4, the viral reads (102 reads) corresponded to varicella zoster virus with a genome coverage of 4.32% Discussion and conclusions The only one who had epilepsy history took antiepileptic drugs, rather than immunosuppressant drugs, which indicated that re- activation of VZV may be a more frequent cause of aseptic meningitis than previously anticipated in im- munocompetent individuals. Koskiniemi et al. reported that 27 and 65% of patients with encephalitis and men- ingitis, respectively, had no skin manifestations, suggest- ing VZV could reactivate independently of vesicular eruptions, and spread directly to the leptomeninges [11]. In the present study, 3 patients (75%) had no cutaneous zosteriform lesions, which was consistence with the pre- vious study [11, 12]. [7]. Till now, many diagnostic assays are based on poly- merase chain reaction (PCR), which relies on sequence- specific primers. NGS has been applied as a diagnostic method to detect the pathogens for CNS infectious dis- eases in recent years. Many successful applications of NGS to the diagnosis with CNS infections have been re- ported [8, 9]. The case series included 4 male adult pa- tients who were diagnosis with VZV meningitis by NGS. Interestingly, all 4 patients presenting in this study were less than 50 years of age, although VZV reactivation were reported more commonly occurred in older adults [10]. This finding was similar to that in another study in which 5 of the 8 patients with VZV meningitis were pre- sented before the fifth decade of life. None of them had traditional risk factors for VZV infection. The only one who had epilepsy history took antiepileptic drugs, rather than immunosuppressant drugs, which indicated that re- activation of VZV may be a more frequent cause of aseptic meningitis than previously anticipated in im- munocompetent individuals. Koskiniemi et al. reported that 27 and 65% of patients with encephalitis and men- ingitis, respectively, had no skin manifestations, suggest- ing VZV could reactivate independently of vesicular eruptions, and spread directly to the leptomeninges [11]. In the present study, 3 patients (75%) had no cutaneous zosteriform lesions, which was consistence with the pre- vious study [11, 12]. The difficulty in meningitis diagnosis is to distinguish whether it’s a viral or bacterial etiology, because this is crucial for treatment decisions. The treatment threshold is usually set low in clinical work, so that antibiotics, even anti-tuberculosis treatment, are often prescribed in cases of doubt, as were patients in this study. The pa- tient of case No. Discussion and conclusions Aseptic meningitis is usually regarded as an uncommon complication of the cutaneous primary infection in patients with impaired cellular immunity. Recently, many studies reported that VZV was an important cause of aseptic infection in cen- tral nervous system (CNS), with the frequencies ranging from 5 to 27% [5, 6]. The sequence-specific PCR identification of VZV was carried out to validate the NGS results for the 4 cases. The specific primers used for the gene amplification were VZV-F2 (GACAATATCATATACATGGAATGT G) and VZV-R2 (GCGGTAGTAACAGAGAATTT CTT). The results showed that the read from Sanger se- quencing was consistent with VZV genome (see Add- itional file 1 for PCR results and Additional file 6 for original images) (Fig. 2). The etiology of acute meningoencephalitis remains un- diagnosed in approximately 60% of cases despite exten- sive clinical laboratory testing for infectious pathogens Chen et al. BMC Infectious Diseases (2020) 20:435 Page 5 of 7 Fig. 2 Sanger sequencing detection of VZV from CSF specimen. M: DNA markers of DL2000 or Trans 2 K Plus. N: negative control. The number 277 bp represent the sample code. Original images were in Additional file 6 in patients with enteroviral infection [12, 13]. Protein levels (median 1.41 g/L) in this study were higher than previously reported. All patients received acyclovir intra- venously for 1–2 weeks resulting in full recovery, sug- gesting that VZV meningitis tends to be mild symptom, good response to treatment and benign prognosis. [7]. Till now, many diagnostic assays are based on poly- merase chain reaction (PCR), which relies on sequence- specific primers. NGS has been applied as a diagnostic method to detect the pathogens for CNS infectious dis- eases in recent years. Many successful applications of NGS to the diagnosis with CNS infections have been re- ported [8, 9]. The case series included 4 male adult pa- tients who were diagnosis with VZV meningitis by NGS. Interestingly, all 4 patients presenting in this study were less than 50 years of age, although VZV reactivation were reported more commonly occurred in older adults [10]. This finding was similar to that in another study in which 5 of the 8 patients with VZV meningitis were pre- sented before the fifth decade of life. None of them had traditional risk factors for VZV infection. Discussion and conclusions 2 had vesicular rash before admission which made the diagnosis with aseptic meningitis rela- tively easy. However, the other 3 patients never showed cutaneous zosteriform lesions. Additionally, the T-cell spot test was positive for the second and fourth cases, meanwhile the protein levels in CSF elevated signifi- cantly for both these 2 patients, which implied possible tuberculosis infection, therefore anti-tuberculosis treat- ment as well as antibiotics were administrated. However, all the cases in our study were pathogen diagnosis within 72 h after admission. Once the sequences of VZV were detected by NGS, inappropriate treatment were stopped. NGS is a rapid and accurate approach for the molecu- lar diagnosis with diseases compared to traditional clin- ical testing. It could dramatically reduce the diagnostic period to less than 3 days [14]. Pathogen-specific PCR is widely used to detect common viruses like herpes sim- plex virus, VZV and enterovirus because of its high sen- sitivity and specificity. The turn-around time, which is the time taken from CSF collection to receipt result re- port, is often 2 days for PCR, and is comparable to that of the NGS test. Most centers around the world would consider NGS only for samples that have been tested negative by pathogen-specific PCR. However, our center opted for direct NGS instead of PCR in this study, for 3 Lumbar puncture revealed increased opening pressure of CSF (235–400 mm H2O) in three patients, and it was also on the high side (165 mm H2O) for the other one. All cases showed elevation in CSF WBC, ranging from 147 to 478 × 106/L, and the CSF cytology indicated lymphocytic inflammation. It was regarded that mild ele- vation in CSF protein levels would been observed in aseptic meningitis. However, the relatively high CSF pro- tein levels were reported seen in patients with VZV in- fection which were significantly higher than those seen Chen et al. BMC Infectious Diseases (2020) 20:435 Chen et al. BMC Infectious Diseases (2020) 20:435 Page 6 of 7 Page 6 of 7 of the cases were difficult to distinguish between viral and bacterial etiology from CSF characteristic or clinical feature. The main limitation of PCR is the level to which assays can be multiplexed, which constrains the number of targets that can be assessed per reaction. Discussion and conclusions For cases of common virus infection, the diagnosis will be quickly confirmed by PCR; for cases of other microorganism in- fection which are out of the PCR test range, the diagno- sis time would be extended. In contrast, NGS is a high- throughput approach that can interrogate all genetic ma- terial in a biologic sample simultaneously [15]. It enable sequencing the total DNA or ribonucleic acid (RNA) from a human sample and identify all possible micro- organism present in the specimen. Besides, NGS is an untarged assay as it can amplify and sequence the entire DNA content of a sample without using any primers or probes. The NGS results were further validated by Sanger sequencing in our cases, which was consistent with our expectation, and indicated the reliability of the results and the great practical guiding value of NGS. Ethics approval and consent to participate All procedures performed in this study involving human participants were in accordance with the ethical standards of Ethics Committee of Northern Jiangsu People’s Hospital. VZV reactivation leading to aseptic meningitis in im- munocompetent adults with or without cutaneous zoster is more common that previous regarded. Relatively high CSF protein levels could be observed in VZV meningitis. This study highlighted the feasibility of using NGS of CSF as a diagnostic tool for CNS infection. Unbiased NGS could facilitate identification of all the potential pathogens in a single assay theoretically, which is of great importance for providing the rapid and accurate diagnosis and the targeted antimicrobial therapy for CNS infection. Funding h k This work was supported by the 333 Project of Jiangsu Province (BRA2015187), and Six kinds of Talent Summit Project funded by Jiangsu Province (WSW-246), for the publication of this article. References 1. McGill F, Griffiths MJ, Bonnett LJ, et al. Incidence, aetiology, and sequelae of viral meningitis in UK adults: a multicentre prospective observational cohort study. Lancet Infect Dis. 2018;18(9):992–1003. 1. McGill F, Griffiths MJ, Bonnett LJ, et al. Incidence, aetiology, and sequelae of viral meningitis in UK adults: a multicentre prospective observational cohort study. Lancet Infect Dis. 2018;18(9):992–1003. Additional file 1. Details of NGS detection process and the results of Sanger sequencing Additional file 2. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 1 Additional file 3. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 2 Additional file 4. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 3 Additional file 5. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 4 Additional file 6. Original images for results of Sanger sequencing detection of VZV from CSF specimen Additional file 1. Details of NGS detection process and the results of Sanger sequencing 2. Wright WF, Pinto CN, Palisoc K, et al. Viral (aseptic) meningitis: a review. J Neurol Sci. 2019;398:176–83. Additional file 2. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 1 3. Granerod J, Ambrose HE, Davies NW, et al. Causes of encephalitis and differences in their clinical presentations in England: a multicentre, population-based prospective study. Lancet Infect Dis. 2010;10(12):835–44. 4. Grahn A, Bergström T, Runesson J, et al. Varicella-zoster virus (VZV) DNA in serum of patients with VZV central nervous system Q6 infections. J Inf Secur. 2016;73(3):254–60. 5. Blauwkamp TA, Thair S, Rosen MJ, et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. 2019;4(4):663–74. 6. Kupila L, Vuorinen T, Vainionpää R, et al. Etiology of aseptic meningitis and encephalitis in an adult population. Neurology. 2006;66(1):75–80. 7. Glaser CA, Honarmand S, Anderson LJ, et al. Beyond viruses: clinical profiles and etiologies associated with encephalitis. Clin Infect Dis. 2006;43(12): 1565–77. Authors’ contributions Y C is the principal investigator who drafted the original manuscript. L C participated in laboratory analysis of CSF and drafted parts of the original manuscript. Y X was involved in case and sample collection as well as analysis or interpretation of data. C L revised the manuscript. H H performed the NGS and Sanger sequencing test. X Z was involved in case and sample collection and analysis of the data. C M was involved in case and sample collection and analysis of the data. H Z analyzed and interpreted the data. All authors have read and approved the manuscript. Availability of data and materials All data generated or analysed during this study are included in this published article. The CSF was detected by mNGS (illumine Nextseq 550, Vision Medical Research Institute). The VZV DNA sequence assembled using the NGS data was submitted to GenBank (accession no. KY062165). Other microbe reads including that of bacterium, fungi, parasite and virus were listed in Additional files 2–5. Supplementary information y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-05155-8. Consent for publication Written informed consents were obtained from patients for publication of this case report. Copies of the written consent for publication from the patients are available for review by the Editor-in-Chief of this journal. Author details 1 1Department of Neurology, Northern Jiangsu People’s Hospital, Medical College of Yangzhou University, Yangzhou 225001, China. 2Department of Neurology, the Second Affiliated Hospital of Soochow University, Suzhou 215004, China. 3Vision Medical Co., Ltd, Guangzhou 510670, China. Received: 25 April 2020 Accepted: 12 June 2020 Received: 25 April 2020 Accepted: 12 June 2020 Competing interests p g The authors declare that they have no competing interests. g The authors declare that they have no competing interests. 8. Wilson MR, O’Donovan BD, Gelfand JM, et al. Chronic meningitis investigated via metagenomic next-generation sequencing. JAMA Neurol. 2018;75(8):947–55. Additional file 1. Details of NGS detection process and the results of Sanger sequencing Additional file 2. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 1 Additional file 3. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 2 Additional file 4. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 3 Additional file 5. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 4 Additional file 6. Original images for results of Sanger sequencing detection of VZV from CSF specimen Abbreviations CNS: Central nervous system; CSF: Cerebrospinal fluid; DNA: Deoxyribonucleic acid; EEG: Electroencephalography; MRI: Magnetic resonance imaging; NGS: Next-generation sequencing; PCR: Polymerase chain reaction; RNA: Ribonucleic acid; VZV: Varicella zoster virus; WBC: White blood cells 8. Wilson MR, O’Donovan BD, Gelfand JM, et al. Chronic meningitis investigated via metagenomic next-generation sequencing. JAMA Neurol. 2018;75(8):947–55. 9. Brown JR, Bharucha T, Breuer J. Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases. J Inf Secur. 2018;76(3):225–40. 9. Brown JR, Bharucha T, Breuer J. Encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases. J Inf Secur. 2018;76(3):225–40. Acknowledgements Not applicable. Not applicable. Page 7 of 7 Chen et al. BMC Infectious Diseases (2020) 20:435 Chen et al. BMC Infectious Diseases (2020) 20:435 10. Meyers JL, Candrilli SD, Rausch DA, et al. Costs of herpes zoster complications in older adults: a cohort study of US claims database. Vaccine. 2019;37(9):1235–44. 10. Meyers JL, Candrilli SD, Rausch DA, et al. Costs of herpes zoster complications in older adults: a cohort study of US claims database. Vaccine. 2019;37(9):1235–44. 11. Koskiniemi M, Piiparinen H, Rantalaiho T, et al. Acute central nervous system complications in varicella zoster virus infections. J Clin Virol. 2002;25(3):293–301. 12. Choi R, Kim GM, Jo IJ, et al. Incidence and clinical features of herpes simplex viruses (1 and 2) and varicella-zoster virus infections in an adult Korean population with aseptic meningitis or encephalitis. J Med Virol. 2014;86(6): 957–62. 13. Ihekwaba UK, Kudesia G, McKendrick MW. Clinical features of viral meningitis in adults: significant differences in cerebrospinal fluid findings among herpes simplex virus, varicella zoster virus, and Enterovirus infections. Clin Infect Dis. 2008;47(6):783–9. 14. Guan H, Shen A, Lv X, et al. Detection of virus in CSF from the cases with meningoencephalitis by next-generation sequencing. J Neuro-Oncol. 2016; 22(2):240–5. 14. Guan H, Shen A, Lv X, et al. Detection of virus in CSF from the cases with meningoencephalitis by next-generation sequencing. J Neuro-Oncol. 2016; 22(2):240–5. 15. Xiao H, Huang K, Li L, et al. Complete genome sequence analysis of human echovirus 30 isolated during a large outbreak in Guangdong Province of China, in 2012. Arch Virol. 2014;159(2):379–83. 15. Xiao H, Huang K, Li L, et al. Complete genome sequence analysis of human echovirus 30 isolated during a large outbreak in Guangdong Province of China, in 2012. Arch Virol. 2014;159(2):379–83. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3201163415	https://aladabj.uobaghdad.edu.iq/index.php/aladabjournal/article/download/1155/1212	Arabic	null	الاسناد الاجتماعي وعلاقته بالاستقرار المهني لدى المرشدين التربويين	Al-ādāb	2,021	cc-by	13,803	0208 / م8113 هـ 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) Social Support and its Relationship to Professional Stability Among Educational Counselors Qais Rashid Khawaf Al-Zaidi University of Kufa - College of Basic Education Rshyeqys66@gmail.com جمةل الآداب / العدد831 (أ يلول) جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ Abstract: The research aims to identify: 1- The level of social support for educational counselors. 1- The level of social support for educational counselors. 2- The level of professional stability of the educational counselors. 2- The level of professional stability of the educational counselors. 3- The correlation between social support and professional stability among educational counselors . 3- The correlation between social support and professional stability among educational counselors . g The current research is determined by educational counselors, male and female, in three general directorates of education in Baghdad Governorate (Rusafa First, Rusafa Second, and Rusafa Three) for the academic year (2017 - 2018) and after applying the standards to the sample members and conducting the data unpacking and dealing with them statistically, the results showed as follows, that the individuals of a sample the research enjoys social attribution, and it also showed that the members of the research sample do not enjoy professional stability, and the results also showed that there is no correlative relationship between the social attribution and the professional stability of the sample . In conclusion, it was recommended that the education directorates should satisfy the work environment and the needs of the individuals working within the school and suggested conducting a study on the school principals for the same concepts . Keywords: social support, occupational stability, educational counsellor. 939 جمةل الآداب / العدد831 (أ يلول) جمةل الآداب / العدد831 (أ يلول) م. د. قيس رشيد خواف الزيدي جامعة الكوفة- كمية التربية االساسية قسم رياض االطفال Rshyeqys66@gmail.com )(مُمَخَّصُ البَحث :يستهدف البحث التعرف بـ 1 . .مستوى اإلسناد االجتماعي لدى المرشدين التربويين 9 . .مستوى االستقرار المهني لدى المرشدين التربويين 3 . .العالقة االرتباطية بين اإلسناد االجتماعي واالستقرار المهني لدى المرشدين التربويين يتحــدد البحــث الحــالي بالمرشــدين ا لتربــويين مــن الــذإلور واإلنــاث دــي تــالث مــديريات عامة لتربية محادظة بغداد (الرصادة االولى والرصادة التانية والرصادة التالتة) للعام الدراسـي ( 9217 - 9218 ) وبعــد تطبيــق المقــاييس علــى ادــراد العينــة واغجــراا التوريــا للبيانــات والتعامــل ,معها احصائيا اظهرت النتائج ما يأتي إ ن ,أدـراد عينـة البحـث يتمتعـون باإلسـناد االجتمـاعي إلما اظهرت ايضا أن أدـراد عينـة البحـث ال يتمتعـون باالسـتقرار المهنـي, إلمـا أظهـرت النتـائج انــال ال توجــد عالقــة ارتباطيــال بــين اإلســناد االجتمــاعي واالســتقرار المهنــي للعينــة ودــي نهايــة البحــث اوصــى علــى مــديريات التربيــة ان تشــب بيئــة العمــل وحاجــات االدــراد العــاملين دا ــل .المدرسة واقترح اجراا دراسة على مدراا المدارس لنوس المواهيم الكممات المفتاحية.: اإلسناد االجتماعي, االستقرار المهني, المرشد التربوي :الفصل االول :مشكمة البحث ,إنّ طبيعة العمل اإلرشادي تؤتر دي المرشد ودرجـة توادقـال دالمرشـد بحاجـة إلـى الشـعور بالرضا والسعادة والشعور باالطمئنـان ناحيـة مسـتقبلال, والتعامـل مـ رؤسـاا وزمـالا يحترمونـال ومســاندة مــن ان ــرين, وتــرتبط بهــم عالقــات ودودة حميمــة وان يطلــب منــال مــا هــو دــي حــدود ,قدراتــــال واســــتعداداتال, ومــــا يوادــــق ميولــــال ور،باتــــال واتجاهاتــــال (عــــوض1987, ص11 - 13 ) ( Eiwad, 1987, P11-13 .) ويـــرى النجوـــورد( Langford ) أن أإلتـــر مجـــاالت العمـــل إتـــارة للضـــغوط تلـــك المجـــاالت التي تمتاز بمواجهة مباشـرة مـ النـاس والتـي يإلـرس ديهـا اأدـراد أنوسـهم ل دمـة ان ـرين متـل المرشــــــدين النوســــــيبن واالجتمــــــاعيين والمعلمــــــين واأطبــــــاا و،يــــــرهم ,(نــــــدى1998 ,ص4 ) 942 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) ( Nada, 1998, P4 ). وتعد مهنة اإلرشاد النوسي واحدة من المهن التي تواجهها صعوبات ومشإلالت نوسية وتؤدي إلى عدم قيام المرشدين النوسيين بدورهم بصورة جيدة, ومـن تـم دهـي تجعــل الــبعض مــنهم ،يــر مســتقرين و،يــر مطمئنــين دــي مهنــتهم ممــا يترتــب عليــال تــار ســلبية تـــنعإلس علـــى عطـــائهم ,وتـــوادقهم النوســـي واالجتمـــاعي والمهنـــي (محمـــد1999, ص196 ) ( Mohammed, 1999, P196 ). :مشكمة البحث دالمرشد الذي يشعر باالستقرار المهني يإلـون أإلتـر قـدرة علــى إحــداث تعــديالت وتغييــرات دــي ســلوك الطلبــة واإلبــر تــأتيرا ً دــي نووســهم, ويعإلــس هــذا االستقرار مدى ما يتمت بال المرشد النوسي من مالمح ش صيتال التي تتسم بالنضج االنوعالي والمرونـــة والقـــدرة علـــى التعـــاون مـــ ان ـــرين واالهتمـــام بهـــم, وهـــذ مـــن مظـــاهر الش صـــية االنبساطية واالنوتاح على ان رين والضمير الحي التي تتمت بها ش صية المرشد بعيدة عن العـــدوان وعـــدم تقبـــل ان ـــرين والحـــزن واالإلتئـــاب وهـــذ مـــن مظـــاهر العصـــ اب وســـوا التوادـــق النوسي والش صي واأسري, وتعد مهنة اإلرشـاد النوسـي مـن المهـن الهامـة لمـا تتسـم بـال مـن ً صائص وما يرتبط بها من واجبات توـرض علـى العـاملين ديهـا أوضـاعا ً قـد تإلـون مصـدرا لإلتيـــر مـــن الجوانـــب الســـلبية والصـــعوبات التـــي تإلتنـــف العمليـــة اإلرشـــادية, وتشـــير اأدبيـــات و ,الدراسات متل دراسة (البلوي9228 ,) ودراسة (ياسين1987 ) و،يرها إلى أن العاملين دي مهنــة اإلرشــاد النوســي هــم مــن أإلتــر المهنيــين المعرضــين لمواقــف مواجئــة وشــعور بالمســؤولية نحــو الطلبــة وأعبــاا عمــل تتســبب لهــذ الوئــة بــالإلتير مــن المشــإلالت النوســية والصــحية التــي تؤتر دي ش صياته.م وتوادقهم واستقرارهم المهني تبرز مشإللة البحث من الل االجابة عن السـؤال االّ تـي: هـل هنـاك عالقـة بـين االسـناد االجتماعي االستقرار المهني لدى المرشدين التربويين؟ أهمية البحث : اتوق المشتغلون دي مجال اإلرشـاد النوسـي علـى أن المهـام اإلرشـادية ينبغـي أن تتمحـور حــول تقــديم المســاعدة واســتتمار القــدرات, والتوادــق مــ البيئــة المدرســية واالجتماعيــة وتحقيــق النمـــو الســـليم للطلبـــة وتبصـــيرهم بعمليـــة ات ـــاذ القـــرار وتشـــجيعهم علـــى المشـــارإلة الواعلـــة دـــي اأنشطة الم تلوة إلإلسابهم مهارة الت طيط واستتمار الورص ودهم جوانب القوة و الضـعف دـي ش صياتهم للوصول إلى تحقيق ذواتهم دضالً إلى تحقيق أهداف المؤسسة التربوية والمجتمـ ,(اإلبراهيمـي9229, ص16 ( ) Al-Ibrahimi, 2002, P16 ). وبنـاا علـى ذلـك دـأن مهنـة اإلرشــاد التربــوي تعــد مــن المهـــن الصــعبة والشــاقة أنهــا تتعامـــل مــ الطبيعــة اإلنســانية ومـــ شريحة واسعة,من أبناا المجتمـ تلـك هـي شـريحة الطلبـة (االسـدي9229, ص995 ( ) Al- Asadi, 2002, P225 .) وقد تبين إن أسوا الضغوط وأإلترها ارتباطا" بالتوتر واالضـطراب النوسـي هـي تلـك التــي تحـدث للوـرد المنعــزل والـذي يوقـد اإلســناد الوجـداني والصـالت واإلســناد 941 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) االجتمـاعي والمـؤازرة, وبعبـارة أ ـرى دالحيـاة مـ الجماعـة واالنتمـاا لمجموعـة مـن الـزمالا أو لشــبإلة مــن العالقـــات االجتماعيــة واأســـرية تعتبــر مــن المصـــادر الرئيســة التـــي تجعــل للحيـــاة معنى ومن تم توجهنا دي عمومها إلى الصحة والعمل والرضـا وتحملنـا دـي نوـس الوقـت علـى ,مقاومـة الضـغوط واإلجهـاد (أرجايـل1993, ص37 - 42 ( ) Argyle, 1993, P37-40 ) . :مشكمة البحث وربما تإلون بدايـة ظهـور اصـطالح اإلسـناد االجتمـاعي حـديتاً دـي العلـوم اإلنسـانية مـ تنـاول علمــاا االجتمــاع لهــذا الموهــوم دــي إطــار تنــاولهم للعالقــات االجتماعيــة إذ صــا،وا اصــطالح ( شـــبإلة العالقـــات االجتماعيـــةSocial Net Work ) الـــذي يعـــد البدايـــة الحقيقيـــة ل ظهـــور ( مصــطلح اإلســناد االجتمــاعيSocial Support ) الــذي يطلــق عليــال الــبعض اســم المــوارد ,االجتماعية واإلمإلانات االجتماعية (الشناوي وعبد الرحمن1994, ص3 ( ) Al-Shenawi and Abdel-Rahman, 1994, P3 ). إال أن اإلسـناد االجتمـاعي حظـي باهتمـام أإلبـر مـن البـاحتين بعـد أن نشـر إلـابلن( Kaplan, 1974 ) دراسـتال التـي تضـمنت أصـناداً متنوعـة مـــن المســـاعدة والعـــون التـــي تقـــدمها أدـــراد اأســـرة واأصـــدقاا والجيـــران وان ـــرون إلـــى الوـــرد ,(ال الدي9228 , ص1 ( ) Al-Khalidi, 2008, P1 .) :أهداف البحث :يستهدف البحث الحالي إلى ما يأتي 1 - التعرف على مستوى اإلسناد االجتماعي لدى المرشدين التربويين2 9 - التعرف على مستوى االستقرار المهني لدى المرشدين التربويين2 3 - معردـــة العالقـــة االرتباطيـــة بـــين اإلســـناد االجتمـــاعي واالســـتقرار المهنـــي لـــدى المرشـــدين التربويين2 :حددددود البحدددث يتحـــدد البحـــث الحـــالي بدراســـة متغيـــر اإلســـناد االجتمـــاعي وعالقتـــال بمتغيـــر االستقرار المهني لدى المرشدين التربويين من الذإلور واإلناث دي تالث مديريات عامة لتربية ( محادظة بغـداد (الرصـادة االولـى والرصـادة التانيـة والرصـادة التالتـة) للعـام الدراسـي9217 - 9218 .) :تحديد المصطمحات Borrios :(1990) "حالـة تقبـل الموظـف أداا الوظيوـة بحـدود إمإلاناتـال وعـدم شـعور - :التعريف النظدري " الـدعم الـذي يتلقـا الوـرد مـن ان ـرين أدـراد أو مؤسسـات دـي البيئـة ."المهنية لتحقيق التوادق المهني والش صي - :التعريف اإلجرائي دهو الدرجة الإللية التي يحصل عليها المرشـد التربـوي مـن إجابتـال .على دقرات مقياس اإلسناد االجتماعي المعد دي هذا البحث اإ :تانيا :االستقرار المهنيVocational stability اإ :تانيا :االستقرار المهنيVocational stability :عرفه كل من 1 . Fayol ( (1949 : ""ويقصـــــد بـــــال توتيـــــق العالقـــــة بـــــين العـــــاملين والمنظمـــــة لمـــــدة طويلـــــة ( Fayol , 1949 , P 53 ) 9 . Herzberg ( 1966 :) "مشــــاعر الوــــرد الحســــنة التــــي يإلونهــــا حــــول المتغيــــرات الماديــــة ( "والمعنوية التي تتعلق بالعمل نوسال والبيئة المحيطة بالHerzberg , 1966 , P85 ) 3 . Deavis ( 1990) : "توازن أو تجـانس العمـل بـين الوحـدات الم تلوـة المترابطـة التـي تقـ تحـــت ظـــروف التغيـــر الـــديناميإلي مـــا دامـــت بيئـــة العمـــل تســـتجيب ل دـــراد علـــى م تلـــف "أنواعهم وللظروف البيئية الم تلوة ,(ديويز1992, ص51 – 59 ( ) Davies, 1990, P51-52 ) 9 . Herzberg ( 1966 :) "مشــــاعر الوــــرد الحســــنة التــــي يإلونهــــا حــــول المتغيــــرات الماديــــة ( "والمعنوية التي تتعلق بالعمل نوسال والبيئة المحيطة بالHerzberg , 1966 , P85 ) 3 . Deavis ( 1990) : "توازن أو تجـانس العمـل بـين الوحـدات الم تلوـة المترابطـة التـي تقـ تحـــت ظـــروف التغيـــر الـــديناميإلي مـــا دامـــت بيئـــة العمـــل تســـتجيب ل دـــراد علـــى م تلـــف "أنواعهم وللظروف البيئية الم تلوة ,(ديويز1992, ص51 – 59 ( ) Davies, 1990, P51-52 ) 9 . Herzberg ( 1966 :) "مشــــاعر الوــــرد الحســــنة التــــي يإلونهــــا حــــول المتغيــــرات الماديــــة ( "والمعنوية التي تتعلق بالعمل نوسال والبيئة المحيطة بالHerzberg , 1966 , P85 ) 3 . Deavis ( 1990) : "توازن أو تجـانس العمـل بـين الوحـدات الم تلوـة المترابطـة التـي تقـ تحـــت ظـــروف التغيـــر الـــديناميإلي مـــا دامـــت بيئـــة العمـــل تســـتجيب ل دـــراد علـــى م تلـــف "أنواعهم وللظروف البيئية الم تلوة ,(ديويز1992, ص51 – 59 ( ) Davies, 1990, P51-52 ) 4 . Borrios :(1990) "حالـة تقبـل الموظـف أداا الوظيوـة بحـدود إمإلاناتـال وعـدم شـعور ( "باضــطراب دــي البيئــة الوظيويــة وارتوــاع إحساســال بالســعادة عمــا يقــوم بــالBorrios , 1990 , P140 ) 4 . :تحديد المصطمحات :تحديد المصطمحات :أوال": اإلسناد االجتماعيSocial Support عرفه كل من: 1 . لن وآخرون(Lin, et al, 1981) : " الــدعم أو التشــجي المــادي أو المعنــوي الــذي يحصــل عليــال الوــرد مــن ان ــرين ــالل " مواقف الحياة اليومية(Lin, et al, 1981, P 74) 9 . ساراسون وآخرون(Sarason, et al, 1983) : :أوال": اإلسناد االجتماعيSocial Support 1 . لن وآخرون(Lin, et al, 1981) : " الــدعم أو التشــجي المــادي أو المعنــوي الــذي يحصــل عليــال الوــرد مــن ان ــرين ــالل " مواقف الحياة اليومية(Lin, et al, 1981, P 74) 9 . ساراسون وآخرون(Sarason, et al, 1983) : 1 . لن وآخرون(Lin, et al, 1981) : " الــدعم أو التشــجي المــادي أو المعنــوي الــذي يحصــل عليــال الوــرد مــن ان ــرين ــالل " مواقف الحياة اليومية(Lin, et al, 1981, P 74) 9 . ساراسون وآخرون(Sarason, et al, 1983) : 949 جمةل الآداب / العدد831 (أ يلول) " وجــود أو تــودر اأشــ اص الــذين يمإلــن أن نرجــ إلــيهم أو نعتمــد علــيهم ونعــرف أنهــم " يهتمون بنا ويقدروننا ويحبوننا(Sarason, et al, 1983, P127-128) 3 . سيم وسكتر(Syme & Schetter, 1987) : " " تواعل الورد دي عالقاتال م ان رين(Syme & Schetter, 1987, P661) 4 . الشناوي وعب( د الرحمن4994 :) " تلــك العالقــات القائمــة بــين الوــرد و ــرين والتــي يــدرإلها علــى أنهــا يمإلــن أن تعاضــد ,عنـــدما يحتـــاج إليهـــا " (الشـــناوي وعبـــد الـــرحمن1994, ص4 ( ) Al-Shenawi and Abdel-Rahman, 1994, P4 ) - :التعريف النظدري " الـدعم الـذي يتلقـا الوـرد مـن ان ـرين أدـراد أو مؤسسـات دـي البيئـة ."المهنية لتحقيق التوادق المهني والش صي - :التعريف اإلجرائي دهو الدرجة الإللية التي يحصل عليها المرشـد التربـوي مـن إجابتـال .على دقرات مقياس اإلسناد االجتماعي المعد دي هذا البحث " وجــود أو تــودر اأشــ اص الــذين يمإلــن أن نرجــ إلــيهم أو نعتمــد علــيهم ونعــرف أنهــم " يهتمون بنا ويقدروننا ويحبوننا(Sarason, et al, 1983, P127-128) وجــود أو تــودر اأشــ اص الــذين يمإلــن أن نرجــ إلــيهم أو نعتمــد علــيهم ونعــرف أنهــم " يهتمون بنا ويقدروننا ويحبوننا(Sarason, et al, 1983, P127-128) 3 . سيم وسكتر(Syme & Schetter, 1987) : " " تواعل الورد دي عالقاتال م ان رين(Syme & Schetter, 1987, P661) 4 . :تحديد المصطمحات الشناوي وعب( د الرحمن4994 :) " تلــك العالقــات القائمــة بــين الوــرد و ــرين والتــي يــدرإلها علــى أنهــا يمإلــن أن تعاضــد ,عنـــدما يحتـــاج إليهـــا " (الشـــناوي وعبـــد الـــرحمن1994, ص4 ( ) Al-Shenawi and Abdel-Rahman, 1994, P4 ) - :التعريف النظدري " الـدعم الـذي يتلقـا الوـرد مـن ان ـرين أدـراد أو مؤسسـات دـي البيئـة ."المهنية لتحقيق التوادق المهني والش صي - :التعريف اإلجرائي دهو الدرجة الإللية التي يحصل عليها المرشـد التربـوي مـن إجابتـال .على دقرات مقياس اإلسناد االجتماعي المعد دي هذا البحث :تانيا :االستقرار المهنيVocational stability :عرفه كل من 1 . Fayol ( (1949 : ""ويقصـــــد بـــــال توتيـــــق العالقـــــة بـــــين العـــــاملين والمنظمـــــة لمـــــدة طويلـــــة ( Fayol , 1949 , P 53 ) 9 . Herzberg ( 1966 :) "مشــــاعر الوــــرد الحســــنة التــــي يإلونهــــا حــــول المتغيــــرات الماديــــة ( "والمعنوية التي تتعلق بالعمل نوسال والبيئة المحيطة بالHerzberg , 1966 , P85 ) 3 . Deavis ( 1990) : "توازن أو تجـانس العمـل بـين الوحـدات الم تلوـة المترابطـة التـي تقـ تحـــت ظـــروف التغيـــر الـــديناميإلي مـــا دامـــت بيئـــة العمـــل تســـتجيب ل دـــراد علـــى م تلـــف "أنواعهم وللظروف البيئية الم تلوة ,(ديويز1992, ص51 – 59 ( ) Davies, 1990, P51-52 ) 4 . Borrios :(1990) "حالـة تقبـل الموظـف أداا الوظيوـة بحـدود إمإلاناتـال وعـدم شـعور ( "باضــطراب دــي البيئــة الوظيويــة وارتوــاع إحساســال بالســعادة عمــا يقــوم بــالBorrios , " تلــك العالقــات القائمــة بــين الوــرد و ــرين والتــي يــدرإلها علــى أنهــا يمإلــن أن تعاضــد ,عنـــدما يحتـــاج إليهـــا " (الشـــناوي وعبـــد الـــرحمن1994, ص4 ( ) Al-Shenawi and Abdel-Rahman, 1994, P4 ) - :التعريف النظدري " الـدعم الـذي يتلقـا الوـرد مـن ان ـرين أدـراد أو مؤسسـات دـي البيئـة ."المهنية لتحقيق التوادق المهني والش صي - :التعريف اإلجرائي دهو الدرجة الإللية التي يحصل عليها المرشـد التربـوي مـن إجابتـال .على دقرات مقياس اإلسناد االجتماعي المعد دي هذا البحث :تانيا :االستقرار المهنيVocational stability :عرفه كل من 1 . Fayol ( (1949 : ""ويقصـــــد بـــــال توتيـــــق العالقـــــة بـــــين العـــــاملين والمنظمـــــة لمـــــدة طويلـــــة ( Fayol , 1949 , P 53 ) 9 . Herzberg ( 1966 :) "مشــــاعر الوــــرد الحســــنة التــــي يإلونهــــا حــــول المتغيــــرات الماديــــة ( "والمعنوية التي تتعلق بالعمل نوسال والبيئة المحيطة بالHerzberg , 1966 , P85 ) 3 . Deavis ( 1990) : "توازن أو تجـانس العمـل بـين الوحـدات الم تلوـة المترابطـة التـي تقـ تحـــت ظـــروف التغيـــر الـــديناميإلي مـــا دامـــت بيئـــة العمـــل تســـتجيب ل دـــراد علـــى م تلـــف "أنواعهم وللظروف البيئية الم تلوة ,(ديويز1992, ص51 – 59 ( ) Davies, 1990, P51-52 ) 4 . :تحديد المصطمحات Borrios :(1990) "حالـة تقبـل الموظـف أداا الوظيوـة بحـدود إمإلاناتـال وعـدم شـعور ( "باضــطراب دــي البيئــة الوظيويــة وارتوــاع إحساســال بالســعادة عمــا يقــوم بــالBorrios , 1990 , P140 ) - التعريف النظري( : اعتمد تعريفHerzberg, 1966 ) .تعريوا ً نظريا ً للبحث الحالي إإ - التعريف النظري( : اعتمد تعريفHerzberg, 1966 ) .تعريوا ً نظريا ً للبحث الحالي - :التعريدددف اإلجرائددددي الدرجـــة الإلليــــة التـــي يحصـــل علي هــــا المرشـــد التربــــوي, علـــى مقيــــاس .االستقرار المهني - التعريف النظري( : اعتمد تعريفHerzberg, 1966 ) .تعريوا ً نظريا ً للبحث الحالي إإ - :التعريدددف اإلجرائددددي الدرجـــة الإلليــــة التـــي يحصـــل علي هــــا المرشـــد التربــــوي, علـــى مقيــــاس .االستقرار المهني 943 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) جمةل الآداب / العدد831 (أ يلول) :الفصل الثاني: اإلطار النظري :اوال: اإلسناد االجتماعي ( Social support ) يالحــظ الــدارس لدســناد االجتمــاعي اهتمامــاً إلبيــراً بهــذا الموهــوم مــن قبــل علمــاا الــنوس اإلرشادي, والعيادي, واالجتماعي و،يرهم وقد شارك علماا النوس دي هذا االهتمام منذ عقود عديدة, دقد إلانت دإلرة تأتير الجماعـة مـن االتجاهـات والسـلوإليات معرودـة جيـداً, دعلـى سـبيل المتــال ازدهــرت دــي اأربعينيــات وال مســينيات مــن القــرن العشــرين نظريــة الجماعــة المرجعيــة ( Reference Group theory ) التي إلانت هددها تقرير إليويـة قيـام الوـ رد بأ ـذ معـايير قـيم اأدـــراد والجماعـــات إلرطـــار مرجعـــي مقـــارن يـــتم مـــن اللـــال تشـــإليل االتجاهـــات والســـلوإليات ( Merton & Rossi, 1968, P195-196 .) وتوتـرض النظريـة أن النـاس ي تـارون أناسـا مـــتلهم (يشـــبهونهم) للمقارنـــة إذ أن جمـــ المعلومـــات مـــن النـــاس الـــذين يشـــبهونهم أإلتـــر دائـــدة للــذات (م ,لإلــوش9222, ص16 ). إمــا المشــارإلات اأإلتــر داللــة ديمــا يتعلــق بموهــوم اإلســناد ( االجتمــاعي, دقــد جــاات مــن نظريــة االنــدماج االجتمــاعيSocial Affiliation theory ) ( لشـــا ترSchachter, 1959 ) ( ودراســـات زاجونـــكZajonc, 1965 ) علـــى التســـهيل ( االجتمـــاعيSocial Facilitation ). وهنــــاك بعـــض اال تالدــــات المهمــــة بــــين االهتمــــام المعاصر لدسناد أالجتماعي, والمواهيم المذإلورة سابقا, ولعل أحد هذ اال تالدـات اأساسـية هــو أنــال متغيــر بيئــي, دــي حــين إلــان علمــاا الــنوس دــي الســتينيات معــرديين وإلــان اهتمــامهم موجهــــاً لــــيس علــــى اأبنيــــة أالجتماعيــــة, ولإلــــن علــــى إليويــــة قيــــام الوــــرد باســــت دام المــــد ل ( االجتماعي لتحسين الذاتSelf-Enhancement .) وأن موهوم اإلسناد االجتماعي مإلون معردـــي بـــالطب , ولإلـــن الســـبب اأســـاس لشـــيوع الموهـــوم حـــديتاً يإلمـــن دـــي إمإلاناتـــال الوقائيـــة ( والعالجية من الل التغيير البيئيHeller & Swindle, 1983, P161 .) إا إ يا نظرية الذات: ( Rogers ) ( يــرى إلــارل روجــرز) أن اإلنســان دــي اأســاس م لــوق واع وعاقــل يحإلمــال اإلدراك التــام ,لذاتــال ال اصــة والمحــيط الــذي يعــيش ديــال (العــاني1989, ص99 ( ) Al-Ani, 1989, P92 ) دبقــدر المســاندة االجتماعيــة التــي يتلقاهــا الوــرد مــن المحيطــين بــال يإلــون نمــو النوســي واالجتمـــاعي صـــحيحاً, وأن النمـــو النوســـي الســـليم لحيـــاة الوـــرد يمإلـــن أن ينـــتج دـــرداً متإلامـــل الوظــائف النوســية, لــذا دهــو يســعى لنمــو وتطــور المجتمــ . وأن الوــرد المحقــق لذاتــال والســاعي ل دمة وتطور مجتمعال يوصف بأنال الورد الذي يإلون على وعي تام بإلامل براتال, أي أنال ال يهمــل أ و يمنـــ أو يشــو هـــذ ال بـــرات أنهــا جميعـــاً ســتغربل وتمـــر مـــن ــالل الـــذات الناميـــة الســليمة الواعلــة إلــذلك يتصــف الوــرد المتإلامــل نوســياً بالقــدرة علــى االســتمتاع بإلــل لحظــة مــن 944 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ حياتــال عــن طريــق المتعــة التــي تجلبهــا ال بــرات الســارة دــي الحيــاة (إلالمســاندة االجتماعيــة مــن الوالــدين واأصــدقا ا والجــنس اأ ــر). إلمــا يتصــف الوــرد المتإلامــل نوســياً بالقــدرة علــى ات ــاذ القرار المناسـب والشـعور بالحريـة مـن أجـل التحـرك دـي أي اتجـا ير،ـب بـال ليإلـون قـادراً علـى ,تحقيـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــق ذاتـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــال (الجنـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــابي9228, ص67 ) ( Al-Janabi, 2008, P67 ). وأن تحقيــق الــذات يعــود إلــى مرحلــة الطوولــة, دــرذا تمتــ ا لطوــل بمســاندة اجتماعيــة مناســبة مــن الوالــدين واأهــل دــأن ذلــك يســاعد علــى أن تنمــو لــدى الصبي الحاجة لما يسميال روجرز االحترام االيجـابي, وربمـا جـاات هـذ الحاجـة نتيجـة الـتعلم على الر،م من أن روجرز يعتقد بأن مصدرها ،ير محدد سواا إلانت دطرية او متعلمة, دهي عامـــة ودائمـــة ومســـت مرة وموجـــودة لـــدى إلـــل البشـــر ويشـــمل االحتـــرام االيجـــابي التقبـــل والحـــب ,واالستحســان مــن لــدن اأشــ اص ان ــرين (شــلتز1983, ص968 - 969 ( ) Schlitz, 1983, P268-269 ), أي أن االحتـــرام االيجــابي هــو مـــا يحظــى بــال الوـــرد مــن المســـاندة االجتماعيــة ممـــن يحيطـــون بـــال وتعـــد اأم المصــدر اأإلتـــر أهميـــة لتحقيـــق االحتـــرام االيجـــابي للـــذات الســـيما دـــي دتـــرة الرضـــاعة, والحصـــول علـــى االحتـــرام االيجـــابي يـــؤدي إلـــى الشـــعور بالرضا, إلما أن عدم الحصول على االحترام االيجابي يؤدي إلى اإلحبـاط, واغذا لـم تمـنح إالم االحتــرام االيجــابي دــأن ميــل الصــبي إلــى تحقيــق الــذات واغترائهــا ســوف يعــاق (شــلت,ز1983 , ص972 ( ) Schlitz, 1983, P268-269 .) 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) ,تحقيـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــق ذاتـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــال (الجنـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــابي9228, ص67 ) ( Al-Janabi, 2008, P67 ). إ يا نظرية الذات: ( Rogers ) وأن تحقيــق الــذات يعــود إلــى مرحلــة الطوولــة, دــرذا تمتــ ا لطوــل بمســاندة اجتماعيــة مناســبة مــن الوالــدين واأهــل دــأن ذلــك يســاعد علــى أن تنمــو لــدى الصبي الحاجة لما يسميال روجرز االحترام االيجـابي, وربمـا جـاات هـذ الحاجـة نتيجـة الـتعلم على الر،م من أن روجرز يعتقد بأن مصدرها ،ير محدد سواا إلانت دطرية او متعلمة, دهي عامـــة ودائمـــة ومســـت مرة وموجـــودة لـــدى إلـــل البشـــر ويشـــمل االحتـــرام االيجـــابي التقبـــل والحـــب ,واالستحســان مــن لــدن اأشــ اص ان ــرين (شــلتز1983, ص968 - 969 ( ) Schlitz, 1983, P268-269 ), أي أن االحتـــرام االيجــابي هــو مـــا يحظــى بــال الوـــرد مــن المســـاندة االجتماعيــة ممـــن يحيطـــون بـــال وتعـــد اأم المصــدر اأإلتـــر أهميـــة لتحقيـــق االحتـــرام االيجـــابي للـــذات الســـيما دـــي دتـــرة الرضـــاعة, والحصـــول علـــى االحتـــرام االيجـــابي يـــؤدي إلـــى الشـــعور بالرضا, إلما أن عدم الحصول على االحترام االيجابي يؤدي إلى اإلحبـاط, واغذا لـم تمـنح إالم االحتــرام االيجــابي دــأن ميــل الصــبي إلــى تحقيــق الــذات واغترائهــا ســوف يعــاق (شــلت,ز1983 , ص972 ( ) Schlitz, 1983, P268-269 .) :نظرية الحاجات ( Maslow ) ينظر ابراهام ماسلو إلى الإلائن البشري نظرة ديها من السـواا والصـحة النوسـية أإلتـر مـن االنحـراف والمـرض, ومــن النمـو والتقــدم أإلتـر مــن التراجـ والعجــز, ومـن القــوة والوضـيلة أإلتــر ,من الضعف والرذيلة, وهو يرى أن اإلنسان دي سعي مستمر لتحقيق ذاتال (الجنابي1998 , ص52 ( ) Al-Janabi, 2008, P67 ). وال يصـــل الوـــرد إلـــى تحقيـــق الـــذات إال إذا مـــر بالحاجــات التمــان اأ ــرى, التــي تتــدرج مــن أســـول او قاعــدة الهــرم صــعودا إلــى قمتــال لـــذلك سميت نظرية ماسلو بالنظرية الهرمية إذ تحتل الحاجات الوسيولوجية قاعدة الهرم وهي تشـمل حاجة الجوع والعطش والجـنس تـم تليهـ ا حاجـات إلـى اأمـن والسـالمة, وهـي تحتـاج إلشـباعها إلــى مســاندة اجتماعيــة معنويــة أي الشــعور بوجــود ان ــرين, تــم تليهــا حاجــة الحــب واالنتمــاا وهــي تحتــاج إلــى انتمــاا إلــى جماعــة, والشــعور باألوــة معهــم وهــي أيضــا مســاندة اجتماعيــة معنوية ودعم عـاطوي ودعـم وجـداني, تـم حاجـات احتـرام الـذ ات والتـي تحتـاج إلـى سلسـلة مـن العالقــــات االجتماعيــــة واغلــــى روابــــط اجتماعيــــة لتحقيقهــــا وهــــي أيضــــا مســــاندة اجتماعيــــة, تــــم , ,الحاجات العقليـة وهـي حاجـات تحققهـا المسـاندة االجتماعيـة للوـرد (العـاني1989, ص96 - 123 ( ) Al-Ani, 1989, P96-103) . إ يا نظرية الذات: ( Rogers ) ويــرى (ادلــر) أن الهــدف اأول الــذي تنــدرج تحتــال إلــل أنمــاط الســلوك اإلنســاني هــو حمايــة الــذات, دإلــل دــرد يبــدأ حياتــال 946 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) ضعيواً ال حيلة لال ويمتلك الدواد الوطرية للتغلـب علـى الشـعور بـالنقص والسـيادة علـى البيئـة ( المحيطــة بــال واســت دم مصــطلح الدونيــة العضــويةOrgauiferiotie ) التــي يمإلــن أن تإلــون ايجابيـــة إذا حصـــل الوـــرد علـــى القبـــول واإلســـناد والتشـــجي مـــن ان ـــرين (اأبـــوين واأصـــدقاا والمحيطــــين بــــال والمتوــــاعلين معــــال) داإلســــناد والقبــــول قــــد تقلــــب الدونيــــة او تتحــــول إلــــى قــــوة ( Mehudirata, 1987, P29 .) إاا ( Mehudirata, 1987, P29 .) نظرية سوليفأن( : Solivan ) ينظر (هاري ستاك سوليوأن) إلى اإلنسان من الل اندماجال وتواعلال م ان رين, وهـو يرى أننا من الصعوبة أن ن درس سمة من سمات الش صية او ظـاهرة نوسـية او اجتماعيـة او ســلوإلية بمعــزل عــن ان ــرين أنــال يــرى أن أســاس دإلــرة المــرا عــن نوســال مبنيــة علــى أســاس ,عالقتــال بــان رين, وأن العزلــة عــن ان ــرين ســببها دقــدان الشــعور بــاأمن (الجنــابي1998 , ص59 ( ) Al-Janabi, 2008, P52 )). ويوتــرض (ســوليوان أن الش صــية تمتــل إليــان درضي ال يمإلن عزلال عن المواقف االجتماعية المتبادلة ويعبـر عنهـا عـن طريـق التواعـل مـ ان رين دقط حتى أن الش ص المنعزل يحمـل معـال ذإلريـات عالقاتـال الش صـية السـابقة التـي تواصل تأتيرها على توإلير وسلوإلال الل العزلة, ويرى سوليوان أن اإلسناد االجتمـاع ي الـذي يأتي من العالقات االجتماعية المحيطـة بـالورد تعمـل علـى وـض حـاالت القلـق والتـوتر التـي ,تصيب الورد (شلتز1983, ص138 – 139 ( ) Schlitz, 1983, P138-139 .) :نظرية التعمم االجتماعيSocial learning Theory تهـتم هـذ النظريـة بقضــايا أساسـية أولهـا: أن النـاس يســتطيعون ت علـم االسـتجابات الجديــد ( لمجــرد مالحظــة ســلوك ان ــرين وهــؤالا النــاس يعــدون مــن الناحيــة التقنيــة نمــاذجmodels ) واإلتســاب االســتجابات مـــن ــالل هــذ المالحظـــة يســمى االقتنــاا بالنمـــاذج. وتانيــا": أن قـــدرة اإلنســان تتوســط بــين مالحظــة اأنمــوذج ومــا يعقــب ذلــك مــن أداا لهــذ الســلوإليات مــن ق بــل ,المالحظ. إ يا نظرية الذات: ( Rogers ) تم تأتي الحاجات الذهنية (العقلية منها ح اجات 945 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ المعردة " األوة, االستإلشاف, التحصيل ") والتي تحتـاج أيضـا لتحقيقهـا مسـاندة اجتماعيـة تـم تـــأتي الحاجـــات الجماليـــة والونيـــة ومنهـــا (الحاجـــة إلـــى النظـــام والجمـــال وال يـــر والعـــدل) وهـــذ الحاجات ال تتحقق إال عن طريق الروابط االجتماعيـة والمسـاندة االجتماعيـة مـن الجماعـة تـم تليهــا الحاجــة لتحقيــق الــذات منهــا الوصــول إلــى أقصــى طاقــة ممإلنــة, والمســاندة االجتماعيــة تساعد الورد على تحقيـق الـذات السـري تـم تـأتي حاجـات السـمو والتأمـل منهـا حاجـات روحيـة تتجاوز حدود السإلون وهذ الحاجات تتحقق للورد إذا تمت بعالقات اجتماعية وروابـط روحيـة اصة بالمجموع ة التي ينتمي إليها الوـرد إذ أن الـروابط الروحيـة للمجموعـة التـي ينتمـي إليهـا الورد تساعد على تحقيق حالة السمو وهذ أيضا مساندة اجتماعية للوـ رد مـن المجموعـة التـي ينتمي إليها ,(زإلري9225, ص964 - 971 ( ) Zakari, 2005, P264-271 .) ا) ) ( قدم ايرك دروم) نظرية التوق وهي نظريـة معرديـة شـاملة ترإلـز علـى السـلوك اال تيـاري للوــرد وهــي مــن أإلتــر النظريــات وضــوحا ودقــة دــي توســير الســلوك وواقعــال, تقــوم هــذ النظريــة علــى مســلمة موادهــا أن ســلوك الوــرد تســبقال عمليــة مواضــلة بــين البــدائل قــد تتمتــل دــي القيــام بالسلوك أو عدم القيام بال إلما قد تتمتـل دـي بـدائل أنمـاط الجهـد الم تلوـة التـي يمإلـن أن يقـوم :بها الورد وتتم هذ المواضلة على اأسس انتية - إن دادعية الورد للقيـام بـأداة عمـل معـين تحإلمـال منـاد العوائـد التـي يحصـل عليهـا مـن ذلـك اأداا وعادة ي تار أإلبر قيمة لتوقع.اتال - إن الورد يوضل عادة عنـد قيامـال بـأي نشـاط تلـك النتـائج التـي تعـود عليـال بـأإلبر نوـ ممإلـن .من جراا ذلك العمل او النشاط - أن التوقعــات لــدى الوــرد تــؤدي دورا" إلبيــرا" دــي محصــلة اال تيــار بــين البــدائل الســلوإليات ,المتاحــة لــدى الوــرد دــي ذلــك الوقــت (زإلــري9225, ص979 - 973 ( ) Zakari, 2005, P272-273 .) يــرى (الوــرد ادلــر) أن ســلوك اإلنســان تحرإلــال أساســا الحــوادز أالجتماعيــة, واإلنســان عنــد (ادلــــر) إلــــائن اجتمــــاعي دــــي أساســــال وهــــو يميــــل إلــــى إقامــــة عالقــــات مــــ ان ــــرين وينشــــغل بنشاطات اجتماعية تعاونية وهو يوضل المصلحة االجتماعية على المصالحة الذاتية. ويعتقد بـــأن االهتمـــام االجتمـــا عي الـــذي يحـــرك الســـلوك البشـــري دطـــري وأن إلانـــت اأنمـــاط النوعيـــة للعالقــات بــين النــاس والــنظم االجتماعيــة تظهــر وتتإلــون وتحــددها طبيعــة المجتمــ الــذي ينشــأ ,ديــال (الهيتــي1985, ص125 ( ) Al-Hiti, 1985, P105 ). إ يا نظرية الذات: ( Rogers ) وتالتا": تتعلق بالجانب االنتقائي دي التعلم للمالحظة (الداهري9223, ص167 - 168 ( ) Al-Dahri, 2003, P167-168 .) رإلز علماا التعلم االجتماعي على البيئة وعواملها دـي تحديـد مـا سـيتم تعلمـال مـن مبـادئ وســلوك, إذ رإلــزوا علــى صــائص النمــوذج والظــروف التــي يظهــر ديهــا دالنمــاذج االجتماعيــة Social Models) ) التــي تتميــز بالــدفا واإلشــباع العــاطوي والتشــابال مــ الشــ ص المــتعلم دتؤتر بصورة اإلبر دي عملية التعلم من النماذج التي ال تتصف بهذ ال صائص, والدراسـات ( الحديتــة التــي أجراهــا بانــدوراBandura ( ) وهارتــب و ــرونHartup, et al ) تويــد دــي نت ائجهـــا أن الطوـــل يميـــل إلـــى أن يتوحـــد او ينســـج ســـلوإلال علـــى منـــوال ســـلوك هـــؤالا النـــاس ,المحيطـــين بــــال دضــــالً عـــن إدــــراد ــــارج حــــدود أســـرتال إلالراشــــدين واأقــــران (مــــوراي1988 , 947 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) جمةل الداب / العدد831 (أيلول) 0208 / م8113 هـ ص183 ( ) Murray, 1988, P183 ). ويضــيف (بانــدورا) إلــى ذلــك بأنــال بعــد أن يلقــي اأدراد التعزيز المناسب من قبل ان ـري ن علـى سـلوإلياتهم النادعـة لي ـرين دـأنهم مـن الممإلـن أن يعـــززوا أدعـــالهم هـــذ بأنوســـهم متـــى مـــا قـــدموا ســـلوإلاً ممـــاتالً لســـلوك النمـــوذج, وقـــد يتمتـــل التعزيـز باالرتيـاح والرضــا عـن الـذات الــذي يلـي القيــام بهـذا السـلوك(Aronson, et at, 2004,P430) . ثانيا: االستقرار المهني: Vocational stability االســتقرار المهنــي نــاتج لمجموعــة إلبيــرة مــن االتجاهــات والمشــاعر ال اصــة بعــدة جوانــب من العمل إلاأجور وظروف العمل و،يرها دضالً عن ذلك دان هذا الموهوم يشتمل على عدة عوامــل ش صـــية أ ـــرى متـــل الر،بـــات والعالقـــات مــ ان ـــرين ومإلانـــة الوـــرد دا ـــل المجتمـــ ( ,حمو9227, ص17 ) ( Hamo, 2007, P17 .) ( ويــرى ريجيــوRegu 1999 ) أن االســتقرار المهنــي هــو حالــة ناتجــة عــن دور بعــض جوانب المهنة أو الوظيوة دي التأتير دي دادعية العمل وحتـى يتسـنى للوـرد الشـعور بـان لعملـال ,معنــى, دالبــد لــال معردــة نتــائج مــا يبذلــال مــن مجهــود (ريجيــو1999, ص949 ) ( Regio, 1999, P242 .) إ يا نظرية الذات: ( Rogers ) وتالتا": تتعلق بالجانب االنتقائي دي التعلم للمالحظة (الداهري9223, ص167 - 168 ( ) Al-Dahri, 2003, P167-168 .) رإلز علماا التعلم االجتماعي على البيئة وعواملها دـي تحديـد مـا سـيتم تعلمـال مـن مبـادئ وســلوك, إذ رإلــزوا علــى صــائص النمــوذج والظــروف التــي يظهــر ديهــا دالنمــاذج االجتماعيــة Social Models)ال ت ل الش التشا ال ال اطو اإلش ا تت ز ال ف ) الت ( ) ينظر (هاري ستاك سوليوأن) إلى اإلنسان من الل اندماجال وتواعلال م ان رين, وهـو يرى أننا من الصعوبة أن ن درس سمة من سمات الش صية او ظـاهرة نوسـية او اجتماعيـة او ســلوإلية بمعــزل عــن ان ــرين أنــال يــرى أن أســاس دإلــرة المــرا عــن نوســال مبنيــة علــى أســاس ,عالقتــال بــان رين, وأن العزلــة عــن ان ــرين ســببها دقــدان الشــعور بــاأمن (الجنــابي1998 , ص59 ( ) Al-Janabi, 2008, P52 )). ويوتــرض (ســوليوان أن الش صــية تمتــل إليــان درضي ال يمإلن عزلال عن المواقف االجتماعية المتبادلة ويعبـر عنهـا عـن طريـق التواعـل مـ ان رين دقط حتى أن الش ص المنعزل يحمـل معـال ذإلريـات عالقاتـال الش صـية السـابقة التـي تواصل تأتيرها على توإلير وسلوإلال الل العزلة, ويرى سوليوان أن اإلسناد االجتمـاع ي الـذي يأتي من العالقات االجتماعية المحيطـة بـالورد تعمـل علـى وـض حـاالت القلـق والتـوتر التـي ,تصيب الورد (شلتز1983, ص138 – 139 ( ) Schlitz, 1983, P138-139 .) يا م تهـتم هـذ النظريـة بقضــايا أساسـية أولهـا: أن النـاس يســتطيعون ت علـم االسـتجابات الجديــد ( لمجــرد مالحظــة ســلوك ان ــرين وهــؤالا النــاس يعــدون مــن الناحيــة التقنيــة نمــاذجmodels ) واإلتســاب االســتجابات مـــن ــالل هــذ المالحظـــة يســمى االقتنــاا بالنمـــاذج. وتانيــا": أن قـــدرة اإلنســان تتوســط بــين مالحظــة اأنمــوذج ومــا يعقــب ذلــك مــن أداا لهــذ الســلوإليات مــن ق بــل ,المالحظ. ثانيا: االستقرار المهني: Vocational stability :التمييز المهنيVocational Benefits يقصد بالتمييز المهني ا تالف المهنة عن ،يرها من المهن اأ ـرى دـي أن هـذ المهنـة أو تلــك ،يــر تقليديــة أو تحقــق عائــدا ً ماديــا ً أو معنويــا, إحــداهما أو إلليهمــا دــي ظــروف أداا ت تلــف عــن ظــروف اأداا دــي المهــ ن اأ ــرى (دــالتمييز المهنــي) ينصــب علــى المهنــة ذاتهــا وهـــو دـــي ذلـــك ي تلـــف عـــن (التمييـــز دـــي المهنـــة) والـــذي يشـــير إلـــى أداا الوـــرد مقارنـــة بـــأداا ان ــرين الـــذين معـــال دــي المهنـــة نوســـها, وان االســتقرار والســـعادة يزيـــد لــدى شـــا،لي اأعمـــال اأإلتر مهـارة التـي تـودر مإلانـة أعلـى, وأإلتـر النـاس شـعورا ً بالسـعادة دـي العمـل هـم أصـحاب المهن التي تحتاج إلى مهارات محددة, بينما يقـل هـذا الشـعور لـدى اأدـراد العـاملين دـي مهـن ( رتيبة ال تتطلب درجة عالية من المهارةBlauner , 1992 , P380 .) 9 . :الشعور بتحقيق الذاتEmotion of self – Actualization ( أن الداد الرئيسي لدنسان عند إلارل روجـرزC. Rogers, 1942 ) هـو الحاجـة إلـى ( تحقيـق الـذاتSelf – Actualization ) الن اإلنسـان تحـت ظـروف مناسـبة يعبـر عـن قـيم أعلـــى لمـــا هـــو متضـــمن دـــي الغرائـــز اأوليـــة لحوـــظ الـــذات أي اللـــذات الحســـية وتجنـــب األـــم ,(الزاروس1981, ص89 ( ) Lazarus, 1981, P89 .) ثانيا: االستقرار المهني: Vocational stability االســتقرار المهنــي نــاتج لمجموعــة إلبيــرة مــن االتجاهــات والمشــاعر ال اصــة بعــدة جوانــب من العمل إلاأجور وظروف العمل و،يرها دضالً عن ذلك دان هذا الموهوم يشتمل على عدة عوامــل ش صـــية أ ـــرى متـــل الر،بـــات والعالقـــات مــ ان ـــرين ومإلانـــة الوـــرد دا ـــل المجتمـــ ( ,حمو9227, ص17 ) ( Hamo, 2007, P17 .) ( ويــرى ريجيــوRegu 1999 ) أن االســتقرار المهنــي هــو حالــة ناتجــة عــن دور بعــض جوانب المهنة أو الوظيوة دي التأتير دي دادعية العمل وحتـى يتسـنى للوـرد الشـعور بـان لعملـال ,معنــى, دالبــد لــال معردــة نتــائج مــا يبذلــال مــن مجهــود (ريجيــو1999, ص949 ) ( Regio, 1999, P242 .) ( وأوضح ماسلوMaslow, 1970 ) موهوم االستقرار المهني دي نظريتال التي تقوم على أســاس عــدة ادتراضــات تعنــي أن العامــل دــي عملــال دــرد يســعى دومــا ً لتحقيــق أهــداف مهنيــة تسمح لال بتلبية حاجاتال الم تلوة إلش ص, دالعامل المستقر مهنيا ً هو عامل استطاع تحقيق ( عدد إلبير من حاجاتالMaslow , 1970 , P68 .) ( أما سوبرSuper ) ديرى االستقرار المهني مرحلة يصلها الورد اسماها مرحلة االستقرار ( أو االحتواظMaintenance Stage ( ) وتمتد هذ المرحلة من السن45 – 65 ) سـنة وديهـا يحـاول الوــرد المحادظـة علــى مـا حققــال أو اإلتســبال مـن المهنــة إلمـا تتواصــل عمليـة التإليــف دــي ,العمل لتحسين مرإلز م الميل إلى عدم تغيير المهنـة (ال طيـب9223, ص938 – 939 ) ( Alkhatib, 2003, P238-239 .) ( وأوضح ماسلوMaslow, 1970 ) موهوم االستقرار المهني دي نظريتال التي تقوم على أســاس عــدة ادتراضــات تعنــي أن العامــل دــي عملــال دــرد يســعى دومــا ً لتحقيــق أهــداف مهنيــة تسمح لال بتلبية حاجاتال الم تلوة إلش ص, دالعامل المستقر مهنيا ً هو عامل استطاع تحقيق ( عدد إلبير من حاجاتالMaslow , 1970 , P68 .) ( أما سوبرSuper ) ديرى االستقرار المهني مرحلة يصلها الورد اسماها مرحلة االستقرار ( أو االحتواظMaintenance Stage ( ) وتمتد هذ المرحلة من السن45 – 65 ) سـنة وديهـا يحـاول الوــرد المحادظـة علــى مـا حققــال أو اإلتســبال مـن المهنــة إلمـا تتواصــل عمليـة التإليــف دــي ,العمل لتحسين مرإلز م الميل إلى عدم تغيير المهنـة (ال طيـب9223, ص938 – 939 ) ( Alkhatib, 2003, P238-239 .) ( أما جينزبرجEli Ginzberg, 1951 ) دقد وجد أن االستقرار المهنـي هـو مرحلـة أ يـرة ( دــي مســيرة النمــو المهنــي للوــرد وهــي مرحلــة الت صــصSpecification ) ودــي هــذ المرحلــة يإلــون الوــرد قــد ا تــار تمامــا العمــل الــذي يريــد والــذي يتوــق مــ ميولــال وقدراتــال وتظهــر بنجــاح الورد وشعور بالهناا والسعادة دي العمل إلما تمتل مرحلة الت صص هذ مرحلة االن راط دـي ( العمل والبقاا ديال واالستوادة من عوائد وبدا اإلنتاجية ديالSavikas and lent , 1994 , P1 – 2 .) 948 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ 0208 / م8113 هـ العوامل المؤثرة في االستقرار المهني: قدمت اأدبيات إلتيرا ً من انراا والنظريات التي تحتوي على مجموعة إلبيرة من العوامل المـــؤترة دـــي تحديـــد مســـار المهنـــة واالســـتقرار المهنـــي صوصـــا ً وتإلـــاد تإلـــون هـــذ العوامـــل متشابهة أو م تلوة دي بع:ض اأحيان ومن هذ العوامل إ يإ المـــؤترة دـــي تحديـــد مســـار المهنـــة واالســـتقرار المهنـــي صوصـــا ً وتإلـــاد تإلـــون هـــذ العوامـــل متشابهة أو م تلوة دي بع:ض اأحيان ومن هذ العوامل 1 . 4 . :نظام األجورWages system أ تعــــد اأجــــور أحــــد أهــــم حــــوادز العمــــل وهــــي موضــــ اهتمــــام إلبيــــر مــــن قبــــل العــــاملين والمنظمــــــات دمــــــن وجهــــــة نظــــــر العــــــاملين تعــــــد وســــــيلة أساســــــية إلشــــــباع حاجــــــاتهم الماديــــــة ,واالجتماعية, وهي من أهم العوامل بل أإلترها تأتيرا ً دـي انـدداع الوـرد للعمـل وزيـادة إنتاجيتـال أو إهمالــال وضــعف إنتاجيتــال, ومــن وجهــة نظــر المنظمــات تمتــل أحــد العنا صــر اأساســية دــي تإلاليف اإلنتاج, إلما أن قيمة ما يحصل عليال اأدراد نقدا ً يساهم دي لق الشعور باالستقرار ,والر،بة دي االستمرار دي العمل (أبـو شـي ال9212, ص179 ( ) Abu Sheikh, 2010, P172 .) ( ويعــد دردريــك تــايلورF. Taylor, 1856 – 1917 ) أول مــن أشــار إلــى أهميــة اســـت دا م الحـــوادز الماليـــة دـــي تشـــجي العـــاملين الـــذين تتزايـــد معـــدالت أدائهـــم عـــن المعـــدالت المعيارية الموضوعة وانال من اأهمية بمإلان وض هيإلل ل جور يتسم بالعدالـة والموضـوعية ويعمــــل علــــى تحقيــــق مســــتوى عــــال ر مــــن اإلشــــباع واالســــتقرار المهنــــي للمــــوارد البشــــرية دــــي المنظمـــة, ويرشـــعر العمـــل د يهـــا بـــان اال ـــتالف دـــي الشـــرائح االجريـــة مرجعـــال الـــرئيس ودرجـــة ,الصـعوبة واأهميــة النســبية بـين الوظــائف (ديســلر9229, ص499 ( ) Disler, 2009, P422 .) 1 . :التمييز المهنيVocational Benefits 1 . :التمييز المهنيVocational Benefits يقصد بالتمييز المهني ا تالف المهنة عن ،يرها من المهن اأ ـرى دـي أن هـذ المهنـة أو تلــك ،يــر تقليديــة أو تحقــق عائــدا ً ماديــا ً أو معنويــا, إحــداهما أو إلليهمــا دــي ظــروف أداا ت تلــف عــن ظــروف اأداا دــي المهــ ن اأ ــرى (دــالتمييز المهنــي) ينصــب علــى المهنــة ذاتهــا وهـــو دـــي ذلـــك ي تلـــف عـــن (التمييـــز دـــي المهنـــة) والـــذي يشـــير إلـــى أداا الوـــرد مقارنـــة بـــأداا ان ــرين الـــذين معـــال دــي المهنـــة نوســـها, وان االســتقرار والســـعادة يزيـــد لــدى شـــا،لي اأعمـــال اأإلتر مهـارة التـي تـودر مإلانـة أعلـى, وأإلتـر النـاس شـعورا ً بالسـعادة دـي العمـل هـم أصـحاب المهن التي تحتاج إلى مهارات محددة, بينما يقـل هـذا الشـعور لـدى اأدـراد العـاملين دـي مهـن ( رتيبة ال تتطلب درجة عالية من المهارةBlauner , 1992 , P380 .) 9 . :الشعور بتحقيق الذاتEmotion of self – Actualization ( أن الداد الرئيسي لدنسان عند إلارل روجـرزC. Rogers, 1942 ) هـو الحاجـة إلـى ( تحقيـق الـذاتSelf – Actualization ) الن اإلنسـان تحـت ظـروف مناسـبة يعبـر عـن قـيم أعلـــى لمـــا هـــو متضـــمن دـــي الغرائـــز اأوليـــة لحوـــظ الـــذات أي اللـــذات الحســـية وتجنـــب األـــم ,(الزاروس1981, ص89 ( ) Lazarus, 1981, P89 .) 3 . :الميول المهنيةVocational Interests تشإلل الميول سمة هامة من سـمات الش صـية التـي اهتمـت بهـا الدراسـات النوسـية أنهـا ترتبط ارتباطا ً وتيقا ً باإلقبال على نواحي النشاط دي المجاالت الم تلوة إلما أنها تسـاهم مـ ،يرهـــا مـــن ســـمات الش صـــية اأ ـــرى متـــل االتجاهـــات والـــداد والقـــيم دـــي التإليـــف التربـــوي والمهنــــي, وتشــــإل ل الميــــول أيضــــا ً احــــد عناصــــر االســــتقرار إذ يالحــــظ انجــــذاب النــــاس إلــــى المواضــي التــي يإلونــون مســتعدين لعملهــا وابتعــادهم عــن تلــك التــي ال يإلونــون مســتعدين لهــا بمعنــى أن الميــول تحــدد مــا يوعلــال الوــرد أإلتــر مــن أن تحــدد الإليويــة التــي يــتم بهــا العمــل (أبــو , شــعير9228, ص79 – 82 ( ) Abu Shaira, 2008, P79-80 ). والميــل شــعور يصاحب انتبا الورد واهتمامال بموضوع ما وهو دـي جـوهر اتجـا نوسـي موجـب يتميـز بترإليـز ,االنتبـــا دـــي موضـــوع معـــين أو ميـــدان ـــاص (ال طيـــب9222, ص95 ( ) Alkhatib, 2003, P25 .) 949 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) جمةل الآداب / العدد831 (أ يلول) 4 . :نظام األجورWages system 5 . :نظام الترقيةPromotion system :العالقة مع جماعة العملRelationship with peers يسود التنظيم مجموعة من العالقات التي تتم بين العاملين إذ يتشارإلون بدرجة إلبيرة من القيم واالتجاهات واأهداف والشعور بوحدة الهوية وتطوير معايير سلوإليال اصة بهـم وتـؤتر هذ العالقات على شعور الورد باأمن والطمأنينة واالسـتقرار ويعتمـد هـذا علـى مقـدار المنـاد التــي يحققهــا الوــرد مــن تلــك العالقــات, دإللمــا إلــان التواعــل القــائم بــين الوــرد والجماعــة يحقــق منوعـــة للوــــرد إلانـــت جماعــــة العمـــل مصــــدرا ً الســـتقرار الوــــرد ورضـــا , أمــــا إذا إلـــان االرتبــــاط مــن اأمــور التــي يهــتم بهــا الموظــف هــو موضــوع الترقيــة إذ أنهــا تمتــل جــزاا مهمــا مــن طموحاتـــال التـــي ي ســـعى إلـــى تحقيقهــــا دـــي حياتـــال الوظيويـــة, دهــــي تجمـــ بـــين المزايـــا الماديــــة والمعنويـــة ويرتوـــ مرإلـــز الـــوظيوي إلمـــا يترتـــب عليهـــا أيضـــا زيـــادة دـــي الراتـــب وتعـــد الحجـــر اأساس دي إيجاد دمة حإلومية مستقرة, ويسعى نظام الترقية إلى تحقيق جملة من اأهداف منها تعزيز روح التواؤل بين الموظ وين وزيادة تقتهم بالمستقبل وتأمين استمرارية العمل واغيجاد جو من التنـادس بـين العـاملين بمـا يسـهم دـي ددعهـم وحوـزهم وإلـذلك شـعور المـوظوين باأمـان ,واالســـتقرار نتيجـــة تحقـــيقهم للتقـــدم المســـتمر دـــي عملهـــم ومســـتوى معيشـــتهم (العـــزاوي وجـــواد 9212, ص494 – 495 ( ) Al-Azzawi and Jawad, 2010, P494-495 .) يسود التنظيم مجموعة من العالقات التي تتم بين العاملين إذ يتشارإلون بدرجة إلبيرة من القيم واالتجاهات واأهداف والشعور بوحدة الهوية وتطوير معايير سلوإليال اصة بهـم وتـؤتر هذ العالقات على شعور الورد باأمن والطمأنينة واالسـتقرار ويعتمـد هـذا علـى مقـدار المنـاد التــي يحققهــا الوــرد مــن تلــك العالقــات, دإللمــا إلــان التواعــل القــائم بــين الوــرد والجماعــة يحقــق منوعـــة للوــــرد إلانـــت جماعــــة العمـــل مصــــدرا ً الســـتقرار الوــــرد ورضـــا , أمــــا إذا إلـــان االرتبــــاط 952 جمةل الآداب / العدد831 (أ يلول) بالجماعة ي لق جوا ً من التو ًتر أو يعوق إشباع حاجات معينة دان هذ العالقة تإلون مصدرا ,لعــدم االرتيــاح واالســتقرار ومــن تــم يــنعإلس علــى أدائــال بالعمــل (حمــادات9228, ص99 – 121 ( ) Hamadat, 2008, P99-101 .) تســـمى نظريـــة العـــاملين أيضـــا ً بنظريـــة الصـــحة والدادعيـــة التـــي تنســـب إلـــى عـــالم الـــنوس ( دردريـك هيرزبـرجFrederick Herzberg, Two–Factor–theory, 1959 ) وهـي مـن ( أشـهر نظريـات الـدواد وأإلترهـا جـدال ً دـي القـرن الماضـيPorter , 1966 , P395 .) ومضمون هذ النظرية يرت إلز على أن لدى جمي الإلائنات البشرية نوعين من الحاجات وهـي الحاجة إلى تجنب األم والحاجة إلى النماا النوسي, وتهـتم اأولـى بالسـالمة والصـحة الماديـة ,بينمـــا تهـــتم اأ ـــرى بحاجـــات النمـــاا النوســـي وتحقيـــق الـــذات (حمـــادات9228, ص135 ) ( Hamadat, 2008, P135 ..) واســتنادا إلــى االعتقــاد الإلــامن بــان عالقــة الوــرد بالعمــل أساســية وانــال يمإلــن تحديــد اتجــا الوــرد نحــو العمــل بشــإلل جيــد وســواا إلــان اتجاهــا ناجحــا أم داشــال, وقــد إلتــب (هيرزبــرج) دــي الســؤال انتــي: مــا الــذي يريــد اأدــراد مــن عملهــم؟ وطلــب مــن اأدــراد وصــف المواقــف التــي شعروا ديهـا بمشـاعر جيـدة أو مشـاع ,ر سـيئة تجـا عملهـم وقـد تـم ترتيـب اإلجابـات وتصـنيوها ومــن ــالل ذلــك توصــل(هيرزبرج) بــان إجابــات اأدــراد الــذين اظهــروا مشــاعر الســعادة تجــا ( عملهـم ت تلـف بداللـال معنويـة عـن تلـك التـي أظهـرت مشـاعر سـيئةRobbins , 2003 , P159 .) 5 . :نظام الترقيةPromotion system مــن اأمــور التــي يهــتم بهــا الموظــف هــو موضــوع الترقيــة إذ أنهــا تمتــل جــزاا مهمــا مــن طموحاتـــال التـــي ي ســـعى إلـــى تحقيقهــــا دـــي حياتـــال الوظيويـــة, دهــــي تجمـــ بـــين المزايـــا الماديــــة والمعنويـــة ويرتوـــ مرإلـــز الـــوظيوي إلمـــا يترتـــب عليهـــا أيضـــا زيـــادة دـــي الراتـــب وتعـــد الحجـــر اأساس دي إيجاد دمة حإلومية مستقرة, ويسعى نظام الترقية إلى تحقيق جملة من اأهداف منها تعزيز روح التواؤل بين الموظ وين وزيادة تقتهم بالمستقبل وتأمين استمرارية العمل واغيجاد جو من التنـادس بـين العـاملين بمـا يسـهم دـي ددعهـم وحوـزهم وإلـذلك شـعور المـوظوين باأمـان ,واالســـتقرار نتيجـــة تحقـــيقهم للتقـــدم المســـتمر دـــي عملهـــم ومســـتوى معيشـــتهم (العـــزاوي وجـــواد 9212, ص494 – 495 ( ) Al-Azzawi and Jawad, 2010, P494-495 .) 6 . ًثانيا:: نظرية التوافق بين الفرد والبيئة ( Pervin, 1968 ) لقــد أشــارت بيــردن (Pervin) أن الســلوك البشــري ال يمإلــن أن يوهــم مــن ــالل البيئــة وحدها أو الورد وحد واغنما يمإلن أن يوهم دي ضوا العالقة المتدا لة بين الورد والبيئة, ولذلك ,دـــان هـــذ النظريـــة ظهـــرت إلـــى الوجـــود لإلـــي تتوـــق مـــ صـــائص الوـــرد و صـــائص البيئـــة دالنظرية تتنب أ بان اأدراد ي تزل لديهم الشعور باالستقرار النوسي والمهنـي عنـدما يإلـون لـديهم سـوا التوادــق بــين صــائص الوــرد وال صــائص المرتبطــة بالمهنــة, دــي حــين يحــدث االســتقرار النوســي والمهنــي عنــدما يإلــون هنــاك توادــق بــين الوــرد وال صــائص المرتبطــة بالعمــل, دعنــدما يحصل الورد على الدعم (ا لمال, الدعم المعنوي, درص االنجاز) دهذا يعد إلاديا بدور إلشباع ,دواد الورد (الدبعي9223, ص57 ( ) Al-Dabaie, 2003, P57 .) 951 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ ( وأشار هاريسـونHarrison, 1978 ) إلـى أن النظريـة طرحـت نـوعين مـن التوادـق بـين الوــرد والبيئــة: النــوع اأول: هــو المــدى الــذي تواإلــب ديــال مهــارات الوــر د وقدراتــال أعبــاا العمــل ومتطلباتــال, والنــوع التــاني: هــو المــدى الــذي تعمــل ديــال بيئــة العمــل علــى إشــباع حاجــات الوــرد ,(عباس9228, ص191 ( ) Abbas, 2008, P121 ). ويتضح مـن ذلـك انـال إذا لـم تشـب بيئة العمل حاجات الورد الذي يمتلك مهارات وقدراتال دان ذلك يسـبب سـوا التوادـق مـ المهنـة ( Harrison , 1978 , P177 .) ي تعد دإلرة التوازن التنظيمي من اإلسهامات التي قدمها (هربرت سايمون) وهي تقـوم علـى أســـاس التـــوازن بـــين المغريـــات التـــي تقـــدمها المنظمــــة للعـــاملين وبـــين مـــا يقدمـــال اأدـــراد مــــن إسهام ات ومجهودات لهذ المنظمـة, دـالورد يسـعى لتحقيـق احتياجاتـال الش صـية ويقبـل العمـل دـي المنظمـة وينوـذ مـا يطلـب منـال مـادام هــذا العمـل يـؤدي إلـى تحقيـق إشـباع حاجاتـال وأهدادــال .الش صية إن التنظيم من وجهة نظر (سايمون) هو نظام موتـوح يسـعى للوصـول إلـى حالـة التـوازن واالستقرار بي ن ما يقدمال الورد من مساهمات وبين ما تقدمال المنظمة من مغريات وحوادز لقاا هذ المساهمات, واغذا ا تل هـذا التـوازن دـان التنظـيم يعجـز عـن االسـتقرار أو البقـاا, وتحـدث حالـة مـن عـدم التـوازن واالســتقرار عنـدما توـوق مسـاهمات اأدــراد ومجهـوداتهم علـى مـا تقدمــال المنظمة من حوادز و مغريات, حيث ينزع اأدراد إلـى التقليـل مـن مسـاهماتهم للمنظمـة, ونوـس الشــــــيا يحــــــدث إذا داقــــــت المغريــــــات والحــــــوادز التــــــي تقــــــدمها المنظمــــــة علــــــى المجهــــــودات والمســـاهمات التـــي يقدمـــال اأدـــراد, إذ إن التنظـــيم دـــي هـــذ الحالـــة يتحمـــل إللوـــة أعلـــى, وبـــذلك تحــدث حالــة عــدم التــوازن, التــي تقــود أيضــا إلــى عــدم اســتمرار أو بقــاا المنظمــة, ولإلــي تبقــى :حالة التوازن التنظيمي دان المنظمة تسعى إلى استعادة هذا التوازن من الل أسلوبين 1 . .زيادة مساهمات اأدراد لتتعادل م حجم المغريات والحوادز التي تقدمها المنظمة 9 . adaptation اهتمت هذ النظرية بدراسة سلوك الموظوين العاملين لمعردة مدى تإليوهم واستقرارهم دـي أعمالهم الوظيوية التي يمارسونها, ويرى أصحاب هذ النظريـة أن سـلوك الموظـف أو العامـل دي الع مل يعتمد على درجة التإلييف والتطابق بين ش صية الموظوين (أي صـوات المـوظوين أو العاملين) وبيئة العمل (أي صوات العمل أو الوظيوة والمنظمـات), ويعـد االنسـجام العامـل الرئيس دي توسير تإليف الورد مـ بيئـة العمـل إلمـا يتجسـد دـي شـعور بالقناعـة واالسـتقرار دـي ,الوظيوة (السالم1997, ص64 – 65 ( ) Alssalim, 1997, P64-65 .) وتؤإلـد نظريـة التإليـف الـوظيوي انـال يجـب علـى اإلدارة أن تهـتم بأبعـاد ش صـية الموظــف الشاملة وذلك الن هذ اأبعاد لهـا دور إلبيـر دـي تحديـد اسـتجابة هـذا الموظـف للمنظمـة التـي ينتمــي إليهــا ســواا إلانــت هــذ االســتجابة ايجابيــة أو ســلبية ويقصــد بأبعــاد الش صــية الصــوات الش صــية إلالحاجــ )ات اإلنســانية, الــدواد , القــدرات (الــذإلاا, المواهــب, االســتعدادات اأ ــرى ,والقــيم والمواقــف واالتجاهــات التــي يعتنقهــا الوــرد ويــؤمن بهــا وإلــذلك الوــروق الورديــة (العــديلي 1995, ص123 – 124 ( ) Al-Adaili, 1995, P103-104 .) اهتمت هذ النظرية بدراسة سلوك الموظوين العاملين لمعردة مدى تإليوهم واستقرارهم دـي أعمالهم الوظيوية التي يمارسونها, ويرى أصحاب هذ النظريـة أن سـلوك الموظـف أو العامـل دي الع مل يعتمد على درجة التإلييف والتطابق بين ش صية الموظوين (أي صـوات المـوظوين أو العاملين) وبيئة العمل (أي صوات العمل أو الوظيوة والمنظمـات), ويعـد االنسـجام العامـل الرئيس دي توسير تإليف الورد مـ بيئـة العمـل إلمـا يتجسـد دـي شـعور بالقناعـة واالسـتقرار دـي ,الوظيوة (السالم1997, ص64 – 65 ( ) Alssalim, 1997, P64-65 .) م (( ) ) وتؤإلـد نظريـة التإليـف الـوظيوي انـال يجـب علـى اإلدارة أن تهـتم بأبعـاد ش صـية الموظــف الشاملة وذلك الن هذ اأبعاد لهـا دور إلبيـر دـي تحديـد اسـتجابة هـذا الموظـف للمنظمـة التـي ينتمــي إليهــا ســواا إلانــت هــذ االســتجابة ايجابيــة أو ســلبية ويقصــد بأبعــاد الش صــية الصــوات الش صــية إلالحاجــ )ات اإلنســانية, الــدواد , القــدرات (الــذإلاا, المواهــب, االســتعدادات اأ ــرى ,والقــيم والمواقــف واالتجاهــات التــي يعتنقهــا الوــرد ويــؤمن بهــا وإلــذلك الوــروق الورديــة (العــديلي 1995, ص123 – 124 ( ) Al-Adaili, 1995, P103-104 .) :الفصل الثالث: منهجية البحث واجراءاته يستعرض هذا ا لوصل اإلجرااات التي قام بها الباحـث, واعتمـد الباحـث المـنهج الوصـوي إلونال يعد من أدضل المناهج دـي دراسـة الظـواهر التـي تعتمـد علـى العالقـات االرتباطيـة وإلمـا :يأتي :أوال: مجتمع البحث تإلــوّن مجتمــ البحــث المرشــدين التربــويين دــي مــديريات تربيــة محادظــة بغــداد (الرصــادة اأو( لــى, والرصــادة التانيــة, والرصــادة التالتــة) مــن (الــذإلور واإلنــاث), والبــالا عــددهم879 ) ( مرشد تربوي بواق391 () ذإلور و481 ( ) اناث للعام الدراسي9217 - 9218 ). ي( ) خامسددا: نظريددة التكيددف الددوظيفي : ( Devid et al, 1966 ) Theory of work adaptation خامسددا: نظريددة التكيددف الددوظيفي : ( Devid et al, 1966 ) Theory of work adaptation ًثانيا:: نظرية التوافق بين الفرد والبيئة ( Pervin, 1968 ) إنقاص المغريات التي تقدمها المنظمـة لتتناسـب مـ حجـم المسـاهمات التـي يقـدمها اأدـراد ,(زويلف والعضايلال1996, ص79 – 82 ( ) Zwailf and Adayleh .) رابع ًا : )نظرية التناقض بين الفرد والتنظيم (نظرية النموذج المختمط: The Mix model (Chris Argyris, 1964) توصـل (إلــرس رجيـرس) إلــى توسـير ســلوك الوـرد دا ــل التنظـيم مــن ـالل دراســ ة مســألة التناقض بين حاجات الورد وحاجات المنظمة, واعتبـر أن أسـاس المشـإلالت دـي المنظمـة هـو التنظــيم الرســمي الهرمــي التقليــدي الــذي تبنتــال المــدارس الإلالســيإلية, وودــق هــذا التنظــيم دــان الورصة محدودة للورد للتطور واإلبداع والمشارإلة, حيث أن التنظيم التقليدي دي رأيال يحاصـر إن التنظيم من وجهة نظر (سايمون) هو نظام موتـوح يسـعى للوصـول إلـى حالـة التـوازن واالستقرار بي ن ما يقدمال الورد من مساهمات وبين ما تقدمال المنظمة من مغريات وحوادز لقاا هذ المساهمات, واغذا ا تل هـذا التـوازن دـان التنظـيم يعجـز عـن االسـتقرار أو البقـاا, وتحـدث حالـة مـن عـدم التـوازن واالســتقرار عنـدما توـوق مسـاهمات اأدــراد ومجهـوداتهم علـى مـا تقدمــال المنظمة من حوادز و مغريات, حيث ينزع اأدراد إلـى التقليـل مـن مسـاهماتهم للمنظمـة, ونوـس الشــــــيا يحــــــدث إذا داقــــــت المغريــــــات والحــــــوادز التــــــي تقــــــدمها المنظمــــــة علــــــى المجهــــــودات والمســـاهمات التـــي يقدمـــال اأدـــراد, إذ إن التنظـــيم دـــي هـــذ الحالـــة يتحمـــل إللوـــة أعلـــى, وبـــذلك تحــدث حالــة عــدم التــوازن, التــي تقــود أيضــا إلــى عــدم اســتمرار أو بقــاا المنظمــة, ولإلــي تبقــى :حالة التوازن التنظيمي دان المنظمة تسعى إلى استعادة هذا التوازن من الل أسلوبين 1 . .زيادة مساهمات اأدراد لتتعادل م حجم المغريات والحوادز التي تقدمها المنظمة 9 . إنقاص المغريات التي تقدمها المنظمـة لتتناسـب مـ حجـم المسـاهمات التـي يقـدمها اأدـراد ,(زويلف والعضايلال1996, ص79 – 82 ( ) Zwailf and Adayleh .) The Mix model (Chris Argyris, 1964) توصـل (إلــرس رجيـرس) إلــى توسـير ســلوك الوـرد دا ــل التنظـيم مــن ـالل دراســ ة مســألة التناقض بين حاجات الورد وحاجات المنظمة, واعتبـر أن أسـاس المشـإلالت دـي المنظمـة هـو التنظــيم الرســمي الهرمــي التقليــدي الــذي تبنتــال المــدارس الإلالســيإلية, وودــق هــذا التنظــيم دــان الورصة محدودة للورد للتطور واإلبداع والمشارإلة, حيث أن التنظيم التقليدي دي رأيال يحاصـر 959 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) ش صــية الوــرد وال يســتطي إشــباع حاجاتــال الســيما الحاجــة إلــى تحقيــق الــذات, وهــو مــا ي لــق حالـــة مـــن التـــوتر واإلحبـــاط وعـــدم اســـتقرار الوـــرد دـــي المنظمـــة, وبـــذلك يحـــدث التنـــاقض بـــين أهـداف الوـرد الــذي يسـعى لتحقيقهـا وأهــداف المنظمـة التــي تسـعى لتحقيقهـا مــن ـالل التنظــيم الهرمي( Argyris , 1964 , P159 .) adaptation :ثانياً: عينة البحث لقد أعتمد الباحث دي ا تيار عينة بحتال على الطريقـة العشـوائية البسـيطة ذات االسـلوب المت ســاوي( اذ بلــا عــدد أدــراد العينــة122 ( ) مرشــد تربــوي بواقــ52 () للــذإلور و52 ) لدنــاث 953 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ ا تيروا من مدارس مديرية تربية الرصادة اأولى والتانية والتالتـة دـي محادظـة بغـداد والجـدول ( 1 .) يوضح ذلك ( جدول4 )عينة البحث االساسية موزعة عمى وفق متغير جنس المدرسة والمرشد جنس المدرسة عدد المدارس عدد المرشدين ذإلور 52 52 إناث 52 52 المجموع 122 122 :ثالثاً: أداتا البحث جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ ا تيروا من مدارس مديرية تربية الرصادة اأولى والتانية والتالتـة دـي محادظـة بغـداد والجـدول ( 1 .) يوضح ذلك ( جدول4 )عينة البحث االساسية موزعة عمى وفق متغير جنس المدرسة والمرشد جنس المدرسة عدد المدارس عدد المرشدين ذإلور 52 52 إناث 52 52 المجموع 122 122 :ثالثاً: أداتا البحث جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ ا تيروا من مدارس مديرية تربية الرصادة اأولى والتانية والتالتـة دـي محادظـة بغـداد والجـدول ( 1 .) يوضح ذلك ( جدول4 )عينة البحث االساسية موزعة عمى وفق متغير جنس المدرسة والمرشد جنس المدرسة عدد المدارس عدد المرشدين ذإلور 52 52 إناث 52 52 المجموع 122 122 :ثالثاً: أداتا البحث تحقيقـــا أهـــداف البحـــث قـــام الباحـــث بتبنـــي مقيـــاس االســـناد االجتمـــاعي المعـــد مـــن قبـــل ,(العامري9229 ( ), وتبني مقياس االسـتقرار المهنـي المعـد مـن قبـل,العيـداني9211), وديمـا :يلي استعراض لدجرااات ,من أجل قياس متغيرات البحث الحالي تبنى الباحـث مقياس االسدناد االجتمداعي المعـد مـــن قبـــل ,(العـــامري9229 ( ), اذ يتـــألف المقيـــاس بصـــيغتال النهائيـــة مـــن32 ) دقـــرة, ويقابلهـــا مس بدائل هي (دائما, ،البا, احيانا, نادرا, ال ابدا) وإلانت درجات( البدائل1,9,3,4,5 ) ( هذا بالنسبة للوقرات االيجابية, اما الوقـرات السـلبية دإلانـت درجـات البـدائل5,4,3,9,1 .) أمـا مقياس االستقرار المهني ( دقد قام الباحث بتبني المقياس المعـد مـن قبـل,العيـداني 9211 ,) ( أذ تــألف المقيــاس مــن52 ,) دقــرة بصــيغتال النهائيــة ويقابلهــا مــس بــدائل هــي (تنطبــق علــي دائما, تنطبق علي ،البا, تنطبق علي احيانا, تنطبق علي نادرا, ال تنطبق علي ابدا) وإلانت ( درجـــات البـــدائل1,9,3,4,5 ) هـــذا بالنســـبة للوقـــرات االيجابيـــة, امـــا الوقـــرات الســـلبية دإلانـــت ( درجات البدائل5,4,3,9,1 .)  ( القيمة الجدولية عند مستوى داللة,0,0 ( ) وبدرجة حرية05 ( ) تساوي50,, ) :صالحية الفقرات )لغرض التعرف على مدى صالحية الوقرات دقد تـم عـرض مقيـاس (االسـناد االجتمـاعي ومقيـاس (االسـتقرار المهنـي) علـى مجموعـة مــن الم تصـين دـي التربيـة وعلـم الـنوس واإلرشــاد النوســـي والتوجيـــال التربـــوي, لتحديـــد مـــدى صـــالحية الوقـــرات, ودـــي ضـــوا اراا الم تصـــين تـــم اإلبقاا على الوقرات التي حصلت على نسـبة اتوـاق82 دـأإلتر وبنـااا علـى ذلـك تـم اإلبقـاا% علــى جميــ الوقــرات دــي المقياســين مــ بعــض التعــديالت دــي الصــيا،ة اللغويــة والمعنــى بمــا .يتالام م طبيعة العينة التي تتم عليها الدراسة الحالية لقــد طبــق مقياســي االســناد االجتمــاعي واال( ســتقرار المهنــي علــى122 ) مرشــد ومرشــدة تربوية واعتمدت هذ العينة إل،راض تحليل الوقرات وإلـذلك اعتمـدت للتطبيـق االساسـي لعينـة 954 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) البحــث, وان الهــدف مــن هــذا االجــراا هــو االبقــاا علــى الوقــرات الجيــدة دــي المقياســين وقــد تــم استعمال اسلوب المجموعتين المتطردتين بوصوال اجراا لتحليل ا:لوقرات وإلما يأتي :المجموعتين المتطرفتين :لغرض اجراا التحليل بهذا االسلوب تم اتباع ال طوات االتية - .تحديد الدرجة الإللية لإلل استمارة من المقياسين - .ترتيب االستمارات من اعلى درجة الى اقل درجة لإلل مقياس - تعيــين97 مــن االســتمارات الحاصــلة علــى الــدرجات العليــا% دــي إلــل مقيــاس و97 مــن% االســتمارات الحاصـــلة علـــى الـــدرجات الـــدنيا, تمـــتالن مجمـــوعتين بـــأإلبر حجـــم واقصـــى تمـــايز ( ممإلن, وإلان عدد االستمارات دي إلل مجموعة97 ) استمارة لإلل مقياس, تم طبـق اال تبـار التائي لعينتين مستقلتين ال تبار الورق بين متوسط درجات المجموعة العليا والمجموعة الـدنيا دــي إلــل دقــرة مــن المقياســين, وعــدت القيمــة التائيــة مؤشــرا لتمييــز إلــل دقــرة بمقارنتهــا بالقيمــة ( الجدولية البالغة9022 )  وقد إلانت الوقرات دـي مقيـاس (االسـناد االجتمـاعي) جميعهـا مميـزة ( عند مسـتوى داللـة2.25 ( ) وبـذلك اصـبح المقيـاس بصـورتال النهائيـة يتإلـون مـن32 ,) دقـرة ( أمــا دقــرات مقيــاس (االســتقرار المهنــي) دقــد إلانــت جميعهــا مميــزة عنــد مســتوى داللــة2025 ) وبـذلك أصــبح المقيــاس بصــورتال النهائيــة يت( إلــون مــن52 ( ) دقــرة. :صالحية الفقرات والجــدولين9 , 3 ) يوضــحا .ذلك ( جدول2 ) القوة التمييزية لفقرات مقياس االسناد االجتماعي ت المجموعة العميا المجموعة الدنيا القيمة التائية المحسوبة مستوى الداللة (0.05) المتوسط الحسابي االنحراف المعياري المتوسط الحسابي االنحراف المعياري 4 40,4 4044 30,8 4034 6034 2 3076 40,4 2099 4045 5044 3 407, ,072 3026 ,095 3054 4 4094 4043 3034 4044 4023 5 40,2 4036 3045 ,098 3088 6 4052 ,078 3099 40,4 40,8 7 3094 4022 204, 40,2 5087 8 3044 4034 2047 4044 4027 9 305, 4048 2034 4044 60,9 955 0208 / م8113 هـ جمةل الداب / العدد831 (أيلول) 0208 / م8113 هـ 956 4, 4057 ,082 3098 4045 4037 44 3044 40,,2 20,5 4042 607, 42 30,4 4042 2044 4045 60,3 43 4039 4042 40,9 40,3 2042 44 3094 4045 2033 4046 3073 45 4058 ,086 3066 40,7 4044 46 3063 4044 2064 4033 6048 47 3044 4035 2059 4028 5045 48 3037 4054 2047 4034 5054 49 4026 40,9 3056 4024 4076 2, 3063 4032 2056 4024 6099 24 3056 4034 2046 4045 7025 22 4025 4043 3042 4036 7044 23 3058 4043 2056 4048 6068 24 4042 402, 3032 4024 5032 25 4027 4044 2063 4035 5046 26 4094 4046 2032 4046 4073 27 4063 4033 2055 4024 7099 28 4094 4023 20,9 4043 6087 29 4063 4033 2055 4024 7099 3, 4056 4035 2045 4045 8025 ( جدول3 ) القوة التمييزية لفقرات مقياس االستقرار المهني ت المجموعة العميا المجموعة الدنيا القيمة التائية المحسوبة مستوى الداللة 0.05 المتوسط الحسابي االنحراف المعياري المتوسط الحسابي االنحراف المعياري 4 3064 203, 2086 4092 3079 2 407, 2034 3076 4084 4079 3 4093 4053 3045 20,2 8046 4 4055 ,096 3076 4033 4034 956 0208 / م8113 هـ جمةل الداب / العدد831 (أيلول) 0208 / م8113 هـ 9 5 4032 4065 3066 4094 3062 6 4075 4074 3047 20,7 6084 7 4038 4028 304, 4068 5092 8 40,4 4054 3047 4074 3044 9 4066 4039 3036 4087 7047 4, 4042 4022 3042 4077 60,4 44 4075 4059 3064 4083 6024 42 4079 4075 3094 2042 4065 43 4077 4062 3036 4087 7085 44 4053 ,083 3085 4048 4065 45 4088 4059 3064 2042 60,9 46 40,4 4057 3045 409, 4096 47 4077 4062 3084 4082 5038 48 4042 4024 3024 4083 70,3 49 40,3 4056 3069 4094 7047 2, 4082 4064 3025 4052 3033 24 4065 4047 3079 4062 50,8 22 4069 4062 3047 4064 2099 23 4025 4096 3046 4044 5059 24 4093 4053 3045 20,2 9046 25 4055 ,096 3076 4033 5034 26 4032 4065 3066 4094 4062 27 4075 4074 3047 20,7 7084 28 4038 4028 304, 4068 6092 29 40,4 4054 3047 4074 4044 3, 4066 4039 3036 4087 8047 34 4042 4022 3042 4077 70,4 32 4075 4059 3064 4083 7024 33 4079 4075 3094 2042 5065 957 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ 34 4077 4062 3036 4087 8085 35 4083 4053 3045 20,2 4,046 36 4054 4096 3076 4033 6034 37 4032 4065 3066 4094 5062 38 4075 4074 3047 20,7 8084 39 4038 4028 304, 4068 7092 4, 40,4 4054 3047 4074 5044 44 4066 4039 3036 4087 9047 42 4042 4022 3042 4077 80,4 43 4075 4059 3064 4083 8024 44 4079 4075 3094 2042 6065 45 4056 4073 3098 4034 9085 46 4093 4053 3045 20,2 4,046 47 4026 40,9 3056 4024 7076 48 3063 4032 2056 4024 9099 49 3056 4034 2046 4045 4,025 5, 4025 4043 3042 4036 4,044 :)عالقة درجة الفقرة بالدرجة الكمية (االتساق الداخمي 0208 / م8113 هـ استعمل الباحث معامل ارتباط بيرسـون السـت راج العالقـة االرتباطيـة بـين درجـة إلـل مـن دقرات المقياس والدرجة الإللية, وقد إلانت معـامالت االرتبـاط دالـة داللـة معنويـة لـدى مقارنتهـا ( بالقيمة الجدولية البالغة20195 ( ) عند مستوى داللة2025 ( ) وبدرجة حرية98 ) دي مقياس (االســـنا( د االجتمـــاعي), وجميعهـــا دالـــة دـــي مقيـــاس (االســـتقرار المهنـــي), والجـــدولين4 , 5 ) .يوضحا ذلك ( جدول4 ) معامالت ارتباط فقرات مقياس االسناد االجتماعي بالدرجة الكمية الفقرة معامل االرتباط الفقرة معامل االرتباط الفقرة معامل االرتباط 4 ,0354 44 ,0379 24 ,0252 2 ,026, 42 ,0364 22 ,0263 3 ,0244 43 ,0343 23 ,028, 4 ,0242 44 ,0233 24 ,0224 958 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ 5 ,0244 45 ,0227 25 ,0273 6 ,0239 46 ,0329 26 ,0242 7 ,0345 47 ,0347 27 ,0363 8 ,0278 48 ,0324 28 ,0258 9 ,0394 49 ,0286 29 ,0359 4, ,0349 2, ,0264 3, ,0327 ( جدول5 ) معامالت ارتباط فقرات مقياس االستقرار المهني بالدرجة الكمية الفقرة معامل االرتباط الفقرة معامل االرتباط الفقرة معامل االرتباط 4 ,0243 48 ,03,, 35 ,03,7 2 ,0245 49 ,0483 36 ,0276 3 ,0338 2, ,0287 37 ,028, 4 ,0234 24 ,048, 38 ,0294 5 ,0473 22 ,0297 39 ,0232 6 ,0264 23 ,0295 4, ,0248 7 ,0288 24 ,0278 44 ,0247 8 ,0272 25 ,03,4 42 ,.542 9 ,.392 26 ,.733 43 ,.6,6 4, ,.427 27 ,.539 44 ,.64, 44 ,.484 28 ,.496 45 ,.533 42 ,.599 29 ,.484 46 ,.529 43 ,.332 3, ,.594 47 ,.544 44 ,.399 34 ,.689 48 ,.423 45 ,.3,9 32 ,.564 49 ,.4,, 46 ,.343 33 ,.48, 5, ,.273 47 ,.288 34 ,.693 959 0208 / م8113 هـ الصدق الظداهريFace Validity : يعـد الصـدق الظـاهري االشـارة الـى مـا يبـدو ان المقيـاس يقــيس مــا وضــ مــن اجلــال اي مــدى مــا يتضــمن دقــرات يبــدو انهــا علــى صــلة بــالمتغير الــذي يقــاس وان مضــمون المقيــاس متوــق مــ الغــرض منــال, وهــو المظهــر العــام للمقيــاس مــن حيــث ,نـوع الموـردات وإليويـة صـيا،تها ومـدى وضـوحها (االمـام1992, ص132 ( ) Al'amam, 1990, P130) وقد تحقق هذا النوع مـن الصـدق دـي هـذ المقياسـين عنـدما عرضـت دقـرات إلــــل مقيــــاس علــــى مجموعــــة مــــن ال بــــراا والم تصــــين للحإلــــم علــــى صــــالحيتهما دــــي قيــــاس .ال اصية المراد قياسها :الثباتReliability تـم حسـاب التبـات لمقياسـيّ البحـث الحـالي بطريقـة ألوـا إلرونبـاخ(Cronbach Alfa) , اذ تعد هذ الطريقة موضـلة لقيـاس التبـا ت دهـي تقـيس االتسـاق الـدا لي والتجـانس بـين دقـرات المقياس(Anstasi, 1997,P95) أي أن الوقرات جميعها تقيس دعالً ال اصـية نوسـها وهـذا يتحقق عندما تإلون الوقرات مترابطة م بعضها البعض دا ل اال تبار إلذلك ارتباط إلـل دقـرة مــ اال تبـــار إللـــال, وتـــم تطبيـــق معادلـــة ألوـــا إلرونبـــاخ( علـــى درجـــات42 ) مرشـــد ومرشـــدة تـــم اســت راج اســتماراتهم عشــوائياً مــن عينــة تطبيــق التحليــل االحصــائي, وبلــا معامــل تبــات ألوــ ا ( 2078 () لمقياس (االسناد االجتماعي), و2076 .)) لمقياس (االستقرار المهني :رابعاً: التطبيق النهائ بعد استإلمال الباحث أداتي البحث والتحقـق مـن صـدقه ما وتباتهمـا وتمييـز الوقـرات, قـام بتطبيقهما بددعة واحدة على عينة البحث من ـالل تقـديم المقياسـين معـاً للموحوصـين والبـالا ( عددهم122 ) مرشد ومرشدة تم ا تيارهم عشوائياً من مدارس مديرية تربية الرصادة (االولـى .ًوالتانية والتالتة) دي محادظة بغداد, إلما ذإلر سابقا ًخامسا:: الوسائل اإلحصائية :استعمل الباحث الوسائل اإلحصائية انتية لتحقيق اهداف البحث  ( اال تبار التائيT-test .) لعينتين مستقلتين  .معامل ارتباط بيرسون  .معادلة ألواإلرونباخ 962 962 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول) جمةل الآداب / العدد831 (أ يلول)  ( اال تبار التائيT-test .) لعينة واحدة  ( اال تبار التائيT-test .) لعينة واحدة  .القيمة التائية لداللة معامالت االرتباط  معادلة النسبة .المئوية ا:لهدف األول: التعرف عمى مستوى االسناد االجتماعي لدى المرشدين التربويين أظهــــرت نتــــائج التحليــــل اإلحصــــائي إن متوســــط درجــــات العينــــة علــــى مقيــــاس االســــناد ( االجتماعي بلا121.18 ( ), وبانحراف معياري مقدار5.49), بينما إلان المتوسط ال ورضي ( للمقيــاس92 ), وباســتعمال اال تبــار التــائي( t-test ) لعينــة واحــدة, ظهــر أن القيمــة التائيــة ( المحســوبة إلانــت13.79 ( ) وعنــد مقارنتهــا بالقيمــة الجدوليــة البالغــة1096 ) تبــين أنهــا دالــة ( إحصائياً عند مستوى2025 ( ) وبدرجة حرية99 ), مما يعني أن أدراد عينة البحث يتمتعـون باإل( سناد أالجتماعي, إلما دي الجدول6 .) ب إل(إي ج و ي جا) ( جدول6 ) نتيجة االختبار التائي لمفرق بين المتوسط الحسابي والفرضي لعينة البحث في االسناد االجتماعي العينة المتوسط الحسابي االنحراف المعياري المتوسط الفرضي القيمة التائية مستوى الداللة ( ,0,5 ) المحسوبة الجدولية 4,, 4,4.48 5.42 9, 43.72 4096 دالة :الهدف الثاني: التعرف عمى مستوى االستقرار المهني لدى المرشدين التربويين إا يا ى ي أظهـــرت نتـــائج التحليـــل اإلحصـــائي إن متوســـط درجـــات العينـــة علـــى مقيـــاس االســـتقرار ( المهنــي بلــا149.88 ( ), وبــانحراف معيــاري مقــدار6.29 ), بينمــا إلــان المتوســط الورضــي ( للمقياس152 ), وباسـتعمال اال تبـار التـائي( t-test ) لعينـة واحـدة, ظهـر أن القيمـة التائيـة ( المحسوبة إلانت1.54) وع( ند مقارنتها بالقيمة الجدولية البالغة1096 ) تبين أنهـا ،يـر دالـة ( إحصـــائياً عنـــد مســـتوى2025 ( ), وبدرجـــة حريـــة99 ), ممـــا يعنـــي أن أدـــراد عينـــة البحـــث ال ( يتمتعون باالستقرار المهني, إلما دي الجدول7 .) ( جدول7 ) نتيجة االختبار التائي لمفرق بين المتوسط الحسابي والفرضي لعينة البحث في االستقرار المهني العينة المتوسط الحسابي االنحراف المعياري المتوسط الفرضي القيمة التائية مستوى الداللة ( ,0,5 ) المحسوبة الجدولية 4,, 442.88 6.,9 45, 4.54 4096 غير دالة 961 جمةل الآداب / العدد831 (أ يلول) بعد تحليل النتائج باستعمال معامل ارتباط بيرسون أظهرت انـال ال توجـد عالقـة ارتباطيـال بــــين اإلســــناد االجتمــــاعي واالســــتقرار المهنــــي للعينــــة إلإلــــل, اذ بلغــــت قيمــــة معامــــل االرتبــــاط ( 20171 ( ), وهــي أصــغر مــن قيمــة بيرســون الجدوليــة البالغــة20195 ), وباســتعمال اال تبــار ( التائي لمعردة داللة معامل االرتباط بلغت القيمة التائية المحسوبة1011 ), وهي أصـغر مـن ( القيمة الجدولية1096 ( ) عند مستوى داللة2025 ( ) ودرجة حرية98 .) :االستنتاجات 1 -ان مواجهــة المشــإلالت التــي تواجــال المرشــدين ودقــدان المســاندة تجعــل مــن الصــع وبة القيــام .بواجباتال اتجا االدراد اال رين 1 -ان مواجهــة المشــإلالت التــي تواجــال المرشــدين ودقــدان المســاندة تجعــل مــن الصــع وبة القيــام .بواجباتال اتجا االدراد اال رين 9 - ان المرشـــد يإلـــون أإلتـــر قـــدرة علـــى اداا واحـــداث تعـــديالت دـــي ســـلوك الطلبـــة إللمـــا شـــعر .المرشد باالستقرار المهني :التوصيات 4 - .على مديريات التربية ان تشب بيئة العمل وحاجات االدراد العاملين دا ل المدرسة 9 - على ادارة المدارس.التعاون م المرشدين التربويين وتقديم المساندة النجاز المهام 3 - علــــى الإلــــادر التدريســــي االتصــــال بالمرشــــدين التربــــويين دــــي طلــــب المســــاعدة دــــي حــــل .مشإلالت الطلبة 4 - اقامــة العالقــات االيجابيــة مــ الطلبــة وذويهــم مــن قبــل المرشــدين التربــويين وتقــديم الــدعم واإلسناد االجتماعي الذي .ينتج من المشارإلة دي الندوات والمقابالت بينهما :المقترحات 1 - .اقامة دراسة مماتلة دي مديريات ا رى ومن محادظات ا رى 9 - .اجراا دراسة على مدراا المدارس لنوس المواهيم 3 - .اجراا دراسة لالستقرار المهني م متغيرات ا رى 4 - اجراا دراسة لدسناد االجتماعي م.متغيرات ا رى :المصادر 1 - اقامة دراسة مماتلة دي مديريات ا رى ومن محادظات ا رى 9 - .اجراا دراسة على مدراا المدارس لنوس المواهيم 3 - .اجراا دراسة لالستقرار المهني م متغيرات ا رى 4 - اجراا دراسة لدسناد االجتماعي م.متغيرات ا رى :المصادر  ( اإلبراهيمــي, جاســم محمــد حلبــوص9229 :) فاعميددة برنددامج أرشددادي لتحسددين المهددارات اإلرشددادية العامددة لممرشدددين التربددويين فددي المدددارس المتوسددطة فددي بغددداد رســالة ماجســتير ،يــر منشــورة كميددة .التربية/ ابن رشد, جامعة بغداد  ( اإلبراهيمــي, جاســم محمــد حلبــوص9229 :) فاعميددة برنددامج أرشددادي لتحسددين المهددارات اإلرشددادية العامددة لممرشدددين التربددويين فددي المدددارس المتوسددطة فددي بغددداد رســالة ماجســتير ،يــر منشــورة كميددة .التربية/ ابن رشد, جامعة بغداد  ( أرجايل, مايإلل1993 :) سيكولوجية السعادة ,, ترجمة ديصل عبد القادر يوسف, سلسلة عالم المعردة رقم175 ., الإلويت  ( أرجايل, مايإلل1993 :) سيكولوجية السعادة ,, ترجمة ديصل عبد القادر يوسف, سلسلة عالم المعردة رقم175 ., الإلويت 969 جمةل الآداب / العدد831 (أ يلول) جمةل الآداب / العدد831 (أ يلول)  ( االسـدي, ســعيد جاســم9229 :)التوافددق االجتمدداعي لدددى طمبددة الكميددات األهميددة , مجلــة إلليــة المــأمون الجامعة ( العدد8 .), العراق  ( االسـدي, ســعيد جاســم9229 :)التوافددق االجتمدداعي لدددى طمبددة الكميددات األهميددة , مجلــة إلليــة المــأمون الجامعة ( العدد8 .), العراق  ( اإلمـــام, مصـــطوى محمـــود, و ـــرون1992 :)التقدددويم والقيددداس ,, جامعـــة بغـــداد وزارة التعلـــيم العـــالي .والبحث العلمي  ( أبو شعيرة, الـد9228 :)التربية المهنية الفاعمة ومعمم الصدف , ط1 , مإلتبـة المجتمـ العربـي للنشـر . جمةل الآداب / العدد831 (أ يلول)  ( ربجيو, ي رونالد1999 :)المدخل إلى عمم النفس الصناعي والتنظيمدي , ترجمـة دـارس حلمـي, ط1 , دار الشروق للنشر وا.لتوزي , عمان, اأردن  ( ربجيو, ي رونالد1999 :)المدخل إلى عمم النفس الصناعي والتنظيمدي , ترجمـة دـارس حلمـي, ط1 , دار الشروق للنشر وا.لتوزي , عمان, اأردن إإ  ( زإلــري, عبــد الطيــف شــهاب9225 :)إدارة سددموك الفددرد فددي المنظمددة والمجتمددع , مرإلــز اأمــين للنشــر .والتوزي , صنعاا, اليمن  ( زويلف, مهدي والعضايلة, علي1996 :)إدارة المنظمة نظريات وسموك , ط1 , دار مجـدالوي للنشـر .والتوزي , عمان, اأردن  ( السالم, سالم محمد1997 :)الرضا الوظيفي لمعاممين في المكتبات الجامعية , مطبوعات مإلتبة الملـك .دهد الوطنية, الرياض, السعودية  ( شـــلتز, داون1983 :)نظريدددات الشخصدددية , ترجمـــة حمـــد دلـــي الإلربـــولي وعبـــد الـــرحمن القيســـي, وزارة .التعليم العالي والبحث العلمي, جامعة بغداد  ( شـــلتز, داون1983 :)نظريدددات الشخصدددية , ترجمـــة حمـــد دلـــي الإلربـــولي وعبـــد الـــرحمن القيســـي, وزارة .التعليم العالي والبحث العلمي, جامعة بغداد ا  الشناوي( , محمد محروس وعبد الرحمن, محمد السـيد1994 :) المساندة االجتماعية والصدحة النفسدية مراجعة نظرية ودراسات تطبيقية , ط1 ., مإلتبة االنجلو المصرية, القاهرة  الشناوي( , محمد محروس وعبد الرحمن, محمد السـيد1994 :) المساندة االجتماعية والصدحة النفسدية مراجعة نظرية ودراسات تطبيقية , ط1 ., مإلتبة االنجلو المصرية, القاهرة  ( العامري, علي محسن ياس9229 :) االسناد االجتماعي وعالقته باإلجهاد لدى المرشدين التربدويين , رسالة ماجستير ،ير منش.ورة, إللية التربية, الجامعة المستنصرية  ( العامري, علي محسن ياس9229 :) االسناد االجتماعي وعالقته باإلجهاد لدى المرشدين التربدويين , رسالة ماجستير ،ير منش.ورة, إللية التربية, الجامعة المستنصرية إ  ( العـــاني, نـــزار محمـــد ســـعيد1989 :) أضدددواء عمدددى الشخصدددية اإلنسدددانية , ط1 , دار الشـــؤون التقاديـــة .العامة, بغداد  ( عباس, علي9228 :)إدارة الموارد البشرية الدولية , إتراا للنشر والتوزي , ط1 ., عمان, اأردن  ( عبد الرحمن, محمد السيد1998 :)نظريات الشخصية., دار قباا للنشر, القاهرة  ( العــديلي, ناصـــر محمـــد1995 :)السدددموك اإلنسددداني والتنظيمدددي , منظـــور إللـــي مقـــارن, اإلدارة العامـــة .للبحوث, جدة, السعودية  ( عباس, علي9228 :)إدارة الموارد البشرية الدولية , إتراا للنشر والتوزي , ط1 ., عمان, اأردن  ( عبد الرحمن, محمد السيد1998 :)نظريات الشخصية., دار قباا للنشر, القاهرة  ( عباس, علي9228 :)إدارة الموارد البشرية الدولية , إتراا للنشر والتوزي , ط1 ., عمان, اأردن  ( عبد الرحمن, محمد السيد1998 :)نظريات الشخصية., دار قباا للنشر, القاهرة  ( العــديلي, ناصـــر محمـــد1995 :)السدددموك اإلنسددداني والتنظيمدددي , منظـــور إللـــي مقـــارن, اإلدارة العامـــة .للبحوث, جدة, السعودية (ي ر ن ب)ي ري ر ر ر ب  ( العــديلي, ناصـــر محمـــد1995 :)السدددموك اإلنسددداني والتنظيمدددي , منظـــور إللـــي مقـــارن, اإلدارة العامـــة .للبحوث, جدة, السعودية  ( العــــزاوي, نجــــم عبــــد اس وجــــواد, عبــــاس حســــين9212 :) تطددددور إدارة المددددوارد البشددددرية المفهددددوم اإلستراتيجية الموقع التنظيمي , ط1 ., دار اليازوري العلمية للنشر والتوزي , عمان, اأردن  ( العــــزاوي, نجــــم عبــــد اس وجــــواد, عبــــاس حســــين9212 :) تطددددور إدارة المددددوارد البشددددرية المفهددددوم اإلستراتيجية الموقع التنظيمي , ط1 ., دار اليازوري العلمية للنشر والتوزي , عمان, اأردن  ( عوض, عباس محمـود1987 :)دراسات في عمم الدنفس الصدناعي والمهندي ,, دار المعردـة الجامعيـة .اإلسإلندرية  ( العيداني, عبد الإلريم محسـن9211 :) االستقرار المهني وعالقته بأنماط الشخصية والتوافق االسدري , .اطروحة دإلتورا ،ير منشورة, إللية التربية, الجامعة المستنصرية  ( العيداني, عبد الإلريم محسـن9211 :) االستقرار المهني وعالقته بأنماط الشخصية والتوافق االسدري , .اطروحة دإلتورا ،ير منشورة, إللية التربية, الجامعة المستنصرية ا  ( الزاروس, ريتشارد1981 :)الشخصية., ترجمة: سيد محمد ،نيم, دار الشروق, بيروت  ( محمــد, يوســف عبــد الوتــاح1999 :)الضددغوط النفسددية لدددى المعممددين وحاجدداتهم اإلرشددادية , مجلــة البحوث التر( بوية, العدد15 .), قطر  ( ملإلــوش, ريــاض9222 :)الدددعم االجتمدداعي والتكيددف الطالبددي لدددى طمبددة الجامعددة األردنيددة , مجلــة ( دراسات العلوم اإلنسانية, مجلد97 ( ), العدد1 .) إ  ( مــوراي, ادوارد, ج1998 " :)الدافعيددة واالنفعددال " ترجمــة د. :الثباتReliability والتوزي  ( أبو شي ة, نادر احمد9212 :)إدارة الموارد البشرية إطار نظري وحداالت عمميدة , دار صـواا للنشـر والتوزي , ط1 ., عمان, اأردن اا  ا( لجنـابي, صـاحب عبـد مـرزوك1998 :)المسددؤولية االجتماعيدة وعالقتهدا بفاعميددة المرشدد التربددوي .أطروحة دإلتورا ،ير منشورة, إللية التربية, جامعة بغداد اا  ( الجنـابي, صـاحب عبـد مـرزوك9228 :)المسددؤولية االجتماعيدة وعالقتهدا بفاعميددة المرشدد التربددوي, ط1 , , دار الضياا للنشر والتوزي.عمان, اأردن  ( حمادات, محمد حسـين9228 :)السدموك التنظيمدي والتحدديات المسدتقبمية فدي المؤسسدات التربويدة , .دار الحامد للنشر, عمان, اأردن  ( حمو, أبو ظريوة و ـرون9227 :)عوامل الرضا لددى أسداتذة الجامعدة , دار الملإليـة للطباعـة والنشـر والتوزي , ط91 ., الجزائر  ال الــدي, جا( جــان9228 :)دور اإلسددناد االجتمدداعي فددي تدددعيم الصددحة النفسددية لممددراهقين , مجلــة انداب, العدد1 ., جامعة الموصل  ( ال طيــب, صــالح احمــد يوســف9222 :) الميددول المهنيددة وعالقتهددا بددبعض أنمدداط الشخصددية كمدددخل لمتوجيه التربوي لطالب المرحمدة الثانويدة بدولدة اإلمدارات العربيدة , رسـالة دإلتـورا ،يـر منشـورة, جامعـة .القاهرة, مصر  ( ـــــــــــــــ9223 :) )اإلرشاد النفسي في المدرسة (أسسه نظرياته تطبيقاته ,دار الإلتـاب الجـامعي, العـين .اإلمارات العربية المتحدة  ( الداهري, صـالح احمـد9223 :)العالقدة بدين مسدتوى الطمدوح والتوافدق النفسدي لددى طدالب الم رحمدة الثانوية فدي منطقدة العدين , دراسـة نوسـية ميدانيـة تربويـة, المجلـة العراقيـة للعلـوم التربويـة والنوسـية وعلـم االجتماع, إللية التربية- .ابن رشد, جامعة بغداد  ( الداهري, صـالح احمـد9223 :)العالقدة بدين مسدتوى الطمدوح والتوافدق النفسدي لددى طدالب الم رحمدة الثانوية فدي منطقدة العدين , دراسـة نوسـية ميدانيـة تربويـة, المجلـة العراقيـة للعلـوم التربويـة والنوسـية وعلـم االجتماع, إللية التربية- .ابن رشد, جامعة بغداد إا  ( الـــدبعي, إلوـــاح ســـعيد ،ـــانم9223 :) الهويدددة االجتماعيدددة واالسدددتقرار النفسدددي وعالقتهدددا بالتنصددديف االجتماعي لدى الموظف ين والموظفدات بددوائر الدولدة الحكوميدة بأماندة صدنعاء , أطروحـة دإلتـورا ،يـر .منشورة, إللية انداب, جامعة بغداد إا  ( الـــدبعي, إلوـــاح ســـعيد ،ـــانم9223 :) الهويدددة االجتماعيدددة واالسدددتقرار النفسدددي وعالقتهدددا بالتنصددديف االجتماعي لدى الموظف ين والموظفدات بددوائر الدولدة الحكوميدة بأماندة صدنعاء , أطروحـة دإلتـورا ،يـر .منشورة, إللية انداب, جامعة بغداد إ  ( ديســـلر, جـــاري9229 :)إدارة المدددوارد البشدددرية , ترجمـــة محمـــد ســـيد احمـــد عبـــد المتعـــال, دار المـــري .للنشر, الرياض, المملإلة العربية السعودية إ  ( ديســـلر, جـــاري9229 :)إدارة المدددوارد البشدددرية , ترجمـــة محمـــد ســـيد احمـــد عبـــد المتعـــال, دار المـــري .للنشر, الرياض, المملإلة العربية السعودية (إل ث199 )م ك ال ل ال ف ان اإلن ا ة ال ل ك ال ظ اإل ا ة ال القا إ  ( ديوــز, إليــث1992 :) السددموك اإلنسدداني فددي العمددل ,, دراســة العالقــات اإلنســانية والســلوك التنظيمــي ,ترجمـــة ســـيد عبـــد الحميـــد مرســـي واغســـماعيل يوســـف, نهضـــة مصـــر للطباعـــة والنشـــر والتوزيـــ , القـــاهرة .مصر 963 جمةل الآداب / العدد831 (أ يلول) References:  Abbas, Ali (2008): International Human Resources Management, Ithraa for Publishing and Distribution, 1st Edition, Amman, Jordan.  Abbas, Ali (2008): International Human Resources Management, Ithraa for Publishing and Distribution, 1st Edition, Amman, Jordan.  Abdel-Rahman, Mohamed El-Sayed (1998): Theories of Personality, Quba Publishing House, Cairo.  Abdel-Rahman, Mohamed El-Sayed (1998): Theories of Personality, Quba Publishing House, Cairo.  Abu Shaira, Khaled (2008): Effective Professional Education and Class Teacher, 1st Edition, Arab Society Library for Publishing and Distribution.  Abu Shaira, Khaled (2008): Effective Professional Education and Class Teacher, 1st Edition, Arab Society Library for Publishing and Distribution.  Abu Sheikha, Nader Ahmad (2010): Human Resources Management, A Theoretical Framework and Practical Cases, Dar Safa for Publishing and Distribution, 1st Edition, Amman, Jordan.  Abu Sheikha, Nader Ahmad (2010): Human Resources Management, A Theoretical Framework and Practical Cases, Dar Safa for Publishing and Distribution, 1st Edition, Amman, Jordan.  Al-Adili, Nasser Muhammad (1995): Human and Organizational Behavior, a Holistic Comparative Perspective, General Administration for Research, Jeddah, Saudi Arabia.  Al-Adili, Nasser Muhammad (1995): Human and Organizational Behavior, a Holistic Comparative Perspective, General Administration for Research, Jeddah, Saudi Arabia.  Al-Amiri, Ali Mohsen Yas (2009): Social Attribution and Its Relation to Stress among Educational Advisors, Unpublished Master Thesis, College of Education, Al-Mustansiriya University.  Al-Amiri, Ali Mohsen Yas (2009): Social Attribution and Its Relation to Stress among Educational Advisors, Unpublished Master Thesis, College of Education, Al-Mustansiriya University.  Al-Ani, Nizar Muhammad Saeed (1989): Spotlight on the Human Personality, 1st Edition, House of General Cultural Affairs, Baghdad.  Al-Ani, Nizar Muhammad Saeed (1989): Spotlight on the Human Personality, 1st Edition, House of General Cultural Affairs, Baghdad.  Al-Asadi, Saeed Jassim (2002): The Social Consensus of Private College Students, Al-Mamoun University College Journal, Issue (8), Iraq.  Al-Asadi, Saeed Jassim (2002): The Social Consensus of Private College Students, Al-Mamoun University College Journal, Issue (8), Iraq.  Al-Azzawi, Najm Abdullah and Jawad, Abbas Hussein (2010): Evolution of Human Resources Management, Concept, Strategy, Organizational Site, 1st Edition, Al-Yazouri Scientific Publishing and Distribution House, Amman, Jordan.  Al-Azzawi, Najm Abdullah and Jawad, Abbas Hussein (2010): Evolution of Human Resources Management, Concept, Strategy, Organizational Site, 1st Edition, Al-Yazouri Scientific Publishing and Distribution House, Amman, Jordan.  ( الهيتي, مصطوى عبد السالم1985 :) عالم الشخصية., مإلتبة الشروق الجديدة, بغداد  ( الهيتي, مصطوى عبد السالم1985 :) عالم الشخصية., مإلتبة الشروق الجديدة, بغداد nces: ( الهيتي, مصطوى عبد السالم1985 :) عالم الشخصية., مإلتبة الشروق الجديدة, بغداد جمةل الآداب / العدد831 (أ يلول) احمــد عبــد العزيــز ســالمة و ــرون, دار الشروق, الق.اهرة  ( مــوراي, ادوارد, ج1998 " :)الدافعيددة واالنفعددال " ترجمــة د. احمــد عبــد العزيــز ســالمة و ــرون, دار الشروق, الق.اهرة 964 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ  ( ندى, يحيى محمـد مسـلم1998 :) مصدادر ومسدتوى الضدغط النفسدي وعالقتهدا بدالروح المعنويدة كمدا يراها معممو وكالدة الغدوث فدي منطقدة ندابمس التعميميدة , رسـالة ماجسـتير ،يـر منشـورة, إلليـة الدراسـات .العليا, جامعة النجاح الوطنية, نابلس جمةل الآداب / العدد831 (أ يلول) References:  Al-Dabai, The Struggle of Saeed Ghanem (2003): Social Identity and Psychological Stability and Its Relation to Social Classification of Employees in Governmental Departments in Sana'a Municipality, unpublished PhD thesis, College of Arts, University of Baghdad.  Al-Dabai, The Struggle of Saeed Ghanem (2003): Social Identity and Psychological Stability and Its Relation to Social Classification of Employees in Governmental Departments in Sana'a Municipality, unpublished PhD thesis, College of Arts, University of Baghdad.  Al-Dahri, Salih Ahmad (2003): The relationship between the level of ambition and psychological compatibility of high school students in the Al-Ain region, a psychological field educational study, Iraqi Journal of Educational and Psychological Sciences and Sociology, College of Education – Ibn Rushd, University of Baghdad.  Al-Dahri, Salih Ahmad (2003): The relationship between the level of ambition and psychological compatibility of high school students in the Al-Ain region, a psychological field educational study, Iraqi Journal of Educational and Psychological Sciences and Sociology, College of Education – Ibn Rushd, University of Baghdad.  Al-Eidani, Abdul-Karim Mohsen (2011): Occupational stability and its relationship to personality patterns and family compatibility, unpublished PhD thesis, College of Education, Al-Mustansiriya University.  Al-Eidani, Abdul-Karim Mohsen (2011): Occupational stability and its relationship to personality patterns and family compatibility, unpublished PhD thesis, College of Education, Al-Mustansiriya University.  Al-Hiti, Mustafa Abd Al-Salam (1985): The World of Personality, New Sunrise Library, Baghdad  Al-Hiti, Mustafa Abd Al-Salam (1985): The World of Personality, New Sunrise Library, Baghdad 965 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ 0208 / م8113 هـ  Al-Ibrahimi, Jassim Muhammad Halbous (2002): The effectiveness of a mentoring program to improve the general counseling skills of educational counselors in middle schools in Baghdad, an unpublished master's thesis, College of Education / Ibn Rushd, University of Baghdad.  Al-Ibrahimi, Jassim Muhammad Halbous (2002): The effectiveness of a mentoring program to improve the general counseling skills of educational counselors in middle schools in Baghdad, an unpublished master's thesis, College of Education / Ibn Rushd, University of Baghdad.  Al-Ibrahimi, Jassim Muhammad Halbous (2002): The effectiveness of a mentoring program to improve the general counseling skills of educational counselors in middle schools in Baghdad, an unpublished master's thesis, College of Education / Ibn Rushd, University of Baghdad.  Al-Imam, Mustafa Mahmoud, and others (1990): Evaluation and Measurement, University of Baghdad, Ministry of Higher Education and Scientific Research. References:  Al-Imam, Mustafa Mahmoud, and others (1990): Evaluation and Measurement, University of Baghdad, Ministry of Higher Education and Scientific Research.  Al-Janabi, Sahib Abd Marzouk (1998): Social Responsibility and Its Relation to the Effectiveness of the Educational Counselor, Unpublished PhD Thesis, College of Education, University of Baghdad.  Al-Janabi, Sahib Abd Marzouk (1998): Social Responsibility and Its Relation to the Effectiveness of the Educational Counselor, Unpublished PhD Thesis, College of Education, University of Baghdad.  Al-Janabi, Sahib Abd Marzouk (2008): Social Responsibility and Its Relation to the Effectiveness of the Educational Counselor, 1st Edition, Dar Al-Dhia for Publishing and Distribution, Amman, Jordan.  Al-Janabi, Sahib Abd Marzouk (2008): Social Responsibility and Its Relation to the Effectiveness of the Educational Counselor, 1st Edition, Dar Al-Dhia for Publishing and Distribution, Amman, Jordan.  Al-Khalidi, Jagan (2008): The Role of Social Support in Supporting Adolescents' Mental Health, Journal of Arts, Issue 1, University of Mosul.  Al-Khalidi, Jagan (2008): The Role of Social Support in Supporting Adolescents' Mental Health, Journal of Arts, Issue 1, University of Mosul.  Al-Khatib, Saleh Ahmed Youssef (2000): Professional tendencies and their relationship to some personality styles as an introduction to educational guidance for high school students in the United Arab Emirates, unpublished PhD thesis, Cairo University, Egypt.  Al-Khatib, Saleh Ahmed Youssef (2000): Professional tendencies and their relationship to some personality styles as an introduction to educational guidance for high school students in the United Arab Emirates, unpublished PhD thesis, Cairo University, Egypt.  Al-Khatib, Saleh Ahmed Youssef (2003): Psychological counseling in the school (based on its theories, applications) University Book House, Al Ain, United Arab Emirates.  Al-Khatib, Saleh Ahmed Youssef (2003): Psychological counseling in the school (based on its theories, applications) University Book House, Al Ain, United Arab Emirates.  Al-Salem, Salem Muhammad (1997): Job satisfaction for university library workers, King Fahd National Library Publications, Riyadh, Saudi Arabia.  Al-Salem, Salem Muhammad (1997): Job satisfaction for university library workers, King Fahd National Library Publications, Riyadh, Saudi Arabia.  Al-Shennawi, Mohamed Mahrous and Abdel-Rahman, Mohamed El-Sayed (1994): Social support and mental health, theoretical review and applied studies, 1st Edition, The Egyptian Anglo Library, Cairo.  Al-Shennawi, Mohamed Mahrous and Abdel-Rahman, Mohamed El-Sayed (1994): Social support and mental health, theoretical review and applied studies, 1st Edition, The Egyptian Anglo Library, Cairo.  Anastasi, A. (1997): Psychological Testing, New york: maccmillow publishing company.  Anastasi, A. References:  Hamou, Abu Zarifa and others (2007): The factors of satisfaction among university professors, Dar Al-Malakiah for Printing, Publishing and Distribution, 21st Edition, Algeria.  Hamou, Abu Zarifa and others (2007): The factors of satisfaction among university professors, Dar Al-Malakiah for Printing, Publishing and Distribution, 21st Edition, Algeria.  Harrison, R. (1978): Person environment fit and Job stress, stress at work, John willey, New York.  Harrison, R. (1978): Person environment fit and Job stress, stress at work, John willey, New York.  Heller, K. & Swindle (1983): social networks, perceived social support, and coping with stress in: R. felner et al, (Eds). Preventive psychology, pergaman press, New York. P:161.  Heller, K. & Swindle (1983): social networks, perceived social support, and coping with stress in: R. felner et al, (Eds). Preventive psychology, pergaman press, New York. P:161.  Herzberg. F (1966): Work and the nature of man, Thomas Y. corn well publisher, New York.  Herzberg. F (1966): Work and the nature of man, Thomas Y. corn well publisher, New York.  Lazarus, Richard (1981): Character, Translated by: Syed Muhammad Ghunaim, Dar Al-Shorouk, Beirut.  Lazarus, Richard (1981): Character, Translated by: Syed Muhammad Ghunaim, Dar Al-Shorouk, Beirut.  Lin, N, & etal. Al. (1983): social support scales: A methodological, note, schizophrenia Bulletin, P:47-74.  Lin, N, & etal. Al. (1983): social support scales: A methodological, note, schizophrenia Bulletin, P:47-74.  Maslow, A. H (1970): Motivation and personality, (2nd), Harper and row publishers Inc, New York.  Maslow, A. H (1970): Motivation and personality, (2nd), Harper and row publishers Inc, New York.  Mehudirata, S. B. (1987): A theoretical mode of Quality in social relation and self, Methuen, p :29.  Mehudirata, S. B. (1987): A theoretical mode of Quality in social relation and self, Methuen, p :29.  Melkouch, Riad (2000): Social support and student adaptation among students at the University of Jordan, Journal of Human Sciences Studies, Vol. (27), Issue (1).  Melkouch, Riad (2000): Social support and student adaptation among students at the University of Jordan, Journal of Human Sciences Studies, Vol. (27), Issue (1).  Merton, R.K. (1968): Social Theory and Social Structure, New York, Prentice- Hall, Inc.  Merton, R.K. (1968): Social Theory and Social Structure, New York, Prentice- Hall, Inc.  Muhammad, Yusef Abdel-Fattah (1999): Psychological stress among teachers and their counseling needs, Journal of Educational Research, Issue (15), Qatar. References: (1997): Psychological Testing, New york: maccmillow publishing company.  Argyle, Michael (1993): The Psychology of Happiness, translated by Faisal Abdul Qadir Youssef, The World of Knowledge Series, No. 175, Kuwait.  Argyle, Michael (1993): The Psychology of Happiness, translated by Faisal Abdul Qadir Youssef, The World of Knowledge Series, No. 175, Kuwait.  Argylis, C (1964): Integrating the Individual and the organization, New York, john wiley and sons lnc.  Argylis, C (1964): Integrating the Individual and the organization, New York, john wiley and sons lnc.  Aronson, E (2004): " Social psychology "Media and research update, by Pearson education, Inc, New Jersey.  Aronson, E (2004): " Social psychology "Media and research update, by Pearson education, Inc, New Jersey.  Awad, Abbas Mahmoud (1987): Studies in Industrial and Occupational Psychology, University Knowledge House, Alexandria.  Awad, Abbas Mahmoud (1987): Studies in Industrial and Occupational Psychology, University Knowledge House, Alexandria.  Blanner, R (1992): work satisfaction and industrial trends in modern society: w. Galensons & S.M. Lopsets (eds.) Labor and trade unions New York.  Blanner, R (1992): work satisfaction and industrial trends in modern society: w. Galensons & S.M. Lopsets (eds.) Labor and trade unions New York.  Borrieos, F (1990): Organizational Behavior, (8th ed), New Jersey, Prentice- Hill, Inc.  Borrieos, F (1990): Organizational Behavior, (8th ed), New Jersey, Prentice- Hill, Inc.  Davis, Keith (1990): Human Behavior at Work, A Study of Human Relations and Organizational Behavior, translated by Syed Abdel Hamid Morsi and Ismail Youssef, Nahdet Misr for Printing, Publishing and Distribution, Cairo, Egypt.  Davis, Keith (1990): Human Behavior at Work, A Study of Human Relations and Organizational Behavior, translated by Syed Abdel Hamid Morsi and Ismail Youssef, Nahdet Misr for Printing, Publishing and Distribution, Cairo, Egypt. 966 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ جمةل الآداب / العدد831 (أ يلول)  Diesler, Gary (2009): Human Resources Management, translated by Muhammad  Diesler, Gary (2009): Human Resources Management, translated by Muhammad Syed Ahmad Abdel Mutaal, Mars Publishing House, Riyadh, Saudi Arabia. Syed Ahmad Abdel Mutaal, Mars Publishing House, Riyadh, Saudi Arabia.  Ebel, P. L (1972): Essentials of Educational Measurement (2nd ed), Prentice- Hill, New Jersey.  Fayol, H (1949): General Industrial management, Loncion: pitman publishing com.  Hamadat, Muhammad Hussein (2008): Organizational behavior and future challenges in educational institutions, Al-Hamed Publishing House, Amman, Jordan.  Porter, L (1966): Personal management, annual review of psychology, vol (17). P: 395-422. References:  Muhammad, Yusef Abdel-Fattah (1999): Psychological stress among teachers and their counseling needs, Journal of Educational Research, Issue (15), Qatar.  Murray, Edward, J. (1998): “Motivation and Emotion,” translated by Dr. Ahmed Abdel Aziz Salama and others, Dar Al-Shorouk, Cairo.  Murray, Edward, J. (1998): “Motivation and Emotion,” translated by Dr. Ahmed Abdel Aziz Salama and others, Dar Al-Shorouk, Cairo.  Nada, Yahya Muhammad Muslim (1998): Sources and level of psychological stress and its relationship to morale as seen by UNRWA teachers in the Nablus Educational District, unpublished master's thesis, College of Graduate Studies, An-Najah National University, Nablus.  Nada, Yahya Muhammad Muslim (1998): Sources and level of psychological stress and its relationship to morale as seen by UNRWA teachers in the Nablus Educational District, unpublished master's thesis, College of Graduate Studies, An-Najah National University, Nablus.  Porter, L (1966): Personal management, annual review of psychology, vol (17). P: 395-422.  Porter, L (1966): Personal management, annual review of psychology, vol (17). P: 395-422. 967 جمةل الآداب / العدد831 (أ يلول) 0208 / م8113 هـ  Reggio, Ronald J. (1999): Introduction to Industrial and Organizational Psychology, translated by Faris Helmy, 1st Edition, Al Shorouk Publishing and Distribution House, Amman, Jordan.  Reggio, Ronald J. (1999): Introduction to Industrial and Organizational Psychology, translated by Faris Helmy, 1st Edition, Al Shorouk Publishing and Distribution House, Amman, Jordan.  Robbins, Stephen. P (2003): Organization behavior by person Education, International, upper saddle river, New Jersey.  Robbins, Stephen. P (2003): Organization behavior by person Education, International, upper saddle river, New Jersey.  Sarason, I G & et al (1983): Assessing social support: the social support questionnaire, Journal of personality and social psychology, vol. (44), No. (1), p:120-141,187.  Sarason, I G & et al (1983): Assessing social support: the social support questionnaire, Journal of personality and social psychology, vol. (44), No. (1), p:120-141,187.  Savikas, Mandlent R. (1994): Over view of career development theory: convergence in career development theories, California: counseling psychologists press Inc.  Savikas, Mandlent R. (1994): Over view of career development theory: convergence in career development theories, California: counseling psychologists press Inc.  Shaltz, Dawn (1983): Theories of Personality, translated by Hamad Delly Al- Karbouli and Abdul-Rahman Al-Qaisi, Ministry of Higher Education and Scientific Research, University of Baghdad. References:  Shaltz, Dawn (1983): Theories of Personality, translated by Hamad Delly Al- Karbouli and Abdul-Rahman Al-Qaisi, Ministry of Higher Education and Scientific Research, University of Baghdad.  Syme, T.R & Schrtter, l. D (1987): Social support, Megraw Hill, New York, p:661.  Syme, T.R & Schrtter, l. D (1987): Social support, Megraw Hill, New York, p:661.  Zakri, Abdul-Taif Shihab (2005): Managing Individual Behavior in the Organization and Society, Al-Amin Center for Publishing and Distribution, Sanaa, Yemen.  Zakri, Abdul-Taif Shihab (2005): Managing Individual Behavior in the Organization and Society, Al-Amin Center for Publishing and Distribution, Sanaa, Yemen.  Zewailf, Mahdi and Adayleh, Ali (1996): Organization Management, Theories and Conduct, 1st Edition, Majdalawi House for Publishing and Distribution, Amman, Jordan.  Zewailf, Mahdi and Adayleh, Ali (1996): Organization Management, Theories and Conduct, 1st Edition, Majdalawi House for Publishing and Distribution, Amman, Jordan. 968 968
https://openalex.org/W4243820137	https://figshare.com/articles/journal_contribution/Fine-Grained_Remote_Monitoring_Control_and_Pre-Paid_Electrical_Service_in_Rural_Microgrids/6468812/1/files/11897366.pdf	English	null	Fine-grained remote monitoring, control and pre-paid electrical service in rural microgrids	null	2,014	cc-by	9,269	Fine-Grained Remote Monitoring, Control and Pre-Paid Electrical Service in Rural Microgrids Maxim Buevich † Dan Schnitzer ‡ Tristan Escalada ∗ Arthur Jacquiau-Chamski ∗ Anthony Rowe † † Electrical and Computer Engineering Department, Carnegie Mellon University ‡ Engineering and Public Policy, Carnegie Mellon University ∗EarthSpark International {mbuevich,dschnitz,agr}@andrew.cmu.edu {tristan,arthur}@earthsparkinternational.org Maxim Buevich † Dan Schnitzer ‡ Tristan Escalada ∗ Arthur Jacquiau-Chamski ∗ Anthony Rowe † † Electrical and Computer Engineering Department, Carnegie Mellon University ‡ Engineering and Public Policy, Carnegie Mellon University ∗EarthSpark International {mbuevich,dschnitz,agr}@andrew.cmu.edu {tristan,arthur}@earthsparkinternational.org Abstract—In this paper, we present the architecture, design and experiences from a wirelessly managed microgrid deployment in rural Les Anglais, Haiti. The system consists of a three-tiered architecture with a cloud-based monitoring and control service, a local embedded gateway infrastructure and a mesh network of wireless smart meters deployed at 52 buildings. Each smart meter device has an 802.15.4 radio that enables remote monitoring and control of electrical service. The meters communicate over a scalable multi-hop TDMA network back to a central gateway that manages load within the system. The gateway also provides an 802.11 interface for an on-site operator and a cellular modem connection to a cloud-backend that manages and stores billing and usage data. The cloud backend allows occupants in each home to pre-pay for electricity at a particular peak power limit using a text messaging service. The system activates each meter within seconds and locally enforces power limits with provisioning for theft detection. We believe that this ﬁne-grained micro-payment model can enable sustainable power in otherwise unfeasible areas. tariffs and weak revenue collection systems (2) inability to limit customer usage to levels below generation output capacity (3) ability to meter usage at a ﬁne enough granularity to quickly diagnose problems and detect theft. In this paper, we present the design and a case-study of a system that addresses these challenges. Many microgrid developers do not utilize meters to track household electricity consumption and issue corresponding bills because meters add cost to systems that are designed to serve very low-income households [4]. Instead, these microgrid operators charge customers a ﬁxed monthly fee. Unfortunately, ﬁxed monthly payments are often priced at levels far below the cost of the energy used by the customer during that time. Operators also face challenges collecting monthly payments on time, as impoverished populations often do not have steady in- come streams to pay ongoing expenses. Fine-Grained Remote Monitoring, Control and Pre-Paid Electrical Service in Rural Microgrids Pre-payment resolves the issue of cost recovery by addressing the issues of both poor pricing and poor collection. Customers are empowered to align their preferences with their budget constraints by purchasing electricity when funds are available and when they need it most. This paper provides a chronology of our deployment and installation strategy that involved GPS-based site mapping along with various network conditioning actions required as the net- work evolved. Finally, we summarize key lessons learned and hypothesis about additional hardware that could be used to ease the tracing of faults like short circuits and downed lines within microgrids. Imposing load limits on customers is crucial for microgrids where consumption may reach the maximum output of the generation source [5]. Under such conditions, the microgrid is forced into a brownout, precluding all users from access to electricity. This situation can arise from a number of cir- cumstances. On some microgrids where the system is sized to provide lighting with CFL bulbs, customers using incandescent light bulbs can drive the microgrid into a brownout. In Bhutan, there is a well-documented case of microgrids browning out when large numbers of customers concurrently power their electric rice cookers in the evening [6]. In response to the threat of brownouts, microgrid operators can employ a number of load management techniques (exhaustively described in [4]), from simple bans on certain loads, to circuit breakers on cus- tomer households, to sophisticated meters with programmable relays. We propose using local monitoring on each of the meters along with a global view of the network with low- latency metering and control to address these concerns. Keywords—Microgrid, Deployment Experience, Electricity Dis- tribution I. INTRODUCTION Haiti Satellite View meters. Monitoring theft is therefore difﬁcult, especially on systems that serve a few hundred households. Theft is most often dealt with through strong local institutions that can impose a credible threat of penalty. However, penalties are often unenforced, and theft persists on many systems. Our system uses time synchronized sampling of power data across the network to aid in loss estimation. scaling up to the remaining 400 households in Les Anglais to be connected to the microgrid. A system that enables pre- payment on such a scale would beneﬁt from: automatic energy readings to update account balances, automatic actuation in response to load limit violations and account balance status, and remote monitoring to enable theft detection. In this paper, we discuss the design, architecture and initial deployment experiences gained by updating and expanding the 14-customer manually controlled microgrid with wireless smart meters on 38 new customers. This effort included the design of custom low-cost wireless energy meters, the develop- ment of a meter networking protocol and involved instrumen- tation for efﬁcient deployment and debugging. The metering hardware communicates back to a local gateway using IEEE 802.15.4 radios and then to a cloud billing management system using an EDGE cellular connection. Wireless meter commu- nication is achieved through a TDMA networking protocol that combines elements of the Flash Flood protocol [7] and the Low-Power Wireless Bus protocol [8]. We chronicle (and critique) our deployment strategy which involved GPS-based packet reception mapping of the town as part of a pre-planning step for tiling forwarders. This also involved a signiﬁcant tuning effort one month after the system was installed. We then provide an evaluation of the system’s performance in terms of packet yield, message delay (critical for load management) and data ﬁdelity. Finally, we discuss lessons learned from the deployment and introduce a hardware extension concept In 2012, EarthSpark International developed a microgrid in the town of Les Anglais, Haiti to demonstrate a sustainable model for microgrid operations encompassing pre-payment, load control and theft detection. Beginning operations on November 20th, 2012, the microgrid provided power to 14 households 24 hours per day. It shares power with the local cellular base transceiver station from a 12 kVA diesel gener- ator. I. INTRODUCTION Nearly 1.3 billion people, mostly living in less developed countries, do not have access to electricity [1]. Thanks to advances in technology, decreases in cost, and improvements to telecommunications and supporting infrastructures, microgrids are becoming an increasingly common means for provisioning electricity in remote areas [2]. In Haiti, less than 25% of the population has access to electricity [3], leaving the vast majority of the country in energy poverty. Although more than 30 microgrids have been developed in Haiti since the mid-1980s, few of them continue to operate sustainably. Powered completely by diesel generators and operated by local municipalities, some of these microgrids operate for as few as 150 hours per year. The unsustainability of these projects and their failure to reliably deliver energy services is symptomatic of the reasons for many microgrids to fail: (1) poor cost recovery due to underpriced Theft is another issue to contend with on remote microgrids [5]. Most often, theft is carried out by making an unauthorized connection to the microgrid distribution line. In other cases, theft is carried out by authorized customers who bypass their ~ ~ P P NUC 802.15.4 802.11 SparkMeter Cellular Modem SparkMeter Gateway Cellphone Tower Complex Breaker Breaker Panel Generator 3-‐Phase Meter Backup Generator Cellular Tower Home Interior Service Drop DistribuMon Line (6 gauge) Home Local Interface 802.11 802.15.4 802.15.4 House Wiring (14 gauge) 802.15.4 Router Router Home Heroku Fig. 1. System Architecture Cuba Jamaica Dominican Republic Les Anglais Haiti Port-au-Prince Cuba Jamaica Dominican Republic Les Anglais Haiti Port-au-Prince Fig. 2. Haiti Satellite View Fig. 3. 3D Model of Deployment ~ ~ P P NUC 802.15.4 802.11 SparkMeter Cellular Modem SparkMeter Gateway Cellphone Tower Complex Breaker Breaker Panel Generator 3-‐Phase Meter Backup Generator Cellular Tower Home Interior Service Drop DistribuMon Line (6 gauge) Home Local Interface 802.11 802.15.4 802.15.4 House Wiring (14 gauge) 802.15.4 Router Router Home Heroku ~ ~ P P NUC 802.15.4 802.11 SparkMeter Cellular Modem SparkMeter Gateway Cellphone Tower Complex Breaker Breaker Panel Generator 3-‐Phase Meter Backup Generator Cellular Tower Home Interior Service Drop DistribuMon Line (6 gauge) Home Local Interface 802.11 802.15.4 802.15.4 House Wiring (14 gauge) 802.15.4 Router Router Home Heroku Backup Generator Fig. 1. System Architecture Fig. 3. 3D Model of Deployment Cuba Jamaica Dominican Republic Les Anglais Haiti Port-au-Prince Cuba Jamaica Dominican Republic Les Anglais Haiti Port-au-Prince Fig. 2. Haiti Satellite View Fig. 3. 3D Model of Deployment Fig. 2. B. Wireless Energy Management Historically, it is not uncommon for grid-level energy man- agement systems to use wireless communication for metering. Many of the new generation of smart meters in use today have PLC or wireless backhauls. Some even have 802.15.4 capabilities for communication with appliances in the home. These systems are typically expensive and not optimized for the challenges found in microgrids because they are built to 100A (or greater) standards and are designed for use over robust distribution or communication systems. Furthermore, while forward-looking demand response programs are investi- gating many of these topics, none have deployed systems with ﬁne-grained control over a non-trivial set of homes. I. INTRODUCTION The cellular tower typically uses between 2 and 5 kVA, leaving ample headroom on the diesel generator to provide power for basic loads such as lighting, cell phone charging and entertainment to a number of households in the community. The most basic level of service provided in our system has a 30W per house limit while the highest consumption level is a 360W limit that can be used for refrigeration. Prior to the im- plementation of the wireless metering system, the EarthSpark microgrid relied on manual meter reading to update pre-paid account balances on a daily basis, miniature circuit breakers to limit loads, and a strong local presence to deter theft. While operations have been smooth since the inauguration of the grid, the manual nature of the system is not well-suited to 802.15.4 Meter / Controller House Terminal Service Drop Terminal (c) SparkMeter BoBom (b) SparkMeter Top (f) 3-‐Phase Meter BoBom (e) 3-‐Phase Meter Top Voltage Inputs CT Inputs Ampliﬁed ATmega128rfa1 BaBery Backup ADE7878 (d) Router (a) SparkMeter Box Fi 4 H d Ph t (c) SparkMeter BoBom (b) SparkMeter Top (f) 3-‐Phase Meter BoBom (e) 3-‐Phase Meter Top Voltage Inputs CT Inputs Ampliﬁed ATmega128rfa1 BaBery Backup ADE7878 (d) Router (c) SparkMeter BoBom (b) SparkMeter Top (d) Router (f) 3-‐Phase Meter BoBom (e) 3-‐Phase Meter Top Voltage Inputs CT Inputs Ampliﬁed ATmega128rfa1 BaBery Backup ADE7878 802.15.4 Meter / Controller House Terminal Service Drop Terminal (a) SparkMeter Box Fig. 4. Hardware Photos House Terminal CT Inputs (c) SparkMeter BoBom (b) SparkMeter Top (a) SparkMeter Box Fig. 4. Hardware Photos Fig. 4. Hardware Photos that we believe will drastically simplify future installations by including automatic distributed fault tracing. systems offer signiﬁcant advantages over both conventional metering systems and less sophisticated pre-paid meters such as those made by Conlog [21]. Those advantages include greater ﬂexibility in billing, remote system monitoring, price- responsive DSM, load-shedding, and dynamic power- and energy-limiting [4]. II. RELATED WORK In this section we examine existing research related to rural microgrid distribution systems. We ﬁrst discuss related projects, followed by relevant support technologies. A. Rural Microgrid Projects Microgrids can be effective means to provide access to electricity in areas of the world where it is ﬁnancially, phys- ically or institutionally difﬁcult to extend the reach of the central grid [5]. They are developed by government agencies, private developers and NGOs around the world. Microgrid developer portfolios range from small deployments such as DESI Power’s four village microgrids in Bihar, India powered by biomass gasiﬁcation [9] to the West Bengal Renewable En- ergy Development Agency’s 18 microgrids powered primarily by solar PV in the Sundarbans [10] to Nepal, which powers 59,000 households with over 300 micro-hydro systems [11]. Multiple research groups and companies have developed similar wireless energy monitoring solutions for buildings. The MIT Plug [22] provides users with power and sensor information by means of a smart surge protector. In [23], the author’s present experiences using the ACme wireless plug sensor in an ofﬁce environment. This hardware platform is quite similar in nature to our solution except that we pro- vide the surrounding eco-system for micro-grid management. Companies like Tendril Inc. [24], AlertMe [25], Trilliant [26] and GreenWave Reality [27] provide solutions that allow home owners to install and monitor their own energy usage. In some cases [24], they are even working with utility companies as part of demand response programs. These are similar technologies, but the installation and use-cases are quite different since micro-grids often require low-latency active management of load. Microgrids vary widely in the services they provide. Some microgrids are designed to provide only the most basic energy services - such as lighting in the evening - while others are designed to power cold storage and other productive commercial or agricultural loads such as mills and irrigation pumps [12]. Unfortunately, many microgrids fall into non-functioning states due to any one of a number of factors, including low levels of tariff collection, poor maintenance, customer over-usage (which causes brownouts), and unmet growth in demand [5]. In recent years, attempts at modeling microgrid operations have shown that a number of interventions can improve microgrid sustainability, such as the use of renewable energy to improve cost-effectiveness [13] [14] [15], energy efﬁciency [15], and the use of demand side management (DSM) strategies and technologies [4] [6]. III. SYSTEM ARCHITECTURE In recent years, microgrid developers are turning to ad- vanced pre-paid meters to solve problems of customer over-use and poor tariff collection. Companies such as Devergy [16], Circutor [17], Inensus [18], Powerhive [19] and Gram Power [20] have each developed such metering systems targeted at microgrids serving low-income customers. While they are in their nascent stages of pilot projects or ﬁeld trials, these In this section we provide a brief overview of the design of our electricity metering and control architecture shown in Figure 1. The main components consist of a cellular phone tower complex which houses two diesel generators that feed the electrical distribution network. Each home is connected to the distribution network using an 802.15.4 wireless metering (a) To House From Service Drop (b) Roof Antenna 802.15.4 Radio Gateway CPU (c) (d) Diesel#Tank# Panel#+#Cellular#Radio# Generator#2# Generator#1# (e) CT Clamps 3-‐Phase Wireless Meter Hand Meters Circuit Breakers (f) ig. 5. Deployment photos: (a) street with forwarder and highlighted service drop (b) sparkmeter on outside of home (c) gateway hardware (d) cellular towe ompound with forwarder (e) cellular tower interior and (f) main electrical panel Roof Antenna 802.15.4 Radio Gateway CPU (c) (a) To House From Service Drop (b) (b) (a) (c) Circuit Breakers CT Clamps 3-‐Phase Wireless Meter Hand Meters Circuit Breakers (f) Diesel#Tank# Panel#+#Cellular#Radio# Generator#2# Generator#1# (e) (d) (e) (f) (d) Fig. 5. Deployment photos: (a) street with forwarder and highlighted service drop (b) sparkmeter on outside of home (c) gateway hardware (d) cellular tower compound with forwarder (e) cellular tower interior and (f) main electrical panel forwarder and highlighted service drop (b) sparkmeter on outside of home (c) gateway hardware (d) cellular tower interior and (f) main electrical panel Fig. 5. Deployment photos: (a) street with forwarder and highlighted service drop (b) sparkmeter on outside of home ( compound with forwarder (e) cellular tower interior and (f) main electrical panel and control device called a SparkMeter. The SparkMeters communicate over multiple hops through powered forwarders mounted on select electrical poles back to a gateway housed in the local management shop. The gateway is responsible for on-site data logging and low-latency management tasks. The gateway also acts as a local WiFi hotspot to serve a management web interface that is used by operators to monitor meters, balance billing and conﬁgure individual devices. III. SYSTEM ARCHITECTURE The gateway has a cellular modem that uses the local EDGE network to communicate with a cloud management backend that stores aggregate data and has a richer set of conﬁguration options for managing multiple connected grids. an ACS712 Hall effect sensor and voltage is read directly using a voltage divider. The output of the Hall effect sensor is passed into two separate ampliﬁcation circuits, one for high- gain (loads less than 100W) and one for low-gain (loads less than 1650W). Each input is fed into different channels of the ATmega’s onboard 10-bit ADC and sampled at 2KHz to compute frequency, RMS voltage, RMS current, true power, apparent power, power factor and energy. The multi-gain inputs allow for high-resolution monitoring of the small loads that are common to microgrid environments (some customers are limited to 30W total per home). The SparkMeter can switch the load on and off using a 15A 240VAC mechanical relay which is more than sufﬁcient for an entire home in rural areas. The power supply for the board produces 5V for the sensors, 3.3V for the micro-controller and 48V for the relay that is switched using a FET. To reduce node costs, we opted to use a PCB antenna and no power ampliﬁer (in retrospect this was a poor choice). Figure 2 shows a satellite view of Haiti, as well as other island nations of the Greater Antillean archipelago (Cayman Islands and Puerto Rico not shown). A 3D model of the physical topology of our deployment in Les Anglais, Haiti can be seen in Figure 3. The cellular tower is shown to the far right. The building shades (colors) indicate different customer service levels. The ﬁrmware running on the SparkMeter is responsible for four main tasks. First, it must sample the raw voltage and current values at 2KHz and integrate voltage and current to produce true power updates. Second, it runs the communica- tion protocol stack described in Section IV. It also periodically stores status and error logging runtime statistics to EEPROM. Finally, it runs a power-limit enforcement task that checks once per second to make sure the meter does not exceed an admin- deﬁned load proﬁle. If the power consumption exceeds these limits, the node will disable power and every 30 seconds switch on the relay to check if the limit is still being violated. III. SYSTEM ARCHITECTURE As part of the load proﬁles, we change the monitoring window based on the allowed power level. This programmability prevents devices with large transients from constantly blowing breakers A. Hardware Components The ﬁeld metering hardware is built upon the FireFly sensor networking platform and consists of a 3-phase meter, the SparkMeter nodes and the forwarder units. Each compo- nent uses an ATmega128RFA1 micro-controller with 16KB of RAM, 128KB of ﬂash memory, running at 16MHz with an integrated on-chip 802.15.4 radio for communication. 1) SparkMeter: The SparkMeter, shown in Figure 4(a), is the interface between the distribution system and each home’s internal wiring. Figure 5(b) shows the board deployed inside a waterproof NEMA enclosure. Current is measured using Heroku SLIP serial Python Client Billing Summary MongoDB 802.15.4 Node Timed Celery task Python Flask Webapp Customer Charts Payment System Customer Management Heroku Sync MOXA Grid Diagnostic Data UDP Ethernet Vendor Laptop WIFI Python Flask Webapp Customer Summary Charts Vendor Payment System Vendor/Customer Management Grid Operator Charts GPRS cell network Operator Laptop Developer Laptop Internet MongoDB Gateway Fig. 6. Software Architecture SLIP serial Python Client Billing Summary MongoDB 802.15.4 Node Python Flask Webapp Customer Charts Payment System Customer Management Heroku Sync MOXA Grid Diagnostic Data UDP Ethernet Vendor Laptop WIFI Gateway like in many other limiting systems. The MAC address of each node is used to delay when the meters enables power after an outage to avoid large inrush currents that can cause additional brownouts. Billing Summary 2) Forwarder Nodes: The forwarder nodes consist of an ATmega128RFA1 with the addition of a CC2591 ampliﬁer that allows for longer-range communication across the network. The main forwarder board shown in Figure 4(d) has an external SMA connector and can be powered from a standard USB power adapter. These nodes are packaged in waterproof NEMA boxes and installed at the tops of utility poles, with direct access to main voltage lines and elevated line-of-sight communication with neighboring forwarders. With the addition of the CC2591 ampliﬁer, these nodes have 3-4 times the range of the SparkMeter nodes and are able to effectively provide coverage for wide areas. 3) 3-Phase Meter: The 3-Phase meter board was de- signed around the ADE7878 3-phase power metering IC. The ADE7878 is highly accurate and used in many industrial metering applications. It connects as a sensor interface to one of the power ampliﬁed ATmega128RFA1 nodes. This board requires at least one voltage input and can support up to three different current transformer inputs. A. Hardware Components Figure 5(f) shows the 3-phase meter installed in the breaker box at the cellular tower and connected to the two input phases of the distribution system. Metering the generator directly allows the system to monitor total microgrid load (even if some SparkMeters are down) and enables the system to perform real-time comparisons of output versus metered load for the purpose of theft detection. Fig. 6. Software Architecture Server Task: The gateway is where most of the main process- ing on the microgrid is hosted. Packets are received by the gateway and transferred to the gateway through UDP/SLIP. Packets are processed by a Python client and written to a MongoDB database. Server Task: The gateway is where most of the main process- ing on the microgrid is hosted. Packets are received by the gateway and transferred to the gateway through UDP/SLIP. Packets are processed by a Python client and written to a MongoDB database. All data processing is controlled using Celery, an asyn- chronous task queue, with a RabbitMQ server as its backend. The Celery tasks operate on data stored in MongoDB. Once written to the database, data is handled by the tasks described below. 4) Gateway: The gateway is located inside our local part- ner’s shop and consists of an Intel NUC mini-PC connected to a MOXA cellular modem and a router node shown in Figure 5(c). In our initial deployment, the NUC contains a 1.8GHz Intel Core i3 processor with 16GB of RAM and a 64GB solid-state drive to simplify remote monitoring and patching. However, our software is also able to run on more resource constrained embedded platforms like the Raspberry Pi which would signiﬁcantly lower costs for future deployments. The gateway has a 20ft coax extension cable which runs to a roof-mounted antenna for improved connectivity with forwarder nodes. Billing Task: Every minute, a billing task is run that sum- marizes all of the readings received since the last task-run. This summary reading includes time-stamped values for power consumed, power factor, frequency, voltage, and current. It also includes the amount of energy consumed in the last minute and the cost of that energy. The one-minute energy costs are then deducted from the customer balances. If a customer’s balance becomes negative, then a command is sent to that customer’s meter to be disabled. A. Hardware Components Cloud Synchronization: Every ﬁfteen minutes, a synchroniza- tion task is run that uploads all of the saved summary readings to Heroku via the GSM / GPRS MOXA modem. This task also checks for any updates in Heroku to see if a grid operator has added any credits to vendor accounts, changed global settings, or made other operational changes from the remote interface. A. Research Challenges grid that cannot be diagnosed with the one-minute summary data displayed in the remote interface, then the vendors can login locally to help the operator. In order to automate and connect a large number of meters, we face the following networking challenges: 1) Node Placement: The provisioning of the network requires careful placement of gateway and forwarder nodes. Peripheral nodes are unreliable when coverage is poor while overprovi- sioning leads to increased network trafﬁc and contention. 2) Heroku Remote Software: The remote, cloud-based soft- ware serves the following core functions: (1) Stores summary data, and (2) Provides a remote interface for microgrid op- erations. Remote microgrid operations include pre-payment of vendor electricity credits, deﬁning tariff levels, monitoring system sensors such as the 3-phase meter, and monitoring and controlling customer meters. 2) Constrained Uplink: Cellular and wired backhauls are both expensive and often intermittent. 3) Reliability: Maintaining meter connections is critical for load management and billing accuracy. 4) Visibility: In order to diagnose and dispatch maintenance work the ability to monitor and evaluate routes within regions that have poor connectivity is critical. The Heroku instance runs on the same codebase as the gateway, but when run in the cloud, a different set of func- tionality is enabled. The main interface dashboard is shown in Figure 7. An operator has access to the key functions listed below. p y 5) Security: The system must be designed to robustly handle issues of tampering and fraud. 6) Scalability: Our target deployment sites often have hun- dreds to thousands of homes. The communication protocol must elegantly scale in a predictable manner to accomodate many nodes. Vendor Management: Local vendors are responsible for selling electricity credits to microgrid customers on a pre- paid basis. The remote interface allows the operator to add or remove vendors, and to add electricity credits to their accounts upon receipt of payment. 7) Latency: The decision to disable a power-meter must be dispatched from the cloud service (not locally at the meter) in case users replenish their charges before the latest quota expires. Maintaining disruption-free service is paramount to keeping users engaged. If a node does not report or suffers a long deactivation delay, the user is able to consume power for free. Long activation delays also prevent usage and negatively impact the effectiveness of the pre-payment model. IV. NETWORK DESIGN Microgrids can easily have tens to hundreds of controllable end-points each of which must be able to report and respond to commands within seconds to avoid brownouts. It is difﬁcult to quantify the exact timing requirements for load balancing and brownout prevention in our target diesel generator, however in practice we see our generator able to operate above nominal capacity for about 10-20 seconds before it browns out or engages safety overrides. Customer List Customer List Fig. 7. Remote Interface B. Software Architecture The SparkMeter system software architecture is divided between the local gateway architecture and the cloud-based architecture as shown in Figure 6. Locally, software runs on an Intel NUC mini-PC connected to a 802.15.4 USB node and a GSM/GPRS MOXA modem, referred to as the gateway. An SSH tunnel is used to connect over EDGE to a Heroku instance. Local Interface: A Python web application serves a website that acts as the local interface. The pre-payment electricity credit vendors can log into the website via WiFi or a direct Ethernet connection to the gateway from their laptops, tablets or smartphones. Once logged in, the vendor is able to see all customer accounts and their current status and usage. When a customer wants to add funds to their balance, they make a payment (cash or mobile money) to the vendor. The vendor enters the payment information through the interface that will increase the customer’s balance, and immediately turn back on their electricity if they had run out of credits. The local interface also provides a webpage of charts displaying the health of the grid in real time. If an issue arises with the 1) Local Gateway Software: The local software serves the following core functions: (1) Manages the sensor network, (2) Stores real-time packet data, (3) Tracks customer account balances, (4) Synchronizes data with the cloud software, and (5) Provides a local interface for microgrid operations. Local microgrid operations include pre-payment of customer electricity credits, and monitoring of real-time data. 3-‐Phase Meter List Tariﬀ List Customer List Vendor List Fig. 7. Remote Interface 3-‐Phase Meter List Tariﬀ List Customer Monitoring and Control: Microgrid operators have access to lists of all customers on each microgrid system. Selecting an individual customer, operators can view summary charts for status variables such as frequency and voltage, consumption charts for power and energy, and ﬁnancial charts for customer account balances, hourly cost of energy used, and bill payments. From each customer webpage, operators can assign customers to their appropriate tariff level. If so desired, operators can also actuate individual customer meters. A. Research Challenges Responsive networking on the order of tens of seconds has a direct impact on the system’s overall quality and operation costs. Since reading data from each meter can take on the order of 10s of seconds, it is important that sampling of power data is synchronous. Synchronous sampling is critical for supporting theft detection since loads may change across a network collection cycle. Tariff Level Management: The remote interface enables mi- crogrid operators to arbitrarily deﬁne an unlimited number of tariff levels. Each tariff level has a corresponding power limit and price and may be assigned to any customer. The interface displays the maximum number of instances for each tariff level given the total capacity of the microgrid, which keeps operators from ”over-subscribing” and putting the microgrid at risk of aggregate over-consumption. System Sensor Monitoring: The SparkMeter system enables microgrid operators to deploy any number of system sensors, such as multi-phase meters on the generation system feeder, meters on the overhead distribution system, and other sensors on inverters or charge controllers if present. The system deployed in Les Anglais uses a 3-phase meter monitoring the two main feeder lines powering the distribution system. Given these design requirements, we decided to base our networking protocol, called mPCF, on a TDMA solution similar to the Low-Power wireless bus protocol described Slot: 0 1 2 3 … G N1 N2 R1 R2 R3 N3 TX RX R1 N1 R2 R3 N3 N2 G Fig. 8. Protocol Transaction Timeline 0 2 4 6 8 number of re-transmitters 0 20 40 60 80 100 packet reception rate Sparse Dense Fig. 9. Impact of forwarder density on PRR G R2 Fig. 9. Impact of forwarder density on PRR Fig. 8. Protocol Transaction Timeline timeline illustrates the ﬁrst three slots of a communication round assuming the connectivity topology graph, where edges represent viable communication links. Nodes N1 through N3 are end-point meter nodes and R1 through R3 represent forwarder nodes. In slot 0, the gateway transmits its beacon which is received by N1, R1 and R2. N1 receives the message uses the TTL and MAX-TTL values in the header to compute its offset from the start of the TDMA cycle which in this case is 0. N2 on the other hand does not receive the master beacon until the third mini-slot within slot 0 and hence must subtract that delta from its next wakeup. D. mPCF Parameters The mPCF header transmitted each cycle by the gateway contains information about how many slots are in each TDMA frame, the length of each slot, the maximum number of hops, the current hop count, and a time-of-day timestamp that can be used for local calendar scheduling. Each node that receives a beacon updates its internal state based on the latest TDMA deﬁnitions, making the protocol highly elastic. If the network grows in terms of nodes, more slots can be added per cycle from the gateway at runtime. Likewise, if the network grows in terms of hop-count, the gateway can increase the slot size and the TTL of each packet. As discussed in Section V-A, these parameters can be tuned to increase redundancy in the presence of poor links. C. Security Each packet is encrypted and authenticated using the ATmega’s built-in 128-bit AES encryption hardware. There is a single shared private key that is set for each network at deployment time. We use the cipher block chaining message authentication code (CBC-MAC) capability provided by the hardware for message integrity. Since data is frequently re- ported by nodes, if they do not respond over a long enough interval, a service request is generated for them to be manually inspected to track physical tampering. In the future, we also intend to track the 3-phase meter output along with the sum of all SparkMeters in order to detect theft. Our network topology consists of many low-powered meter nodes services by a small number of high-powered forwarder nodes. Flash Flood is used to provide meter nodes with contention-free one-hop communication to the nearest for- warder. This combination of techniques allows our system to beneﬁt from many of the timing and performance advantages of low-power wireless bus while at the same time allowing us to collect real-time route and packet-loss diagnostics from Flash Flood. Flash Flood strives to balance load with con- tention such that the capture effect still allows concurrent radio transmissions. We show in Section IV-D that when the density is relatively low, this approach provides good packet reception rates while being extremely robust in terms of routing. A. Research Challenges This is accomplished by subtracting the packet transmission time Tp times the number of hops based on TTL from the slot length Ts. in [8]. In [8] the authors use the Glossy [28] time syn- chronization primitive to establish the beginning of TDMA communication slots. Each node in the system is then given a scheduled collision-free time to reply with a message that again uses Glossy to return to the gateway. Unfortunately, Glossy requires that all messages be identical so that receivers capture time-synchronized transmissions from two different nodes as a single message can still be decoded. Glossy is powerful in terms of performance, but this restriction means that forwarders cannot append unique route or diagnostic data at each hop to trace the exact trajectory through the network. To avoid this problem, we leverage the Flash Flood protocol [7] that capitalized on the radio capture effect to ﬂood messages across the network. Using Flash Flood instead of Glossy for ﬂooding, each end-node responds to a downstream start- of-frame message from the gateway using a ﬁxed TDMA schedule. Replies are then ﬂooded back again using Flash Flood within a ﬁxed window time. By using ﬁxed hop-counts and low-level time stamping (described in FTSP [29]), each meter is able to estimate and subtract message propagation delays so that it can accurately transmit at the beginning of its time-slot, even if it received the sync message multiple hops into the network. B. mPCF Protocol Figure 8 outlines an example transaction timeline of the mPCF protocol. The gateway downstream message contains a header used for time synchronization, a nounce for seeding packet encryption, TDMA schedule tuning and any pending control messages for the meters. In Figure 8, transmitted messages are shown next to each node on the timeline as a dark grey box while received messages are light grey. The In our 52 node deployment, each slot was 25ms with a TTL of 3. A maximum 802.15.4 packet takes 4ms to transmit, meaning that each ﬂooding cycle takes at most 12 ms of the slot time. Our TDMA cycle length was set to 64 slots such that each node would report once every 1.6 seconds. With additional generation capacity (through sources like solar) we (a) (b) 25 37 12 08 34 14 13 03 23 01 18 16 11 28 27 33 10 19 09 38 41 22 15 42 06 49 26 43 52 07 50 20 02 04 24 17 51 30 (c) Fig. 10. GPS RSSI site survey with (a) gateway to forwarder coverage and (b) meter to forwarder coverage (c) ﬁnal node placement with links 25 37 12 08 34 14 13 03 23 01 18 16 11 28 27 33 10 19 09 38 41 22 15 42 06 49 26 43 52 07 50 20 02 04 24 17 51 30 (c) 25 37 12 08 34 14 13 03 23 01 18 16 11 28 27 33 10 19 09 38 41 22 15 42 06 49 26 43 52 07 50 20 02 04 24 17 51 30 (c) (b) (a) (b) (c) (a) approximates to tile the town with ﬁve forwarders as shown in Figure 10(c). The dark lines indicate links used for forwarding trafﬁc. plan to eventually connect the remaining 450 homes in Les Anglais. We estimate this would require 25 forwarders with a TTL of 6 and a slot length of 40ms which yields an update rate of approximately once per minute. The installation process took approximately three weeks: one for installing distribution lines, one for installing service drops to homes and one for installing the meter hardware and forwarders. When installing the meters, we used a utility to conﬁgure the node channel, MAC address and AES encryption key, all of which are stored in EEPROM. B. mPCF Protocol After installing the system, we left each home in the off-state for one month while we monitored the network performance. As discussed in Section V-A the performance degraded signiﬁcantly and required both additional forwarders as well as ﬁne tuning of device placement to remain consistent. To ensure that Flash Flood is able to balance contention with load, we ran a simple experiment in a high-contention conﬁguration (using Flash Flood version 1) to determine the worst-case limit of our forwarders. Figure 9 shows how the packet reception rate (PRR) is inﬂuenced by the number of for- warders. The bottom line shows the case where all forwarders are very close to each other in a high-density conﬁguration that is the worst scenario for the capture effect. The top line shows the performance as the same nodes are spread apart 10 meters from each other. The source and gateway were out of range of each other and hence required forwarders to communicate. We see that even in the highly congested scenario, reception slowly increases up to 3 or 4 transmitters before it begins to fall off due to contention. In the more spread out scenario, PRR remains stable at more than 8 forwarders at a density higher than what we would see in practice. This indicates that with correct tiling of forwarders, even the simple Flash Flood protocol is quite effective. V. SYSTEM PERFORMANCE As described in Section IV-D, our ﬁnal deployment trans- mitted a packet once every 1.6 seconds from each of the 52 meters. At the time of writing this paper, the network has been operating smoothly for almost six months, and has been supplying power to customers for nearly four months. E. Deployment Methodology Immediately after our installation (before we energized the grid), we saw relatively good performance. As shown in Figure 11, the performance of the network signiﬁcantly decreased over the ﬁrst month of installation. Before activating the grid, we returned to Les Anglais and adjusted the positions of many of the SparkMeters as well as added an additional forwarder node in the southwest corner of the network. We also ran extensive tests with different TDMA parameters to rule out protocol malfunctions (for example packets arriving out of order). We determined that poorly performing nodes were solely a result of weak or intermittent signal strength. The ﬁnal map in Figure 11 shows the network performance one week after the tuning. Since then, the network has remained stable. Our deployment strategy was comprised of two main phases. First, we planned the physical network topology based on a GPS site survey that estimated device coverage. These ranges were use to place locations on satellite photos. Next, we installed the devices, collected data for approximately one month and then returned to iterate on the network topology. Since our system has two classes of radio (high-powered forwarders and low-powered end-nodes), it was important to establish range estimates. We placed a gateway node set to continuously broadcast at the local operations facility. We then walked around with a forwarder mounted on top of a 15 ft pole that included GPS coordinates with its received signal values. Figure 10(a) shows a heat map of the signal strength around the town. Black areas denote regions where there was GPS but no gateway signal. Next, we repeated this experiment with the low-powered node mounted in the gateway location and the forwarder node being moved around at ground-level shown in Figure 10(b). This approximated the case where a meter needs to transmit to the nearest forwarder. We then used these The other critical metric to evaluate overall system per- formance is the node reporting latency. As described in Sec- tion IV, the reporting latency has a direct impact on the accu- racy of billing and the overall quality of the user experience. Figure 12 shows the average and worst-case latency of packets received over a 24 hour period. E. Deployment Methodology Data was collected on the day 60% 0% 100% 80% 20% Packet Reception 40% (a) (b) (c) 0 5 10 15 20 25 30 35 40 45 node number 0 0.2 0.4 0.6 0.8 1 PRR 0 5 10 15 20 25 30 35 40 45 node number 0 0.2 0.4 0.6 0.8 1 PRR 0 5 10 15 20 25 30 35 40 45 node number 0 0.2 0.4 0.6 0.8 1 PRR Fig. 11. PRR over a 1 hour window post-installation, 1 month later and then after tuning (a) 0 5 10 15 20 25 30 35 40 45 node number 0 0.2 0.4 0.6 0.8 1 PRR 60% 0% 100% 80% 20% Packet Reception 40% (c) 0 5 10 15 20 25 30 35 40 45 node number 0 0.2 0.4 0.6 0.8 1 PRR (b) 0 5 10 15 20 25 30 35 40 45 node number 0 0.2 0.4 0.6 0.8 1 PRR (a) (b) (c) Fig. 11. PRR over a 1 hour window post-installation, 1 month later and then after tuning 1 10 100 average latency (seconds) 0 0.2 0.4 0.6 0.8 1 ratio of nodes 1 10 100 average latency (seconds) 0 0.2 0.4 0.6 0.8 1 ratio of nodes ratio of nodes 0 s) 1 10 100 1000 worst-case latency (seconds) 0 0.2 0.4 0.6 0.8 1 ratio of nodes Fig. 12. CDF of average/worst meter latency 10 100 1000 rst-case latency (seconds) a 10-hour data set consisting of 11,000 points. The ﬁrst set of traces (left) shows true power for both power phases as read by the 3-phase meter. At 2.3 hours, there is a signiﬁcant dip in load caused by lights being shut off. The second set of traces (middle) compares voltage levels sensed by the 3- phase meter and an isolated SparkMeter. The last set of traces (right) compares frequency data between the 3-phase meter and a single SparkMeter. Traces indicate a high correlation in readings between the two meters and show the SparkMeter can be used to accurately estimate the state of the microgrid. In both sets, a shift in voltage and frequency occurs from 2.3 hours to 4 hours. This shift is caused by changes in load and are less pronounced in the SparkMeter because they are occurring on the opposite phase. In addition, oscillations in voltage and frequency are present after 6 hours. VI. LESSONS LEARNED of a tropical storm to show the most extreme variations. We see that 40% of nodes have an average latency of 2 seconds or less and 80% of nodes have an average latency of 10 seconds or less. In the worst-case, 60% of nodes reply in less than 100 seconds with an overall worst case time of around 30 minutes which was likely due heavy rain. Deploying microgrids in remote regions requires extensive planning and coordination of both personnel and supplies. We performed extensive functional testing in our lab before deploying the system, which paid off in that the system essentially performed as designed. What we could not test in the lab was how the system would respond under the volatile environmental conditions at the deployment site. Homes in Les Anglais have tin roofs which signiﬁcantly attenuated the signal depending on where meters were mounted. Since it was difﬁcult to energize the grid on a node-by-node basis, we had to install most of the nodes before being able to test connectivity. Having the option to operate nodes off of batteries (even if E. Deployment Methodology These are caused by the activation of the cell tower cooling system in response to increased ambient temperature. 1 10 100 1000 worst-case latency (seconds) 0 0.2 0.4 0.6 0.8 1 ratio of nodes Figure 14 shows pairwise voltage and frequency error spread for the 52 SparkMeters on the microgrid, sampled over a 10-hour window. The ﬁrst plot (top) shows the bi- modal nature of voltage values, a result of the fact that each SparkMeter serves one of two dedicated phases of generator output. Within each phase, 90% of samples fall within 2V of the average for that phase. The second plot (bottom) displays the error in frequency, which does not depend on the phase selected. Frequency values were found to be highly consistent, with 85% of samples falling within 0.5Hz of the average. Fig. 12. CDF of average/worst meter latency B. Data Fidelity The most important aspect of our system is its ability to accurately meter generation sources and loads. Figure 13 compares traces from a single SparkMeter with high-resolution traces from the 3-phase meter. The traces were computed from 0 2 4 6 8 10 time (hours) 107.5 108 108.5 109 109.5 0 2 4 6 8 10 time (hours) 111.5 112 112.5 113 0 2 4 6 8 10 time (hours) 63 63.5 64 64.5 65 65.5 0 2 4 6 8 10 time (hours) 63 64 65 66 0 2 4 6 8 10 time (hours) 160 180 200 220 240 260 280 voltage (V) apparent power (VA) frequency (Hz) Fig. 13. True power for two phases over a 10 hour period (left). Voltage measured at the generator (middle-top) as compared to an isolated SparkMeter (middle-bottom) along with frequency measured at the generator (right-top) compared to the SparkMeter (right-bottom). 0 2 4 6 8 10 time (hours) 63 63.5 64 64.5 65 65.5 0 2 4 6 8 10 time (hours) 63 64 65 66 frequency (Hz) 0 2 4 6 8 10 time (hours) 160 180 200 220 240 260 280 apparent power (VA) Fig. 13. True power for two phases over a 10 hour period (left). Voltage measured at the generator (middle-top) as compared to an isolated SparkMeter middle-bottom) along with frequency measured at the generator (right-top) compared to the SparkMeter (right-bottom). -0.5 0 0.5 1 ir-wise frequency error (Hz) Hot In -‐ + + -‐ R R R R R R R R + -‐ Power Supply Current Sensor Voltage Sensor SparkMeter Low Resistance Meter Front-‐End 802.15.4 ATmega 128RFA1 Neutral In Hot Out Neutral Out Low–Power Relay BaNery Fig. 15. Short-circuit measurement schematic -10 -5 0 5 10 pair-wise voltage error (V) 0 500 1000 1500 numbers of samples 0 500 1000 1500 2000 numbers of samples 5 10 ror (V) -1 -0.5 0 0.5 1 pair-wise frequency error (Hz) 0 500 1000 1500 2000 numbers of samples Fig. 14. Pairwise error of voltage and frequency across 52 SparkMeters -10 -5 0 5 10 pair-wise voltage error (V) 0 500 1000 1500 numbers of samples Neutral In Low Resistance Meter Front-‐End pair-wise voltage error (V) -1 -0.5 0 0.5 1 pair-wise frequency error (Hz) 0 500 1000 1500 2000 numbers of samples Neutral Out Neutral Out Fig. 15. B. Data Fidelity Short-circuit measurement schematic When we ﬁrst powered-up the grid with all of the SparkMe- ters attached, the main breaker immediately tripped. It became clear that there was a short circuit somewhere in the system. Shorts can be traced with either an RF carrier transmitter like those used to trace circuits in homes or a sensitive multi-meter used to periodically probe resistance. RF transmitters often bleed past shorts providing inaccurate readings. Measuring resistance can be a tedious process if the network has multiple branches. It took our team nearly two full days of tracking down what turned out to be three faults in the wiring before we were able to eliminate all problems. In one case, the linemen climbing the pole to check for faulty wiring stepped on a bracket that caused another short. For this reason we propose updating our SparkMeter design to include functionality for tracing resistance in the event of a fault in the system. Fig. 14. Pairwise error of voltage and frequency across 52 SparkMeters temporarily) would have saved time. We also saw that the RF characteristics changed as the network aged. We believe this change happened slowly, in which case careful trend monitoring could help preventatively isolate issues. Through this experience we also validated the power of visualizing geographically-based network information. Seeing node statis- tics in a simpliﬁed view on GPS coordinate maps became an invaluable debugging tool as the deployment progressed. Figure 15 shows a conceptual design of a circuit that would add low-value resistance measurement into the meters in the event of a power loss. The sensing front-end uses a set of relays to switch over to battery power during such an event. This is a major advantage of wireless as compared to PLC. Since meters inject voltage to determine resistance they cannot operate concurrently. One solution would be to leverage our TDMA protocol to avoid measurement collisions. Each meter can take a resistance measurement and then transmit the data back to the gateway for analysis. Another unanticipated outcome learned during our deploy- ment was how automation impacts user interaction with the system. Our energy enforcement with quick reset and test has signiﬁcantly increased per-user energy consumption compared to the previous manual circuit-breaker system. In the upgraded system, since enforcement happens on a minute-ly rather than daily basis, users are less discouraged from trying to consume closer to their limit. VII. CONCLUSIONS AND FUTURE WORK VII. CONCLUSIONS AND FUTURE WORK [6] T. Quetchenbach, M. Harper, J. Robinson IV, K. Hervin, N. Chase, C. Dorji, and A. Jacobson, “The gridshare solution: a smart grid approach to improve service provision on a renewable energy mini-grid in bhutan,” Environmental Research Letters, vol. 8, no. 1, p. 014018, 2013. This paper showed the viability of using wireless sensor networking technology to manage microgrids in rural environ- ments. As compared to solutions like power line communica- tion, wireless offers advantages in terms of ease-of-deployment and robustness in the presence of faults. We discuss the performance of our deployment in Les Anglais, Haiti that includes a backend for ﬁne-grained pre-payment of electricity along with a ﬁeld-deployed metering and control infrastructure. This prototype system is currently powering 52 homes that no longer require expensive and toxic kerosene for lighting and are able to charge cellular phones and operate fans. In addition, it presents the opportunity for local storage of medicines that require refrigeration. [7] J. Lu and K. Whitehouse, “Flash ﬂooding: Exploiting the capture effect for rapid ﬂooding in wireless sensor networks,” in in Proceedings of IEEE INFOCOM, Rio de Janeriro, 2009. [8] F. Ferrari, M. Zimmerling, L. Mottola, and L. Thiele, “Low- power wireless bus,” in Proceedings of the 10th ACM Conference on Embedded Network Sensor Systems, ser. SenSys ’12. New York, NY, USA: ACM, 2012, pp. 1–14. [Online]. Available: http://doi.acm.org/10.1145/2426656.2426658 [9] “DESI Power:http://www.desipower.com/downloads/DESI-Power- Company-Proﬁle.pdf.” [10] Chaudhuri, “Renewable energy in the sundarbans,” New Delhi: The Energy and Resources Institute, 2009. [11] UNDP, “Case studies of sustainable development in practice: Triple wins for sustainable development.” United Nations Development Pro- gram, 2012. While this paper focuses on the hardware, software and networking components of the system, there are signiﬁcant additional challenges related to the policy and economics of making systems like this viable. As future work, we intend to reﬁne and scale the system such that it can be easily duplicated across regions that suffer from energy poverty. Scaling will require a management framework that can balance demand with multiple generation sources like solar panels and storage banks. We also need to design the hardware infrastructure in such a manner that it can easily be upgraded to higher power capacity metering systems to support future modernization of the infrastructure. B. Data Fidelity This has resulted in increasing the total load on the system by 500% of what we anticipated. The additional consumption increases the revenue potential while still bounding overall usage limits. Though beyond the scope of this paper, we believe there are techniques that can be used if approximate wire length estimates are known in order to localize microgrid shorts. Con- versely, the circuit can also be used to trace open-circuits due to downed lines. Seeing a gradient map of resistances across the network would have drastically reduced our debugging time. VIII. ACKNOWLEDGMENTS [21] “Conlog http://www.conlog.co.za/pages/ProductsServices/Single- Phase-Meters.html (viewed 10/10/2013).” This research was funded in part by the Link Foun- dation Energy Program, the Bertucci Graduate Fellowship program and TerraSwarm, one of six centers of STARnet, a Semiconductor Research Corporation program sponsored by MARCO and DARPA. We would also like to thank the Carnegie Mellon Electricity Industry Center (CEIC) for their support. [22] Lifton, J., Feldmeier, M., Ono, Y., Lewis, C., Paradiso, J., “A Platform for Ubiquitous Sensor Deployment in Occupational and Domestic Environments,” International Conference on Information Processing in Sensor Networks (IPSN), April 2007. [23] Jiang X., Ly M V., Taneja J., Dutta P., and Culler D., “Experiences with a High-Fidelity Wireless Building Energy Auditing Network,” SenSys, November 2009. [24] “http://www.tendrilinc.com/ (viewed 2/20/2009).” [25] “http://www.alertme.com/ (viewed 2/20/2009).” VII. CONCLUSIONS AND FUTURE WORK As part of this effort, our next generation meters will include our proposed fault diagnostics circuit and will ﬁt the standard enclosure ﬁxtures used around the world. As the microgrid grows, the low-cost meters will be replaced with the latest variety of smart meter. Finally, we plan to evaluate different theft detection and management schemes using this deployment as a testbed. [12] K. D. Kirubi C, Jacobson A and A. Mills., “Community-based electric micro-grids can contribute to rural development: Evidence from kenya.” World Development 37:12081221., 2009. [13] M. Khan and M. Iqbal., “Pre-feasibility study of stand-alone hybrid energy systems for applications in newfoundland,” Renewable Energy, 2005. [14] S. S. Rana S., Chandra R. and M. Sodha, “Optimal mix of renewable energy resources to meet the electrical energy demand in villages of madhya pradesh.” Energy Conversion and Management, 1998. [15] D. M. Kammen and C. E. Casillas, “The delivery of low-cost, low- carbon rural energy services,” 2011. [16] “Devergy: http://www.devergy.com (viewed 10/10/2013).” [17] “Circutor 2012a. Renewable Energies: http://circutor.com (viewed 2/22/2013).” [18] “INENSUS: http://www.inensus.com/en/products5.htm (viewed 10/10/2013).” [19] “Power Hive: http://powerhive.com (viewed 10/10/2013).” [20] “GRAM Power: http://www.grampower.com/ (viewed 10/10/2013).” REFERENCES [26] “http://www.trilliantinc.com (viewed 2/20/2009).” [1] I. E. Agency, “World energy outlook database,” 2012. [1] I. E. Agency, “World energy outlook database,” 2012. [27] “http://www.greenwavereality.com (viewed 3/20/2010).” [2] R. Deshmukh, J. P. Carvallo, and A. Gambhir, “Sustainable development of renewable energy mini–grids for energy access: A framework for policy design,” 2013. [28] F. Ferrari, M. Zimmerling, L. Thiele, and O. Saukh, “Efﬁcient network ﬂooding and time synchronization with glossy,” in Information Process- ing in Sensor Networks (IPSN), 2011 10th International Conference on, 2011, pp. 73–84. [3] G. of Haiti, “Haiti energy sector development plan 2007-2017,” 2006. [4] M. Harper, “Review of strategies and technologies for demand–side management on isolated mini–grids,” 2013. [29] M. Mar´oti, B. Kusy, G. Simon, and A. L´edeczi, “The ﬂooding time synchronization protocol,” in Proceedings of the 2nd International Conference on Embedded Networked Sensor Systems, ser. SenSys ’04. New York, NY, USA: ACM, 2004, pp. 39–49. [Online]. Available: http://doi.acm.org/10.1145/1031495.1031501 [5] M.-G. D. Manual, “Esmap technical paper 007,” Energy Sector Manage- ment Assistance Programme (ESMAP) UNDP/World Bank–April, 2000.
https://openalex.org/W4293394555	https://link.springer.com/content/pdf/10.1007/s12185-022-03441-6.pdf	English	null	Post-remission measurable residual disease directs treatment choice and improves outcomes for patients with intermediate-risk acute myeloid leukemia in CR1	International journal of hematology	2,022	cc-by	7,102	Abstract Objectives This study retrospectively investigated in which cycle measurable residual disease (MRD) is associated with prognosis in patients in first complete remission (CR1) of intermediate-risk acute myeloid leukemia (AML). p g pi p y Methods The study enrolled 235 younger patients with intermediate-risk AML. MRD was evaluated by multiparameter flow cytometry after the 1st, 2nd, and 3rd chemotherapy cycles (MRD1–3, respectively).i Methods The study enrolled 235 younger patients with intermediate-risk AML. MRD was evaluated by multiparameter flow cytometry after the 1st, 2nd, and 3rd chemotherapy cycles (MRD1–3, respectively). Results No significant association was detected after the 1st and 2nd cycles. However, the 5-year incidence of relapse was higher in the MRD3-positive group (n = 99) than in the negative group (n = 136) (48.7% vs. 13.7%, P = 0.005), while 5-year disease-free survival (DFS) and overall survival (OS) were lower in the MRD3-positive group than in the negative group (43.2% vs. 81.0% and 45.4% vs. 84.1%; P = 0.003 and 0.005, respectively). Allogeneic hematopoietic stem cell transplantation led to a lower 5-year relapse, and higher DFS and OS rates than chemotherapy in the MRD3-positive group (22.3% vs. 71.5%, 65.9% vs. 23.0%, and 67.1% vs. 23.9%; P < 0.001, 0.002, and 0.022, respectively), but did not affect the MRD-negative group. Conclusions MRD3 could serve as an indicator for post-remission treatment choice and help improve outcomes for intermediate-risk AML in CR1. Keywords Acute myeloid leukemia · Intermediate-risk · Measurable residual disease · Hematopoietic stem cell transplantation Keywords Acute myeloid leukemia · Intermediate-risk · Measurable residual disease · Hematopoietic stem cell transplantation Keywords Acute myeloid leukemia · Intermediate-risk · Measurable residual disease · Hematopoietic stem cell transplantation https://doi.org/10.1007/s12185-022-03441-6 International Journal of Hematology (2022) 116:892–901 https://doi.org/10.1007/s12185-022-03441-6 International Journal of Hematology (2022) 116:892–901 ORIGINAL ARTICLE Post‑remission measurable residual disease directs treatment choice and improves outcomes for patients with intermediate‑risk acute myeloid leukemia in CR1 Lijie Han1 · Yilu Li1 · Jiaying Wu1 · Jie Peng2 · Xiaolin Han3 · Hongmian Zhao4 · Chen He5 · Yuanyuan Li6 · Weimin Wang1 · Mengmeng Zhang1 · Yafei Li5 · Hui Sun1 · Haixia Cao1 · Li’na Sang1 · Zhongxing Jiang1 · Jifeng Yu1 Lijie Han1 · Yilu Li1 · Jiaying Wu1 · Jie Peng2 · Xiaolin Han3 · Hongmian Zhao4 · Chen He5 · Yuanyuan Li6 · Weimin Wang1 · Mengmeng Zhang1 · Yafei Li5 · Hui Sun1 · Haixia Cao1 · Li’na Sang1 · Zhongxing Jiang1 · Jifeng Yu1 Received: 17 May 2022 / Revised: 20 August 2022 / Accepted: 21 August 2022 © The Author(s) 2022 / Published online: 28 August 2022 Received: 17 May 2022 / Revised: 20 August 2022 / Accepted: 21 August 2022 © The Author(s) 2022 / Published online: 28 August 2022 Introduction Lijie Han, Yilu Li, Jiaying Wu, and Jie Peng have contributed equally to this work. For adult patients with acute myeloid leukemia (AML), complete remission (CR) rates are approximately 70–80%. However, approximately 60% of these patients experience a relapse in their first CR (CR1) despite intensive chemotherapy [1]. To improve the leukemia-free survival of patients with AML, distinct therapeutic strategies for post- remission consolidation have been developed. Treatment decisions are based on cytogenetic risk profiles, and, recently, on the measurable residual disease (MRD) [2, 3]. In the treatment of intermediate-risk AML (IR-AML), post-remission relapse is a major concern; but no consensus on post-remission treatment exists. Some reports suggest that high-dose combined chemotherapy is a viable option for patients with IR-AML [4, 5]. Other studies have indicated that allogeneic hematopoietic stem cell transplantation (allo- HSCT) was associated with better survival compared with chemotherapy when applied as post-remission therapy (PRT) For adult patients with acute myeloid leukemia (AML), complete remission (CR) rates are approximately 70–80%. However, approximately 60% of these patients experience a relapse in their first CR (CR1) despite intensive chemotherapy [1]. To improve the leukemia-free survival of patients with AML, distinct therapeutic strategies for post- remission consolidation have been developed. Treatment decisions are based on cytogenetic risk profiles, and, recently, on the measurable residual disease (MRD) [2, 3]. * Lijie Han hanlijiede@126.com * Lijie Han hanlijiede@126.com * Jifeng Yu yujifengzzu@163.com 1 Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 1 Department of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2 Department of Oncology, The Second Affiliated Hospital of Guizhou Medical University, Kaili, China 3 Department of Hematology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China 4 Department of Hematology, Huaihe Hospital of Henan University, Kaifeng, China 5 Laboratory of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 6 School of Foreign Languages, Henan University of Chinese Medicine, Zhengzhou, China 2 Department of Oncology, The Second Affiliated Hospital of Guizhou Medical University, Kaili, China In the treatment of intermediate-risk AML (IR-AML), post-remission relapse is a major concern; but no consensus on post-remission treatment exists. Some reports suggest that high-dose combined chemotherapy is a viable option for patients with IR-AML [4, 5]. Patients Younger patients in our centers (the First Affiliated Hospital of Zhengzhou University, the First Affiliated Hospital of Xinxiang Medical University, and Huaihe Hospital of Henan University) who had a confirmed diagnosis of IR-AML between January 2015 and December 2018 were enrolled if they met the following criteria: (1) newly diagnosed with de novo IR-AML, (2) between the ages of 14 and 60 years, (3) received ≥3 cycles of chemotherapy and in CR1. The study is in accordance with the principles of the Declaration of Helsinki and was approved by the ethical committee of the First Affiliated Hospital of Zhengzhou University, the First Affiliated Hospital of Xinxiang Medical University, and Huaihe Hospital of Henan University. Introduction Other studies have indicated that allogeneic hematopoietic stem cell transplantation (allo- HSCT) was associated with better survival compared with chemotherapy when applied as post-remission therapy (PRT) 3 Department of Hematology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China 3 Department of Hematology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China 4 Department of Hematology, Huaihe Hospital of Henan University, Kaifeng, China 4 Department of Hematology, Huaihe Hospital of Henan University, Kaifeng, China 5 Laboratory of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 5 Laboratory of Hematology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 6 School of Foreign Languages, Henan University of Chinese Medicine, Zhengzhou, China 6 School of Foreign Languages, Henan University of Chinese Medicine, Zhengzhou, China Vol:.1 :.(1234567 3 Post-remission measurable residual disease directs treatment choice and improves outcomes… 893 [6–9]. Interestingly, the HOVON/SAKKAML 42A study mentioned that MRD plays a crucial role in deciding which PRT options would be the most effective [10]. Another study, which investigated standard-risk AML in CR1 according to the second cycle MRD status, could not indicate whether allo-HSCT was significantly useful in MRD-positive cases (95% confidence interval (CI) 0.31–1.69) [11]; in the IR-AML subgroup following the GIMEMA AML1310 protocol [12], no significant differences in survival between the second cycle (one induction and one consolidation cycle) MRD-positive receiving allo-HSCT and MRD-negative receiving auto-HSCT subgroups were found. However, the selection of strategy (including allo-HSCT, auto-HSCT, and chemotherapy) for IR-AML, and the MRD cycle for deciding the therapeutic strategy remains controversial. Thus, which specific cycles of MRD are closely associated with prognosis should be clarified; this would be helpful in determining post-remission treatment strategies for IR-AML in CR1. We conducted a retrospective study examining younger adults with de novo IR-AML in CR1 who underwent either chemotherapy, auto-HSCT, or allo-HSCT as consolidation therapies in our centers. Comprehensive Cancer Net-work 2019 recommendations version 3 as follows: (1) mutated NPM1 and FLT3-ITD high; (2) wild type NPM1 without FLT3-ITD or with FLT3-ITD low (without adverse-risk genetic lesions); (3) t(9;11) (p21.3;q23.3); MLLT3-KMT2A, or (4) cytogenetic abnormalities not classified as favorable or adverse [16]. Treatment Induction chemotherapy was administered following one of the three regimens: (1) idarubicin (8–10 mg/m2 for 3 days) combined with cytarabine (Ara-C, 100 mg/m2 for 7 days); (2) daunorubicin (45 mg/m2 for 3 days) in combination with Ara-C, or (3) homoharringtonine (2 mg/m2), Ara-C (100 mg/m2) and aclarubicin (20 mg) for 7 days. Induction chemotherapy was performed for two cycles if the patients achieved CR or partial remission (PR) in the first cycle. Otherwise, those who had no-remission (NR) after the first cycle received FLAG (30 mg/m2 fludarabine on days 1–5, 2 g/m2 Ara-C on days 1–5, and 300 µg G-CSF on days 0–5) or CLAG (5 mg/m2 cladribine on days 1–5, 2 g/m2 Ara-C on days 1–5, and 300 µg G-CSF on days 0–5 [17, 18]. After two cycles of induction chemotherapy, patients who were NR were administered decitabine + CAG (cytarabine, aclarubicin, and G‐CSF) or were enrolled in a clinical trial, and the CR/complete remission with incomplete count recovery (CRi) patients were given consolidation chemotherapy, which consisted of one cycle of IDAC (i.e., intermediate-dose cytarabine, 2 g/m2 q12h for 3 days). Subsequently, the CR/CRi patients were administered further consolidation chemotherapy (IDAC*3–4 cycles) or allo-HSCT, which consisted of matched sibling donor (MSD) transplantation, matched unrelated donor (MUD) transplantation, haploidentical donor (HID) transplantation, or auto-HSCT based on MRD and donor availability (Fig. 1). In auto-HSCT, peripheral blood stem cells were harvested after mobilization with IDAC. In allo-HSCT, Busulfan-based myeloablative conditioning regimens were administered to all patients as previously described [19]. Diagnostic methods CR was defined as follows: <5% BM blasts; absence of blasts with Auer rods; absence of extramedullary disease; absolute neutrophil count >1.0 × 109/L; platelet (PLT) count >100 × 109/L, and independence of red cell transfusions. CRi corresponded to all CR criteria except for neutropenia (<1.0 × 109/L) or thrombocytopenia (<100 × 109/L). PR was defined as 5% <BM blasts <25% and a decrease in the pre-treatment bone marrow blast by at least 50%. NR was defined as BM blasts ≥25%; relapse was a recurrence of ≥5% BM blasts, the reappearance of blasts in AML was diagnosed as previously described [13]. Immunophenotyping was performed on diagnostic bone marrow (BM) aspirate samples by CD45/SSC gated 8-color flow cytometry [14]. The cytogenetic examination was performed using standard techniques. Molecular screening for fusion genes and gene mutations was performed using real-time quantitative-polymerase chain reaction, and sequence analysis was applied to all patients [15]. The classification of IR-AML was based on the National 1 L. Han et al. 894 Fig. 1 Diagram showing patients’ enrollment in this study. MRD3, measurable residual disease after the third cycle of chemotherapy (two induction cycles and one consolidation cycle) achievement of the first remission, and OS was measured from the date of diagnosis. The χ2 analyzed categorical variables or Fisher’s exact test, and differences between distributions of continuous variables were evaluated using the Mann–Whitney U test. The area under the receiver operating curve constructed from logistic regression analysis was used to present the associations between MRD and relapse. Based on the cut-off value for relapse, MRD was defined as negative or positive. achievement of the first remission, and OS was measured from the date of diagnosis. The χ2 analyzed categorical variables or Fisher’s exact test, and differences between distributions of continuous variables were evaluated using the Mann–Whitney U test. The area under the receiver operating curve constructed from logistic regression analysis was used to present the associations between MRD and relapse. Based on the cut-off value for relapse, MRD was defined as negative or positive. the blood, or the development of extramedullary disease, and non-CR included NR and PR as described previously [13]. Monitoring and definition of MRD BM samples were collected to monitor MRD using 8-color multiparameter flow cytometry (MFC) after each course of chemotherapy (induction and each cycle of consolidation). MRD was detected using a panel of eight antibody combinations [14]. LAIP (leukemia-associated immunophenotype) defined at AML diagnosis was used for MFC-MRD detection. The different-from-normal immunophenotype was performed to monitor MFC-MRD when LAIP was not available at diagnosis. The sensitivity of MFC-MRD detection was 0.1%, and any level of measurable MRD ≥0.1% was considered positive, and ≤0.1% was negative. MRD1–3 refers to the MRD after the 1st, 2nd, and 3rd cycles of chemotherapy, respectively. i The Cox proportional hazards model was applied to evaluate the association between patient characteristics and outcomes in multivariate analysis. Variables associated with relapse or survival with P-values less than 0.10 in univariate analysis or variables (e.g., age, high white blood cell (WBC), cycles required to achieve CR ≥2) known to influence outcomes were included in the final models. P-values of <0.05 whereas considered statistically significant. SPSS 22.0 (Mathsoft, Seattle, WA), GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA), and R software (http://cran.R- project.org) were used for all data analysis. Patient characteristics From January 2015 to December 2018, 337 consecutive younger adult patients were diagnosed with IR-AML in our centers (Fig. 1). Of the 337 patients, 21 lacked complete cytogenetic data at diagnosis, and 25 were not treated; both were excluded from the study. Of the remaining 291 patients, 32 were excluded from this study due to death (n = 19) or loss to follow-up (n = 13) during the two cycles of induction chemotherapy. Of the remaining 259 patients, those with non-CR (n = 12), loss to follow-up (n = 4), missing MRD measures (n = 6), or mortality (n = 2) were excluded after another cycle of chemotherapy consolidation. The remaining 235 patients were enrolled. p After the first induction, 184 (78.3%) patients were MRD1- positive while 51 (21.7%) were MRD1-negative. Among the MRD1-positive patients, 91 (49.5%), 63 (34.2%), and 30 (16.3%) were administered chemotherapy, allo-HSCT, and auto-HSCT, respectively; correspondingly, 23 (45.1%), 16 (31.4%), and 12 (23.5%) MRD1-negative patients underwent chemotherapy, allo-HSCT, and auto-HSCT, respectively. After the second induction, 153 (65.1%) patients were MRD2- positive while 82 (34.9%) were MRD2-negative. Among the MRD2-positive patients, 73 (47.7%), 54 (35.3%), and 26 (17.0%) underwent chemotherapy, allo-HSCT, and auto- HSCT, respectively. Among the MRD2-negative patients, 41 (50.0%), 25 (30.5%), and 16 (19.5%) underwent chemotherapy, allo-HSCT, and auto-HSCT, respectively. After the third cycle of chemotherapy, 43.4% of patients were MRD3-positive while 56.6% were MRD3-negative. According to the MRD3 status, the patients were divided into MRD3-positive (n = 102) and -negative (n = 133) groups. The MRD3-positive group contained 55 patients in chemotherapy consolidation and 47 in Allo-HSCT subgroups; and the MRD3-negative group contained 59 in chemotherapy, 32 in allo-HSCT, and 42 in auto-HSCT subgroups (Fig. 1). Allo-HSCT Allogeneic hematopoietic stem cell transplantation, Auto- HSCT Autologous HSCT, CR Complete remission, CRi CR with incomplete blood count recovery, MRD Measurable residual disease, MRD1–3 MRD after the 1st–3rd chemotherapy cycle, respectively The characteristics of the patients are summarized in Table 1. The median age of the patients was 36 years, and the WBC count at diagnosis was ≥100 × 109/L in 29 patients. After the first induction cycle, 181 patients achieved CR/CRi, including 51 MRD-negative cases (MRD1 <0.1%); after the second induction cycle, 215 achieved CR/CRi including 82 MRD2-negative cases; and after the third cycle, 133 (56.6%) patients were MRD3-negative. The median follow-up period for this cohort was 18 (3–72) months. Endpoints and statistical methods Cumulative incidence of relapse (CIR) was the primary endpoint of the study. Secondary endpoints included disease- free survival (DFS) and overall survival (OS), as estimated using the Kaplan–Meier method and compared by the log- rank test. Both CIR and DFS were measured from the date of l:.(1234567 3 l:.(1234567 1 3 Post-remission measurable residual disease directs treatment choice and improves outcomes… 895 Table 1 Patient clinical characteristics Allo-HSCT Allogeneic hematopoietic stem cell transplantation, Auto- HSCT Autologous HSCT, CR Complete remission, CRi CR with incomplete blood count recovery, MRD Measurable residual disease, MRD1–3 MRD after the 1st–3rd chemotherapy cycle, respectively Characteristic No. (%) Age, years Median (range) 36 (14–60) ≤36 122 (51.9) >36 113 (48.1) Sex (%) Male 123 (52.3) Female 112 (47.7) WBC count at diagnosis (range) ×109/L High WBC (>100) 29 (12.3) WBC (≤100) 206 (87.7) Cytogenetics and gene mutations Normal cytogenetics only 58 (24.7) NPM1 and FLT3-ITDhigh 14 (6.0) Wild NPM1 and FLT3-ITDlow 21 (8.9) t(8;21) with c-Kit 15 (6.4) inv(16) with c-Kit 7 (3.0) t(9;11) 6 (2.6) Others without favorable and poor-risk cytogenetics and genetic lesions 114 (48.4) Induction regimen IA 76 (32.3) DA 132 (56.2) HAA 27 (11.5) CR/CRi achieved After cycle1 181 (77.0) After cycle2 215 (91.5) MRD- (FCM <0.1%) MRD1- 51 (23.7) MRD2- 82 (34.9) MRD3- 133 (56.6) Consolidation Chemotherapy 114 (48.5) Allo-HSCT 79 (33.6) Auto-HSCT 42 (17.9) Table 1 Patient clinical characteristics Association between different MRD cycles and relapse and survival As shown in Fig. 2, in the context of MRD1, the estimated 5-year CIR (MRD1 positive vs. negative: 32.3% vs. 18.2%, P = 0.419), DFS (60.0% vs. 76.0%, P = 0.339), and OS (61.4% vs. 79.3%, P = 0.500) were not significantly different between the groups. Similarly, after the 2nd cycle (MRD2), 5-year relapse incidence, DFS, and OS were not significantly different between the groups (MRD2 positive vs. negative: 34.6% vs. 17.2%, 58.8% vs. 74.4%, and 59.7% 1 L. Han et al. 896 Fig. 2 Cumulative relapse, DFS, and OS based on MRD for different chemotherapy cycles. The estimated 5-year cumulative incidence of relapse, DFS, and OS according to MRD1 A, MRD2 B, and MRD3 C. MRD1–3, measurable residual disease after the 1st, 2nd, and 3rd cycles of chemotherapy respectively L. Han et al. 896 Fig. 2 Cumulative relapse, DFS, and OS based on MRD for different chemotherapy cycles. The estimated 5-year cumulative incidence of relapse, DFS, and OS according to MRD1 A, MRD2 B, and MRD3 C. MRD1–3, measurable residual disease after the 1st, 2nd, and 3rd cycles of chemotherapy respectively relapse were smaller (AUC = 0.58 and 0.56, P = 0.111 and 0.239, respectively, Figure S1). vs. 75.8%; P = 0.257, 0.469, and 0.373; respectively). However, after the 3rd cycle (MRD3), the 5-year CIR was higher in the MRD3-positive group than in the negative group (48.7% vs. 13.7%, P = 0.005). Furthermore, the 5-year DFS and OS were lower in the MRD3-positive than in the negative group (43.2% vs. 81.0% and 45.4% vs. 84.1%; P = 0.003 and 0.005, respectively). vs. 75.8%; P = 0.257, 0.469, and 0.373; respectively). However, after the 3rd cycle (MRD3), the 5-year CIR was higher in the MRD3-positive group than in the negative group (48.7% vs. 13.7%, P = 0.005). Furthermore, the 5-year DFS and OS were lower in the MRD3-positive than in the negative group (43.2% vs. 81.0% and 45.4% vs. 84.1%; P = 0.003 and 0.005, respectively). ol:.(1234567 1 3 Multivariate prognostic analysis The univariate and multivariate analyses of relapse, DFS, and OS are shown in Table2. For all patients, age >36 years, MRD3 positivity, and consolidation chemotherapy (compared with allo-HSCT) was independent risk factors for relapse [P = 0.023, 0.001 and <0.001; hazard ratio (HR) = 2.200, 3.937 and 4.560; and 95% CI 1.116–4.338, 1.692–9.159 and 1.967–10.575, respectively]. No other Additionally, the area under the curve (AUC) of MRD3 for predicting relapse was the largest (AUC = 0.72, P < 0.001, cut-off value, 0.1%), with sensitivity and specificity of 0.77 and 0.56, respectively (Figure S1). The corresponding AUCs for MRD1 and MRD2 for predicting ol:.(1234567 1 3 ol:.(1234567 1 3 Post-remission measurable residual disease directs treatment choice and improves outcomes… Table 2 Multivariate analysis of outcomes Allo Allogeneic transplantation, Auto Autologous transplantation, Chem Chemotherapy, CI Confidence Interval, DFS Disease-free survival, HR Hazard ratio, MRD Measurable residual disease, MRD3 MRD after the third chemotherapy cycle, OS Overall survival Factors Relapse DFS OS Univariate Multivariate (P (HR, 95% CI)) Univariate Multivariate (P (HR, 95% CI)) Univariate Multivariate (P (HR, 95% CI)) Patient age ≤36 vs. >36 years 0.268 0.023 (2.200, 1.116– 4.338) 0.348 0.030 (1.886, 1.065– 3.341) 0.665 0.525 (1.235, 0.644–2.371) High WBC vs. WBC 0.452 0.604 (1.369, 0.418– 4.485) 0.634 0.847 (1.096, 0.432– 2.782) 0.741 0.975 (1.017 0.358–2.884) Cycles required to achieve CR 1 vs. ≥2 0.709 0.523 (1.266, 0.614– 2.610) 0.925 0.805 (1.082, 0.578– 2.207) 0.343 0.367 (1.366, 0.693–2.693) MRD3− vs.MRD3+ 0.007 0.001 (3.937, 1.692– 9.159) 0.004 0.003 (2.643, 1.381– 5.058) 0.006 0.016 (2.582, 1.197–5.567) Treatment choice Chem vs. Allo 0.005 <0.001 (4.560, 1.967– 10.575) 0.035 0.001 (2.872, 1.499– 5.503) 0.082 0.025 (2.267, 1.107–4.645) Chem vs. Auto 0.138 0.859 (1.103, 0.372– 3.237) 0.016 0.235 (1.787, 0.685– 4.661) 0.056 0.400 (1.631, 0.522–5.102) Allo vs. Auto 0.333 0.026 (0.242, 0.069– 0.845) 0.792 0.371 (0.622, 0.220– 1.758) 0.523 0.594 (0.719, 0.214–2.417) Post-remission measurable residual disease directs treatment choice and improves outcomes… 897 897 Allo Allogeneic transplantation, Auto Autologous transplantation, Chem Chemotherapy, CI Confidence Interval, DFS Disease-free survival, HR Hazard ratio, MRD Measurable residual disease, MRD3 MRD after the third chemotherapy cycle, OS Overall survival Allo Allogeneic transplantation, Auto Autologous transplantation, Chem Chemotherapy, CI Confidence Interval, DFS Hazard ratio, MRD Measurable residual disease, MRD3 MRD after the third chemotherapy cycle, OS Overall survival The interaction between MRD1-3 and HSCT and its effects on patient outcomes were assessed. Multivariate prognostic analysis Among chemotherapy, allo-HSCT, and auto-HSCT subgroups of MRD1-positive and -negative patients, the estimated CIR, and survival are shown in the Supplementary Material; the 5-year cumulative OS was not significantly different (P > 0.05, Figure S2). Among chemotherapy, allo-HSCT, and auto-HSCT subgroups of MRD2-positive and -negative patients, CIR and survival are shown in the Supplementary Material; the estimated 5-year OS was not significantly different (P > 0.05, Figure S2). The effects of MRD3 and HSCT on patient outcome were also evaluated (Fig. 3). The results indicated that outcomes were improved by allo-HSCT (n = 47) compared with consolidation chemotherapy (n = 55) for patients with MRD3 positivity. For MRD3-negative patients, no significant difference between different therapies was found in 5-year cumulative relapse (13.9% vs. 9.5% vs. 17.3% in chemotherapy, allo-HSCT, and auto-HSCT subgroups, respectively; P = 0.469, Fig. 3). Similarly, there was no statistically significant difference in the estimated 5-year DFS and OS rates among the three subgroups (DFS: 78.1% vs. 83.0% vs. 82.7%; OS: 79.0% vs. 87.7% vs. 85.6%; P = 0.805 and 0.940; respectively, Fig. 3). However, for MRD3-positive patients, the 5-year CIR was lower in the allo-HSCT than in the chemotherapy subgroup (22.3% vs. 71.5%, P < 0.001, Fig. 3); the 5-year cumulative DFS and OS were higher in the allo-HSCT than in the chemotherapy subgroup (65.9% vs. 23.0%, and 67.1% vs. 23.9%; P = 0.002 and 0.022, respectively, Fig. 3). factors, including high WBCs and ≥2 cycles required to achieve CR, were significantly associated with relapse in multivariate analysis (P > 0.05). For DFS, patient age >36 years, MRD3 positivity and consolidation chemotherapy were also independent risk factors (P = 0.030, 0.003 and 0.001; HR = 1.886, 2.643 and 2.872; 95% CI 1.065–3.341, 1.381–5.058 and 1.499–5.503, respectively), whereas other factors demonstrated no association with DFS (P > 0.05). For OS, MRD3 positivity and consolidation chemotherapy were also independent risk factors (P = 0.016 and 0.025; HR = 2.582 and 2.267; 95% CI 1.197–5.567 and 1.107–4.645, respectively), while patient age >36 years, high WBCs, and ≥2 cycles required to achieve CR had no significant influence (P > 0.05). Discussion status. These patients should preferentially be offered myeloablative rather than reduced-intensity conditioning allo-HSCT [29–31]. Nevertheless, the timepoint at which MRD is most closely associated with prognosis remains controversial. A few studies showed that MRD after cycle 1 or 2 was associated with prognosis, while another study suggested that MRD was an independent prognostic factor after several cycles by next-generation sequencing (such as pre-transplantation) [10, 11, 32].l AML is a heterogeneous clonal disease with dismal survival, largely due to high relapse incidence [20]. Although more than 70% of patients will achieve CR1 after induction chemotherapy, many may relapse [21]. In previous studies, MRD status was proven to be an independent prognostic predictor for oncologic outcomes [10, 22, 23]. The precise timepoint at which MRD is associated with prognosis remains controversial. In this study, we demonstrated that MRD3-positive patients (i.e., after the third chemotherapy cycle, two inductions and one consolidation) were more likely to relapse compared to those with MRD3 negativity in the population of younger patients with de novo IR-AML. Additionally, MRD3-positive patients had lower 5-year DFS and OS. However, these unfavorable outcomes were ameliorated more effectively by allo-HSCT than chemotherapy. Therefore, our results indicated that MRD3 (after the third cycle of chemotherapy) was an appropriate indicator of PRT choice for IR-AML in CR1. Several factors influence MRD status and the subsequent outcomes of patients with IR-AML, including the chemotherapy intensity, infection, HSCT application, and survival [33–35]. Recently, a study indicates that high- intensity relative to low-intensity chemotherapy results in higher rates of MRD-negative cases [35]; another study indicates that induction therapy of IDAC with daunorubicin and omacetaxine mepesuccinate increases survival in patients with AML aged 15–55 years relative to conventional-dose cytarabine [36]. Luca Maurillo reported that MRD negativity is not less in the standard-dose group as compared to high-dose cytarabine after both induction and consolidation [37]. Three doses of cytarabine consolidation (1.5 g/m2, 2 g/m2, and high-dose cytarabine (HiDAC) 3 g/ m2) were administered to intermediate- and poor-risk AML patients, and showed no significant differences in DFS and OS; however; septic shock was significantly higher after HiDAC 3 g/m2 administration as compared to the IDAC regimen [38]. The present study indicated that MRD direction and HSCT selection in the PRT were vital for improving the survival of IR-AML patients. Interaction between MRD and beneficial effects of HSCT in CR1 In this study, 42 patients who were MRD3 negative underwent auto-HSCT in CR1. Among the other patients, consolidation chemotherapy and allo-HSCT were administered to 114 and 79 cases respectively. The allo- HSCT subgroup included 34 patients (43.0%) with a human leukocyte antigen (HLA)-MSD transplant (10/10 HLA- matched), 21 patients (26.6%) with a matched or mismatched unrelated donor transplant (10/10–9/10 HLA-matched), and 24 patients (30.3%) with an HLA-mismatched related donor transplant (1/10–5/10 HLA-mismatched). 1 3 L. Han et al. 898 Fig. 3 Interaction between MRD3 and beneficial effects of HSCT in AML with CR1. The estimated cumulative incidence of relapse A, DFS B, and OS C according to the MRD level after the 3rd cycle of chemotherapy (≥0.1% or <0.1%) in patients with intermediate- risk acute myeloid leukemia in the first complete remission. Allo allogeneic hematopoietic stem cell transplantation, Auto autologous hematopoietic stem cell transplantation, Chem Chemotherapy consolidation, DFS Disease-free survival, MRD Measurable residual disease, OS Overall survival Fig. 3 Interaction between MRD3 and beneficial effects of HSCT in AML with CR1. The estimated cumulative incidence of relapse A, DFS B, and OS C according to the MRD level after the 3rd cycle of chemotherapy (≥0.1% or <0.1%) in patients with intermediate- risk acute myeloid leukemia in the first complete remission. Allo allogeneic hematopoietic stem cell transplantation, Auto autologous hematopoietic stem cell transplantation, Chem Chemotherapy consolidation, DFS Disease-free survival, MRD Measurable residual disease, OS Overall survival ction between MRD3 and beneficial effects of HSCT in References 1. Klco JM, Miller CA, Griffith M, Petti A, Spencer DH, Ketkar- Kulkarni S, et al. Association between mutation clearance after induction therapy and outcomes in acute myeloid leukemia. JAMA. 2015;314:811–22. 2. Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med. 2016;374:2209–21. 3. Jongen-Lavrencic M, Grob T, Hanekamp D, Kavelaars FG, Al Hinai A, Zeilemaker A, et al. Molecular minimal residual disease in acute myeloid leukemia. N Engl J Med. 2018;378:1189–99. f This study had some limitations. First, our study was not randomized, and the number of patients was not large. f This study had some limitations. First, our study was not randomized, and the number of patients was not large. f This study had some limitations. First, our study was not randomized, and the number of patients was not large. Second, the possibility of treatment bias exists, as patients with MRD positivity may be less likely to receive auto- HSCT due to the high rate of relapse [46]. In addition, older patients were not included in this study because of their inability to receive HSCT. Therefore, larger, prospective randomized clinical trials are required to validate, modify, or disprove our results with more certainty. 4. Kantarjian H, Kadia T, DiNardo C, Daver N, Borthakur G, Jabbour E, et al. Acute myeloid leukemia: current progress and future directions. Blood Cancer J. 2021;11:41. 5. Walter RB, Appelbaum FR, Estey EH. Optimal dosing of cytarabine in induction and post-remission therapy of acute myeloid leukemia. Leukemia. 2021;35:295–8. 6. Ustun C, Le-Rademacher J, Wang HL, Othus M, Sun Z, Major B, et al. Allogeneic hematopoietic cell transplantation compared to chemotherapy consolidation in older acute myeloid leukemia (AML) patients 60–75 years in first complete remission (CR1): an alliance (A151509), SWOG, ECOG-ACRIN, and CIBMTR study. Leukemia. 2019;33:2599–609. In summary, our results indicate that MRD3, rather than MRD1 or MRD2, could be used, not only as a potent predictor of outcomes, but also as an indicator of optimal subsequent treatment strategies for patients with de novo IR-AML in CR1. The findings of this study also suggest that patients with IR-AML could be further categorized based on MRD3 status, which could improve personalized PRT. 7. Versluis J, In ’t Hout FE, Devillier R, van Putten WL, Manz MG, Vekemans MC, et al. Declarations Conflict of interest The authors declare that they have no conflicts of interest. Conflict of interest The authors declare that they have no conflicts of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Our results also demonstrated that MRD3, but not MRD1 or MRD2 was closely related to relapse and survival. MRD3 could be interpreted as insufficient ‘depth of remission’ from the first two chemotherapy cycles, and patients should undergo or prepare for allo-HSCT to prevent relapse and protect organs at the time of the fourth chemotherapy cycle. We also found that a 0.1% cut-off value for MRD was appropriate to discriminate between patients with significantly different CIRs. In addition, a recent study by Paiva et al. [43] indicated that a cut-off value of 0.1% was suitable to discriminate patients with different relapse rates, although other reports showed different values [44, 45]. Discussion For patients with IR-AML who have achieved CR after induction therapy, additional PRT is an essential component for maintaining a disease-free status. Three PRT alternative strategies, including conventional chemotherapy, auto- HSCT, and allo-HSCT, could be chosen as consolidation therapy for post-induction. The centers have different choices that depend on factors such as patient characteristics, availability of a donor, and doctors’ discretion [24–27]. Auto-HSCT has higher availability and a lower rate of non- relapse mortality than allo-HSCT. However, it is associated with a higher relapse rate, due to a lack of a graft-versus- leukemia effect [28]. Allo-HSCT following a myeloablative conditioning regimen has been considered an effective option for reducing the risk of relapse for patients with AML in CR1, particularly in younger patients with MRD-positive In our study, we explored the role of MRD1-3 in directing the selection of PRT for patients with de novo IR-AML in CR1, although the selection for transplant was dependent on MRD status and donor availability. As demonstrated by our results for patients who were MRD3 negative, outcomes l:.(1234567 1 3 ol:.(1234567 1 3 Post-remission measurable residual disease directs treatment choice and improves outcomes… 899 did not differ among high-dose chemotherapy, auto-HSCT, and allo-HSCT treatment. Venditti et al. [12] also reported that auto-HSCT exerts an effect similar to that of allo-HSCT on patients with IR-AML and MRD negativity. However, for our patients with MRD3 positivity, allo-HSCT yielded a better outcome than consolidation chemotherapy; it significantly decreased relapse rates and increased DFS and OS. Additionally, different outcomes were not found among the MSD, MUD, and HID transplant subgroups, although the sample sizes were limited. This agrees with results from recent studies, which have also indicated that clinical outcomes for HID are equivalent to MSD and MUD transplantation [39–42]. Therefore, consolidation chemotherapy and auto-HSCT could be considered a valuable alternative after induction therapy, which may be preferred for patients with IR-AML and MRD3 negativity in the absence of a suitably matched donor. In the presence of low resources, allo-HSCT should be reserved for patients with IR-AML and MRD3 positivity. Jointly Sponsored Project of Henan Medical Science and Technology Research Plan (LHGJ20190040), Key scientific research projects of colleges and universities in Henan Province (20B320048), Medical Science and Technology Project of Henan Province (SB201901106), and National Natural Science Foundation of China (82000163). References 2015;29:1041–50. 13. Döhner H, Estey EH, Amadori S, Appelbaum FR, Büchner T, Burnett AK, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115:453–74. 28. Takami A. Hematopoietic stem cell transplantation for acute myeloid leukemia. Int J Hematol. 2018;107:513–8. 29. Cornelissen JJ, van Putten WL, Verdonck LF, Theobald M, Jacky E, Daenen SM, et al. Results of a HOVON/SAKK donor versus no-donor analysis of myeloablative HLA-identical sibling stem cell transplantation in first remission acute myeloid leukemia in young and middle-aged adults: benefits for whom? Blood. 2007;109:3658–66. 14. Chang YJ, Wang Y, Liu YR, Xu LP, Zhang XH, Chen H, et al. Haploidentical allograft is superior to matched sibling donor allograft in eradicating pre-transplantation minimal residual disease of AML patients as determined by multiparameter flow cytometry: a retrospective and prospective analysis. J Hematol Oncol. 2017;10:134. 30. Gilleece MH, Labopin M, Yakoub-Agha I, Volin L, Socié G, Ljungman P, et al. Measurable residual disease, conditioning regimen intensity, and age predict outcome of allogeneic hematopoietic cell transplantation for acute myeloid leukemia in first remission: a registry analysis of 2292 patients by the Acute Leukemia Working Party European Society of Blood and Marrow Transplantation. Am J Hematol. 2018;93:1142–52. 15. Yu J, Li Y, Li T, Li Y, Xing H, Sun H, et al. Gene mutational analysis by NGS and its clinical significance in patients with myelodysplastic syndrome and acute myeloid leukemia. Exp Hematol Oncol. 2020;9:2. 16. Tallman MS, Wang ES, Altman JK, Appelbaum FR, Bhatt VR, Bixby D, et al. Acute myeloid leukemia, version 3.2019, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 2019;17:721–49. 31. Hourigan CS, Dillon LW, Gui G, Logan BR, Fei M, Ghannam J, et al. Impact of conditioning intensity of allogeneic transplantation for acute myeloid leukemia with genomic evidence of residual disease. J Clin Oncol. 2020;38:1273–83. 17. Burnett AK, Hills RK, Nielsen OJ, Freeman S, Ali A, Cahalin P, et al. A comparison of FLAG-Ida and daunorubicin combined with clofarabine in high-risk acute myeloid leukaemia: data from the UK NCRI AML17 Trial. Leukemia. 2018;32:2693–7.f 32. Tsai CH, Tang JL, Tien FM, Kuo YY, Wu DC, Lin CC, et al. Clinical implications of sequential MRD monitoring by NGS at 2 time points after chemotherapy in patients with AML. Blood Adv. 2021;5:2456–66. 18. References Comparative value of post-remission treatment in cytogenetically normal AML subclassified by NPM1 and FLT3-ITD allelic ratio. Leukemia. 2017;31:26–33. 8. Stelljes M, Krug U, Beelen DW, Braess J, Sauerland MC, Heinecke A, et al. Allogeneic transplantation versus chemotherapy as postremission therapy for acute myeloid leukemia: a prospective matched pairs analysis. J Clin Oncol. 2014;32:288–96. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s12185-022-03441-6. 9. Keating A, DaSilva G, Pérez WS, Gupta V, Cutler CS, Ballen KK, et al. Autologous blood cell transplantation versus HLA- identical sibling transplantation for acute myeloid leukemia in first complete remission: a registry study from the Center for Acknowledgements We would like to thank Editage (www.editage.cn) for the English language editing. This work was partially funded by the 1 3 1 3 900 L. Han et al. International Blood and Marrow Transplantation Research. Haematologica. 2013;98:185–92. 24. Döhner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Büchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129:424–47. 10. Terwijn M, van Putten WL, Kelder A, van der Velden VH, Brooimans RA, Pabst T, et al. High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: data from the HOVON/SAKK AML 42A study. J Clin Oncol. 2013;31:3889–97. 25. Ferrara F, Picardi A. Is there still a role for autologous stem cell transplantation for the treatment of acute myeloid leukemia? Cancers (Basel). 2019. https://doi.org/10.3390/cancers12010059. 11. Freeman SD, Hills RK, Virgo P, Khan N, Couzens S, Dillon R, et al. Measurable residual disease at induction redefines partial response in acute myeloid leukemia and stratifies outcomes in patients at standard risk without NPM1 mutations. J Clin Oncol. 2018;36:1486–97.i 26. Saraceni F, Labopin M, Gorin NC, Blaise D, Tabrizi R, Volin L, et al. Matched and mismatched unrelated donor compared to autologous stem cell transplantation for acute myeloid leukemia in first complete remission: a retrospective, propensity score- weighted analysis from the ALWP of the EBMT. J Hematol Oncol. 2016;9:79. 12. Venditti A, Piciocchi A, Candoni A, Melillo L, Calafiore V, Cairoli R, et al. GIMEMA AML1310 trial of risk-adapted, MRD- directed therapy for young adults with newly diagnosed acute myeloid leukemia. Blood. 2019;134:935–45. 27. Cornelissen JJ, Versluis J, Passweg JR, van Putten WL, Manz MG, Maertens J, et al. Comparative therapeutic value of post-remission approaches in patients with acute myeloid leukemia aged 40–60 years. Leukemia. References Patzke CL, Duffy AP, Duong VH, El Chaer F, Trovato JA, Baer MR, et al. Comparison of high-dose cytarabine, mitoxantrone, and pegaspargase (HAM-pegA) to high-dose cytarabine, mitoxantrone, cladribine, and filgrastim (CLAG-M) as first-line salvage cytotoxic chemotherapy for relapsed/refractory acute myeloid leukemia. J Clin Med. 2020. https://doi.org/10.3390/ jcm9020536. 33. Stemler J, de Jonge N, Skoetz N, Sinkó J, Brüggemann R, Busca A, et al. Antifungal prophylaxis in adult patients with acute myeloid leukaemia treated with novel targeted therapies: a systematic review and expert consensus recommendation from the European Hematology Association. Lancet Haematol. 2022;9:e361–73.fi 34. Griffiths J, White P, Thompson A, da Fonseca D, Pickering R, Ingram W, et al. A novel strategy to identify haematology patients at high risk of developing Aspergillosis. Front Immunol. 2021;12: 780160. 19. Han L, Zhao K, Li Y, Han H, Zhou L, Ma P, et al. A gut microbiota score predicting acute graft-versus-host disease following myeloablative allogeneic hematopoietic stem cell transplantation. Am J Transplant. 2020;20:1014–27. 35. Hochman M, Othus M, Walter R, Shaw C, Gardner K, Percival M, et al. Effect of post-treatment MRD status on subsequent outcomes according to chemotherapy intensity in acute myeloid leukemia (AML). Leuk Lymphoma. 2021;62:1532–5. 20. Ngai LL, Kelder A, Janssen J, Ossenkoppele GJ, Cloos J. MRD tailored therapy in AML: what we have learned so far. Front Oncol. 2020;10: 603636. 21. Koreth J, Schlenk R, Kopecky KJ, Honda S, Sierra J, Djulbegovic BJ, et al. Allogeneic stem cell transplantation for acute myeloid leukemia in first complete remission: systematic review and meta- analysis of prospective clinical trials. JAMA. 2009;301:2349–61. 36. Wei H, Wang Y, Gale R, Lin D, Zhou C, Liu B, et al. Randomized trial of intermediate-dose cytarabine in induction and consolidation therapy in adults with acute myeloid leukemia. Clin Cancer Res. 2020;26:3154–61. 22. Chen X, Xie H, Wood BL, Walter RB, Pagel JM, Becker PS, et al. Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol. 2015;33:1258–64. 37. Maurillo L, Buccisano F, Piciocchi A, Del Principe M, Sarlo C, Di Veroli A, et al. Minimal residual disease as biomarker for optimal biologic dosing of ARA-C in patients with acute myeloid leukemia. Am J Hematol. 2015;90:125–31. 23. Freeman SD, Virgo P, Couzens S, Grimwade D, Russell N, Hills RK, et al. References Prognostic relevance of treatment response measured by flow cytometric residual disease detection in older patients with acute myeloid leukemia. J Clin Oncol. 2013;31:4123–31. 38. Chanswangphuwana C, Polprasert C, Owattanapanich W, Kungwankiattichai S, Rattarittamrong E, Rattanathammethee T, et al. Comparison of three doses of cytarabine consolidation Vol:1 :.(1234567 3 901 Post-remission measurable residual disease directs treatment choice and improves outcomes… for intermediate- and adverse-risk acute myeloid leukemia: real world evidence from Thai acute myeloid leukemia registry. Clin Lymphoma Myeloma Leuk. 2022. https://doi.org/10.1016/j.clml. 2022.06.005. 43. Paiva B, Vidriales MB, Sempere A, Tarín F, Colado E, Benavente C, et al. Impact of measurable residual disease by decentralized flow cytometry: a PETHEMA real-world study in 1076 patients with acute myeloid leukemia. Leukemia. 2021. https://doi.org/10. 1038/s41375-021-01126-3. 39. Han LJ, Wang Y, Fan ZP, Huang F, Zhou J, Fu YW, et al. Haploidentical transplantation compared with matched sibling and unrelated donor transplantation for adults with standard-risk acute lymphoblastic leukaemia in first complete remission. Br J Haematol. 2017;179:120–30. 44. Rossi G, Minervini MM, Melillo L, di Nardo F, de Waure C, Scalzulli PR, et al. Predictive role of minimal residual disease and log clearance in acute myeloid leukemia: a comparison between multiparameter flow cytometry and Wilm’s tumor 1 levels. Ann Hematol. 2014;93:1149–57. 40. Wang Y, Liu QF, Xu LP, Liu KY, Zhang XH, Ma X, et al. Haploidentical versus matched-sibling transplant in adults with Philadelphia-negative high-risk acute lymphoblastic leukemia: a biologically phase III randomized study. Clin Cancer Res. 2016;22:3467–76. 45. Sui JN, Chen QS, Zhang YX, Sheng Y, Wu J, Li JM, et al. Identifying leukemia-associated immunophenotype-based individualized minimal residual disease in acute myeloid leukemia and its prognostic significance. Am J Hematol. 2019;94:528–38. 41. Nishiwaki S, Atsuta Y, Tanaka J. Allogeneic hematopoietic cell transplantation from alternative sources for adult Philadelphia chromosome-negative ALL: what should we choose when no HLA-matched related donor is available? Bone Marrow Transplant. 2013;48:1369–76. i 46. Zhu HH, Zhang XH, Qin YZ, Liu DH, Jiang H, Chen H, et al. MRD-directed risk stratification treatment may improve outcomes of t(8;21) AML in the first complete remission: results from the AML05 multicenter trial. Blood. 2013;121:4056–62. 42. Gorin NC, Labopin M, Blaise D, de Groot M, Socié G, Bourhis JH, et al. References Stem cell transplantation from a haploidentical donor versus a genoidentical sister for adult male patients with acute myelogenous leukemia in first remission: a retrospective study from the acute leukemia working party of the European Society for Blood and Marrow Transplantation. Cancer. 2020;126:1004–15. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3
https://openalex.org/W2891453476	https://www.frontiersin.org/articles/10.3389/fphar.2018.01023/pdf	English	null	Delay-Dependent Impairments in Memory and Motor Functions After Acute Methadone Overdose in Rats	Frontiers in pharmacology	2,018	cc-by	12,263	ORIGINAL RESEARCH published: 10 September 2018 doi: 10.3389/fphar.2018.01023 Edited by: Antonella Gasbarri, University of L’Aquila, Italy Reviewed by: Diego Andolina, Università degli Studi di Roma “La Sapienza”, Italy Amir Mohammad Alizadeh, KU Leuven, Belgium Reviewed by: Diego Andolina, Università degli Studi di Roma “La Sapienza”, Italy Amir Mohammad Alizadeh, KU Leuven, Belgium Correspondence: Carlos Tomaz ctomaz@ceuma.br; ctomaz@unb.br Abbas Haghparast haghparast@sbmu.ac.ir; haghparast@yahoo.com Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Keywords: methadone, naloxone, learning and memory performance, motor coordination, overdose, rat Received: 27 January 2018 Accepted: 23 August 2018 Published: 10 September 2018 Leila Ahmad-Molaei1, Hossein Hassanian-Moghaddam2,3, Fariba Farnaghi4, Carlos Tomaz5 and Abbas Haghparast1* Leila Ahmad-Molaei1, Hossein Hassanian-Moghaddam2,3, Fariba Farnaghi4, Carlos Tomaz5* and Abbas Haghparast1* 1 Neuroscience Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 2 Department of Clinical Toxicology, Loghman-Hakim Hospital, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 3 Social Determinants of Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 4 Department of Pediatric Clinical Toxicology, Loghman-Hakim Hospital, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 5 Neuroscience Research Program, CEUMA University, São Luís, Brazil Methadone is used as a substitution drug for the treatment of opioid dependence and chronic pain. Despite its widespread use and availability, there is a serious concern with respect to the relative safety of methadone. The purpose of this study was to characterize how acute methadone overdose affects the cognitive and motor performance of naïve healthy rats. The methadone overdose was induced by administering an acute toxic dose of methadone (15 mg/kg; ip; the equivalent dose of 80% of LD50) to adolescent rats. Resuscitation using a ventilator pump along with a single dose of naloxone (2 mg/kg; ip) was administered following the occurrence of apnea. The animals which were successfully resuscitated divided randomly into three apnea groups that evaluated either on day 1, 5, or 10 post-resuscitation (M/N-Day 1, M/N-Day 5, and M/N-Day 10 groups) in the Y-maze and novel object memory recognition tasks as well as pole and rotarod tests. The data revealed that a single toxic dose of methadone had an adverse effect on spontaneous behavior. In addition, Recognition memory impairment was observed in the M/N-Day 1, 5, and 10 groups after methadone-induced apnea. Further, descending time in the M/N-Day 5 group increased signiﬁcantly in comparison with its respective Saline control group. The overall results indicate that acute methadone-overdose-induced apnea produced delay-dependent cognitive and motor impairment. We suggest that methadone poisoning should be considered as a possible cause of delayed neurological disorders, which might be transient, in some types of memory or motor performance in naïve healthy rats. Keywords: methadone, naloxone, learning and memory performance, motor coordination, overdose, rat Ahmad-Molaei L, Hassanian-Moghaddam H, Farnaghi F, Tomaz C and Haghparast A (2018) Delay-Dependent Impairments in Memory and Motor Functions After Acute Methadone Overdose in Rats. Front. Pharmacol. 9:1023. doi: 10.3389/fphar.2018.01023 Edited by: Antonella Gasbarri, University of L’Aquila, Italy Drugs h In the present study, Methadone hydrochloride 5 mg/ml (Darou- Pakhsh Pharmaceutical Company, Tehran, Iran) and Naloxone 0.4 mg/ml (Tolid-Darou Pharmaceutical Company, Tehran, Iran) were used. Citation: Methadone is a long-acting, synthetic mu-opioid agonist having multiple actions and pharmacologic properties that are similar to morphine (Barbosa Neto et al., 2015). Methadone has long been used for the treatment of opioid dependence and detoxiﬁcation or maintenance in cases of opioid addiction because of its long eﬃcacy and low cost (Kleber, 2007). In addition, like other opioids such as buprenorphine, fentanyl, morphine, and oxycodone, methadone is used to alleviate severe pain (Argoﬀand Silvershein, 2009). Despite its considerable therapeutic applications, acute methadone intoxication may lead to morbidity and death (Shields et al., 2007; Soltaninejad et al., 2014). In the United States, opioid drugs were involved in 61% of all drug Ahmad-Molaei L, Hassanian-Moghaddam H, Farnaghi F, Tomaz C and Haghparast A (2018) Delay-Dependent Impairments in Memory and Motor Functions After Acute Methadone Overdose in Rats. Front. Pharmacol. 9:1023. doi: 10.3389/fphar.2018.01023 September 2018 \| Volume 9 \| Article 1023 1 Frontiers in Pharmacology \| www.frontiersin.org Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. MATERIALS AND METHODS overdose deaths and caused more than 28,000 deaths in 2014 (Rudd et al., 2016). Acute poisoning with methadone continues to occur after therapeutic, recreational or accidental use (Jones et al., 2012). In Iran, opium was the drug of choice in 50% of all drug abuse from 2006 to 2009, but the prevalence of methadone toxicity has increased signiﬁcantly from 2.26% in 2006 to 24.72% in 2011 (Hassanian-Moghaddam et al., 2014). overdose deaths and caused more than 28,000 deaths in 2014 (Rudd et al., 2016). Acute poisoning with methadone continues to occur after therapeutic, recreational or accidental use (Jones et al., 2012). In Iran, opium was the drug of choice in 50% of all drug abuse from 2006 to 2009, but the prevalence of methadone toxicity has increased signiﬁcantly from 2.26% in 2006 to 24.72% in 2011 (Hassanian-Moghaddam et al., 2014). Frontiers in Pharmacology \| www.frontiersin.org Experimental Design and Drug Administration In order to induce acute methadone overdose, rats intraperitoneally (i.p.) received a single toxic dose of 15 mg/kg of methadone at equivalent doses (80% of the LD50) which was chosen based on Chevillard study (Chevillard et al., 2009). Slow and diﬃcult breathing, dizziness, cold and clammy skin, motionlessness, drowsiness, straub tail, muscular rigidity, plantar cyanosis, and irritability were seen after the administration of a single toxic dose of methadone in adolescent rats. It has been noted that the primary signs of opioid intoxication include: pinpoint pupils, respiratory depression, and confusion/unconsciousness, referred to as the opioid overdose triad (Ford, 2001). In 35% of all rats, methadone- induced apnea and caused death if they were left untreated, but the rest of the animals regained normal respiration rate after a few hours without any intervention which was randomly selected as methadone group. In order to evaluate cognitive and motor functions in the rats which experienced apnea (cessation of respiration was for 20 s) (Gaspari and Paydarfar, 2007), an acute single dose of naloxone (2 mg/kg; i.p.) (Farahmandfar et al., 2010; Zamani et al., 2015) was administered following methadone-induced apnea. In addition, resuscitation procedure was performed by a respirator pump to serve artiﬁcial respiration (Model V5KG, Narco-Biosystems Inc., Houston, TX, United States). Naloxone administration which was done concomitantly with resuscitation, recovered apnea in 67% of all rats in which had cessation of respiration for 20 s. Therefore, animals which have been successfully resuscitated, were randomly divided into three groups so-called “M/N” groups (rats which received naloxone after methadone overdose) to measure neurological functions either on day 1st, 5th, or 10th post- resuscitation (including M/N-Day 1, M/N-Day 5, and M/N-Day 10 groups; Figure 1). In other groups all behavioral ( g ) Few studies have examined cognitive and sensorimotor performance after an acute dose of methadone-induced toxicity in clinical or experimental trials in healthy volunteers. Most studies have examined the eﬀect of the prolonged use of methadone, which can result in neuropsychological impairment as compared to opioid-naïve, healthy controls (Prosser et al., 2006). There is considerable evidence that chronic exposure to methadone in animals can have an adverse eﬀect on memory processes (Hepner et al., 2002; Verdejo et al., 2005). Animal One-month- old male Wistar rats, (Pasteur Institute, Tehran, Iran) weighting 50-80 g, were kept under the standard laboratory conditions (22◦C, 12-h light/12-h dark cycle) and randomly allocated to diﬀerent experimental groups. All rats habituated to their new environment for 5 days before the experimental procedure started. The tests were performed between 8:00 and 16:00 h. All procedures were conducted according to the Guide for the care and use of laboratory animals (National Institutes of Health Publication No. 80–23, revised 1996) and were approved by the Research and Ethics Committee of School of Medicine, Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1395.33), Tehran, Iran. g Since the opiate naïve patients have no tolerance to opiates, (Drummer et al., 1992; Milroy and Forrest, 2000) the stabilization phase should be carefully assessed to reduce the risk of overdose during the induction period to avert the risk of toxicity and death in methadone maintenance treatment (MMT) programs (Morgan et al., 2006; Modesto-Lowe et al., 2010). Several studies indicate a 10-fold increase in methadone-induced toxicity and related death after the increase in the number of methadone maintenance clinics and its arbitrary consumption in recent decades (Shields et al., 2007; Graham et al., 2008). The incidence of poisoning with methadone in children is common due to the availability of this drug as used by family members (Pragst et al., 2013; Shadnia et al., 2013). Methadone poisoning should be considered as a serious threat to naïve, healthy subjects, especially children, as very low doses can cause severe complications or death due to its toxicity (Modesto-Lowe et al., 2010; Jabbehdari et al., 2013; Hassanian-Moghaddam et al., 2017). Indeed, some studies have associated therapeutic doses of methadone with the occurrence of sudden death due to respiratory apnea or cardiac arrest (Chugh et al., 2008). Experimental Design and Drug Administration Moreover, patients undergoing the MMT program usually experience limited short-term memory and deﬁcits in working memory (Sjøgren et al., 2000; Mintzer and Stitzer, 2002), visuospatial attention, long-term memory (Prosser et al., 2006) and general cognitive speed (Mintzer et al., 2005) which are in part due to white matter abnormalities (Lin et al., 2012). It has been shown that acute administration of methadone impairs sensorimotor abilities and memory retrieval in rats (Tramullas et al., 2007). Because it has a signiﬁcantly long half-life of 25–52 h, even a single acute administration of methadone can cause delayed clinical manifestations, including respiratory depression, apnea and unexpected death (LoVecchio et al., 2007). Despite the fact that, in recent years, methadone overdose has increased, little data is available about the adverse manifestations of methadone overdose in experimentally naïve animals. In addition, behavioral research in human subjects is extremely rare because of ethical considerations. The present study aimed to investigate whether or not a single toxic dose of methadone will result in apnea-caused impairment on cognitive and/or motor functions in adolescent rats. September 2018 \| Volume 9 \| Article 1023 2 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. Novel Object Recognition Test (NOR) The task procedure consisted of three distinct phases: habituation, familiarization, and test. The NOR task was performed to measure non-spatial memory. An open ﬁeld box (40 × 40 × 40 cm) (length × width × height) was made of black wood used as an apparatus to test recognition memory. Rats were allowed 1 h of accustomed to the test room before starting each phase. All rats were given a 10-min session to explore apparatus with no objects as a habituation phase in two consecutive days. During the familiarization phase, two identical objects (A1 and A2) were attached to the ﬂoor at an equal distance, 10 cm from the walls while positioned in the two adjacent corners. Each rat was placed in the box facing the wall opposite the two identical objects allowed to explore freely for 3 min. If the total exploration time was less than 12 s for the novel and familiar objects during familiarization phase, the rat excluded from the data analysis. Then, object A1 or A2 was replaced with object B before starting the test phase. Pole Test Pole test was ﬁrst introduced by Ogawa (Ogawa et al., 1985) to evaluate movement impairment and coordination in mice indicating a practical task for the basal ganglia dysfunction. The apparatus consisted of a 90 cm vertical wooden pole length and 5 cm in diameter which covered with the rough surface that led into their home cage. All animals received training sessions on two consecutive days (10-trials/day) before the test day where they were placed with the head facing upward right below the top. During the ﬁrst trial on the ﬁrst day, if the rat failed to climb down, it was gently turned around on the pole and thus forced to return to its home cage. On the test day, three parameters were measured; t-turn (the time to turn downward), descending time (the time to descend the pole) and total time (the time to turn downward and descent the pole to reach the ﬂoor). When the animal failed to turn downward after 120 s, it was taken as a default value. The animals were tested on 3 trials on the test day and the average time was used as the pole test score. Experimental Design and Drug Administration Evaluation of short-term memory was conducted 90-min later in which the familiar object and the new object (object B) located in the open ﬁeld. Rats were allowed to explore freely for 3-min in the box. After 24 h, object B was replaced with object C for testing long-term memory in a 3-min period to explore the box. The time spent exploring both objects (familiar and novel) was recorded by a video tracking system. The preference index was calculated as the exploration time for the novel and familiar objects relative to the total time (Antunes and Biala, 2012; Cohen and Stackman, 2015). tests were carried out only 1 day after the drug administration. Methadone group was selected randomly from the animals which re-obtained normal respiration rate after administration of a single toxic dose of methadone without any intervention. There was another group without apnea (M/N-Sedate) in which they received the same dose of methadone but naloxone administration was delivered at the beginning of the sedation state. Naloxone group with a single administration dose was designated to test memory and motor functions exclusively in a separated group. Control (Saline) group received an equal volume of saline 0.9% and behavioral assessment was performed 1 day after saline injection. It has been noted that data in the M/N-Day 1, 5 and 10 groups were compared with the Saline control-Day 1, Day 5 and Day 10 groups, respectively. Each group consisted of 6–14 rats while were grouped in 10 experimental groups. tests were carried out only 1 day after the drug administration. Methadone group was selected randomly from the animals which re-obtained normal respiration rate after administration of a single toxic dose of methadone without any intervention. There was another group without apnea (M/N-Sedate) in which they received the same dose of methadone but naloxone administration was delivered at the beginning of the sedation state. Naloxone group with a single administration dose was designated to test memory and motor functions exclusively in a separated group. Control (Saline) group received an equal volume of saline 0.9% and behavioral assessment was performed 1 day after saline injection. It has been noted that data in the M/N-Day 1, 5 and 10 groups were compared with the Saline control-Day 1, Day 5 and Day 10 groups, respectively. Each group consisted of 6–14 rats while were grouped in 10 experimental groups. Behavioral Training For ﬁve consecutive days, rats were handled for 5 min before starting any test procedure. All rats had multiple behavioral tests including the Y-maze, novel object memory recognition (NOR) tests as well as pole, rotarod tasks to investigate neurological functions. Animal behaviors were observed by a researcher who was blind to the experimental groups. The order of tests was the same for all animals. In order to avoid the eﬀect of any confounding factors or minimize the inﬂuence of stress on animals for each task, the order of behavioral tests was as follow; (1) Y-maze test, (2) NOR test, (3) pole test, and (4) rotarod test. In addition, locomotor activity was measured for each rat during a 5-min period on the test day (Figure 6). Spontaneous Alternation Behavior Test (Y-Maze) The Y-maze test can be used as a measure of spatial working memory in rodents. It is applied to evaluate the natural tendency of animals to explore new places by recording spontaneous alternation behavior. In this study, the Y-maze apparatus consisted of the Y-shaped maze with three identical arms at 120 degrees to each other which was made of gray- painted Plexiglass. Rats were placed at the end of the one arm and allowed to navigate the maze during an 8-min trial. The sequence and number of the total arm entries were manually registered. An arm entry was deﬁned when four paws were within the arm. An alternation behavior was determined from consecutive entries into the three diﬀerent arms. The percentage of alternation was calculated as the following equation: {(number of alternation)/(total number of arm entries −2)} × 100 Rotarod apparatus is used to evaluate motor coordination and skills in rodents (Dunham and Miya, 1957; Deacon, 2013). Animals were placed on a 2.5 cm diameter drum supported 25 cm above the base of the apparatus. Rats were trained 5 trials a day, separated by 30 min inter-trial intervals on the two successive days. Animals were placed in the testing room for 1 h before starting the test to acclimate to the testing. The rats were held by their tails while facing away from the direction of rotation Total number of arm entries were recorded as well. In addition, animals with 8 arm entries or less were omitted from analysis during an 8-min session (Holcomb et al., 1998; Ma et al., 2007; Farhadinasab et al., 2009). September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 3 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. FIGURE 1 \| Schematic illustration of the experimental schedule. (A) Protocol overview of the study. After 2 days of behavioral training (rotarod and pole tasks), all rats were administered drug injection on day 0. According to the time of the behavioral test, (starting on day 1, 5, or day 10 after drug administration) they represented here in three parts. (B) After drug application, six separated animal groups (Saline, Methadone, Naloxone, M/N-Sedate, Saline control-Day 1, and M/N-Day 1) were used to evaluate different behavioral tests including Y-Maze, Rotarod test and Pole test on day 1 after drug administration and Novel object recognition (NOR) test from day 1 to day 4 post-treatment. (C) In two other separated groups (Saline control-Day 5, M/N-Day 5), after administration of saline alone or methadone + naloxone on day 0, Y-Maze, Pole test, and Rotarod test were carried out on day 5 followed by NOR test from day 5 to day 8. (D) In Saline control-Day 10 and M/N-Day 10 groups, 10 days after administration of saline alone or methadone + naloxone, Y-Maze, Pole test and Rotarod test were carried out on day 10 followed by NOR test from day 10 to day 13. FIGURE 1 \| Schematic illustration of the experimental schedule. (A) Protocol overview of the study. After 2 days of behavioral training (rotarod and pole tasks), all rats were administered drug injection on day 0. Locomotor Activity Total numbers of infrared beam break automatically were recorded. Rats were placed in a box (40 × 40 × 40 cm) to evaluate locomotion. Locomotor activity was tracked by a 5 × 5 photobeam conﬁguration for each rat in which sensed infrared beam interruption caused by movement of the animal in real time for 5 min (Zhang and Kong, 2017). The novel object recognition task is the ability to distinguish the novel from familiar stimuli which is directly dependent on the prefrontal cortex and hippocampus function (Banks et al., 2012; Pezze et al., 2017). In Figure 3A, the data obtained analyzed using paired t-test exhibited that animals spent equal time to explore both object A1 and A2 and there were not any signiﬁcant preference in exploring two objects in familiarization phase in the M/N-Day 1 [t(6) = 0.7381, P = 0.4883; ns], Day 5 [t(6) = 0.5558, P = 0.5984; ns] and Day 10 [t(6) = 0.5176, P = 0.6109; ns] groups as compared with their respective Saline control groups (right panel). In Figure 3B which shown short- term memory phase, unpaired t-test analysis indicated that a single toxic dose of methadone (apnea groups) signiﬁcantly impaired recognition memory in the M/N-Day 1 [t(12) = 2.785, P < 0.01] and M/N-Day 5 [t(9) = 3.032, P < 0.01] when compared with their respective Saline control groups (right panel). One-way ANOVA followed by Newman–Keuls post hoc test exhibited that administration of an acute toxic dose of methadone (15 mg/kg; i.p.) with subsequent naloxone (2 mg/kg; i.p.) administration in sedation state (M/N-Sedate group), three M/N-treated groups, as well as methadone and naloxone groups did not have attenuating eﬀects on short-term memory when compared to the Saline group [F(6,46) = 3.871, P = 0.0010], while this parameter shown signiﬁcant reduction in the M/N-Day 5 groups as compared with the Saline group. In Figure 3C, the data obtained for long-term memory test revealed detrimental eﬀect of methadone overdose on long-term memory in the M/N- Day 1 [t(11) = 3.903, P = 0.0025] and Day-5 [t(9) = 4.512, P < 0.001] groups which have been continued on day 13 in M/N- Day 10 group [t(11) = 4.285, P < 0.001] when compared with their respective Saline control groups (right panel). {(number of alternation)/(total number of arm entries −2)} × 100 According to the time of the behavioral test, (starting on day 1, 5, or day 10 after drug administration) they represented here in three parts. (B) After drug application, six separated animal groups (Saline, Methadone, Naloxone, M/N-Sedate, Saline control-Day 1, and M/N-Day 1) were used to evaluate different behavioral tests including Y-Maze, Rotarod test and Pole test on day 1 after drug administration and Novel object recognition (NOR) test from day 1 to day 4 post-treatment. (C) In two other separated groups (Saline control-Day 5, M/N-Day 5), after administration of saline alone or methadone + naloxone on day 0, Y-Maze, Pole test, and Rotarod test were carried out on day 5 followed by NOR test from day 5 to day 8. (D) In Saline control-Day 10 and M/N-Day 10 groups, 10 days after administration of saline alone or methadone + naloxone, Y-Maze, Pole test and Rotarod test were carried out on day 10 followed by NOR test from day 10 to day 13. September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. revealed no signiﬁcant reduction in the number of total arm entries in the M/N-Sedate, as well as M/N-Day 1 and Day 10 groups (with apnea, right panel), methadone or naloxone groups when compared with the Saline group [F(6,47) = 2.232, P < 0.05] but not for the M/N-Day 5 groups which showed signiﬁcant reduction as compared with the Saline group. the drum such that animals released on the horizontal rod while walking forward to keep their balance. The Rotation speed was set at 20 rpm in the training and testing sessions. If the rat failed to grasp rod properly and fell before 5 s, the procedure would start again to keep the balance. During the test session, animals were assessed by placing on the rod until either they fell oﬀor reached a maximum 300 s. The mean values of the 3 test trials were calculated for each rat. Locomotor Activity Moreover, administration of an acute toxic dose of methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone as well as M/N-treated rats (right panel) and M/N-Sedate groups did not show any signiﬁcant deﬁcit in long-term memory when compared to the Saline group [F(6,46) = 2.384, P = 0.0434]. Statistical Analysis All data were represented as mean ± SEM (standard error of mean) and were analyzed by commercially available software GraphPad Prism R⃝5.0. In order to compare data between two groups in familiarization phase in NOR, data in apnea groups which have been compared with their respective Saline control groups, paired or unpaired t-test were used, respectively. For multiple comparisons between groups, one-way analysis of variance (ANOVA) followed by post hoc Newman–Keuls test was applied as needed. The level of statistical signiﬁcance was set at P-value less than 0.05 (P < 0.05). Effect of a Single Acute Toxic Dose of Methadone Administration on Spatial Working Memory in the Y-Maze Test g y As shown in Figure 2A, unpaired t-test analysis revealed that administration of an acute toxic dose of methadone (15 mg/kg; i.p.) which caused apnea and subsequent naloxone injection (2 mg/kg; i.p.), impaired the percentage of the spontaneous alternation behavior in the M/N-Day 5 [t(9) = 2.908, P < 0.01] and M/N-Day 10 groups [t(10) = 2.695, P = 0.0225] when compared with their respective Saline control groups (right panel) while this parameter was not diﬀerent in the M/N-Day 1 as compared to its Saline control group [t(12) = 0.9745, P = 0.3491; ns]. As depicted in Figure 2A, one way ANOVA followed by Newman–Keuls post hoc analysis showed that there was not signiﬁcant deﬁcient in the alternation behavior in M/N- Sedate, M/N-treated rats (with apnea, right panel), methadone (without apnea) or naloxone groups as compared with the Saline [F(6,46) = 1.571, P = 0.1773] group. Moreover, in Figure 2B, unpaired t-test analysis manifested that the number of arm entries were not diﬀerent in the M/N-Day 1 [t(12) = 1.947, P = 0.0754; ns], Day 5 [t(9) = 0.8357, P = 0.4249; ns] and Day 10 [t(10) = 1.010, P = 0.3363; ns] groups as compared with their respective Saline control groups (right panel). In addition, One-way ANOVA followed by Newman–Keuls post hoc analysis RESULTS Effect of a Single Acute Toxic Dose of Methadone Administration on Spatial Working Memory in the Y-Maze Test Effect of an Acute Toxic Dose of Effect of an Acute Toxic Dose of Methadone Administration on Motor Functions in Pole Test in Adolescent Rats As exhibited in Figure 4A (right panel), unpaired t-test analysis showed that there was no signiﬁcant diﬀerence in t-turn between the M/N-Day 1 [t(18) = 1.674, P = 0.1115; ns], Day 5 [t(10) = 1.820, P = 0.0988; ns] and their respective Saline control groups but this parameter increased in the M/N-Day 10 group as compared with its respective Saline control group [t(12) = 2.180, September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 5 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. FIGURE 2 \| (A) Spontaneous alternation behavior and (B) Total number of arm entries were recorded in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and spontaneous alternation behavior was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 6), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups; n = 6 right panel), M/N-Sedate (n = 8; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 12, methadone; n = 8 and naloxone; n = 7 (saline, methadone or naloxone were administered alone in separated groups in which spontaneous alternation behavior and the number of arm entries were recorded only 1 day after drug administration during an 8-min trial in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both (apnea groups) in a single dose. Each bar shows the mean ± SEM for 6–12. ∗P < 0.05 different from the Saline group. +P < 0.05 and ++P < 0.01 different from their respective Saline control groups. Effect of an Acute Toxic Dose of Methadone Overdose, Cognitive, and Motor Functions Performance of recognition memory in the novel object recognition task in three sessions as follow; (A) Familiarization (rats were allowed to explore freely l objects A1 and A2 for 3-min), (B) Short-term memory (object A1 or A2 was replaced with object B while rats were allowed to explore for 3-min), m memory (object B was replaced with object C which provided rats explored freely two objects for 3-min) in different groups, including; M/N-treated ngle dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and recognition memory was ther on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 7), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control 6); right panel), M/N-Sedate (n = 7; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 10, methadone; n = 11, naloxone; n = 8 (Saline, methadone or ere administered alone in separated groups which recognition memory was evaluated only 1 day after the drug administration in adolescent rats) groups. eived methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6–11. ∗P < 0.05 and different from the Saline group. ++P < 0.01 and +++P < 0.001 different from their respective Saline control groups. Ahmad-Molaei et al. Effect of an Acute Toxic Dose of FIGURE 2 \| (A) Spontaneous alternation behavior and (B) Total number of arm entries were recorded in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and spontaneous alternation behavior was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 6), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups; n = 6 right panel), M/N-Sedate (n = 8; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 12, methadone; n = 8 and naloxone; n = 7 (saline, methadone or naloxone were administered alone in separated groups in which spontaneous alternation behavior and the number of arm entries were recorded only 1 day after drug administration during an 8-min trial in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both (apnea groups) in a single dose. Each bar shows the mean ± SEM for 6–12. ∗P < 0.05 different from the Saline group. +P < 0.05 and ++P < 0.01 different from their respective Saline control groups. September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 6 i et al. Effect of an Acute Toxic Dose of valuation of motor performance in pole test such that three parameters were measured including; (A) time to turn downward (t-turn), (B) descending move downward to reach the ﬂoor and (C) total time (time to turn and descending the pole to reach the ﬂoor) in different groups, including; M/N-treated le dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor behavior was er on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 11), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 7) groups and their respective Saline control right panel), M/N-Sedate; n = 9 (a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of ehavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 8 and naloxone; n = 8 (saline, methadone or e administered alone in separated groups in which motor function was evaluated 1 day after drug administration during pole test in adolescent rats) als received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6-14. Effect of an Acute Toxic Dose of Methadone Overdose, Cognitive, and Motor Functions FIGURE 3 \| Performance of recognition memory in the novel object recognition task in three sessions as follow; (A) Familiarization (rats were allowed to explore freely two identical objects A1 and A2 for 3-min), (B) Short-term memory (object A1 or A2 was replaced with object B while rats were allowed to explore for 3-min), (C) long-term memory (object B was replaced with object C which provided rats explored freely two objects for 3-min) in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and recognition memory was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 7), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups (n = 6); right panel), M/N-Sedate (n = 7; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 10, methadone; n = 11, naloxone; n = 8 (Saline, methadone or naloxone were administered alone in separated groups which recognition memory was evaluated only 1 day after the drug administration in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6–11. ∗P < 0.05 and ∗∗P < 0.01 different from the Saline group. ++P < 0.01 and +++P < 0.001 different from their respective Saline control groups. September 2018 \| Volume 9 \| Article 1023 7 Frontiers in Pharmacology \| www.frontiersin.org Ahmad-Molaei et al. Methadone Overdose, Cognitive, and Motor Functions al. Effect of an Acute Toxic Dose of Methadone Overdose, Cognitive, and Motor Functions luation of motor performance in pole test such that three parameters were measured including; (A) time to turn downward (t-turn), (B) descending ve downward to reach the ﬂoor and (C) total time (time to turn and descending the pole to reach the ﬂoor) in different groups, including; M/N-treated dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor behavior was on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 11), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 7) groups and their respective Saline control ght panel), M/N-Sedate; n = 9 (a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of havioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 8 and naloxone; n = 8 (saline, methadone or administered alone in separated groups in which motor function was evaluated 1 day after drug administration during pole test in adolescent rats) received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6-14. ∗∗∗P < 0.001 different from the Saline group. ††P < 0.01 and †††P < 0.001 different from the M/N-Sedate group. +P < 0.05 different from their e control groups. Effect of an Acute Toxic Dose of URE 4 \| Evaluation of motor performance in pole test such that three parameters were measured including; (A) time to turn downward (t-turn), (B) descending (time to move downward to reach the ﬂoor and (C) total time (time to turn and descending the pole to reach the ﬂoor) in different groups, including; M/N-treated ps (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor behavior was uated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 11), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 7) groups and their respective Saline control ps; n = 6; right panel), M/N-Sedate; n = 9 (a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of ation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 8 and naloxone; n = 8 (saline, methadone or xone were administered alone in separated groups in which motor function was evaluated 1 day after drug administration during pole test in adolescent rats) ps. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6-14. < 0.01 and ∗∗∗P < 0.001 different from the Saline group. ††P < 0.01 and †††P < 0.001 different from the M/N-Sedate group. +P < 0.05 different from their ective Saline control groups. DISCUSSION The purpose of this research was to investigate the cognitive and motor eﬀects of a single toxic dose of methadone on three random groups of naïve adolescent rats tested on either day 1, 5, and 10 after drug administration as depicted in Figure 1. The ﬁndings showed that (i) Administration of an acutely toxic dose of methadone induced apnea in 35% of treated rats, (ii) Naloxone as a non-speciﬁc opioid receptor antagonist resuscitated 67% of the rats which experienced apnea, (iii) Delay-dependent impairment in cognitive and motor functions was observed in diﬀerent behavioral tests, (iv) Transient motor impairment following methadone-induced apnea, and (v) Motor deﬁcient in descending time on day 5 after administration of an acute toxic dose of methadone overdose in pole test was observed. The purpose of this research was to investigate the cognitive and motor eﬀects of a single toxic dose of methadone on three random groups of naïve adolescent rats tested on either day 1, 5, and 10 after drug administration as depicted in Figure 1. The ﬁndings showed that (i) Administration of an acutely toxic dose of methadone induced apnea in 35% of treated rats, (ii) Naloxone as a non-speciﬁc opioid receptor antagonist resuscitated 67% of the rats which experienced apnea, (iii) Delay-dependent impairment in cognitive and motor functions was observed in diﬀerent behavioral tests, (iv) Transient motor impairment following methadone-induced apnea, and (v) Motor deﬁcient in descending time on day 5 after administration of an acute toxic dose of methadone overdose in pole test was observed. It has been documented that opioids suppress respiration in humans and animals (Van Der Schier et al., 2014). Methadone is a long-acting opioid agonist used for therapy and as medication for abuse/dependence and to treat severe refractory cancer pain (Leppert, 2009; Keane, 2013; Schuckit, 2016). The extensive prescription of methadone has enhanced the risk of life- threatening overdoses in diﬀerent countries (Paulozzi et al., 2006; Rudd et al., 2016). Buprenorphine, like methadone, is used in the treatment of opioid addiction, but as a partial agonist, displays a ceiling eﬀect; after a certain point, an increase in the dosage will not enhance its eﬀects (Dahan et al., 2006). Rotarod Test Unpaired t-test analysis indicated that there were no signiﬁcant detrimental eﬀect of methadone overdose on motor coordination in rotarod test in the M/N-Day 1 [t(14) = 1.915, P = 0.0762; ns], Day 5 [t(11) = 2.153, P = 0.0543; ns] and Day 10 [t(13) = 1.689, P = 0.1150; ns] groups when compared with their respective Saline control groups (right panel). In addition, One-way ANOVA with Newman–Keuls post hoc test showed that in methadone group (without apnea) there was signiﬁcant impairment in motor coordination as compared with the Saline group [F(6,49) = 3.386, P = 0.0071]. However, in M/N-Sedate and naloxone groups as well as three M/N-treated groups, no signiﬁcant diﬀerences revealed on motor coordination when compared with the Saline group (Figure 5). In the Y-maze task, which is a measure of spatial working memory, impairment was revealed in the alternation behavior after methadone overdose. Consistent with our results, Hepner et al. (2002) indicated that methadone which acutely administered impaired the working memory version of Morris water task in rats. In the current study, we did not measure the concentration of methadone in blood or brain tissues, but Andersen et al. (2011) indicated that no methadone detected in brain tissue on the test day which showed memory impairment after the drug administration. The long-lasting impairment in learning or memory after acute or chronic opioid administration DISCUSSION In contrast, methadone is a full opioid agonist which has the potential to be abused, misused or used non-medically, making overdose-related death, especially due to respiratory depression, a big problem (Ayatollahi et al., 2011; Whelan and Remski, 2012; Soltaninejad et al., 2014). In recent years, methadone has been extensively prescribed in the MMT programs or to relieve pain, giving rise to methadone overdoses by adults using supratherapeutic amounts or by accidental ingestion in the pediatric population (Soltaninejad et al., 2014). Effect of an Acute Toxic Dose of d ∗∗∗P < 0 001 different from the Saline group ††P < 0 01 and †††P < 0 001 different from the M/N-Sedate group +P < 0 05 different from their FIGURE 4 \| Evaluation of motor performance in pole test such that three parameters were measured including; (A) time to turn downward (t-turn), (B) descending time (time to move downward to reach the ﬂoor and (C) total time (time to turn and descending the pole to reach the ﬂoor) in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor behavior was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 11), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 7) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate; n = 9 (a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 8 and naloxone; n = 8 (saline, methadone or naloxone were administered alone in separated groups in which motor function was evaluated 1 day after drug administration during pole test in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Each bar shows the mean ± SEM for 6-14. ∗∗P < 0.01 and ∗∗∗P < 0.001 different from the Saline group. ††P < 0.01 and †††P < 0.001 different from the M/N-Sedate group. +P < 0.05 different from their respective Saline control groups. September 2018 \| Volume 9 \| Article 1023 8 Frontiers in Pharmacology \| www.frontiersin.org Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. P < 0.05]. Moreover, one-way ANOVA followed by Newman– Keuls post hoc test showed that in the M/N-Sedate, methadone and naloxone groups no change has been observed in t-turn values when compared with the Saline group [F(6,57) = 6.007, P < 0.0001] but signiﬁcant increase revealed in the M/N-Day 10 when compared with the Saline or the M/N-Sedate groups. Effect of an Acute Toxic Dose of In Figure 4B (right panel), unpaired t-test analysis indicated that there was no signiﬁcant diﬀerence in descending time in the M/N-Day 1 [t(18) = 1.793, P = 0.0898; ns] and Day 10 [t(11) = 1.442, P = 0.1772; ns] groups as compared with their respective Saline control groups, but in the M/N-Day 5 group, the impairment was obvious in descending time when compared with its respective Saline control group [t(10) = 2.209, P < 0.05]. However, as shown in Figure 4B, one way ANOVA revealed that in the M/N-Sedate, the M/N-Day 1 and Day 10 groups as well as methadone (without apnea) and naloxone groups, motor functions were not impaired as compared with the Saline group [F(6,56) = 4.221, P = 0.0014] but signiﬁcant increase in descending time was observed in the M/N-Day 5 when compared with the Saline or M/N-Sedate groups. Additionally, in Figure 4C (right panel), unpaired t-test analysis showed that there was no signiﬁcant impairment in motor function in the M/N-Day 1 [t(18) = 2.008, P = 0.0599; ns] and Day 10 [t(12) = 1.307, P = 0.2155; ns] groups as compared with their respective Saline control groups, but in the M/N-Day 5, the detrimental eﬀect of methadone overdose was seen in motor activity when compared with its respective Saline control group [t(10) = 2.217, P < 0.05]. Furthermore, As depicted in Figure 3C, One-way ANOVA followed by Newman–Keuls post hoc test revealed that motor function did not impair in the M/N-Sedate as well as in methadone and naloxone groups as compared with the Saline group [F(6,56) = 4.605, P = 0.0007] but total time increased signiﬁcantly in the M/N-Day 5 and Day 10 groups in comparison with the Saline or M/N-Sedate groups. locomotor activity in the M/N-Day 1 [t(15) = 0.3083, P = 0.7621; ns], Day 5 [t(11) = 0.6218, P = 0.5468; ns] and Day 10 [t(10) = 0.0126, P = 0.9902; ns] groups when compared to their respective Saline control groups (right panel). In addition, One- way ANOVA by Newman–Keuls post hoc analysis showed that there was no signiﬁcant diﬀerence in locomotor activity in the M/N-Sedate group, M/N-treated rats (right panel, with apnea) as well as methadone and naloxone groups when compared with the Saline group [F(6,62) = 1.084, P = 0.3817]. September 2018 \| Volume 9 \| Article 1023 Effect of Acute Toxic Dose of Methadone on Locomotor Activity As shown in Figure 6, unpaired t-test analysis showed that a single dose of naloxone (2 mg/kg; i.p.) administration following methadone overdose (15 mg/kg; i.p.) in animals which experienced apnea did not displayed signiﬁcant deﬁcit in September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 9 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. FIGURE 5 \| Evaluation of motor coordination and balance in rotarod test during a 5-min trial in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor coordination was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 9), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 8) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate (n = 8; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 10, methadone; n = 9 and naloxone; n = 6 (saline, methadone or naloxone was administered alone in separated groups in which coordination assessment was evaluated only 24 h following the drug administration in a 5-min trial in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. The time each rat stay on the rod before falling was recorded. Each bar shows the mean ± SEM for 6–10. ∗∗P < 0.01 different from the Saline group. Effect of Acute Toxic Dose of Methadone on Locomotor Activity FIGURE 5 \| Evaluation of motor coordination and balance in rotarod test during a 5-min trial in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and motor coordination was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 9), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 8) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate (n = 8; a single dose of naloxone was administered following methadone overdose, immediately in the initial stage of sedation, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 10, methadone; n = 9 and naloxone; n = 6 (saline, methadone or naloxone was administered alone in separated groups in which coordination assessment was evaluated only 24 h following the drug administration in a 5-min trial in adolescent rats) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. The time each rat stay on the rod before falling was recorded. Each bar shows the mean ± SEM for 6–10. ∗∗P < 0.01 different from the Saline group. might be associated with the persistent impairment of diﬀerent brain functions through several mechanisms, including changes in central signaling proteins (Lou et al., 1999), activation of apoptosis signaling pathway (Emeterio et al., 2006) or impaired synaptic plasticity (Pu et al., 2002). Previous results suggest that the hippocampus and prefrontal cortex are involved in the NOR task (Banks et al., 2012; Pezze et al., 2017). The current ﬁndings indicate impairment of short-term memory in the NOR task on days 3, 7, and 12 (timeline was shown in Figure 1) after methadone overdose. In addition, the reduced recognition memory in the methadone (without apnea) group and the M/N groups which experienced apnea might be due to the activation or changes in protein signaling or the apoptosis pathways related to methadone overdose which have been induced by a high-dose application of opioids (Tramullas et al., 2007; Andersen et al., 2012). Frontiers in Pharmacology \| www.frontiersin.org Effect of Acute Toxic Dose of Methadone on Locomotor Activity It has been proposed that memory impairment might be the result of the direct toxicity of methadone that overstimulates the opioid receptors in the hippocampus and limbic system related to particular forms of learning and memory, including spontaneous object recognition memory (Pertschuk and Sher, 1975; Wehner et al., 2000). Recognition impairment has been observed in previous studies that have described damage to the hippocampus as suﬃcient to create impairment of recognition memory (Broadbent et al., 2010). group. In the current study, the transient impairment of motor performance after methadone overdose suggests that perhaps alternative strategies with other brain regions involved in the processing of sensorimotor performance. Another explanation is, administration of naloxone (reversing methadone overdose) may partly reduce the motor disabilities following injection of a toxic dose of methadone with unknown mechanisms. In the present study, the rotarod test for evaluation of motor coordination and balance showed mild or no deﬁciency in animals which experienced apnea. It is important to note that the lack of signiﬁcant impairment in motor performance in the rotarod test could be in part due to the small number of rats which experienced apnea that had executed the test. It should be noted that other conditions such as test protocol, laboratory environmental factors, and rod diameter could have inﬂuenced the sensitivity of the test for detecting subtle deﬁciencies in motor function or balance following methadone-induced apnea. Nevertheless, several previous reports indicated the lack of motor coordination, executive function, and ataxia which were observed following methadone overdose (Tramullas et al., 2007; Cottencin et al., 2009). The current results showed no changes in locomotor activity after a single toxic dose of methadone, which is inconsistent with the results of previous studies on the attenuating or increasing Pole test measurements reﬂected the deterioration of motor function in the M/N-Day 5 but not in the M/N-Day 1 September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 10 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. Effect of Acute Toxic Dose of Methadone on Locomotor Activity FIGURE 6 \| Locomotor activity was recorded by using photobeam activity system during a 5-min session in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and locomotor activity was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 10), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate (n = 11; a single dose of naloxone was administered after methadone overdose immediately in the initial stage of sedation state, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 12 and naloxone; n = 8 (saline, methadone or naloxone was administered alone in separated groups in which locomotor activity was evaluated 1 day after drug administration) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Administration of toxic dose of methadone did not change locomotor activity in all groups. Each bar shows the mean ± SEM for 6–14. FIGURE 6 \| Locomotor activity was recorded by using photobeam activity system during a 5-min session in different groups, including; M/N-treated groups (a single dose of naloxone was administered after methadone overdose in apnea stage, in animals which experienced apnea and locomotor activity was evaluated either on day 1, 5, or 10 day post-resuscitation; M/N-Day 1 (n = 10), M/N-Day 5 (n = 6) and M/N-Day 10 (n = 6) groups and their respective Saline control groups; n = 6; right panel), M/N-Sedate (n = 11; a single dose of naloxone was administered after methadone overdose immediately in the initial stage of sedation state, so behavioral evaluation was carried out only 1 day after drug administration), Saline; n = 14, methadone; n = 12 and naloxone; n = 8 (saline, methadone or naloxone was administered alone in separated groups in which locomotor activity was evaluated 1 day after drug administration) groups. Animals received methadone (15 mg/kg; i.p.) or naloxone (2 mg/kg; i.p.) alone or both in a single dose. Administration of toxic dose of methadone did not change locomotor activity in all groups. Each bar shows the mean ± SEM for 6–14. tasks (Ebert et al., 1995; Tsien et al., 1996; Nylander et al., 2016). Frontiers in Pharmacology \| www.frontiersin.org Effect of Acute Toxic Dose of Methadone on Locomotor Activity eﬀect of motor activity following acute or chronic administration of methadone in rats (Mendez and Trujillo, 2008). Diﬀerent routes of administration, patterns and doses of methadone prescribed, and the duration of recording of locomotion, as well as the diﬀerent time point measurements, might aﬀect the outcomes and produce diﬀerent results (Allouche et al., 2013). Delayed leukoencephalopathy was described for the ﬁrst time after anoxic injury with symmetrical necrotic lesions of the central white matter, along with the damage to gray matter caused delayed neurological deterioration after initial recovery (Lin et al., 2012; Meyer, 2013). There are several reports of severe methadone-induced leukoencephalopathy which can be recognized by magnetic resonance imaging ﬁndings (Mittal et al., 2010; Cerase et al., 2011). The exact mechanism remains uncertain, but one possible hypothesis is that it is in part due to the defect in energy metabolism caused by demyelination following respiratory depression/arrest after methadone overdose (Weinberger et al., 1994). Direct damage or activation of immunological responses to brain tissue is another hypothesis which explains the pathogenesis of methadone- induced leukoencephalopathy (Mills et al., 2008; Mittal et al., 2010; Cerase et al., 2011; Rando et al., 2016). The serum half-life of morphine administered to an opioid-naïve patient was nearly 2–3 h, while this for methadone is approximately 150 h (Ciejka et al., 2016). With respect to methadone toxicity in cell culture, it has been suggested that methadone- induced cell death uncouples mitochondria, resulting in impairment of ATP synthesis (Perez-Alvarez et al., 2010; It also has been suggested that administration of naloxone after methadone overdose may modulate the detrimental eﬀects of opioid receptor activation on both locomotor activity and motor coordination in the rotarod task. The results showed that administration of a single dose of naloxone had no eﬀect on memory or motor performance. Hayward and Low reported that naloxone dose-dependently decreased motor activity, which is inconsistent with the current ﬁndings (Hayward and Low, 2005). It appears that the short duration of action of naloxone (Aghabiklooei et al., 2013) did not cause alterations in motor function at 24 h post- injection. Administration of NMDA receptor antagonists like AP5 and MK-801 could impair spatial working memory. REFERENCES Banks, P. J., Bashir, Z. I., and Brown, M. W. (2012). Recognition memory and synaptic plasticity in the perirhinal and prefrontal cortices. Hippocampus 22, 2012–2031. doi: 10.1002/hipo.22067 Aghabiklooei, A., Hassanian-Moghddam, H., Zamani, N., Shadnia, S. H., Rahimi, M., Nasouhi, S., et al. (2013). Eﬀectiveness of naltrexone in the prevention of delayed respiratory arrest in opioid-naive methadone- intoxicated patients. Biomed Res. Int. 2013:903172. doi: 10.1155/2013/ 903172 Barbosa Neto, J. O., Garcia, M. A., and Garcia, J. B. S. (2015). Revisiting methadone: pharmacokinetics, pharmacodynamics and clinical indication. Rev. Dor 16, 60–66. doi: 10.5935/1806-0013.20150012 Broadbent, N. J., Gaskin, S., Squire, L. R., and Clark, R. E. (2010). Object recognition memory and the rodent hippocampus. Learn. Mem. 17, 5–11. doi: 10.1101/lm.1650110 Allouche, S., Le Marec, T., Noble, F., and Marie, N. (2013). Diﬀerent patterns of administration modulate propensity of methadone and buprenorphine to promote locomotor sensitization in mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 40, 286–291. doi: 10.1016/j.pnpbp.2012.10.013 Cerase, A., Leonini, S., Bellini, M., Chianese, G., and Venturi, C. (2011). Methadone-induced toxic leukoencephalopathy: diagnosis and follow-up by magnetic resonance imaging including diﬀusion-weighted imaging and apparent diﬀusion coeﬃcient maps. J. Neuroimaging 21, 283–286. doi: 10.1111/ j.1552-6569.2010.00530.x Andersen, J. M., Klykken, C., and Mørland, J. (2012). Long-term methadone treatment reduces phosphorylation of CaMKII in rat brain. J. Pharm. Pharmacol. 64, 843–847. doi: 10.1111/j.2042-7158.2012.01469.x j Chevillard, L., Mégarbane, B., Risède, P., and Baud, F. J. (2009). Characteristics and comparative severity of respiratory response to toxic doses of fentanyl, methadone, morphine, and buprenorphine in rats. Toxicol. Lett. 191, 327–340. doi: 10.1016/j.toxlet.2009.09.017 Andersen, J. M., Olaussen, C. F., Ripel, Å., and Mørland, J. (2011). Long-term methadone treatment impairs novelty preference in rats both when present and absent in brain tissue. Pharmacol. Biochem. Behav. 98, 412–416. doi: 10.1016/j. pbb.2011.02.017 Antunes, M., and Biala, G. (2012). The novel object recognition memory: neurobiology, test procedure, and its modiﬁcations. Cogn. Process. 13, 93–110. doi: 10.1007/s10339-011-0430-z Chugh, S. S., Socoteanu, C., Reinier, K., Waltz, J., Jui, J., and Gunson, K. (2008). A community-based evaluation of sudden death associated with therapeutic levels of methadone. Am. J. Med. 121, 66–71. doi: 10.1016/j.amjmed.2007. 10.009 Argoﬀ, C. E., and Silvershein, D. I. (2009). A comparison of long-and short- acting opioids for the treatment of chronic noncancer pain: tailoring therapy to meet patient needs. Mayo Clin. Proc. 84, 602–612. doi: 10.1016/S0025-6196(11) 60749-0 Ciejka, M., Nguyen, K., Bluth, M. H., and Dubey, E. (2016). ACKNOWLEDGMENTS The authors greatly appreciate the Neuroscience Research Center members for the kindly collaboration. FUNDING This study was carried out as a part of Ph.D. thesis written by LA-M funded (Grant/Registration no: 70) by the School of Medicine, Shahid Beheshti University of Medical Sciences. This study was carried out as a part of Ph.D. thesis written by LA-M funded (Grant/Registration no: 70) by the School of Medicine, Shahid Beheshti University of Medical Sciences. The important limitation of this study means, it remains unclear whether the impairments relate directly to methadone toxicity or cerebral hypoxia. Moreover, we did not measure the concentration of methadone in blood or brain tissue on test days due to the limited ﬁnancial resource at the time of doing research. Effect of Acute Toxic Dose of Methadone on Locomotor Activity As a result, the antagonistic action of methadone on the NMDA receptors might conﬁrm the hypothesis that methadone mediates through both opioid and NMDA receptors to exert adverse/neurotoxic eﬀects on memory and motor function in diﬀerent behavioral September 2018 \| Volume 9 \| Article 1023 11 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. AUTHOR CONTRIBUTIONS All authors designed the study, analyzed and interpreted the data, and wrote the paper. CONCLUSION Nylander et al., 2016). Although these ﬁndings are not speciﬁc, such symptoms are in part consistent with the current behavioral results. Acute cerebellitis (Mills et al., 2008; Rando et al., 2016) or basal ganglia (Cottencin et al., 2009; Corliss et al., 2013) damage involvement following methadone overdose may explain the motor impairment observed in the pole and rotarod tasks caused in part by overstimulation of opioid receptors in these brain regions. Nylander et al., 2016). Although these ﬁndings are not speciﬁc, such symptoms are in part consistent with the current behavioral results. Acute cerebellitis (Mills et al., 2008; Rando et al., 2016) or basal ganglia (Cottencin et al., 2009; Corliss et al., 2013) damage involvement following methadone overdose may explain the motor impairment observed in the pole and rotarod tasks caused in part by overstimulation of opioid receptors in these brain regions. In contrast to the majority of studies on the neurological consequences of MMT patients, the current study has shown that acute exposure to a toxic dose of methadone in naïve healthy rats impaired cognitive and/or motor function. The deﬁcient was reversible in motor function but not for memory during an observation period of nearly 2 weeks. The exact mechanisms remain uncertain, but further studies are required to elucidate the diﬀerent pathophysiological mechanisms of methadone-induced neurotoxicity. Despite its limitation, our ﬁndings indicate that following acute methadone overdose, reporting and follow-up assessment with the use of brain-imaging techniques after relative initial recovery should be performed. Utilization of plausible neurotoxicity biomarkers would allow continual monitoring to explore complications and possible damage to the central nervous system as well. It is suggested that methadone overdose should be considered to be a possible cause of delayed neurological disorders which require accurate monitoring for adverse reactions or signs to aid diagnosis of the risk of complications to the nervous system in hospital poison centers for healthy subjects, especially children. It has been noted that respiratory depression should be considered in patients using opioids for the ﬁrst time, but not in chronic users because they develop a tolerance to opioid drugs. It has been suggested that overdose with long-acting opioids such as methadone by a naïve individual may require a longer observation period in the hospital to reduce delayed complications or possible long-term sequelae. REFERENCES Drug toxicities of common analgesic Medications in the emergency department. Clin. Lab. Med. 36, 761–776. doi: 10.1016/j.cll.2016.07.003 Ayatollahi, V., Behdad, S., Oliwiaie, H., Hajiesmaili, M. R., Dehghan, M., and Mehrpour, O. (2011). Characteristic features of patients hospitalized with Narcotic poisoning in Yazd, Iran. Iran. J. Toxicol. 4, 362–366. Cohen, S. J., and Stackman, R. W. Jr. (2015). Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav. Brain Res. 285, 105–117. doi: 10.1016/j.bbr.2014. 08.002 September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 12 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. Keane, H. (2013). Categorising methadone: addiction and analgesia. Int. J. Drug Policy 24, e18–e24. doi: 10.1016/j.drugpo.2013.05.007 Corliss, R. F., Mandal, R., and Soriano, B. J. (2013). Bilateral acute necrosis of the globi pallidi and rhabdomyolysis due to combined methadone and benzodiazepine toxicity. Am. J. Forensic Med. Pathol. 34, 1–4. doi: 10.1097/PAF. 0b013e31823a8b1e Kleber, H. D. (2007). Pharmacologic treatments for opioid dependence: detoxiﬁcation and maintenance options. Dialogues Clin. Neurosci. 9, 455–470. Cottencin, O., Guardia, D., Warembourg, F., Gaudry, C., and Goudemand, M. (2009). Methadone overdose, auto-activation deﬁcit, and catatonia: a case study. Prim. Care Companion J. Clin. Psychiatry 11, 275–276. doi: 10.4088/PCC. 08l00691 Leppert, W. (2009). The role of methadone in cancer pain treatment–a review. Int. J. Clin. Pract. 63, 1095–1109. doi: 10.1111/j.1742-1241.2008.01990.x Lin, W.-C., Chou, K.-H., Chen, C.-C., Huang, C.-C., Chen, H.-L., Lu, C.-H., et al. (2012). White matter abnormalities correlating with memory and depression in heroin users under methadone maintenance treatment. PLoS One 7:e33809. doi: 10.1371/journal.pone.0033809 Dahan, A., Yassen, A., Romberg, R., Sarton, E., Teppema, L., Olofsen, E., et al. (2006). Buprenorphine induces ceiling in respiratory depression but not in analgesia. Br. J. Anaesth. 96, 627–632. doi: 10.1093/bja/ael051 Lou, L., Zhou, T., Wang, P., and Pei, G. (1999). Modulation of Ca2+/calmodulin- dependent protein kinase II activity by acute and chronic morphine administration in rat hippocampus: diﬀerential regulation of α and β isoforms. Mol. Pharmacol. 55, 557–563. Deacon, R. M. (2013). Measuring motor coordination in mice. J. Vis. Exp. 75:e2609. doi: 10.3791/2609 Drummer, O. H., Opeskin, K., Syrjanen, M., and Cordner, S. M. (1992). Methadone toxicity causing death in ten subjects starting on a methadone maintenance program. Am. J. Forensic Med. Pathol. 13, 346–350. doi: 10.1097/00000433- 199212000-00017 LoVecchio, F., Pizon, A., Riley, B., Sami, A., and D’incognito, C. (2007). Onset of symptoms after methadone overdose. Am. J. Emerg. Med. REFERENCES 25, 57–59. doi: 10.1016/j.ajem.2006.07.006 Dunham, N., and Miya, T. (1957). A note on a simple apparatus for detecting neurological deﬁcit in rats and mice. J. Pharm. Sci. 46, 208–209. doi: 10.1002/ jps.3030460322 Ma, M., Chen, Y., He, J., Zeng, T., and Wang, J. (2007). Eﬀects of morphine and its withdrawal on Y-maze spatial recognition memory in mice. Neuroscience 147, 1059–1065. doi: 10.1016/j.neuroscience.2007.05.020 Ebert, B., Andersen, S., and Krogsgaard-Larsen, P. (1995). Ketobemidone, methadone and pethidine are non-competitive N-methyl-D-aspartate (NMDA) antagonists in the rat cortex and spinal cord. Neurosci. Lett. 187, 165–168. doi: 10.1016/0304-3940(95)11364-3 Mendez, I. A., and Trujillo, K. A. (2008). NMDA receptor antagonists inhibit opiate antinociceptive tolerance and locomotor sensitization in rats. Psychopharmacology 196, 497–509. doi: 10.1007/s00213-007- 0984-8 Meyer, M. A. (2013). Delayed post-hypoxic leukoencephalopathy: case report with a review of disease pathophysiology. Neurol. Int. 5:e13. doi: 10.4081/ni.2013.e13 Emeterio, E. P. S., Tramullas, M., and Hurlé, M. A. (2006). Modulation of apoptosis in the mouse brain after morphine treatments and morphine withdrawal. J. Neurosci. Res. 83, 1352–1361. doi: 10.1002/jnr. 20812 Mills, F., MacLennan, S. C., Devile, C. J., and Saunders, D. E. (2008). Severe cerebellitis following methadone poisoning. Pediatr. Radiol. 38, 227–229. doi: 10.1007/s00247-007-0635-6 Farahmandfar, M., Karimian, S. M., Naghdi, N., Zarrindast, M.-R., and Kadivar, M. (2010). Morphine-induced impairment of spatial memory acquisition reversed by morphine sensitization in rats. Behav. Brain Res. 211, 156–163. doi: 10.1016/ j.bbr.2010.03.013 Milroy, C. M., and Forrest, A. R. W. (2000). Methadone deaths: a toxicological analysis. J. Clin. Pathol. 53, 277–281. doi: 10.1136/jcp.53.4.277 Mintzer, M. Z., Copersino, M. L., and Stitzer, M. L. (2005). Opioid abuse and cognitive performance. Drug Alcohol Depend. 78, 225–230. doi: 10.1016/j. drugalcdep.2004.10.008 Farhadinasab, A., Shahidi, S., Najaﬁ, A., and Komaki, A. (2009). Role of naloxone as an exogenous opioid receptor antagonist in spatial learning and memory of female rats during the estrous cycle. Brain Res. 1257, 65–74. doi: 10.1016/j. brainres.2008.12.043 Mintzer, M. Z., and Stitzer, M. L. (2002). Cognitive impairment in methadone maintenance patients. Drug Alcohol Depend. 67, 41–51. doi: 10.1016/S0376- 8716(02)00013-3 Ford, M. D. (2001). Clinical Toxicology. Philadelphia, PA: WB Saunders Company. Mittal, M., Wang, Y., Reeves, A., and Newell, K. (2010). Methadone-induced delayed posthypoxic encephalopathy: clinical, radiological, and pathological ﬁndings. Case Rep. Med. 2010:716494. doi: 10.1155/2010/716494 Gaspari, R. J., and Paydarfar, D. (2007). Pathophysiology of respiratory failure following acute dichlorvos poisoning in a rodent model. Neurotoxicology 28, 664–671. doi: 10.1016/j.neuro.2007.02.002 Graham, N. A., Merlo, L. J., Goldberger, B. REFERENCES A., and Gold, M. S. (2008). Methadone- and heroin-related deaths in Florida. Am. J. Drug Alcohol. Abuse 34, 347–353. doi: 10.1080/00952990802010892 Modesto-Lowe, V., Brooks, D., and Petry, N. (2010). Methadone deaths: risk factors in pain and addicted populations. J. Gen. Intern. Med. 25, 305–309. doi: 10.1007/s11606-009-1225-0 Morgan, O., Griﬃths, C., and Hickman, M. (2006). Association between availability of heroin and methadone and fatal poisoning in England and Wales 1993–2004. Int. J. Epidemiol. 35, 1579–1585. doi: 10.1093/ije/dyl207 Hassanian-Moghaddam, H., Hakiminejhad, M., Farnaghi, F., Mirafzal, A., Zamani, N., and Kabir, A. (2017). Eleven years of children methadone poisoning in a referral center: a review of 453 cases. J. Opioid Manag. 13, 27–36. doi: 10.5055/jom.2017.0365 Nylander, E., Grönbladh, A., Zelleroth, S., Diwakarla, S., Nyberg, F., and Hallberg, M. (2016). Growth hormone is protective against acute methadone- induced toxicity by modulating the NMDA receptor complex. Neuroscience 339, 538–547. doi: 10.1016/j.neuroscience.2016.10.019 Hassanian-Moghaddam, H., Zamani, N., Rahimi, M., Shadnia, S., Pajoumand, A., and Sarjami, S. (2014). Acute adult and adolescent poisoning in Tehran, Iran; the epidemiologic trend between 2006 and 2011. Arch. Iran. Med. 17, 534–538. Ogawa, N., Hirose, Y., Ohara, S., Ono, T., and Watanabe, Y. (1985). A simple quantitative bradykinesia test in MPTP-treated mice. Res. Commun. Chem. Pathol. Pharmacol. 50, 435–441. Hayward, M. D., and Low, M. J. (2005). Naloxone’s suppression of spontaneous and food-conditioned locomotor activity is diminished in mice lacking either the dopamine D 2 receptor or enkephalin. Mol. Brain Res. 140, 91–98. doi: 10.1016/j.molbrainres.2005.07.016 Paulozzi, L. J., Budnitz, D. S., and Xi, Y. (2006). Increasing deaths from opioid analgesics in the United States. Pharmacoepidemiol. Drug Saf. 15, 618–627. doi: 10.1002/pds.1276 Hepner, I. J., Homewood, J., and Taylor, A. J. (2002). Methadone disrupts performance on the working memory version of the Morris water task. Physiol. Behav. 76, 41–49. doi: 10.1016/S0031-9384(02)00695-9 Perez-Alvarez, S., Cuenca-Lopez, M. D., De Mera, R. M. M.-F., Puerta, E., Karachitos, A., Bednarczyk, P., et al. (2010). Methadone induces necrotic- like cell death in SH-SY5Y cells by an impairment of mitochondrial ATP synthesis. Biochim. Biophys. Acta 1802, 1036–1047. doi: 10.1016/j.bbadis.2010. 07.024 Holcomb, L., Gordon, M. N., McGowan, E., Yu, X., Benkovic, S., Jantzen, P., et al. (1998). Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes. Nat. Med. 4, 97–100. doi: 10.1038/nm0198-097 Jabbehdari, S., Farnaghi, F., Shariatmadari, S. F., Jafari, N., Mehregan, F.-F., and Karimzadeh, P. (2013). REFERENCES Accidental children poisoning with methadone: an Iranian pediatric sectional study. Iran. J. Child Neurol. 7, 32–34. Pertschuk, L., and Sher, J. (1975). Demonstration of methadone in the human brain by immunoﬂuorescence. Res. Commun. Chem. Pathol. Pharmacol. 11, 319–322. Pezze, M. A., Marshall, H. J., Fone, K. C., and Cassaday, H. J. (2017). Role of the anterior cingulate cortex in the retrieval of novel object recognition memory after a long delay. Learn. Mem. 24, 310–317. doi: 10.1101/lm.04 4784.116 Jones, J. D., Mogali, S., and Comer, S. D. (2012). Polydrug abuse: a review of opioid and benzodiazepine combination use. Drug Alcohol Depend. 125, 8–18. doi: 10.1016/j.drugalcdep.2012.07.004 September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 13 Methadone Overdose, Cognitive, and Motor Functions Ahmad-Molaei et al. Tsien, J. Z., Huerta, P. T., and Tonegawa, S. (1996). The essential role of hippocampal CA1 NMDA receptor–dependent synaptic plasticity in spatial memory. Cell 87, 1327–1338. doi: 10.1016/S0092-8674(00)81827-9 Pragst, F., Broecker, S., Hastedt, M., Herre, S., Andresen-Streichert, H., Sachs, H., et al. (2013). Methadone and illegal drugs in hair from children with parents in maintenance treatment or suspected for drug abuse in a German community. Ther. Drug Monit. 35, 737–752. doi: 10.1097/FTD.0b013e31829a78c3 Van Der Schier, R., Roozekrans, M., Van Velzen, M., Dahan, A., and Niesters, M. (2014). Opioid-induced respiratory depression: reversal by non-opioid drugs. F1000prime Rep. 6:79. doi: 10.12703/P6-79 Prosser, J., Cohen, L. J., Steinfeld, M., Eisenberg, D., London, E. D., and Galynker, I. I. (2006). Neuropsychological functioning in opiate-dependent subjects receiving and following methadone maintenance treatment. Drug Alcohol Depend. 84, 240–247. doi: 10.1016/j.drugalcdep.2006.02.006 Verdejo, A., Toribio, I., Orozco, C., Puente, K. L., and Pérez-García, M. (2005). Neuropsychological functioning in methadone maintenance patients versus abstinent heroin abusers. Drug Alcohol Depend. 78, 283–288. doi: 10.1016/j. drugalcdep.2004.11.006 Pu, L., Bao, G.-B., Xu, N.-J., Ma, L., and Pei, G. (2002). Hippocampal long-term potentiation is reduced by chronic opiate treatment and can be restored by re- exposure to opiates. J. Neurosci. 22, 1914–1921. doi: 10.1523/JNEUROSCI.22- 05-01914.2002 Wehner, F., Wehner, H.-D., Schieﬀer, M. C., and Subke, J. (2000). Immunohistochemical detection of methadone in the human brain. Forensic Sci. Int. 112, 11–16. doi: 10.1016/S0379-0738(00)00157-2 Rando, J., Szari, S., Kumar, G., and Lingadevaru, H. (2016). Methadone overdose causing acute cerebellitis and multi-organ damage. Am. J. Emerg. Med. 34, 343.e1–343.e3. doi: 10.1016/j.ajem.2015.06.032 Weinberger, L., Schmidley, J., Schafer, I., and Raghavan, S. (1994). Delayed postanoxic demyelination and arylsulfatase-A pseudodeﬁciency. Neurology 44, 152–154. REFERENCES doi: 10.1212/WNL.44.1.152 Rudd, R. A., Aleshire, N., Zibbell, J. E., and Matthew Gladden, R. (2016). Increases in drug and opioid overdose deaths—United States, 2000–2014. Am. J. Transplant. 16, 1323–1327. doi: 10.1111/ajt.13776 Whelan, P. J., and Remski, K. (2012). Buprenorphine vs methadone treatment: a review of evidence in both developed and developing worlds. J. Neurosci. Rural Pract. 3, 45–50. doi: 10.4103/0976-3147.91934 Schuckit, M. A. (2016). Treatment of opioid-use disorders. N. Engl. J. Med. 375, 357–368. doi: 10.1056/NEJMra1604339 Zamani, N., Hassanian-Moghaddam, H., Bayat, A. H., Haghparast, A., Shadnia, S., Rahimi, M., et al. (2015). Reversal of opioid overdose syndrome in morphine- dependent rats using buprenorphine. Toxicol. Lett. 232, 590–594. doi: 10.1016/ j.toxlet.2014.12.007 Shadnia, S., Rahimi, M., Hassanian-Moghaddam, H., Soltaninejad, K., and Noroozi, A. (2013). Methadone toxicity: comparing tablet and syrup formulations during a decade in an academic poison center of Iran. Clin. Toxicol. 51, 777–782. doi: 10.3109/15563650.2013.830732 Zhang, J.-J., and Kong, Q. (2017). Locomotor activity: a distinctive index in morphine self-administration in rats. PLoS One 12:e0174272. doi: 10.1371/ journal.pone.0174272 Shields, L. B., Hunsaker, J. C. III, Corey, T. S., Ward, M. K., and Stewart, D. (2007). Methadone toxicity fatalities: a review of medical examiner cases in a large metropolitan area. J. Forensic Sci. 52, 1389–1395. doi: 10.1111/j.1556-4029. 2007.00565.x Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Sjøgren, P., Thomsen, A. B., and Olsen, A. K. (2000). Impaired neuropsychological performance in chronic nonmalignant pain patients receiving long-term oral opioid therapy. J. Pain Symptom Manage. 19, 100–108. doi: 10.1016/S0885- 3924(99)00143-8 Copyright © 2018 Ahmad-Molaei, Hassanian-Moghaddam, Farnaghi, Tomaz and Haghparast. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Soltaninejad, K., Hassanian-Moghaddam, H., and Shadnia, S. (2014). Methadone related poisoning on the rise in Tehran. Iran. APJMT 3, 104–109. Tramullas, M., Martínez-Cué, C., and Hurlé, M. A. (2007). Chronic methadone treatment and repeated withdrawal impair cognition and increase the expression of apoptosis-related proteins in mouse brain. Psychopharmacology 193, 107–120. September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org REFERENCES doi: 10.1007/s00213-007-0751-x September 2018 \| Volume 9 \| Article 1023 Frontiers in Pharmacology \| www.frontiersin.org 14
https://openalex.org/W2624292923	https://europepmc.org/articles/pmc5461770?pdf=render	English	null	Bayesian hierarchical piecewise regression models: a tool to detect trajectory divergence between groups in long-term observational studies	BMC Medical research methodology	2,017	cc-by	15,088	RESEARCH ARTICLE Open Access Bayesian hierarchical piecewise regression models: a tool to detect trajectory divergence between groups in long-term observational studies Marie-jeanne Buscot1, Simon S. Wotherspoon2, Costan G. Magnussen1,3, Markus Juonala3,4, Matthew A. Sabin5,6, David P. Burgner5,6,7, Terho Lehtimäki8, Jorma S. A. Viikari3,4, Nina Hutri-Kähönen9,10, Olli T. Raitakari3,4 and Russell J. Thomson11* Buscot et al. BMC Medical Research Methodology (2017) 17:86 DOI 10.1186/s12874-017-0358-9 RESEARCH ARTICLE Open Access Bayesian hierarchical piecewise regression models: a tool to detect trajectory divergence between groups in long-term observational studies Marie-jeanne Buscot1, Simon S. Wotherspoon2, Costan G. Magnussen1,3, Markus Juonala3,4, Matthew A. Sabin5,6, David P. Burgner5,6,7, Terho Lehtimäki8, Jorma S. A. Viikari3,4, Nina Hutri-Kähönen9,10, Olli T. Raitakari3,4 and Russell J. Thomson11* Buscot et al. BMC Medical Research Methodology (2017) 17:86 DOI 10.1186/s12874-017-0358-9 Buscot et al. BMC Medical Research Methodology (2017) 17:86 DOI 10.1186/s12874-017-0358-9 Open Access Bayesian hierarchical piecewise regression models: a tool to detect trajectory divergence between groups in long-term observational studies Marie-jeanne Buscot1, Simon S. Wotherspoon2, Costan G. Magnussen1,3, Markus Juonala3,4, Matthew A. Sabin5,6, David P. Burgner5,6,7, Terho Lehtimäki8, Jorma S. A. Viikari3,4, Nina Hutri-Kähönen9,10, Olli T. Raitakari3,4 and Russell J. Thomson11* © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated * Correspondence: russell.thomson@westernsydney.edu.au 11Centre for Research in Mathematics, School of Computing, Engineering & Mathematics, Western Sydney University, Sydney, Australia Full list of author information is available at the end of the article Abstract Background: Bayesian hierarchical piecewise regression (BHPR) modeling has not been previously formulated to detect and characterise the mechanism of trajectory divergence between groups of participants that have longitudinal responses with distinct developmental phases. These models are useful when participants in a prospective cohort study are grouped according to a distal dichotomous health outcome. Indeed, a refined understanding of how deleterious risk factor profiles develop across the life-course may help inform early-life interventions. Previous techniques to determine between-group differences in risk factors at each age may result in biased estimate of the age at divergence. Methods: We demonstrate the use of Bayesian hierarchical piecewise regression (BHPR) to generate a point estimate and credible interval for the age at which trajectories diverge between groups for continuous outcome measures that exhibit non-linear within-person response profiles over time. We illustrate our approach by modeling the divergence in childhood-to-adulthood body mass index (BMI) trajectories between two groups of adults with/ without type 2 diabetes mellitus (T2DM) in the Cardiovascular Risk in Young Finns Study (YFS). Results: Using the proposed BHPR approach, we estimated the BMI profiles of participants with T2DM diverged from healthy participants at age 16 years for males (95% credible interval (CI):13.5–18 years) and 21 years for females (95% CI: 19.5–23 years). These data suggest that a critical window for weight management intervention in preventing T2DM might exist before the age when BMI growth rate is naturally expected to decrease. Simulation showed that when using pairwise comparison of least-square means from categorical mixed models, smaller sample sizes tended to conclude a later age of divergence. In contrast, the point estimate of the divergence time is not biased by sample size when using the proposed BHPR method. Conclusions: BHPR is a powerful analytic tool to model long-term non-linear longitudinal outcomes, enabling the identification of the age at which risk factor trajectories diverge between groups of participants. The method is suitable for the analysis of unbalanced longitudinal data, with only a limited number of repeated measures per participants and where the time-related outcome is typically marked by transitional changes or by distinct phases of change over time. Background medication), or it could be a later health outcome (i.e. disease status in mid-adulthood). When participants are grouped accord- ing to a distal dichotomous health outcome, longitudinal data provide the foundation to understand pathways to deleterious risk factor profiles, which may help inform the timing of interventions [8, 14, 15]. In contrast, continuous time models such as individual- based trajectory modeling methods, including mixed effect [12], hierarchical [40], multilevel [41] and the closely re- lated structural equation and Latent Growth Curve models [42], have become invaluable tools to understand the natural history of health outcome as well as risk fac- tor/determinant trajectories [14, 43–45]. They have advantages over traditional approaches to repeated- measure data analysis; their main feature being that they allow summarizing each participant’s outcome trajectory with a few trajectory parameters [39, 46]. In addition, they permit the explicitly modeling of inter-individual differ- ences in intra-individual change, permitting inference regarding the average response trajectory over time and how this evolution may vary with participant characteris- tics (i.e. participant-level predictors) [47–50]. When it is established that groups of interest start with similar initial outcome levels, but do not change similarly overtime, it is often of interest to determine the point in time or age at which they start diverging in their trajectories [16–20]. Being able to determine how and when the change manifests between groups of partici- pants is important, since it can help pinpoint periods in the life course that are critical in the development of ab- normal risk factor profiles [21]. However, there is little methodological guidance in the literature on statistical techniques to achieve this, and several studies have noted a lack of relevant methods to investigate trajectory divergence between groups [20–22]. Despite their flexibility, these models are not often used to analyse sparse long-term observational data since accelerated longitudinal designs [14, 22, 51] and non-linear response overtime [44, 52–54] both introduce significant complexity into the growth curve modeling approach [55–58]. Indeed, being able to represent non- linear patterns with a relatively small number of meas- urement occasions per participants (often <10 time points) and be specific about where between-participant heterogeneity appears in those patterns is a statistical challenge. A common attempt is to fit a mixed model with time (or age) treated as categorical variable (i.e. non time- ordered/ordinated [23]) to retrieve linear predictions at each age for each group of interest from this model (i.e. Background group difference materializes in the LS-means [23, 27, 28]. Several studies have used this approach to determine at “what times the groups means are different” (e.g. between- subject effect or post-hoc pairwise group comparison, if there are more than two groups) and/or ‘at what times the means differ’ within each group (within-subject effect test- ing) [27, 29, 30]. However, even when adjustments are ap- plied for multiple tests [27, 31–33], many authors advise against the unrestricted use of multiple comparisons among marginal means due to well-documented multiple testing issues, especially the increase in false positive rate as the number of hypothesis tests increases [30, 34–37]. Mixed models that assume an unstructured mean response by treating age or time as categorical variables tend to be over parameterized and may be inefficient at detecting main effects [38]. Another crucial disadvantage of this approach, is that it only tests for the difference in means between groups at each time point and does not provide any informa- tion on subject-specific response evolution in time [13, 39], so that the age (or point in time) at which the group differ- ence manifests is ultimately a question of sample size and statistical power. Child- to-adult trajectories of health markers are likely to have implications for the risk of chronic diseases in later life, such as obesity, type 2 diabetes mellitus (T2DM) and cardiovascular diseases; it is therefore im- portant to understand their development throughout the life-course [1–4]. Long-running observational studies that follow the same subjects participants across the life-course are es- pecially suited to studying adult onset disorders, such as cardiometabolic disease, since they allow characterizing the development of normal vs. pathological processes overtime. A goal of such studies is often to determine how a number of patient characteristics, modifiable risk factors profiles [1, 5], their interactions and normal aging may impact the onset and progression of disease over time [6–8] in order to identify time periods of di- vergence in these factors [9–11]. A key statistical issue in these studies is often to deter- mine whether the risk factor levels vary over time be- tween and within groups of participants, and whether different groups are changing in a similar or different fashion over time [12, 13]. Depending on the study, the stratification of participants into groups can relate to participants’ characteristics or exposure (i.e. smoking status), intervention arm (i.e. control vs. Abstract Keywords: Piecewise model, Hierarchical regression, Non-linear trajectory model, Accelerated longitudinal design, Cohort effect, Group divergence * Correspondence: russell.thomson@westernsydney.edu.au 11Centre for Research in Mathematics, School of Computing, Engineering & Mathematics, Western Sydney University, Sydney, Australia Full list of author information is available at the end of the article * Correspondence: russell.thomson@westernsydney.edu.au 11Centre for Research in Mathematics, School of Computing, Engineering & Mathematics, Western Sydney University, Sydney, Australia Full list of author information is available at the end of the article Page 2 of 15 Page 2 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 Buscot et al. BMC Medical Research Methodology (2017) 17:86 No-covariate model To accommodate the curvilinear developmental pattern in an individual continuous response over time while providing an adequate representation of its developmen- tal theory, we consider a linear-linear piecewise regres- sion model as the functional form of change in the trajectory model. The ‘change point’ (CP) represents the age (or time) at which the transition to a different growth rate occurs. We consider the following uncondi- tional (no covariates) multilevel model: Level 1 model: Level 1 model: Reponseij ¼ b0i þ b1iageij: 1−uCPi ageij þb2i ageij−CPi :uCPi ageij þ εij ð1:1Þ ð1:1Þ Level 2 model: b0i ¼ β00 þ v0i b1i ¼ β10 þ v1i b2i ¼ β20 þ v2i CPi ¼ CP þ vCPi ð1:2Þ v0i v1i v2i vCPi 0 B B B @ 1 C C C AeN 0 0 0 0 0 B B @ 1 C C A; σv02 … ⋯ … σv01 σv12 … … σv02 σv12 σv2 2 … σv0CP σv1CP σv2CP σCP 2 !# 2 6666666666664 ð1:3Þ ð1:2Þ p Few studies have, however, investigated the inclusion of categorical covariates or grouping variables as level 2 predictors of the variability in the change point, and the random Bayesian change point model has not, to our knowledge, been formulated to test specifically for the existence of a ‘trajectory divergence’ between two (or more) known groups of participants that have longitu- dinal responses characterised by distinct developmental phases. In this paper we illustrate the use of Bayesian hierarchical piecewise regression modeling to detect tra- jectory divergence between groups of participants using longitudinal BMI data from the Cardiovascular Risk in Young Finns (YFS) Study, a well phenotyped prospective cohort with measures from multiple time-points. Previ- ously published work on this data, based on categorical mixed modeling, suggested that BMI levels became sta- tistically different between those who develop T2DM in adulthood and those who did not from the age of 15 years [87]. Background means of least squares predictions, aka LS-means [24–26]), and to test for a group difference in these predictions using a number of contrasts (i.e. post-hoc pairwise comparisons). In this case, the age at which the difference between-groups emerge is often the age at which a significant between- Many applications often relied on higher order time (or age) polynomials or latent basis coefficients [14, 20, 44, 59–61], which strengths and limitations have been described elsewhere [9, 46, 62–65]. In the context of our study the polynomial parameterisation of the growth Page 3 of 15 Page 3 of 15 Page 3 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 model does not specifically yield an age or point in time when the growth pattern is changing within-and between-groups. Alternatively, piecewise models, also known as linear splines or broken stick models, can be used to break up a non-linear or curvilinear growth tra- jectory into several separate linear components [66]. They are particularly useful to compare growth rates in different periods over time if the functional form of the response is characterised by different phases of develop- ment, or if there is a shift in the outcome trajectory at some point in the event window (i.e. an acceleration or a deceleration in the response change rate from one point in time (or age)) [67–73]. Piecewise linear trajectory models have been used to model ‘multiphase’ develop- mental processes primarily with ‘fixed’ transition points in a variety of applications in the frequentist multilevel [40, 45, 69, 74, 75] and structural equation modeling framework [42, 76]. Bayesian applications of these pro- cesses are often referred to as ‘random change point model’ where the position of individual breakpoints is also estimated, allowing for between-person variability in the transition points [77–86]. Methods model does not specifically yield an age or point in time when the growth pattern is changing within-and between-groups. Alternatively, piecewise models, also known as linear splines or broken stick models, can be used to break up a non-linear or curvilinear growth tra- jectory into several separate linear components [66]. They are particularly useful to compare growth rates in different periods over time if the functional form of the response is characterised by different phases of develop- ment, or if there is a shift in the outcome trajectory at some point in the event window (i.e. an acceleration or a deceleration in the response change rate from one point in time (or age)) [67–73]. Piecewise linear trajectory models have been used to model ‘multiphase’ develop- mental processes primarily with ‘fixed’ transition points in a variety of applications in the frequentist multilevel [40, 45, 69, 74, 75] and structural equation modeling framework [42, 76]. Bayesian applications of these pro- cesses are often referred to as ‘random change point model’ where the position of individual breakpoints is also estimated, allowing for between-person variability in the transition points [77–86]. No-covariate model We re-analyse this data set to demonstrate how the Bayesian method can be used to (1) model the BMI profiles to better understand the natural history of the BMI trajectories in those who do and do not develop T2DM in adulthood while controlling for potential co- hort effects, and (2) obtain a refined estimate and confi- dence interval of the age at which the two groups start diverging from one another, translating into significantly different BMI from a certain age onwards. In addition, we conduct a series of short simulations to illustrate the difference in the estimates of age at divergence when using the traditional approach (i.e. pairwise comparisons of marginal means from a categorical mixed model) vs. the proposed trajectory modeling approach. ð1:3Þ Where at age j for participant i, Responseij is the re- peated continuous outcome measures, and ageij is the corresponding time related variables centered around its grand mean. uCPi ageij is a unit heavyside step function where uCPi ageij =1 if ageij ≥CPi and uCPi ageij ¼ 0 if ageij < CPi. The random trajectory parameters b0i, b1i and b2i correspond to the individual intercept, slope be- fore and slope after the person-specific change point CPi, respectively. For each person i, b0i controls the indi- vidual baseline level (or initial status) for the response and its interpretation depends on the centering of the age variable (e.g. if age is centered around 25 years, b0i will be the expected participant-level response at 25 years of age given they are in the first phase of growth b1i). b1i, b2i and CPi, are the expected linear increase per year of age in the first phase of growth, the expected linear rate of increase after the change point, and age at which the linear perturbation to the initial trend occurs, re- spectively. εij is the level-1 residual (i.e. random within- person error for person i at age j) and is independent Page 4 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 parameters as random effects only (as in 1.2). No-covariate model For each of the p + 1 individual growth parameters, additional participant-specific covariates (TICs) can be included in a similar fashion to have multiple predictors at level 2 as follows: bpi ¼ βp0 þ XQp q¼1βpqxqi þ upi , with xqi, the qth measured TIC; βpq,the effect of the TIC xqi on the (p + 1)th trajectory parameter; and upi, the (p + 1)th random effect. The set of p + 1 random effects for person i as- sumed to be multivariate normally distributed with co- variance matrix of dimension (p + 1) * (p + 1), although simpler variance-covariance structures of the random effects can be considered during model building (i.e. mutual independence of the random effects). It is advis- ory to standardize TVCs in order to stabilize the vari- ance, improve normality of errors and linearity of the mean [88]. The common assumption for the error struc- ture is εij ~ iid N(0, σe 2) but it can be relaxed to include time specific variances or residual error correlation such as AR1 errors. and normally distributed (i.e. εij ~ iid N(0, σe 2)). v0i, v1i, v2i and vCPi are the level-2 random effects, multivariate nor- mally distributed with zero mean and variances σv0 2 , σv1 2 , σv2 2 and σCP 2 respectively and full covariance matrix as shown in 1.3 . β00, β10, β20 and CP are the fixed effects (i.e. population average of each trajectory parameter). In this model, the level 1 residual variance σe 2 can be inter- preted as the deviations around an individual’s trajectory and level-2 residuals as between-participant variability in the overall intercept (σv0 2 ), in the rate of change before and after the change point CPi (σv1 2 and σv2 2 respectively), and in the change point itself (σCP 2 ), respectively. Model with group-effect To explore heterogeneity in individual trajectories be- tween groups of interests, the unconditional segmented growth model can be expanded by including time- varying covariates (TVCs) at level-1 and time invariant covariates (TICs) at level 2, while simultaneously adjust- ing for the effects of variables measured on participants at all time points. Whereas TICs directly predict the growth parameters, TVCs directly predict the repeated measures while controlling for the influence of the growth parameters [43, 88]. If the TIC variable is a bin- ary dummy grouping factor (“GRPi”), identifying partici- pants coming from two identified groups, the model can be rewritten as follows: The same approach can be used to expand the hier- archical piecewise trajectory model with grouping fac- tors that have more than 2 levels. This is one of the possible approaches to test for a cohort-effect on the de- velopment of curvilinear responses over time when data arises from multi-cohort or accelerated longitudinal de- signs [89, 90]. If study participants belong to one of k possible birth cohorts, k-1 binary dummy variables are created to identify observations coming from people born in the same calendar year, and as in 2.2, these new k-1 grouping variables are introduced as level 2 predic- tors of the different trajectory parameters in the model. The binary dummy variables are introduced to sequen- tially shift the conditional means of each of the different trajectory parameters. The fixed effects will be the aver- age trajectory parameters for the cohort chosen as the reference cohort in the study sample, and each (βcohort)1.. k −1 coefficient will thus be interpreted as the variation in growth parameters in the corresponding k-1th cohort compared to the reference cohort. Level 1 model: Level 1 model: Responseij ¼ b0i þ b1iageij: 1−uCPi ageij þb2i ageij−CPi :uCPi ageij þTVCij þ εij Responseij ¼ b0i þ b1iageij: 1−uCPi ageij þb2i ageij−CPi :uCPi ageij þTVCij þ εij ð2:1Þ Level 2 model: ð2:1Þ Level 2 model: b0i ¼ β00 þ β0grpGRPi þ v0i b1i ¼ β10 þ β1grpGRPi þ v1i b2i ¼ β20 þ β2grpGRPi þ v2i CPi ¼ CP þ βCPGRPi þ vCPi ð2:2Þ b0i ¼ β00 þ β0grpGRPi þ v0i b1i ¼ β10 þ β1grpGRPi þ v1i b2i ¼ β20 þ β2grpGRPi þ v2i CPi ¼ CP þ βCPGRPi þ vCPi ð2:2Þ ð2:2Þ Trajectory divergence mechanisms Where β00, β10, β20 and CP are the expected trajectory parameters for the reference group (at zero values for other potential covariates); β0grp, β1grp, β2grp and βCP are the expected intergroup variations in these parameters for participants in the second group (i.e. respectively, in the mean response, in the linear age effect, in the devi- ation from linear rate after the CP and in the CP tim- ing); and v0i, v1i, v2i and vCPi are the level-2 residuals person i for intercept, slopes, and age at the change point after controlling for group differences. To test for a between-group difference in one trajectory parameter only, ‘GRP’ can be included as a level-2 predictor for the parameter of interest, and model all other growth The equation 2.2 above, allows for between-group differ- ence in each of the 4 trajectory parameters of the piece- wise model. (i.e. intercept, slope before and after the change point (CP), and the change point itself). If the focus is to determine and model the divergence in the trajectories between group, then model 2.2 can be modi- fied by forcing the intercept and slope before the CP to be invariant across groups by setting β0grp and β1grp to zero at level 2 in equation 2.2. As illustrated in Fig. 1, we identify 3 possible ways in which continuous out- comes trajectories can diverge over time between groups: (1) type 1: the two groups have different slope Buscot et al. BMC Medical Research Methodology (2017) 17:86 Page 5 of 15 Fig. 1 Three hypothetical models of between-group divergence in curvilinear response trajectories over time. Red and black solid lines indicate the average response curve of participants belonging to one or the other group; dashed lines show the position and age at change point(s) for the two groups of participants, or the age at which trajectories diverge between the two groups. Graph obtained using simulated data Fig. 1 Three hypothetical models of between-group divergence in curvilinear response trajectories over time. Red and black solid lines indicate the average response curve of participants belonging to one or the other group; dashed lines show the position and age at change point(s) for the two groups of participants, or the age at which trajectories diverge between the two groups. Significance of group-differences in trajectory parameters Significance of group-differences in trajectory parameters Significance of group-differences in trajectory parameters Testing for group-differences in trajectory parameters is equivalent to investigating the significance of the group- ing covariates parameters at level 2 in the hierarchical change point model. In the Bayesian context, this is done by looking at the posterior probability density for the " βgrp” parameters in 2.2. (i.e. β0grp, β1grp., β2grp.and βCP) of the estimated covariate parameters. For example, the effect of ‘GRP’ on each trajectory parameter is sig- nificant if the 95% Bayesian credible intervals (CI) of the estimated regressors (i.e. each “βgrp”) exclude zero, in which case, the estimated “βgrp” can be interpreted as the shifts in each trajectory parameter in one group compared to the other group [77, 92, 94]. ResponseijeNormal muij; σ2 muij ¼ v0i þ β0grpGRPi þ v1i þ β1grpGRPi ageij þ v2i þ β2grpGRPi ageij−vCPi þ CPgrpGRPi þ ResponseijeNormal muij; σ2 muij ¼ v0i þ β0grpGRPi þ v1i þ β1grpGRPi ageij þ v2i þ β2grpGRPi ageij−vCPi þ CPgrpGRPi þ ResponseijeNormal muij; σ2 To ensure that the effect of ‘group’ on each trajectory parameter can be either positive or negative and that the prior information does not dominate the likelihood, un- informative priors for the fixed group effects β0grp, β1grp, β2grp, CPgrp can be set as N ~ (0, 104). In vector notation, the random effects vi = (voi, v1i , v2i , vcpi )T are assumed to follow a multivariate normal distribution with mean β and unstructured 4 x4 variance-covariance matrix φ as in 1.3, where β = (β0, β1, β2, CP)T, the vector of popula- tion means. Traditionally in Bayesian analysis for ran- dom effects, InvWishart(Σ,k) is used as a conjugate prior to the unknown variance-covariance matrix of Trajectory divergence mechanisms Graph obtained using simulated data after the CP, (2) Type 2: the two groups have different change points, and (3) Type 3: the two groups have different CP and post-CP slopes. To test for group- difference at different stages of the outcome develop- ment, our approach consists in fitting these 3 possible conditional Bayesian hierarchical models to the data and comparing model fit to determine which mechanisms provides the best representation of the underlying devel- opment of the outcome between groups of participants. multivariate normal distributions, where Σ is a positive definite inverse scale matrix of degree of freedom k [93]. Inverse-Gamma (λ1, λ2) is often used as the conjugate prior to the variance of univariate normal distribution (i.e. for mutually independent random effects, and model error variance σ2). Alternative prior distributions may be used for level 2 variances of independent random effects or for the variance components of multivariate normal distributions [41, 92, 93]. Bayesian estimation of the hierarchical piecewise model Bayesian estimation of the hierarchical piecewise model We used a Bayesian approach to estimate and summarize the parameters of interest in the conditional multilevel piecewise model (formula 2.2) [86, 91]. In our illustrative example, all models were fit in RJAGS and R2JAGS in R.. In combined Bayesian notation, the tra- jectory model with a binary grouping status ‘GRP’ as the TIC covariate interacting with all 4 trajectory parameters can be written as follows: Illustrative data In contrast, the proposed hierarchical piecewise regression approach considers and makes full use of individual trajectory in- formation to test for group-differences at specific stages of BMI development from childhood to adulthood. Unlike categorical approaches, the proposed growth model provides a clearer representation of the under- lying pathological BMI development among those who develop T2DM in adulthood. Visual inspection of the sex-specific smoothed BMI trajectories confirms the presence of a divergence be- tween the two groups in adolescence (Additional file 3: Figure S2). Compared to participants who remain healthy, those who develop T2DM seem to have greater average BMI levels by the time they are young adults, al- though it is unclear whether this divergence results from a group-difference in the timing at which the transition to a slower BMI growth rate happens (Type II diver- gence) from a group-difference in rate itself after pu- berty (Type I divergence), or from both (Type III divergence). Although the distal outcome of ‘adult T2DM’ is the grouping factor of interest in our illustrative trajectory divergence analyses, we also demonstrate how the same modeling approach can be used to investigate potential inter-cohort variation in childhood to adulthood BMI trajectories by considering models with ‘year of birth’ as a categorical level 2 predictor of each of the 4 trajectory parameters. Individual age- and sex-specific BMI Z- scores at the first clinic (in 1980) were also included as level 2 predictors of each BMI trajectory parameters to investigate if systematic deviation from participants of comparable age and sex at baseline had any influence on the development of BMI trajectories later in life. All continuous covariates used in the analyses were stan- dardized in order to stabilize the variance, improve nor- mality of errors and linearity of the mean. p In our illustration, we include 2540 YFS participants (1401 females and 1139 males) followed-up a maximum of six times between 1980 and 2011 (Additional file 2: Table S1). Information on adult T2DM status was col- lected on participants at their latest individual adult follow-up (i.e. dichotomous outcome coded 0 for partici- pants without T2DM, and 1 for those with T2DM in 2001, 2007, or 2011). Included participants had at least one BMI measure available in childhood (i.e. in 1980, 1983 or 1986 between age 3 and 18 years). Illustrative data the age at adiposity rebound, which usually occurs be- fore age 6 years in normal weight children [99, 100]. Using BMI data collected on participants aged 6 years and over, we expect that most participants had reached this important childhood milestone, and that a linear trend was thus an appropriate functional form to ap- proximate childhood BMI growth from that age (Additional file 3: Figure S1). the age at adiposity rebound, which usually occurs be- fore age 6 years in normal weight children [99, 100]. Using BMI data collected on participants aged 6 years and over, we expect that most participants had reached this important childhood milestone, and that a linear trend was thus an appropriate functional form to ap- proximate childhood BMI growth from that age (Additional file 3: Figure S1). We illustrate the application of the proposed Bayesian piecewise modeling approach by using it to investigate the divergence in child-to adult trajectories of BMI between participants who do and do not develop adult T2DM in a well-studied ongoing population-based prospective co- hort, the Cardiovascular Risk in YFS [15]. Details on study design and on the collection of cardiovascular risk factors between 1980 and 2011 are published elsewhere [98] and summarized in Additional file 1. Since sex differences in childhood growth and pubertal timing have been demonstrated [101, 102], subsequent BMI trajectory modeling between age 6 and 49 years was conducted among males and females separately [103]. BMI, especially in adulthood, is slightly right skewed, but using log10 transformed BMI in the model- ing approach presented below did not alter our conclu- sions. For ease of interpretation, we thus present results using untransformed BMI only. In a previously published work on the YFS cohort, ele- vated BMI in children between 9 and 18 years was asso- ciated with an increased risk of developing T2DM in adulthood [87]. Additionally, a sex- and insulin-adjusted mixed model incorporating participants ages as a cat- egorical variable, suggested that differences in average BMI values between those who do and those who do not develop adult T2DM tended to emerge during adoles- cence, becoming marginally significant from the age of 15 years onwards. In this approach, the between-group difference at each age groups was assessed by pairwise comparisons of the predicted marginal means (i.e. LS- means), and did not incorporate BMI trajectory informa- tion at the individual or population level. Model convergence, fit and adequacy The choice of the best model among the suite of candi- date (conditional) Bayesian hierarchical models can be based on two criteria: (1) the deviance information criterion DIC [95, 96], an index of quality of fit that is commonly used for Bayesian model comparison [97], and (2), the Bayesian posterior predictive p-value (PP p- value), obtained through posterior predictive checking of the likelihood of each potential model (the sum of resid- uals was used as a as a discrepancy measure) [41]. Page 6 of 15 Page 6 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 Divergence of BMI profiles in T2DM and non-T2DM YFS participants p p Following the modeling approach presented in the Methods and the priors and their corresponding hyper- parameters (Additional file 4: Table S1) we fitted the fol- lowing set of conditional Bayesian hierarchical piecewise models for each sex: unconditional (Model A), adult T2DM status adjusted intercept (Model B), adult T2DM status adjusted childhood slope (Model C), adult T2DM status adjusted adult slope (Model D), adult T2DM status adjusted change point (CP) (Model E), adult T2DM status adjusted CP and adult slope (Model F), adult T2DM status adjusted change point, childhood and adult slopes (Model G), adult T2DM status adjusted intercept, and change point (Model G), and a model with all four parameters ad- justed for adult T2DM status (Model H). As mentioned above, previous research on this data set suggested BMI levels were not significantly different between the two groups in childhood [87]. Models C (i.e. group difference in childhood slopes) and B (i.e. BMI response consistently higher in one group across the life course) were thus fitted to demonstrate our modeling approach. An annotated ex- tract showing the RJAGS code syntax used to fit Model E is available in Additional file 6. g For the other participants varying variable included in the analysis, sex- and age-specific BMI z-scores at the first visit and birth cohort, priors were set to N ~ (0,0.001) for all cor- responding parameters (i.e. all βcohort priors and βinitialBMI − z −score). To remain consistent with previous analyses of this data set [87], time-varying measures of fasting insulin were log-transformed and standardized before being included as a level-1 predictor in the Bayesian hierarchical models to improve right skewedness and to linearize its relationship with BMI About 17% of the insulin measures were not available in the data. The missing data mechanism for ‘insu- lin’ was considered non-informative, as we have no reason to believe that the probability of an individual insulin meas- ure being missing depends on the true value of this missing insulin observation (although it may be related to other ob- served variables for that individual). We thus consider that insulin is missing at random (MAR), and we specify a prior for this covariate [104]. Since log (insulin) is approximately normally distributed, we specify a N ~ (μlog(insulin), τlog(insu- lin) ) likelihood for log(insulin)i and place a vague prior on its variance (i.e. τlog(insulin) ~ Gamma(0.001,0,001) ). Illustrative data Participants had on average 4.98 repeated measures of BMI over the study period, with 90.7% of participants having 4 or more BMI measures (Additional file 2: Figure S1). 88 in- cluded participants (3.5%, 44 females and 44 males) had T2DM in adulthood. We excluded BMI observations made among those aged 3 years in 1980 so that the ages of participants considered in the trajectory analysis ranged from 6 to 49 years. 3 year olds were not included in the analysis since only 3 participants in this birth co- hort developed T2DM in adulthood. Furthermore, the lack of BMI measures between 3 and 6 years, prevented modeling the downwards slope from infancy peak, nor Specific values for the hyperparameters used in our il- lustrative analyses are given in Additional file 4. While in principle φ can be unstructured, in our application, convergence for some parameters could not be reached when considering an unrestricted covariance structure between all four random effects in the unconditional change point model (equations 1.1 and 1.2), probably due to over parameterisation. Because initial analyses sug- gested a correlation between the slopes before the change Page 7 of 15 Page 7 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 point (b1i) and the difference in slopes after the change point (b2i), we constrained the model by including a non- zero correlation between these two random effects but setting independence for all other random effects, leading to a block diagonal structure of φ (Additional file 4). Based on DIC, this covariance structure was preferred over mu- tually independent random effects for both males and fe- males (Additional file 4), and used when expending the trajectory models with level 2 predictors. In our applica- tion, we investigated prior sensitivity by fitting the uncon- ditional BMI trajectory model using three sets of priors for the hyperparameters (Additional file 4). Because we found that the choice of hyperparameters had a minor in- fluence on the marginal posterior distributions, for subse- quent (conditional) analyses, we chose to report posterior estimates of parameters estimated from the set of priors that yielded the lowest DIC in the sensitivity analyses (Additional file 4). Illustrative data In this set the priors for the means of the change points were based on the sex-specific estimates that maximized the profile log likelihood for the fixed (population-average) breakpoints in the unconditional model (estimated at 16 years for females and 22 for males, see estimation method in Additional file 5). Using these priors for the change point means also kept computation running times reasonable. did not suggest any systematic patterns of change in insulin levels at the intra-individual level as people age. That is, the age smoother estimate obtained by fitting a generalized additive mixed model had an estimated degree of freedom (edf) close to 1 and was not significant (p-value >0.3), which did not suggest a non-linear relationship between log(insulin) and age [105]. Approximate posterior distributions of the parameters of the models considered throughout the analyses are obtained via MCMC simulations. Each model ran with 4 independent parallel chains of the Gibbs sampler (see Appendix 3 for an example of code). For each model, the first 50000 iterations were discarded in a burn-in run, and the draws from the posterior were thinned by a factor of 10 to reduce serial correlation of the chains. The following 20000 iterations were used to obtain pos- terior distributions of model parameters by mixing the 4 sequences. Model convergence was assessed through MCMC iterations traceplots and Gelman-Rubin diag- nostic [92], and residual errors were plotted to confirm they approximately followed a normal distribution. Divergence of BMI profiles in T2DM and non-T2DM YFS participants However, females who developed T2DM reached their developmental transition in BMI rate on average 12.37 years later (Table 2). compared to childhood (i.e. 0.67- vs. 0.18 -, and 0.61- vs. 0.15 kg/m2 per year in childhood and adulthood for females and males respectively), and participants who developed T2DM had similar BMI yearly rates in adult- hood compared to those who remained healthy (β2T2DM effect not significant in model F for both sex Table 2). However, females who developed T2DM reached their developmental transition in BMI rate on average 12.37 years later (Table 2). The effect of the time-varying covariate of insulin at level 1 was significant for both males and females, with a 1-sd increase in log(Insulin) resulting in a BMI observa- tion increased by 2.6 and 2.8 kg/m2 respectively (i.e. exp(βlog(insulin)), Table 2). To assess potential differences in the magnitude of the insulin effect as a function of between-person characteristics, we expanded model E by including an interaction between ‘adult T2DM status’ and log(insulin). For each sex, the estimated parameters were not significant (95% CI included 0), suggesting that the effect of insulin on BMI was homogenous between the two groups and across genders. Similarly for males, estimated BMI growth rates were not markedly different between the two T2DM groups in childhood or in adulthood, and comparable to those estimated in females (Table 2). But again, compared with healthy adults, those who developed T2DM reached their slower BMI growth rate on average 6.47 years later. The estimates of the variance-covariance parameters of model E showed that the correlation between an indi- vidual’s BMI growth rate in childhood and adulthood is equal to 0.61 for females and 0.47 for males, suggesting that children who have greater yearly BMI increase rates also have greater adult rates of increase (correlation estimated as: σβ1β2= ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ σ2 β1σ2 β2 p , Table 2). The between- participant variation around the change point σCP was comparable between males and females (Table 2). Divergence of BMI profiles in T2DM and non-T2DM YFS participants Under this parametrization, the posterior predictive distribution for μlog(insulin) and τlog(insulin) will be informed by the observed part of the data only. Although individual insulin measure- ments change at each data collection point, by adding log(insulin) as a level 1 covariate in the multilevel model, the estimated relationship between insulin and BMI devel- opment remains constant across time [45]. This is a reason- able assumption in our application, since data exploration For both sex, the lowest DIC was obtained when fit- ting model E, which was also the best fitting model with PP p-values close to 0.5 (Table 1). This supported the type II divergence mechanism where a difference in BMI levels emerged between the two groups due to a group difference in the change point timing. BMI growth rate in adulthood for both sexes was decreased by two-thirds Buscot et al. BMC Medical Research Methodology (2017) 17:86 Page 8 of 15 Page 8 of 15 Table 1 Analyses of the divergence in BMI trajectories between T2DM adults and non-T2DM adults: assessment of Bayesian model complexity (effective number of parameters pD), and fit (deviance information criteria DIC) for each candidate model Model Females PP p-val Males PP p-val Unconditional A 26910 (2544) 0.47 19837 (2223) 0.55 T2DMgroup (int β0) B 26670 (2366) 0.45 19741 (2270) 0.43 T2DMgroup (childhood slope β1) C 26780 (2510) 0.6 19865 (2247) 0.58 T2DMgroup (Adulthood slope β2) D 26701 (2401) 0.58 19828 (2242) 0.62 T2DMgroup (change point CP) E 26076 (2777) 0.52 19762 (2213) 0.51 T2DMgroup (CP + β2) F 26504(2430) 0.6 19860 (2271) 0.54 T2DMgroup (CP + β0) G 26436 (2751) 0.55 19896 (2242) 0.45 T2DMgroup (all 4 parameters) H 26532 (2978) 0.52 19920 (2435) 0.51 Reported for each model are DIC (pD), and posterior predictive p-values (PP p-val). Best fitting models are indicated in bold characters Table 1 Analyses of the divergence in BMI trajectories between T2DM adults and non-T2DM adults: assessment of Bayesian model complexity (effective number of parameters pD), and fit (deviance information criteria DIC) for each candidate model compared to childhood (i.e. 0.67- vs. 0.18 -, and 0.61- vs. 0.15 kg/m2 per year in childhood and adulthood for females and males respectively), and participants who developed T2DM had similar BMI yearly rates in adult- hood compared to those who remained healthy (β2T2DM effect not significant in model F for both sex Table 2). Divergence of BMI profiles in T2DM and non-T2DM YFS participants Table 2 Posterior mean parameter estimates for Bayesian hierarchical Piecewise BMI trajectory for best fitting trajectory divergence models in males and females (Models E) Females Model E Males Model E β0 I 26.5 (0.20) 27.46 (0.16) β1 S1 0.67 (0.012) 0.61 (0.01) β2 S2 −0.49 (0.015) −0.46 (0.06) CP CP 16.02 (0.29) 21.62 (0.42) CP T2DM CP 12.37 (1.21) 6.47 (1.23) σβ0 2.07 (0.05) 2.36 (0.07) σβ1 0.02 (0.005) 0.06 (0.004) σβ2 0.07 (0.006) 0.05 (0.004) σβ1β2 0.11 (0.05) 0.14 (0.03) σCP 3.1 (0.26) 4.3 (0.2) σ 1.33 (0.02) 1.21 (0.01) βlog(insulin) 1.01 (0.04) 0.98 (0.03) Posterior standard deviations (uncertainty in the parameters) are reported in brackets. Reported β0 coefficients are in kg/m2, β1 and β2 are in kg/m2 per year, CP and CP T2DM are in years. All σ coefficients are standard deviations for the corresponding growth parameters and the residual error. βlog(insulin) coefficients are in kg/m2 for a 1 sd increase in log(insulin) level Table 2 Posterior mean parameter estimates for Bayesian hierarchical Piecewise BMI trajectory for best fitting trajectory divergence models in males and females (Models E) Figure 2 shows the estimated population-average prototypical trajectories for each sex and T2DM group obtained from the estimated parameters for Model E, along with 100 trajectories predicted for each sex and T2DM group from Model E by Monte Carlo simulation. This illustrates a range of credible individual profiles generated under this model (see Appendix for code). For each sex and adult T2DM status group, Fig. 3. shows a box and whiskers plot of the estimated individual BMI slopes obtained from Model E after the average change point in the healthy group and before the T2DM groups reach their average CP (i.e. slopes between 16.02 and 28.4 years in females, and slopes between 21.62 and 28.09 years in males). Figure 3 illustrates that individual rates of change after puberty provides better discrimin- ation of participants who went on to develop T2DM from those who did not, compared to punctual individual BMI levels at age 15 or 18 for females, and ages 21 and 24 for males (Additional file 2: Figure S2). While the Buscot et al. BMC Medical Research Methodology (2017) 17:86 Page 9 of 15 Fig. Divergence of BMI profiles in T2DM and non-T2DM YFS participants 2 Sex-specific population average prototypical BMI trajectories for healthy and T2DM adults in the YFS cohort (solid blue and solid red lines, respectively) and prediction of 200 individual trajectories for each sex (100 per T2DM status group). The dashed trajectories were obtained by MCMC simulation using sex-specific posterior estimates of mean and variance of growth parameters for the best fitting models (Model E). In these predictions, time varying measures of log(insulin) were set to the average log(Insulin) observed in the cohort Fig. 2 Sex-specific population average prototypical BMI trajectories for healthy and T2DM adults in the YFS cohort (solid blue and solid red lines, respectively) and prediction of 200 individual trajectories for each sex (100 per T2DM status group). The dashed trajectories were obtained by MCMC i l i i ifi i i f d i f h f h b fi i d l (M d l E) I h Fig. 2 Sex-specific population average prototypical BMI trajectories for healthy and T2DM adults in the YFS cohort (solid blue and solid red lines, respectively) and prediction of 200 individual trajectories for each sex (100 per T2DM status group). The dashed trajectories were obtained by MCMC simulation using sex-specific posterior estimates of mean and variance of growth parameters for the best fitting models (Model E). In these predictions, time varying measures of log(insulin) were set to the average log(Insulin) observed in the cohort the childhood BMI slope, with a 1-sd increase in BMI z-score associated with a 0.056 (sd = 0.012) and a 0.038 (sd =0.009) increase in childhood (in kg/m2 per year) for male, and females respectively. This suggests that in the YFS sample, higher age- and sex-adjusted BMI at first visit in childhood were associated with faster BMI in- crease in childhood, but not with the age at transition in BMI development nor the change rate in adulthood. distribution of BMI levels at age groups surrounding the age at divergence overlaps considerably (Additional file 2: Figure S2), individual slopes allow to differentiate partici- pants who have switched to a rate consistent with a nor- mal slowing down of BMI development after puberty, from those who are still on the trajectory of increasing BMI development consistent with the rate from childhood. Effect of age-and sex-specific childhood Z-score on BMI trajectories The results show that most of the between cohort variation for females is due to slight trajectory differences in two specific birth cohorts: those born in 1968, who reached the transition to adult BMI growth rate on average 2.89 years later than the cohort (year of birth 1971), and those born in 1974, who had adult BMI yearly rates increased by 0.06 (e.g. adult slopes of 0.24 compared to 0.18 kg/m2 per year on average for the other 5 cohorts) (Additional file 7). depart from the population-average childhood slope rep- resents the age at which the two groups of participants diverge in their outcome trajectories (i.e. the second group maintains this rate of change for 12.30 years lon- ger). To resemble the YFS BMI data, we randomly sam- pled baseline ages from the YFS cohort subtracted by 25 years as ages at the first visit for each participant, with 6 non-missing repeated measures 3, 6, 9, 21, 27 and 31 years later for each participant. We considered 3 sce- narios of sample sizes for the number of participants in each group (group 1/group 2): (1) 100/100, (2) 50/100, and (3) 30/100. For each of the three scenarios, we simu- lated 100 datasets and fitted both a mixed model with age as a categorical variable and the Type II divergence Bayesian Hierarchical piecewise model using the set of priors defined in Additional file 4. For each piecewise model, we recorded the posterior estimate for the “CP” parameter, and for each fitted categorical mixed model, we applied pairwise comparison of the least-square means (LS-means) with Tukey adjustment for multipli- city to retrieve: (1) the earliest age at which the group- difference in means was found significant (p < 0.05), and (2) the midway point between two consecutive ages that had a minimum number of non-significant differences in means before and significant differences in means after the “midway point” method (2) is a potential alternative definition of age at which the group-difference appears in the LS means. Compared to the “earliest age with p- val <0.05” method (1), the “midway” point definition minimises the impact of simulations where some tests show significance at a young age, even though tests for the surrounding ages are not. For each scenario, esti- mated ages at divergence using the 3 methods were aver- aged across the 100 simulations. Effect of age-and sex-specific childhood Z-score on BMI trajectories To test whether ‘year of birth’ was associated with between-participant heterogeneity in the development of BMI from age 6 to 49 years, five binary dummy variables identifying BMI observations of people born in different years (i.e. 62, 65, 71, 74 and 77) were introduced as level 2 When including individual age-and sex-specific BMI z- scores at the first clinic as continuous level 2 predictors of each of the four growth parameters in sex-specific Models E, the only significant effect observed was for Fig. 3 Box and whiskers plot of fitted individuals random slopes between 16.02 and 28.4 years for females (a) and between 21.62 and 28.09 years for males (b). Individual random slopes are estimated from the Bayesian hierarchical random change point model E. Solid lines in the boxplot indicate the group-specific median for the slopes (equivalent to the 50th percentiles of the posterior distribution) Fig. 3 Box and whiskers plot of fitted individuals random slopes between 16.02 and 28.4 years for females (a) and between 21.62 and 28.09 years for males (b). Individual random slopes are estimated from the Bayesian hierarchical random change point model E. Solid lines in the boxplot indicate the group-specific median for the slopes (equivalent to the 50th percentiles of the posterior distribution) Page 10 of 15 Page 10 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 predictors of BMI growth parameters in sex-specific models (with year 1971 as the reference level) (Table 3). Increasing the complexity of the model did not improve model fit for males, and the lowest DIC was obtained for the unconditional model (Model A, Table 3) suggesting that their life-course BMI trajectory is more stable across cohorts. For females, model E marks a significant im- provement in model fit, suggesting that the most signifi- cant predictor of between-cohort variations resides in the timing of the CP, although the best model was obtained when adjusting for a cohort effect on both the adult BMI growth rate and change point. For each sex, the posterior mean parameter estimates of the best fitting model are presented in Additional file 7. Effect of age-and sex-specific childhood Z-score on BMI trajectories Figure 4 presents the simulation results in term of the quartiles distribution and means of these estimates of age at divergence across the 100 simulations. When sample size decreases for one group of participants, the pairwise LS mean com- parison method will tend to overestimate the age at di- vergence, with significant variability in the estimates Reported are: DIC (pD), and posterior predictive p-values (PP p-val). Best fitting models for each sex indicated in bold characters. (Convergence was not reached for the most complex model where all 4-trajectory parameters (i.e.β0, β1, β2, and CP) were adjusted for birth cohort effects) Simulations 4 Boxplots and mean “Age at divergence” (x) estimated across 100 simulations using the three methods. Bottom and top of the boxes are the lower (Q1) and upper quartiles (Q3), respectively; the bands near the middle of the boxes are the medians, the lengths of the boxes represent the interquartile range (IQR = Q3-Q1); the upper whiskers are defined as min(max(x), Q3 + 1.5 * IQR) and the lower whiskers as max(min(x), Q1 – 1.5 * IQR). Means of age at divergence across the 100 simulations for each scenario are indicated with empty circles. The horizontal dashed line indicates the true age at divergence set in the simulations (16.02 years old) Fig. 4 Boxplots and mean “Age at divergence” (x) estimated across 100 simulations using the three methods. Bottom and top of the boxes are the lower (Q1) and upper quartiles (Q3), respectively; the bands near the middle of the boxes are the medians, the lengths of the boxes represent the interquartile range (IQR = Q3-Q1); the upper whiskers are defined as min(max(x), Q3 + 1.5 * IQR) and the lower whiskers as max(min(x), Q1 – 1.5 * IQR). Means of age at divergence across the 100 simulations for each scenario are indicated with empty circles. The horizontal dashed line indicates the true age at divergence set in the simulations (16.02 years old) Fig. 4 Boxplots and mean “Age at divergence” (x) estimated across 100 simulations using the three methods. Bottom and top of the boxes are the lower (Q1) and upper quartiles (Q3), respectively; the bands near the middle of the boxes are the medians, the lengths of the boxes represent the interquartile range (IQR = Q3-Q1); the upper whiskers are defined as min(max(x), Q3 + 1.5 * IQR) and the lower whiskers as max(min(x), Q1 – 1.5 * IQR). Means of age at divergence across the 100 simulations for each scenario are indicated with empty circles. The horizontal dashed line indicates the true age at divergence set in the simulations (16.02 years old) In contrast, piecewise regression is particularly suited to model BMI across different life-stages as its parame- ters map onto what is known about the natural develop- ment of BMI over time [116]. Since ‘change points’ (i.e. Simulations A short series of simulations was conducted to compare difference in estimates of the age at which the groups di- verge when using the proposed Bayesian piecewise growth modeling approach compared to a more trad- itional approach based on pairwise comparison of LS- means estimated from a categorical mixed model. We simulated repeated measure data from a Type II diver- gence model (i.e. group-difference in the change point timing only), using the posterior estimates of mean growth parameters for the model fitted for females (average parameters are set to: βO = 26.5, β1 = 0.67, CP = 16.02, = βGroupCP =12.37, β2 = −0.49, matching Model E posterior estimates for females in Table 2), and both a participant-level random effect (σerror 2 = 2.77) and an observation-level residual error (σerror 2 = 2.47). Under this model, “CP”, the change point for the first group to Table 3 Analyses of inter-cohort differences in BMI trajectories: assessment of Bayesian model complexity (effective number of parameters pD), and fit (deviance information criteria DIC) for each candidate model Model Females PP p-val Males PP p-val Unconditional A 26910 (2544) 0.72 197837(2223) 0.52 Birth cohort (int β0) B 26811 (2455) 0.70 19872 (2232) 0.70 Birth cohort (childhood slope β1) C 26759 (2489) 0.34 19849 (2175) 0.63 Birth cohort (Adulthood slope β2) D 26645 (2358) 0.67 19857 (2263) 0.68 Birth cohort (change point CP) E 26395 (2599) 0.60 19862 (2211) 0.63 Birth cohort (CP and β2) F 26390 (2671) 0.49 19877 (2255) 0.43 Birth cohort (CP, β2 and β1) G 26783 (2775) 0.48 19945 (2342) 0.53 Reported are: DIC (pD), and posterior predictive p-values (PP p-val). Best fitting models for each sex indicated in bold characters. (Convergence was not reached for the most complex model where all 4-trajectory parameters (i.e.β0, β1, β2, and CP) were adjusted for birth cohort effects) Table 3 Analyses of inter-cohort differences in BMI trajectories: assessment of Bayesian model complexity (ef parameters pD), and fit (deviance information criteria DIC) for each candidate model Reported are: DIC (pD), and posterior predictive p-values (PP p-val). Best fitting models for each sex indicated in bold characters. (Convergence was not reached for the most complex model where all 4-trajectory parameters (i.e.β0, β1, β2, and CP) were adjusted for birth cohort effects) Buscot et al. BMC Medical Research Methodology (2017) 17:86 Page 11 of 15 Fig. Simulations milestones in the case of BMI) are model parameters in the piecewise model, there is no need to use elaborate techniques to retrieve these points of interest [59, 112, 117]. Piecewise models are also often preferable to more general continuous non-linear models if the number of repeated measurements per participant is small (i.e. 3 to 6 data points each as in [16, 109]) as is often the case in long-running observational prospective studies [99, 112]. Moreover piecewise multi-level regression models may be used to characterize the divergence mechanisms in non-linear responses between groups by modeling change points as random parameters and introducing grouping factors as predictors of the between-person heterogeneity in responses over time. arising due to random variation, especially when age at significance is determined using the first age at which a p-value <0.05 occurs (Fig. 4.). In contrast, the hierarch- ical Bayesian piecewise model was less sensitive to sam- ple size, and the true age at divergence was consistently within the estimated interquartile range of the produced estimates, indicating that the Bayesian trajectory diver- gence model outperforms the LS mean method in both accuracy and precision, regardless of the way “age at di- vergence” is defined from the model output. Discussion Using the repeated BMI data from the YFS study, we demonstrated how Bayesian hierarchical piecewise re- gression (BHPR) modeling may be used to investigate between-group trajectory divergence in non-linear longi- tudinal outcomes. The non-linearity in BMI development across the life- course is well documented in the literature, with changes in BMI corresponding to a number of identified develop- mental phases [101, 106, 107]. In particular, BMI rate de- celerates after puberty once people reach their adult height, translating to a leveling-off of the BMI trajectory in adulthood [108, 109]. Although many recent applica- tions have relied on such approaches [99, 102, 110–113], traditional polynomial parameterizations of growth curve models are not well suited to analyse BMI development [114, 115], especially if the focus is to identify transitional changes or determine divergence between groups. Although our main goal was to characterize how and when the developmental patterns of BMI diverged be- tween those who did and did not develop T2DM in the YFS, we also demonstrated the utility of the method to investigate cohort effects in the outcome response. Pre- vious analyses of the YFS BMI and T2DM data consid- ered categorical mixed models and tested for differences in the estimated BMI levels between the two T2DM groups at different ages by pairwise comparisons of the BMI predicted marginal means (i.e. Least-Square means) averaged over sex while adjusting for multiple testing (i.e. Page 12 of 15 Page 12 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 Tukey adjustment). This approach suggested that from age 15 years, the T2DM group had significantly higher BMI levels than those without T2DM. However, these analyses ignored the potential confounding effect of birth cohorts, and each existing “age” was treated as a non time-ordered categorical variable so that no inference could be made on individual or group-specific age-related BMI trajectories. Some age groups comprised those from up to five separate birth cohorts, while others only comprised those from a unique birth cohort (i.e. those aged 3 and 27 years). Having substantially fewer participants in one or both T2DM status groups at some age points results in a decreased power to detect a significant difference between groups (i.e. the ob- served difference at age 27 years was not significant in ei- ther sex-averaged or sex-adjusted LS-means, Additional file 8: Tables S1, S2 and Figure S1, S2). Conclusions S d h Studying within-person and between-person differences in the development of continuous outcomes as a func- tion of age in long-running multi-cohort observational studies is crucial to better understand the natural history of healthy vs. pathological risk factor profiles. Due to the typically unbalanced data designs, loss to follow-up and expected non-linear responses, it remains methodologic- ally challenging to analyse such data. When the substan- tial focus is on when and how two or more groups of participants grouped according to a distal dichotomous health outcome have diverged in their response trajec- tories, traditional parameterisations of curvilinear growth model do not allow to identify an age at which the group that developed the condition moved onto a different path compared to the group that remained healthy. In contrast, the hierarchical piecewise multi- level modeling enables the separation of multiple aspects of change in complex developmental processes such as individual and group differences in the rates of change at different periods, and potential heterogeneity in the timing at which individuals from identified groups enter each developmental phase, providing a powerful tool to help inform intervention The methodology we illustrate here focuses on a response with only one developmental change point, but it could easily be extended to more complex non-linear responses with multiple transitions. In contrast, the method we illustrate here is not sensi- tive to sample size and uses developmental theory to in- form a model that allows between-group differences in within-person BMI trajectories at four possible levels for males and females to be examined (i.e. the overall BMI level, the childhood BMI growth rate, the adult BMI growth rate, and the age at which the transition between the two phases of change occurs). Applied to the example data set, our method allowed us to characterise group differences in the non-linear devel- opment of BMI and to identify a critical age window at which weight intervention programs might be best applied to help reduce or delay the incidence of T2DM in adult- hood. Our findings support the theory that girls who keep on gaining weight at the same rate they did in childhood past the age of 16 years are more likely to develop T2DM in adulthood. Similarly, for males, the natural deceleration in BMI velocity occurs, on average, at 21 years of age. Conclusions S d h Those who stay on their childhood BMI trajectory past that age may be at increased risk of developing T2DM. Discussion Because BMI develop- ment is known to progress differently in males and females, and the oldest and youngest cohorts in the YFS sample are almost a generation apart (~15 years), not taking these con- founders into account may result in biased inferences. In fact, when estimating the LS-means separately for each sex, the age at which the difference between T2DM groups be- comes significant is not as clear since in males the differ- ence is not significant at age 21 and 24 years, suggesting the true divergence in BMI between T2DM groups for males occurs more around those ages (Additional file 8: Table S2 and Figure S2). approach, serial measures of patient’s weight and height, often routinely collected in paediatric, general practice, and healthy or well child clinics, could be used to deter- mine if an individual is on a path to an healthy adult weight status, or if their BMI trajectory places them in a category more susceptible to develop adult metabolic con- ditions such as T2DM. Ethics approval and consent to participate Ethics approval and consent to participate YFS Participants or their parents provided written informed consent, and the study was approved by local ethics committees (The Ethics Committee of the Hospital District of Southwest Finland) in agreement with the Declaration of Helsinki. Additional file 7: Posterior mean parameter estimates for best fitting birth cohort-adjusted trajectory model for each sex (Model E for females and Model A for males). (DOCX 16 kb) Additional file 7: Posterior mean parameter estimates for best fitting birth cohort-adjusted trajectory model for each sex (Model E for females and Model A for males). (DOCX 16 kb) Additional file 8: Results of mixed models with age as a categorical predictor and log-insulin as a continuous predictor: LS means contrasts (No adult T2DM vs. adult T2DM) and significance at each age averaged over- (Table S1.) or adjusted for the levels of sex (Table S2.) and pairwise comparisons of Least-square means of BMI and 95% CIs at each age in each T2DM status group averaged over levels of sex (Figure S1.) at each age in each T2DM status group and sex group combination (M = males, F-females, 1 = No adult T2DM, 2 = adult T2DM) (Figure S2.) and adjusted for log(insulin). (DOCX 549 kb) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Funding g The Young Finn study was financially supported by the Academy of Finland (grants 126925, 121584, 124282, 129378, 117797, 265877 and 41071), the Social Insurance Institution of Finland, the Turku University Foundation, Paavo Nurmi Foundation, Juho Vainio Foundation, Sigrid Juselius Foundation, Maud Kuistila Foundation, Research funds from the Kuopio, Turku and Tampere University Hospitals, the Finnish Foundation of Cardiovascular Research, the Finnish Medical Foundation, the Orion-Farmos Research Foundation, and the Finnish Cultural Foundation. This work was partly funded by the National Health and Medical Research Council Project Grant (APP1098369). CGM was supported by a National Heart Foundation of Australia Future Leader Fellowship (100849). Abbreviations BHPR: Bayesian hierarchical piecewise regression; BMI: Body mass index; CI: Credible interval; CP: Change point; CVD: Cardiovascular disease; DIC: Deviance information criteria; MCMC: Markov Chain Monte Carlo; PP p-value: Posterior predictive p-value; T2DM: Type 2 Diabetes mellitus; TIC: Time-independent covariate; TVC: Time-varying covariate; YFS: Cardiovascular risk in Young Finn study References 1. Power C, Kuh D, Morton S. From Developmental Origins of Adult Disease to Life Course Research on Adult Disease and Aging: Insights from Birth Cohort Studies. Annu Rev Public Health. 2013;34:7–28. 1. Power C, Kuh D, Morton S. From Developmental Origins of Adult Disease to Life Course Research on Adult Disease and Aging: Insights from Birth Cohort Studies. Annu Rev Public Health. 2013;34:7–28. 1. Power C, Kuh D, Morton S. From Developmental Origins of Adult Disease to Life Course Research on Adult Disease and Aging: Insights from Birth Cohort Studies. Annu Rev Public Health. 2013;34:7–28. 2. Færch K, Witte DR, Tabák AG, Perreault L, Herder C, Brunner EJ, Kivimäki M, Vistisen D. Trajectories of cardiometabolic risk factors before diagnosis of three subtypes of type 2 diabetes: a post-hoc analysis of the longitudinal Whitehall II cohort study. Lancet Diabetes Endocrinol.1:43–51. 3. Kuh D, Ben-Shlomo Y, Lynch J, Hallqvist J, Power C. Life course epidemiology. J Epidemiol Community Health. 2003;57:778–83. 4. Narayan KMV, Boyle JP, Thompson TJ, Gregg EW, Williamson DF. Effect of BMI on Lifetime Risk for Diabetes in the U.S. Diabetes Care. 2007;30:1562–6. Availability of data and materials 5. Ben-Shlomo Y, Kuh D. A life course approach to chronic disease epidemiology: conceptual models, empirical challenges and interdisciplinary perspectives. Int J Epidemiol. 2002;31:285–93. The dataset supporting the conclusions of this article were obtained from the Cardiovascular Risk In Young Finns study (YFS) after submission and approval of our study plan by the Young Finns Study coordinators. The YFS dataset comprises health related participant data and their use is therefore restricted under the regulations on professional secrecy (Act on the Openness of Government Activities, 612/1999) and on sensitive personal data (Personal Data Act, 523/1999, implementing the EU data protection directive 95/46/EC). Due to these ethical restrictions, the data from this study cannot be stored in public repositories or otherwise made publicly available. However, data access may be permitted on a case to case basis upon request only. Data requests should be addressed to the project coordinator, Dr. Olli Raitakari (Email:olli.raitakari@utu.fi), Tel: +358 2 333 7220 Fax: +358 2 333 7270. Written requests should be sent to the following postal address: Research Centre of Applied and Preventive Cardiovascular, Medicine University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, FINLAND. 6. Dudina A, Cooney MT, Bacquer DD, Backer GD, Ducimetiere P, Jousilahti P, Keil U, Menotti A, Njolstad I, Oganov R, et al. Relationships between body mass index, cardiovascular mortality, and risk factors: a report from the SCORE investigators. Eur J Cardiovasc Prev Rehabil. 2011;18:731–42. 7. Chen Y, Copeland WK, Vedanthan R. Association between body mass index and cardiovascular disease mortality in east Asians and south Asians: pooled analysis of prospective data from the Asia Cohort Consortium. BMJ. 2013; 347:f5446. 7. Chen Y, Copeland WK, Vedanthan R. Association between body mass index and cardiovascular disease mortality in east Asians and south Asians: pooled analysis of prospective data from the Asia Cohort Consortium. BMJ. 2013; 347:f5446. 8. Freedman DS, Khan LK, Dietz WH, Srinivasan SR, Berenson GS. Relationship of childhood obesity to coronary heart disease risk factors in adulthood: the Bogalusa Heart Study. Pediatrics. 2001;108:712–8. 9. Twisk JWR. Applied Longitudinal Data Analysis for Epidemiology: A Practical Guide. 2nd ed. Cambridge: Cambridge medicine; 2013. 10. Diggle PJ, Heagerty P, Liang KY, Zeger SL. Analysis of Longitudinal Data, Oxford Statistical Science Series. 2002. ISBN 978-0-19-852484-7. 10. Diggle PJ, Heagerty P, Liang KY, Zeger SL. Analysis of Longitudinal Data, Oxford Statistical Science Series 2002 ISBN 978 0 19 852484 7 Author details 1Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia. 2Institute of Marine and Antarctic Studies, University of Tasmania, Hobart, Australia. 3Research Centre of Applied and Preventive Cardiovascular Medicine, University of Turku, Turku, Finland. 4Department of Medicine, University of Turku and Division of Medicine, Turku University Hospital, Turku, Finland. 5Murdoch Childrens Research Institute, The Royal Children’s Hospital, Melbourne, Australia. 6Department of Paediatrics, University of Melbourne, Melbourne, Australia. 7Department of Paediatrics, Monash Medical Centre, Melbourne, Australia. 8Department of Clinical Chemistry, Fimlab Ltd and University of Tampere School of Medicine, Tampere, Finland. 9Department of Pediatrics, University of Tampere School of Medicine, Tampere, Finland. 10Tampere University Hospital, Tampere, Finland. 11Centre for Research in Mathematics, School of Computing, Engineering & Mathematics, Western Sydney University, Sydney, Australia. Acknowledgements We thank the clinic and administrative staff for their contribution to data collection. Above all, we thank the participants of the Cardiovascular Risk in Young Finns Study. Received: 13 October 2016 Accepted: 10 May 2017 Consent for publication Not applicable. Consent for publication Not applicable. Additional file 6: Annotated RJAGS sample code to fit a type 1 trajectory divergence model with a fully unstructured 4 by 4 covariance matrix for the random growth parameters. (DOCX 14 kb) Additional files Additional file 1: Additional information on BMI, T2DM status and fasting insulin information collection in the YFS subset used in the illustrative analyses. (DOCX 17 kb) Additional file 2: Subset of the YFS cohort used for the BMI trajectory analysis. Reported are the total number (No.) of participants seen at each clinic year and their ages (Figure S1.) Density plot of the number of BMI measures per YFS participants in the subset of the cohort used for the BMI trajectory analysis (Figure S2.) and average BMI values in kg/m2 at each age stratified by T2DM group (pink, no adult T2DM; blue, Adult T2DM), with error bars representing the mean BMI ± SD (standard deviation) (Figure S3.) (DOCX 180 kb) Longitudinal studies often aim to make inferences on differences among average population health marker tra- jectories. Typically, this involves comparing change rates (or slope differences) in healthy participants vs. those with pathological development, specific treatment conditions, or groups following certain lifestyle patterns [118–120]. Using our Bayesian hierarchical piecewise regression Additional file 3: Spaghetti plot of the individual trajectories of those with T2DM in adulthood (N = 88) and those who did not develop T2DM in adulthood (N = 2452). Red solid line: loess smoother curve indicating the average longitudinal trend in each group (Figure S1.) and scatterplot of the life-course BMI data (in kg/m2) stratified by sex. Solid lines and gray bands: loess smoothed average trajectories and confidence intervals for each group (adult T2DM vs. non-T2DM group); dashed lines: age-specific averages of BMI levels (Figure S2.) (DOCX 1918 kb) Page 13 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 Competing interests Additional file 4: Prior sensitivity analyses methods and results. (DOCX 25 kb) Additional file 5: Log-likelihood profiling method and R-code for the choice of priors of the changepoint mean. (μcp). (DOCX 14 kb) Additional file 6: Annotated RJAGS sample code to fit a type 1 trajectory divergence model with a fully unstructured 4 by 4 covariance matrix for the random growth parameters. (DOCX 14 kb) Additional file 7: Posterior mean parameter estimates for best fitting birth cohort-adjusted trajectory model for each sex (Model E for females and Model A for males). (DOCX 16 kb) Additional file 8: Results of mixed models with age as a categorical predictor and log-insulin as a continuous predictor: LS means contrasts (No adult T2DM vs. Additional files adult T2DM) and significance at each age averaged over- (Table S1.) or adjusted for the levels of sex (Table S2.) and pairwise comparisons of Least-square means of BMI and 95% CIs at each age in each T2DM status group averaged over levels of sex (Figure S1.) at each age in each T2DM status group and sex group combination (M = males, F-females, 1 = No adult T2DM, 2 = adult T2DM) (Figure S2.) and adjusted for log(insulin). (DOCX 549 kb) Additional file 4: Prior sensitivity analyses methods and results. (DOCX 25 kb) Additional file 5: Log-likelihood profiling method and R-code for the choice of priors of the changepoint mean. (μcp). (DOCX 14 kb) Additional file 6: Annotated RJAGS sample code to fit a type 1 trajectory divergence model with a fully unstructured 4 by 4 covariance matrix for the random growth parameters. (DOCX 14 kb) p g The authors declare that they have no competing interests. Authors’ contributions Hox J, Stoel RD. Multilevel and SEM approaches to Growth Curve modeling. In: Everitt BS, Howell DC, editors. Encyclopedia of Statistics in Behavioral Science, vol. 3. Chichester: John Wiley & Sons; 2005. 17. Stuart B, Panico L. Early-childhood BMI trajectories: evidence from a prospective, nationally representative British cohort study. Nutr Diabetes. 2016;6:e198. 45. Singer JD, Willett JB. Applied Longitudinal Data Analysis: Modeling change and event occurence. New York: Oxford University Press; 2003. 18. Finkel D, Reynolds CA, McArdle JJ, Pedersen NL. Cohort differences in trajectories of cognitive aging. J Gerontol B Psychol Sci Soc Sci. 2007;62: P286–294. 46. Fitzmaurice G, Laird NM, Ware JH. Applied Longitudinal Analysis. 2nd ed. New Jersey: John Wiley & Sons; 2011. 19. Gimeno D, Ferrie JE, Elovainio M, Pulkki-Raback L, Keltikangas-Jarvinen L, Eklund C, Hurme M, Lehtimaki T, Marniemi J, Viikari JS, et al. When do social inequalities in C-reactive protein start? A life course perspective from conception to adulthood in the Cardiovascular Risk in Young Finns Study. Int J Epidemiol. 2008;37:290–8. 47. Preacher KJ. Latent growth curve models. In: Muelle GRHRO, editor. The reviewer's guide to quantitative methods in the social sciences (pp 185– 198). London: Routledge; 2010. p. 185–98. 48. Mirman D. Growth Curve Analysis and Visualization Using R. Boca Raton: Chapman and Hall/CRC; 2014. 20. Howe LD, Tilling K, Galobardes B, Smith GD, Gunnell D, Lawlor DA. Socioeconomic differences in childhood growth trajectories: at what age do height inequalities emerge? J Epidemiol Community Health. 2012;66:143–8. 49. Mirman D, Dixon JA, Magnuson JS. Statistical and computational models of the visual world paradigm: Growth curves and individual differences. J Mem Lang. 2008;59:475–94. 21. Carles S, Charles M-A, Forhan A, Slama R, Heude B, Botton J, group Emcs. A Novel Method to Describe Early Offspring Body Mass Index (BMI) Trajectories and to Study Its Determinants. PLoS One. 2016;11:e0157766. 50. Heo M, Faith MS, Mott JW, Gorman BS, Redden DT, Allison DB. Hierarchical linear models for the development of growth curves: an example with body mass index in overweight/obese adults. Stat Med. 2003;22:1911–42. 22. Grajeda LM, Ivanescu A, Saito M, Crainiceanu C, Jaganath D, Gilman RH, Crabtree JE, Kelleher D, Cabrera L, Cama V, Checkley W. Modeling subject- specific childhood growth using linear mixed-effect models with cubic regression splines. Emerging Themes in Epidemiology. 2016;13:1. 51. Tilling K, Macdonald-Wallis C, Lawlor DA, Hughes RA, Howe LD. Modeling Childhood Growth Using Fractional Polynomials and Linear Splines. Authors’ contributions Oxford Statistical Science Series. 2002. ISBN 978-0-19-852484-7. 11. Mattsson N, Ronnemaa T, Juonala M, Viikari JS, Raitakari OT. Childhood predictors of the metabolic syndrome in adulthood. The Cardiovascular Risk in Young Finns Study. Ann Med. 2008;40:542–52. Analyzed the data: MJB SSW. Wrote the paper: MJB CGM RJT. Designed the study: MJB CGM RJT OTR. Collected the clinical data: JSAV TL MJ NHK OTR. Revised several drafts of the manuscript for critically for important intellectual content: DPB MAS JSAV, NHK, TL MJ CGM RJT OTR. Reviewed and approved the final manuscript: MJB SSW CGM RJT JSAV TL MJ NHK DPB MAS OTR. 12. Verbeke G, Molenberghs G. Linear Mixed Models for Longitudinal Data. New York: Springer; 2000. Page 14 of 15 Page 14 of 15 Page 14 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 13. Fitzmaurice G, Davidian M, Verbeke G, Molenberghs G. Longitudinal data analysis. Boca Raton: Chapman and Hall/CRC; 2008. 39. Goldstein H, Browne W, Rabash J. Multilevel modeling of medical data. Stat Med 2002: 21:3291–315. Stat Med. 2002;21:3291–315. 40. Raudenbush SW, Bryk AS. Hierarchical linear models: Applications and data analysis methods. 2. Thousand Oaks: Sage Publications; 2002. 14. Tu YK, Tilling K, Sterne JA, Gilthorpe MS. A critical evaluation of statistical approaches to examining the role of growth trajectories in the developmenta origins of health and disease. Int J Epidemiol. 2013;42:1327–39. analysis methods. 2. Thousand Oaks: Sage Publications; 2002. 41. Gelman A, Hill J. Data Analysis Using Regression and Multilevel/Hierarchical 41. Gelman A, Hill J. Data Analysis Using Regression and Mul Models. Cambridge: Cambridge University Press; 2007. 41. Gelman A, Hill J. Data Analysis Using Regression and Mul Models. Cambridge: Cambridge University Press; 2007. 15. Raitakari OT, Juonala M, Ronnemaa T, Keltikangas-Jarvinen L, Rasanen L, Pietikainen M, Hutri-Kahonen N, Taittonen L, Jokinen E, Marniemi J, et al. Cohort profile: the cardiovascular risk in Young Finns Study. Int J Epidemiol. 2008;37:1220–6. 42. Duncan TE, Duncan SC, Strycker LA. An Introduction to Latent Variable Growth Curve Modeling: Concepts, Issues, and Application, Second Edition Mahwah: Lawrence Erlbaum Associates; 2006. 43. Curran PJ, Obeidat K, Losardo D. Twelve Frequently Asked Questions About Growth Curve Modeling. J Cogn Dev. 2010;11:121–36. 16. Li L, Hardy R, Kuh D, Lo Conte R, Power C. Child-to-adult body mass index and height trajectories: a comparison of 2 British birth cohorts. Am J Epidemiol. 2008;168:1008–15. Growth Curve Modeling. J Cogn Dev. 2010;11:121–36. 44. Authors’ contributions Ann Nutr Metab. 2014;65:129–38. 23. Locascio JJ, Atri A. An overview of longitudinal data analysis methods for neurological research. Dement Geriatr Cogn Dis Extra. 2011;1:330–57. 52. Sterba S. Fitting Non linear Latent growth Curve models with Inidvidually varying Time point. Struct Equ Model Multidiscip J. 2014;21:630–47. 24. Lenth RV. Least-Squares Means.R package version 2.22. In: Book Least- Squares Means.R package version 2.22. 2016. 53. Cudeck R, du Toit SHC. A nonlinear form of quadratic regression with interpretable parameters. Multivar Behav Res. 2002;37:501–19. 25. Goodnight JH, Harvey WR. Least-Squares Means in the Fixed-Effects General Linear Models.Technical Report R-103, Book Least-Squares Means in the Fixed-Effects General Linear Models.Technical Report R-103. NC: SAS Institute Inc; 1978. 54. Harring JR, Cudeck R, du Toit SH. Fitting Partially Nonlinear Random Coefficient Models as SEMs. Multivariate Behav Res. 2006;41:579–96. 55. Frenk SM, Yang YC, Land KC. Assessing the Significance of Cohort and Period Effects in Hierarchical Age-Period-Cohort Models: Applications to Verbal Test Scores and Voter Turnout in U.S. Presidential Elections. Soc Forces. 2013;92:221–48. 26. Harvey WR. Use of the HARVEY Procedure, Book Use of the HARVEY Procedure. NC: SAS Institute Inc; 1976. 27. Liu C, Cripe TP, Kim MO. Statistical issues in longitudinal data analysis for treatment efficacy studies in the biomedical sciences. Mol Ther. 2010;18:1724–30. 56. Raudenbush SW, Chan WS. Application of a hierarchical linear model to the study of adolescent deviance in an overlapping cohort design. J Clin Consulting Psychol. 1993;61:941–51. 28. Diggle PJ, Heagerty P, L K-Y, Zeger SLO. Analysis of Longitudinal Data. 2nd ed. Oxford: Oxford University Press; 2002. 57. Yang Y, Land KC. A mixed models approach to the age-period-cohort analysis of repeated cross-section surveys, with an application to data on trends in verbal test scores. Sociol Methodol. 2006;36:75–97. 29. Kreft IGG. Are multilevel techniques necessary? An overview, including simulation studies, Book Are multilevel techniques necessary? An overview, including simulation studies. Los Angeles: California State University at Los Angeles; 1996. 58. Raudenbush SW, Chan WS. Growth Curve Analysis in Accelerated Longitudinal Designs. Criminology Penology. 1992;29:387–411. 30. Kowalchuk RK, Keselman HJ. Mixed-model pairwise multiple comparisons of repeated measures means. Psychol Methods. 2001;6:282–96. 59. Howe LD, Tilling K, Benfield L, Logue J, Sattar N, Ness AR, Smith GD, Lawlor DA. Changes in ponderal index and body mass index across childhood and their associations with fat mass and cardiovascular risk factors at age 15. PLoS One. 2010;5(12):e15186. 31. Sullivan LM. Repeated measures. Circulation. 2008;117:1238–43. Authors’ contributions Second-generation structural equation modeling with a combination of categorical and continuous latent variables: New opportunities for latent class–latent growth modeling. Washington: American Psychological Association; 2001. 107. Rogol AD, Clark PA, Roemmich JN. Growth and pubertal development in children and adolescents: effects of diet and physical activity. Am J Clin Nutr. 2000;72:521–8. 77. Congdon P. Bayesian statistical modeling. New York: Wiley; 2001. 108. Ostbye T, Malhotra R, Landerman LR. Body mass trajectories through adulthood: results from the National Longitudinal Survey of Youth 1979 Cohort (1981–2006). Int J Epidemiol. 2011;40:240–50. 78. Jacqmin-Gadda H, Commenges D, Dartigues JF. Random changepoint model for joint modeling of cognitive decline and dementia. Biometrics. 2006;62:254–60. 109. Li L, Hardy R, Kuh D, Power C. Life-course body mass index trajectories and blood pressure in mid life in two British birth cohorts: stronger associations in the later-born generation. Int J Epidemiol. 2015;44:1018–26. 79. Lange N, Carlin BP, Gelfand AE. Hierarchical Bayes models for the progression of HIV infection using longitudinal CD4 T-cell numbers. J Am Stat Assoc. 1992;87:615–26. 110. Guo SS, Huang C, Maynard LM, Demerath E, Towne B, Chumlea WC, Siervogel RM. Body mass index during childhood, adolescence and young adulthood in relation to adult overweight and adiposity: the Fels Longitudinal Study. Int J Obes Relat Metab Disord. 2000;24:1628–35. 80. Slate EH, Turnbull BW. Statistical models for longitudinal biomarkers of disease onset. Stat Med. 2000;19:617–37. 81. Ghosh P, Vaida F. Random changepoint modeling of HIV immunologic responses. Stat Med. 2007;26:2074–87. 111. Williams S, Davie G, Lam F. Predicting BMI in young adults from childhood data using two approaches to modeling adiposity rebound. Int J Obes Relat Metab Disord. 1999;23:348–54. 82. Dominicus A, Ripatti S, Pedersen NL, Palmgren J. A random change point model for assessing variability in repeated measures of cognitive function. Stat Med. 2008;27:5786–98. 112. Wen X, Kleinman K, Gillman MW, Rifas-Shiman SL, Taveras EM. Childhood body mass index trajectories: modeling, characterizing, pairwise correlations and socio-demographic predictors of trajectory characteristics. BMC Med Res Methodol. 2012;29:12–38. 83. Hall CB, Lipton RB, Sliwinski M, Stewart WF. A change point model for estimating the onset of cognitive decline in preclinical Alzheimer's disease. Stat Med. 2000;19:1555–66. 84. Hall CB, Ying J, Kuo L, Lipton RB. Bayesian and profile likelihood change point methods for modeling cognitive function over time. Comput Stat Data Anal. 2003;42:91–109. 113. Besharat Pour M, Bergstrom A, Bottai M, Magnusson J, Kull I, Moradi T. Authors’ contributions Plummer M. Penalized loss functions for Bayesian model comparison. Biostatistics. 2008;9:523–39. 97. Burnham K, Anderson D. Model selection and multimodel inference: a practical information-theoretic approach. New York: Springer; 2002. 68. Cudeck R, Klebe KJ. Multiphase mixed-effects models for repeated measures data. Psychol Methods. 2002;7:41–63. 69. Muggeo MR, Atkins DC, Gallop RJ, Dimidjan S. Segmented mixed models with random changepoints: a maximum likelihood approach with application to treatment for depression study. Stat Model. 2014;14(4):293–313. 98. Juonala M, Viikari JS, Raitakari OT. Main findings from the prospective Cardiovascular Risk in Young Finns Study. Curr Opin Lipidol. 2013;24:57–64. 99. Chivers P, Hands BP, Parker H, Beilin L, Kendall G, Bulsara M. Longitudinal modeling of body mass index from birth to 14 years. Obes Facts. 2009;2:302–10. 70. Flora DB. Specifying piecewise latent trajectory models for longitudinal data. Struct Equ Model. 2008;15:513–33. 100. Rolland-Cachera MF, Deheeger M, Maillot M, Bellisle F. Early adiposity rebound: causes and consequences for obesity in children and adults. Int J Obes (Lond). 2006;30 Suppl 4:S11–17. 71. Kohli N, Harring JR. Modeling growth in latent variables using a piecewise function. Multivar Behav Res. 2013;48:370–97. Karlberg J. On the modeling of human growth. Stat Med. 1987;6:185– 72. Willett JB, Singer JD, Martin NC. The design and analysis of longtiduinal studies of development and psychopathology in context: statistical models and methodological recommendations. Dev Psychopathol. 1998;10:395–426. 102. Silverwood RJ, De Stavola BL, Cole TJ, Leon DA. BMI peak in infancy as a predictor for later BMI in the Uppsala Family Study. Int J Obes (Lond). 2009; 33:929–37. y 73. Li L, Hardy R, Kuh D, Lo Conte R, Power C. Child-to-Adult body mass index and height ttrajectories : A comparison of 2 british cohorts. 2008. 103. Kuczmarski RJ. 2000 CDC Growth Charts for the United States: methods and development. Vital Health Statistics1. 2002;11:1–190. 74. Muggeo MR. Segmented mixed models with random changepoints in R. Working Paper. 2016. https://www.researchgate.net/publication/292629179_ Segmented_mixed_models_with_random_changepoints_in_R. 104. Lunn D, Jackson C, Best N, Thomas A, Spiegelhalter D. The BUGS Book: A Practical Introduction to Bayesian Analysis. Boca Raton: CRC press; 2012. 105. Wood S. Generalized additive models: an introduction with R. Boca Raton: CRC press; 2006. 75. Muggeo MR. Modeling temperature effects on mortality: multiple segmented relationships with common break points. Biostatistics. 2008;9:613–20. 106. Dietz WH. Critical periods in childhood for the development of obesity. Am J Clin Nutr. 1994;59:955–9. 76. Muthen B. Authors’ contributions 32. Greenhouse SW, Geisser S. On methods in the analysis of profile data. Psychometrika. 1959;24:95–112. 60. Long J, Ryoo J. Using fractional polynomials to model non-linear trends in longitudinal data. Br J Math Stat Psychol. 2010;63:177–203. 33. Huynh H, Feldt LS. Estimation of the Box Correction for Degrees of Freedom from Sample Data in Randomized Block and Split-Plot Designs. J Educ Behav Stat. 1976;1:69–82. 61. Tilling K, Sterne JAC, Wolfe CDA. Multilevel growth curve models with covariate effects: application to recovery after stroke. Stat Med. 2001;20:685–704. 34. Li D, Dye TD. Power and stability properties of resampling-based multiple testing procedures with applications to gene oncology studies. Comput Math Methods Med. 2013;2013:610297. 62. Liu X, Engel CE. Methods and Applications of Longitudinal D Elsevier Science Publishing Company Incorporated; 2015 63. Royston P, Altman DG. Regression using fractional polynomials of continuous covariates: parsimonious parametric modeling. Appl Stat-J R Stat Soc. 1994;43:29–67. 35. Westfall PJ, Young SS. Resampling-Based Multiple Testing. New York: Willey; 1993. 64. Royston P, Ambler G, Sauerbrei W. The use of fractional polynomials to model continuous risk variables in epidemiology. Int J Epidemiol. 1999;28(5): 964–74. 36. Pe'er I, Yelensky R, Altshuler D, Daly MJ. Estimation of the multiple testing burden for genomewide association studies of nearly all common variants. Genet Epidemiol. 2008;32:381–5. 37. Westfall PH, Tobias RD, Rom D, Wolfinger RD, Hochberg Y. Multiple Comparisons and Multiple Tests Using the SAS System, Book Multiple Comparisons and Multiple Tests Using the SAS System. NC: SAS Institute Inc; 1999. 65. Verbeke G. Linear mixed models for longitudinal data, Book Linear mixed models in practice. New York: Springer; 1997. 66. Lawrence FR, Blair C. Factorial invariance in preventive intervention: modeling the development of intelligence in low birth weight, preterm infants. Prev Sci. 2003;4:249–61. 38. Donohue MC, Aisen PS. Mixed model of repeated measures versus slope models in Alzheimer's disease clinical trials. J Nutr Health Aging. 2012;16:360–4. Page 15 of 15 Page 15 of 15 Buscot et al. BMC Medical Research Methodology (2017) 17:86 67. Howe LD, Tilling K, Matijasevich A, Petherick ES, Santos AC, Fairley L, Wright J, Santos IS, Barros AJ, Martin RM, et al. Linear spline multilevel models for summarising childhood growth trajectories: A guide to their application using examples from five birth cohorts. Stat Methods Med Res. 2013;4:249–61. 95. Spiegelhalter DJ, Best NG, Carlin BP. Bayesian measure of model complexity and fit. J R Stat Soc Ser B. 2002;64:583–639. 96. Authors’ contributions Age at adiposity rebound and body mass index trajectory from early childhood to adolescence; differences by breastfeeding and maternal immigration background. Pediatr Obes. 2016;30 Suppl 4:S11–17. 85. Kiuchi A, Hartigan JA, Holford TR. Change points in the series of T4 counts prior to AIDS. Biometrics. 1995;51:236–48. 114. Royston P, Altman DG. Regression using fractional polynomials of continuous covariates: parsimonious parametric modeling. J R Stat Soc, Appl Stat. 1994;43:429–67. 86. Muniz-Terrera G, Van den Hout A, Matthews FE. Random change point models: investigating cognitive decline in the presence of missing data. J Appl Stat. 2011;38:705–16. 115. McArdle JJ, Wang L. Modeling age-based turning points in longitudinal life- span growth curves of cognition. New York: Routledge/Taylor & Francis Group edn; 2008. 87. Sabin MA, Magnussen CG, Juonala M, Shield JPH, Kähönen M, Lehtimäki T, Rönnemaa T, Koskinen J, Loo B-M, Knip M, et al. Insulin and BMI as Predictors of Adult Type 2 Diabetes Mellitus. Pediatrics. 2015;135:e144–51. 116. Ram N, Grimm K. Using simple and complex growth models to articulate developmental change: Matching theory to method. Int J Behav Dev. 2007; 31:303–16. 88. McCoach DB, Kaniskan B. Using Time-Varying Covariates in Multilevel Growth Models. Front Psychol. 2010;1(17):1–12. 89. Miyazaki Y, Raudenbush SW. Tests for linkage of multiple cohorts in an accelerated longitudinal design. Psychol Methods. 2000;5:44–63. 117. Tilling K, Davies NM, Nicoli E. Associations of growth trajectories in infancy and early childhood with later childhood outcomes. Am J Clin Nutr 2011;94 Suppl 6:1808s-13s. Am J Clin Nutr. 2011;94:1808s–13s. 90. Galbraith S, Bowden J, Mander A. Accelerated longitudinal designs: An overview of modeling, power, costs and handling missing data. Stat Methods Med Res. 2014;92:221–48. 118. Magee CA, Caputi P, Iverson DC. Identification of distinct body mass index trajectories in Australian children. Pediatr Obes. 2013;8:189–98. 91. Mostafa AA, Ghorbal A. Bayesian and non-Bayesian analysis for random changepoint problem using standard computer packages. Int Mathematical Archive. 2011;2:1963–79. 119. Ekberg J, Angbratt M, Valter L, Nordvall M, Timpka T. History matters: childhood weight trajectories as a basis for planning community-based obesity prevention to adolescents. Int J Obes. 2012;36:524–8. 92. Gelman A, Carlin JB, Stern HS, Rubin DB. Bayesian Data Analysis. 2. Boca Raton: Chapman & Hall/CRC; 2004. 120. Huang DY, Lanza HI, Wright-Volel K, Anglin MD. Developmental trajectories of childhood obesity and risk behaviors in adolescence. J Adolesc. 2013;36: 139–48. 93. Gelman A. Prior distributions for variance parameters in hierarchical models. Bayesian Anal. 2006;1:515–33. 94. Buscot et al. BMC Medical Research Methodology (2017) 17:86 Authors’ contributions Gelman A, Hill J. Data Analysis Using Regression and Multilevel/Hierarchical Models. Cambridge: Cambridge University Press; 2006.
https://openalex.org/W2072082343	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0056276&type=printable	English	null	Expression of TP53 Isoforms p53β or p53γ Enhances Chemosensitivity in TP53null Cell Lines	PloS one	2,013	cc-by	9,353	Abstract The carboxy-terminal truncated p53 alternative spliced isoforms, p53b and p53c, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53b and p53c protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53b and p53c congenic H1299 was accompanied by increased p21(CIP1/WAF1), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53c. The proteasome inhibitor bortezomib substantially increased basal p53c protein level, while the level of p53b protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53c protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53b and p53c expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53b and p53c share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level. Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America Received February 3, 2012; Accepted January 12, 2013; Published February 11, 2013 Received February 3, 2012; Accepted January 12, 2013; Published February 11, 2013 Copyright: 2013 Silden et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Norwegian Cancer Society with Solveig og Ove Lund’s legacy and the Weste Authority. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscr supported by grants from the Norwegian Cancer Society with Solveig og Ove Lund’s legacy and the Western Norway Regional Health ad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following conflicts: D.M. is an employee of BergenBio. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. Abstract * E-mail: bjorn.gjertsen@med.uib.no correlation between an accumulation of full-length p53 protein and inferior outcome [7]. In breast cancer, patients with mutated p53 have more than three times increased risk of recurrence and death compared to patients with wild-type p53, but co-expression of p53c and mutated p53 leads to similar beneficial prognostic outcomes as those expressing wild-type p53 only [8]. p53b is over- expressed in renal cell carcinomas compared to normal tissue and the p53b mRNA level is significantly associated with tumor stage in these cancers [9]. In addition the p53b concentration is associated with poorly differentiated ovarian cancer and in patients with functionally active p53, expression of p53b correlated with worse recurrence-free survival [10]. Furthermore, a frequent loss of p53b and p53c has been reported in head and neck squamous carcinoma [11]. These studies suggest that p53b and p53c may influence carcinogenesis and drug-sensitivity in an organ- and ratio-dependent manner, and emphasize the need to discern their individual function and regulation. Expression of TP53 Isoforms p53b or p53c Enhances Chemosensitivity in TP53null Cell Lines 1 Hematology Section, Institute of Medicine, University of Bergen, Bergen, Norway, 2 Inserm-European Associated Laboratory Inserm U858, Department of Surgery and Molecular Oncology, University of Dundee Medical School, Dundee, Scotland, United Kingdom, 3 Institute of Biomedicine, University of Bergen, Bergen, Norway, 4 Hematology Section, Department of Internal Medicine, Haukeland University Hospital, Bergen, Norway February 2013 \| Volume 8 \| Issue 2 \| e56276 Citation: Silden E, Hjelle SM, Wergeland L, Sulen A, Andresen V, et al. (2013) Expression of TP53 Isoforms p53b or p53c Enhances Chemosensitivity in TP53null Cell Lines. PLoS ONE 8(2): e56276. doi:10.1371/journal.pone.0056276 Introduction The gene of the tumor suppressor p53 is shown to encode at least 12 different p53 protein isoforms through alternative splicing, promoter and translational initiation (reviewed in [1,2]) (Figure 1A). The differential expression of several of these isoforms has recently been established in cancer, [3,4] though their functional role is not fully understood. Their structural character- istics may indicate isoform specific mechanisms. p53b and p53c lack the oligomerization domain (Figure 1A) that is required for p53 tetramerization and thus influence p53 DNA binding and transcriptional activity. However, p53b has been shown to bind certain p53 promoters and form protein complexes with full-length p53. Furthermore, p53b and p53c is expressed in a tissue-specific manner, which may suggest diverse tissue-determined functions that may be reflected in cancer [5]. This complicates a simple understanding of p53 function, but may support future use of p53 isoform profiles in prediction of outcome and drug sensitivity in cancers. The major negative regulator of full-length p53 protein is a family of E3 ubiquitin ligases, including Mdm2, which mark p53 for proteasomal degradation through polyubiquitination of lysines on the carboxy-terminal tail of p53 [12]. These lysines are lost in the carboxy-terminally truncated p53b and p53c. Recently it has been demonstrated that the expression level of both p53b and We have previously reported that acute myeloid leukemia patients with high expression of p53b and p53c protein relative to full-length p53 protein respond better to intensive chemotherapy and have a significant longer survival after treatment [6]. Similarly, in chronic lymphocytic leukemia, there is a strong February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 1 February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org p53b or p53c Enhances Chemosensitivity Figure 1. p53 isoforms and experimental setup. (A) The human TP53 gene have alternative promoters (P1, P19, P2) and several alternat splicing sites (full-length p53 (a), b, c) generating p53 isoforms. Alternative splicing of intron 9 leads to expression of the p53 protein isoforms p5 and p53c with a truncated carboxy-terminal terminating with 10 and 15 additional amino acids, respectively. Binding site of p53 antibodies Bp53 (recognizes amino-acid region 16–25), DO-1 (amino-acids 20–25) and DO-7 (amino-acids 37–45) is indicated. NLS: nuclear localization signal. tetramerization domain. BD: basic domain. Stable expression of p53b and p53c in p53null H1299 lung carcinoma cells In order to study isoform specific biology, retroviral constructs/ vectors containing p53b, p53c or full-length p53 (Figure 1B) were generated to either retrovirally transduce or transiently transfect p53null cancer cell lines. The p53null lung carcinoma H1299 cell line was retrovirally transduced and FACS-sorted to generate stably expressing p53b, p53c or full-length p53 H1299 cell lines. Employing a retroviral vector containing the p53 isoform and a fluorescent protein marker (tdTomato), transduction and sorting of tdTomato+ cells was performed twice (Figure 1B; see Materials and Methods for experimental details). Sorted cells were evaluated for tdTomato expression by flow cytometry and fluorescence microscopy (Figure 1C), and re-sorted for tdTomato expression if needed. Considerably fewer tdTomato+ fluorescent p53c+ cells were observed following all transductions when compared to transduction efficiencies obtained with p53b+ cells (not shown). Full-length p53+ congenic H1299 cells could not be established, presumably due to the cytotoxic effect of p53 expression. p53 immunofluorescence showed a predominantly nuclear localization of p53b and both nuclear and cytoplasmic localization of p53c (Figure 1C). p53b or p53c in H1299 cells was confirmed by PCR of TP53 segment sequencing (exon 1–12) of both strands and immunoblot of p53 protein isoforms (Figure 2A; for details see Materials and Methods section). Immunoblot showed that p53b was expressed at considerably higher levels compared to p53c (Figure 2A). Immunoblots of camptothecin- and doxorubicin-treated H1299 p53b, H1299 p53c and H1299 vector control cells showed a significant decrease in p53c and p53b expression indicating degradation of p53b and p53c protein (Figure 4A). Camptothecin and doxorubicin treatment in H1299 p53b cells amplified the Bax and p21(CIP1/WAF1) response (Figure 4B,C). p53c+ cells showed a significant decrease in Bax after doxorubicin treatment while p21(CIP1/WAF1) was significantly elevated after camptothecin treatment (Figure 4B,C). Furthermore, an increase in basal level of the kinase Chk1 was detected with p53b. Chk1 was significantly reduced upon camptothecin treatment (Figure 4D) and no clear changes in Puma protein levels were seen between the groups (Figure 4E). Decreased protein levels of Tigar (TP53-induced glycolysis and apoptosis factor) protein in p53b+ and p53c+ cells after doxorubicin treatment were observed (Figure 4F). Increased basal Mdm2 levels were observed in both p53b and p53c, and reduced in all cells after chemotherapy treatments (Figure 4G). 3H-thymidine DNA-incorporation was measured to investigate the proliferative capacity of the transduced cells. Introduction (B) p53null H1299 lung carcinoma cell line were retrovirally transduced with plasmid vector containing p isoforms p53b, p53c or p53FL and a tdTomato reporter. TdTomato expression allows FACS sorting of successfully transduced cells. (C) Fluoresce PLOS ONE \| www plosone org 2 February 2013 \| Volume 8 \| Issue 2 \| e562 Figure 1. p53 isoforms and experimental setup. (A) The human TP53 gene have alternative promoters (P1, P19, P2) and several alternative splicing sites (full-length p53 (a), b, c) generating p53 isoforms. Alternative splicing of intron 9 leads to expression of the p53 protein isoforms p53b and p53c with a truncated carboxy-terminal terminating with 10 and 15 additional amino acids, respectively. Binding site of p53 antibodies Bp53-12 (recognizes amino-acid region 16–25), DO-1 (amino-acids 20–25) and DO-7 (amino-acids 37–45) is indicated. NLS: nuclear localization signal. TD: tetramerization domain. BD: basic domain. (B) p53null H1299 lung carcinoma cell line were retrovirally transduced with plasmid vector containing p53 isoforms p53b, p53c or p53FL and a tdTomato reporter. TdTomato expression allows FACS sorting of successfully transduced cells. (C) Fluorescence PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 2 p53b or p53c Enhances Chemosensitivity microscopy confirms tdTomato expression (red) of FACS sorted H1299 cells. Scale bar: 100 mm. p53 (DO-1) immunofluorescence staining (green) show mainly nuclear localization of p53b and both nuclear and cytoplasmic localization of p53c. DAPI (blue) DNA stain visualize the nucleus. Scale bar: 20 mm. doi:10 1371/journal pone 0056276 g001 microscopy confirms tdTomato expression (red) of FACS sorted H1299 cells. Scale bar: 100 mm. p53 (DO-1) immunofluorescence staining (green) show mainly nuclear localization of p53b and both nuclear and cytoplasmic localization of p53c. DAPI (blue) DNA stain visualize the nucleus. Scale bar: 20 mm. doi:10 1371/journal pone 0056276 g001 doi:10.1371/journal.pone.0056276.g001 p53c are regulated by the proteasome and although Mdm2 was found to interact with both isoforms, the degradation of both isoforms was independent of Mdm2 [13]. Therefore, p53b and p53c may be degraded through an alternative pathway [14]. Interestingly, it has been described that p53 may be degraded by the 20S proteasome by default, independently of ubiquitination. Also, the NAD(P)H quinone oxidoreductase NQO1, localized to the S20 proteasome, is able to stabilize full-length p53, thereby protecting it from ubiquitin-independent proteasomal degradation [15]. Both p53b and p53c sensitize cells to chemotherapy Both p53b and p53c sensitize cells to chemotherapy Following the observation that the isoforms may activate p53- responsive genes, we examined the response of the H1299 p53b and H1299 p53c cell lines to chemotherapeutics with a colony- formation assay. The pyrimidine antagonist arabinofuranosyl cytidine (cytarabine, Ara-C), the cytotoxic antibiotic and topo- isomerase II inhibitor doxorubicin (Dox) and the topoisomerase I inhibitor camptothecin (CPT) were tested. In both H1299 p53b and H1299 p53c, a significantly decreased colony formation compared to vector control was observed when treated with doxorubicin (Figure 3A–C). Treatment with Ara-C and campto- thecin (not shown) showed less effect on colony formation than doxorubicin, but nevertheless revealed a tendency towards reduced number of colonies. Decreased proliferation of H1299 p53b and H1299 p53c after treatment with doxorubicin and camptothecin (especially at higher dose) was also identified by a 3H-thymidine incorporation assay (Figure 3D). Transient trans- fection of p53null SAOS-2 osteosarcoma cell line with p53b- and p53c-tdTomato construct followed by treatment with 0.5 mM doxorubicin for 24 hours, also showed significant reduced 3H- thymidine DNA incorporation in p53b+ cells (data not shown). This was not seen with the p53c or full-length p53, and we propose that this lack of doxorubicin toxicity may be caused by high cell death caused by the cell death induction potential of p53c or full-length p53. Introduction hypoxic conditions also demonstrated a reduced tendency of colony formation compared with vector control (mean 6 SEM for vector control+ cells; 93613, p53b+ cells; 7264, p53c+ cells; 6864 (n = 3) (not shown)). Immunoblots illustrated an increased basal- level of p21(CIP1/WAF1) in both H1299 p53b and H1299 p53c cells, while little change in Bax was noted (Figure 2E, 2D). A p53 promoter reporter assay was performed to determine if p53b or p53c would be able to activate the genes DDI2, ARG2, CDKN1A, E2F7, SERPINE1, TP53INP1 or TP73. However, no significant activation was detected in untreated or treated (camptothecin, doxorubicin) H1299 p53b or H1299 p53c cells (not shown). However, a transient transfection of H1299 p53b and H1299 p53c cells with a 136p53 responsive element (RE) coupled to a GFP reporter gene, and subsequent analysis for GFP expression by flow cytometry, indicated that p53b and p53c both may activate this p53 consensus responsive element (Figure 2F). It is not known if p53b and p53c direct biological effects alone, or if they require full-length p53 to function [14]. Therefore, we expressed p53b and p53c individually in a p53null background using the H1299 lung carcinoma and SAOS-2 osteosarcoma cell lines. We compared functional implications of individual expressed isoforms on clonogenicity, examined the proteasomal route of degradation, and tested functional impact on chemosensitivity in vitro and tumor growth in a xenograft model. p53b and p53c were found to have a chemosensitizing effect as well as an increased tumor growth potential in vivo. Stable expression of p53b and p53c in p53null H1299 lung carcinoma cells A small, but significant reduction in proliferation was noted in H1299 p53b and H1299 p53c compared with H1299 transduction control (p,0.01; Figure 2B). The reduced proliferation of both p53b+ and p53c+ cells was also perceived by electric cell-substrate impedance sensing (ECIS) when compared to proliferation of vector control cells 30 hours after plating (p,0.05; Figure 2C). Performing a colony formation assay of H1299 p53b and H1299 p53c under February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 3 Figure 2. Basal characteristics of H1299 p53b and H1299 p53c. (A) p53 immunoblot (Bp53-12) of H1299 cells transduced with p53b- tdTomato and p53c-tdTomato construct. b-actin was used as loading control. (B) 3H-thymidine incorporation assay of H1299 p53b, H1299 p53c and H1299 control cells (tdTomato vector control). The graph shows results from four individual experiments (average of 18 wells each). The experiments have been normalized. ** P-value,0.01. Error bars: Standard Error of the Mean (SEM). (C) Cell proliferation measured by Electric-Substrate Impedance Sensing. Impedance values are normalized after initial cell stabilization and shown as ratio of normalization value. The graph shows results from four measurements of vector control cells and six measurements of p53b+ and p53c+ cells from two separate experiments. Standard error of mean is denoted by dotted lines. Highest variation in cell proliferation occurred after 30 hours after initiation, P-value,0.05 calculated by paired Students t- test. (D) and (E) show immunoblot of basal level of Bax and p21, in both p53b+ and p53c+ H1299 cells. GAPDH act as loading control, and the ratio of p21 or Bax to loading control with control value set to 1.0 is indicated. (F) Transient transfection of H1299 p53b and H1299 p53c with the 136p53RE- GFP construct and H1299 wt cells with both p53FL-construct and 136p53RE-GFP were analyzed by flow cytometry (n = 2). Results are presented as a ratio of GFP positive cells in H1299 p53b, H1299 p53c and H1299 cells transiently transfected with full-length p53 to H1299 vector control. Error bars: standard error of mean. Student’s t-test give P-value 0.053 of p53b cells versus vector control, and P-value 0.21 of p53c versus vector control. doi:10.1371/journal.pone.0056276.g002 p53b or p53c Enhances Chemosensitivity p53b or p53c Enhances Chemosensitivity Figure 2. Basal characteristics of H1299 p53b and H1299 p53c. (A) p53 immunoblot (Bp53-12) of H1299 cells transduced with p53b- tdTomato and p53c-tdTomato construct. b-actin was used as loading control. Stable expression of p53b and p53c in p53null H1299 lung carcinoma cells (B) 3H-thymidine incorporation assay of H1299 p53b, H1299 p53c and H1299 control cells (tdTomato vector control). The graph shows results from four individual experiments (average of 18 wells each). The experiments have been normalized. * P-value,0.01. Error bars: Standard Error of the Mean (SEM). (C) Cell proliferation measured by Electric-Substrate Impedance Sensing. Impedance values are normalized after initial cell stabilization and shown as ratio of normalization value. The graph shows results from four measurements of vector control cells and six measurements of p53b+ and p53c+ cells from two separate experiments. Standard error of mean is denoted by dotted lines. Highest variation in cell proliferation occurred after 30 hours after initiation, P-value,0.05 calculated by paired Students t- test. (D) and (E) show immunoblot of basal level of Bax and p21, in both p53b+ and p53c+ H1299 cells. GAPDH act as loading control, and the ratio of p21 or Bax to loading control with control value set to 1.0 is indicated. (F) Transient transfection of H1299 p53b and H1299 p53c with the 136p53RE- GFP construct and H1299 wt cells with both p53FL-construct and 136p53RE-GFP were analyzed by flow cytometry (n = 2). Results are presented as a ratio of GFP positive cells in H1299 p53b, H1299 p53c and H1299 cells transiently transfected with full-length p53 to H1299 vector control. Error bars: standard error of mean. Student’s t-test give P-value 0.053 of p53b cells versus vector control, and P-value 0.21 of p53c versus vector control. doi:10.1371/journal.pone.0056276.g002 Proteasomal inhibition increases p53c but not p53b (C) Each bar represents the number of colonies within each treatment group, and statistics have been calculated based on the untreated control within each cell subtype (p53b, p53c, wt (not transduced) or vector control (ctrl, tdTomato vector control). (D) 3H- thymidine incorporation assay of p53b+ and p53c+ cells exposed for 8 hrs to 0.5 mM Dox (n = 6 experiments), 0.4 mM camptothecin (CPT; n = 2), or vehicle (DMSO) (each separate experiments have six parallels each). Cytarabine (AraC) at 0.1 mM gave no significant response. Columns represent the ratio of treated to control (DMSO) 3H-thymidine uptake. P-value,0.05, P-value,0.01, * P-value,0.001. Error bars: standard error of mean. doi:10.1371/journal.pone.0056276.g003 Figure 3. H1299 colony formation and proliferation after chemotherapy. (A) Colony formation assay of H1299 p53b, H1299 p53c and H1299 vector control demonstrated significantly decreased colony formation in p53b+ and p53c+ cells after seven-days treatment with 25 nM doxorubicin (Dox). Insert shows a magnification of selected colonies from the doxorubicin treated plates. (B) illustrates count of treated colonies relative to colony count of untreated cells from (A). Controls within each group have been normalized to 100 colonies, and the treatment groups have been subsequently adjusted. (C) Each bar represents the number of colonies within each treatment group, and statistics have been calculated based on the untreated control within each cell subtype (p53b, p53c, wt (not transduced) or vector control (ctrl, tdTomato vector control). (D) 3H- thymidine incorporation assay of p53b+ and p53c+ cells exposed for 8 hrs to 0.5 mM Dox (n = 6 experiments), 0.4 mM camptothecin (CPT; n = 2), or vehicle (DMSO) (each separate experiments have six parallels each). Cytarabine (AraC) at 0.1 mM gave no significant response. Columns represent the ratio of treated to control (DMSO) 3H-thymidine uptake. * P-value,0.05, P-value,0.01, * P-value,0.001. Error bars: standard error of mean. doi:10.1371/journal.pone.0056276.g003 other hand, was to a higher degree degraded after treatment with nutlin-3 compared to doxorubicin, indicating that Mdm2 is involved in p53b stability (Figure 5C). To investigate whether p53c could be regulated by NQO1, as reported for full-length p53, p53c+- cells were treated with the NQO1-inhibitor dicoumarol. A dose dependent degradation of p53c after dicoumarol exposure demonstrates that NQO1 is indeed involved in the regulation of p53c. p53b was less affected by dicoumarol-treatment and only the high-dose treatment resulted in a minor reduction in the p53b protein level (Figure 5D). Proteasomal inhibition increases p53c but not p53b Proteasomal inhibition increases p53c but not p53b with bortezomib was needed in order to inhibit p53c degradation since treatment for 1 hour, 2 hours and 4.5 hours did not result in an inhibition of p53c degradation and also no difference in the expression level of p53b was observed using these time points (data not shown). To further investigate the subcellular localization of p53c and p53b after bortezomib treatment as compared to untreated cells, the cells was investigated using a higher magnification where the untreated p53c cells were overexposed in order to capture the localization (Figure 5B ii)). It is evident in both untreated and bortezomib treated cells that p53c is concentrated in the nucleus, excluded from the nucleoli and only observed diffusely in the cytoplasm. Without treatment, the p53b protein was concentrated in the nucleus, localized in the nucleoli and in a speckled nucleoplasmic pattern, in addition to diffusely in The p53c protein was considerably degraded after doxorubicin exposure (Figure 4A). To investigate the mechanism of degrada- tion, H1299 p53b and H1299 p53c cells were treated with either the proteasome inhibitor bortezomib (Bzm) or the lysosome inhibitor chloroquine (Chq). Immunoblotting demonstrated that while p53c levels were elevated considerably subsequent to treatment with bortezomib, p53b displayed stable protein levels after bortezomib and chloroquine treatment (Figure 5A). These findings was further confirmed at the subcellular level through immunofluorescence of the H1299 p53b and H1299 p53c cells (Figure 5B) where an increase in fluorescence was observed for p53c after 8 hours of bortezomib treatment while the level of p53b appeared unaffected (Figure 5B (i)). An incubation time of 8 hours February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 4 p53b or p53c Enhances Chemosensitivity Figure 3. H1299 colony formation and proliferation after chemotherapy. (A) Colony formation assay of H1299 p53b, H1299 p53c and H1299 vector control demonstrated significantly decreased colony formation in p53b+ and p53c+ cells after seven-days treatment with 25 nM doxorubicin (Dox). Insert shows a magnification of selected colonies from the doxorubicin treated plates. (B) illustrates count of treated colonies relative to colony count of untreated cells from (A). Controls within each group have been normalized to 100 colonies, and the treatment groups have been subsequently adjusted. Proteasomal inhibition increases p53c but not p53b (A) Immunoblot analysis of p53 levels in H1299 p53c and H1299 p53b cells after treatment with 0.5 mM doxorubicin (Dox) and 0.2 mM camptothecin (CPT) (8 hrs incubation time). Bar graphs represent the mean of four different experiments. * P-value,0.05, ** P-value,0.01. (B–G) Immunoblot analysis of Bax, p21, Chk1, Puma, Tigar and Mdm2 levels in H1299 p53b, H1299 p53c and H1299 vector control cells after treatment with 0.5 mM Dox and 0.2 mM CPT (8 hrs). b-actin act as loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to value of untreated vector control cells (set to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or three (Puma) different experiments. Error bars: standard error of mean. Protein levels were compared using Student’s t-test. * P-value,0.05, ** P-value,0.01. Brackets represent significant changes in basal protein levels relative to untreated H1299 vector control cells. doi:10.1371/journal.pone.0056276.g004 Figure 4. p53b and p53c protein modulation after treatment with camptothecin or doxorubicin. (A) Immunoblot analysis of p53 levels in H1299 p53c and H1299 p53b cells after treatment with 0.5 mM doxorubicin (Dox) and 0.2 mM camptothecin (CPT) (8 hrs incubation time). Bar graphs represent the mean of four different experiments. * P-value,0.05, ** P-value,0.01. (B–G) Immunoblot analysis of Bax, p21, Chk1, Puma, Tigar and Mdm2 levels in H1299 p53b, H1299 p53c and H1299 vector control cells after treatment with 0.5 mM Dox and 0.2 mM CPT (8 hrs). b-actin act as loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to value of untreated vector control cells (set to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or three (Puma) different experiments. Error bars: standard error of mean. Protein levels were compared using Student’s t-test. * P-value,0.05, ** P-value,0.01. Brackets represent significant changes in basal protein levels relative to untreated H1299 vector control cells. doi:10.1371/journal.pone.0056276.g004 treated with doxorubicin in combination with dicoumarol, which resulted in a further degradation of p53c (Figure 5E). This degradation was partly rescued by bortezomib treatment. by a significantly increased tumor growth was found of both p53c and p53b congenic H1299 cells, compared to vector control (Figure 6A). Proteasomal inhibition increases p53c but not p53b This was in contrast to in vitro findings whereby the 3H-thymidine-incorporation assay showed only a minor decrease in proliferation of p53b+ and p53c+ cells (Figure 2B). To evaluate if the growth factors present in the matrigel (for example TGF-b, epidermal growth factor, insulin-like growth factor and fibroblast growth factor) injected with the tumor cells could influence growth of p53b+ and p53c+ tumors, the same assay was performed without matrigel. However, the same pattern was observed with these tumors (Figure 6A, top left graph). Immunohistochemistry of Proteasomal inhibition increases p53c but not p53b In order to further map the degradation pathway of p53c after doxorubicin treatment, p53c+ cells were the cytoplasm. After bortezomib treatment the nuclear localization of p53b appeared more diffuse. No change in p53c and p53b stability or subcellular localization was observed after exposure to chloroquine at the time points described above (data not shown). the cytoplasm. After bortezomib treatment the nuclear localization of p53b appeared more diffuse. No change in p53c and p53b stability or subcellular localization was observed after exposure to chloroquine at the time points described above (data not shown). To investigate if Mdm2 could have a role in degradation of p53b or p53c, the H1299 cells were co-treated with the p53- Mdm2 inhibitor nutlin-3 in addition to doxorubicin (Figure 5C). The nutlin-3 treatment alone had no effect on p53c stability and the doxorubicin-induced degradation of p53c was not rescued by treatment with nutlin-3, indicating a Mdm2 independent protea- somal degradation of p53c (Figure 5C). The p53b protein, on the To investigate if Mdm2 could have a role in degradation of p53b or p53c, the H1299 cells were co-treated with the p53- Mdm2 inhibitor nutlin-3 in addition to doxorubicin (Figure 5C). The nutlin-3 treatment alone had no effect on p53c stability and the doxorubicin-induced degradation of p53c was not rescued by treatment with nutlin-3, indicating a Mdm2 independent protea- somal degradation of p53c (Figure 5C). The p53b protein, on the February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 p53b or p53c Enhances Chemosensitivity Figure 4. p53b and p53c protein modulation after treatment with camptothecin or doxorubic H1299 p53c and H1299 p53b cells after treatment with 0.5 mM doxorubicin (Dox) and 0.2 mM camptothec represent the mean of four different experiments. * P-value,0.05, ** P-value,0.01. (B–G) Immunoblot Mdm2 levels in H1299 p53b, H1299 p53c and H1299 vector control cells after treatment with 0.5 mM loading control, and the ratio of p53, Bax, p21,Chk1, Puma, Tigar or Mdm2 to loading control compared to to 1.0) is indicated. Bar graphs represent the mean of five (Bax), four (p21, Chk1, Tigar, Mdm2) or thre standard error of mean. Protein levels were compared using Student’s t-test. * P-value,0.05, ** P-value, in basal protein levels relative to untreated H1299 vector control cells. doi:10.1371/journal.pone.0056276.g004 Figure 4. p53b and p53c protein modulation after treatment with camptothecin or doxorubicin. Growth advantage of p53b and p53c expressing H1299 cells in vivo but not in vitro To examine the function of p53b and p53c protein expression in a more realistic cancer environment exhibiting hypoxia and low nutrition, we examined the growth of subcutaneous H1299 in NSG mice. Surprisingly, an early tumor growth initiation followed PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org February 2013 \| Volume 8 \| Issue 2 \| e56276 6 p53b or p53c Enhances Chemosensitivity Figure 5. Protein stabilization of p53b and p53c. (A) Immunoblot of p53c and p53b protein after treatment with 50 nM proteasome inhibit bortezomib (Bzm) or 100 mM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53c and H1299 p53b2 +bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences expression levels (106magnification), while ii) enlightens the subcellular localization (636magnification). (C) Treatment of H1299 p53c (top) and H129 p53b (bottom) with doxorubicin (0.5 mM) and nutlin-3 (10 mM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading contr Figure 5. Protein stabilization of p53b and p53c. (A) Immunoblot of p53c and p53b protein after treatment with 50 nM proteasome inhibitor bortezomib (Bzm) or 100 mM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53c and H1299 p53b2/ +bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences in expression levels (106magnification), while ii) enlightens the subcellular localization (636magnification). (C) Treatment of H1299 p53c (top) and H1299 p53b (bottom) with doxorubicin (0.5 mM) and nutlin-3 (10 mM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading control compared to the untreated vector control cells (set to 1.0) is indicated. (D) Treatment of H1299 p53c (left) and H1299 p53b (right) with NQO1-inhibitor dicoumarol for 8 hrs. Quantification of protein signal presented as ratio of p53 to b-actin in lower panel (n = 2 immunoblots). b-actin was included as a loading control. Error bars: standard error of mean. (E) Protein levels of p53c and Mdm2 after treatment of H1299 p53c with 0.5 mM doxorubicin (Dox), 200 mM dicoumarol (Dic) and 50 nM bortezomib (Bzm) for 8 hours (a representative immunoblot shown). GAPDH is included as a loading control. doi:10.1371/journal.pone.0056276.g005 Figure 5. Protein stabilization of p53b and p53c. (A) Immunoblot of p53c and p53b protein after treatment with 50 nM proteasome inhibitor bortezomib (Bzm) or 100 mM lysosome inhibitor chloroquine (Chq) for 8 hrs. (B) Immunofluorescence images of H1299 p53c and H1299 p53b2/ +bortezomib treatment (Bzm; 50 nM, 8 hrs). p53 (DO-1) immunofluorescence staining in green. DAPI (blue): nucleic acid stain. i) shows the differences in expression levels (106magnification), while ii) enlightens the subcellular localization (636magnification). (C) Treatment of H1299 p53c (top) and H1299 p53b (bottom) with doxorubicin (0.5 mM) and nutlin-3 (10 mM) for 8 hrs. GAPDH is included as a loading control. The ratio of p53 to loading control compared to the untreated vector control cells (set to 1.0) is indicated. (D) Treatment of H1299 p53c (left) and H1299 p53b (right) with NQO1-inhibitor dicoumarol for 8 hrs. Quantification of protein signal presented as ratio of p53 to b-actin in lower panel (n = 2 immunoblots). b-actin was included as a loading control. Error bars: standard error of mean. (E) Protein levels of p53c and Mdm2 after treatment of H1299 p53c with 0.5 mM doxorubicin (Dox), 200 mM dicoumarol (Dic) and 50 nM bortezomib (Bzm) for 8 hours (a representative immunoblot shown). GAPDH is included as a loading control. doi:10.1371/journal.pone.0056276.g005 February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 7 p53b or p53c Enhances Chemosensitivity Figure 6. Tumor growth of H1299 p53b and H1299 p53c cells. (A) H1299 in vivo s.c. tumors were measured in 3 independent experiments. In total: p53b+ tumors n = 17; p53c+ tumors n = 17; Control vector (Ctrl; tdTomato+) n = 20. The tumor sizes of p53b+ and p53c+ tumors were compared to ctrl tumors using Students t-test. * P-value,0.05, P-value,0.001, * P-value,0.0001. Insert (top left corner) shows tumor measurements of tumors that were injected without matrigel. Error bars: standard error of mean. (B) Immunohistochemistry of p53 (DO-7) of s.c. tumors. Top: p53- negative vector control tumor (Ctrl). Middle: p53c+ tumor. Bottom: p53b+ tumor. Scale bar left images 1 mm, right images 50 mm, middle insert 10 mm. doi:10.1371/journal.pone.0056276.g006 Figure 6. Tumor growth of H1299 p53b and H1299 p53c cells. (A) H1299 in vivo s.c. tumors were measured in 3 independent experiments. In total: p53b+ tumors n = 17; p53c+ tumors n = 17; Control vector (Ctrl; tdTomato+) n = 20. The tumor sizes of p53b+ and p53c+ tumors were compared to ctrl tumors using Students t-test. * P-value,0.05, P-value,0.001, * P-value,0.0001. Insert (top left corner) shows tumor measurements of tumors that were injected without matrigel. Error bars: standard error of mean. (B) Immunohistochemistry of p53 (DO-7) of s.c. tumors. Top: p53- negative vector control tumor (Ctrl). Middle: p53c+ tumor. Bottom: p53b+ tumor. Scale bar left images 1 mm, right images 50 mm, middle insert 10 mm. d i 10 1371/j l 0056276 006 doi:10.1371/journal.pone.0056276.g006 doi:10.1371/journal.pone.0056276.g006 p53 in tumors (Figure 6B) reflected the previous in vitro observations by immunofluorescence (Figure 1C) demonstrating strong p53b signals (bottom images) with a major localization to the nucleus (insert), and p53c signals of predominantly cytoplasmic origins. was only indicated and not significant (data not shown). However, the basal level of p21(CIP1/WAF1) protein was found to be increased by p53b and p53c expression (Figure 2E). Furthermore, the basal levels of Mdm2 were also found to be increased by p53b and p53c expression (Figure 4G). Although a p53 promoter reporter assay did not detect activation of the genes tested (data not shown, see Materials and Methods section for details), a weak positive signal by a 136p53 responsive element reporter was observed (Figure 2E). Thus, we cannot rule out p53b and p53c modulation of these p53 targeted genes at the posttranslational level or a weak gene induction not detected by the promoter reporter assay. Discussion Stably transduced H1299 cells demonstrated enhanced chemo- sensitivity to doxorubicin and camptothecin after introduction of p53b and p53c (Figures 3, 4). This was particularly evident in the colony formation assays, which reflects the total sum of all proliferative, differentiation, senescence and cell death effects [16]. Immunoblot analysis demonstrated an upregulation of p21(CIP1/ WAF1) and Bax after exposure to doxorubicin and camptothecin, apparently through a p53-independent mechanism, but with an enhanced p21(CIP1/WAF1) response in p53b+ and p53c+ cells especially in response to camptothecin. It has previously been reported that p53b may bind to the p21(CIP1/WAF1) promoter sequences [5]. p21(CIP1/WAF1) promotes cellular arrest, but may also promote apoptosis through both p53-dependent and inde- pendent mechanisms under certain cellular stress (reviewed in [17]), dependent upon upregulation of pro-apoptotic Bax [18]. This may also explain the reduced 3H-thymidine incorporation observed in CPT-treated p53b cells. We found that p53b and p53c may have an effect on an optimized p53-responsive element (Fig. 2F), but a direct activation of p21(CIP1/WAF1) promoter assay We find that resting cells stably transduced with p53c only express low levels of p53 isoform protein, corresponding with previous reports that p53c may be cytotoxic [19]. We propose that this low level is sufficient to induce muted levels of p21(CIP1/WAF1) protein and is accompanied by a tendency towards decreased proliferation and clonogenicity in vitro. Further experiments are needed to determine if the isoforms respond specifically to different chemotherapeutics. Protein levels of p53b and p53c decreased after treatment with camptothecin or doxorubicin, and p53c was particularly attenu- ated following therapy with doxorubicin. Through treatment with proteasome-, lysosome- and a NQO1-inhibitor, we suggest that the stability of p53b and p53c are differentially regulated (Figure 5). Treatment with nutlin-3, a Mdm2-binding inhibitor of the Mdm2-p53 interaction, did not result in an increased level of p53b or p53c, suggesting that Mdm2 is not a negative regulator February 2013 \| Volume 8 \| Issue 2 \| e56276 February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 8 p53b or p53c Enhances Chemosensitivity USA), bortezomib (Millenium Pharmaceuticals, Cambridge, MA, USA) and chloroquine (Sigma-Aldrich), as indicated in the text. USA), bortezomib (Millenium Pharmaceuticals, Cambridge, MA, USA) and chloroquine (Sigma-Aldrich), as indicated in the text. of p53b/c and consistent with previous reports [13,19]. However, our experiments indicate that p53c is degraded by the proteasome (Figure 5). Discussion Conflicting reports exist on proteasomal degradation of p53b and p53c [13] [20]. It is also reported that Mdm2 interact with both isoforms, but only promote ubiquitination of p53b. However, Mdm2-promoted stabilization of p53b is suggested through neddylation [13]. This could explain the decreased level of p53b we observed after treatment with nutlin-3 (Figure 5C). It was recently suggested that p53 may be proteasomally degraded by default in an Mdm2 and ubiquitin-independent manner, and that p53 is stabilized by the NAD(P)H quinone oxidoreductase NQO1 [15]. Treatment of H1299 p53c and p53b cells with the NQO1 inhibitor dicuomarol (Figure 5) resulted in a dose dependent degradation of p53c but not p53b, suggesting that NQO1 may be an important enzyme in proteasomal processing of p53c. This was further confirmed with an increased degradation of p53c after combining doxorubicin and dicuomarol treatment (Figure 5E). Together, these observations emphasize the impor- tance to further characterize the route of degradation of p53b and p53c. Transient transfection of cells H1299 (26103) or SAOS-2 cells (7.56103) were seeded in 96- well plates and transfected with the p53 isoform -tdTomato constructs or 136p53RE-GFP reporter plasmid [27,28] (a generous gift from Professor M Laiho, University of Helsinki, Finland) using FuGENE 6 Transfection Reagent (Roche Diag- nostics, GMbH, Mannheim, Germany) according to manufactur- ers instructions. In summary, we suggest that p53b and p53c individually imply functional effects in cancer cell lines. Future studies are needed to investigate if the function of p53b and p53c at defined expression levels, and to delineate the mechanisms of p53 isoform regulation in cancer growth and chemotherapy. Retroviral transduction of NCI-H1299 cells NCI-H1299 (p532/2) cells made to stably express p53b, p53c and control by retroviral transduction with the p53b-tdTomato vector, p53c-tdTomato vector, and tdTomato only vector (transfection control). Production of infectious retroviral vector particles in 293-based Phoenix A packaging cells and infection of cells were carried out as described [26]. Sequencing The p53 isoform- tdTomato construct were sequenced both prior to transduction and after transduction into H1299 cells to confirm correct TP53 isoform sequence. DNA was purified from the cells using DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA, USA), and the concentration was calculated by NanoDrop UV-Vis Spectrophotometer (Thermo Scientific, Wil- mington, DE, USA). By PCR using a forward primer for p53 (59- 39) a reverse primer for p53b (59- 39) and a reverse primer for p53c (59- 39) [5] the p53 isoform product were amplified. This product was characterized by agarose gel separation to confirm segment, and purified using ExoSAP-IT according to suppliers instructions (USB Corporation, Cleveland, Ohio USA). Sequencing PCR was performed using the abovementioned primers in addition to primers towards the middle of the p53 sequence to make sure that the sequencing reaction detects the whole segment: p53 forward primer (59 gg ccc atc ctc acc atc atc-39) and reverse primer (59-c agg gga gta cgt gca agt-39), with the BigDye Terminator v1.1 Cycle sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using DNA sequencing chromatogram Design of p53isoform-tdTomato constructs Expression vectors for p53 isoforms p53FL, p53b, and p53c were generously provided by Dr. Bourdon (University of Dundee, Scotland, UK). The p53 segments from these plasmids were cloned into an MMLV retroviral vector (L335, D.R. Micklem, unpublished) upstream of an IRES-tdTomato reporter gene. This vector drives constitutive transcription of a bicistronic mRNA comprising the cloned gene followed by an internal ribosome entry site and the red fluorescent protein tdTomato. The predicted sequence of the vector is available upon request. p53 isoforms were amplified by PCR using a forward primer containing EcoRI and SfiI sites (at gaa ttc ggc cat tac ggc cac acc ATG GAG GAG CCG CAG TCA GAT) and reverse primers containing BamHI and SfiI sites (p53FL: ta gga tcc ggc cga ggc ggc cat ata TCA GTC TGA GTC AGG CCC TTC; p53b: ga gga tcc ggc cga ggc ggc cat cta AGG CAA AGT CAT AGA ACC ATT; p53c: gc gga tcc ggc cga ggc ggc cga ata CAC GGA TAA TAT TTT CAA CTT; overlap with the p53 gene in capitals). After digestion with SfiI, the p53 isoforms were cloned into matching SfiI sites upstream of the IRES-tdTomato reporter gene. Correct inserts were confirmed by sequencing. p53 is proposed to play a role in metabolic regulation of tumor growth, including tumor responses to hypoxia and nutritional deprivation [21,22]. Therefore, we compared tumor growth in a subcutaneous xenograft. Both H1299 p53c and H1299 p53b lead to significantly increased size of tumors compared to vector control cells. A critical stage of early tumor progression is an adaptation to hypoxic and acidic conditions and a change to aerobic glycolysis to promote further tumor expansion [23,24]. Thus, the finding that p53b and p53c cells caused a significantly earlier initiation of tumor growth indicates that they play a role in the adaptive response to metabolic stress. It has recently been suggested that p53 isoforms may have more specific metabolic functions that promote metabolic adaptation [21], and tumor growth advantage in serum-nutrient starvation is suggested through modulation of p21(CIP1/WAF1) [25]. Engraftment without the use of matrigel resulted in the same tumor size profile, eliminating interference by the matrigel basement membrane matrix injected with the cells (Figure 6, insert). Further investigation of the role of the various isoforms and their response to metabolic stress may contribute to additional understanding of their role in cancer. Immunoblot analysis NCI-H1299 cells were lysed according to Shieh et al [29] before analysis with one-dimensional gel electrophoresis, according to standard procedures described in [30]. p53 protein was detected using Bp53-12 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), p21(CIP1/WAF1) protein by EA10 p21 antibody (ab16767, Abcam, Cambridge, UK), Bax by Bax 2D2 antibody (sc-20067, Santa Cruz Biotechnology), Chk1 by Chk1 2G11D5 antibody (sc-56288, Santa Cruz Biotechnology), Puma by a polyclonal Puma Antibody (#4976, Cell Signaling Technology, Inc., Danvers, MA, USA), Tigar by Tigar antibody Woody-1 (ALX-804-872C100, Enzo Life Sciences Inc., Farmingdale, NY, USA), Mdm2 by Mdm2 antibody Ab-2 (2A10) (OP115, Merck Millipore, Darmstadt, Germany), GAPDH by GAPDH mAbcam 9484 (Abcam) and b-actin was detected by anti-b-actin, sc-47778 (Santa Cruz Biotechnology). Primary antibodies were followed by secondary horseradish peroxidase conjugated mouse or rabbit antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Membranes were visualized using Pico Stable peroxide solution and luminol enhancer solution (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein bands were detected by Kodak Image Station 4000R (Eastman Kodak Company, Rochester, NY, USA), and quantified using the Carestream MI (Carestream Molecular Imaging, Woodbridge, CT, USA) analysis software. Data were exported to Excel spreadsheet and corrected for background and loading control signal, prior to statistical analysis in the Graphpad PRISM software, using the paired Student’s t-test to compare two and two groups. Cell culture and reagents g NCI-H1299 non-small cell lung carcinoma cell line (DSMZ, The German Resource Centre for Biological Material, Braunsch- weig, Germany) were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma-Aldrich, Inc. St. Louis, MO, USA) and SAOS-2 osteosarcoma cell line (DSMZ) was cultured in McCoy’s 5A medium (Sigma-Aldrich) supplemented with 10% and 15%, respectively, of heat-inactivated Fetal Bovine Serum (FBS) (PAA Laboratories GmbH, Pasching, Austria), 1% penicillin/streptomycin (PS) (Sigma-Aldrich) and 1% L-glutamine (Sigma-Aldrich) and the cells were incubated in a 5% humidified atmosphere at 37uC. Cells were treated with camptothecin (Sigma-Aldrich), doxorubicin (Pfizer Inc., New York, NY, USA), arabinofuranosyl cytidine (Sigma-Aldrich), dicoumarol (Sigma- Aldrich), nutlin-3 (Cayman Chemical Company, Ann Arbor, MI, February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 9 p53b or p53c Enhances Chemosensitivity trace viewer FinchTV v1.4.0 (Geospiza Inc., Seattle, WA, USA) and EMBOSS Pairwise Sequence Alignment Matcher. trace viewer FinchTV v1.4.0 (Geospiza Inc., Seattle, WA, USA) and EMBOSS Pairwise Sequence Alignment Matcher. concentration in 8W10E+ plates coated with cysteine, and cultured in RPMI supplemented with 10% FBS. Impedance was measured at 64 kHz every 30 seconds for two days. Flow cytometric analysis 6 1–56106 transduced H1299 cells were washed twice in 16PBS and suspended in 16PBS at a concentration of 56106 cells/ml. Stably expressing tdTomato+ cells were isolated by a Fluorescence Activated Cell Sorter (FACSAria, BD Biosciences) using a 532 nm laser. TdTomato expression was regularly evaluated on an Accuri (Accuri Cytometers Ltd., St. Ives, Cambs UK) flow cytometer to keep TdTomato expression equal between the three H1299 subclones, and cells were re-sorted if needed. 3H-thymidine DNA incorporation H1299 cells (26103 or 56103) were seeded in 96 well plates and left to settle for 20 hours prior to treatment. The cells were treated for 8 or 12 hrs, and 3H -thymidine (1 mCi per well; TRA310, Amersham International, Amersham, UK) was added the last 6 or 10 hours of the treatment period, respectively. For basal prolifer- ation experiments the cells were incubated with 3H-thymidine for 10 hours. Cells were harvested and DNA synthesis was deter- mined by 3H-thymidine incorporation assays as described [31]. Statistical analysis was performed using GraphPad PRISM (version 5.0b, GraphPad Software, Inc., La Jolla, CA, USA) software. Groups were compared using paired Student’s t-test. Luciferase reporter assay A p53 reporter assay, p53 Biomarker Set (genes DDI2, ARG2, CDKN1A, E2F7, SERPINE1, TP53INP1 or TP73) (SwitchGear Genomics, CA, USA), was transiently transfected into H1299 p53b, H1299 p53c and H1299 transduction control cells using FuGENE 6 (Roche Diagnostics, GMbH, Mannheim, Germany) according to manufacturers instructions. In a 96-well format, 2500 cells were seeded into each well and transfected after 24 hrs incubation using 0.05 mg of each individual construct. Treatments (camptothecin, doxorubicin) or vehicle control was added to the wells 12 hrs after transfection. After additional 24 hrs incubation Steady-Glo (Promega, WI, USA) was added to the wells and luminescence was measured in an Infinite M200 luminometer (TECAN, Ma¨nnedorf, Switzerland). p53 Immunofluorescence 26105 H1299 p53b, H129 A total of 500 cells were seeded per 10 cm2 dish in 8 ml of RPMI supplemented with 10% FBS, 1% penicillin/streptomycin and 1% L-glutamine. Treatment with 5 nM camptothecin, 25 nM doxorubicin, and 0.1 mM arabinofuranosyl cytidine (cytarabine, Ara-C) was initiated following 3 days of culture, and the assay terminated at day 10. Colonies were washed twice in 16PBS before 3 ml of 95% methanol was added for 3 minutes, washed in 16PBS and stained with 1:5 solution of tryphan blue stain solution (Thermo Fisher Scientific, Hanover Park, IL, USA) in ddH2O. Colony assay was also performed at hypoxic conditions with 1.5% O2. p53 Immunofluorescence 26105 H1299 p53b, H1299 p53c and H1299 control cells (tdTomato only) were grown on coverslips immersed in 0.5 ml RPMI medium with 10% FBS and 1% L-glutamine. The cells were fixed and permeabilized with 4% paraformaldehyde and ice- cold 99% methanol, respectively, before blocking with 0.5% BSA (Roche Diagnostics GmbH) in 16PBS. Next, cells were treated with primary p53 antibody (1:100 or 1:50, mouse anti-human p53, cat. 554293 BD Pharmingen) diluted in 16PBS with 0.5% BSA and incubated at 4uC over night before incubation with secondary antibody (1:5,000 of Alexa 488 goat anti-mouse (Invitrogen Molecular Probes)) diluted in 16PBS with 0.5% BSA, and incubated in the dark for 1 hour at room temperature. Last, the coverslip was washed 16PBS and mounted in 5 ml Fluoro-gel II with DAPI (Electron Microscopy Sciences, PA, USA). Images of cell fluorescence were acquired with a Zeiss Axio Observer Z1 inverted microscope (Carl Zeiss Microimaging GmbH, Germany) and analyzed by the AxioVision 4.8.2 software. General animal care and ethics statement All experiments were approved by The Norwegian Animal Research Authority under study permit number 2008 070, and conducted according to The European Convention for the Protection of Vertebrates Used for Scientific Purposes. Mice capable of engrafting human cancer cell lines NOD/LtSz- Prkdcscid/IL2Rcnull mice (abbreviated as NSG) [34,35] originally from Dr. Leonard Schultz, Jackson Laboratories, Bar Harbour, ME, USA) were housed in groups of five or less in individually ventilated cages (Techniplast, Buguggiate, Italy) and kept on a 12 hrs dark/light schedule at a constant temperature of 21uC and 50% relative humidity. The mice had continuous access to food and autoclaved water. Electric-Substrate Impedance Sensing cell proliferation assay References 26. Swift S, Lorens J, Achacoso P, Nolan GP (2001) Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell- based systems. Curr Protoc Immunol Chapter 10: Unit 10 17C. 9. Song W, Huo SW, Lu JJ, Liu Z, Fang XL, et al. (2009) Expression of p53 isoforms in renal cell carcinoma. Chin Med J (Engl) 122: 921–926. 27. Peltonen K, Colis L, Liu H, Jaamaa S, Moore HM, et al. (2010) Identification of novel p53 pathway activating small-molecule compounds reveals unexpected similarities with known therapeutic agents. PLoS ONE 5: e12996. 10. Hofstetter G, Berger A, Fiegl H, Slade N, Zoric A, et al. (2010) Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer. Oncogene 29: 1997–2004. 28. el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B (1992) Definition of a consensus binding site for p53. Nat Genet 1: 45–49. 11. Boldrup L, Bourdon JC, Coates PJ, Sjostrom B, Nylander K (2007) Expression of p53 isoforms in squamous cell carcinoma of the head and neck. Eur J Cancer 43: 617–623. 29. Shieh SY, Ikeda M, Taya Y, Prives C (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91: 325–334. 30. Wergeland L, Sjoholt G, Haaland I, Hovland R, Bruserud O, et al. (2007) Pre- apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells. Mol Cancer 6: 33. 12. Rodriguez MS, Desterro JM, Lain S, Lane DP, Hay RT (2000) Multiple C- terminal lysine residues target p53 for ubiquitin-proteasome-mediated degrada- tion. Mol Cell Biol 20: 8458–8467. 31. Tronstad KJ, Berge K, Flindt EN, Kristiansen K, Berge RK (2001) Optimization of methods and treatment conditions for studying effects of fatty acids on cell growth. Lipids 36: 305–313. 13. Camus S, Menendez S, Fernandes K, Kua N, Liu G, et al. (2012) The p53 isoforms are differentially modified by Mdm2. Cell Cycle 11: 1646–1655. g p 32. Giaever I, Keese CR (1993) A morphological biosensor for mammalian cells. Nature 366: 591–592. y y y 14. Marcel V, Dichtel-Danjoy ML, Sagne C, Hafsi H, Ma D, et al. (2011) Biological functions of p53 isoforms through evolution: lessons from animal and cellular models. Cell Death Differ 18: 1815–1824. 33. References 19. Khoury MP, Bourdon JC (2011) p53 Isoforms: An Intracellular Microprocessor? Genes Cancer 2: 453–465. 1. Khoury MP, Bourdon JC (2010) The isoforms of the p53 protein. Cold Spring Harb Perspect Biol 2: a000927. 1. Khoury MP, Bourdon JC (2010) The isoforms of the p53 protein. Cold Spring Harb Perspect Biol 2: a000927. 2. Marcel V, Perrier S, Aoubala M, Ageorges S, Groves MJ, et al. (2010) Delta160p53 is a novel N-terminal p53 isoform encoded by Delta133p53 transcript. FEBS Lett. 584:4463–4468. 20. Graupner V, Schulze-Osthoff K, Essmann F, Janicke RU (2009) Functional characterization of p53beta and p53gamma, two isoforms of the tumor suppressor p53. Cell Cycle 8: 1238–1248. pp p y 21. Maddocks OD, Vousden KH (2011) Metabolic regulation by p53. J Mol Med (Berl) 89: 237–245. 3. Bourdon JC (2007) p53 and its isoforms in cancer. Br J Cancer 97: 277–282. 4. Wei J, Zaika E, Zaika A (2012) p53 Family: Role of Protein Isoforms in Human Cancer. J Nucleic Acids 2012: 687359. 22. Lane D, Levine A (2010) p53 Research: the past thirty years and the next thirty years. Cold Spring Harb Perspect Biol 2: a000893. 5. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, et al. (2005) p53 isoforms can regulate p53 transcriptional activity. Genes Dev 19: 2122–2137. 23. Dang CV, Semenza GL (1999) Oncogenic alterations of metabolism. Trends Biochem Sci 24: 68–72. 6. Anensen N, Hjelle SM, Van Belle W, Haaland I, Silden E, et al. (2012) Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia. Oncogene, 31:1533–1545. 24. Gatenby RA, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4: 891–899. therapy response in acute myeloid leukemia. Oncogene, 31:1533–1 25. Braun F, Bertin-Ciftci J, Gallouet AS, Millour J, Juin P (2011) Serum-nutrient starvation induces cell death mediated by Bax and Puma that is counteracted by p21 and unmasked by Bcl-x(L) inhibition. PLoS One 6: e23577. py p y g 7. Sellmann L, Carpinteiro A, Nuckel H, Scholtysik R, Siemer D, et al. (2012) p53 protein expression in chronic lymphocytic leukemia. Leuk Lymphoma. 53.1282– 1288 y p21 and unmasked by Bcl-x(L) inhibition. PLoS One 6: e2357 8. Bourdon JC, Khoury MP, Diot A, Baker L, Fernandes K, et al. (2011) p53 mutant breast cancer patients expressing p53gamma have as good a prognosis as wild-type p53 breast cancer patients. Breast Cancer Res 13: R7. Acknowledgments We thank Wenche Eilifsen, Siv Lise Bedringaas and Edith Fick for expert technical assistance and Dr. Per Øyvind Enger for generously providing the ECIS instrument. Subcutaneous tumor model 56106 NCI-H1299 p53b-tdTomato, NCI-H1299 p53c-tdTo- mato, NCI-H1299 tdTomato and NCI-H1299 wt cells were suspended in 100 ml sterile 16PBS with 10% Matrigel (BD MatrigelTM Basement Membrane Matrix, BD Biosciences) for s.c. inoculation, and injected with a 28 G syringe. Animals were monitored closely for tumor growth, and tumor volume was measured twice weekly using digital calipers. The mice were euthanized when tumor size reached 1000 mm3. 56106 NCI-H1299 p53b-tdTomato, NCI-H1299 p53c-tdTo- mato, NCI-H1299 tdTomato and NCI-H1299 wt cells were suspended in 100 ml sterile 16PBS with 10% Matrigel (BD MatrigelTM Basement Membrane Matrix, BD Biosciences) for s.c. inoculation, and injected with a 28 G syringe. Animals were monitored closely for tumor growth, and tumor volume was measured twice weekly using digital calipers. The mice were euthanized when tumor size reached 1000 mm3. Histology Tumor samples collected following euthanasia were transferred to a tube containing 4% formalin for paraffin embedding, cryosectioning and subsequent immunohistochemistry of the samples. Sections were stained with hematoxylin and eosin (H&E) and p53 (DO-7, Dako) (appearing as brown stain). Results Author Contributions Conceived and designed the experiments: ES BTG SMH LW VA EM JCB. Performed the experiments: ES SMH LW AS VA. Contributed with vector-design: DM JCB. Revised the paper: ES BTG SMH EM LW VA AS. Analyzed the data: ES SMH LW VA BTG. Contributed reagents/ materials/analysis tools: JCB DM. Wrote the paper: ES BTG. Electric-Substrate Impedance Sensing cell proliferation assay Growth potential of the H1299 cells were assessed using by Electric-Substrate Impedance Sensing (ECIS; Applied Biophysics, Troy, NY) [32,33]. 26104 cells were seeded at 56104/ml February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 10 p53b or p53c Enhances Chemosensitivity were analyzed by standard light microscopy (Olympus BX51, Olympus Corp., Tokyo 163-0914, Japan). were analyzed by standard light microscopy (Olympus BX51, Olympus Corp., Tokyo 163-0914, Japan). References Wegener J, Keese CR, Giaever I (2000) Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 259: 158–166. 15. Asher G, Tsvetkov P, Kahana C, Shaul Y (2005) A mechanism of ubiquitin- independent proteasomal degradation of the tumor suppressors p53 and p73. Genes Dev 19: 316–321. p 34. McCormack E, Bruserud O, Gjertsen BT (2005) Animal models of acute myelogenous leukaemia - development, application and future perspectives. Leukemia 19: 687–706. 16. Brown JM, Attardi LD (2005) The role of apoptosis in cancer development and treatment response. Nat Rev Cancer 5: 231–237. 35. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, et al. (2005) Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174: 6477–6489. 17. Abbas T, Dutta A (2009) p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 9: 400–414. 18. Gartel AL (2005) The conflicting roles of the cdk inhibitor p21(CIP1/WAF1) in apoptosis. Leuk Res 29: 1237–1238. February 2013 \| Volume 8 \| Issue 2 \| e56276 PLOS ONE \| www.plosone.org 11
https://openalex.org/W2574986000	https://iris.unife.it/bitstream/11392/2371194/1/PhysRevLett.118.111803.pdf	Latin	null	Observation of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msubsup><mml:mi>B</mml:mi><mml:mi>c</mml:mi><mml:mo>+</mml:mo></mml:msubsup><mml:mo stretchy="false">→</mml:mo><mml:msup><mml:mi>D</mml:mi><mml:mn>0</mml:mn></mml:msup><mml:msup><mml:mi>K</mml:mi><mml:mo>+</mml:mo></mml:msup></mml:math> Decays	Physical review letters	2,017	cc-by	8,548	DOI: 10.1103/PhysRevLett.118.111803 The decay Bþ →¯D0πþ is used for normalization. Since the ratio of production rates for Bþc and Bþ mesons within the LHCb acceptance, fc=fu, is unknown, the measured observables are The Bþc meson is the only ground-state meson consisting of two heavy quarks of different flavor, namely a ¯b and a c quark. As such, its formation in pp collisions is suppressed relative to the lighter B mesons. Unlike B0, Bþ and B0s mesons, the b-quark decay accounts for only ∼20% of the Bþc width [1]. Around 70% of its width is due to c-quark decays, where the c-quark transition has been observed with Bþc →B0sπþ decays [2]. This leaves ∼10% for ¯bc →Wþ →¯qq annihilation amplitudes, which can be unambiguously probed in charmless final states. No charm- less Bþc decays have been reported to date, although searches show an indication at the level of 2.4 standard deviations (σ) [3]. RDðÞ0h ¼ fc fu × BðBþc →DðÞ0hþÞ; ð1Þ ð1Þ where h is π or K and BðBþc →DðÞ0hþÞ represents the corresponding branching fraction. The four observables are measured with a simultaneous fit to the D0πþ and D0Kþ invariant mass distributions. Theoretical estimates for BðBþc →J=ψπþÞ range from 6.0 × 10−4 [9] to 1.8 × 10−3 [10], which implies fc=fu values in the range 0.004–0.012 using the production ratio measured in Ref. [5] and the branching fraction BðBþ →J=ψKþÞ [11]. Estimates for BðBþc →D0KþÞ vary from 1.3 × 10−7 [6] to 6.6 × 10−5 [8], while estimates for BðBþc →D0πþÞ vary from 2.3 × 10−7 [6] to 2.3 × 10−6 [7]. Using Eq. (1), the expectation for RD0π is seen to cover the range 9 × 10−10 −3 × 10−8, while RD0K covers the range 5 × 10−10 −8 × 10−7. To test QCD factorization and explore the new physics potential of Bþc decays, rarer decays such as suppressed tree-level b →u transitions and b →s loop-mediated (penguin) decays can be studied, where the charm quantum number remains unchanged. The simplest decay is the color-allowed Bþc →DðÞ0πþ decay, illustrated in Fig. 1(a). The expected branching fraction for this decay is a factor jVub=Vcbj2 ≈0.007 lower than the favored b →c and color-allowed Bþc →J=ψπþ decay [4,5], placing this mode at the limit of sensitivity with current LHCb data. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) Full author list given at the end of the article. Observation of Bþc →D0Kþ Decays R. Aaij et al. (LHCb Collaboration) j (LHCb Collaboration) ( ) (Received 7 January 2017; revised manuscript received 17 February 2017; published 15 March 2017) (Received 7 January 2017; revised manuscript received 17 February 2017; published 15 March 2017) Using proton-proton collision data corresponding to an integrated luminosity of 3.0 fb−1, recorded by the LHCb detector at center-of-mass energies of 7 and 8 TeV, the Bþc →D0Kþ decay is observed with a statistical significance of 5.1 standard deviations. By normalizing to Bþ →¯D0πþ decays, a measurement of the branching fraction multiplied by the production rates for Bþc relative to Bþ mesons in the LHCb acceptance is obtained, RD0K ¼ ðfc=fuÞ × BðBþc →D0KþÞ ¼ ð9.3þ2.8 −2.5 0.6Þ × 10−7, where the first uncertainty is statistical and the second is systematic. This decay is expected to proceed predominantly through weak annihilation and penguin amplitudes, and is the first Bþc decay of this nature to be observed. DOI: 10.1103/PhysRevLett.118.111803 DOI: 10.1103/PhysRevLett.118.111803 A two-dimensional optimization is performed to determine the second stage BDT requirements for the two-body and four-body modes, where the signal S is compared to the number of background events B in data using a figure of merit S=ð ﬃﬃﬃﬃ B p þ 3=2Þ [25]. The value of B is determined within 50 MeV=c2 of the known Bþc mass. No PID information is used in the BDT training, so that the efficiency for B →D0Kþ and B →D0πþ decays is similar. The use of BDTs to select signal decays was validated by comparing the efficiency of the BDT requirements for Bþ →¯D0πþ decays in data and simulation, where close agreement was found across a wide range of BDT cuts. The purity of the selection is further improved by requiring all kaons and pions in the D0 decay to be identified with a PID selection that has an efficiency of about 85% per particle. RDðÞ0h ¼ N ðBþc →DðÞ0hþÞ N ðBþ →¯D0πþÞ × BðBþ →¯D0πþÞ × ξ; ð2Þ ð2Þ where N ðBþc →DðÞ0hþÞ represents the Bþc →DðÞ0hþ yield, N ðBþ →¯D0πþÞ represents the yield of Bþ →¯D0πþ normalization decays, BðBþ →¯D0πþÞ is the normalization mode branching fraction [11], and ξ is the ratio of efficiencies for reconstructing and selecting Bþ and Bþc mesons decaying to these final states. The LHCb detector is a single-arm forward spectrometer covering the pseudorapidity range 2 < η < 5, described in detail in Refs. [12,13]. The detector allows the reconstruction of both charged and neutral particles. For this analysis, the ring-imaging Cherenkov (RICH) detectors [14], distinguishing pions, kaons, and protons, are particu- larly important. Simulated events are produced using the software described in Refs. [15–22]. After reconstruction of the D0 meson candidate, the same selection is applied to the Bþc and Bþ candidates. The invariant mass of the D0 candidate must be within 25 MeV=c2 of its known value [11]. The other hadron originating from the B decay must have transverse momen- tum (pT) in the range 0.5–10.0 GeV=c and momentum (p) in the range 5–100 GeV=c, ensuring that the track is within the kinematic coverage of the RICH detectors that provide particle identification (PID) information. A kinematic fit is performed to each decay chain [23], with vertex constraints applied to both the B and D vertices, and the D0 candidate mass constrained to its known value. DOI: 10.1103/PhysRevLett.118.111803 However, this expectation may be enhanced by penguin and weak annihilation amplitudes, which will be more pronounced in the Bþc →DðÞ0Kþ mode [see Fig. 1(b) and 1(c)]. This motivates a search for the Bþc →DðÞ0Kþ and Bþc → DðÞ0πþ decays, particularly as the branching fraction estimates in the literature vary considerably [6–8]. This Letter reports a search for Bþc →D0πþ and Bþc → D0Kþ decays in pp collision data corresponding to FIG. 1. Tree (a), penguin (b), and weak annihilation (c) diagrams for the decays studied. In each case, the meson appearing before the comma denotes the favored decay. 111803-1 © 2017 CERN, for the LHCb Collaboration 111803-1 0031-9007=17=118(11)=111803(9) week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) combinations from data with invariant mass in the range 5900–7200 MeV=c2. For the first BDT, background can- didates with a D0 invariant mass more than 30 MeV=c2 away from the known D0 mass are used. In the second BDT, background candidates with a D0 invariant mass within 25 MeV=c2 of the known D0 mass are used. A loose cut on the classifier response of the first BDT is applied before training the second one. This focuses the second BDT training on backgrounds enriched with fully reconstructed D0 mesons. integrated luminosities of 1.0 and 2.0 fb−1 taken by the LHCb experiment at center-of-mass energies of 7 and 8 TeV, respectively, where the D0 meson is reconstructed in the Cabibbo-favored final states D0 →K−πþ or D0 →K−πþπ−πþ (inclusion of charge-conjugate proc- esses is implied throughout). Partially reconstructed Bþc →ðD0 →D0fπ0; γgÞhþ decays, where the neutral particle indicated in braces is not considered in the invariant mass calculation, are treated as additional signal channels. The number of Bþc decays is normalized by comparison to the number of Bþ →½ ¯D0 →Kþπ−ðπþπ−Þπþ decays. A fit to the invariant mass distribution of DðÞ0hþ candidates in the range 5800–6900 MeV=c2 enables a measurement of The inputs to all BDTs include properties of each particle (p, pT, and the IP significance) and additional properties of the B and D0 candidates (decay time, flight distance, decay vertex quality, radial distance between the decay vertex and the PV, and the angle between the reconstructed momentum vector and the line connecting the production and decay vertices). DOI: 10.1103/PhysRevLett.118.111803 The red solid curve illustrates Bþc →D0Kþ decays, the red dashed curve illustrates Bþc →D0Kþ decays, the green dashed curve represents Bþc →D0πþ decays, the gray shaded region represents partially reconstructed background decays, the cyan dashed line represents the combinatorial background, and the total PDF is displayed as a blue solid line. The small drop visible in the total Bþc →DðÞ0πþ PDF around the Bþc mass arises from the fact that the fit finds a small negative value for the Bþc →D0πþ yield. Partially reconstructed decays form a background at invariant masses lower than that of the signal peak. This background is described by a combination of parametric PDFs, with yield and shape parameters that are allowed to vary. A linear function describes the combinatorial back- ground. The yield of Bþ →¯D0Kþ decays, where the kaon is misidentified as a pion, is fixed using a simultaneous fit to correctly identified Bþ →¯D0Kþ events. Using a data- driven analysis of approximately 20 million Dþ decays reconstructed as Dþ →D0πþ, D0 →K−πþ, the proba- bility of kaon misidentification is determined to be 32%. The invariant mass fits to Bþ →ð ¯D0 →Kþπ−Þπþ and Bþ →ð ¯D0 →Kþπ−πþπ−Þπþ decays determine a total observed yield N ðBþ →¯D0πþÞ ¼ 309462 550. two particles are missed, with shape parameters taken from simulated Bþ →D0πþπ0 decays and scaled to account for the different momenta of the decay products in Bþc and Bþ decays. y ð Þ To measure N ðBþc →DðÞ0hþÞ, a simultaneous invariant mass fit to the Bþc →D0πþ and Bþc →D0Kþ samples is performed in the region 5800–6900 MeV=c2. Two-body and four-body D -decay candidates are included, where a Gaussian PDF describes the fully reconstructed Bþc signals. The mean of this Gaussian is fixed to the known Bþc mass [11]. The width of the Bþc →D0πþ PDF is taken from a fit to suppressed Bþ →ð ¯D0 →πþK−Þπþ decays, scaled up by a factor 1.3 to account for the difference in momenta of the decay products in Bþc →D0πþ and Bþ →¯D0πþ decays. The width of the Bþc →D0Kþ peak is related to that of Bþc →D0πþ decays by the ratio of the widths of the Bþ →¯D0Kþ and Bþ →¯D0πþ peaks found in the normali- zation mode fits. DOI: 10.1103/PhysRevLett.118.111803 The Bþ (Bþc ) meson candidates with an invariant mass in the interval 5080–5900 MeV=c2 (5800–6900 MeV=c2) and with a proper decay time above 0.2 ps are retained. Each B candidate is associated with the primary vertex (PV) to which it has the smallest impact parameter (IP), defined as the distance of closest approach of the candidate’s trajec- tory to a given PV. p Simulated signal samples are used to evaluate the relative efficiency for selecting Bþc and Bþ decays. The efficiency ratio is ξ ¼ ϵðBþÞ=ϵðBþc Þ, where ϵðBþÞ and ϵðBþc Þ re- present the combined efficiencies of detector acceptance, trigger, reconstruction, and offline selection. As both Bþc and Bþ mesons are required to decay to the same final-state particles, differences between ϵðBþÞ and ϵðBþc Þ arise due to differences in their masses and lifetimes. The Bþc meson lifetime is ð0.507 0.009Þ ps, which is 3.2 times shorter than that of the Bþ meson [11]. This results in a lower Bþc efficiency relative to Bþ by a factor 2.4, due to the proper decay time cut. The Bþc meson is heavier than the Bþ, which reduces by a factor 1.3 the fraction of Bþc decays in which all final-state particles are within the detector acceptance. However, as the BDTs are trained specifically on Bþc simulated decays, the offline selection efficiency is lower for Bþ decays, contributing a relative efficiency of 0.94. Overall, the efficiency ratio is ξ ¼ 3.04 0.16 ð2.88 0.15Þ for the two-body (four-body) D0 decay. The uncertainties are systematic, arising from the use of Two boosted decision tree (BDT) discriminators [24] are used for further background suppression. They are trained using simulated Bþc →½D0 →K−πþðπþπ−Þhþ signal decays and a sample of wrong-sign Kþπ−ðπþπ−Þhþ 111803-2 111803-2 ] 2 c ) [MeV/ ± h 0 D ( m 5800 6000 6200 6400 6600 6800 5 10 15 20 25 ± π 0 D → ± c B LHCb ) 2 c Candidates / (40 MeV/ 5 10 15 20 25 ± K 0 D → ± c B LHCb FIG. 2. Results of the simultaneous fit to the D0Kþ (top plot) and D0πþ (bottom plot) invariant mass distributions in the Bþc mass region, including the D0 →K−πþ and D0 →K−πþπ−πþ final states. Inclusion of the charge conjugate decays is implied. DOI: 10.1103/PhysRevLett.118.111803 The red solid curve illustrates Bþc →D0Kþ decays, the red dashed curve illustrates Bþc →D0Kþ decays, the green dashed curve represents Bþc →D0πþ decays, the gray shaded region represents partially reconstructed background decays, the cyan dashed line represents the combinatorial background, and the total PDF is displayed as a blue solid line. The small drop visible in the total Bþc →DðÞ0πþ PDF around the Bþc mass arises from the fact that the fit finds a small negative value for the Bþc →D0πþ yield. V I E W L E T T E R S week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) ] 2 c ) [MeV/ ± h 0 D ( m 5800 6000 6200 6400 6600 6800 5 10 15 20 25 ± π 0 D → ± c B LHCb ) 2 c Candidates / (40 MeV/ 5 10 15 20 25 ± K 0 D → ± c B LHCb 17 MARCH 2017 finite simulated samples and possible mismodeling of the simulated Bþc lifetime and production kinematics. To measure N ðBþ →¯D0πþÞ, binned maximum like- lihood fits to the invariant mass distributions of selected Bþ candidates are performed, where separate fits are employed for the two-body and four-body ¯D0 modes. The total probability density function (PDF) is built from four contributions. The Bþ →¯D0πþ decays are modeled by the sum of two modified Gaussian functions with asym- metric power-law tails and an additional Gaussian function as used in Ref. [26], all of which share a common peak position. Misidentified Bþ →¯D0Kþ candidates have an incorrect mass assignment and form a distribution dis- placed downward in mass, with a tail extending to lower invariant masses. They are modeled by the sum of two modified Gaussian PDFs with low-mass power-law tails. All PDF parameters are allowed to vary, with the exception of the tail parameters which are fixed to the values found in simulation. FIG. 2. Results of the simultaneous fit to the D0Kþ (top plot) and D0πþ (bottom plot) invariant mass distributions in the Bþc mass region, including the D0 →K−πþ and D0 →K−πþπ−πþ final states. Inclusion of the charge conjugate decays is implied. DOI: 10.1103/PhysRevLett.118.111803 π 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb K 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb decay. The value of RD0K is at the high end of theoretical predictions [6–8] and an expectation based on the observed Bþc →J=ψπþ yield at LHCb [28]. From Refs. [5] and [11], RJ=ψπ ¼ ð7.0 0.3Þ × 10−6 is obtained. As fc=fu is common to both RJ=ψπ and RD0K, the ratio of branching fractions is measured to be BðBþc →D0KþÞ= BðBþc →J=ψπþÞ ¼ 0.13 0.04 0.01 0.01, where the first uncertainty is statistical, the second is systematic, and the third comes from RJ=ψπ. FIG. 3. CLs p-value distributions for the RD0h observables. The dashed line represents the expected CLs values, where the 1σ and 2σ contours are indicated by the green (dark) and yellow (light) shaded regions, respectively. Upper limits are determined by the points at which the observed CLsþb p-values (black points connected by straight lines) fall below 5% (red solid line). Also displayed are the corresponding CLs ¼ CLsþb=CLb values (blue points connected by straight dotted lines). The absence of the Bþc →D0πþ mode shows that the Bþc →D0Kþ amplitude is not dominated by the tree-level b →u transition shown in Fig. 1(a), but rather by the penguin 1(b) and/or weak annihilation 1(c) diagrams. This result constitutes the first observation of such amplitudes in the decay of a Bþc meson. We express our gratitude to our colleagues in the CERN accelerator departments for the excellent performance of the LHC. We thank the technical and administrative staff at the LHCb institutes. We acknowledge support from CERN and from the national agencies: CAPES, CNPq, FAPERJ and FINEP (Brazil); NSFC (China); CNRS/IN2P3 (France); BMBF, DFG and MPG (Germany); INFN (Italy); FOM and NWO (Netherlands); MNiSW and NCN (Poland); MEN/IFA (Romania); MinES and FASO (Russia); MinECo (Spain); SNSF and SER (Switzerland); NASU (Ukraine); STFC (United Kingdom); NSF (USA). We acknowledge the computing resources that are provided by CERN, IN2P3 (France), KIT and DESY (Germany), INFN (Italy), SURF (Netherlands), PIC (Spain), GridPP (United Kingdom), RRCKI and Yandex LLC (Russia), CSCS (Switzerland), IFIN-HH (Romania), CBPF (Brazil), PL-GRID (Poland) and OSC (USA). We are indebted to the communities behind the multiple open source software packages on which we depend. DOI: 10.1103/PhysRevLett.118.111803 Partially reconstructed Bþc →D0hþ signal decays are modeled using a combination of para- metric PDFs, with yield and shape parameters that are allowed to vary. These decays contribute at lower invariant masses than the fully reconstructed signal decays, as a result of not considering the natural particle in the invariant mass calculation. An additional background component at low invariant mass is included to describe Bþc decays where Misidentified Bþc →D0πþðKþÞ decays in the Bþc →D0KþðπþÞ sample are modeled using the same PDFs as the normalization fits, with widths and peak positions scaled for the decay momentum difference. These shapes are fixed in the fit. Signal decays are split into separate samples with correct and incorrect kaon identification, with a kaon misidentification rate of 7% and a corresponding pion identification efficiency of 91% fixed using the data-driven Dþ analysis described above. An exponential function describes the combinatorial back- ground, which is fitted independently in the Bþc →D0πþ and Bþc →D0Kþ samples. The combinatorial yields, signal yields, and partially reconstructed Bþc →D0hþfπ0g and Bþc →D0hþfπ0g background yields are all free to vary. The fit to data is shown in Fig. 2, where a Bþc →D0Kþ yield of 20 5 events is found. All other signal yields are consistent with zero. To test the significance of each signal yield, CLs hypothesis tests [27] are performed. Upper limits at 95% confidence level (C.L.) are determined by the point 111803-3 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) π 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb K 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb FIG. 3. CLs p-value distributions for the RD0h observables. The dashed line represents the expected CLs values, where the 1σ and 2σ contours are indicated by the green (dark) and yellow (light) shaded regions, respectively. Upper limits are determined by the points at which the observed CLsþb p-values (black points connected by straight lines) fall below 5% (red solid line). Also displayed are the corresponding CLs ¼ CLsþb=CLb values (blue points connected by straight dotted lines). [1] I. P. Gouz, V. V. Kiselev, A. K. Likhoded, V. I. Romanovsky, and O. P. Yushchenko, Prospects for the Bc studies at LHCb, Phys. At. Nucl. 67, 1559 (2004). y [2] R. Aaij et al. (LHCb Collaboration), Observation of the Decay Bþc →B0sπþ, Phys. Rev. Lett. 111, 181801 (2013). [3] R. Aaij et al. (LHCb Collaboration), Study of Bþc decays to the KþK−πþ final state and evidence for the decay Bþc →χc0πþ, Phys. Rev. D 94, 091102 (2016). [1] I. P. Gouz, V. V. Kiselev, A. K. Likhoded, V. I. Romanovsky, and O. P. Yushchenko, Prospects for the Bc studies at LHCb, Phys. At. Nucl. 67, 1559 (2004). [2] R. Aaij et al. (LHCb Collaboration), Observation of the Decay Bþc →B0sπþ, Phys. Rev. Lett. 111, 181801 (2013). [3] R. Aaij et al. (LHCb Collaboration), Study of Bþc decays to the KþK−πþ final state and evidence for the decay Bþc →χc0πþ, Phys. Rev. D 94, 091102 (2016). DOI: 10.1103/PhysRevLett.118.111803 This is the first observation of the Bþc →D0Kþ 111803-4 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) [18] D. J. Lange, The EvtGen particle decay simulation package, Nucl. Instrum. Methods Phys. Res., Sect. A 462, 152 (2001). [4] V. M. Abazov et al. (D0 Collaboration), Observation of the Bc Meson in the Exclusive Decay Bc →J=ψπ, Phys. Rev. Lett. 101, 012001 (2008). [5] R. Aaij et al. (LHCb Collaboration), Measurement of Bþc Production at ﬃﬃﬃs p ¼ 8 TeV, Phys. Rev. Lett. 114, 132001 (2015). [19] J. Allison et al. (Geant4 Collaboration), Geant4 develop- ments and applications, IEEE Trans. Nucl. Sci. 53, 270 (2006). ( ) [6] H. F. Fu, Y. Jiang, C. S. Kim, and G.-L. Wang, Probing non- leptonic two-body decays of Bc meson, J. High Energy Phys. 06 (2011) 15. [20] S. Agostinelli et al. (Geant4 Collaboration), Geant4: A simulation toolkit, Nucl. Instrum. Methods Phys. Res., Sect. A 506, 250 (2003). y ( ) [7] D.-S. Du and Z.-T. Wei, Space-like Penguin effects in Bc decays, Eur. Phys. J. C 5, 705 (1998). [21] M. Clemencic, G. Corti, S. Easo, C. R Jones, S. Miglioranzi, M. Pappagallo, and P. Robbe, The LHCb simulation application, Gauss: Design, evolution and experience, J. Phys. Conf. Ser. 331, 032023 (2011). [8] J. Zhang and X.-Q. Yu, Branching ratio and CP violation of Bc →DK decays in the perturbative QCD approach, Eur. Phys. J. C 63, 435 (2009). [22] C.-H. Chang, C. Driouichi, P. Eerola, and X.-G. Wu, BCVEGPY: An event generator for hadronic production of the Bc meson, Comput. Phys. Commun. 159, 192 (2004). [9] D. Ebert, R. N. Faustov, and V. O. Galkin, Weak decays of the Bc meson to charmonium and D mesons in the relativistic quark model, Phys. Rev. D 68, 094020 (2003). [23] W. D. Hulsbergen, Decay chain fitting with a Kalman filter, Nucl. Instrum. Methods Phys. Res., Sect. A 552, 566 (2005). [10] C.-H. Chang and Y.-Q. Chen, Decays of the Bc meson, Phys. Rev. D 49, 3399 (1994). [11] C. Patrignani et al. (Particle Data Group Collaboration), Review of particle physics, Chin. Phys. C 40, 100001 (2016). [24] B. P. Roe, H.-J. Yang, J. Zhu, Y. Liu, I. Stancu, and G. DOI: 10.1103/PhysRevLett.118.111803 Individual groups or members have received support from AvH Foundation (Germany), EPLANET, Marie Skłodowska-Curie Actions and ERC (European Union), Conseil Général de Haute- Savoie, Labex ENIGMASS and OCEVU, Région Auvergne (France), RFBR and Yandex LLC (Russia), GVA, XuntaGal and GENCAT (Spain), Herchel Smith Fund, The Royal Society, Royal Commission for the Exhibition of 1851 and the Leverhulme Trust (United Kingdom). at which the p-value falls below 5%. All free variables in the fit are considered as nuisance parameters in this procedure. The p-value distributions for each RD0h meas- urement are shown in Fig. 3. The Bþc →D0hþ modes demonstrate no excess, and the RD0h CLs confidence intervals are determined similarly to that of RD0π. The upper limits at 95% confidence level found for RD0π, RD0π, and RD0K are RD0π < 3.9 × 10−7; RD0π < 1.1 × 10−6; RD0K < 1.1 × 10−6: RD0π < 3.9 × 10−7; RD0π < 1.1 × 10−6; RD0K < 1.1 × 10−6: The systematic uncertainties affecting the measurements are found to be much smaller than the statistical uncer- tainty, and do not alter the above upper limits. In the case of RD0K, the observed signal is of much higher significance. To determine the full uncertainty for RD0K, the systematic uncertainties affecting the measure- ment are accounted for. A systematic uncertainty of 1.1 × 10−8 is incurred from the use of fixed terms in the invariant mass fit. According to Eq. (2), several terms with associated relative uncertainties scale the measured signal yield: ξ with 5.3% uncertainty, BðBþ →¯D0πþÞ with 3.1% uncertainty [11], and N ðBþ →¯D0πþÞ with 0.14% uncer- tainty. The total systematic uncertainty, given by the sum in quadrature, is 6.2%. To determine the significance of the Bþc →D0Kþ peak, a likelihood scan is performed. The resulting −Δ logðLÞ value for the RD0K ¼ 0 hypothesis corresponds to a statistical significance of ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ −2Δ logðLÞ p ¼ 5.1σ for the signal. The final result is RD0K ¼ ð9.3þ2.8 −2.5 0.6Þ × 10−7; [3] R. Aaij et al. (LHCb Collaboration), Study of Bþc decays to the KþK−πþ final state and evidence for the decay Bþc →χc0πþ, Phys. Rev. D 94, 091102 (2016). where the first uncertainty is statistical and the second is systematic. R. Aaij,40 B. Adeva,39 M. Adinolfi,48 Z. Ajaltouni,5 S. Akar,59 J. Albrecht,10 F. Alessio,40 M. Alexander,53 S. Ali,43 G. Alkhazov,31 P. Alvarez Cartelle,55 A. A. Alves Jr.,59 S. Amato,2 S. Amerio,23 Y. Amhis,7 L. An,3 L. Anderlini,18 G. Andreassi,41 M. Andreotti,17,g J. E. Andrews,60 R. B. Appleby,56 F. Archilli,43 P. d’Argent,12 J. Arnau Romeu,6 A. Artamonov,37 M. Artuso,61 E. Aslanides,6 G. Auriemma,26 M. Baalouch,5 I. Babuschkin,56 S. Bachmann,12 J. J. Back,50 A. Badalov,38 C. Baesso,62 S. Baker,55 V. Balagura,7,c W. Baldini,17 R. J. Barlow,56 C. Barschel,40 S. Barsuk,7 W. Barter,56 F. Baryshnikov,32 M. Baszczyk,27 V. Batozskaya,29 B. Batsukh,61 V. Battista,41 A. Bay,41 L. Beaucourt,4 J. Beddow,53 F. Bedeschi,24 I. Bediaga,1 L. J. Bel,43 V. Bellee,41 N. Belloli,21,i K. Belous,37 I. Belyaev,32 E. Ben-Haim,8 G. Bencivenni,19 S. Benson,43 A. Berezhnoy,33 R. Bernet,42 A. Bertolin,23 C. Betancourt,42 F. Betti,15 M.-O. Bettler,40 M. van Beuzekom,43 Ia. Bezshyiko,42 S. Bifani,47 P. Billoir,8 T. Bird,56 A. Birnkraut,10 A. Bitadze,56 A. Bizzeti,18,u T. Blake,50 F. Blanc,41 J. Blouw,11,† S. Blusk,61 V. Bocci,26 T. Boettcher,58 A. Bondar,36,w N. Bondar,31,40 W. Bonivento,16 I. Bordyuzhin,32 A. Borgheresi,21,i S. Borghi,56 M. Borisyak,35 M. Borsato,39 F. Bossu,7 M. Boubdir,9 T. J. V. Bowcock,54 E. Bowen,42 C. Bozzi,17,40 S. Braun,12 M. Britsch,12 T. Britton,61 J. Brodzicka,56 E. Buchanan,48 C. Burr,56 A. Bursche,2 J. Buytaert,40 S. Cadeddu,16 R. Calabrese,17,g M. Calvi,21,i M. Calvo Gomez,38,m A. Camboni,38 P. Campana,19 D. H. Campora Perez,40 L. Capriotti,56 A. Carbone,15,e G. Carboni,25,j R. Cardinale,20,h A. Cardini,16 P. Carniti,21,i L. Carson,52 K. Carvalho Akiba,2 G. Casse,54 L. Cassina,21,i L. Castillo Garcia,41 M. Cattaneo,40 G. Cavallero,20 R. Cenci,24,t D. Chamont,7 M. Charles,8 Ph. Charpentier,40 G. Chatzikonstantinidis,47 M. Chefdeville,4 S. Chen,56 S. -F. Cheung,57 V. Chobanova,39 DOI: 10.1103/PhysRevLett.118.111803 McGregor, Boosted decision trees as an alternative to artificial neural networks for particle identification, Nucl. Instrum. Methods Phys. Res., Sect. A 543, 577 (2005). [12] A. A. Alves Jr. et al. (LHCb Collaboration), The LHCb detector at the LHC, J. Instrum. 3, S08005 (2008). [25] G. Punzi, in Statistical Problems in Particle Physics, Astrophysics, and Cosmology, edited by L. Lyons, R. Mount, and R. Reitmeyer (Stanford Linear Accelerator Center, Stanford, California, 2003), p. 79. [13] R. Aaij et al. (LHCb Collaboration), LHCb detector performance, Int. J. Mod. Phys. A 30, 1530022 (2015). [14] M. Adinolfi et al., Performance of the LHCb RICH detector at the LHC, Eur. Phys. J. C 73, 2431 (2013). [26] R. Aaij et al. (LHCb Collaboration), Measurement of CP observables in B →DK and B →Dπ with two- and four-body D meson decays, Phys. Lett. B 760, 117 (2016). [15] T. Sjöstrand, S. Mrenna, and P. Skands, PYTHIA 6.4 physics and manual, J. High Energy Phys. 05 (2006) 026. [16] T. Sjöstrand, S. Mrenna, and P. Skands, A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178, 852 (2008). [27] L. Moneta et al., The RooStats Project, Proc. Sci., ACAT20102010 (2010) 057. [17] I. Belyaev et al., Handling of the generation of primary events in Gauss, the LHCb simulation framework, J. Phys. Conf. Ser. 331, 032047 (2011). [28] R. Aaij et al. (LHCb Collaboration), Measurement of the branching fraction ratio BðBþc →ψð2SÞπþÞ= BðBþc →J=ψπþÞ, Phys. Rev. D 92, 072007 (2015). R. Aaij,40 B. Adeva,39 M. Adinolfi,48 Z. Ajaltouni,5 S. Akar,59 J. Albrecht,10 F. Alessio,40 M. Alexander,53 S. Ali,43 G. Alkhazov,31 P. Alvarez Cartelle,55 A. A. Alves Jr.,59 S. Amato,2 S. Amerio,23 Y. Amhis,7 L. An,3 L. Anderlini,18 G. Andreassi,41 M. Andreotti,17,g J. E. Andrews,60 R. B. Appleby,56 F. Archilli,43 P. d’Argent,12 J. Arnau Romeu,6 A. Artamonov,37 M. Artuso,61 E. Aslanides,6 G. Auriemma,26 M. Baalouch,5 I. Babuschkin,56 S. Bachmann,12 J. J. Back,50 A. Badalov,38 C. Baesso,62 S. Baker,55 V. Balagura,7,c W. Baldini,17 R. J. Barlow,56 C. Barschel,40 S. Barsuk,7 W. Barter,56 F. Baryshnikov,32 M. Baszczyk,27 V. Batozskaya,29 B. Batsukh,61 V. Battista,41 A. Bay,41 L. Beaucourt,4 J. Beddow,53 F. Bedeschi,24 I. Bediaga,1 L. J. Bel,43 V. Bellee,41 N. Belloli,21,i K. Belous,37 I. Belyaev,32 E. Ben-Haim,8 G. Bencivenni,19 S. Benson,43 A. Berezhnoy,33 R. Bernet,42 A. Bertolin,23 C. Betancourt,42 F. Betti,15 M.-O. Bettler,40 M. van Beuzekom,43 Ia. Bezshyiko,42 S. Bifani,47 P. Billoir,8 T. Bird,56 A. Birnkraut,10 A. Bitadze,56 A. M. Chrzaszcz,42,27 X. Cid Vidal,39 G. Ciezarek,43 P. E. L. Clarke,52 M. Clemencic,40 H. V. Cliff,49 J. Closier,40 V. Coco,59 J. Cogan,6 E. Cogneras,5 V. Cogoni,16,40,f L. Cojocariu,30 G. Collazuol,23,o P. Collins,40 A. Comerma-Montells,12 A. Contu,40 A. Cook,48 G. Coombs,40 S. Coquereau,38 G. Corti,40 M. Corvo,17,g C. M. Costa Sobral,50 B. Couturier,40 G. A. Cowan,52 D. C. Craik,52 A. Crocombe,50 M. Cruz Torres,62 S. Cunliffe,55 R. Currie,55 C. D’Ambrosio,40 F. Da Cunha Marinho,2 E. Dall’Occo,43 J. Dalseno,48 P. N. Y. David,43 A. Davis,3 K. De Bruyn,6 S. De Capua,56 M. De Cian,12 J. M. De Miranda,1 L. De Paula,2 M. De Serio,14,d P. De Simone,19 C. T. Dean,53 D. Decamp,4 M. Deckenhoff,10 L. Del Buono,8 M. Demmer,10 A. Dendek,28 D. Derkach,35 O. Deschamps,5 F. Dettori,40 B. Dey,22 A. Di Canto,40 H. Dijkstra,40 F. Dordei,40 M. Dorigo,41 A. Dosil Suárez,39 A. Dovbnya,45 K. Dreimanis,54 L. Dufour,43 G. Dujany,56 K. Dungs,40 P. Durante,40 R. Dzhelyadin,37 A. Dziurda,40 A. Dzyuba,31 N. Déléage,4 S. Easo,51 M. Ebert,52 U. Egede,55 V. Egorychev,32 S. Eidelman,36,w S. Eisenhardt,52 U. Eitschberger,10 R. Ekelhof,10 L. Eklund,53 S. Ely,61 S. Esen,12 H. M. Evans,49 T. Evans,57 A. Falabella,15 N. Farley,47 S. Farry,54 R. Fay,54 D. Fazzini,21,i D. Ferguson,52 A. Fernandez Prieto,39 F. Ferrari,15,40 F. Ferreira Rodrigues,2 M. Ferro-Luzzi,40 S. Filippov,34 R. A. Fini,14 M. Fiore,17,g M. Fiorini,17,g M. Firlej,28 C. Fitzpatrick,41 T. Fiutowski,28 F. Fleuret,7,b K. Fohl,40 M. Fontana,16,40 F. Fontanelli,20,h D. C. Forshaw,61 R. Forty,40 V. Franco Lima,54 M. Frank,40 C. Frei,40 J. Fu,22,q W. Funk,40 E. Furfaro,25,j C. Färber,40 A. Gallas Torreira,39 D. Galli,15,e S. Gallorini,23 S. Gambetta,52 M. Gandelman,2 P. Gandini,57 Y. Gao,3 L. M. Garcia Martin,69 J. García Pardiñas,39 J. Garra Tico,49 L. Garrido,38 P. J. Garsed,49 D. Gascon,38 C. Gaspar,40 L. Gavardi,10 G. Gazzoni,5 D. Gerick,12 E. Gersabeck,12 M. Gersabeck,56 T. Gershon,50 Ph. Ghez,4 S. Gianì,41 V. Gibson,49 O. G. Girard,41 L. Giubega,30 K. Gizdov,52 V. V. Gligorov,8 D. Golubkov,32 A. Golutvin,55,40 A. Gomes,1,a I. V. Gorelov,33 C. Gotti,21,i R. Graciani Diaz,38 L. A. Granado Cardoso,40 E. Graugés,38 E. Graverini,42 G. Graziani,18 A. Grecu,30 P. Griffith,47 L. Grillo,21,40,i B. R. Gruberg Cazon,57 O. Grünberg,67 E. Gushchin,34 Yu. Guz,37 T. Gys,40 C. Göbel,62 T. Hadavizadeh,57 C. Hadjivasiliou,5 G. Haefeli,41 C. Haen,40 S. C. Haines,49 B. Hamilton,60 X. Han,12 S. Hansmann-Menzemer,12 N. Harnew,57 S. T. Harnew,48 J. Harrison,56 M. Hatch,40 J. He,63 T. Head,41 A. Heister,9 K. Hennessy,54 P. Henrard,5 L. Henry,8 E. van Herwijnen,40 M. Heß,67 A. Hicheur,2 D. Hill,57 C. Hombach,56 H. Hopchev,41 W. Hulsbergen,43 T. Humair,55 M. Hushchyn,35 D. Hutchcroft,54 M. Idzik,28 P. Ilten,58 R. Jacobsson,40 A. Jaeger,12 J. Jalocha,57 E. Jans,43 A. Jawahery,60 F. Jiang,3 M. John,57 D. Johnson,40 C. R. Jones,49 C. Joram,40 B. Jost,40 N. Jurik,57 S. Kandybei,45 M. Karacson,40 J. M. Kariuki,48 S. Karodia,53 M. Kecke,12 M. Kelsey,61 M. Kenzie,49 T. Ketel,44 E. Khairullin,35 B. Khanji,12 C. Khurewathanakul,41 T. Kirn,9 S. Klaver,56 K. Klimaszewski,29 S. Koliiev,46 M. Kolpin,12 I. Komarov,41 R. F. Koopman,44 P. Koppenburg,43 A. Kosmyntseva,32 A. Kozachuk,33 M. Kozeiha,5 L. Kravchuk,34 K. Kreplin,12 M. Kreps,50 P. Krokovny,36,w F. Kruse,10 W. Krzemien,29 W. Kucewicz,27,l M. Kucharczyk,27 V. Kudryavtsev,36,w A. K. Kuonen,41 K. Kurek,29 T. Kvaratskheliya,32,40 D. Lacarrere,40 G. Lafferty,56 A. Lai,16 G. Lanfranchi,19 C. Langenbruch,9 T. Latham,50 C. Lazzeroni,47 R. Le Gac,6 J. van Leerdam,43 A. Leflat,33,40 J. Lefrançois,7 R. Lefèvre,5 F. Lemaitre,40 E. Lemos Cid,39 O. Leroy,6 T. Lesiak,27 B. Leverington,12 T. Li,3 Y. Li,7 T. Likhomanenko,35,68 R. Lindner,40 C. Linn,40 F. Lionetto,42 X. Liu,3 D. Loh,50 I. Longstaff,53 J. H. Lopes,2 D. Lucchesi,23,o M. Lucio Martinez,39 H. Luo,52 A. Lupato,23 E. Luppi,17,g O. Lupton,40 A. Lusiani,24 X. Lyu,63 F. Machefert,7 F. Maciuc,30 O. Maev,31 K. Maguire,56 S. Malde,57 A. Malinin,68 T. Maltsev,36 G. Manca,16,f G. Mancinelli,6 P. Manning,61 J. Maratas,5,v J. F. Marchand,4 U. Marconi,15 C. Marin Benito,38 M. Marinangeli,41 P. Marino,24,t J. Marks,12 G. Martellotti,26 M. Martin,6 M. Martinelli,41 D. Martinez Santos,39 F. Martinez Vidal,69 D. Martins Tostes,2 L. M. Massacrier,7 A. Massafferri,1 R. Matev,40 A. Mathad,50 Z. Mathe,40 C. Matteuzzi,21 A. Mauri,42 E. Maurice,7,b B. Maurin,41 A. Mazurov,47 M. McCann,55,40 A. McNab,56 R. McNulty,13 B. Meadows,59 F. Meier,10 M. Meissner,12 D. Melnychuk,29 M. Merk,43 A. Merli,22,q E. Michielin,23 D. A. Milanes,66 M. -N. Minard,4 D. S. Mitzel,12 A. Mogini,8 J. Molina Rodriguez,1 I. A. Monroy,66 S. Monteil,5 M. Morandin,23 P. Morawski,28 A. Mordà,6 M. J. Morello,24,t O. Morgunova,68 J. Moron,28 A B M i 52 R M t i 61 F M h i 52 M M ld 43 M M i i 15 D Müll 56 J Müll 10 K Müll 42 V Müll 10 DOI: 10.1103/PhysRevLett.118.111803 Bizzeti,18,u T. Blake,50 F. Blanc,41 J. Blouw,11,† S. Blusk,61 V. Bocci,26 T. Boettcher,58 A. Bondar,36,w N. Bondar,31,40 W. Bonivento,16 I. Bordyuzhin,32 A. Borgheresi,21,i S. Borghi,56 M. Borisyak,35 M. Borsato,39 F. Bossu,7 M. Boubdir,9 T. J. V. Bowcock,54 E. Bowen,42 C. Bozzi,17,40 S. Braun,12 M. Britsch,12 T. Britton,61 J. Brodzicka,56 E. Buchanan,48 C. Burr,56 A. Bursche,2 J. Buytaert,40 S. Cadeddu,16 R. Calabrese,17,g M. Calvi,21,i M. Calvo Gomez,38,m A. Camboni,38 P. Campana,19 D. H. Campora Perez,40 L. Capriotti,56 A. Carbone,15,e G. Carboni,25,j R. Cardinale,20,h A. Cardini,16 P. Carniti,21,i L. Carson,52 K. Carvalho Akiba,2 G. Casse,54 L. Cassina,21,i L. Castillo Garcia,41 M. Cattaneo,40 G. Cavallero,20 R. Cenci,24,t D. Chamont,7 M. Charles,8 Ph. Charpentier,40 G. Chatzikonstantinidis,47 M. Chefdeville,4 S. Chen,56 S. -F. Cheung,57 V. Chobanova,39 111803-5 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) J. Cogan,6 E. Cogneras,5 V. Cogoni,16,40,f L. Cojocariu,30 G. Collazuol,23,o P. Collins,40 A. Comer L. De Paula,2 M. De Serio,14,d P. De Simone,19 C. T. Dean,53 D. Decamp,4 M. Deckenhoff,10 L. A. Dendek,28 D. Derkach,35 O. Deschamps,5 F. Dettori,40 B. Dey,22 A. Di Canto,40 H. Dijkstra, g y N. Farley,47 S. Farry,54 R. Fay,54 D. Fazzini,21,i D. Ferguson,52 A. Fernandez Prieto,39 F. Ferrari,15,40 F. Ferreira Rodrigues,2 M. Ferro-Luzzi,40 S. Filippov,34 R. A. Fini,14 M. Fiore,17,g M. Fiorini,17,g M. Firlej,28 C. Fit pp j p F. Fleuret,7,b K. Fohl,40 M. Fontana,16,40 F. Fontanelli,20,h D. C. Forshaw,61 R. Forty,40 V. Franco Lima,54 M. Frank,40 C. Frei,40 J. Fu,22,q W. Funk,40 E. Furfaro,25,j C. Färber,40 A. Gallas Torreira,39 D. Galli,15,e S. Gallorini,23 S. Gambetta,52 M. Lucio Martinez,39 H. Luo,52 A. Lupato,23 E. Luppi,17,g O. Lupton,40 A. Lusiani,24 X. Lyu,63 F. Machefert,7 F. Maciuc,30 J. F. Marchand,4 U. Marconi,15 C. Marin Benito,38 M. Marinangeli,41 P. Marino,24,t J. Marks,12 G. Martellotti,26 M. Martin,6 M. Martinelli,41 D. Martinez Santos,39 F. Martinez Vidal,69 D. Martins Tostes,2 L. M. Massacrier,7 A. Massafferri,1 R. Matev,40 A. Mathad,50 Z. Mathe,40 C. Matteuzzi,21 A. Mauri,42 E. Maurice,7,b B. Maurin,41 A. Mazurov,47 M. McCann,55,40 A. McNab,56 R. McNulty,13 B. Meadows,59 F. Meier,10 M. Meissner,12 D. Melnychuk,29 M. Merk,43 A M li 22 q E Mi hi li 23 D A Mil 66 M N Mi d 4 D S Mi l 12 A M i i 8 J M li R d i 1 M. DOI: 10.1103/PhysRevLett.118.111803 Viaud,7 D. Vieira,63 M. Vieites Diaz,39 H. Viemann,67 X. Vilasis-Cardona,38,m M. Vitti,49 V. Volkov,33 A. Vollhardt,42 B. Voneki,40 A. Vorobyev,31 V. Vorobyev,36,w C. Voß,9 J. A. de Vries,43 C. Vázquez Sierra,39 R. Waldi,67 C. Wallace,50 R. Wallace,13 J. Walsh,24 J. Wang,61 D. R. Ward,49 H. M. Wark,54 N. K. Watson,47 D. Websdale,55 A. Weiden,42 M. Whitehead,40 J. Wicht,50 G. Wilkinson,57,40 M. Wilkinson,61 M. Williams,40 M. P. Williams,47 M. Williams,58 T. Williams,47 F. F. Wilson,51 J. Wimberley,60 J. Wishahi,10 W. Wislicki,29 M. Witek,27 G. Wormser,7 S. A. Wotton,49 K. Wraight,53 K. Wyllie,40 Y. Xie,65 Z. Xing,61 Z. Xu,4 Z. Yang,3 Y. Yao,61 H. Yin,65 J. Yu,65 X. Yuan,36,w O. Yushchenko,37 K. A. Zarebski,47 M. Zavertyaev,11,c L. Zhang,3 Y. Zhang,7 Y. Zhang,63 A. Zhelezov,12 Y. Zheng,63 X. Zhu,3 V. Zhukov,33 and S. Zucchelli15 (LHCb Collaboration) 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil 2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3Center for High Energy Physics, Tsinghua University, Beijing, China 4LAPP, Université Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, France 5Clermont Université, Université Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 7LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France 8LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 9I. Physikalisches Institut, RWTH Aachen University, Aachen, Germany 10Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany 11Max-Planck-Institut für Kernphysik (MPIK), Heidelberg, Germany 12Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 13School of Physics, University College Dublin, Dublin, Ireland 14Sezione INFN di Bari, Bari, Italy 15Sezione INFN di Bologna, Bologna, Italy 16Sezione INFN di Cagliari, Cagliari, Italy DOI: 10.1103/PhysRevLett.118.111803 McCann,55,40 A. McNab,56 R. McNulty,13 B. Meadows,59 F. Meier,10 M. Meissner,12 D. Melnychuk,29 M. Merk,43 A. Merli,22,q E. Michielin,23 D. A. Milanes,66 M. -N. Minard,4 D. S. Mitzel,12 A. Mogini,8 J. Molina Rodriguez,1 I. A. Monroy,66 S. Monteil,5 M. Morandin,23 P. Morawski,28 A. Mordà,6 M. J. Morello,24,t O. Morgunova,68 J. Moron,28 , , , , , , , , , P. Naik,48 T. Nakada,41 R. Nandakumar,51 A. Nandi,57 I. Nasteva,2 M. Needham,52 N. Neri,22 S. Neubert,12 N. Neufeld,40 M. Neuner,12 T. D. Nguyen,41 C. Nguyen-Mau,41,n S. Nieswand,9 R. Niet,10 N. Nikitin,33 T. Nikodem,12 A. Nogay,68 A. Novoselov,37 D. P. O’Hanlon,50 A. Oblakowska-Mucha,28 V. Obraztsov,37 S. Ogilvy,19 R. Oldeman,16,f C. J. G. Onderwater,70 J. M. Otalora Goicochea,2 A. Otto,40 P. Owen,42 A. Oyanguren,69 P. R. Pais,41 A. Palano,14,d A. Novoselov, D. P. O Hanlon, A. Oblakowska Mucha, V. Obraztsov, S. Ogilvy, R. Oldeman, C. J. G. Onderwater,70 J. M. Otalora Goicochea,2 A. Otto,40 P. Owen,42 A. Oyanguren,69 P. R. Pais,41 A. Palano,14,d M. Palutan,19 A. Papanestis,51 M. Pappagallo,14,d L. L. Pappalardo,17,g W. Parker,60 C. Parkes,56 G. Passaleva,18 g g p S. Perazzini,40 P. Perret,5 L. Pescatore,47 K. Petridis,48 A. Petrolini,20,h A. Petrov,68 M. Petruzzo,22,q E. Picatoste Olloqui,38 111803-6 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) S. Poslavskii,37 C. Potterat,2 E. Price,48 J. D. Price,54 J. Prisciandaro,39,40 A. Pritchard,54 C. Prouve,48 V. Pugatch,46 A. Puig Navarro,42 G. Punzi,24,p W. Qian,50 R. Quagliani,7,48 B. Rachwal,27 J. H. Rademacker,48 M. Rama,24 C. Sanchez Mayordomo,69 B. Sanmartin Sedes,39 R. Santacesaria,26 C. Santamarina Rios,39 M. Santimaria,19 E. Santovetti,25,j A. Sarti,19,k C. Satriano,26,s A. Satta,25 D. M. Saunders,48 D. Savrina,32,33 S. Schael,9 M. Schellenberg,10 C. Sanchez Mayordomo,69 B. Sanmartin Sedes,39 R. Santacesaria,26 C. Santamarina Rios,39 M. Santimaria,19 E. Santovetti,25,j A. Sarti,19,k C. Satriano,26,s A. Satta,25 D. M. Saunders,48 D. Savrina,32,33 S. Schael,9 M. Schellenberg,10 M. Schiller,53 H. Schindler,40 M. Schlupp,10 M. Schmelling,11 T. Schmelzer,10 B. Schmidt,40 O. Schneider,41 A. Schopper,40 K. Schubert,10 M. Schubiger,41 M. -H. Schune,7 R. Schwemmer,40 B. Sciascia,19 A. Sciubba,26,k A. Semennikov,32 6 5 5 U. Uwer,12 C. Vacca,16,f V. Vagnoni,15,40 A. Valassi,40 S. Valat,40 G. Valenti,15 R. Vazquez Gomez,19 P. Vazquez Regueiro,39 S. Vecchi,17 M. van Veghel,43 J. J. Velthuis,48 M. Veltri,18,r G. Veneziano,57 A. Venkateswaran,61 M. Vernet,5 M. Vesterinen,12 J. V. Viana Barbosa,40 B. 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil 2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3Center for High Energy Physics, Tsinghua University, Beijing, China 4LAPP, Université Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, France 5Clermont Université, Université Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 7LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France 8LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 9I. Physikalisches Institut, RWTH Aachen University, Aachen, Germany 10Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany 11Max-Planck-Institut für Kernphysik (MPIK), Heidelberg, Germany 12Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 13School of Physics, University College Dublin, Dublin, Ireland 14Sezione INFN di Bari, Bari, Italy 15Sezione INFN di Bologna, Bologna, Italy 16Sezione INFN di Cagliari, Cagliari, Italy F. Polci,8 A. Poluektov,50,36 I. Polyakov,61 E. Polycarpo,2 G. J. Pomery,48 A. Popov,37 D. Popov,11,40 B. Popovici,30 S. Poslavskii,37 C. Potterat,2 E. Price,48 J. D. Price,54 J. Prisciandaro,39,40 A. Pritchard,54 C. Prouve,48 V. Pugatch,46 A. Puig Navarro,42 G. Punzi,24,p W. Qian,50 R. Quagliani,7,48 B. Rachwal,27 J. H. Rademacker,48 M. Rama,24 M. Ramos Pernas,39 M. S. Rangel,2 I. Raniuk,45 F. Ratnikov,35 G. Raven,44 F. Redi,55 S. Reichert,10 A. C. dos Reis,1 C. Remon Alepuz,69 V. Renaudin,7 S. Ricciardi,51 S. Richards,48 M. Rihl,40 K. Rinnert,54 V. Rives Molina,38 P. Robbe,7,40 A. B. Rodrigues,1 E. Rodrigues,59 J. A. Rodriguez Lopez,66 P. Rodriguez Perez,56,† A. Rogozhnikov,35 S. Roiser,40 A. Rollings,57 V. Romanovskiy,37 A. Romero Vidal,39 J. W. Ronayne,13 M. Rotondo,19 M. S. Rudolph,61 T. Ruf,40 P. Ruiz Valls,69 J. J. Saborido Silva,39 E. Sadykhov,32 N. Sagidova,31 B. Saitta,16,f V. Salustino Guimaraes,1 C. Sanchez Mayordomo,69 B. Sanmartin Sedes,39 R. Santacesaria,26 C. Santamarina Rios,39 M. Santimaria,19 E. Santovetti,25,j A. Sarti,19,k C. Satriano,26,s A. Satta,25 D. M. Saunders,48 D. Savrina,32,33 S. Schael,9 M. Schellenberg,10 M. Schiller,53 H. Schindler,40 M. Schlupp,10 M. Schmelling,11 T. Schmelzer,10 B. Schmidt,40 O. Schneider,41 A. Schopper,40 K. Schubert,10 M. Schubiger,41 M. -H. Schune,7 R. Schwemmer,40 B. Sciascia,19 A. Sciubba,26,k A. Semennikov,32 A. Sergi,47 N. Serra,42 J. Serrano,6 L. Sestini,23 P. Seyfert,21 M. Shapkin,37 I. Shapoval,45 Y. Shcheglov,31 T. Shears,54 L. Shekhtman,36,w V. Shevchenko,68 B. G. Siddi,17,40 R. Silva Coutinho,42 L. Silva de Oliveira,2 G. Simi,23,o S. Simone,14,d M. Sirendi,49 N. Skidmore,48 T. Skwarnicki,61 E. Smith,55 I. T. Smith,52 J. Smith,49 M. Smith,55 H. Snoek,43 l. Soares Lavra,1 M. D. Sokoloff,59 F. J. P. Soler,53 B. Souza De Paula,2 B. Spaan,10 P. Spradlin,53 S. Sridharan,40 F. Stagni,40 M. Stahl,12 S. Stahl,40 P. Stefko,41 S. Stefkova,55 O. Steinkamp,42 S. Stemmle,12 O. Stenyakin,37 H. Stevens,10 S. Stevenson,57 S. Stoica,30 S. Stone,61 B. Storaci,42 S. Stracka,24,p M. Straticiuc,30 U. Straumann,42 L. Sun,64 W. Sutcliffe,55 K. Swientek,28 V. Syropoulos,44 M. Szczekowski,29 T. Szumlak,28 S. T’Jampens,4 A. Tayduganov,6 T. Tekampe,10 G. Tellarini,17,g F. Teubert,40 E. Thomas,40 J. van Tilburg,43 M. J. Tilley,55 V. Tisserand,4 M. Tobin,41 S. Tolk,49 L. Tomassetti,17,g D. Tonelli,40 S. Topp-Joergensen,57 F. Toriello,61 E. Tournefier,4 S. Tourneur,41 K. Trabelsi,41 M. Traill,53 M. T. Tran,41 M. Tresch,42 A. Trisovic,40 A. Tsaregorodtsev,6 P. Tsopelas,43 A. Tully,49 N. Tuning,43 A. Ukleja,29 A. Ustyuzhanin,35 U. Uwer,12 C. Vacca,16,f V. Vagnoni,15,40 A. Valassi,40 S. Valat,40 G. Valenti,15 R. Vazquez Gomez,19 P. Vazquez Regueiro,39 S. Vecchi,17 M. van Veghel,43 J. J. Velthuis,48 M. Veltri,18,r G. Veneziano,57 A. Venkateswaran,61 M. Vernet,5 M. Vesterinen,12 J. V. Viana Barbosa,40 B. Viaud,7 D. Vieira,63 M. Vieites Diaz,39 H. Viemann,67 X. Vilasis-Cardona,38,m M. Vitti,49 V. Volkov,33 A. Vollhardt,42 B. Voneki,40 A. Vorobyev,31 V. Vorobyev,36,w C. Voß,9 J. A. de Vries,43 C. Vázquez Sierra,39 R. Waldi,67 C. Wallace,50 R. Wallace,13 J. Walsh,24 J. Wang,61 D. R. Ward,49 H. M. Wark,54 N. K. Watson,47 D. Websdale,55 A. Weiden,42 M. Whitehead,40 J. Wicht,50 G. Wilkinson,57,40 M. Wilkinson,61 M. Williams,40 M. P. Williams,47 M. Williams,58 T. Williams,47 F. F. Wilson,51 J. Wimberley,60 J. Wishahi,10 W. Wislicki,29 M. Witek,27 G. Wormser,7 S. A. Wotton,49 K. Wraight,53 K. Wyllie,40 Y. Xie,65 Z. Xing,61 Z. Xu,4 Z. Yang,3 Y. Yao,61 H. Yin,65 J. Yu,65 X. Yuan,36,w O. Yushchenko,37 K. A. Zarebski,47 M. Zavertyaev,11,c L. Zhang,3 Y. Zhang,7 Y. Zhang,63 A. Zhelezov,12 Y. Zheng,63 X. Zhu,3 V. Zhukov,33 and S. Zucchelli15 P H Y S I C A L R E V I E W L E T T E R S Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom 49Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 50Department of Physics, University of Warwick, Coventry, United Kingdom 51STFC Rutherford Appleton Laboratory, Didcot, United Kingdom 52School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 53School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 54Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 55Imperial College London, London, United Kingdom 56School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 57Department of Physics, University of Oxford, Oxford, United Kingdom 58Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 59University of Cincinnati, Cincinnati, Ohio, USA 60University of Maryland, College Park, Maryland, USA 61Syracuse University, Syracuse, New York, USA 62Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, associated with Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 63University of Chinese Academy of Sciences, Beijing, China, associated with Center for High Energy Physics, Tsinghua University, Beijing, China 64School of Physics and Technology, Wuhan University, Wuhan, China, associated with Center for High Energy Physics, Tsinghua University, Beijing, China 65Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China, associated with Center for High Energy Physics, Tsinghua University, Beijing, China 66Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia, associated with LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 67Institut für Physik, Universität Rostock, Rostock, Germany, associated with Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 68National Research Centre Kurchatov Institute, Moscow, Russia, associated with Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia Imperial College London, London, United Kingdom 58Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 59 59University of Cincinnati, Cincinnati, Ohio, USA 60 61Syracuse University, Syracuse, New York, USA 62Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, 63University of Chinese Academy of Sciences, Beijing, China, f g gy y g y j g 64School of Physics and Technology, Wuhan University, Wuhan, China, 66Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia, associated with LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, Fran 67 associated with LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, Fra 67Institut für Physik Universität Rostock Rostock Germany with Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 68 68National Research Centre Kurchatov Institute, Moscow, Russia, associated with Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 111803-8 111803-7 111803-7 week ending 17 MARCH 2017 PRL 118, 111803 (2017) P H Y S I C A L R E V I E W L E T T E R S 17Sezione INFN di Ferrara, Ferrara, Italy 18Sezione INFN di Firenze, Firenze, Italy 19Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy 20Sezione INFN di Genova, Genova, Italy 21Sezione INFN di Milano Bicocca, Milano, Italy 22Sezione INFN di Milano, Milano, Italy 23Sezione INFN di Padova, Padova, Italy 24Sezione INFN di Pisa, Pisa, Italy 25Sezione INFN di Roma Tor Vergata, Roma, Italy 26Sezione INFN di Roma La Sapienza, Roma, Italy 27Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland 28AGH - University of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, Poland 29National Center for Nuclear Research (NCBJ), Warsaw, Poland 30Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania 31Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 32Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 33Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia 34Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russia 35Yandex School of Data Analysis, Moscow, Russia 36Budker Institute of Nuclear Physics (SB RAS), Novosibirsk, Russia 37Institute for High Energy Physics (IHEP), Protvino, Russia 38ICCUB, Universitat de Barcelona, Barcelona, Spain 39Universidad de Santiago de Compostela, Santiago de Compostela, Spain 40European Organization for Nuclear Research (CERN), Geneva, Switzerland 41Institute of Physics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 42Physik-Institut, Universität Zürich, Zürich, Switzerland 43Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands 44Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, Netherlands 45NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine 46Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine 47University of Birmingham, Birmingham, United Kingdom 48H.H. 111803-8 week ending 17 MARCH 2017 PRL 118, 111803 (2017) PRL 118, 111803 (2017) 69Instituto de Fisica Corpuscular, Centro Mixto Universidad de Valencia - CSIC, Valencia, Spain, associated with ICCUB, Universitat de Barcelona, Barcelona, Spain 70Van Swinderen Institute, University of Groningen, Groningen, Netherlands, associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands †Deceased. aUniversidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. b bLaboratoire Leprince-Ringuet, Palaiseau, France. cP.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia d cP.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS) d dUniversità di Bari, Bari, Italy. eUniversità di Bologna, Bologna, Italy. f fUniversità di Cagliari, Cagliari, Italy. fUniversità di Cagliari, Cagliari, Italy. gUniversità di Ferrara, Ferrara, Italy. h gUniversità di Ferrara, Ferrara, Italy. h hUniversità di Genova, Genova, Italy. I IUniversità di Milano Bicocca, Milano, Italy. j IUniversità di Milano Bicocca, Milano, Italy. j jUniversità di Roma Tor Vergata, Roma, Italy. k jUniversità di Roma Tor Vergata, Roma, Italy. k kUniversità di Roma La Sapienza, Roma, Italy. l Università di Roma La Sapienza, Roma, Italy. lAGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. mLIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. p , , y lAGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. mLIFAELS L S ll U i it t R Ll ll B l S i lAGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. mLIFAELS L S ll U i i R Ll ll B l S i lAGH - University of Science and Technology, Faculty of Computer Science, Electronics a mLIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. LIFAELS, La Salle, Universitat Ramon Llull, Barc nHanoi University of Science, Hanoi, Viet Nam. nHanoi University of Science, Hanoi, Viet Nam. oUniversità di Padova, Padova, Italy. pUniversità di Pisa, Pisa, Italy. pUniversità di Pisa, Pisa, Italy. qUniversità degli Studi di Milano, Milano, Italy. r qUniversità degli Studi di Milano, Milano, Italy. rUniversità di Urbino, Urbino, Italy. rUniversità di Urbino, Urbino, Italy. sUniversità della Basilicata, Potenza, Italy. sUniversità della Basilicata, Potenza, Italy. tScuola Normale Superiore, Pisa, Italy. tScuola Normale Superiore, Pisa, Italy. uUniversità di Modena e Reggio Emilia, Modena, Italy. uUniversità di Modena e Reggio Emilia, Modena, Italy. vIligan Institute of Technology (IIT), Iligan, Philippines. vIligan Institute of Technology (IIT), Iligan, Philippines. wNovosibirsk State University, Novosibirsk, Russia. wNovosibirsk State University, Novosibirsk, Russia. 111803-9
https://openalex.org/W2290782402	https://europepmc.org/articles/pmc5685250?pdf=render	English	null	Gender Confirmation Surgery: An Update for the Primary Care Provider	Transgender health	2,016	cc-by	5,716	Transgender Health Volume 1.1, 2016 DOI: 10.1089/trgh.2015.0006 Transgender Health Volume 1.1, 2016 DOI: 10.1089/trgh.2015.0006 REVIEW ARTICLE ª Loren S. Schechter et al. 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Abstract Increased advocacy efforts and expanded third-party insurance coverage have improved access to healthcare for transgender individuals. In conjunction with mental health and medical professionals, gender surgeries offer an important step in allowing individuals to realize their true selves. To provide quality multidisciplinary care, primary care doctors need to understand challenges facing transgender individuals and treatment options available to them. In this article, we will review the role of the surgeon and the goals of various gender conﬁrming surgeries. In addition, we will provide an overview of the available surgical options. Key words: gender conﬁrmation surgery; gender nonconforming; metoidioplasty; phalloplasty; transgender; vaginoplasty y g vaginoplasty The WPATH developed the Standards of Care (SOC) to help provide the highest standards of care for transgender individuals. Since WPATH published the ﬁrst version of SOC in 1979, the guidelines have been updated seven times, illustrating the complex and developing needs in caring for the transgender population. SOC state that the overarching treatment goal is ‘‘lasting personal comfort with the gendered self to maximize overall health, psychological well- being, and self-fulﬁllment.’’2 Toward this end, gender conﬁrmation surgery provides the appropriate physical morphology. Congruent genitalia allow an individual to experience harmony between one’s body and self- identity, appear nude in social situations without vio- lating taboos (i.e., health clubs, physician ofﬁces), and, in some states, have legal identiﬁcation concordant with one’s physical appearance.3 Loren S. Schechter* Loren S. Schechter* University Plastic Surgery, Morton Grove, Illinois. Address correspondence to: Loren S. Schechter, MD, FACS, University Plastic Surgery, 9000 Waukegan Road, #210 Morton Grove, IL 60053, E-mail: lss@univplastics.com Address correspondence to: Loren S. Schechter, MD, FACS, University Plastic Surgery, 9000 Waukegan Road, #210 Morton Grove, IL 600 Address correspondence to: Loren S. Schechter, MD, FACS, University Plastic Surgery, 9000 Wauke Introduction The terms gender dysphoria and gender incongruence describe a heterogeneous group of individuals who ex- press dissatisfaction with their anatomic gender and the wish to have the secondary sexual characteristics of the opposite sex.1 While not all transgender individ- uals suffer gender dysphoria, many do. In recent years, there have been signiﬁcant advances in the understand- ing, management, and care of transgender persons. These developments encompass psychological, med- ical, and surgical approaches to therapy to help allevi- ate gender dysphoria. In addition, social and political changes over the past 35 years have brought more atten- tion to this underserved and diverse population. In fact, in 2010, the World Professional Association for Transgender Health (WPATH) released a statement recommending the de-psycho-pathologization of gen- der nonconformity, stating that the expression of gen- der characteristics, including identities, which are not stereotypically associated with one’s assigned sex at birth, is a common and culturally diverse human phe- nomenon that should not be judged as inherently path- ological or negative.2 For many transfemales, a successful surgical result involves the creation of a natural-appearing vagina and mons pubis, which are sensate and functional. This includes removal of the stigmatizing scrotum, cre- ation of feminine-appearing labia majora and minora, construction of a sensate neoclitoris, and development 32 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 33 mains responsible for any diagnosis on the basis of which he or she performs surgical interventions.5 Com- munication between the surgeon and mental health pro- fessional(s) is recommended. This serves to educate the surgeon and to aid with his or her understanding of each patient’s unique needs. It also helps to prevent possible falsiﬁcation of letters of recommendation. Communica- tion should not be limited to the evaluation phase. It is the responsibility of the surgeon to communicate perti- nent operative ﬁndings as well as postoperative instruc- tions with the relevant members of the healthcare team.6 This includes mental health professionals, primary care providers, endocrinologists, mid-level practitioners, and other surgeons involved in an individual’s care. of adequate vaginal depth and introital width for inter- course. Additional desirable qualities include a moist appearance to the labia minora, clitoral hooding, and lubrication for intercourse. Aside from genital reconstruction, breast augmenta- tion, thyroid chondroplasty (tracheal shave), and facial feminization offer additional procedures designed to feminize one’s appearance. Hormone Therapy Many,butnotall,individualswithgenderdysphoriadesire hormone therapy to transition. Endocrinologists or pri- mary care providers typically guide therapy. As with sur- gery, hormone therapy requires a tailored approach. Not all gender surgery (i.e., chest surgery) requires preopera- tive hormone therapy. However, some surgeries, such as metoidioplastyorphalloplasty,requireadequatehormonal therapy to allow for clitoral virilization. Often times, third- party payers require documentation of 12 months of hor- mone therapy, or an explanation as to why a patient does not take hormones, before authorizing surgery. g g y q Although multiple studies conﬁrm the efﬁcacy of surgery and low complication rates,7,8,9–13 the surgeon must actively investigate potential risk factors that may increase the risk of postoperative complications before proceeding with surgery. Based upon questionnaire data from 19 gender clinics in Europe and North America, conditions that could result in delay or denial of surgery included psychosocial instability, married status, substance abuse, chronic or psychotic illness, and antisocial behavior.14 In addition, in a retrospec- tive review of 136 patients who underwent sex reassign- ment in Sweden, several preoperative factors were identiﬁed and reported to be associated with higher rates of unsatisfactory surgical outcomes. These in- cluded personal and social instability, unsuitable body build, and age over 30 years at operation. Additionally, in this study, adequate family and social support were noted to be important for postoperative functioning.15 Introduction For transmen, as outlined by Professor Stan Monstrey and his team at the University of Ghent, (Belgium), phal- lic reconstruction should result in an aesthetic phallus with both tactile and erogenous sensations, the ability to void while standing, minimal morbidity of the surgical intervention and donor site, an aesthetic scrotum, and the ability to experience sexual satisfaction postoperatively.4 Severalmodelsforpreoperativeevaluationexist.Forex- ample,somecentersfollowaninformedconsent,orshared decision-making model, in which the healthcare provider and the patient make healthcare decisions together, taking into account best clinical practices and the preferences of the patient. However, most surgeons and third-party pay- ers follow guidelines as provided in the WPATH SOC. While phalloplasty represents the most complete geni- toperineal transformation, it requires complex staged pro- cedures, the use of tissue from remote sites resulting in scarring at the donor site, and increased risk of compli- cations associated with urethral reconstruction and im- plantable prostheses. For these reasons, some individuals undergo metoidioplasty. This entails lengthening of the virilized clitoris and may be performed with, or without, urethral lengthening to allow for urination while standing. As per the WPATH guidelines, evaluation by behav- ior health professionals is central in preoperative assess- ment. These professionals can come from a variety of backgrounds, including psychology, psychiatry, social work, mental health counseling, nursing, or medicine, and should have speciﬁc and ongoing training in evalu- ating and caring for transgender individuals.3 The number of referral letters needed from mental health providers depends on the procedure requested. In gen- eral, nongenital surgery requires one referral letter, whereas genital surgery requires two referral letters. In addition to genital surgery, chest surgery, involving bilateral subcutaneous mastectomies and contouring of the chest, is commonly performed in transmen. Chest surgery also includes repositioning and resizing of the nipple–areola complex when necessary. Preoperative Evaluation The diagnosis of gender dysphoria is generally made by mental health providers who then refer individuals for surgical evaluation. Before performing gender conﬁr- mation surgery, the surgeon must verify that the diag- nosis or gender dysphoria or gender incongruence is accurate. As noted by Dr. J.J. Hage, the surgeon re- Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 34 The surgical options for vaginoplasty consist of one of three options: penile inversion vaginoplasty, intestinal transplantation, or nongenital ﬂaps. Most centers per- form primary vaginoplasty with the penile inversion vaginoplasty using an anteriorly pedicled penile skin ﬂap combined with a posteriorly based scrotal-perineal ﬂap and/or skin graft. However, intestinal transposition, typically reserved for revision cases, is a ﬁrst-line surgi- cal therapy at some centers. The advantage of intestinal transposition is the creation of a vascularized 12–15cm vagina with a moist lining. This may lessen the require- ments for postoperative vaginal dilation as well as the need for lubrication during intercourse. However, the drawbacks of intestinal transposition include the need for an intra-abdominal operation with a bowel anasto- mosis and the potential for neovaginal secretions. Non- genital ﬂaps are typically considered for reconstruction following oncologic resections, traumatic repair, or re- construction following infection. Although understanding potential preoperative risk factors is important, their presence is not necessarily a contraindication to surgery. It must be emphasized that the SOC are not intended as barriers to surgery, but rather as a means of identifying patients who would beneﬁt from surgical therapy. In addition to mental health evaluation, additional input from a patient’s primary care doctor or endocri- nologist is useful. Documentation of hormone therapy, if applicable, and conﬁrmation that a patient is medically ﬁt for surgery are important before planning surgery. Once the surgeon is satisﬁed that the diagnosis has been established, surgical therapy is considered. A preop- erative surgical consultation is obtained, during which the procedure and postoperative course are described, the potential risks and beneﬁts of surgery are reviewed, and the patient’s questions are answered. Equally important is a discussion of the patient’s expectations as well as an understanding of the limitations of surgery. In a study of 55 transgender patients treated in Belgium, De Cuypere et al.9 noted that transgender persons’ expectations were met at an emotional and social level, but less so at the physical and sexual level. Preoperative Evaluation This occurred despite an indi- cated improvement in sex life and sexual excitement after reassignment surgery.9 Based upon these ﬁndings, it was recommended that discussion regarding sexual expecta- tions be entertained before surgery. Regardless of the technique utilized, the author rec- ommends that hormones are discontinued for 2 weeks out of concern for risk of venous thromboem- bolism (VTE). Some centers opt to continue hor- mones or change to the transdermal route before surgery; however, there are no studies evaluating clin- ical outcomes with these protocols. In addition, a pre- operative bowel prep is administered. Before surgery, sequential compression devices are placed and intrave- nous antibiotics are administered. Following induction of general anesthesia, chemoprophylaxis for VTE is ad- ministered subcutaneously (either fractionated or unfrac- tionated heparin, depending upon institutional policies), the patient is positioned in lithotomy, and an indwelling urinary catheter is placed. If an individual decides to proceed with surgery, written documentation of informed consent should be included in the patient’s chart. Gender Surgeries Transfemale genitalia surgery Transfemale genitalia surgery l f h Surgical conversion of the genitalia of transwomen has evolved since the use of skin grafts for creation of a neovagina in cases of vaginal agenesis.16 The use of pedicled penile and scrotal ﬂaps was described over 40 years ago and remains the foundation for neovaginal construction.17–19 The vascular basis of these ﬂaps is derived from one of two sources: (1) the femoral artery (deep and superﬁcial external pudendal arteries) and (2) the internal pudendal artery (perineal branches). Penile inversion vaginoplasty Penile inversion vaginoplasty Hair removal, whether by electrolysis or laser, is com- pleted as thoroughly as possible from the penile shaft and central perineum and scrotum (Fig. 1) before pe- nile inversion vaginoplasty. Preoperative depilation helps to prevent intravaginal hair growth. Adequate hair removal can take 3–6 months to complete and should not be performed within 2 weeks of surgery. Although a functional vaginoplasty is performed in a single stage, labiaplasty can be performed at a second sur- gical stage. Labiaplasty, which can be performed under local anesthesia as an outpatient procedure 3 months after vaginoplasty, creates a convergent anterior commis- sure and provides additional clitoral hooding. However, with recent trends toward hair removal of the mons, fewer individuals opt to proceed with the labiaplasty. Although a variety of technical modiﬁcations for penile inversion vaginoplasty are described in the litera- ture, the penile disassembly and inversion technique uti- lizes the penile skin and a second, posteriorly based, scrotal-perineal ﬂap to construct the vaginal cavity.16 The author’s preferred technique is described herein (Fig. 2): the labia majora are formed from the lateral Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 35 aspects of the scrotum, the neoclitoris is formed from the dorsal glans penis, and the labia minora are formed from the penile skin and urethral ﬂap. The penile ure- thra is shortened, spatulated, and everted to create the neourethral meatus. Depending upon the length of the penis and previous surgical history (i.e., circumci- sion), skin grafts may be required for additional vagi- nal depth. Full-thickness skin grafts may be harvested from discarded portions of the scrotum. If this is insuf- ﬁcient, additional full-thickness skin grafts may be har- vested with a Pfannenstiel incision.20 Alternatively, split- thickness skin grafts may also be harvested from the lower abdomen or mons region. FIG. 1. Approximate area of depilation for scrotoperineal ﬂap. FIG. 1. Approximate area of depilation for scrotoperineal ﬂap. The postoperative care consists of a variable period of bedrest, during which a vaginal stent or packing is used to maintain the vaginal cavity. A urinary catheter remains in place until the vaginal packing is removed and ambulation is initiated, typically 5–6 days after sur- gery. Once the vaginal stent is removed, a regimen of vaginal dilation with a prosthesis is begun. Additionally, intermittent vaginal douching with a dilute povidone–io- dine solution is performed to remove intravaginal debris. Penile inversion vaginoplasty Vaginal intercourse may begin 6–8 weeks after surgery. An annual speculum and prostate examinations are also recommended. FIG. 1. Approximate area of depilation for scrotoperineal ﬂap. Early postoperative complications include bleed- ing, infection, and delayed wound healing. Additional early or late complications include rectovaginal ﬁs- tula, urinary stream abnormalities, inadequate vaginal depth or constricted introitus, partial ﬂap loss, loss of neoclitoral sensation, and an unsatisfactory cosmetic appearance. FIG. 2. Postoperative penile inversion vaginoplasty. FIG. 2. Postoperative penile inversion vaginoplasty. Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Intestinal vaginoplasty The advantage of intestinal transposition, especially in revision cases, is the provision of a reliable length of vas- cularized tissue with mucus secretion providing lubrica- tion for vaginal intercourse. Intestinal transposition may utilize either the small or large intestine; however, the sig- moid colon is the most commonly used. The advantage of the sigmoid colon is the larger luminal diameter and less copious secretions compared with that of jejunum or ileum. Before performing the sigmoid vaginoplasty, a preoperative colonoscopy is performed soas toevaluate for pre-existing colorectal malignancies. The sigmoid vaginoplasty (Fig. 3) is also performed in the lithotomy position in conjunction with general surgery. A combined abdominal and perineal ap- proach is utilized and allows visualization and protec- tion of the bladder and urethra anteriorly and the FIG. 2. Postoperative penile inversion vaginoplasty. Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 36 FIG. 3. Postoperative intestinal vaginoplasty using sigmoid colon. and possible malodorous discharge with the large intes- tine. Additional concerns include the possibility of di- version colitis in the defunctionalized sigmoid colon as well as the risk of gastrointestinal malignancies. Finally, the colonic mucosa may be somewhat friable, and small amounts of postcoital bleeding may occur. Transfemale breast augmentation and facial feminization Additional feminizing procedures include both breast augmentation and facial feminization. The goal of these procedures is to remove the secondary sexual character- istics and stigmata associated with thebiological male ap- pearance. The timing of these surgeries in relation to genitalsurgerymayvarybetweencentersaswellaswithin individual centers. It is not uncommon for these feminiz- ing procedures to be performed before genital surgery so as to improve an individual’s sense of well-being.7 Following hormonal therapy, there is frequently some breast growth in the transwoman. However, the degree of breast growth is often inadequate, and indi- viduals may continue to wear external prostheses or padded bras. As such, augmentation mammaplasty may be requested. Anatomic differences between the male and female chests are relevant with regard to im- plant selection, incision choice, and pocket location.21 FIG. 3. Postoperative intestinal vaginoplasty using sigmoid colon. The male chest is not only wider than the female chest but the pectoral muscle is also usually more de- veloped. Intestinal vaginoplasty Furthermore, the male areola is smaller than the female areola, the distance between the nipple and inframammary crease is less, and there is less ptosis in the natal male breast, even after hormonal therapy.22 Based upon these characteristics, a larger implant is commonly chosen (Fig. 4). Pocket location and incision choice depend upon the individual and the degree of breast growth in response to hormonal therapy. A sub- glandular, subfascial, or subpectoral pocket may be used. Most often, silicone implants and, more recently, form-stable silicone implants are utilized. rectum posteriorly. Most recently, the harvest of the sigmoid colon was performed in a minimally invasive manner with the use of the operating robot and/or laparoscopic assistance. The sigmoid colon is har- vested by the general surgery team, and the perineal dissection is performed concurrently by the plastic surgery team. A 12–15-cm segment of sigmoid colon is transferred in an isoperistaltic manner. The defunc- tionalized sigmoid colon is sutured to the introitus of the neovagina. Additionally, the mesentery of the defunctionalized sigmoid colon may be sewn to the pelvis to prevent torsion of the vascular pedicle. An end-to-end colonic anastomosis is performed, and the distal stump of the neovagina is separated from the colorectal anastomosis so as to reduce the risk of ﬁstulization. In addition to breast augmentation, surgery to fem- inize the face of transwomen may be requested. A vari- ety of characteristics have been identiﬁed as male and are often associated with the forehead, nose, malar re- gion, mandible, and thyroid cartilage. The neovagina is packed with nonadherent gauze, and the patient remains on bedrest with a urinary cath- eter for 1–2 days. Upon return of bowel function and oral intake, the patient is discharged from the hospital. The neovagina is packed with nonadherent gauze, and the patient remains on bedrest with a urinary cath- eter for 1–2 days. Upon return of bowel function and oral intake, the patient is discharged from the hospital. The potential drawbacks of intestinal-based ﬂaps in- clude secretions, most notably with the small intestine, Because the female eyebrow is located above the su- praorbital rim and has a more arched appearance than the male, typical procedures for facial feminization in- clude a brow lift with advancement of the frontal hair- line and frontal bone reduction (Fig. 5). Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Transmale chest surgery Chest wall contouring is an important early surgical step in the process of gender conﬁrmation for transmen and mayhelptofacilitatetransition.Thegoalsofchestsurgery include the esthetic contouring of the chest by removal of breast tissue and skin excess, reduction and repositioning of the nipple–areola complex, release of the inframam- mary crease, and minimization of chest scars.28 FIG. 7. Preoperative transmale before chest surgery. Chest surgery in transmen presents an aesthetic challenge due to breast volume, breast ptosis, nipple– areola size and position, degree of skin excess, and po- tential loss of skin elasticity (Fig. 7). Choice of incision is largely determined by degree of breast ptosis and skin quality/elasticity, as well as position of the nipple–are- ola complex.29 Incisions may range from a periareolar incision in small breasts with a small areola and good skin elasticity, to circumareolar incisions, to transverse inframammary crease incisions with free nipple grafts. Liposuction may be used as an adjunct to excisional techniques. In general, the nipple areola is positioned just medial to the lateral border of the pectoralis major muscle, *2–3 cm above the inferior insertion of the pectoralis major muscle. Postoperative manage- ment includes drains and elastic compression. Secon- dary revisions related to the scar and or nipple– areolar complex are not uncommon. Metoidioplasty, described in 1996 by Hage, has been offered as an alternative to microsurgical or pedicled ﬂap phalloplasty in transmen (Fig. 8). The procedure entails lengthening the hormonally hypertrophied cli- toris by release of the suspensory ligament and resec- tion of the ventral chordee and lengthening of the female urethra with the aid of labia minora and/or vag- inal musculomucosal ﬂaps.29 Additionally, buccal mucosa grafts have been utilized to aid with urethral extension. Urethral reconstruction is the major challenge associated with metoidioplasty, and most complications involve ei- ther urethral ﬁstulae or strictures. The operative technique may involve concomitant re- moval of the female genitalia (vaginectomy) in addition to metoidioplasty.30 Most often, a hysterectomy and oophorectomy are performed before the metoidioplasty. A caudally based anterior vaginal wall ﬂap incorporating the muscularis of the ventral vaginal wall may be used. This ﬂap can be used to reconstruct the ﬁxed portion of the neourethra.31 The clitoral shaft is degloved and re- leased by detaching the suspensory ligament from the pubic bone. On the ventral aspect of the clitoris, the ure- thral plate is dissected from the clitoral bodies. Intestinal vaginoplasty Although the brow lift may be performed with an endoscope, The potential drawbacks of intestinal-based ﬂaps in- clude secretions, most notably with the small intestine, Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 37 37 FIG. 4. Breast augmentation in a transwoman. FIG. 4. Breast augmentation in a transwoman. reduction of the frontal bone and lateral brow, as well as the advancement of the frontal hairline, is facilitated with the open approach. Depending upon the thickness of the anterior table in relation to the degree of frontal bossing, craniofacial techniques may be employed for the desired correction. performed through intraoral incisions. Other proce- dures, such as upper lip shortening, facelift, blepharo- plasty, malar implants, hair transplantation, injectable ﬁllers, and skin resurfacing, may also be performed. Reduction thyroid chondroplasty may be reques- ted to reduce the appearance of the Adam’s apple or prominent thyroid cartilage (pomus Adamus) (Fig. 6). The procedure is typically performed as an outpatient under general or local anesthesia with sedation. The pro- cedure is performed through a transverse incision in a naturally occurring skin crease. Following vertical divi- sion of the middle cervical fascia, the sternothyroid and thyrohyoid muscles are retracted laterally. On A feminizing rhinoplasty typically involves dorsal hump reduction, cephalic trim, elevation of the nasal tip, and osteotomies to narrow the nasal pyramid (Fig. 6). Individuals may also request feminization of the chin and mandible. Based upon an individual’s anat- omy, either chin implants or osteoplastic genioplasty may be required. In addition, reduction of the masseter muscle or contouring of the mandibular angle may be FIG. 5. Frontal bone reduction with brow lift, upper lip shortening, and lip augmentation in a transwoman. FIG. 6. Rhinoplasty and thyroid chondroplasty in a transfemale. FIG. 6. Rhinoplasty and thyroid chondroplasty in a transfemale. FIG. 6. Rhinoplasty and thyroid chondroplasty in a transfemale. FIG. 5. Frontal bone reduction with brow lift, upper lip shortening, and lip augmentation in a transwoman. FIG. 5. Frontal bone reduction with brow lift, upper lip shortening, and lip augmentation in a transwoman. FIG. 5. Frontal bone reduction with brow lift, upper lip shortening, and lip augmentation in a transwoman. FIG. 5. Frontal bone reduction with brow lift, upper lip shortening, and lip augmentation in a transwoman. FIG. 6. Rhinoplasty and thyroid chondroplasty in a transfemale. Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 38 FIG. 7. Preoperative transmale before chest surgery. Transmale chest surgery The ure- thral plate is divided so as to release the ventral clitoral Intestinal vaginoplasty the posterior surface of the cartilage, subperichondrial dissection is performed inferiorly to the thyroepiglot- tic ligament. Identiﬁcation of the insertion of the vocal cords may be facilitated with ﬁberoptic laryngoscopy by the anesthesthiologist.23 Resection of the thyroid cartilage is performed between the superior thyroid notch in the midline and the superior thyroid tubercle superolaterally.24 Voice surgery, designed to raise vocal pitch, may be requested by individuals following voice therapy, al- though its efﬁcacy is debated.25,26 Hormonal interven- tion does not commonly affect vocal pitch and this may represent a residual stigma of masculinity. As such, various techniques to shorten the vocal cords, increase vocal cord tension, or reduce vibrating vocal cord mass may be performed.27 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Surgery for gender nonconforming/expansive individuals Phalloplasty represents the most complete genito- perineal transformation for transmen (Fig. 9). Phallo- plasty techniques may be divided into pedicled ﬂaps and free ﬂaps. Pedicle ﬂaps transfer tissue, typically of the thigh, groin, or lower abdomen, to reconstruct the penis, while free ﬂaps involve the microsurgical transfer of tissue from a remote location. It has been increasingly recognized that many individuals withgenderdysphoriadonotseethemselvesintraditional male or female categories. Gender nonconformity or gen- der expansive describes a difference between an individu- al’s gender identity, role, or expression and that of cultural norms. For these individuals, varying degrees of hormone therapy and surgical intervention may be helpful for allowing them to become their true selves. Similar to other transgender persons, a customized multidisciplin- ary approach is necessary to provide appropriate medical and surgical care to gender nonconforming individuals. The most common technique for phallic reconstruc- tion is the radial forearm-free ﬂap. This procedure transfers tissue, including blood vessels and nerves, from the forearm to reconstruct the penis and urethra. This ﬂap allows single-stage reconstruction of a sensate phallus and glans penis. Potential drawbacks of this technique include the visibility of the donor site on the forearm and the need for microsurgical skills. Transmale genitalia surgery The goal of genital surgery in transmen requires indi- vidualization. Surgery may range from clitoral release (metoidioplasty) with or without urethral lengthening (to allow for voiding while standing) to a phalloplasty, capable of sexual penetration.29 Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 39 FIG. 8. Postoperative metoidioplasty in a transman. FIG. 9. Postoperative phalloplasty in a transman. FIG. 8. Postoperative metoidioplasty in a transman. FIG. 8. Postoperative metoidioplasty in a transman. FIG. 9. Postoperative phalloplasty in a transman. curvature, allowing straightening and lengthening of the clitoris.30 Additional lengthening of the urethra is per- formed with ﬂaps developed from the labia minora. Additional phalloplasty techniques include the use of tissue from the back, known as the musculocutane- ous latissimus dorsi ﬂap. One notable downside of this ﬂap is the lack of a sensory nerve to the tissue that will be used to construct the phallus. Scrotoplasty, constructed with bilateral labia majora ﬂaps, may be performed at the time of metoidioplasty. Testicular implants are placed at a secondary surgical procedure so as to reduce the risk of infection and ure- thral complications. Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 Author Disclosure Statement No competing ﬁnancial interests exist. 29. Hage JJ, van Turnhout AA, Dekker JJ, Karim RB. Saving labium minus skin to treat possible urethral stenosis in female-to-male transsexuals. Ann Plast Surg. 2006;56:456–459. Measuring Outcomes The ﬁeld of gender conﬁrming surgery has grown with increased awareness, need, and third-payer coverage. However, there are still no formal training programs or speciﬁc board certiﬁcations for surgeons performing gender conﬁrming surgery. This will likely change in the coming years. Currently, surgery is performed by recon- structive plastic surgeons, urologists, and gynecologists, often in a collaborative approach. In addition, methods to accurately measure patient outcomes are still under development. Working groups are currently participat- ing with the National Institutes of Health to develop sys- tems to capture and report these important data. Perhaps the next most common technique for phallo- plasty is the use of tissue from the thigh, known as the anterolateral thigh ﬂap. Similar to the forearm technique, tissue, including nerves, may be transferred from the thigh to construct the penis. Depending upon the indi- vidual’s distribution of subcutaneous fat, a second ﬂap may be required for urethral reconstruction. For both the forearm and thigh techniques, the ure- thra is formed by a skin-lined tube. As such, preopera- tive electrolysis may be required for depilation of the urethra. Schechter; Transgender Health 2016, 1.1 http://online.liebertpub.com/doi/10.1089/trgh.2015.0006 40 Conclusion 19. Pandya NJ, Stuteville OH. A one-stage technique for constructing female external genitalia in male transsexuals. Br J Plast Surg. 1973;26:277–282. Signiﬁcant advances in social, medical, and surgical care have led to improved access to care for transgen- der individuals. Gender surgery presents one facet of a complex multidisciplinary approach to allow trans- gender individuals to become who they know them- selves to be. Continued collaboration between the surgeon, mental health professional, and medical phy- sician in the framework provided by the WPATH SOC guidelines is important in providing quality care to transgender individuals. In addition, continued re- search focused on objective parameters and reporting of outcome data will foster innovation and continued improvements in surgical techniques. 20. Hage JJ, Karim RB. Abdominoplastic secondary full-thickness skin graft vaginoplasty for male-to-female transsexuals. Plast Reconstr Surg. 1998;101:1512–1515. 21. Kanhai RC, Hage JJ, Bloemena E, et al. Mammary ﬁbroadenoma in a male- to-female transsexual. Histopathology. 1999;35:183–185. 22. Laub DR. Discussion: augmentation mammaplasty in male-to-female transsexuals. Plast Reconstr Surg. 1999;104:550–551. 23. Spiegel JH, Rodriguez G. Chondrolaryngoplasty under general anesthesia using a ﬂexible ﬁberoptic laryngoscope and laryngeal mask airway. Arch Otolaryngol Head Neck Surg. 2008;134:704–708. 24. Wolfort FG, Dejerine ES, Ramos DJ, Parry RG. Chondrolaryngoplasty for appearance. Plast Reconstr Surg. 1990;86:464–469; discussion 470. 25. Lechien JR, Delvaux V, Huet K, et al. [Transgender voice and communi- cation treatment: review of the literature]. Rev Laryngol Otol Rhinol (Bord). 2014;135:97–103. 26. McNeill EJ. Management of the transgender voice. J Laryngol Otol. 2006;120:521–523. 27. Ettner R. Principles of Transgender Medicine and Surgery. New York: The Haworth Press, 2007. 28. Monstrey S, Selvaggi G, Ceulemans P, et al. Chest-wall contouring surgery in female-to-male transsexuals: a new algorithm. Plast Reconstr Surg. 2008;121:849–859. References 30. Djordjevic ML, Bizic M, Stanojevic D, et al. Urethral lengthening in metoidioplasty (female-to-male sex reassignment surgery) by com- bined buccal mucosa graft and labia minora ﬂap. Urology. 2009;74: 349–353. 1. Brown GR. A review of clinical approaches to gender dysphoria. J Clin Psychiatry. 1990;51:57–64. 1. Brown GR. A review of clinical approaches to gender dysphoria. J Clin Psychiatry. 1990;51:57–64. 2. World Professional Association for Transgender Health. Press release May 26, 2010. Available at: www.wpath.org/uploaded_ﬁles/140/ﬁles/de- psychopathologisation%205-26-10%20on%20letterhead.pdf (accessed October 1, 2015). 2. World Professional Association for Transgender Health. Press release May 26, 2010. Available at: www.wpath.org/uploaded_ﬁles/140/ﬁles/de- psychopathologisation%205-26-10%20on%20letterhead.pdf (accessed October 1, 2015). 31. Hage JJ, Torenbeek R, Bouman FG, Bloem JJ. The anatomic basis of the anterior vaginal ﬂap used for neourethra construction in female-to-male transsexuals. Plast Reconstr Surg. 1993;92:102–108; discussion 109. 31. Hage JJ, Torenbeek R, Bouman FG, Bloem JJ. The anatomic basis of the anterior vaginal ﬂap used for neourethra construction in female-to-male transsexuals. Plast Reconstr Surg. 1993;92:102–108; discussion 109. 3. World Professional Association for Transgender Health. Standards of Care. 2011. Available at http://www.wpath.org/uploaded_ﬁles/140/ﬁles/ Standards of Care, V7 Full Book.pdf (accessed October 1, 2015). 3. World Professional Association for Transgender Health. Standards of Care. 2011. Available at http://www.wpath.org/uploaded_ﬁles/140/ﬁles/ Standards of Care, V7 Full Book.pdf (accessed October 1, 2015). 4. Monstrey S, Hoebeke P, Selvaggi G, et al. Penile reconstruction: is the radial forearm ﬂap really the standard technique? Plast Reconstr Surg. 2009;124:510–518. 4. Monstrey S, Hoebeke P, Selvaggi G, et al. Penile reconstruction: is the radial forearm ﬂap really the standard technique? Plast Reconstr Surg. 2009;124:510–518. Abbreviations Used SOC ¼ Standards of Care VTE ¼ venous thromboembolism WPATH ¼ World Professional Association for Transgender Health 5. Hage JJ, van Turnhout AA. Long-term outcome of metaidoioplasty in 70 female-to-male transsexuals. Ann Plast Surg. 2006;57:312–316. 5. Hage JJ, van Turnhout AA. Long-term outcome of metaidoioplasty in 70 female-to-male transsexuals. Ann Plast Surg. 2006;57:312–316. 6. Schechter LS. The surgeon’s relationship with the physician prescribing hormones and the mental health professional: review for version 7 of the World Professional Association for Transgender Health’s Standards of Care. Int J Transgend. 2009;11:222–225. g 7. Monstrey S, Hoebeke P, Dhont M, et al. Surgical therapy in transsexual patients: a multi-disciplinary approach. Acta Chir Belg. 2001;101:200–209. 8. Bowman CGJ. Care of the patient undergoing sex reassignment surgery. Int J Transgend. 2006;9:135–165. 7. Monstrey S, Hoebeke P, Dhont M, et al. Surgical therapy in transsexual patients: a multi-disciplinary approach. References Acta Chir Belg. 2001;101:200–209. Cite this article as: Schechter LS (2016) Gender conﬁrmation surgery: an update for the primary care provider, Transgender Health 1:1, 32–40, DOI: 10.1089/trgh.2015.0006. Cite this article as: Schechter LS (2016) Gender conﬁrmation surgery: an update for the primary care provider, Transgender Health 1:1, 32–40, DOI: 10.1089/trgh.2015.0006. 8. Bowman CGJ. Care of the patient undergoing sex reassignment surgery. Int J Transgend. 2006;9:135–165. 9. De Cuypere G, T’Sjoen G, Beerten R, et al. Sexual and physical health after sex reassignment surgery. Arch Sex Behav. 2005;34:679–690. 10. Lawrence A. Factors associated with satisfaction or regret following male- to-female sex reassignment surgery. Arch Sex Behav. 2003;32:299–315. 11. Lobato MI, Koff WJ, Manenti C, et al. Follow-up of sex reassignment sur- gery in transsexuals: a Brazilian cohort. Arch Sex Behav. 2006;35:711–715. Publish in Transgender Health -Immediate, unrestricted online access -Rigorous peer review -Compliance with open access mandates -Authors retain copyright -Highly indexed -Targeted email marketing liebertpub.com/trgh 12. Nagai ATE, Nanba Y, et al. Sex reassignment surgery for male to female transsexuals: initial experience at Okayma University Hospital. Acta Med Okayama. 2005;59:231–233. 13. Smith YVG, Kuiper A, et al. Sex reassignment: outcomes and predictors of treatment for adolescent and adult transsexuals. Psychol Med. 2005;35:89–99. -Immediate, unrestricted online access -Rigorous peer review -Compliance with open access mandates -Authors retain copyright -Highly indexed -Targeted email marketing 14. Petersen ME, Dickey R. Surgical sex reassignment: a comparative survey of international centers. Arch Sex Behav. 1995;24:135–156. 15. Eldh J. Construction of a neovagina with preservation of the glans penis as a clitoris in male transsexuals. Plast Reconstr Surg. 1993;91:895–900; discussion 901–903. 16. McIndoe AH, Bright J. An operation for the cure of congenital absence of the vagina. J Obstet Gynaecol Br Commonw. 1938;5:490–494. 17. Jones HW Jr, Schirmer HK, Hoopes JE. A sex conversion operation for males with transsexualism. Am J Obstet Gynecol. 1968;100:101–109. 18. Edgerton MT, Bull J. Surgical construction of the vagina and labia in male transsexuals. Plast Reconstr Surg. 1970;46:529–539.
W4226230312.txt	https://zenodo.org/records/6365482/files/3-BELBAHI,%20IMESSAOUDENE-11-19.pdf	fr		LA REMEDIATION DE L’ECRIT : EMPLOI DES PREPOSITIONS FRANÇAISES DANS LES PRODUCTIONS ECRITES DES APPRENANTS DU FLE EN ALGERIE / REMEDIATION OF THE WRITTEN WORD: THE USE OF FRENCH PREPOSITIONS IN THE WRITTEN PRODUCTIONS OF FLE LEARNERS IN ALGERIA	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	5,073	Studii şi cercetări filologice. Seria Limbi Străine Aplicate LA REMEDIATION DE L’ECRIT : EMPLOI DES PREPOSITIONS FRANÇAISES DANS LES PRODUCTIONS ECRITES DES APPRENANTS DU FLE EN ALGERIE / REMEDIATION OF THE WRITTEN WORD: THE USE OF FRENCH PREPOSITIONS IN THE WRITTEN PRODUCTIONS OF FLE LEARNERS IN ALGERIA 1 Résumé: La langue maternelle (Langue arabe) est la première langue (L1) que l'enfant algérien apprend auprès de ses parents et à l'école. Le sentiment de maîtrise et de facilité de cette langue entraîne une certaine sensation de sécurité linguistique et affective chez les apprenants. Parallèlement, dans les autres langues étrangères, comme la langue française, certains apprenants algériens ont tendance à se retrouverdans des situations stressantes face à des problèmes plus complexes comme par exemple l’emploi des prépositions françaises à l’écrit. Ainsi, l’enseignement de la langue arabe permet aux apprenants du FLE la maîtrise totale de l'expression écrite et orale. Cet enseignement, qui est un facteur très important participe au développement de leurs personnalités, mais, il doit les doter d'un instrument linguistique de base pour faciliter l'apprentissage des langues étrangères. Mots clés: Langue cible, langue source, interlangue, préposition, analogie Abstract: Native language (Arabic language) is the first language (L1) that the Algerian child learns from his parents and at school. The feeling of mastery and ease of this language leads to a certain feeling of linguistic and emotional security in the learners. On the contrary, in other foreign languages, as is the case with the French language, some Algerian learners tend to find themselves in stressful situations facing more complex problems taking as an example the use of French prepositions in writing. Thus, the teaching of the Arabic language allows FFL learners complete mastery of written and oral expression. This teaching, which is a very important factor, participates in the development of their personalities, but it must equip them with a basic linguistic instrument to penetrate the other different disciplines including the learning of foreign languages. Keywords: Target language, source language, interlanguage, preposition, analogy Introduction La langue française occupe une place prépondérante en Algérie. Elle est considérée, en effet,comme première langue étrangère occupant une place de choix dans plusieurs secteurs sensibles : politique, éducation ou médiatique...Il faut dire que la langue française a toujours existé dans le monde économique algérien et parfois même dans la conversation quotidienne chez certaines familles dans lesmilieux urbains. Dans le domaine de l'enseignement, le français est enseigné dès le primaire, à partir de la 3ème année de scolarisation. Toutefois, la majorité des apprenants sont confrontés à de nombreux obstacles dans l'apprentissage du F.L.E compte tenu de l'environnementsociolinguistique, des conditions matérielles et d’autres facteurs inhérents à chaque individu... Ainsi, parmi les difficultés linguistiquesobservées chezles apprenants du F.L.E, aucours de nos expériences pédagogiques au cycle moyen, nous pouvons signaler la récurrence de''fautes'' sur une catégorie grammaticale qui est très importante dans le domaine de laconstruction du langage français. En effet, la redondance de ces fautes, à travers les productions écrites et lesdifférents exercices réalisés par les élèves, a éveillé notre intérêt pour enfaire l'objet de notre étude. Du point de vue épistémologique des descriptions grammaticales, l'objet « préposition » attire vite l'attention de l'observateur : la préposition est un objet de langage que l'on reconnaît à son fonctionnement mais dont onne sait pas très exactement définir la nature et la place à l'intérieur du discours. Les prépositions jouent un rôle essentiel dans les structures analytiques de la langue.Elles servent à exprimer des rapports entre des choses, des idées…A vrai dire, l'emploi d'une préposition à la placed'une autre peut être considéré comme une erreur. Éléments diachroniques de base Taieb BELBAHI, Université d’Oran 2, Algérie, belbahitaieb@gmail.com, Mohamed Fouzi IMESSAOUDENE, Université d’Ahmed Benyahiya Elwancharissi – Tissemsilt, Algérie, ifoozy@yahoo.fr 1 11 Studii şi cercetări filologice. Seria Limbi Străine Aplicate Le travail que nous présentons sert à recenser les erreurs communes commises par les apprenants du FLE en essayant de présenter des stratégies de remédiation à l’écrit quant à l’emploi correct des prépositions.Pour cela, nous nous inspironsdes sciences du langage et plus précisément de la linguistique contrastive. Si la linguistique se définit comme une branche de la psychologie cognitive dont latâche est la caractérisation rigoureuse du savoir linguistique des locuteurs particuliers, ilserait nécessaire d'effectuer des recherches profondes pour savoir et comprendre comment cesavoir linguistique se développe chez ces locuteurs. Ainsi, « si la formule contemporaine de centration sur l'apprenant n'était pas encore enCirculation, Tisanière soulignait régulièrement l'importance de suivre le rythme des élèveset de répondre à leurs besoins et demandes »1. C'est à Ferdinand De Saussure que l'on peut faire remonter la linguistique synchronique,qui nous permet d'étudier le français contemporain car les études précédentes se focalisentprincipalement sur les problèmes traitant de l'évolution des langues. En effet, c'est grâce à DeSaussure que l'on peut désormais considérer comme acquis qu'une langue fasse l'objet d'uneétude à un moment donné de son développement. Ferdinand De Saussure a donné un acte de naissance à la synchronie pour ouvrir unenouvelle ère dans l'étude des langues. Cette étude permet de voir la languehorizontalement, c'est à dire voir les relations internes qui s'établissent entre les signes qui lacomposent à un moment donné de son histoire. Nous considérons, effectivement, l'approche synchroniquecomme étant susceptible de sous-tendre notre travail. La langue est considérée comme « un système de signes dont il existe un ensemble derègles qui lient les uns par rapport aux autres, si bien que, si l'un des termes est modifié,l'équilibre du système est affecté »2. Ainsi, ces signes ne peuvent être définis que par opposition. L'ensemble de cesoppositions constitue dans chaque langue « un système, ou plutôt un système desystèmes ».3 dont le système syntaxique qui fait partie de notre travail. Les relations dont on a parlé précédemment sont les règles qui constituenttelle ou telle langue. Donc, on peut considérer que « toute langue est un systèmeparticulier de règles »4. Ceci nous amène à estimer que la langue est unsystème de règles,autrement dit, la langue est régie par une loi, cette loi est ce qu'onappelle la syntaxe. Cette dernière est composée d'un stock réduit de mots et phrasesà partir desquelles toutes autres énoncés peuvent et doivent en être dérivés par desopérations transformationnelles.De ce fait, notre travail essaie de vérifier le degréd'application des règles qui régissent la langue française dans un champ linguistiqueprécis :il s’agit de l'emploi des prépositions à l'écrit en classe de quatrième Année Moyenne. Notre expérience nous a permis de remarquer que les apprenants algériens duF.L.E utilisent de manière particulière les prépositions françaises : Ils commettentdes erreurs quant à l'emploi des prépositions, certains apprenants ne produisent pas lapréposition là où elle est attendue, parfois elle apparaît là où elle ne doit pas êtreproduite, et là où une forme est attendue, une autre forme est plutôt rendue.Les erreurs les plus courantes se trouvent au niveau des prépositions : dans - sur -avec - par - à - de - en, …et avec l'emploi de quelques locutions prépositives pourexprimer les rapports exprimantla cause, le but, le temps, etc. Cette situation a attiré notre attention en nous rendant compte que ce mauvaisemploi est loin d'être de simples fautes ou erreurs. Des lors, plusieurs questionsont émergé: -Quelles sont les erreurs commises par ce groupe d'apprenants du F.L.E ? Madray-Lesigne, F., et Richard-Zappella, J., (1995), : « Lucien Tesniereaujourd’hui », Editions, Peeters. Paris.P. 80. 2 Baylon, C. ; Fabre, P., (2005), :« Initiation à la linguistique, Cours et applications corrigés »., ArmandColin.2èmeEdition, P.19 3 Ibid. P. 19. 4 Ibid.P.20. 1 12 Studii şi cercetări filologice. Seria Limbi Străine Aplicate - Pourquoi commettent-ils des erreurs syntaxiquesquant à l'emploi des prépositions en production écrite ? - Comment peut-on réduire l'écart entre la langue de cesapprenants et la norme ? Objectifs de l’expertise -Recenser les fautes des apprenants algériens du F.L.E en classe de 4AM quant àl'emploi des prépositions françaises à l'écrit. -Identifier les différentes formes de déviances relatives à l'emploi des prépositions etidentifier leurs causes. -Montrer l'influence de la langue maternelle L1 sur l'apprentissage du F.L.E L2 (Langue cible). -Repérer les difficultés qui entravent l'apprentissage de la langue étrangère (Cas desapprenants du F.L.E au cycle moyen en Algérie : Emploi de la préposition à l'écrit). -Réduire l'écart qui existe entre la langue des apprenants et la norme(langue à laquelle tout apprenant du F.L.E doit y faire référence). Ainsi, Nous tentons de démontrer : -L'influence de l’approche du dialecte idiosyncrasique (D.I) des apprenants quiparticipe à l’élargissement de l’écart par rapport à la norme dans la construction de lalangue chez les apprenants. -Découvrir les stratégies utilisées par ces apprenants pour communiquer en FLE etsavoir comment ils réorganisent le système syntaxique de la langue cible (Français). Pour concrétiser notre étude, nous avons émis les hypothèses suivantes : 1- Les erreurs commises par ces apprenants informateurs sont dues au phénomène d’interférence (influence par la langue maternelle L.1). Là, on parle de l'influence d'une ou de plusieurs langues connues desapprenants dans le processus d'apprentissage de la langue cible (L.2). -L'arabe régulier. -L'arabe dialectal. -Le tamazight. 2- La différence entre l'interlangue des apprenants et la langue cible peut participer àl'élargissement de l'écart entre les productions fautives (les déviances) et la norme(la production correcte). 3-Les productions déviantes des prépositions sont relatives aux difficultés de lalangue cible (la langue française).Là, on vise à étudier les difficultés du système syntaxique de la langue cible telles que ladiversité des catégories et les sous-catégories des prépositions, l'analogie, laconcurrence entre deux prépositions proches de sens,etc. Exemple : 1-La Confusion entre le sens des prépositions de localisation « à, dans,sur, de, en,avec, après, par, pour » : -* Elle est dans la fenêtre - Elle est à la fenêtre. 2-La concurrence entre deux prépositions qui possèdent presque le même sens(parenté de sens). Pour vérifier les hypothèses émises, nous recueilleronsdes productions linguistiques des apprenants pour cerner les erreurs ou les fautes relatives à l’emploi desprépositions chez des apprenants en français langue étrangère « F.L.E » parl’intermédiaire de la technique « analyse d’erreurs ». Méthode Pour ce faire, nous noussommes penchés sur : Des productions écrites :A ce niveau, nous proposons d’étudier lesproductionsécrites de deux classes de 4A.M en situation d’apprentissage du françaislangue étrangère : Un groupe de 80 apprenants sera choisi. 13 Studii şi cercetări filologice. Seria Limbi Străine Aplicate L’enquête s’est déroulée au début du 1er trimestre de l’année scolaire, les apprenants sontinvités alors à produire des paragraphes dans lesquels ils décrivent un lieu où ils doiventfaire sa publicité pour une éventuellevisitetouristique. L'objectif à atteindre est la production écrite d'un texte descriptif(description itinérante) à visée argumentative. Dans un tel sujet les apprenants doiventemployer les prépositions qui indiquent l'espace (le lieu) ou la destination telles que : à, chez, dans, devant, derrière, parmi, sous, sur, vers ; Celles qui marquent le but : Envers, pour, afin de, etc. La lecture des productions écrites des apprenants nous a permis de relever des constructions déviantes.Les erreurs commises s'ordonnent autour des propriétés de la langue : emploi du nom, accord de l'adjectif qualificatif, accord du verbe avec son sujet et l'emploi de la préposition.La correction des copies a permis de repérer deux formes de déviances : certaines sont d’ordre systématique (les erreurs), d'autres sont occasionnelles(lesfautes). a- L'emploi des prépositions dans/à L'item 01 * - « Oran se situe dans l'ouest algérien » ainsi que l’item 03* « Nous habitons dans Tissemsilt » sont des constructions fautives.Ces formes présentent un écart par rapport à la norme de la langue française.Les deux formes sont considérées comme des constructions déviantes qui s'opposent à l'usage courant ou à la forme la plus attendue. Les apprenants devaient écrire : + Oran se situe à l'ouest algérien au lieu de * Oran se situe dans l'ouest algérien.Du point de vue sémantique, la différence entre à et dans concerne la propriété de la localisation spatiale.Avec la préposition à, le lieu est conçu comme un point mais avec la préposition dans, on conçoit l'intériorité du lieu. b- L'emploi des prépositions en/vers * " Je suis parti en forêt '' est un item fautif qui devait amener à un autre item correct : + Je suis parti vers la forêt.Là, la préposition vers devait remplacer la préposition en car la transgression linguistique ici porte sur la nécessité de marquer la propriété '' destination '' alors que celle qui a été donnée par l'apprenant est ''statique ''. L'emploi de la préposition ''en'' par les apprenants dans cette phrase peut se justifier par la définition du dictionnaire Larousse : " préposition qui marque le lieu, letemps, l’état, lamanière, etc.''. L'item 05 * Il grimpe O l’arbre.Cet item est aussi fautif.La majorité des élèves ont supprimé la préposition au point de dire que c'est un exercice facile. Nous avons constaté que la transgression ici porte sur la structure. La question se posait sur la transitivité et la non transitivité du verbe grimper dans cette phrase.Ici, le verbe est utilisé comme une forme transitive justifiée par le système de la langue maternelle (languearabe) dont le verbe grimper(talaa) en arabe est transitif et ne nécessite pas l'emploi de la préposition Or la forme correcte est : + Il grimpe à l'arbre. Les transgressions dans ce genre de constructions s'effectuent généralement sur la propriété du verbe « Cette propriété se caractérise par le fait de la suppression de la préposition. Elle amène un emploi acceptable et sémantiquement très voisin de l'emploi intransitif » » 1. Les élèves appliquent fautivement cette propriété en raison de la proximité sémantique de l'emploi des verbes '' grimper, monter, venir, voler, survoler, …'' dans des structures transitives comme le cas du verbe escalader dans escalader une montagne,et monter dans monter un cheval,etc. 1 Guy, F., (1987), : « le français scolaire en Algérie ». Office des Publications Universitaires. Alger.p74. 14 Studii şi cercetări filologice. Seria Limbi Străine Aplicate De la même façon, on peut former plusieurs phrases sous la forme :Sujet + Verbe + Complément d'objet direct en langue arabe.Nous proposons quelques exemples pour mieux éclaircir la situation. -+ J'ai visité Alger : S+V+COD. -+ J'ai pris le bus : S+V+COD. -* Il frappe la porte ou * Il frappe dans la porte, au lieu de + il frappe à la porte. -* Il joue un match au lieu de + il joue à un match : (S+V+COI). L'analyse de l'item 05 nous permet de classer ce genre de fautes dans la catégorie portée sur la propriété'' constructions transitives ou constructions intransitives du verbe '' dans la langue cible. Grimper : " V.T, monter en s'aidant des pieds et des mains : grimper aux arbres // monter sur un point élevé sur une petite pente raide ; Grimper au sommet d'un piton rocheux // s'élever en pente raide, chemin qui grimpe dans la montagne."Ainsi, Larousse présente une deuxième définition du verbe grimper qui est la suivante : V.T.Escalader : Grimper un escalier à toute vitesse.Cette définition peur participer à l'élargissement de l'écart par rapport à la norme de la langue cible dans l'emploi d'un tel verbe dans les productions écrites des apprenants.Face à cette situation, l'élève se trouve dans l'ambiguïté : transitivité ou non transitivité du verbe grimper.Ainsi, la langue arabe, qui est très proche de la langue française du point de vue sociolinguistique (coexistence), peut élargir la fréquence d'écart par le phénomène appelé par les linguistes '' contamination linguistique ''. L'analyse des formes déviantes sur l'emploi des prépositions à et dans nous indique que l'écart existant par rapport aux règles de l'emploi des prépositions en français est considéré comme des transgressions des règles de la langue cible que Guy Fève les rattache au phénomène linguistique qu'il appelle ''Analogie''.Guy Fève donne le même exemple que nos informateurs ont commis comme erreur: Il grimpe l'arbre. Il explique que cet item est produit suite à une analogie avec une autre structure voisine de langue cible sous la formule : Sujet +Verbe +Complément d'objet direct comme dans l'exemple suivant : IL grimpe l'escalier.L'analogie est définie comme « un rapport de ressemblance établi entre deux ou plusieurs choses ou personnes : Analogie de forme, de goûts » 1.Sous cet effet, un apprenant écrira Il a grimpé l'arbre comme on écrivait +Il grimpait l'escalier. C- Emploi des prépositions à / de La préposition'' à'' s'emploie après les verbes à un complément indirect ou après les verbes à deux compléments indirects.Parmi ces verbes qui admettent un complément indirect, on cite : -Mentirà quelqu'un -Sourireà quelqu'un -arriverà quelqu'un -Manquerà quelqu'un -Resterà quelqu'un -Plaire à quelqu'un -Ressembler à quelqu'un -S'intéresser à quelqu'un -Penser à quelqu'un -Tenir à quelqu'un -Parler à quelqu'un -Se plaindreà quelqu'un -Servirà quelqu'un -Pardonnerà quelqu'un -Ecrire à quelqu'un -Répondre àquelqu'un -Télégraphier àquelqu’un -Téléphonerà quelqu'un 1 Le petit Larousse., (1986), : « Dictionnaire de la langue française »,Librairie Larousse. P. 4 15 Studii şi cercetări filologice. Seria Limbi Străine Aplicate Il est bien utile de noter que ces verbes n'admettent que des objets animés.Exemple: Il écrit une lettre à son père.Le SN ''son père'' est un nom animé.Le SP '' à son père" est un complément d'objet indirect. Nous présentons également une autre liste de verbes qui s'emploient avec la préposition '' à '' + Complément indirect (objets inanimés) sous la formule suivante: Verbe +préposition+ N. inanimé. -Arriver à quelque chose. -Manquer à qqch. -Ressembler à qqch. -S'intéresser à qqch. -Se mettre à qqch. -Penser à qqch. -Tenir à qqch. -Servir à qqch. Pour mieux expliciter cette présentation, nous proposons les exemples suivants sur l'emploi de ces verbes avec la préposition ''à ''. -Il va à la plage. -Je n'arrive à rien. -Il tient à être là. Nous signalons aussi que certains verbes admettent deux compléments indirects grâce à l'emploi des deux prépositions : "à " et "de" comme dans l'exemple suivant : « Pierre parle de Paul à Marie» 1.Parmi ces verbes qui admettent deux compléments indirects, nous citons : 1-Les verbes : Parler, se plaindre, Exemple :'' Pierre parle(se plaint) de Jean à Marie "2. 2-Le verbe " servir" comme dans l’énoncé :-ça sert aux chasseurs, ça leur sert (leur = à qqn). » 3 On remarque que le verbe " servir " dans la phrase suivante présente un ensemble de constructions particulier, à partir du syntagme : ça sert = c'est utile. Bilan d’analyse des résultats obtenus L'analyse des productions écrites nous a permis de recenser les erreurs commises parces apprenants sur l'emploi des prépositions françaises relatives à la description itinérante. Nous avons pu déterminer les propriétés qui n'ont pas été respectées et que nous classons en trois catégories : 1-Des constructions fautives relatives qui sont portées sur la propriété distributionnelle(Lieu- source- destination- statique-direction). 2-Des constructions fautives qui sont portées sur la propriété transformationnelle (Confusion syntaxique ou morpho-syntaxique). 3-L'absence de relation entre la sémantique et la grammaire. Après l'analyse des productions écrites, nous avons opté pour des exercices de types scolaires enproposant au même public des activités sousforme de Questions à Choix Multiples (QCM). Nous avons placéces apprenants en situation contraignante. En effet, ce deuxième exercice a agi comme un contre - test des productions écrites libres.Par un tel exercice, nous avons vouluvoir si nous allions trouver les mêmesrégularités de fautes que celles qui existent dans les productions écrites. Nousattendons à ce que l’analyse des réponses des apprenants montre les différentesdifficultés que les apprenants éprouvent en français. Résultats et discussion La langue maternelle L1 agit sur les habitudes linguistiques des apprenants du F.L.E. des mécanismes inconscients interviennent sur la langue étrangère, ce qui fait qu'il ya une 1 Le Goffic, P., McBride, N-C., Gross, M., Debyser, F.,(1975), : « Les constituants fondamentales du français »., Librairies Hachette et Larousse. Paris. France..P.53. 2 Ibid 3 Ibid 16 Studii şi cercetări filologice. Seria Limbi Străine Aplicate spontanéité entre la pensée et l'interprétation linguistique. Alors, que faut-ilfaire dans une telle situation d’apprentissage ? Les résultats obtenus à travers l'analyse des productions écrites et du test (Q.C.M)affirment l'empreinte de la langue maternelle sur les habitudes linguistiques acquisespar les apprenants de la langue française. En revanche, la langue étrangère peut être acquise sans avoir le recours à cettespontanéité entre langue et pensée. C'est la raison pour laquelle D.Seleskovitch a ditque lorsqu'on parle sa langue maternelle, on plie la langue à sa pensée ; Lorsqu'onparle une langue étrangère, on plie sa pensée à la langue. D'aprèsSeleskovitch, la langue maternelle joue un rôle négatif en ce qui concernel'apprentissage et la maîtrise d'une langue étrangère car elle est la cause des différentes interférences.Cette hypothèse suppose que la langue maternelle interfère sur l'énoncé etparticipe à l'élargissement de l'écart de l'interlangue des apprenants du FLE parrapport à la norme de la langue cible. L'interférence se manifeste au niveau profond de la compétence de l'apprenant duFLE et agit sur la stratégie qu'il met en place pour énoncer en langue étrangère.Notre rôle est de procéder à séparer les langues en prenant conscience de laspécificité et la démarche propre à chacune des deux langues en contact en évitant lesinterférences.Ainsi, les enseignants de la langue étrangère FLE doivent lutter contre cephénomène linguistique qui s'impose. Les deux langues L1 et LE peuvent agir négativement l'une sur l'autre, mais on peuts'en servir pour activer et enrichir l'expressivité. Ainsi, l’on se pose la question suivante : comment faire de l'erreur un élément dynamisant de l'apprentissage ? Tout d'abord, il faut partir du principe que chaque apprenant a droit à l'erreur.L'apprenant du FLE produit spontanément des messages approximatifs, plus oumoins éloignés des normes en vigueur. Il a le droit de se tromper puisqu'il est en traind'apprendre une langue qui est étrangère pour lui. « Il faut accepter le risque de l'erreur comme une phase normale etconstructive des connaissances à acquérir »1.Ce que nous proposons dans une telle situation d'apprentissage c'est d'inciter lesapprenants à écrire car c'est en écrivant qu'on apprend à écrire.Tout est prétexte de l'écriture. Et c'est à travers les écrits qu'on peut savoir comments'organise l'apprentissage d'une telle ou telle langue (maternelle ou étrangère).S’ensuit l'étape qui consiste à repérer les erreurs des apprenants: (Phase de repérage deserreurs), puis vient l'étape de dialogue, d'analyse, d'explication, de reformulation etde réécriture:(La phase de remédiation).L'enseignant peut se servir de l'erreur de l'apprenant comme prétexte pourl'inciter à l'apprentissage de la langue.L'erreur pourrait être un élément adjuvant dans le processus d'apprentissage si on s'ensert justement. Si certaines langues sont proches, d'autres par contre s'éloignent radicalement lesunes des autres.C'est par la connaissance de cet éloignement que l'enseignant va pouvoir comprendreles difficultés de ses apprenants et c'est par sa prise en compte qu'il va pouvoir construirele cadre didactique de son intervention au plus proche des besoins de chacun d'eux. Il faut dire que l'apprenant algérien emploie involontairement ce qu'il a appris en sa languematernelle (L1) dans la langue cible (LE).Et comme ce crible ne convient pas pour la langue étrangère, il se produit denombreuses erreurs et incompréhensions.Ainsi, lorsqu'on apprend une langue étrangère, on fait tout d'abord le parallèle avecnotre langue maternelle au niveau de la syntaxe. Les interférences de la langue maternelle sur la langue cible peuvent êtrephonétiques, morphologiques, syntaxiques, lexicales ou sémantiques.Ce qui touche notre travail, c'est l'influence de la langue maternelle sur la languecible, phénomène qui se place sur deux plans: - Sur le plan lexical - Sur le plan des structures (le plan syntaxique) : ici, l'apprenant transfère directement dans la nouvelle langueétrangère la règle connue dans sa langue maternelle. Il s'agit de la constitution del’interlangue.Cette action passe par trois étapes: L'apprenant s'engage dans une action mentale consciente pour trouver du sens à lanouvelle langue (la phase cognitive). 1 Ferrand, N. (1993), :« Actes de lecture », Atelier ACLER, N° : 42. Maison de la promotion sociale.P.03 17 Studii şi cercetări filologice. Seria Limbi Străine Aplicate L'apprenant associe à tout ce qui est déjà connu : c'est l'émergence de l'interlangue(la phase associative). Quand l'apprenant utilise la langue étrangère, il se rapproche de plus en plus desa langue maternelle : Il ne pense plus aux structures de la langue cible. (La phased'autonomisation). Nous avons démontré que, dans certains cas, ce phénomène peut être conscient :L'apprenant construit des énoncés linguistiques sur le modèle syntaxique de la languematernelle.L'opération se fait par des représentations mentales, l'erreur devient donc nécessaire dansl'apprentissage d'une langue étrangère.C'est grâce aux erreurs que les enseignants peuvent définir et programmer desapprentissages adéquats à leurs apprenants. Un autre point peut être ajouter et qui favorise l'apprentissage d'une langueétrangère, et plus particulièrement le français.En général, tout système éducatif a pour mission de développer la personnalité desapprenants(citoyens) afin de les préparer à la vie active enleur faisantacquérir des connaissances générales, scientifiques et technologiques et en répondant auxbesoins de la nation et de la patrie.Partant de ces principes, il faudrait penser à préparer nos enfants à cette réalité dèsque possible.L'enseignement au préscolaire est l'une des mesures qui a été prise par le systèmeéducatif algérien dans le cadre des réformes pédagogiques.Cet enseignement est destiné aux enfants qui n'ont pas atteint l’âge scolaire obligatoire.Les études et les travaux de recherche effectuées dans ce domaine ont démontré quele préscolaire est devenu un espace de socialisation de petite enfance. Ainsi, les travaux menés sur le préscolaire, attestent de l'effet positif d'une prise en charge pédagogique précoce avant six ans.De ce fait, la préscolarisation des enfants participe effectivement à laconstruction des savoirs et le développement du langage en arabe et en français. Nous citonsci-dessous quelques prépositions qui causent le plus souvent des problèmes chez les apprenants informateurs auprès de qui nous avons collectés notre corpus : A- La préposition : -à : Préposition qui « exprime un rapport de lieu, de temps, de destination, de possession, de moyen, de manière, de prix, etc. »1. Cette préposition introduit un complément d'objet indirect, un complément d'attribution, un complément du nom ou un complément de l'adjectif. Ainsi, elle introduit un complément marquant un rapport de direction, de destination, de lieu, d'appartenance, de moyen, de manière ou de temps. B- La préposition : -Dans : C'est une préposition qui marque « lelieu, le temps, la manière, l'évaluation… »2.Les exemples donnés par le dictionnaire Larousse, n'enlèvent pas l'ambiguïté existant entre les deux prépositions à et dans car les deux prépositions marquent le lieu et le temps. C- Les prépositions - à et de : elles sont les prépositions les plus fréquentes en français, elles sont amenées par un verbe (il vient de sortir, il pense à son père), un nom (l'arrivée de l’hiver) ou un adjectif (il est fier de soi). Elles introduisent un complément circonstanciel de temps (Il est sorti à cinq heures). Elles se trouvent dissimulées sous des formes contractées au, aux, du et des (Il va au village, il vient du collège). La préposition '' à '' indique : -l'appartenance (Ce livre appartient à Mourad) -une manière d'être ou une fonction (Venir à plusieurs, Une boite à outils) -des rapports de direction (aller à l'école). des rapports de position ou de lieu (vivre à l'étranger). La préposition « de » : Elle indique une relation précise, comme par exemple : -la provenance (Il vient de Palestine). Le temps et la durée (de septembre à octobre). -La manière (de l'avis de tous). -La matière (une table de verre). -L'appartenance (les patates de Rovigo). -Le moyen ou l'instrument (Il est armé d'un fusil). -La mesure (avancer de deux crans). Ainsi, la préposition ''de'' est vide de sens dans certains emplois (elle introduit tout simplement un complément) comme dans l'exemple suivant : La ville de Tlemcen. Comme on vient de le constater, les valeurs des deux prépositions sont très proches, d'où ces confusions qui perturbentles apprenants lorsqu’ils veulent les employer à l’écrit. 1 Le petit Larousse., (1986), : « Dictionnaire de la langue française », Librairie Larousse, P.01. Ibid.P.280 2 18 Studii şi cercetări filologice. Seria Limbi Străine Aplicate Conclusion et perspectives pédagogiques Si certaines langues étrangères sont proches de la langue arabe d'autres par contre sont éloignées.En contexte d’enseignement du FLE, la connaissance de cettedistance s’avère nécessaire pourla compréhensiondes difficultés de des apprenants et c'est par leurprise en considération qu’il va pouvoir suivre une approchecurriculaire qui prend en considérationles besoins identifiés à travers les erreurs. A vrai dire l'apprenant algérien puise involontairement de sa languematernelle (L1) lorsqu’il s’exprimeen langue cible (LE).Et comme ce crible ne convient pas pour la langue étrangère, il se produit denombreuses erreurs et incompréhensions : tel est le cas du mauvais emploi des prépositions françaises par les apprenants algériens du FLE. Conséquemment, nous préconisons àune prise en charge des apprenants du FLE dès le premier âge.Les études et les travaux de recherche effectuées dans ce domaine ont montré que le préscolaire est devenu un espace de socialisation de petite enfance. Ainsi, les travaux menés sur le préscolaire, attestent de l'effet positif d'une prise en charge pédagogique précoce avant six ans. De ce fait, nouspensons quela préscolarisation des enfants participe effectivement à laconstruction des savoirs et le développement du langage en arabe et en français.Ceci prouve l'importance de la langue maternelle, qui est condition nécessaire etpréalable pour apprendre une langue étrangère.Dans ce sens, l'apprentissage précoce d'une langue étrangère autre que la languematernelle, ne peut être qu'un facteur d'enrichissement individuel ou collectif; Ilpermet au pluralisme d'exister et il est prometteur d'ouverture sur la culture de l'autremonde.En fin de cette étape, nous proposons l’intégration du FLE au cycle préscolaire, âge de découverte et d’acquisition de premières compétenceslinguistiques. Bibliographie Baylon, C. ; Fabre, P., (2005), :« Initiation à la linguistique, Cours et applications corrigés »., ArmandColin.2èmeEdition. Ferrand, N. (1993), :« Actes de lecture »,Atelier ACLER, N° : 42.Maison de la promotionsociale. Guy, F.,(1987), :« le français scolaire en Algérie ». Office des Publications Universitaires. Alger. Madray-Lesigne, F., et Richard-Zappella, J., (1995), : « Lucien Tesniereaujourd’hui »,Editions, Peeters. Paris. Le petit Larousse., (1986), : « Dictionnaire de la langue française »,Librairie Larousse. Le Goffic, P., McBride, N-C., Gross, M., Debyser, F.,(1975),: « Les constituants fondamentales du français ».,Librairies Hachette et Larousse.Paris.France. Seleskovitch, D., (1975),: « Langage, Langues et mémoire, étude de la prise de notes en interprétation consécutive », Minard Lettres Modernes, Paris. Taieb BELBAHI, enseignant et Maitre-Assistant au département de français à l'université d’Ahmed Ben Yehia Elwancharissi de Tissemsilt- ALGERIE. Il est titulaire de doctorat en lettres arabes et prépare un doctorat en didactique et sciences du langage à l'université d'Oran2 - Algérie. Il était membre du laboratoire de l'analyse du discours à l'université Oran1. Il a déjà publié des articles en arabe et en français dans des revues nationales en Algérie. Mohamed Fouzi IMESSAOUDENE, docteur en langue et littérature française, maitre de conférences exerçant comme enseignant-chercheur au département de français de la faculté des lettres et des langues à l’université de Tissemsilt–Algérie. Ses recherches et publications traitent du domaine didactique en l’occurrence les langues spécialisées, le Français sur Objectifs Spécifiques/universitaires/professionnels (FOS) (FOU) (FLP) et de l’ingénierie de formation en contexte du Français Langue Etrangère (FLE) 19
https://openalex.org/W2280113084	https://thescipub.com/pdf/jcssp.2012.393.397.pdf	English	null	An Analysis and Overview of Wireless Routing Protocols	Journal of computer sciences/Journal of computer science	2,012	cc-by	3,428	INTRODUCTION discussed the requirements of the NGN and Chandra and Baskaran (2011) proposed barrier free service architecture for providing reliable transmission in the NGN. This study is further focusing in the routing protocol of the wireless communication. In the past few years, the wireless communication emerges enormously in the computer communication industry. Based on its higher demands and necessity, there are many research issues in the wireless communication such as Optimal routing protocols, Traffic Management, Providing QoS, Offering reliable services, Effective Transport Control Protocol (TCP), Medium Access Scheme, Energy Management, Scalability, Load Balancing, Efficient Node Deployment, Self-organization and Service Discovery. The wireless communication has two major types of network, which are (1) infrastructure based network and (2) adhoc network. Both of these wireless networks spreads its importance in everyone life through cellular communication and Mobile Adhoc Network (MANET). In which, the cellular communication is a infrastructure based communication and the MANET is an adhoc network. The cellular communication is evolved as 1G for circuit switching, 2G for circuit and packet switching, 3G for video conference and4G for IP based networks. The NGN is in the current research in the cellular communication which is also termed as 5G+ (5G and above). There are many recent researches are proposed (Akyildiz et al., 2005; Lee and Knignt, 2005; Chandra and Baskaran, 2011) in the NGN, in which Akyildiz et al. (2005) proposed architecture and design of the next generation network, Lee and Knignt (2005) The routing protocol lies in the network layer, the task of wired routing protocol is simply exchanging route information and finding feasible path, but in the wireless routing there are some more tasks are added to meet the wireless environment which includes minimum power requirement, utilizing minimal network resources like bandwidth, gathering and updating link failures. Therefore, in order to provide optimal routing in the wired cum wireless environment, the routing protocols is need to fulfill the following major challenges and requirements. The challenges of wireless routing are suitability, mobility, bandwidth constraint, Error-prone and shared channel, Location dependent contention and Resource Constraint like battery power, buffer size. The requirements of routing protocol in the wireless network are quick route configuration, loop free routing, minimum route acquisition delay, distributed routing approach, minimum control overhead, scalability, QoS, time sensitive traffic and security. Journal of Computer Science 8 (3): 393-397, 2012 ISSN 1549-3636 © 2012 Science Publications Journal of Computer Science 8 (3): 393-397, 2012 ISSN 1549-3636 © 2012 Science Publications An Analysis and Overview of Wireless Routing Protocols 1Dhanalakshmi, N. and 2P. Alli 1NPR College of Engineering and Technology, Dindigul, India 2Department of CSE, Velammal College of Engineering, Madurai, India 1Dhanalakshmi, N. and 2P. Alli Abstract: Problem statement: In the past, the main focus of the research in the wireless network is to provide optimal routes between source and destination nodes. The wireless routing is required additional computational effort than wired routing in order to fulfill the major wireless characteristics such as battery power constraints, frequent mobility and less processing. Approach: In addition to wired routing the wireless routing requires some characteristics, such as scalability, higher throughput, lesser packet loss and providing QoS. The wireless routing protocol is categorized based on routing update mechanism, based on routing topology and based on special resources like energy aware and location aware. Results: Routing in the wireless network is classified as proactive, reactive and hybrid routing protocols. These routing protocols are discussed in this study with the experimental result. Conclusion: In this study, an analysis is described on the existing routing methodology which is proposed for solving various routing issues in the wireless communication. Key words: Computer communication, wireless networks, mobile adhoc network, routing protocol MATERIALS AND METHODS Routing Information Protocol (RIP) and Open Shortest Path First (OSPF) using Distance Vector (DV) and Link State (LS) respectively are the major traditional routing protocol which is used for wired single path routing. Dijkstra-Old-Touch-First (D-OTF) with multipath routing extension is an extended version of conventional Dijikstra’s shortest path algorithm that computing all lexicographic-lightest paths from a source to every other node in the network. OSPF version 2 (IGI, 2000) and OSPF- optimized multi-path (Villamizar, 1998) are some of the extended version of multi path routing protocol using traditional OSPF. In wireless, as every node may forward data from other nodes, the network resources usage like limited energy, limited bandwidth andlimited processing power should be fairly distributed across the networks nodes to avoid the high consumption of the resources in some network nodes and low consumption in other nodes. The routing algorithm should deal with the rapid changes in the network and should have the ability to optimize more than one network parameters in the network. Therefore, the goal for designing an optimal routing protocol for wireless environment may be termed as; it must be fully distributed, adaptive to frequent topology changes, loop free, localized control and must provide QoS. The AODV is optimal for intra segment routing and the DSR is optimal for inter segment routing. Therefore, Bai and Singhal (2006) proposed an extended hybrid version of AODV and DSR, called DOA (DSR over AODV). In which, DSR is proposed for inter-segment and AODV is proposed for intra- segment routing for improving packet delivery ratio. However, it requires more control overhead and complexity when implementing in the real time. Instead of segment, a zone based routing protocol are also proposed for wireless network, in which Zone- based Hierarchical LS(ZHLS) and Boundary mapping and boundary state (BSR) are the major routing protocols. The ZHLS is a hybrid routing protocol, which uses the geographical information for route discovery and route maintenance. The ZHLS applies the proactive approach inside the geographical zone and reactive approach beyond the zone. Lemmon and Musumeci (2008) proposed the BSR for MANET which also uses the geographical information for route discovery and route maintenance, which consists of two components. The first component is an improved forwarding strategy called as Greedy-Bounded Compass and the second component is a Boundary Mapping Protocol (BMP). MATERIALS AND METHODS The mathematical model for routing problem is as follows. Let V = {a, …, z} be a set of cities, A = {(r, s) gineering and Technology, Dindigul, India Tel: +91-9486294011 g 393 J. Computer Sci., 8 (3): 393-397, 2012 : r, s € V} be the edge set andδ(r, s) = δ(s, r) be a cost measure associated with edge (r, s) € A. The routing is the problem of finding a minimal cost closed tour that visits each city once, also called Traveling Salesman Problem (TSP). source initiated on demand routing protocol such as AODV, DSR and Temporally Ordered Routing (TORA). Another category is called hybrid routing protocols which offers the best features of both table driven and on demand routing protocols. The DV and LS is perfectly suit in the wired routing but it is not an optimal routing protocol for wireless routing. These routing protocols are suitable for smaller networks with low mobility but do not scale well in larger dynamic environments due to the periodic and global dissemination of topology updates. The on- demand routing uses a query response mechanism like route request and route reply for discovering and maintaining routes for each individual session. This provides scalability than the DV and LS, however, flooding of route queries limits the performance under conditions of high mobility and high traffic loads. The hybrid routing uses a cluster or hierarchical network architecture for group of nodes in the clustered architecture, the cluster may be formed statically and dynamically and then apply different routing strategies within and between these clusters. The hybrid routing also provides scalability in static clustered networks or situations involving group mobility but it occupies an excessive control overhead for route maintenance in order to choose cluster head. In this case, the cities r € V are given by their co- ordinates (xr, yr) and δ(r, s) is the Euclidean distance between r and s, then it is Euclidean TSP. If δ(r, s) ≠ δ(s, r) for at least some (r, s) then the TSP becomes an Asymmetric TSP. Choosing a single feasible solution is called a single path while choosing all possible feasible solution is called a multi-path. In which, the multi path routing avoids traffic and helps to improves the network efficiency. RESULTS In the last few years, the interest in the studies of Swarm Intelligence (SI) based optimization techniques is increasing. In SI, the organisms of animals and insects are studied for solving optimization problems and patterns generation. SI groups those techniques inspired by the collective behavior of social insect colonies, as well as other animal societies that are able to solve large-scale distributed problems. Like ACO, the Artificial Bee Colony (ABC) provides optimality in many aspects for variety of engineering problems. Load Balanced Ad Hoc Routing Protocols (LAOR) proposed by Toh et al. (2009), is delay-based load balancing scheme which is an extension of AODV routing. It has many similarities with AODV such as it also has two phases, route discovery and route maintenance. LAOR achieves load balancing by minimizing the estimated total route delay and route hop count. A node initiates route discovery by sending a Route Request (RREQ) when a valid route to the destination is not available in the routing table. The major difference than AODV is, if an intermediate node receives a duplicate RREQ with a smaller total delay and hop count, it updates this route in the routing table. The destination node then sends a reply message (RREP) when it receives the first RREQ. If duplicate RREQs received at the destination node have smaller total delay and hop count than previous ones, it sends an RREP message to the source node to change the chosen route immediately. When the source node receives the RREP, it initiates data packet transmission. Honey bees are insects that live in large colonies (around 50,000 bees as a colony) usually containing one queen and her progeny, some 20,000-40,000 female workers and 200-300 male drones. Meisel et al. (2010) is a detailed study of honeybee in the biological aspect and about the foraging behaviour. There are many syndromes observed like aggression syndrome, waggling dance, from the honey bee colony which is used for solving optimization problems. Although honey bees are depicted in many cave paintings dated from 6000BC, the first recorded observations of bee behaviour were made by Aristotle. In the bee colony, Queen Bee is the head of the operation and maintenance and the scout bees and working bees are the labour. Initially, scout bees are assigned to find out best flower batches which contain more nectar than its neighbouring flower batches. MATERIALS AND METHODS The first component is forwarding data packets around the defined boundaries 3 There are variety of wireless routing protocols (Murthy, 2004) such as Dynamic Source Routing (DSR), Destination Sequenced Distance Vector (DSDV), Adhoc On-demand Distance Vector (AODV), Wireless Routing Protocol (WRP), Cluster-head Gateway Source Routing (CGSR), Source Tree Adaptive Routing (STAR), Optimized Link State Routing(OLSR), Flow Oriented Routing (FSR), Hierarchical State Routing (HSR), Associatively Based Routing (ABR) and Signal Stability based Adaptive Routing (SSA) are included in IEEE802 family standard. These algorithms can be classified into two groups: table driven routing protocol (DSDV) and 394 J. Computer Sci., 8 (3): 393-397, 2012 J. Computer Sci., 8 (3): 393-397, 2012 without looping and the BMP is used to maintain LS information for boundaries. proposed ACO for wireless routing using priority model which is formed based on the availability of the wireless nodes. Lopez-Ibanez and Blum (2010), the beam search is applied with existing ACO for adhoc routing. Li et al. (2011), the author proposes a energy efficient routing for wireless routing. The SI techniques are proved optimal in many engineering applications (Chandra and Baskaran, 2011) and also in the wireless routing than the above discussed methodologies. This study further explains the overview of ABC which may be applied for wireless routing in the future. Proposed cost matrix agent based routing algorithm which uses the minimum-cost matrix. The next-node matrices are also calculated from the typical adjacency- cost matrix which is used to represents the link costs of the network. For efficient routing methodology, this study uses the link costs instead of hop counts as a routing metric. All the nodes in networks maintain the same information on the minimum-cost matrix. When a node needs to transmit data to a destination, the path can be quickly calculated using the maintained minimum-cost and the next-node matrices. These calculations are taken place using mobile agent which floods in the network and collects information about the network or subnet. A leader node which works as the agent calculates the minimum-cost matrix and the next- node matrices and transmits them to all the other nodes in a network. And the stop criteria of the system is Eq. 4: And the stop criteria of the system is Eq. 4: This dance is essential for colony communication in order to inform the working bees about the selected flower batches. This waggling dance contains information regarding flower patches like the direction of the food source, its distance from the hive and its quality rating. i i th N (Q) N (E) H − ≥ (4) Where: Ni (Q) = Represents the values of nectar of Queen Ni (E) = Represents the values of nectar of Elite bee i i th N (Q) N (E) H − ≥ (4) Where: N (Q) R h l f f Q (4) Ni (Q) = Represents the values of nectar of Queen Ni (E) = Represents the values of nectar of Elite bee The algorithm of ABC requires a number of parameters to be set, namely: number of scout bees (n), number of elite bees (e), number of patches selected out of n visited points (m), number of bees recruited for patches visited by “elite bees” (nep), number of bees recruited for the other (m-e) selected patches (nsp), size of patches (ngh) and stopping criterion. The algorithm starts with the n scout bees being placed randomly in the search space. And Hth represents the minimum threshold value of the Hive. At the end of iteration, each selected patch by the scout bee is identified. This promising algorithm can be applied to wireless like environment for routing issues. DISCUSSION The bees search for food sources in a way that maximizes the ratio Eq. 1: The honey bee is a diffuse creature which can extend itself over long distances in multiple directions in order to find a large number of food sources and at the same time to find the best food source from the collection of food sources. For example, the food source of the bees are designated as flower patches, in which plentiful amounts of nectar or pollen is available. This nectar or pollen is the food of the bees and the task is collecting the enormous amount of nectar with less effort and visited by more bees, whereas patches with less nectar or pollen should receive fewer bees. E F( ) T θ = (1) Where: E = The energy obtained T = The time spent for foraging (1) Where: E = The energy obtained E = The energy obtained T = The time spent for foraging Here ‘E’ is proportional to the nectar amount of food sources. In a maximization problem, the goal is to find the maximum of the objective function F (θ), θ ∈ RP. RP represents the region of search area. Assume that θi is the position of the ith food source; F (θi) represents the nectar amount of the food source located at θi and it is proportional to the energy E (θi). CONCLUSION The objective of this study is, to provide short description on various existing protocols which is proposed till date for wireless routing in order to help the current researchers for a well start. Based on this objective, this study analyses, classifies and discuss the advantages and pitfalls of existing wireless routing protocols. This study also describes the mathematical model for routing, the difference in wired and wireless routing and the requirements of wireless routing such as random mobility. This study provided recent research in the wireless routing such as ACO, ABC. Therefore, it is concluded that the objective of the study is fulfilled and this study will fulfil the needs of researcher those initialized their research in wireless routing. Let P(C) = {θi(C) \| i = 1, 2... S} represent the population of food sources being visited by bees, in which, C is cycle andS is number of food sources around the hive. The preference of a food source by the worker bee depends on the nectar amount F (θ) of that food source. As the nectar amount of the food source increases, the probability with the preferred source by the worker bee increases proportionally. Therefore, the probability with the food source located at θi will be chosen by a bee can be expressed as Eq. 2: i i s k k 1 F( ) P F( ) = θ = θ ∑ (2) (2) RESULTS When the scout bees identified such flower batches, the working bees are directed to collect nectar from the selected flower batches. Scout bees search randomly from one patch to another and the best flower batches are rated based on the combinations of some constituents, such as sugar content deposited in the nectar. Therefore a best flower batch is the food which has above certain threshold value of nectar presented in the flower batches. When a scout bee identifies such a flower batches and it go to the “dance floor” to perform a dance known as the “waggle dance”. p The swarm intelligence algorithms like Ant Colony Optimization (ACO), Artificial Bee Colony (ABC), Fire Flies and Particle Swarm Optimization (PSO) are attracts the recent research for optimization problems. There are many routing protocols (Chandra and Baskaran, 2011; Mohan and Baskaran, 2011a; Lopez- Ibanez and Blum, 2010; Li et al., 2011) are proposed in the recent years using ACO for wireless communication. In which, (Chandra and Baskaran, 2011) reviewed the research on variety of ACO implementation in many engineering application like Travelling Salesman Problem, Routing, Scheduling, Image Processing and Load optimization in electrical engineering. Mohan and Baskaran (2011b), the author 395 J. Computer Sci., 8 (3): 393-397, 2012 REFERENCES Akyildiz, I.F., S. Mohanty and J. Xie, 2005. A ubiquitous mobile communication architecture for next-generation heterogeneous wireless systems. IEEE Commun. Mag., 43: S29-S36. DOI: 10.1109/MCOM.2005.1452832 The position of the selected neighbour food source is calculated as the following Eq. 3: 3 i i (C 1) (C) θ + −θ (3) (3) i i (C 1) (C) θ + −θ 396 J. Computer Sci., 8 (3): 393-397, 2012 Chandra, M.B. and R. Baskaran, 2011. Survey on recent research and implementation of ant colony optimization in various engineering applications. Int. J. Comput. Intell. Syst., 4: 566-582. DOI: 10.2991/ijcis.2011.4.4.14 Lopez-Ibanez, M. and C. Blum, 2010. Beam-ACO for the travelling salesman problem with time windows. Comput. Operat. Res., 37: 1570-1583. DOI: 10.1016/j.cor.2009.11.015 Meisel, M., V. Pappas and L. Zhang, 2010. A taxonomy of biologically inspired research in computer networking. Comput. Netw., 54: 901-916. DOI: 10.1016/j.comnet.2009.08.022 Mohan, B.C. and R. Baskaran, 2011a. Priority and compound rule based routing using ant colony optimization. Int. J. Hybrid Intel. Syst., 8: 93-97. Mohan, B.C. and R. Baskaran, 2011b. Reliable Barrier- Free Services (RBS) for heterogeneous next generation network. Adv. Power Elect. Instrum. Eng., 148: 79-82. DOI: 10.1007/978-3-642-20499- 9_13 IGI, 2000. Cascade Communications Corporation: International Competitive Benchmarks and Financial Gap Analysis. 1st Edn., Icon Group International, Incorporated, ISBN-10: 0597023115, pp: 21. Lemmon, C.J. and P. Musumeci, 2008. Boundary mapping and Boundary-State Routing (BSR) in ad hoc networks. IEEE Trans. Mobile Comput., 7: 127-139. DOI: 10.1109/TMC.2007.70722 Bai, R. and M. Singhal, 2006. DOA: DSR over AODV routing for mobile ad hoc networks. IEEE Trans. Mobile Comput., 5: 1403-1416. DOI: 10.1109/TMC.2006.150 Lee, C.S. and D. Knignt, 2005. Realization of the next- generation network. IEEE Commun. Mag., 43: 34- 41. DOI: 10.1109/MCOM.2005.1522122 Murthy, 2004. Ad Hoc Wireless Networks: Architectures and Protocols. 1st Edn., Pearson Education India, ISBN: 8131706885, pp: 878. Li, H., X. Zhang and Y. Liu, 2011. Energy efficient routing based on ant colony algorithm in mine equipment monitoring. Proceedings of the International Conference on Consumer Electronics, Communications and Networks, Apr. 16-18, IEEE Xplore Press, XianNing, pp: 314-319. DOI: 10.1109/CECNET.2011.5768645 Toh, C.K., A.N. Le and Y.Z. Cho, 2009. Load balanced routing protocols for ad hoc mobile wireless networks. IEEE Commun. Mag., 47: 78-85. DOI: 10.1109/MCOM.2009.5181896 Villamizar, C., 1998. OSPF Optimized Multipath (OSPF-OMP). Internet Draft. 397
https://openalex.org/W2796548289	https://zenodo.org/records/1823923/files/article.pdf	English	null	Whimland. An Operetta for Children	Musical times and singing class circular	1,901	public-domain	2,836	Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/3365632 Accessed: 11-11-2015 14:45 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Musical Times Publications Ltd. is collaborating with JSTOR to digitize, preserve and extend access to The Musical Times and Singing Class Circular. http://www.jstor.org SONGS. Shuttle and Loom. The Yacobite. Words and Music byWilliamWallace ( ) [Novello and Company, Limited.] Cradle Song. Calm at Sea. Huntsman's Song. By Alfred S. Henry [Oppenheimer Bros.] p y The first of the above evening services was written for the twenty-seventh annual festival of the London Church Choir Association, held in St. Paul's Cathedral, and as may be anticipated the work is of important design. Mr. Lemare's setting at once attracts attention by his having allied the first word to the first beat in the bar, accentuating these by the personal element in the text. The part writing will prove attractive to a well-trained choir. The Nunc Dimittis is opened impressively by the tenors and basses in unison, pianissimo. Mr. Boyton Smith's composition is opened by sopranos in unison, with a bright and melodious phrase. The verse beginning 'He remembering his mercy,' set for soprano solo, forms a good contrast to the dignified music of the Gloria. The Nunc Dimittis is allied to most impressive music of devotional character. The first of the Introits by Mr. Lee Williams opens with a soprano solofollowed by a quartet. The music is eminently reverential. The second is an excerpt from the composer's cantata 'The Last Night at Bethany,' and consists of a quartet and chorus of fervent expression. Mr. Selby's Kyrie Eleison provides an alternative setting for his service in F, and will doubtless prove acceptable to many choir- masters. It consists of two principal strains, and the music is simple and flowing. g g y pp In this trio of songs the composer shows considerable versatility and originality. His harmonic schemes are bold, but they are manifestly dictated by desire to suggest the spirit of the text. The 'Cradle Song' is very pleasing, and a vocalist with dramatic perception could make 'Calm at Sea' impressive. The spirited and lively 'Huntsman's Song 'may be said to express the spirit underlying the Nimrodic remark, 'It is a lovely day, let us go and kill something.' It should be added that the original German text of these songs-severally by Oscar Meyer, Goethe, and Ludwig Uhland-is given, and that the English versions are respectively by Miss Constance Bache, Mr. E. A. Bowring, and Mr. Frederick Corder. y Whimland. An Operetta for children. Words by R. G. Waddington. Music by S. P. Waddington. ce. [G. Ricordi and Co.] Both these compositions well merit the attention of vocalists. The former is a song of a mother to her ' bairnie, whose ' laugh is as free as the wind in the wheat'; and music and words are instinct with poetry and healthy senti- ment. The 'Jacobite' is a stirring ditty, breathing destruction and death to all, save those who ' stand on the heather, Where the clan fought together '-a song, in short, that, delivered with appropriate warlike spirit, would be sure to delighta peacefuland law-abidingaudience. y gg [Novello and Company, Limited.] p y Mr. Higgs is well-known as an earnest and accom- plished musician, and his setting in C of the Communion Service reflects these estimable qualities very distinctly Three alternative settings are provided for the responses to the Commandments, in addition to the final one, and their smooth and flowing character is indicative of the rest of the music. The composer has made his greatest effort in the Nicene Creed, which is set in a very scholarly manner. It contains a short tenor solo, and the part-writing demands a well-trained choir for its satisfactory delivery. The Benedictus and Agnus Dei, as well as an offertory sentence, the Sanctus and the Gloria in Excelsis are included, but although thus complete, the setting is by no means bordering on the land of prolixity, and this fact, combined with the reverential and devotional spirit of the music, will doubtless cause the composition to be widely accepted. How Mendelssohn would have enjoyed that critic's Christianization of his Baal chorus ! Again:- g There are composers and conductors who are eaten up with vanity, and who appear to believe that the universe was created for the sole purpose of producing them. Some of them will appear at Gloucester during the week. As there were eight conductors of the Festival omnibus, this critical fare may be regarded as a passing jeer. A well-patronised hostelry at Gloucester during the Festival week was the RAM--very homely sound- ing quarters for Sir Alexander Mackenzie, and any others reigning in or hailing from Tenterden Street. DOTTED CROTCHET. g g g DOTTED CROTCHET. REVIEWS. by William Wallace. [G. Ricordi and Co.] CHURCH MUSIC. Magnificat and Nunc Dimittis in B flat. By Edwin H. Lemare; Magnificat and Nunc Dimittis in E Flat. By Boyton Smith. Magnificat and Nunc Dimittis in B flat. By Edwin H. Lemare; Magnificat and Nunc Dimittis in E Flat. By Boyton Smith. y I am not worthy. The Heavenly Word. Introit By C. Lee Williams. y Kyrie Eleison in F. By B. Luard Selby. (Novello'sParish Choir Book.) This content downloaded from 134.176.129.147 on Wed, 11 Nov 2015 14:45:37 UTC All use subject to JSTOR Terms and Conditions 676 THE MUSICAL TIMES.- OCTOBER I, 1901. O God, Thou art my God. By Ferris Tozer. Suffer not the little children. By Basil Harwood. And God shall wipe away. By Frederick R. Greenish. (Novello'sOctavo Anthems.) Mr. Frederick Evans, to whom the above letter belongs, was the solo boy at Gloucester when Wesley became organist. He was the first to sing the solo in 'Blessed be the God and Father' under Wesley, and he well remembers the agony of that trying rehearsal-its length, the exacting composer, and the tears that were shed. But Wesley slipped a two-shilling piece into the boy's hand and kindly said, 'There, don't cry.' During his residence in London, Mr. Evans was a member of the Sacred Harmonic Society, but on his return to Gloucester (in 1876) he became a lay clerk of the Cathedral. He has had the distinction of singing the tenor part in a quartet with Madame Albani. This content downloaded from 134.176.129.147 on Wed, 11 Nov 2015 14:45:37 UTC All use subject to JSTOR Terms and Conditions (Novello s Anthems.) [Novello and Company, Limited.] p y Dr. Tozer's anthem, composed for this year's festival of the Exeter Diocesan Choral Association, is appropriately bright and confident in character. It contains a pleasing section for first and second sopranos, and concludes with an effectively developed fugue built upon a melodious subject. The anthem by Dr. Basil Harwood is intended for baptismal services, and to be sung unaccompanied It will require a well-trained choir to do justice to the music, which, however, is not difficult to read, and more- over is interesting to sing. The third anthem on our list is the quartet from Dr. Greenish's cantata ' The Church Triumphant.' The music is melodious, and the part- writing is flowing and expressive. With smooth and careful singing, its impressiveness would be undoubted. In lighter vein, are two choice specimens of provincial musical criticism :- .a. and the " Take all the prophets of Baal, bring all and slay them," by its determined energy, seemed to show that both choir and conductor had thoroughly grasped the Christian spirit of the words. Communion Service in C. By H. M. Higgs. [Novello and Company, Limited.] Communion Service in C. By H. M. Higgs. [ d ] ( ) [Novello and Company, Limited.] [Novello and Company, Limited.] p y ' Whimland' is one of the latest of the admirable series of Novello's School Music, and the whimsical title is justified by the character both of the libretto and music. The work consists of thirteen numbers, comprising three solos, two duets, seven choruses, and two dances. The story enforces This content downloaded from 134.176.129.147 on Wed, 11 Nov 2015 14:45:37 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES.-OCTOBER I, 1901. 677 a moral in a humorous fashion, and the music is brigh and pleasing, and at the same time well designed for young folks' voices. It may be added that the character consist of Fairy Patience, a wizard called Watto, and his attendant Thomo, and small parts for seven children. The dresses will be easy to provide, and the principal stage property is a tree, or, as it is modestly stated, ' the stump of one.' Thus it leaves very little to be desired, or provided a moral in a humorous fashion, and the music is brigh and pleasing, and at the same time well designed for young folks' voices. It may be added that the character consist of Fairy Patience, a wizard called Watto, and his attendant Thomo, and small parts for seven children. The dresses will be easy to provide, and the principal stage property is a tree, or, as it is modestly stated, ' the stump of one.' Thus it leaves very little to be desired, or provided t Dawson, all under the direction of Mr. Vert, who is also responsible for three Kruse QuartetConcerts. t Dawson, all under the direction of Mr. Vert, who is also responsible for three Kruse QuartetConcerts. t Dawson, all under the direction of Mr. Vert, who is also responsible for three Kruse QuartetConcerts. p Quartet Concerts. Vocal recitals will be given by Madame Marchesi, Herr Van Rooy, and Mr. Denis O'Sullivan. The Saturday Evening Orchestral Concerts under the direction of Mr. Edward O'Brien, will re-commence on the 5th inst. p Quartet Vocal recitals will be given by Madame Marchesi Herr Van Rooy, and Mr. Denis O'Sullivan. The Saturday Evening Orchestral Concerts under the direction of Mr. Edward O'Brien, will re-commence on the 5th inst. Mdlle. Ilona Eibenschi~tz will give recitals at Bechstein Hall, and the Chaplin Trio will appear at Steinway Hall, in November. Twelve Two-part Songs. For female voices. PROVINCIAL SOCIETIES, information with regard to some of the earlier announce- ments will be found under the news supplied from various centres by our own correspondents. g by correspondents. Next month we shall hope to give a list of the doings of other Associations. g g y correspondents. Next month we shall hope to give a list of the doings of other Associations. ROYAL CHORAL SOCIETY claims premier attention. As in preceding years, eight con- certs are announced (commencing on November 7), seven of these being Subscription concerts, and one being the usual extra performance on Good Friday of 'The Messiah.' Some advance has been made in the introduction of fresh material by the announcement of Coleridge-Taylor's Leeds cantata ' The Blind Girl of Castel Cuille,' and Handel's 'L'Allegro,' while the success of the two previous performances of the former composer's ' Hiawatha' induces a repetition of that beautiful trilogy. The prospectus also includes Dvorak's 'Spectre's Bride,' Mendelssohn's 'Walpurgis Night,' and Sir F. Bridge's Gloucester cantata 'The Forging of the Anchor.' 'The Messiah,' 'Elijah,' ' Redemption,' and 'Golden Legend,' complete an attractive programme. Opera The Bermondsey Settlement Choral Society will give among other works Beethoven's ' Mount of Olives,' and Schumann's ' Paradise and the Peri.' s The Streatham Choral Society will give three concerts one of which will be the ' Hiawatha' Trilogy. Mr. Quance's Choral Society (Streatham) includes ' Hiawatha's Departure,' Bridge's 'Forging of the Anchor,' Gade's 'Crusaders,' and Mackenzie's ' Bride'; and the Clapham Choral Society includes Elgar's ' Dream of Gerontius' in the works named for practice. practice. For the six concerts announced by the South London Choral Association, the only quasi novelty named is 'Hiawatha's Departure.' GADE'S ' ERL-KING'S DAUGHTER' DRAMATISED. d t Those who attended the opening day of the Industria Exhibition held in connection with the Westbourne Park Institute, on the I7th ult., had the novel experience of listening to and seeing a dramatised version of Gade's ' Erl King's Daughter.' The music was performed exactly as published, but Mr. Sam James, the conductor, had made the addition of very pretty costumes and scenery and dramatic action, with the result of a hearty acceptance by a considerable audience. Mr. T. Harry Bull sang and acted the part of Sir Oluff very creditably, his fine voice telling well; the parts of the Mother and the Erl-King's Daughter were undertaken by Miss Mabel Todd, who sang sweetly. The pianist, Mr. Frank M. Jephson, did his work well, and the 'appointments' were under the management of Mr. H. James. The choral singing was good. g19. Foremost amongst the numerous performances at SUBURBAN SOCIETIES. t The Alexandra Palace Choral and Orchestral Society announces eleven concerts, and these include Sullivan's ' Martyr of Antioch,' Stanford's ' Revenge' and 'Last Post.' The Finsbury Choral Association will give three concerts, and here indeed meritorious enterprise is shown in the choice of Elgar's' King Olaf,' Stanford's ' Last Post,' Mackenzie's ' Dream of Jubal' and Coleridge- Taylor's ' Hiawatha' (complete). Among the four concerts to be given by the People's Palace Choral Society, we find Parry's 'Judith.' These three Societies are now all under the experienced direction of Mr. Allen Gill. QUEEN'S HALL. Departure. The West Norwood Choral and Orchestral Society proposes to include Bridge's ' Flag of England' and Mackenzie's 'Bride,' and the Beckenham Choral Society will give Parts I and 2 of ' Hiawatha.' Four Saturday Symphony Concerts will be given (the first on October 26) before Christmas, and four after, under Mr. Robert Newman's direction. The 'Golden Legend' will be performed on November 22, and 'Elijah' on December 12. Three recitals are to be given by Mr. Mark Hambourg; Messrs. Ysaye and Busoni will collaborate on November 7, and again on February 15, and these two artists will be joined by Herr Becker in three recitals, the first taking place on November 14, while later Signor Busoni will give two recitals alone, as also will Herr Becker on February 8. Mr. Edwin H. Lemare is announced to give five organ recitals, commencing November 13, while a novelty will be the inauguration of a series of Promenade Concerts on Boxing Night, extending to February I. The London Ballad Concerts will be resumed on November 2. Under Mr. Vert's direction Madame Clara Butt and Mr. Kennerley Rumford will give a concert at this Hall on the 12th inst., and Herr Kubelik a recitalon Novemberg19 g The Ealing Choral Society, with which is associated the Ealing Orchestral Society, announces the ' Hiawatha' Trilogy and Dvorak's ' Spectre'sBride.' gy Spectre s Concerning the movements of THE COMING SEASON. The advanced guard of the autumn season--the Promenade Concerts-was this year so far in advance of the general army of concerts that their commencemen has already been chronicled in our last issue. On account of its importance, and its regal title, the experienced Gill. The Highbury Philharmonic Society, now in its twenty fourth season, announces but one actual novelty-a part- song, 'Firm in her native strength,' by A. C. Mackenzie, whose ' Britannia' overture will also be given. There will also be a concert recital of Sullivan's Opera' Ivanhoe.' ( ) [Novello and Company, Limited.] Words by Edward Oxenford. Music by Battison Haynes. [Novello and Company, Limited.] At St. George's Hall, Mr. Charles Fry will this year increase his Shakespearian Costume Recitals to six, in which the music will as usual be an important feature Mr. Arthur Fox has written some incidental music for the ' Winter's Tale,' and special interest to musicians will attach to the introduction of Dr. Boyce's Masque music to the 'Tempest.' One of the talents of the late Mr. Battison Haynes was aptitude for teaching, and he who teaches gains a lively knowledge of requirements in his art. While these Twelve Two-part Songs are eminently suited for school use-for which they were primarily composed--their melodious charm and musicianly qualities will make them equally acceptable in the home as well as to some children of older growth. ST. JAMES'S HALL the Richter Concerts command notice. There will be three of them, the first taking place on the evening of the 26th inst. The Saturday Popular Concerts will be resumed on the same afternoon, and will be continued until December 14. The St. James's Hall Ballad Concerts will be re-commence on November 6. The Soldat Ladies' Quartet announces three concerts. Numerous pianoforte recitals are announced, prominen amongst the givers of these being Miss Fanny Davies (Three), Mr. Donald F. Tovey (Four), and Mr. Frederick This content downloaded from 134.176.129.147 on Wed, 11 Nov 2015 14:45:37 UTC All use subject to JSTOR Terms and Conditions
https://openalex.org/W4315978886	http://repository.uinsu.ac.id/18503/1/jurnal%20Almira%20cantik.pdf	Indonesian	null	Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal	Al-Manhaj/Al-Manhaj : Jurnal Hukum dan Pranata Sosial Islam	2,022	cc-by	5,611	ARTICLE INFO Imported products in Indonesia have been highly developed and become people's needs such as food, cosmetics, medicines, and so on. There are so many imported products that attract the attention of consumers because of the rapid and easily accessible advances in technology and information for consumers. However, the large number of imported products that are distributed to various places in Indonesia are not in accordance with the regulations. Islam obliges its people to consume/use halal products and stay away from all unclean products. The problem is, how is the enforcement of halal certificates for imported products in Indonesia based on the MUI fatwa number 4 of 2003. This study uses a normative juridical research method with a statutory approach and analyzes literature sources related to this research. From the results of the author's research, it was found that many products had received halal certification from the country of origin but did not register them with the Halal Product Assurance Organizing Agency (BPJPH). So it can be concluded that imported products that are halal certified in the country of origin, if they have made adjustment cooperation with Indonesia, do not apply for halal certificates in Indonesia again, but register at the Halal Product Assurance Organizing Agency or abbreviated as BPJPH (which is under the auspices of the Ministry of Religion) to get a halal registration number to comply with the Standard Operating Procedure (SOP) contained in the MUI Fatwa. Law; Accreditation Standards; Overseas Halal Certificate; MUI fatwa Article history: Received 2022-06-20 Revised 2022-09-21 Accepted 2022-12-24 This is an open access article under the CC BY license. Corresponding Author: Almira Kemala Dewi Almira Kemala Dewi1, Tetty Marlina Tarigan2 1 Universitas Islam Negeri Sumatera Utara, Indonesia; almirakemaladewi03@gmail.com 2 Universitas Islam Negeri Sumatera Utara, Indonesia; tettymarlinatarigan02@gmail.com Almira Kemala Dewi This is an open access article under the CC BY license. Universitas Islam Negeri Sumatera Utara, Indonesia; almirakemaladewi03@gmail.com 1. PENDAHULUAN At-Thabrani) Disebutkan juga didalam Hadis riwayat At-Thabrani : Yang Artinya “ Setiap tubuh yang tumbuh dari (makanan) yang haram, maka api neraka lebih utama baginya (lebih layak membakarnya).” ( HR. At-Thabrani) Produk halal adalah suatu produk yang sudah dinyatakan halal sesuai dengan syariat Islam (Rahmadani, 2015). Produk halal belakangan ini sedang menjadi tren gaya hidup, bukan hanya pada penduduk mayoritas muslim tetapi pada penduduk yang minoritas muslim. Indonesia sebagai penduduk Muslim terbanyak di dunia seharusnya bias dipenuhi hak-haknya untuk dapat mengkonsumsi atau menggunakan produk halal. Produk yang pada saat ini tersebar luas di masyarakat tidak dapat dijamin semuanya halal, untuk menentukan suatu produk halal atau tidak dibuktikan dengan sertifikat halal. g Terdapat sebuah hadis yang menjelaskan tentang semua makanan dan minuman boleh (halal) dikonsumsi sampai terdapat dalil yang menyatakannya haram, diriwayatkan oleh Ibnu Majah dan at- Tirmidz. Yang Artinya “ Apa yang dihalalkan oleh Allah dalam Kitab-Nya adalah halal dan apa yang diharamkan Allah didalam Kitab-Nya adalah haram, dan apa yang didiamkan (tidak diterangkan), maka barang itu termasuk yang dimaafkan.” dalam (Putri, 2021). Standardisasi merupakan senjata yang dapat memenangkan persaingan terhadap pasar global. Standardisasi ini pun tidak bisa berdiri sendiri, perlu ada 3 pillar infrasutruktur atau mutu nasional yang terdapat dalam standardisasi yakni : metrology, standardisasi, dan penilaian kesesuaian. Hanya Negara yang mempunyai daya sainglah yang mempunyai manfaat besar dalam meningkatkan kesejahteraan negara itu sendiri, dan Negara yang tidak mempunyai daya saing akan menjadi korban dari segala potensi perdagangan itu (Yuanitasari & Muchtar, 2018). Akreditasi Lembaga Pemeriksa Halal (LPH) adalah sebuah bentuk kegiatan pengakuan formal untuk penilaian kesesuaian, kompetinsi, dan kelayakan LPH. Sertifikat halal merupakan sebuah fatwa yang tertulis dari MUI untuk menyatakan kehalalan produk sesuai dengan syariat islam. Gunanya agar konsumen bisa lebih tenang dalam mengkonsumsi atau menggunakan produk yang mengandung unsur haram (Ihatec, 2022). Namun pada saat ini masih sangat banyak ditemukan sertifikat halal pada produk yang diragukan legalitasnya, seperti menggunakan label halal palsu yang dibuat tanpa proses pengajuan sertifikat halal ke Badan Penyelenggaraan Jaminan Produk Halal (BPJPH). BPJPH merupakan sebuah Lembaga yang dibawah naungan Kementrian Agama yang bertugas untuk menyelenggarakan jaminan produk halal di Indonesia (Suparto, Djanurdi, Yuanitasari, & Suwandono, 2016), menggantikan LPPOM-MUI, BPJPH dibentuk agar setiap produk yang akan masuk, diedarkan, atau diperdagangkan di wilayah Indonesia terjamin kehalalannya (BPJPH, 2022). Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal 1. PENDAHULUAN Peredaran produk impor kini sudah menyebar luas di Indonesia, dimana produk impor sudah menjadi bagian penting dikehidupan masyarakat Indonesia mulai dari pangan, obat-obatan, kosmetik, Peredaran produk impor kini sudah menyebar luas di Indonesia, dimana produk impor sudah menjadi bagian penting dikehidupan masyarakat Indonesia mulai dari pangan, obat-obatan, kosmetik, https://ejournal.insuriponorogo.ac.id/index.php/almanhaj https://ejournal.insuriponorogo.ac.id/index.php/almanhaj Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 632 of 640 632 of 640 dan lainnya. Tidak sedikit masyarakat yang mudah tergiur dengan mengkonsumsi atau menggunakan produk impor dikarenakan kemajuan teknologi dan informasi sehingga masyarakat sebagai konsumen sangat mudah mendapatkan produk impor di wilayah Indonesia. Batas terhadap antar Negara sepertinya tidak menjadi suatu penghalang. Sebagai konsekuensi nya produk impor akan lebih banyak dijumpai di wilayah Indonesia, bertarung dengan produk local. Jual beli menurut Bahasa adalah tukar menukar secara mutlak (Sabiq, 1995). dan lainnya. Tidak sedikit masyarakat yang mudah tergiur dengan mengkonsumsi atau menggunakan produk impor dikarenakan kemajuan teknologi dan informasi sehingga masyarakat sebagai konsumen sangat mudah mendapatkan produk impor di wilayah Indonesia. Batas terhadap antar Negara sepertinya tidak menjadi suatu penghalang. Sebagai konsekuensi nya produk impor akan lebih banyak dijumpai di wilayah Indonesia, bertarung dengan produk local. Jual beli menurut Bahasa adalah tukar menukar secara mutlak (Sabiq, 1995). ( q ) Sejak terbentuknya MEA ( Masyarakat Ekonomi Asean ) pada tahun 2015 negara yang ada di Asia Tenggara dapat melakukan ekspor- impor dengan berkurangnya hambatan seperti yang terjadi sebelumnya. Sangat banyak produk impor yang tersebar luas, namun juga banyak pelaku usaha yang mengabaikan hak konsumen, seperti jaminan kehalalan terhadap produk yang dijual. Halal dan haram tidaklah hal yang sederhana bagi konsumen muslim, tetapi itu merupakan hal yang sangat penting dan diatur dalam syariat islam (Thobieb, 2003). Halal merupakah aspek yang sangat penting dalam kehidupan umat muslim, yang dimana didalamnya terkait hubungan manusia dengan Allah SWT. Seperti Firman Allah dalam surat Al-Baqarah ayat 168, yaitu : Yang Artinya “ Hai Sekalian manusia, makanlah yang halal lagi baik dari apa yang terdapat di bumi dan janganlah kamu mengikuti langkah-langkah syaiton, karena sesungguhnya syaiton itu musuh yang nyata “ Yang Artinya “ Hai Sekalian manusia, makanlah yang halal lagi baik dari apa yang terdapat di bumi dan janganlah kamu mengikuti langkah-langkah syaiton, karena sesungguhnya syaiton itu musuh yang nyata “ Disebutkan juga didalam Hadis riwayat At-Thabrani : Yang Artinya “ Setiap tubuh yang tumbuh dari (makanan) yang haram, maka api neraka lebih utama baginya (lebih layak membakarnya).” ( HR. 1. PENDAHULUAN Pasal 6 ayat (d) Undang-Undang Nomor 33 Tahun 2014 tentang Jaminan Produk Halal menyatakan bahwa “ Produk Luar Negeri yang beredar di Indonesia harus melakukan registrasi Sertifikat Halal” (Indonesia, 2014). Kepala BPJPH, Sukoso, mengatakan bahwa diperlukan registrasi sertifikat Halal Luar Negeri sebagai dasar pelaksanaan kewenangan BPJPH dari produk halal yang sudah Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 633 of 640 633 of 640 tersertifikasi oleh Lembaga halal luar negeri (Kemenag, 2022a). Bagi pelaku usaha yang memproduksi produk tidak halal, tetap bisa mengedarkannya di Indonesia dengan mencantumkan keterangan tidak halal pada kemasan produk berupa gambar, tulisan, atau tanda yang dapat dilihat oleh konsumen. Hal itu dilakukan untuk memberikan perlindungan bagi masyarakat terkait produk yang akan mereka konsumsi atau gunakan. Penelitian dari Baihaqi (2022) menunjukan hasil yang ditinjau dari Fatwa MUI Nomor 4 Tahun 2003 masih terdapat penamaan produk makanan yang mengandung unsur kebatilan dan kekufuran, penyembelihan hewan yang masih samar dan sebagian saja yang terjamin prosesnya, dan kebersihan yang dapat dikatakan terjamin sesuai dengan fatwa yang dikeluarkan oleh Majelis Ulama Indonesia. Adapun penelitian dari Nisaa (2021) hasil penelitian ini dapat disimpulkan bahwa implementasi terhadap proses pembuatan brem Industri Rumah Tangga Brem Suling Istimewa di Madiun sudah sesuai dengan ketentuan Fatwa MUI No.4 Tahun 2003. Bahan-bahan dan alat-alat yang digunakan bersih dan halal tanpa ada kontaminasi silang dengan bahan atau sesuatu yang haram sehingga prosesnya sesuai dengan ketentuan fatwa. Adapun terkait dengan standar kehalalan produk olahan brem Industri Rumah Tangga Brem Suling Istimewa di Madiun, sudah sesuai dengan ketentuan fatwa. Selain dari sertifikat dalam negeri di Indonesia, Adapun dari sertifikat luar negeri di Indonesia yang dilakukan oleh (Firma Aditya & Al-Fatih (2017) dengan Indonesia masih bersifat semi-pemerintah, namun pada prakteknya pemerintah banyak membentuk produk hukum berdasar substansi dari fatwa MUI. Salah satunya adalah Undang-undang No. 33 Tahun 2014 Tentang Jaminan Produk Halal merupakan salah satu bentuk dari tindak lanjut pemerintah terhadap Fatwa halal MUI. Ketika Fatwa MUI sudah dituangkan dalam bentuk undang-undang, maka setiap masyarakat harus mematuhinya. Atas dasar ini, maka dapat dilihat bahwa fatwa MUI di Indonesia tidak hanya terbatas pada kehalalan suatu produk akan tetapi jauh lebih luas, karena pemerintah jauh lebih responsif dalam menindaklanjuti fatwa MUI. Sedangkan di luar negeri, meskipun lembaga pemberi fatwa merupakan lembaga publik milik pemerintah namun Fatwa yang dikeluarkan oleh lembaga-lembaga tersebut memiliki kekuatan hukum yang relatif lemah karena tidak dapat mengikat masyarakat secara luas. 1. PENDAHULUAN Berdasarkan penjelasan diatas, tujuan penulis membuat penelitian ini untuk mengetahui bagaimana pemberlakuan sertifikat halal luar negeri di Indonesia ditinjau dalam perspektif fatwa mui nomor 4 tahun 2003. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal 2. METODE Dari penjelasan yang telah diuraikan maka jenis penelitian yang digunakan adalah penelitian hukum yuridis normatif yaitu penelitian hukum kepustakaan ( Library Research ) dengan cara meneliti bahan-bahan atau kepustakaan atau data sekunder dan primer (Soekanto, 2007), Pendekatan yang digunakan dalam penelitian ini yaitu meliputi Fatwa MUI nomor 4 tahun 2003, peraturan perundang- undangan (statute approach), artikel, buku yang merupakan sumber literatur yang berhubungan dengan penelitian ini. Dimana masih banyak pelaku usaha yang tidak mengikuti peraturan yang telah di buat. Sumber data yang digunakan adalah data sekunder yang diperoleh melalui studi kepustakaan (library research) berupa buku-buku literatur dan pengaksesan e-journal dan internet yang berkaitan dengan penelitian. Penelitian ini bersifat deskriptif analisis yakni suatu penelitian yang melukiskan, memaparkan, menuliskan, melaporkan, menjelaskan, atau mengambarkan suatu keadaan, gejala, atau kelompok tertentu dengan proses penyederhanaan data penelitian yang amat besar jumlahnya menjadi informasi yang masih sederhana agar lebih mudah dipahami dengan apa adanya yang terjadi dilapangan. Teknik pengumpulan data dilakukan secara triangulasi, analisis data bersifat induktif. Teknik analisis dengan cara menganalisis data yang bersumber dari bahan hukum berdasarkan kepada konsep, teori, peraturan perundang-undangan, doktrin, prinsip hukum, pendapat pakar atau pandangan peneliti sendiri. 634 of 640 Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 3.1 Pengertian Hukum Menurut Para Ahli 3.1 Pengertian Hukum Menurut Para Ahli Dalam Undang-undang dasar 1945 pasal 1 ayat 3 dijelaskan bahwa Indonesia merupakan Negara hokum dan setiap warga negara Indinesia harus mematuhi setiap aturan hokum yang berlaku di Indonesia. Hukum yang ada di setiap Negara merupakan peraturan yang secara adat, resmi dan mengikat. Hukum ialah suatu aturan yang dibuat oleh manusia untuk membatasi tingkah laku manusia supaya dapat terkontrol, hukum merupakan aspek yang terpenting dalam setiap pelaksanaan atas rangkaian kekuasaan suatu Lembaga. Hukum harus menjamin adanya kepastian hukum didalam masyarakat, oleh sebab itu masyarakat mempunyai hak untuk mendapatkan pembelaan didepan hukum (Yuhelson, 2017). Hukum dapat diartikan sebagai ketetapan/ ketentuan yang tertulis maupun tidak tertulis untuk mengatur kehidupan masyarakat dan mempunyai sanksi bagi setiap orang yang melanggar hukum. Pengertian hukum menurut beberapa ahli yaitu menurut R. Soeroso dalam Rosifany (2020) menyatakan bahwa hukum adalah himpunan sebuah peraturan yang dibuat oleh pihak yang berwenang yang berfungsi untuk mengatur tata kehidupan masyarakat yang mempunyai ciri melarang, memerintah, memaksa dengan menjatuhkan sanksi hukuman bagi siapa yang melanggarnya. Pengertian hukum menurut E. Utrecht dalam Arrasjid (2008) menjelaskan hukum merupakan himpunan peraturan ( perintah / larangan ) yang mengatur tentang tata tertib dalam suatu masyarakat dan apabila tidak dipatuhi dapat menimbulkan tindakan dari pihak pemerintah masyarakat itu. Penjelasan diuraikan juga menurut M. H. Tirtaatmidjaja dalam Wantu (2015) menguraikan bahwa hukum ialah semua aturan/ norma yang harus ditaati pada tingkah laku tindakan- tindakan dalam pergaulan hidup dengan ancaman harus mengganti kerugian jika melanggar aturan itu akn berdampak pada membahayakan diri sendiri atau harta, seperti contoh orang yang kehilangan kemerdekaannya, dedenda atau sebaginya. Pengertian hukum menurut Thomas Hobbes dalam Zainal (2021) diuraikan bahwa hukum merupakan perintah dari seseorang yang mempunyai kekuasaan untuk memerintah dan memkasakan oerintahnya terhadap orang lain. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal 3.2 Sejarah Sertifikasi Halal di Indonesia Sebelum adanya sertifikasi halal yang di pegang oleh Majelis Ulama Indonesia (MUI), telah ada labelisasi halal untuk produk pangan di Indonesia yang berdiri pada tahun 1976 oleh Kementrian Kesehatan. Pada waktu itu semua makanan yang mengandung babi ataupun turunanya harus membuat identitas peringatan pada produk tersebut bahwa mengandung babi. Tanda peringatan itu bisa menggunakan gambar babi kemudian menuliskan “ MENGANDUNG BABI” yang diberi warna merah. Ketika itu pemilihan label haram lebih efektif dibandingkan label halal. Hal itu diatur dalam Surat Keputusan Mentri Kesehatan RI Nomor 280/Men. Kes/ Per/XI/ 76 mengenai peraturan pengedaran dan penandaan pada makanan yang mengandung babi (Faridah, 2019). Pada sekitar tahun 1987 Dosen Universitas Brawijaya Dr. Ir. Tri Susantu melakukan penelitian terhadap beberapa produk yaitu mie, susu, makanan ringan. Dari penelitian yang beliau lakukan terdapat bahwa produk tersebut mengandung gelatin, shortening, dan lechitin yang besar kemungkinan berasal dari babi. Atas dasar itu terjadi penurunan minat pembeli yang hampir mengakibatkan lumpuhnya perekonomian nasional. Peristiwa itu menjadi factor utama didirikannya LPPOM MUI ( Lembaga Pengkajian Pangan Obat-Obatan dan Kosmetika – Majelis Ulama Indonesia) untuk menjalankan tugas MUI dalam mengawasi makanan, obat dan kosmetika yang belum jelas kehalalannya. LPPOM – MUI didirikan pada 06 Januari 1989 / 26 Jumadil Awal 1409 H yang diketuai oleh Prof. Dr. Ir. M. Amin Aziz dan selanjutnya Prof. Dr. Hj. Aisjah Girindra (LPPOM MUI Sumut, 2022). Pada tahun 2017 Mentri Agma Lukman Hakim Saifuddin meresmikan Badan Penyelenggara Jaminan Produk Halal (BPJPH) yang berlandaskan Undang-undang Nomor 33 Tahun 2014 tentang Jaminan Produk Halal (JPH). Hal ini untuk memberikan keamanan, keselamatan, dan kepastian dalam ketersediaan Produk Halal bagi masyarakat dalam mengkonsumsi ataupun menggunakan suatu produk. Kehadiran BPJPH sebagai penguat sertifikasi yang awalnya bersifat voluntary sekarang Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 635 of 640 635 of 640 menjadi obligatory, artinya sesuatu yang diwajibkan berdasarkan Undang-undang untuk kemaslahatan seluruh bangsa. Kemudian setelah beroperasinya BPJPH dibawah naungan kemenag, peran MUI juga masih sangat penting yaitu memberikan fatwa penetapan kehalalan produk yang setelah itu disampaikan kepada BPJPH untuk dasar penerbitan sertifikat halal (Kemenag, 2022b). p p p g Sertifikat halal bukan hanya memberikan manfaat perlindungan hukum dan hak-hak konsumen muslim saja, namun juga memberikan peningkatan terhadap nilai jual produk. Dengan adanya sertifikat halal konsumen sudah tidak ragu dalam menggunakan atau mengkonsumsi suatu produk, karena sudah sesuai dengan standar syariat islam (Syafrida, 2016). Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal 3.2 Sejarah Sertifikasi Halal di Indonesia Terdapat 11 kriteria dalam sistem jaminan halal (SJH) sebagai berikut, organisasi manajemen halal, pelatihan, Bahan (dengan memperhatikan titik krisis bahan), produk, fasilitas produksi, prosedur tertulis aktivitas krisis, kemampuan telusur, penanganan produk yang tidak memenuhi kriteria, audit internal, dan kaji ulang manajemen. Menurut LPPOM MUI bahan baku merupakan faktor yang sangat penting untuk diteliti karena harus terhindar dari unsur babi ataupun unsur yang mengharamkan suatu produk (Raisqi, 2022). Gambar 1 Alur proses Sertifikasi Halal Dalam mengajukan sertifikat halal sebuah produk, terdapat langkah-langkah yang harus dilewati oleh setiap pelaku usaha, yang mana pelaku usaha mengajukan permohonan sertifikasi halal ke Badan Penyelenggara Jaminan Produk Halal (BPJPH) yang bertugas melaksanakan penyelenggaraan jaminan produk halal sesuai dengan ketentuan yang ada di dalam peraturan perundang-undangan. Langkah pertama, pelaku usaha mengajukan permohonan sertifikasi halal kepada BPJPH dengan melampirkan dokumen-dokumen pelengkap seperti, data pelaku usaha, nama dan jenis produk yang akan di sertifikasi, daftar produk dan bahan yang digunakan oleh pelaku usaha dalam membuat suatu produk, tata cara pengolahan produk, dan yang terakhir pelaku usaha harus melampirkan dokumen sistem aminan produk halal. Setelah proses permohonan sertifikasi halal sudah dilakukan oleh pelaku usaha, yang kedua BPJPH memeriksa kelengkapan dokumen-dokumen permohonan pelaku usaha, apabila dokumen yang telah dibutuhkan lengkap maka pengajuan sertifikasi dapat dilanjutkan, namun Jika terdapat dokumen/data yang kurang, maka BPJPH akan meminta pelaku usaha untuk melengkapi dokumen yang belum lengkap dalam jangka waktu 2 hari kerja, apabila hal ini tidak di penuhi oleh Dalam mengajukan sertifikat halal sebuah produk, terdapat langkah-langkah yang harus dilewati oleh setiap pelaku usaha, yang mana pelaku usaha mengajukan permohonan sertifikasi halal ke Badan Penyelenggara Jaminan Produk Halal (BPJPH) yang bertugas melaksanakan penyelenggaraan jaminan produk halal sesuai dengan ketentuan yang ada di dalam peraturan perundang-undangan Langkah Dalam mengajukan sertifikat halal sebuah produk, terdapat langkah-langkah yang harus dilewa Dalam mengajukan sertifikat halal sebuah produk, terdapat langkah-langkah yang harus dilewati oleh setiap pelaku usaha, yang mana pelaku usaha mengajukan permohonan sertifikasi halal ke Badan Penyelenggara Jaminan Produk Halal (BPJPH) yang bertugas melaksanakan penyelenggaraan jaminan produk halal sesuai dengan ketentuan yang ada di dalam peraturan perundang-undangan. Langkah pertama, pelaku usaha mengajukan permohonan sertifikasi halal kepada BPJPH dengan melampirkan dokumen-dokumen pelengkap seperti, data pelaku usaha, nama dan jenis produk yang akan di sertifikasi, daftar produk dan bahan yang digunakan oleh pelaku usaha dalam membuat suatu produk, tata cara pengolahan produk, dan yang terakhir pelaku usaha harus melampirkan dokumen sistem jaminan produk halal. Setelah proses permohonan sertifikasi halal sudah dilakukan oleh pelaku usaha, yang kedua BPJPH memeriksa kelengkapan dokumen-dokumen permohonan pelaku usaha, apabila dokumen yang telah dibutuhkan lengkap maka pengajuan sertifikasi dapat dilanjutkan, namun Jika terdapat dokumen/data yang kurang, maka BPJPH akan meminta pelaku usaha untuk melengkapi dokumen yang belum lengkap dalam jangka waktu 2 hari kerja, apabila hal ini tidak di penuhi oleh pelaku usaha maka permohonan akan ditolak. 3.3 Tata Cara Pengajuan Sertifikasi dan Registrasi Sertifikat Halal Luar Negeri g j g g Pengajuan halal bagi pelaku usaha yang tinggal diluar Negeri dan ingin produknya di pasarkan di Indonesia, maka produk tersebut dapat diaudit oleh Lembaga sertifikasi yang berada diluar negeri, cara tersebut juga meminimalisir waktu serta lebih praktis dikarenakan letak LSH berada sangat dekat dengan tempat produksi produksehingga pengujian dapat dilakukan lebih mudah, LSH merupakan kepanjangan dari Lembaga Sertifikasi Halal. Jika produk yang sudah diuji tadi dan telah tersertifikasi oleh LSH, maka pelaku usaha hanya perlu melakukan registrasi kembali di BPJPH. Namun terdapat beberapa hal yang perlu diperhatikan pada saat pelaku usaha ingin menggunakan sertifikasi halal yang dikeluarkan oleh LSH luar Negeri yaitu, LSH luar negeri yang melakukan sertifikasi halal, baik itu untuk pangan, kosmetik, ataupun obat sibentuk oleh organisasi keislaman yang telah diakui secara hukum, selanjutnya organisasi keislaman itu sudah memiliki kantor yang permanen dan telah didukung oleh sumber daya manusia (SDM) yang berkualitas dan memiliki kualifikasi. Selanjutnya organisasi keislaman itu juga harus memiliki dewan ataupun komisi fatwa, yang nantinya dewan inilah yang akan memberikan fatwa halal, serta terdapat tim ilmuwan yang telah ahli didalam melakukan audit halal. Selanjutnya, LSH itu juga harus memiliki standard operating procedurs ( SOP) contohnya seperti prosedur pendaftaran, administrasi, pemeriksaan halal ke pabrik, laporan, serta komisi fatwa. Selanjutnya berkas yang sudah dikeluarkan oleh LSH harus tertata dengan baik, baik itu berupa formulir pendaftraan, data perusahaan, ataupun dokumen lain yang fungsinya agar lebih mudah nantinya menelusuri informasi yang dibutuhkan. Selanjutnya, hal yang perlu diperhatikan mengenai LSH harus mempunyai jaringan yang luas dan merupakan anggota dari World Halal Food Council (WHFC). Dan yang terakhir LSH harus memiliki jalinan kerjasama yang baik dengan MUI dalam melakukan audit ataupun pengawasan terhadap produk halal. Setelah pelaku usaha menilai LSH yang terdapat pada Negaranya, selanjutnya pelaku usaha mengunggah dokumen-dokumen yang diperlukan, nantinya BPJPH akan melakukan verifikasi dan mengeluarkan invoice sesuai skema pengakuan sertifikat halal atau akreditasi. Selanjutnya Lembaga Halal Luar Negeri ( LHLN ) melakukan pembayaran sebesar 17.500.000 (diluar biaya akomodasi dan tarnspor tim asesor), setelah itu menyampaikan bukti pembayaran kepada BPJPH. Kemudian tim assessment atau tim akreditasi melakukan penilaian untuk pengakuan sertifikat halal / proses akreditasi oleh tim akreditasi BPJPH (Hidayati & Primadhany, 2020). Syarat- syarat yang harus dipenuhi LSH luar negeri sama seperti LPH yang berada di Indonesia. Hal tersebut dilakukan untuk memastikan prosedur-prosedur sertifikasi kehalalan produk dapat terjamin dengan baik. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal Ketiga, setelah BPJPH telah memverifikasi dan memeriksa kelengkapan data pelaku usaha, selanjutnya BPJPH akan menetapkan Lembaga pemeriksa halal berdasarkan pilihan pemohon (pelaku usaha) dengan syarat LPH yang pelaku usaha pilih wajib memiliki akreditasi dan kompetensi untuk melakukan sertifikasi produk. Keempat, Lembaga Pengujian Halal atau yang disingkat dengan LPH yang telah ditunjuk akan melakukan pemeriksaan atau pengujian kehalalan produk yang akan di sertifikasi dalam kurun waktu 15 hari kerja, pengujian tersebut dilakukan oleh auditor halal yang telah tersertivikasi di lokasi usaha pada saat proses produksi produk, apabila selama pengujian terdapat bahan yang meragukan maka auditor halal selanjutnya melakukan pemeriksaan ke laboratorium. Kelima, setelah dilakukan pengujian oleh LPH,kemudian hasilnya diberikan ke BPJPH melakukan pengecekan terhadap tahapan yang sudah diselesaikan, adapun lampiran dokumen yang harus diserahkan oleh LPH yaitu produk dan bahan yang digunakan si pelaku usaha, PPH, hasil analisis atau spesifikasi, berita acara pemeriksaan,dan Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 636 of 640 rekomendasi. Keenam, setelah semua sesuai dengan aturan yang berlaku maka BPJPH selanjutnya akan melimpahkan proses selanjutnya terhadap Majelis Ulama Indonesia (MUI) untuk menyelenggarakan sidang fatwa halal dan sekaligus menerbitkan keputusan penetapan kehalalan produk, MUI melakukan verifikasi terhadap dokumen yang diserahkan oleh BPJPH, adapun pihak- pihak yang terlibat dalam sidang fatwa ini seperti pakar, unsur kementrian terkait, dan Lembaga yang terkait, ketika terdapat informasi tambahan yang dibutuhkan oleh MUI untuk keperluan sidang fatwa halal maka pelaku usaha mempunyai waktu selama 3 hari kerja. Ketujuh, pada tahap akhir ini setelah MUI sudah menetapkan kehalalan produk maka MUI akan mengembalikan pengerjaanya kepada BPJPH untuk menerbitkan sertifikat berdasarkan keputusan penetapan kehalalan produk yang ditetapkan oleh MUI yang memiliki jangka waktu selama 1 hari kerja. 3.3 Tata Cara Pengajuan Sertifikasi dan Registrasi Sertifikat Halal Luar Negeri 3.4 Analisis hukum tentang Pemberlakuan sertifikat halal luar negeri perspektif fatwa MUI nomor 4 tahun 2003 3.4 Analisis hukum tentang Pemberlakuan sertifikat halal luar negeri perspektif fatwa MUI nomor 4 tahun 2003 Produk impor yang masuk ke dalam Indonesia dibagi menjadi dua jenis, produk impor yang telah besertifikat halal dari Negara asal dan Produk impor yang sama sekali belum bersertifikat halal. Produk impor yang bersertifikat halal dari Negara asal dibagi menjadi dua lagi, yaitu produk impor yang bersertifikat halal dan sudah sesuai dengan sertifikat halal BPJPH dan produk impor yang telah bersertifikat halal dari Negara asal tetapi belum sesuai dengan sertifikat halal BPJPH. Produk impor yang telah mendapatkan sertikat halal dari Negara asal dan sudah berkesesuaian (bekerjasama) dengan sertifikat halal BPJPH diwajibkan untuk melakukan registrasi, gunanya untuk mendapatkan nomor registrasi yang nantinya akan dicantumkan berdekatan dengan label halal Negara asal. Untuk produk impor yang sudah bersertifikat halal dari Negara asal namun belum melangsungkan kerjasama kesesuain dengan BPJPH ataupun produk impor tersebut belum bersertifikat halal maka pelaku usaha melakukan pengajuan sertifikat halal sesuai dengan prosedur yang berlaku di Indonesia secara tertulis maupun on-line ke Lembaga BPJPH sesuai dengan dokumen yang dibutuhkan. Setelah semua dokumen terpenuhi LPH akan menunjuk auditor halal yang akan melakukan pemeriksaan/pengujian pada saat proses produksi. Ketika selama proses pengujian ada ditemukan keraguan atas kehalalan suatu produk, maka produk itu akan dilanjutkan dengan pemeriksaan ke laboraturium. Setelah selesai hasil pemeriksaan auditor halal maka LPH menyerahkan kepada BPJPH, kemudian BPJPH menyerahkannya kepada MUI untuk mengadakan sidang fatwa halal untuk menentukan produk yang diajukan halal atau tidak. Kurang lebih dalam waktu 30 hari sidang fatwa MUI harus memutuskan dan menetapkan kehalalan produk. Dan setelah MUI menerbitkan sertifikat halal, BPJPH mempunyai kewenangan untuk mempublikasi sertifikat halal produk. Pada saat ini masih banyak pelaku usaha yang tidak mengikuti standar peraturan yang telah dibuat. seperti produk impor yang tidak meregistrasikan sertifikat halal luar negeri ke BPJPH namun sudah diedarkan di wilayah Indonesia. Hal ini menunjukkan masih rendahnya kewajiban pelaku usaha mengikuti peraturan tentang standardisasi produk halal luar Negeri dan longgarnya peran BPJPH dalam memeriksa segala produk impor yang beredar di wilayah Indonesia. Di dalam fatwa MUI Nomor 4 tahun 2003 menjelaskan : “ Perlu ada standard akreditasi dalam hal SOP dan fatwanya. Jika diragukan kebenarannya, harus diteliti ulang” Indonesia, fatwa halal atas sebuah produk dikeluarkan oleh Majelis Ulama Indonesia (MUI). Sedangkan di beberapa negara lain, fatwa halal di keluarkan oleh lembaga yang berbeda-beda. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal 3.3 Tata Cara Pengajuan Sertifikasi dan Registrasi Sertifikat Halal Luar Negeri Mengenai tata cara registrasi sertifikat halal luar negeri ini sudah diatur didalam Peraturan Pemerintah (PP) Nomor 39 Tahun 2021. Produk impor halal yang sertifikat halalnya diterbitkan oleh Lembaga Luar Negeri yang telah melangsungkan kerjasama saling pengakuan sertifikat halal dengan Badan Penyelenggaraan Jaminan Produk Halal (BPJPH) tidak perlu melakukan permohonan sertifikat halal kembali, namun diwajibkan bagi pelaku usaha melakukan registrasi sebelum produk tersebut diedarkan di Indonesia. Untuk mengajukan registrasi, adapun yang harus dilakukan oleh pelaku usaha mengajukan permohonan kepada BPJPH dengan melampirkan beberapa berkas, pertama yaitu berupa Salinan sertifikat halal luar negeri produk yang telah disahkan oleh perwakilan Indonesia yang berada di luar negeri. Kedua, pelaku usaha melampirkan daftar barang-barang yang akan diimpor ke Indonesia yang Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 637 of 640 dilengkapi dengan kode sistem harmonisasi. Ketiga, surat pernyataan bahwa yang disampaikan oleh pelaku usaha benar dan sah (Indonesia, 2019). Setelah semua berkas telah pelaku usaha siapkan, maka pelaku usaha dapat melakukan permohonan menggunakan sistem elektronik ataupun manual. Setelah semua berkas dilampirkan, BPJPH akan menerbitkan nomor registrasi bagi pelaku usaha yang telah memenuhi persyaratan. Kemudian pelaku usaha yang telah mendapatkan nomor registrasi diwajibkan mencantumkan nomor registrasi berdekatan dengan label halal pada bagian tertentu. Ketika sertifikat halal luar negeri yang diterbitkan oleh Lembaga halal luar negeri tetapi belum melakukan kerja sama dengan BPJPH, maka pelaku usaha harus membuat sertifikat halal sesuai dengan peraturan perundang-undangan di Indonesia. 3.4 Analisis hukum tentang Pemberlakuan sertifikat halal luar negeri perspektif fatwa MUI nomor 4 tahun 2003 MUI telah memantau fatwa halal yang dikeluarkan oleh beberapa lembaga di luar negeri (Firma Aditya & Al-Fatih, 2017). Penelitian yang dilakukan oleh Desriani (2021) menunjukkan bahwa Fatwa MUI Nomor 4 Tahun 2003 maka analisis penulis terhadap pemberian nama makanan olahan yang tidak boleh yaitu, mie iblis, kumis naga, dan ayam sambal setan karena nama makanan tersebut bertentangan dengan Fatwa MUI Nomor 4 Tahun 2003 bagian keempat masalah Penggunaan Nama Dan Bahan yaitu tidak boleh mengkonsumsi dan menggunakan nama dan/atau simbol-simbol makanan/minuman yang mengarah kepada kekufuran dan kebatilan. Menurut Firma Aditya & Al-Fatih (2017) Sertifikasi halal belum diterbitkan, Diawali dengan MUI akan mengeluarkan fatwa halal untuk produk yang akan disertifikasi. Menariknya, MUI sebagai lembaga semi pemerintah adalah sebuah lembaga yang bersifat nirlaba. Sangat berbeda dengan lembaga sertifikasi halal di negara Lain. Di luar negeri biasanya berbentuk badan usaha bukan lembaga Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 638 of 640 Non-komersial. Pada kenyataannya, ada banyak hal yang perlu dipertimbangkan tentang bagaimana proses itu bekerja. Skema asuransi halal dapat dilaksanakan atas dasar keyakinan yang benar. Lembaga Sertifikat harus dikaitkan dan disetujui oleh lembaga Uramah Islam negara tersebut. Tertarik dan didukung oleh para ahli penelitian Halal. Dari ketentuan fatwa MUI diatas MUI sudah mengeluarkan Standar aturan tentang bagaimana mekanisme masuknya produk impor ke Indonesia, namun pelaku usaha impor yang tidak menaati aturan tersebut. Dengan ditemukannya banyak produk yang sudah mendapatkan sertifikasi halal dari Negara asal namun tidak meregistrasikan nya ke Badan Penyelenggaraan jaminan Produk Halal (BPJPH) tersebut mengindikasikan bahwa impelementasi Fatwa MUI dan UU JPH belum sepenuhnya dipatuhi oleh pelaku usaha dan apabila sebuah produk diragukan kehalalannya maka produk itu harus diteliti ulang. Undang-undang No. 33 Tahun 2014 Tentang Jaminan Produk Halal merupakan salah satu bentuk dari tindak lanjut pemerintah terhadap fatwa MUI nomor 4 tahun 2003, karena Undang-Undang tersebut sebagian besar substansinya berdasar pada fatwa MUI nomor 4 tahun 2003. Ketika fatwa MUI nomor 4 tahun 2003 sudah dituangkan dalam bentuk undang-undang, maka setiap masyarakat harus mematuhinya. 4. KESIMPULAN Dari pembahasan yang telah dipaparkan diatas, maka penulis menarik kesimpulan produk impor yang sudah bersertifikat halal dari Negara asal dan sudah melakukan kerjasama penyesuaian dengan BPJPH maka sertifikat halalnya berlaku di Indonesia, namun diwajibkan untuk melakukan registrasi agar produknya bisa di edarkan di wilayah Indonesia. Apabila produk impor sudah mendapatkan sertifikat halal dari Negara asal tetapi belum melakukan kerjasama penyesuaian dengan BPJPH, maka pelaku usaha harus melakukan pengajuan sertifikat halal ke BPJPH, namun banyak ditemukan produk yang sudah mendapatkan sertifikasi halal dari Negara asal namun tidak meregistrasikan nya ke Badan Penyelenggaraan jaminan Produk Halal (BPJPH) ketika fatwa MUI nomor 4 tahun 2003 sudah dituangkan dalam bentuk undang-undang, maka setiap masyarakat harus mematuhinya. Sehingga Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal untuk pelaksanaanya semakin baij. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal REFERENSI Arrasjid, C. (2008). Dasar-dasar Ilmu hukum. Baihaqi, W. S. (2022). Jaminan Produk Makanan Halal Kaki Lima Di Stadion Maguwoharjo Yogyakarta Perspektif Undang–Undang No. 33 Tahun 2014 Ditinjau Dari Fatwa Mui No. 4 Tahun 2003. BPJPH. (2022). No Title. Desriani, D. (2021). Pemberian nama makanan olahan di Kota Padangsidimpuan ditinjau dari Fatwa MUI Nomor 4 Tahun 2003. IAIN Padangsidimpuan. Faridah, H. D. (2019). Sertifikasi halal di Indonesia: sejarah, perkembangan, dan implementasi. Journal of Halal Product and Research, 2(2), 68–78. Firma Aditya, Z., & Al-Fatih, S. (2017). Perbandingan Hukum Fatwa Halal Di Beberapa Negara: Kajian Yuridis Fatwa Halal Mui Dan Fatwa Halal Dari Lembaga Lain Di Luar Negeri (Comparative Law Of Halal Fatwa Among Several Countries: The Juridical Study Of Mui Halal Fatwa With A Halal Fatwa From Othe. Prosiding Konferensi Nasional “Perbandingan Hukum Dan Perkembangan Sistem Hukum: Konvergensi Atau Divergensi. Hidayati, T., & Primadhany, E. F. (2020). Sistem Jaminan Produk Halal: sertifikasi halal dan peran pemerintah daerah dalam melindungi UMKM di Kalimantan Tengah. LP2M IAIN Palangka Raya Press. Ih (2022) S ifik i H l l S U Ihatec. (2022). Sertifikasi Halal Secara Umum. nesia. (2014). Pasal 6 Undang-Undang Nomor 33 Tahun 2014 tentang Jaminan Produk Halal. Sekretaria Negara. Indonesia. (2019). Pasal 65 ayat (1-2) Peraturan Pemerintah Republik Indonesia Nomor 31 tahun 2019 tentang Peraturan Pelaksanaan Undang-Undang Nomor 33 Tahun 2014 tentang Jaminan Produk Halal. Sekretariat Negara. g Kemenag. (2022a). BPJPH atur tata cara Registrasi Sertifikat Halal Luar Negeri. 639 of 640 639 of 640 Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. 4, 2 (Desember, 2022): 631-640 Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal Kemenag. (2022b). Negara dan Sertifikasi Halal Indonesia. Kemenag. (2022b). Negara dan Sertifikasi Halal Indonesia. LPPOM MUI Sumut. (2022). Sejarah LPPOM MUI. j Nisaa, M. D. C. (2021). Implementasi Fatwa MUI No. 4 Tahun 2003 Tentang Standardisasi Fatwa Halal terhadap Pembuatan Produk Olahan Brem di Madiun (Studi Kasus di Industri Rumah Tangga Brem Suling Istimewa). IAIN Ponorogo. g g Putri, S. D. (2021). Analisis Deskriptif Hadis tentang Halal Food. Jurnal Riset Agama, 1(2), 285–2 ahmadani, G. (2015). Halal dan Haram dalam Islam. Jurnal Ilmiah Penegakan Hukum, 2(1), 20–26. Raisqi, N. R. (2022). Implementasi Sertifikasi Halal pada Produk Pangan dalam Rangka Perlindungan Konsumen (Studi Kasus Dominos Pizza). AL-IQTISHADIYAH: EKONOMI SYARIAH DAN HUKUM EKONOMI SYARIAH, 8(1), 38–51. Rosifany, O. (2020). Perlindungan Hukum Terhadap Pekerja Perempuan Menurut Ketentuan Undang- Undang Ketenagakerjaan. LEGALITAS: Jurnal Ilmiah Ilmu Hukum, 4(2), 36–53. Sabiq, S. (1995). Fikih Sunnah. Alma’rif. q ( ) Soekanto, S. (2007). Penelitian hukum normatif: Suatu tinjauan singkat. Raja Grafindo Persada. Suparto, S., Djanurdi, D., Yuanitasari, D., & Suwandono, A. (2016). Harmonisasi Dan Sinkronisasi Pengaturan Kelembagaan Sertifikasi Halal Terkait Perlindungan Konsumen Muslim Indonesia. Mimbar Hukum-Fakultas Hukum Universitas Gadjah Mada, 28(3), 427–438. Syafrida, S. (2016). Sertifikat Halal Pada Produk Makanan Dan Minuman Memberi Perlindungan Dan Kepastian Hukum Hak-Hak Konsumen Muslim. ADIL: Jurnal Hukum, 7(2), 159–174. Thobieb, A.-A. (2003). Bahaya Makanan Haram Bagi Kesehatan Jasmani dan Kesucian Rohani. In PT. Al-Mawardi Prima. PT. Al-Mawardi Prima. Wantu, F. M. (2015). Pengantar Ilmu Hukum. REVIVA CENDEKIA. ( ) g Yuanitasari, D., & Muchtar, H. N. (2018). Aspek Hukum Standarisasi Produk di Indonesia dalam Rangka Masyarakat Ekonomi ASEAN. Jurnal Hukum Ius Quia Iustum, 25(3), 538–559. Yuhelson, Y. (2017). Pengantar Ilmu Hukum. IDEAS PUBLISHING. Zainal, A. (2021). Pengantar Ilmu Hukum. Rajawali Pres, Jakarta. Yuanitasari, D., & Muchtar, H. N. (2018). Aspek Hukum Standarisasi Produk di Indonesia Yuanitasari, D., & Muchtar, H. N. (2018). Aspek Hukum Standarisasi Produk di Indonesia dalam Rangka Masyarakat Ekonomi ASEAN. Jurnal Hukum Ius Quia Iustum, 25(3), 538–559. Yuhelson, Y. (2017). Pengantar Ilmu Hukum. IDEAS PUBLISHING. Rangka Masyarakat Ekonomi ASEAN. Jurnal Hukum Ius Quia Yuhelson, Y. (2017). Pengantar Ilmu Hukum. IDEAS PUBLISHING. Rangka Masyarakat Ekonomi ASEAN. Jurnal Hukum Ius Quia I Yuhelson, Y. (2017). Pengantar Ilmu Hukum. IDEAS PUBLISHING. Zainal, A. (2021). Pengantar Ilmu Hukum. Rajawali Pres, Jakarta. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal Al-Manhaj: Jurnal Hukum dan Pranata Sosial Islam,Vol. Almira Kemala Dewi, Tetty Marlina Tarigan / Analisis Hukum Standar Akreditasi Terhadap Sertifikat Halal Luar Negeri Perspektif Fatwa MUI Nomor 4 Tahun 2003 Tentang Standardisasi Fatwa Halal Kemenag. (2022b). Negara dan Sertifikasi Halal Indonesia. 4, 2 (Desember, 2022): 631-640 640 of 640 640 of 640
https://openalex.org/W2225990999	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0146146&type=printable	English	null	Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation	PloS one	2,016	cc-by	7,320	Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation Maria Cheng1, Hayato Yoshiyasu1, Kenji Okano1, Hisao Ohtake1, Kohsuke Honda1,2* 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Sui Osaka 565–0871, Japan, 2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 7 Gobancho, Chiyoda-ku, Tokyo 102–0076, Japan Maria Cheng1, Hayato Yoshiyasu1, Kenji Okano1, Hisao Ohtake1, Kohsuke Honda1,2* Maria Cheng1, Hayato Yoshiyasu1, Kenji Okano1, Hisao Ohtake1, Kohsuke Honda1,2* 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2–1 Yamadaoka, Suita, Osaka 565–0871, Japan, 2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 7 Gobancho, Chiyoda-ku, Tokyo 102–0076, Japan * honda@bio.eng.osaka-u.ac.jp OPEN ACCESS Citation: Cheng M, Yoshiyasu H, Okano K, Ohtake H, Honda K (2016) Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation. PLoS ONE 11(1): e0146146. doi:10.1371/journal.pone.0146146 Editor: Luis Menéndez-Arias, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), SPAIN Editor: Luis Menéndez-Arias, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), SPAIN Received: October 21, 2015 Accepted: December 13, 2015 Published: January 5, 2016 Copyright: © 2016 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Luis Menéndez-Arias, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), SPAIN Editor: Luis Menéndez-Arias, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), SPAIN Received: October 21, 2015 Accepted: December 13, 2015 Published: January 5, 2016 Copyright: © 2016 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Luis Menéndez-Arias, Centro de Biología Molecular Severo Ochoa (CSIC-UAM), SPAIN Received: October 21, 2015 Accepted: December 13, 2015 Published: January 5, 2016 Copyright: © 2016 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2016 Cheng et al. This is an open access article distributed under the terms of the Copyright: © 2016 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetalde- hyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofac- tor-enzyme intermediate and a shift in the reaction specificity of the enzyme. RESEARCH ARTICLE Introduction Enzymes have been recognized as a powerful tool in chemical manufacturing processes and are replacing conventional metallo- and organocatalysts [1]. Thermostable enzymes, in particular, are attracting much attention owing to their inherent stability and compatibility with high- temperature and harsh industrial processes, and therefore bioprospecting effort has been devoted to obtain novel thermostable enzymes with desired catalytic properties [2,3]. To date, there have been two major strategies to obtain thermostable enzymes. The first is by increasing the thermostability of mesophilic enzymes by random mutation [4–8] and by rational design based on known stabilization mechanisms [9–14]. The second strategy is by mining thermo- philic and hyperthermophilic microorganisms for their indigenous enzymes. Enzymes from these (hyper)thermophiles have been reported to display a higher tolerance not only to high temperatures but also to denaturants, such as detergents and organic solvents, than their meso- philic counterparts and are therefore of interest in chemical manufacturing [15]. Studies on the stabilization mechanisms of these thermophilic enzymes have made it possible to design enzymes with higher thermostability [16–18]. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Japan Science and Technology Agency (JST), CREST program, and the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant (26450088). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase formation of a central and highly reactive intermediate, 2-hydroxyethyl-TPP. In acetolactate synthase, the carboligation between 2-hydroxyethyl-TPP and the second pyruvate molecule leads to the liberation of the reaction product, acetolactate, and the catalytic cycle is completed. On the other hand, protonation of the 2-hydroxyethyl-TPP intermediate preferably occurs in pyruvate decarboxylase, yielding acetaldehyde as a reaction product. On the basis of these structural and functional similarities, we aimed to generate a thermo- stable pyruvate decarboxylase through the alteration of the reaction specificity of a thermo- philic acetolactate synthase by random mutagenesis. Gene cloning and construction of mutant library The expression vector for A. pasteurianus pyruvate decarboxylase (ApPDC) was constructed as described previously [22]. The plasmid encoding the acetolactate synthase large subunit (TtALS; Genbank accession number, YP_144479.1) was obtained from the RIKEN Thermus thermophilus HB8 expression plasmid set [33] and used as a gene source. The TtALS gene was amplified by PCR using ALS-F and -R primers (Table 1), gel-purified, and digested with EcoRI and HindIII. The DNA fragment was introduced to the corresponding restriction site of pUC- 18 (designated as pUC-TtALS) and then transformed into Escherichia coli JM109. Random mutagenesis was introduced to the TtALS gene by error-prone PCR. The PCR mix- ture contained GoTaq Green Master Mix (Promega KK, Tokyo, Japan), 0.2 mM M13-F and M13-R primers (Table 1), 0.1 mM MnCl2, and approximately 10 ng of pUC-TtALS in a total vol- ume of 12.5 μl. PCR was carried out with 95°C preheating for 5 min, 30 cycles of 95°C for 30 s, 55°C for 1 min, 72°C for 1 min 40 s, followed by an elongation step at 72°C for 7 min. This opera- tion resulted in the 1–4 mutation points in the 1,689-bp long TtALS gene. PCR products were purified, restricted, ligated to pUC-18, and transformed into E. coli JM109, as described above. Blue/white selection on an LB agar supplemented with 100 μg ml-1 ampicillin, 40 μg ml-1 5-bromo-4-chloro-3 -indolyl-β-D-galactoside (X-gal, Wako Pure Chemical, Osaka, Japan), and 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Wako Pure Chemical) was performed to select positive transformants. White colonies were picked and stored as a mutant library. Table 1. Oligonucleotide primers used in this study. Primer Sequence (5´-3´) Purpose ALS-F TCGAATTCaGAAGGGAGCGGAGGCACTTTTA Vector construction ALS-R CCAAGCTTbTCACGCCCCCACCTCCTCCT Vector construction M13-F TGTAAAACGACGGCCAGT Error-prone PCR M13-R CAGGAAACAGCTATGAC Error-prone PCR Y35N-F GCCCTCAcACGACAGCCCCATCCGCCAC Site-directed mutagenesis Y35N-R GCTGTCGTTcGAGGGCGTCGTAGGTGGG Site-directed mutagenesis K139R-F GGTGGTGAGcGGAGGCCTTCCACATCGC Site-directed mutagenesis K139R-R GCCTCCCcTCACCACCCGGGGGATCTCG Site-directed mutagenesis V172A-F TTGACGCcGAAGCTGGACCTCCCCGGGT Site-directed mutagenesis V172A-R CCAGCTTCGcCGTCAAAGCTCCCCGTGA Site-directed mutagenesis H474R-F TCTTCCGcCGCCAAGCGCTACAGCGAGG Site-directed mutagenesis H474R-R GCTTGGCGCcGGAAGAGGTCCTGCCACT Site-directed mutagenesis a EcoRI restriction sight is underlined. b HindIII restriction sight is underlined. c Substituted nucleotides are underlined. doi:10.1371/journal.pone.0146146.t001 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 3 / 13 Table 1. Oligonucleotide primers used in this study. doi:10.1371/journal.pone.0146146.t001 Development of Thermophilic Pyruvate Decarboxylase In this study, we propose an alternative approach to obtaining a thermostable enzyme. Pyru- vate decarboxylases (EC 4.1.1.1) are the class of enzymes catalyzing the non-oxidative decarbox- ylation of pyruvate to acetaldehyde, which serves as a primary precursor for the production of ethanol and acetyl-CoA. Pyruvate decarboxylases and their genes are widely distributed in yeast, fungi, and higher plants but are relatively rare in prokaryotes [19]. Among the prokaryotic pyru- vate decarboxylases, those from mesophilic bacteria, including Zymomonas mobilis [20] and Acetobacter pasteurianus [21], have been well characterized. However, BLAST searches of the fully sequenced genomes of (hyper)thermophiles gave no hits when the amino acid sequences of these mesophilic pyruvate decarboxylases were used as queries [19,22]. On the other hand, data- base searches have revealed that many thermophilic enzymes, which are annotated as acetolactate synthase (EC 2.2.1.6), share a certain level of similarity with pyruvate decarboxylases. Acetolactate synthase and pyruvate decarboxylase are both thiamin pyrophosphate (TPP)- dependent enzymes that use pyruvate as a substrate, but they produce different products (Fig 1). Whereas pyruvate decarboxylase catalyzes the non-oxidative decarboxylation of pyruvate to acetaldehyde [23–26], acetolactate synthase, which is involved in the biosynthesis of branched amino acids (Val, Leu, Ile), catalyzes the carboligation between two pyruvate molecules to form an acetolactate molecule and carbon dioxide [23,27–29]. Despite differences in their apparent enzymatic function, the amino acid sequence comparison of several acetolactate synthases and pyruvate decarboxylases showed that they have comparable sequence similarity as well as con- served amino acids (S1 Fig). In addition, the conversion of pyruvate by acetolactate synthase and pyruvate decarboxylase proceeds via the formation of a common substrate-cofactor- enzyme complex (Fig 1) [25,26,30–32]. The carbonyl addition of pyruvate to TPP yields a pre- decarboxylation intermediate followed by the elimination of carbon dioxide, resulting in the Fig 1. Schematic representation of the catalytic cycle of acetolactate synthase and pyruvate decarboxylase. doi:10.1371/journal.pone.0146146.g001 Schematic representation of the catalytic cycle of acetolactate synthase and pyruvate decarboxylase. Fig 1. Schematic representation of the catalytic cycle of acetolactate synthase and pyruvate decarboxylase. d i 10 13 1/j l 0146146 001 Fig 1. Schematic representation of the catalytic cycle of acetolactate synthase and pyruvate decarboxylase. doi:10.1371/journal.pone.0146146.g001 doi:10.1371/journal.pone.0146146.g001 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 2 / 13 Mutant library screening A colorimetric screening system was developed to evaluate the acetaldehyde-forming activity of mutant enzymes. The reaction was coupled with NAD+-dependent acetaldehyde dehydroge- nase from T. thermophilus HB8 (TtALDH; Genbank accession number, YP_145486.1) [22]. The TtALDH gene was obtained from the RIKEN plasmid set and the enzyme was prepared in E. coli Rosetta 2 (DE3) as described elsewhere [22]. Clones in the mutant TtALS library were aerobically cultivated in a 96-deep-well plate at 30°C for 15 hours. Each well contained 500 μl of LB medium supplemented with 100 μg ml-1 ampicillin and 0.1 mM IPTG. Cells were pelleted by centrifugation at 1,500 × g and 4°C, for 10 min. The average weight of the cell pellet in each well was calculated from the increase in the total weight of the multiwell plate, and the cells were resuspended in 50 mM potassium phos- phate buffer (pH 7.0) containing 10 mM MgCl26H2O at an average cell concentration of 50 mg wet cells ml-1. An aliquot (100 μl) of the cell suspension was transferred to a 200 μl PCR tube and heated at 70°C for 30 min with T3000 thermocycler (Biometra, Göttingen, Germany). The cell suspension was then mixed with a 100 μl reaction mixture consisting of 5 mM sodium pyruvate, 12 μM 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS, Dojindo, Kumamoto, Japan), 0.3 mM 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Nacalai Tesque, Kyoto, Japan), 2 mM NAD+ (Oriental Yeast, Tokyo, Japan), 0.2 mM thiamine pyrophosphate (TPP; Wako Pure Chemical), and 100 mg wet cells ml-1 of the heat- treated (70°C for 30 min) cell suspension of E. coli with TtALDH [22]. The mixture was incu- bated at 60°C for 30 min and the formation of MTT formazan was visually monitored. Gene cloning and construction of mutant library Primer Sequence (5´-3´) Purpose ALS-F TCGAATTCaGAAGGGAGCGGAGGCACTTTTA Vector construction ALS-R CCAAGCTTbTCACGCCCCCACCTCCTCCT Vector construction M13-F TGTAAAACGACGGCCAGT Error-prone PCR M13-R CAGGAAACAGCTATGAC Error-prone PCR Y35N-F GCCCTCAcACGACAGCCCCATCCGCCAC Site-directed mutagenesis Y35N-R GCTGTCGTTcGAGGGCGTCGTAGGTGGG Site-directed mutagenesis K139R-F GGTGGTGAGcGGAGGCCTTCCACATCGC Site-directed mutagenesis K139R-R GCCTCCCcTCACCACCCGGGGGATCTCG Site-directed mutagenesis V172A-F TTGACGCcGAAGCTGGACCTCCCCGGGT Site-directed mutagenesis V172A-R CCAGCTTCGcCGTCAAAGCTCCCCGTGA Site-directed mutagenesis H474R-F TCTTCCGcCGCCAAGCGCTACAGCGAGG Site-directed mutagenesis H474R-R GCTTGGCGCcGGAAGAGGTCCTGCCACT Site-directed mutagenesis a EcoRI restriction sight is underlined. b HindIII restriction sight is underlined. c Substituted nucleotides are underlined. doi:10.1371/journal.pone.0146146.t001 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 3 / 13 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase Site-directed mutagenesis PrimeStar mutagenesis kit (Takara Bio, Ohtsu, Japan) was used to create single-amino-acid substituted mutants (Y35N, K139R, V172A, and H474R). PCR was performed in accordance with the manufacturer’s instructions using pUC-TtALS as a template DNA and the primers listed in Table 1. The DNA sequence was confirmed with the 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Enzyme preparation E. coli cells having the wild-type and mutant TtALS were aerobically cultivated at 37°C in a 500 ml Erlenmeyer flask containing LB medium supplemented with 100 μg ml-1 ampicillin. Gene expression was induced by adding 0.1 mM IPTG in the late-log phase. The cells were harvested by centrifugation and resuspended in 20 mM Tris-HCl (pH 8.0) at a cell concentration of 200 mg wet cells ml-1. The cells were disrupted by ultrasonication and centrifuged to remove the cell debris. The soluble fraction was collected as a crude lysate and the total protein concentra- tion was measured with Bio-Rad protein assay kit II (Bio-Rad, Hercules, CA). The crude lysate was then heated at 70°C for 30 min and centrifuged to remove denatured proteins. The result- ing supernatant was used as a heat-treated crude extract. The heat-treated extract of E. coli with ApPDC was prepared in the same manner except that the heat treatment was done at 50°C for 30 min owing to the relatively low thermal stability of the enzyme. Enzyme assays The acetaldehyde-forming activity of the enzyme was determined by coupling with TtALDH. The reaction mixture was composed of 50 mM potassium phosphate buffer (pH7.0), 10 mM sodium pyruvate, 240 μM 1-methoxy PMS, 6 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium (WST-1; Dojindo), 0.67 mM NAD+, 0.1 mM TPP, 10 mM 4 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase β-mercaptoethanol, 10 mM MgCl2, and the heat-treated extract of E. coli with TtALDH. After pre- incubation at 60°C for 2 min, reaction was initiated by adding an appropriate amount of TtALS or its mutants. The reduction of WST-1 to soluble formazan was monitored at 438 nm with UV- 2600 spectrophotometer (Shimadzu, Kyoto, Japan). Enzyme activity was calculated using the molar extinction coefficient of the soluble formazan (37×103 cm-1 at 438 nm). One unit of enzyme activity was defined as the amount of enzyme required to form 1 μmol formazan per minute. β-mercaptoethanol, 10 mM MgCl2, and the heat-treated extract of E. coli with TtALDH. After pre- incubation at 60°C for 2 min, reaction was initiated by adding an appropriate amount of TtALS or its mutants. The reduction of WST-1 to soluble formazan was monitored at 438 nm with UV- 2600 spectrophotometer (Shimadzu, Kyoto, Japan). Enzyme activity was calculated using the molar extinction coefficient of the soluble formazan (37×103 cm-1 at 438 nm). One unit of enzyme activity was defined as the amount of enzyme required to form 1 μmol formazan per minute. Acetolactate-forming activity was assessed by mixing the enzyme solution with 50 mM potassium phosphate buffer (pH7.0), 20 mM sodium pyruvate, 0.1 mM TPP, and 10 mM MgCl2. After incubation at 60°C for 30 minutes, 250 μl of the sample was taken, mixed with 50 μl of 50% H2SO4, and incubated at 37°C for 30 min to promote the decarboxylation of acet- olactate to acetoin. Subsequently, 500 μl each of 0.5% creatin, 5% α-naphtol, and 2.5 M NaOH were added to the mixture and incubated at 37°C for 30 min. Acetoin concentration was deter- mined by measuring the absorbance at 540 nm. The concentration was calculated from a stan- dard curve obtained using serially diluted authentic acetoin (Tokyo Chemical Industry, Tokyo, Japan). One unit of enzyme activity was defined as the amount of enzyme required to form 1 μmol acetoin per minute. Enzyme assays Acetolactate-forming activity was assessed by mixing the enzyme solution with 50 mM potassium phosphate buffer (pH7.0), 20 mM sodium pyruvate, 0.1 mM TPP, and 10 mM MgCl2. After incubation at 60°C for 30 minutes, 250 μl of the sample was taken, mixed with 50 μl of 50% H2SO4, and incubated at 37°C for 30 min to promote the decarboxylation of acet- olactate to acetoin. Subsequently, 500 μl each of 0.5% creatin, 5% α-naphtol, and 2.5 M NaOH were added to the mixture and incubated at 37°C for 30 min. Acetoin concentration was deter- mined by measuring the absorbance at 540 nm. The concentration was calculated from a stan- dard curve obtained using serially diluted authentic acetoin (Tokyo Chemical Industry, Tokyo, Japan). One unit of enzyme activity was defined as the amount of enzyme required to form 1 μmol acetoin per minute. Selection and characterization of model enzymes The protein BLAST search using T. thermophilus HB8 acetolactate synthase (TtALS) large sub- unit as query resulted in 90–100% sequence identity with other known thermophilic acetolac- tate synthases, suggesting high sequence similarities among these enzymes. Since the genomic sequence and gene-expression plasmid set of T. thermophilus HB8 are available [33], enzymes from this organism are more feasible compared to those from other thermophilic sources. In addition, when compared to amino acid sequence of several pyruvate decarboxylases, TtALS also showed relatively high similarities (S1 Fig). Considering these facts, we chose TtALS as a model thermophilic acetolactate synthase. The reaction specificity of TtALS was assessed and compared with that of a bacterial pyru- vate decarboxylase from A. pasteurianus (ApPDC) (Table 2). ApPDC exhibited not only physi- ological activity (acetaldehyde formation) but also a significant acetolactate-forming activity. In contrast, the reaction catalyzed by TtALS was more specific to acetolactate formation. Multiple alignment and structural modeling Multiple alignment of amino acid sequences was performed with the ClustalW2 online tool [34]. The addition of a secondary structure element and the rendering of similarities from aligned sequences were conducted with ESPript (http://espript.ibcp.fr) online tool [35]. Model structures of TtALS and its mutants were generated by SWISS-MODEL homology modeling [36]. The enzyme structure was visualized with the PyMOL Molecular Graphics System (Ver- sion 1.3, Schrödinger, LLC). The structures of the proteins were compared by the secondary structure matching (SSM) method using the Coot program [37]. Development of Thermophilic Pyruvate Decarboxylase Table 2. Specific enzyme activities. Enzyme Speciﬁc activity (x 10−3 U mg-1 total protein)a,b Ratioc Acetolactate formation Acetaldehyde formation ApPDC d 10.8 ± 0.47 8.12 ± 1.8 0.76 TtALS_WT e 12.6 ± 0.63 1.45 ± 0.19 0.12 TtALS_quadruple e 7.96 ± 1.5 4.51 ± 0.42 0.57 TtALS_Y35N e 10.6 ± 0.46 2.00 ± 0.28 0.19 TtALS_K139R e 12.2 ± 3.0 2.23 ± 0.46 0.18 TtALS_V172A e 10.2 ± 2.6 1.27 ± 0.18 0.12 TtALS_H747R e 8.13 ± 2.0 5.52 ± 0.87 0.68 Table 2. Specific enzyme activities. Table 2. Specific enzyme activities. Table 2. Specific enzyme activities. Enzyme Speciﬁc activity (x 10−3 U mg-1 total protein)a,b Ratioc Acetolactate formation Acetaldehyde formation ApPDC d 10.8 ± 0.47 8.12 ± 1.8 0.76 TtALS_WT e 12.6 ± 0.63 1.45 ± 0.19 0.12 TtALS_quadruple e 7.96 ± 1.5 4.51 ± 0.42 0.57 TtALS_Y35N e 10.6 ± 0.46 2.00 ± 0.28 0.19 TtALS_K139R e 12.2 ± 3.0 2.23 ± 0.46 0.18 TtALS_V172A e 10.2 ± 2.6 1.27 ± 0.18 0.12 TtALS_H747R e 8.13 ± 2.0 5.52 ± 0.87 0.68 a Speciﬁc enzyme activity was measured using the heat-treated extracts of E. coli with indicated enzymes and normalized by the protein concentration of the corresponding non-heated crude lysate. The enzyme activities were calculated by subtracting those detected in control experiments, which were conducted using the heat-treated extracts of E. coli without the expression vector. b The enzyme assays were performed at least in triplicate; mean±standard deviations are shown. c Speciﬁc acetaldehyde-forming activities were divided by the acetolactate-forming ones. d Heat-treated extract was prepared by incubating the crude lysate of the recombinant E. coli at 50°C for 30 min. e Heat-treated extracts were prepared by incubating the crude lysate of the recombinant E. coli at 70°C for 30 min. doi:10.1371/journal.pone.0146146.t002 Speciﬁc activity (x 103 U mg-1 total protein)a,b insoluble formazan, which is a blue-colored compound, in the presence of an electron mediator, 1-methoxy PMS. About 5,000 transformants were screened using this system, and 12 mutants were selected for their relatively high signal intensities. The specific acetolactate-forming activities of these mutants were then spectrophotometrically assessed under standard assay conditions; and consequently, one mutant was confirmed to exhibit a 3.1-fold higher activity than that of wild-type TtALS (TtALS_WT) (Table 2). On the other hand, the acetolactate-forming activity of the mutant decreased to 63% of that of TtALS_WT. Site-directed mutagenesis Single-point mutants with either one of the four amino-acid substitutions found in TtALS_- quadruple were constructed by site-directed mutagenesis to examine their contributions to the alteration of the reaction specificity of the enzyme. Among the resulting mutants, the H474R mutant exhibited a higher acetolactate-forming activity than the quadruple mutant (Table 2). In contrast, the reaction-specificity profiles of the other single-point mutants were not signifi- cantly different from that of TtALS_WT, demonstrating that the improved acetaldehyde-form- ing activity of TtALS_quadruple was largely due to the H474R mutation. The ratio of the specific acetaldehyde-forming to acetolactate-forming activity of the H474R mutant reached 0.68, approaching that of ApPDC (0.76). The ratio of the specific acetaldehyde-form- ing activity to the acetolactate-forming activity of the resulting mutant was 4.9-fold higher than that of TtALS_WT. Sequencing analysis revealed that there are four amino acid substitutions (Y35N, K139R, V172A and H474R) in the mutant; thus, it was designated as TtALS_quadruple. As expected, TtALS_quadruple exhibited a similar thermal stability to TtALS (Fig 2). Although ApPDC lost more than 80% of its initial activity after the incubation at 60°C for 90 min, the wild- type and the quadruple mutant of TtALS remained almost intact upon the same treatment. Mutant library screening and characterization of the positive mutant A colorimetric, high-throughput assay system was developed to screen the TtALS-mutant library for improved acetaldehyde-forming activity. In this system, enzymatically generated acetaldehyde was further converted by another enzyme, the aldehyde dehydrogenase from Thermus thermophilus HB8 (TtALDH) [22], with a concomitant reduction of NAD+ to NADH. NADH was then used as an electron donor for the reduction of MTT to a water- 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 doi:10.1371/journal.pone.0146146.t002 Structural modeling analysis Model structures of the wild-type and the H474R mutant TtALS were built on the basis of pro- tein homology using the SWISS MODEL program (Fig 3). The crystal structure of the 6 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase Fig 2. Thermal stability of TtALS_WT (red squares), TtALS_quadruple (yellow triangles), and ApPDC (green diamonds). Enzyme activity was determined by measuring the acetolactate-formation activity of each enzyme after incubation at 60°C for the indicated time period. The assays were performed at least in triplicate; mean±standard deviations (error bars) are depicted. doi:10.1371/journal.pone.0146146.g002 Fig 2. Thermal stability of TtALS_WT (red squares), TtALS_quadruple (yellow triangles), and ApPDC (green diamonds). Enzyme activity was determined by measuring the acetolactate-formation activity of each enzyme after incubation at 60°C for the indicated time period. The assays were performed at least in triplicate; mean±standard deviations (error bars) are depicted. doi:10.1371/journal.pone.0146146.g002 doi:10.1371/journal.pone.0146146.g002 doi:10.1371/journal.pone.0146146.g002 acetohydroxy-acid synthase from Arabidopsis thaliana (AtAHAS; PDB ID, 1Z8N), was chosen by the program as the best template among the available protein structures. The model struc- ture of TtALS implied that both the wild-type and the H474R mutant function as a homotetra- mer, similarly to other structurally characterized acetolactate synthases [29,32,38]. As expected, the model revealed structural similarity between TtALS and ApPDC (RMSD 2.08 Å, S2 Fig), supporting the validity of our concept of engineering TtALS to develop a thermostable pyruvate decarboxylase. , y y y [ , , ] expected, the model revealed structural similarity between TtALS and ApPDC (RMSD 2.08 Å, S2 Fig), supporting the validity of our concept of engineering TtALS to develop a thermostable pyruvate decarboxylase. The model structure also showed that the H474R mutation is located at the γ-domain (Figs 3 and 4), i.e., the TPP-binding domain, of TtALS. The helix containing H474R is located on the pro- tein surface in the interfacial area between two monomers of TtALS and forms an active site with the neighboring monomer (Fig 4). The TtALS_H474R model showed that two newly generated hydrogen bonds, which are also located on the protein surface, are present in the mutant protein. This might result in the increased hydrophilicity in the local area where TPP is bound and also make it easier for H+ to attack the substrate-TPP-enzyme complex intermediate in the enzymatic reaction, thus allowing the mutant to function as a pyruvate-decarboxylase-like protein. Recently, Meyer et al. PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Structural modeling analysis [31] reported the structural analysis on the transitional states of the 2-hydroxyethyl-TPP intermediate (carbanion-enamine intermediate) in TPP-dependent enzymes, leading us to another possible mechanism for the altered reaction specificity of the H474R mutant. The positive charge provided by Arg side chain might contribute to the stabili- zation of the negatively charged carbanion intermediate and result in the redirection of the reaction specificity of the enzyme. Previous works with Zymomonas mobilis pyruvate decar- boxylase also demonstrated the importance of the amino acid residues in the active site of the enzyme for proper substrate binding [39,40]. Considering the position of H474R in the 7 / 13 Development of Thermophilic Pyruvate Decarboxylase Fig 3. Comparison of the model structure of TtALS (left panel) and the crystal structure of AtAHAS (right panel). N- and C-terminal of the proteins are shown by the letters N and C, respectively. The α-, β-, and γ-domains, and the C-terminal tail are shown in light blue, light yellow, pink, and light green, respectively. In the AtAHAS structure, Mg2+ is shown as a yellow sphere while the backbones of other cofactors are shown in yellow with oxygen and nitrogen atoms colored red and blue, respectively. The detailed location of the H474R mutation in TtALS is shown in the inset with the backbone of the mutated residue shown in gray. Fig 3. Comparison of the model structure of TtALS (left panel) and the crystal structure of AtAHAS (right panel). N- and C-terminal of the proteins are shown by the letters N and C, respectively. The α-, β-, and γ-domains, and the C-terminal tail are shown in light blue, light yellow, pink, and light green, respectively. In the AtAHAS structure, Mg2+ is shown as a yellow sphere while the backbones of other cofactors are shown in yellow with oxygen and nitrogen atoms colored red and blue, respectively. The detailed location of the H474R mutation in TtALS is shown in the inset with the backbone of the mutated residue shown in gray. doi:10.1371/journal.pone.0146146.g003 enzyme, it is likely that the positively charged group from Arg formed an ion pair with the carbanion intermediate and facilitated the proton addition. PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Discussion TPP is an important cofactor involved in various types of enzyme reactions, including the decarboxylation, dehydrogenation, and carboligation of keto acids and their derivatives [23,26]. Some of them have recently attracted biotechnological interest as a catalytic module to construct an engineered metabolic pathway. Atsumi et al. demonstrated the non-fermentative production of isobutanol by an engineered E. coli, in which the heterologously expressed aceto- lactate synthase of Bacillus subtilis served as a key enzyme [41]. Opgenorth et al. reported the in vitro reconstitution of thermophilic pyruvate dehydrogenase complexes with different nico- tinamide-cofactor specificities [42]. The reconstituted enzyme complexes were used to con- struct a molecular purge valve system for maintaining the intrapathway NADP+/NADPH balance in in vitro artificial pathways for polyhydroxybutyryate and isoprene production. Among TPP-dependent enzymes, pyruvate decarboxylases play a key role in fermentative pro- duction of ethanol and related compounds. In vitro synthesis of ethanol from glucose has been demonstrated by applying a pyruvate decarboxylase at 50°C [43]. Similarly, we constructed cofactor-balanced, oxygen-insensitive artificial pathway for the in vitro conversion of glucose 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase Fig 4. TPP binding site of the model structure of TtALS. TtALS_WT, TtALS_H474R, and the neighboring monomer are shown in gray, purple, and cyan, respectively. TPP and FAD are shown in yellow, whereas Mg2+ is represented as a yellow sphere. Oxygen atoms are shown in red whereas nitrogen atoms are in blue. New hydrogen bonds are formed between the side chain of H474R with the side chain of S479 and the main chain of M28, which belongs to the neighboring monomer (shown by dotted lines). The cavity through which substrates seem to access the catalytic site is indicated by a black arrow. p p y y Fig 4. TPP binding site of the model structure of TtALS. TtALS_WT, TtALS_H474R, and the neighboring monomer are shown in gray, purple, and cyan, respectively. TPP and FAD are shown in yellow, whereas Mg2+ is represented as a yellow sphere. Oxygen atoms are shown in red whereas nitrogen atoms are in blue. New hydrogen bonds are formed between the side chain of H474R with the side chain of S479 and the main chain of M28, which belongs to the neighboring monomer (shown by dotted lines). The cavity through which substrates seem to access the catalytic site is indicated by a black arrow. PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase are inherently oxygen-sensitive enzymes, hampering their use in biotechnological applications. This limited availability of thermophilic pyruvate-decarboxylating enzymes motivated us to develop a thermostable pyruvate decarboxylase by redirecting the reaction specificity of a func- tionally and structurally related thermophilic enzyme, acetolactate synthase. Through the ran- dom-mutant-library screening followed by site-directed mutagenesis experiments, we confirmed that the single-point mutant with an amino acid substitution of H747R exhibits markedly improved pyruvate-decarboxylase-like activity. However, it should be noted that the H747R mutant still exhibits a significant acetolactate-forming activity, which may hamper the selective, high-yield production of a target compound when the enzyme is applied to a biocata- lytic chemical manufacturing process. Further work focusing on decreasing the acetolactate activity would be indispensable for the application of this mutant. In contrast to our study, Ser- gienko and Jordan demonstrated that the reaction specificity of a yeast pyruvate decarboxylase could be shifted toward the carboligating direction by the mutations at Asp28 and Glu477 resi- dues in the catalytic center of the enzyme [49]. Similarly, the Glu473Gln mutant of Zymomonas mobilis pyruvate decarboxylase was shown to catalyze an enantio-selective carboligation between pyruvate and an aromatic aldehyde and was applied to the asymmetric production of (R)-phenylacetyl carbinol [30]. In fact, the sequence alignment revealed that these amino acid residues are well conserved among pyruvate decarboxylases but not among acetolactate synthases (S1 Fig), suggesting that alteration of the corresponding residues of TtALS (i.e., Gly37 and Val487) may lead to the development of a mutant enzyme with further improved specificity to pyruvate decarboxylation. are inherently oxygen-sensitive enzymes, hampering their use in biotechnological applications. This limited availability of thermophilic pyruvate-decarboxylating enzymes motivated us to develop a thermostable pyruvate decarboxylase by redirecting the reaction specificity of a func- tionally and structurally related thermophilic enzyme, acetolactate synthase. Through the ran- dom-mutant-library screening followed by site-directed mutagenesis experiments, we Supporting Information S1 Fig. Multiple sequence alignment of acetolactate synthases and pyruvate decarboxylases. Amino acid sequences of Arabidopsis thaliana acetohydroxyacid synthase (AtAHAS; PDB ID, 1Z8N) and Thermus thermophilus HB8 acetolactate synthase (TtALS) are aligned with those of acetolactate synthases from Klebsiella pneumonia (KpALS; PDB ID, 1OZG) and Bacillus subti- lis (BsALS; PDB ID, 4RJJ), and pyruvate decarboxylases from Zymomonas mobilis (ZmPDC; PDB ID, 2WVA), Acetobacter pasteurianus (ApPDC; PDB ID, 2VBI), and Saccharomyces cere- visiae (ScPDC; PDB ID, 1PVD). Symbols above the alignment represent the structure of AtA- HAS. α-Helices and 310-helices (η) are indicated by curved lines. Black arrows and TT letters represent β-strands and β-turns, respectively. Black dots above the AtAHAS sequence mark the sequence every 10 residues. Similar residues in the alignment are shown in blue boxes, while those printed in white on red are conserved residues. The position of H474 in the TtALS sequence is indicated by a red arrow. The positions of D28 and E477 in ScPDC, whose substitu- tions caused the alteration of the reaction specificity of the enzyme [49], are indicated by green asterisks. (PDF) S2 Fig. Superimposition of TtALS model structure with the crystal structure of ApPDC (PDB ID 2VBI). The TtALS model structure is colored purple whereas ApPDC is shown in green. S2 Fig. Superimposition of TtALS model structure with the crystal structure of ApPDC (PDB ID 2VBI). The TtALS model structure is colored purple whereas ApPDC is shown in green. (PDF) Discussion doi:10.1371/journal.pone.0146146.g004 to 1-butanol [22] and N-acetylglutamate [44] by employing ApPDC at 50°C. Thermostable pyruvate decarboxylases would be a promising enzyme module to establish more feasible in vitro bioconversion systems as well as to develop an engineered thermophile for consolidated bioprocessing at high temperatures [43,45,46]. However, the number of reports on thermophilic TPP-dependent enzymes is still limited. In particular, to the best of our knowledge, there have been no reports on (hyper)thermophile- derived pyruvate decarboxylase [19,22]. Although several pyruvate ferredoxin oxidoreductases from hyperthermophilic archaea have been reported to catalyze the analogous reaction, i.e., the non-oxidative decarboxylation of pyruvate to acetaldehyde, in the absence of CoA [47,48], they 9 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 References 1. Liese A. Replacing chemical steps by biotransformations: Industrial application and processes using biocatalysis. Enzyme Catalysis in Organic Synthesis. Wiley-VCH Verlag GmbH; 2008. pp. 1419–1459. doi: 10.1002/9783527618262.ch19 2. Bruins ME, Janssen AE, Boom RM. Thermozymes and their applications: a review of recent literature and patents. Appl Biochem Biotechnol. 2001; 90: 155–86. Available: http://www.ncbi.nlm.nih.gov/ pubmed/11297390. PMID: 11297390 3. Atomi H, Sato T, Kanai T. Application of hyperthermophiles and their enzymes. Curr Opin Biotechnol. 2011; 22: 618–26. doi: 10.1016/j.copbio.2011.06.010 PMID: 21741818 4. Labrou NE. Directed enzyme evolution: Bridging the gap between natural enzymes and commercial applications. Biomol Eng. 2005; 22: vii–ix. doi: 10.1016/j.bioeng.2005.02.001 5. Koyama Y, Hidaka M, Nishimoto M, Kitaoka M. Directed evolution to enhance thermostability of galacto-N-biose/lacto-N-biose I phosphorylase. Protein Eng Des Sel. 2013; 26: 755–61. doi: 10.1093/ protein/gzt049 PMID: 24065834 6. Akbulut N, Tuzlakoğlu Öztürk M, Pijning T, İşsever Öztürk S, Gümüşel F. Improved activity and thermo- stability of Bacillus pumilus lipase by directed evolution. J Biotechnol. 2013; 164: 123–9. doi: 10.1016/j. jbiotec.2012.12.016 PMID: 23313890 7. Yamada R, Higo T, Yoshikawa C, China H, Ogino H. Improvement of the stability and activity of the BPO-A1 haloperoxidase from Streptomyces aureofaciens by directed evolution. J Biotechnol. 2014; 192PA: 248–254. doi: 10.1016/j.jbiotec.2014.10.030 8. Denard CA, Ren H, Zhao H. Improving and repurposing biocatalysts via directed evolution. Curr Opin Chem Biol. 2015; 25C: 55–64. doi: 10.1016/j.cbpa.2014.12.036 9. Fágáin CÓ. Understanding and increasing protein stability. Biochim Biophys Acta—Protein Struct Mol Enzymol. 1995; 1252: 1–14. doi: 10.1016/0167-4838(95)00133-F 10. Yun HS, Park HJ, Joo JC, Yoo YJ. Thermostabilization of Bacillus subtilis lipase A by minimizing the structural deformation caused by packing enhancement. J Ind Microbiol Biotechnol. 2013; 40: 1223–9. doi: 10.1007/s10295-013-1330-2 PMID: 24005991 11. Ding H, Gao F, Liu D, Li Z, Xu X, Wu M, et al. Significant improvement of thermal stability of glucose 1- dehydrogenase by introducing disulfide bonds at the tetramer interface. Enzyme Microb Technol. 2013; 53: 365–72. doi: 10.1016/j.enzmictec.2013.08.001 PMID: 24315638 12. Sakai G, Kojima K, Mori K, Oonishi Y, Sode K. Stabilization of fungi-derived recombinant FAD-depen- dent glucose dehydrogenase by introducing a disulfide bond. Biotechnol Lett. 2015; doi: 10.1007/ s10529-015-1774-8 13. Naderi M, Moosavi-Movahedi AA, Hosseinkhani S, Nazari M, Bohlooli M, Hong J, et al. Implication of disulfide bridge induced thermal reversibility, structural and functional stability for luciferase. Protein Pept Lett. 2015; 22: 23–30. Available: http://www.ncbi.nlm.nih.gov/pubmed/25159509. PMID: 25159509 14. Acknowledgments This work was supported in part by the Japan Science and Technology Agency (JST), as part of the CREST program. This work was also partly supported by the Japan Society for the Promo- tion of Science (JSPS), KAKENHI Grant (26450088). 10 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase Author Contributions Conceived and designed the experiments: HO KH. Performed the experiments: MC HY. Ana- Conceived and designed the experiments: HO KH. Performed the experiments: MC HY. Ana- lyzed the data: KO HO KH. Wrote the paper: MC KH. lyzed the data: KO HO KH. Wrote the paper: MC KH. PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 References Structure and mechanism of inhibition of plant acetohydroxya- cid synthase. Plant Physiol Biochem. 2008; 46: 309–24. doi: 10.1016/j.plaphy.2007.12.004 PMID: 18234503 28. Chipman D, Barak Z, Schloss J V. Biosynthesis of 2-aceto-2-hydroxy acids: acetolactate synthases and acetohydroxyacid synthases. Biochim Biophys Acta—Protein Struct Mol Enzymol. 1998; 1385: 401–419. doi: 10.1016/S0167-4838(98)00083-1 29. Chipman DM, Duggleby RG, Tittmann K. Mechanisms of acetohydroxyacid synthases. Curr Opin Chem Biol. 2005; 9: 475–81. doi: 10.1016/j.cbpa.2005.07.002 PMID: 16055369 30. Meyer D, Walter L, Kolter G, Pohl M, Müller M, Tittmann K. Conversion of pyruvate decarboxylase into an enantioselective carboligase with biosynthetic potential. J Am Chem Soc. American Chemical Soci- ety; 2011; 133: 3609–16. doi: 10.1021/ja110236w PMID: 21341803 31. Meyer D, Neumann P, Koers E, Sjuts H, Lüdtke S, Sheldrick GM, et al. Unexpected tautomeric equilib- ria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versa- tility of thiamin. Proc Natl Acad Sci U S A. 2012; 109: 10867–72. doi: 10.1073/pnas.1201280109 PMID: 22730460 32. Tittmann K, Golbik R, Uhlemann K, Khailova L, Schneider G, Patel M, et al. NMR analysis of covalent intermediates in thiamin diphosphate enzymes. Biochemistry. 2003; 42: 7885–91. doi: 10.1021/ bi034465o PMID: 12834340 33. Yokoyama S, Matsuo Y, Hirota H, Kigawa T, Shirouzu M, Kuroda Y, et al. Structural genomics projects in Japan. Prog Biophys Mol Biol. 2000; 73: 363–376. doi: 10.1016/S0079-6107(00)00012-2 PMID: 11063781 34. McWilliam H, Li W, Uludag M, Squizzato S, Park YM, Buso N, et al. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res. 2013; 41: W597–600. doi: 10.1093/nar/gkt376 PMID: 23671338 35. Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014; 42: W320–4. doi: 10.1093/nar/gku316 PMID: 24753421 36. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014; 42: W252–8. doi: 10.1093/nar/gku340 PMID: 24782522 37. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. International Union of Crystallography; 2010; 66: 486–501. doi: 10.1107/ S0907444910007493 PMID: 20383002 38. Pang SS, Duggleby RG, Schowen RL, Guddat LW. The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate. J Biol Chem. 2004; 279: 2242–53. doi: 10.1074/jbc.M304038200 PMID: 14557277 39. Huang CY, Chang AK, Nixon PF, Duggleby RG. References Rojkova AM, Galkin AG, Kulakova LB, Serov AE, Savitsky PA, Fedorchuk V V., et al. Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices. FEBS Lett. 1999; 445: 183–188. doi: 10.1016/S0014-5793(99)00127-1 PMID: 10069397 15. Atomi H. Recent progress towards the application of hyperthermophiles and their enzymes. Curr Opin Chem Biol. 2005; 9: 166–173. doi: 10.1016/j.cbpa.2005.02.013 PMID: 15811801 16. Kimura S, Nakamura H, Hashimoto T, Oobatake M, Kanaya S. Stabilization of Escherichia coli ribonu- clease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart. J Biol Chem. 1992; 267: 21535–42. Available: http://www.ncbi.nlm.nih.gov/pubmed/1328237. PMID: 1328237 17. Berezovsky IN, Shakhnovich EI. Physics and evolution of thermophilic adaptation. Proc Natl Acad Sci U S A. 2005; 102: 12742–7. doi: 10.1073/pnas.0503890102 PMID: 16120678 18. Jorda J, Yeates TO. Widespread disulfide bonding in proteins from thermophilic archaea. Archaea. 2011; 2011: 409156. doi: 10.1155/2011/409156 PMID: 21941460 19. Eram MS, Ma K. Decarboxylation of pyruvate to acetaldehyde for ethanol production by hyperthermo- philes. Biomolecules. Multidisciplinary Digital Publishing Institute; 2013; 3: 578–96. doi: 10.3390/ biom3030578 PMID: 24970182 11 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase 20. Dawes EA, Ribbons DW, Large PJ. The route of ethanol formation in Zymomonas mobilis. Biochem J. 1966; 98: 795–803. Available: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid= 1264921&tool=pmcentrez&rendertype=abstract. PMID: 4287842 21. Chandra Raj K, Ingram LO, Maupin-Furlow JA. Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus. Arch Microbiol. 2001; 176: 443–51. doi: 10. 1007/s002030100348 PMID: 11734888 22. Krutsakorn B, Honda K, Ye X, Imagawa T, Bei X, Okano K, et al. In vitro production of n-butanol from glucose. Metab Eng. 2013; 20: 84–91. doi: 10.1016/j.ymben.2013.09.006 PMID: 24055789 23. Bunik VI, Tylicki A, Lukashev N V. Thiamin diphosphate-dependent enzymes: from enzymology to met- abolic regulation, drug design and disease models. FEBS J. 2013; 280: 6412–42. doi: 10.1111/febs. 12512 PMID: 24004353 24. Pei X-Y, Erixon KM, Luisi BF, Leeper FJ. Structural insights into the prereaction state of pyruvate decar- boxylase from Zymomonas mobilis. Biochemistry. American Chemical Society; 2010; 49: 1727–36. doi: 10.1021/bi901864j PMID: 20099870 25. Jordan F. Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions. Nat Prod Rep. The Royal Society of Chemistry; 2003; 20: 184–201. doi: 10.1039/b111348h PMID: 12735696 26. Kluger R, Tittmann K. Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates. Chem Rev. 2008; 108: 1797–833. doi: 10.1021/cr068444m PMID: 18491870 27. Duggleby RG, McCourt JA, Guddat LW. PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 References Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence. Eur J Biochem. 2001; 268: 3558–65. Available: http://www.ncbi.nlm.nih.gov/pubmed/11422387. PMID: 11422387 40. Meyer D, Neumann P, Parthier C, Friedemann R, Nemeria N, Jordan F, et al. Double duty for a con- served glutamate in pyruvate decarboxylase: evidence of the participation in stereoelectronically 12 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0146146 January 5, 2016 Development of Thermophilic Pyruvate Decarboxylase controlled decarboxylation and in protonation of the nascent carbanion/enamine intermediate. Bio- chemistry. 2010; 49: 8197–212. doi: 10.1021/bi100828r PMID: 20715795 41. Atsumi S, Hanai T, Liao JC. Non-fermentative pathways for synthesis of branched-chain higher alco- hols as biofuels. Nature. 2008; 451: 86–9. doi: 10.1038/nature06450 PMID: 18172501 42. Opgenorth PH, Korman TP, Bowie JU. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nat Commun. 2014; 5: 4113. doi: 10.1038/ncomms5113 PMID: 24936528 43. Guterl J-K, Garbe D, Carsten J, Steffler F, Sommer B, Reiße S, et al. Cell-free metabolic engineering: production of chemicals by minimized reaction cascades. ChemSusChem. 2012; 5: 2165–72. doi: 10. 1002/cssc.201200365 PMID: 23086730 44. Krutsakorn B, Imagawa T, Honda K, Okano K, Ohtake H. Construction of an in vitro bypassed pyruvate decarboxylation pathway using thermostable enzyme modules and its application to N-acetylglutamate production. Microb Cell Fact. 2013; 12: 91. doi: 10.1186/1475-2859-12-91 PMID: 24099461 45. Cripps RE, Eley K, Leak DJ, Rudd B, Taylor M, Todd M, et al. Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production. Metab Eng. 2009; 11: 398–408. doi: 10.1016/j. ymben.2009.08.005 PMID: 19703579 46. Van Zyl LJ, Taylor MP, Eley K, Tuffin M, Cowan DA. Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius. Appl Microbiol Biotech- nol. 2013; 98: 1247–1259. doi: 10.1007/s00253-013-5380-1 PMID: 24276622 47. Ma K, Hutchins A, Sung SJ, Adams MW. Pyruvate ferredoxin oxidoreductase from the hyperthermo- philic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase. Proc Natl Acad Sci U S A. 1997; 94: 9608–13. Available: http://www.pubmedcentral.nih.gov/articlerender. fcgi?artid=23233&tool=pmcentrez&rendertype=abstract. PMID: 9275170 48. Eram MS, Oduaran E, Ma K. The bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreduc- tase from Thermococcus guaymasensis. Archaea. 2014; 2014: 349379. doi: 10.1155/2014/349379 PMID: 24982594 49. Sergienko EA, Jordan F. Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products. Biochemistry. 2001; 40: 7369–81. Available: http://www.ncbi.nlm.nih.gov/pubmed/11412091. 49. Sergienko EA, Jordan F. Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products. Biochemistry. 2001; 40: 7369–81. Available: http://www.ncbi.nlm.nih.gov/pubmed/11412091. PMID: 11412091 References PMID: 11412091 13 / 13
https://openalex.org/W4324393292	https://www.emerald.com/insight/content/doi/10.1108/IJCST-04-2022-0050/full/pdf?title=differences-in-fabric-hand-perceptions-among-japanese-and-chinese-individuals	English	null	Differences in fabric hand perceptions among Japanese and Chinese individuals	International journal of clothing science and technology	2,023	cc-by	8,367	Differences in fabric hand perceptions among Japanese and Chinese individuals Received 6 April 2022 Revised 17 August 2022 23 November 2022 Accepted 13 December 2022 y y School of Engineering, Utsunomiya University, Utsunomiya, Japan © Tomoharu Ishikawa, Junki Tsunetou, Yoshiko Yanagida, Mutsumi Yanaka, Minoru Mitsui, Kazuya Sasaki and Miyoshi Ayama. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http:// creativecommons.org/licences/by/4.0/legalcode This work was supported by JSPS KAKENHI [Grant number: 18H03317]. International Journal of Clothing Science and Technology Vol. 35 No. 3, 2023 pp. 334-349 Emerald Publishing Limited 0955-6222 DOI 10.1108/IJCST-04-2022-0050 g y g This work was supported by JSPS KAKENHI [Grant number: 18H03317]. The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/0955-6222.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/0955-6222.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/0955-6222.htm Introduction The use of online shopping for clothing and fabrics is increasing, especially amid the COVID-19 pandemic (Aston et al., 2020; Koch et al., 2020). However, there is often a discrepancy between the texture of the cloth judged from a presented image vs that of the actual cloth. To address this issue, it is essential to clarify the mechanisms by which consumers judge fabric texture and thus employ images in online shopping that enable consumers to easily recognize fabric textures. 335 People’s evaluations of fabric hand are shaped by several factors, including the fabric attributes, such as its weave or the thickness of the yarn, the perceptual information (such as tactile or visual information) regarding these attributes and the person’s expertise in fabrics. There is a large body of literature on fabric hand. The pioneering studies in this area are those by Kawabata (1980) and Niwa (1990). Using standard fabric hand values provided by skilled technicians, these authors developed the “Kawabata Evaluation System for Fabrics” (KES- FB) to evaluate fabric hand based entirely on the mechanical properties of fabric. Subsequent studies examined how fabric hand evaluations are shaped by evaluator attributes, fabric attributes and perceptual modality and how these variables interact (Kobayashi, 1972; Nishimatsu and Sakai, 1987; Yashima et al., 2017). However, few studies have considered the influence of the evaluator’s expertise together with fabric and perceptual variables. A previous study (Tsunetou et al., 2018) attempted to address this issue by examining how fabric hand evaluations were shaped by the evaluator’s expertise, fabric attributes and perceptual modality. Participants were divided into experienced and less experienced groups and evaluated the fabric hand of varying attributes via two perceptual modalities: tactile and visuotactile. It was found that expertise level influenced fabric hand evaluations. Specifically, for both modalities, the hand evaluations of the experienced participants were likelier to result from an accurate analysis of the key fabric attributes and the tactile and visuotactile hand evaluations exhibited similarities. However, the study was limited in that the sample was solely Japanese. y J p Clothing industries aiming to globalize their production and sales may be interested in cross-cultural differences in fabric hand evaluations. For example, Stean et al. (1988) surveyed the summer-wear hand evaluations of judges in Japan, Australia, New Zealand, India, the US and China. National differences in fabric hand feel Abstract Abstract Purpose – The study aimed to clarify differences in fabric hand perceptions among Japanese and Chinese participants and implement online shopping strategies that enable consumers to easily recognize fabric texture. Purpose – The study aimed to clarify differences in fabric hand perceptions among Japanese and Chinese participants and implement online shopping strategies that enable consumers to easily recognize fabric texture. Design/methodology/approach – Forty (20 Japanese and 20 Chinese) participants knowledgeable about clothing and fabric were recruited. Participants evaluated fabric by sight and touch in a visuotactile experiment (VTE). The stimulus material comprised 39 fabric samples representing a broad range of fabric attributes (7 fibers, 5 weaving/knitting techniques and 3 yarn thicknesses and density). A Mann–Whitney U test and a factor analysis were conducted to determine differences in responses for the different fabric variables. Findings – The fabric hand perceptions factors were similar between both groups. Japanese participants showed a stronger preference for fabrics that felt wet. Japanese participants’ fabric hand perceptions had a 3-factor structure, while Chinese participants had a 2-factor structure. Chinese participants regarded “crisp” as perceptually and linguistically equivalent to “stretchy.” Originality/value – The study’s findings suggest that Chinese people have stronger preferences in fabrics than Japanese people do. Japanese people evaluate fabric hand in a more nuanced manner than Chinese individuals, including discerning different fabric attributes, such as fiber and yarn thickness and density. Thus, nationality may influence fabric hand perceptions more than fabric knowledge does. Specifically, in evaluating “crispness,” the results required further analysis because differences in nationality may have affected evaluations regarding perception and linguistic perspectives. The findings provide design guidelines for implementing online shopping strategies adapted to each participant group. Keywords Fabric, Chinese, Japanese, Fabric hand perceptions Keywords Fabric, Chinese, Japanese, Fabric hand perceptions Introduction According to a factor analysis, the Japanese and Chinese hand evaluations exhibited a factor structure that differed from the evaluations in other countries, and this was attributable to the mechanical properties of the fabrics. Moreover, Kim and Winakor (1996) examined the adjectives that American and South Korean consumers used to describe fabric hand. They reported that fabric perceptions were shaped by gender, culture and language, and that this was especially true for fabric preferences. Suehiro et al. (2014) examined how mechanical properties informed Japanese and Chinese participants’ perceptions of a fabric’s “shittori,” which is defined as a sensation similar to moisturized skin and semantically described as “baby’s skin.” Japanese participants perceived fabric as “shittori” if it used a knitting technique, featured a surface with frictional resistance and exhibited rigid shear, bending and low breathability. Contrastingly, Chinese participants perceived fabric as “shittori” if it featured gentle shear and bending. However, these studies focused on either a limited set of fabric attributes or a few evaluation parameters; for example, participants only evaluated the fabric regarding its intended use. Consequently, the effect of nationality on hand evaluations for fabrics of varying attributes remains unclear. IJCST 35,3 Figure 2. Experimental procedure Method Th The present study sought to determine cross-cultural differences in fabric hand perceptions. Twenty Japanese and 20 Chinese individuals, knowledgeable about clothing, were recruited. China was selected as the comparison nationality because the Chinese account for the largest proportion of the world’s population and have many connections to Japan. Participants undertook one visuotactile experiment (VTE). The participants’ responses were analyzed to determine intergroup differences regarding fabric attributes. proportion of the world’s population and have many connections to Japan. Participants undertook one visuotactile experiment (VTE). The participants’ responses were analyzed to determine intergroup differences regarding fabric attributes. Experimental method The fabric stimuli were stored for over 24 h in a standardized environment, with a temperature of 208C and 65% humidity (Figure 1). Tests used a Judge II lighting booth (X-Rite, Grand Rapids). Each participant sat before the booth and evaluated the fabric therein. The booth was illuminated with a standard light source (D65). A total of 40 individuals participated: 20 Japanese (19 women, 1 man; aged 20–23 years) and 20 Chinese participants (19 women, 1 man; aged 21–30 years). Participants were students of Bunka Gakuen University and had formally studied fabrics and clothing. Informed written consent was obtained from all participants. 336 p p Figure 2 shows the experimental procedure. Participants washed their hands in preparation for the evaluation experiment, in which 39 types of fabrics were evaluated. One session was conducted with 13 types of fabric evaluation, and there were a total of three sessions, with 5-min breaks between them. The participants followed a standardized procedure when providing tactile evaluations of the fabric hand. This procedure mirrored End Rest (5minutes) Evaluation (13 types) Explanation of experiment Start Washing hands 13 types × 3 = total 39 types VTE: Visual Tactile Experiment Viewing distance 40~50cm Judge II Standard light source (D65) Fabric sample Figure 2. Experimental procedure Figure 1. National differences in fabric hand feel Method Th Experimental environment VTE: Visual Tactile Experiment Viewing distance 40~50cm Judge II Standard light source (D65) Fabric sample VTE: Visual Tactile Experiment Viewing distance 40~50cm Judge II Standard light source (D65) Fabric sample Standard light source (D65) Judge II Viewing distance 40~50cm Fabric sample VTE: Visual Tactile Experiment End Rest (5minutes) Evaluation (13 types) Explanation of experiment Start Washing hands 13 types × 3 = total 39 types End Rest (5minutes) Evaluation (13 types) Explanation of experiment Start Washing hands 13 types × 3 = total 39 types Saito and Harada’s (1987) three-step process (Figure 3). Upon completing these three steps, the participants rated how the fabric felt to touch. Participants could feel the fabric again and reconsider their rating if necessary, because we aimed for fabric hand ratings to be as accurate as possible. The scoring system consisted of a set of fabric hand descriptors rated on a 5-point scale, ranging from 0 5 disagree to 4 5 completely agree (Inoue, 2002, Figure 4). Nine descriptors were taken from Ishikawa et al. (2015): thin, thick, flat, rustic, soft, crisp, wet, dry and stretchy. We also added two thermal descriptors, two descriptors assessing participants’ preference for the fabric and a visual descriptor: warm and cool, likeable and comfortable and shiny, respectively. The participants were provided sheets on which to state their ratings, with evaluation words randomly written for each subject. In addition, to control the meaning of each evaluation word, similar words examined in previous research (Ishikawa et al., 2015) were summarized in a table and explained. For Japanese participants, the text was provided entirely in Japanese. For Chinese participants, the content was provided in Japanese, accompanied by a Chinese translation. The Chinese translation was carefully conducted by a Chinese-native clothing expert to preserve the meaning in the Japanese clothing field as much as possible. 337 3. Bending and stroking the edges of the fabric with dominant hands 1. Stroking the fabric surface in the dominant hand 3. Bending and stroking the edges of the fabric with dominant hands Results 338 esu ts Evaluations results Mann–Whitney U test. To compare the ratings between the Japanese and Chinese participants, a Mann–Whitney U test was conducted on the responses for the different fabric variables. Table 2 shows the results of the Mann–Whitney U test by fabric attribute. The results indicate significant intergroup differences in ratings for five of the descriptors (crisp, wet, cool, likeable and comfortable) regarding any or all fabric attributes. The significant intergroup differences for the descriptors are discussed below. Wet. Figure 5 shows the Japanese and Chinese participants’ average and standard deviation of “wet” ratings for each fiber represented by the symbols and error bars, respectively. The Japanese ratings and deviations are on the X-axis and that of the Chinese are represented on the Y-axis. The data suggest that Japanese participants mostly rated the fabrics as wetter than the Chinese participants did. However, the Japanese participants’ standard deviation of evaluation for each fabric was larger than that of the Chinese participants. Only hemp fabric (indicated by a diamond in Figure 5) was rated as less wet by the Japanese participants. J p p p Likeable. Figure 6 shows the intergroup responses for “likeable.” The graph is plotted identically to the format in Figure 5. Figure 6 indicates that the Chinese rated more fabrics as “likeable” than the Japanese participants did; however, both groups’ standard deviation of evaluation for each fabric material were large. Similarly, the Chinese participants deemed more fabrics with many fibers to be “comfortable” than did their Japanese counterparts. Accordingly, weexaminedwhether theresponses for “likeable” correlated with theresponses for semantically similar descriptors. Table 3 shows the four comparative descriptors (soft, wet, comfortable and dry) that exhibited a strong correlation with “likeable” (the absolute value of the Pearson’s correlation coefficient was >0.6 in each case). “Likeable” was strongly positively correlated with “soft,” “wet,” and “comfortable,” and strongly negatively correlated with “dry,” regardless of nationality and sensory modality (Table 3). Crisp. Figure 7 show the intergroup responses for “crisp.” The Japanese participants tended to perceive fabric as crisper if it was woven (plain, twill, or satin weave) than if it was stitched (plain or rib stitch). However, the Japanese participants’ standard deviation of evaluation for each fabric weaving technique was larger than that of the Chinese participants. IJCST 35,3 yarns/inch). To control the influence of color on visual impression, only beige fabric samples were used. The dimensions were also standardized at 20 cm2. Table 1 shows the mechanical properties of all 39 fabrics. These characteristics were measured using KES-FB Series (Katotech) [1]. yarns/inch). To control the influence of color on visual impression, only beige fabric samples were used. The dimensions were also standardized at 20 cm2. Table 1 shows the mechanical properties of all 39 fabrics. These characteristics were measured using KES-FB Series (Katotech) [1]. Results The “crisp” ratings of Chinese participants were strongly positively correlated with their ratings for “stretchy.” Figure 8 depicts the Japanese and Chinese “crisp” and “stretchy” ratings for all 39 fabrics. The symbols represent the different weaving/knitting techniques. The figure shows correlations between each group’s responses for “crisp” and “stretchy;” specifically, it was negative among the Japanese group (R_J 5 0.76) and strongly positive among the Chinese group (R_C 5 0.97). IJCST 35,3 Fabrics The study used a total of 39 fabric stimuli representing various combinations of three fabric attribute variables: fiber, weaving/knitting and yarn thickness and density. The fibers used were cotton, hemp, wool, silk, cupro, nylon, or polyester. The weaving/knitting variable was plain weave (PW), twill weave (T), satin weave (S), plain stitch (PS), or rib stitch (R). The yarn thickness and density were classified as thick (T; >500 dtex and 38.3 ± 19.34 yarns/inch), medium (M; 250–500 dtex and 56.6 ± 23.98 yarns/inch), or fine (F: <250 dtex and 94.2 ± 45.05 1. Stroking the fabric surface in the dominant hand 2. Holding the fabric with both hands and stroking with your fingertips 3. Bending and stroking the edges of the fabric with dominant hands 0 1 2 3 4 Disagree Slightly agree Agree Agree strongly Agree Very strongly Evaluation word Figure 3. Procedures of touching the fabric Figure 4. Evaluation scale 1. Stroking the fabric surface in the dominant hand 3. Bending and stroking the edges of the fabric with dominant hands 1. Stroking the fabric surface in the dominant hand 2. Holding the fabric with both hands and stroking with your fingertips Figure 3. Procedures of touching the fabric Figure 3. Procedures of touching the fabric 0 1 2 3 4 Disagree Slightly agree Agree Agree strongly Agree Very strongly Evaluation word Factors determining fabric hand perceptions g f p p To determine how fabric hand perceptions were influenced by nationality (Japanese/Chinese), a factor analysis was performed on the results for each fabric according to nationality. The factor scores (termed “fabric hand spaces”) were then analyzed. Table 1. Fabric samples and mechanical characteristics Factors determining fabric hand perceptions Specifically, we analyzed the nationality-related differences within a fabric hand space, derived from a combination (“VTE space”) of Japanese (“Japanese space”) and Chinese participants’ (“Chinese space”) Fiber Weaving knitting Thickness of yarn Tensile Bending Shear Compression Surface Thickness Weight LT WT RT G 2HG 2HG5 B 2HB LC WC RC MIU MMD SMD TO W – gf$cm/ cm2 % gf/ cm$degree gf/ cm gf/ cm gf$cm2/ cm gf$cm/ cm – gf$cm/ cm2 % – – μm mm mg/m2 Cotton PW F 0.66 10.51 46.22 1.08 1.23 4.55 0.04 0.03 0.33 0.12 52.11 0.14 0.02 2.44 0.35 11.21 PW M 0.71 10.54 47.72 2.29 2.77 7.60 0.09 0.08 0.34 0.12 50.52 0.13 0.04 4.70 0.47 17.23 PW T 0.62 9.46 48.10 2.25 2.44 7.25 0.20 0.12 0.33 0.17 45.85 0.16 0.02 6.26 0.66 25.65 T F 0.66 10.67 44.60 1.68 3.30 8.26 0.08 0.08 0.41 0.28 36.53 0.16 0.01 2.00 0.60 16.25 T M 0.63 10.11 46.13 0.86 1.04 3.47 0.11 0.07 0.31 0.16 47.27 0.15 0.01 1.80 0.57 19.50 T T 0.60 10.63 44.09 1.00 1.26 3.77 0.17 0.14 0.39 0.16 46.29 0.15 0.01 2.67 0.70 26.89 S F 0.62 9.36 43.32 0.51 0.84 2.14 0.06 0.03 0.31 0.13 46.11 0.14 0.01 1.40 0.41 12.00 S M 0.65 9.43 45.19 1.11 1.91 4.60 0.14 0.10 0.33 0.18 44.28 0.15 0.01 1.54 0.67 23.53 PS F 0.83 9.28 32.02 0.80 2.77 3.05 0.02 0.02 0.32 0.26 39.44 0.17 0.01 2.86 0.74 13.94 PS M 0.87 8.01 32.22 0.77 3.18 3.47 0.03 0.03 0.32 0.29 37.07 0.20 0.01 3.44 0.86 16.03 R F 0.84 15.39 37.14 0.61 2.02 2.10 0.03 0.03 0.32 0.28 41.71 0.19 0.02 7.44 0.87 15.28 R M 0.96 8.54 33.59 1.12 4.66 5.33 0.14 0.13 0.31 0.32 39.33 0.22 0.04 7.17 1.36 31.19 Hemp PW M 0.59 13.16 29.46 0.26 0.16 0.53 0.16 0.09 0.28 0.18 37.74 0.16 0.03 7.81 0.57 15.84 PW T 0.61 14.30 35.28 0.30 0.19 0.66 0.22 0.11 0.42 0.26 41.62 0.19 0.03 9.22 0.69 21.90 T F 0.49 13.31 32.94 0.22 0.11 0.23 0.11 0.04 0.34 0.20 41.50 0.21 0.02 4.02 0.59 13.80 Wool PW M 0.59 12.22 65.99 0.53 0.31 1.13 0.06 0.02 0.33 0.08 72.25 0.17 0.04 5.99 0.39 16.06 PW T 0.51 11.68 56.41 0.54 0.94 1.83 0.18 0.06 0.39 0.17 62.42 0.16 0.01 5.32 0.74 25.64 T M 0.69 12.70 62.43 0.55 0.50 1.31 0.11 0.03 0.35 0.11 63.55 0.16 0.01 2.42 0.61 21.70 T T 0.51 10.44 52.65 0.49 0.97 1.63 0.14 0.05 0.42 0.26 66.73 0.16 0.01 2.90 0.83 24.62 S M 0.68 10.01 61.65 0.71 0.94 2.06 0.15 0.06 0.40 0.17 68.90 0.16 0.01 2.67 0.79 25.86 PS M 0.94 8.75 50.67 1.03 2.46 3.10 0.12 0.08 0.39 0.29 68.74 0.16 0.01 3.39 1.14 29.04 PS T 0.85 12.65 38.84 0.56 2.22 2.34 0.21 0.18 0.43 0.93 56.73 0.28 0.02 7.34 2.33 27.67 R M 0.74 7.18 54.36 0.57 1.01 1.19 0.07 0.04 0.40 0.74 61.97 0.26 0.02 8.69 1.90 26.19 Silk PW F 0.60 18.27 46.60 0.22 0.03 0.17 0.01 0.00 0.47 0.05 81.81 0.17 0.02 2.61 0.22 6.56 T F 0.58 3.93 68.37 0.23 0.18 0.40 0.04 0.02 0.46 0.04 76.61 0.16 0.01 0.89 0.16 6.39 S F 0.58 9.56 53.50 0.20 0.04 0.17 0.02 0.01 0.49 0.04 76.79 0.19 0.01 1.46 0.21 7.07 Cupro PW F 0.69 7.19 65.33 2.84 2.99 9.22 0.10 0.04 0.29 0.04 75.46 0.17 0.03 8.03 0.27 12.94 T F 0.72 4.24 71.40 12.17 23.65 30.24 0.79 0.27 0.50 0.03 91.45 0.19 0.04 7.49 0.33 20.29 S F 0.73 4.17 67.35 6.89 7.99 18.70 0.36 0.17 0.49 0.03 78.03 0.22 0.01 1.40 0.33 18.41 (continued) IJCST 35,3 (continued) 339 Fiber Weaving knitting Thickness of yarn Tensile Bending Shear Compression Surface Thickness Weight LT WT RT G 2HG 2HG5 B 2HB LC WC RC MIU MMD SMD TO W – gf$cm/ cm2 % gf/ cm$degree gf/ cm gf/ cm gf$cm2/ cm gf$cm/ cm – gf$cm/ cm2 % – – μm mm mg/m2 Nylon PW F 0.63 19.02 42.34 0.27 0.64 0.66 0.00 0.00 0.50 0.22 56.78 0.27 0.01 9.67 0.60 8.53 PW M 0.59 6.60 70.62 0.31 0.25 0.79 0.03 0.01 0.39 0.05 62.11 0.23 0.02 3.81 0.21 6.42 T F 0.64 10.90 57.34 1.16 1.36 4.63 0.04 0.03 0.31 0.05 65.21 0.29 0.01 0.65 0.32 17.45 S F 0.67 13.29 58.33 0.80 1.26 3.27 0.22 0.10 0.55 0.09 57.11 0.28 0.01 2.44 0.56 20.10 Polyester PW F 0.64 4.18 67.87 0.35 0.35 1.19 0.05 0.04 0.48 0.04 75.95 0.30 0.01 1.04 0.18 9.39 T F 0.77 25.04 55.47 0.39 1.01 1.02 0.01 0.01 0.50 0.15 48.51 0.35 0.02 2.73 0.68 23.28 T M 0.87 6.39 49.45 0.91 2.11 2.30 0.01 0.01 0.54 0.15 53.65 0.28 0.01 5.87 0.64 13.66 S F 0.70 6.18 64.03 0.25 0.21 0.57 0.05 0.02 0.42 0.02 129.00 0.20 0.02 1.30 0.10 6.60 PS F 0.66 4.69 68.56 0.22 0.12 0.35 0.04 0.01 0.36 0.02 115.13 0.17 0.02 1.40 0.10 6.66 R F 0.66 3.72 65.95 0.28 0.16 0.63 0.11 0.04 0.51 0.02 97.29 0.19 0.01 0.78 0.15 10.56 IJCST 35,3 340 ratings. Using the principal factor method, factors exhibiting an eigenvalue of >1 were extracted, and varimax rotation was applied. Tables 4–6 show the results for each analysis, with bold font indicating factor loadings of descriptors that were closely associated with specific factors. The Japanese space presented a 3-factor structure: moist/preference, thermal sensation and flexibility. The Chinese space presented a 2-factor structure: thermal sensation and flexibility/preference. The VTE space presented a 3-factor structure: flexibility/ preference, thermal sensation and surface. These factors were named based on the Category Fiber Weaving/knitting Thickness of yarn Thin – – – Thick – – – Flat – – – Rustic – – – Soft – – – Crisp – * * Wet * Dry – – – Stretchy – – – Warm – – – Cool * – – Likeable Comfortable Glossy – – – Note(s): (p < 0.05) (p < 0.01) Table 2. Fabric hand spaces Figures 9 and 10 show the Japanese and Chinese VTE space results, respectively. Although the VTE space had a 3-factor structure, we displayed only the first two factors in Figures 9 and 10 for Figure 7. Evaluation result of “Crisp” (continued) Results of Mann– Whitney U test Figure 5. Evaluation result of “Wet” National differences in fabric hand feel 341 Category Fiber Weaving/knitting Thickness of yarn Thin – – – Thick – – – Flat – – – Rustic – – – Soft – – – Crisp – * Wet * Dry – – – Stretchy – – – Warm – – – Cool * – – Likeable Comfortable Glossy – – – Note(s): (p < 0.05) (p < 0.01) Table 2. Results of Mann– Whitney U test National differences in fabric hand feel 341 Category Fiber Weaving/knitting Thickness of yarn Thin – – – Thick – – – Flat – – – Rustic – – – Soft – – – Crisp – * Wet * Dry – – – Stretchy – – – Warm – – – Cool * – – Likeable Comfortable Glossy – – – Note(s): (p < 0.05) (p < 0.01) Table 2. Results of Mann– Whitney U test National differences in fabric hand feel 341 Figure 5. Evaluation result of “Wet” ratings. Using the principal factor method, factors exhibiting an eigenvalue of >1 were extracted, and varimax rotation was applied. Tables 4–6 show the results for each analysis, with bold font indicating factor loadings of descriptors that were closely associated with specific factors. The Japanese space presented a 3-factor structure: moist/preference, thermal sensation and flexibility. The Chinese space presented a 2-factor structure: thermal sensation and flexibility/preference. The VTE space presented a 3-factor structure: flexibility/ preference, thermal sensation and surface. These factors were named based on the descriptors with the largest loadings. Fabric hand spaces Figures 9 and 10 show the Japanese and Chinese VTE space results, respectively. Although the VTE space had a 3-factor structure, we displayed only the first two factors in Figures 9 and 10 for Figure 6. Evaluation result of “likeable” IJCST 35,3 342 Figure 6. Evaluation result of “likeable” IJCST 35,3 342 IJCST 35,3 Subjects Experiments Soft Wet Comfortable Dry Japanese TE 0.87 0.88 0.96 0.8 VTE 0.8 0.79 0.87 0.78 Chinese TE 0.82 0.68 0.92 0.6 VTE 0.84 0.67 0.91 0.53 Figure 6. Evaluation result of “likeable” Table 3. Correlations between “likeable” and other evaluation words Figure 7. (continued) Different values for a given fiber attribute are linked by each line to indicate their distribution range, termed “fiber spaces.” The X-axis and Y-axis represent the Factor 1 and Factor 2 scores, respectively. The fabric symbols are as follows: - 5 cotton, ◆5 hemp, ▲5 wool, C 5 silk, 5 cupro, 3 5 nylon and þ 5 polyester. Table 7 shows the factor scores for Factors 1 and 2 for each fabric, that is, the x- and y-axis coordinates for each fabric, respectively, in Figures 9 and 10. The portion of a factor axis occupied by a fiber space describes how much the fabric attributes influenced the ratings. That is, if a fiber space extends across large sections of the axes, it implies that the participants keenly discerned these attributes when evaluating a specific fabric. To investigate further, the fiber spaces for the Japanese and Chinese participants were compared for each of the three factors (“factor width”) in the VTE. Table 8 shows the results for VTE spaces. Differences in the overall area occupied by the fiber spaces were also examined. Table 8 displays the average Figure 8. Evaluation result of “crisp” and “stretchy” National differences in fabric hand feel 343 Figure 8. Evaluation result of “crisp” and “stretchy” National differences in fabric hand feel 343 Figure 8. Evaluation result of “crisp” and “stretchy” National differences in fabric hand feel 343 clarity. Both figures show the fiber-specific factor scores plotted against the factor axes. Different values for a given fiber attribute are linked by each line to indicate their distribution range, termed “fiber spaces.” The X-axis and Y-axis represent the Factor 1 and Factor 2 scores, respectively. The fabric symbols are as follows: - 5 cotton, ◆5 hemp, ▲5 wool, C 5 silk, * 5 cupro, 3 5 nylon and þ 5 polyester. Table 7 shows the factor scores for Factors 1 and 2 for each fabric, that is, the x- and y-axis coordinates for each fabric, respectively, in Figures 9 and 10. The portion of a factor axis occupied by a fiber space describes how much the fabric attributes influenced the ratings. That is, if a fiber space extends across large sections of the axes, it implies that the participants keenly discerned these attributes when evaluating a specific fabric. (continued) Evaluation result of “Crisp” IJCST 35,3 342 Subjects Experiments Soft Wet Comfortable Dry Japanese TE 0.87 0.88 0.96 0.8 VTE 0.8 0.79 0.87 0.78 Chinese TE 0.82 0.68 0.92 0.6 VTE 0.84 0.67 0.91 0.53 Table 3. Correlations between “likeable” and other evaluation words Subjects Experiments Soft Wet Comfortable Dry Japanese TE 0.87 0.88 0.96 0.8 VTE 0.8 0.79 0.87 0.78 Chinese TE 0.82 0.68 0.92 0.6 VTE 0.84 0.67 0.91 0.53 Table 3. Correlations between “likeable” and other evaluation words Figure 7. Evaluation result of “Crisp” clarity. Both figures show the fiber-specific factor scores plotted against the factor axes. Different values for a given fiber attribute are linked by each line to indicate their distribution range, termed “fiber spaces.” The X-axis and Y-axis represent the Factor 1 and Factor 2 scores, respectively. The fabric symbols are as follows: - 5 cotton, ◆5 hemp, ▲5 wool, C 5 silk, * 5 cupro, 3 5 nylon and þ 5 polyester. Table 7 shows the factor scores for Factors 1 and 2 for each fabric, that is, the x- and y-axis coordinates for each fabric, respectively, in Figures 9 and 10. The portion of a factor axis occupied by a fiber space describes how much the fabric attributes influenced the ratings. That is, if a fiber space extends across large sections of the axes, it implies that the participants keenly discerned these attributes when evaluating a specific fabric. To investigate further, the fiber spaces for the Japanese and Chinese participants were compared for each of the three factors (“factor width”) in the VTE. Table 8 shows the results for VTE spaces. Differences in the overall area occupied by the fiber spaces were also examined. Table 8 displays the average Evaluation words Moist preference factor Thermal sensation factor Flexible factor Wet 0.870 0.186 0.392 Dry 0.858 0.027 0.364 Rustic 0.834 0.440 0.013 Comfortable 0.782 0.258 0.510 Flat 0.699 0.598 0.254 Like 0.685 0.050 0.598 Cool 0.065 0.892 0.102 Thick 0.299 0.880 0.170 Thin 0.354 0.875 0.270 Warm 0.062 0.868 0.465 Crisp 0.155 0.003 0.953 Soft 0.437 0.147 0.871 Stretchy 0.147 0.288 0.782 Eigenvalue 4.219 3.850 3.573 Contribute ratio 32.45% 29.62% 27.49% Cumulative contribute ratio 32.45% 62.07% 89.56% Figure 8. Evaluation result of “crisp” and “stretchy” Table 4. Factor analysis result of Japanese space National differences in fabric hand feel 343 clarity. Both figures show the fiber-specific factor scores plotted against the factor axes. Figure 8. Evaluation result of “crisp” and “stretchy” (continued) To investigate further, the fiber spaces for the Japanese and Chinese participants were compared for each of the three factors (“factor width”) in the VTE. Table 8 shows the results for VTE spaces. Differences in the overall area occupied by the fiber spaces were also examined. Table 8 displays the average Evaluation words Moist preference factor Thermal sensation factor Flexible factor Wet 0.870 0.186 0.392 Dry 0.858 0.027 0.364 Rustic 0.834 0.440 0.013 Comfortable 0.782 0.258 0.510 Flat 0.699 0.598 0.254 Like 0.685 0.050 0.598 Cool 0.065 0.892 0.102 Thick 0.299 0.880 0.170 Thin 0.354 0.875 0.270 Warm 0.062 0.868 0.465 Crisp 0.155 0.003 0.953 Soft 0.437 0.147 0.871 Stretchy 0.147 0.288 0.782 Eigenvalue 4.219 3.850 3.573 Contribute ratio 32.45% 29.62% 27.49% Cumulative contribute ratio 32.45% 62.07% 89.56% Table 4. Factor analysis result of Japanese space aluation words Moist preference factor Thermal sensation factor Flexible factor results for factor width, factor width difference and area difference. Both factor width difference and area difference were determined by subtracting the average for the Chinese participants from that of the Japanese participants. Accordingly, a positive value describes differences in the factor width and fiber space for Japanese participants compared to Chinese participants. Conversely, a negative value describes such differences among Chinese participants compared to Japanese participants. The data in Figures 9 and 10 and Table 8 indicate that the factor width and area difference were greater among Japanese participants in all cases. Evaluation words Flexible preference factor Thermal sensation factor Surface factor Soft 0.955 0.179 0.104 Comfortable 0.846 0.163 0.360 Like 0.784 0.060 0.228 Stretchy 0.775 0.191 0.275 Crisp 0.203 0.086 0.069 Cool 0.134 0.888 0.194 Thick 0.266 0.855 0.304 Warm 0.322 0.849 0.403 Thin 0.391 0.834 0.337 Flat 0.140 0.475 0.819 Rustic 0.330 0.354 0.779 Glossy 0.062 0.414 0.725 Dry 0.633 0.180 0.672 Wet 0.603 0.161 0.633 Eigenvalue 4.125 3.623 3.333 Contribute ratio 29.47% 25.88% 23.81% Cumulative contribute ratio 29.47% 55.35% 79.16% Evaluation words Thermal sensation factor Flexible preference factor Cool 0.930 0.091 Flat 0.920 0.092 Thin 0.909 0.143 Thick 0.894 0.013 Rustic 0.886 0.224 Warm 0.876 0.396 Dry 0.842 0.349 Wet 0.788 0.461 Stretchy 0.129 0.869 Comfortable 0.432 0.824 Crisp 0.199 0.808 Like 0.324 0.804 Soft 0.480 0.802 Eigenvalue 6.797 3.957 Contribute ratio 52.29% 30.44% Cumulative contribute ratio 52.29% 82.73% Table 6. Factor analysis result of VTE space Table 5. (continued) Factor analysis result of Chinese space IJCST 35,3 344 Evaluation words Thermal sensation factor Flexible preference factor Cool 0.930 0.091 Flat 0.920 0.092 Thin 0.909 0.143 Thick 0.894 0.013 Rustic 0.886 0.224 Warm 0.876 0.396 Dry 0.842 0.349 Wet 0.788 0.461 Stretchy 0.129 0.869 Comfortable 0.432 0.824 Crisp 0.199 0.808 Like 0.324 0.804 Soft 0.480 0.802 Eigenvalue 6.797 3.957 Contribute ratio 52.29% 30.44% Cumulative contribute ratio 52.29% 82.73% Table 5. Factor analysis result of Chinese space IJCST 35,3 344 Evaluation words Flexible preference factor Thermal sensation factor Surface factor Soft 0.955 0.179 0.104 Comfortable 0.846 0.163 0.360 Like 0.784 0.060 0.228 Stretchy 0.775 0.191 0.275 Crisp 0.203 0.086 0.069 Cool 0.134 0.888 0.194 Thick 0.266 0.855 0.304 Warm 0.322 0.849 0.403 Thin 0.391 0.834 0.337 Flat 0.140 0.475 0.819 Rustic 0.330 0.354 0.779 Glossy 0.062 0.414 0.725 Dry 0.633 0.180 0.672 Wet 0.603 0.161 0.633 Eigenvalue 4.125 3.623 3.333 Contribute ratio 29.47% 25.88% 23.81% Cumulative contribute ratio 29.47% 55.35% 79.16% Table 6. Factor analysis result of VTE space results for factor width, factor width difference and area difference. Both factor width difference and area difference were determined by subtracting the average for the Chinese participants from that of the Japanese participants. Accordingly, a positive value describes differences in the factor width and fiber space for Japanese participants compared to Chinese participants. Conversely, a negative value describes such differences among Chinese participants compared to Japanese participants. The data in Figures 9 and 10 and Table 8 indicate that the factor width and area difference were greater among Japanese participants in all cases. results for factor width, factor width difference and area difference. Both factor width difference and area difference were determined by subtracting the average for the Chinese participants from that of the Japanese participants. Accordingly, a positive value describes differences in the factor width and fiber space for Japanese participants compared to Chinese participants. Conversely, a negative value describes such differences among Chinese participants compared to Japanese participants. The data in Figures 9 and 10 and Table 8 indicate that the factor width and area difference were greater among Japanese participants in all cases. results for factor width, factor width difference and area difference. Both factor width difference and area difference were determined by subtracting the average for the Chinese participants from that of the Japanese participants. (continued) Accordingly, a positive value describes differences in the factor width and fiber space for Japanese participants compared to Chinese participants. Conversely, a negative value describes such differences among Chinese participants compared to Japanese participants. The data in Figures 9 and 10 and Table 8 indicate that the factor width and area difference were greater among Japanese participants in all cases. Figure 10. Chinese VTE space Figure 9. Japanese VTE space Intergroup differences in strength of preference Intergroup differences in strength of preference The Chinese participants rated the fabrics as more likeable than the Japanese participants did (Figure 6), indicating that the participants’ nationality affected their preferences for the fabrics. This is consistent with Kim and Winakor (1996), who reported that American and Korean individuals differed regarding their strength of preference for fabrics. ver, both Japanese and Chinese participants associated likeability ratings with es of “soft,” “wet,” and “comfortable,” and the opposite was true for “dry” (Table 3). onally, the associations of “likeable” with “wet” and “dry” were stronger among the se participants. This may suggest that Chinese participants preferred soft fabrics, Figure 10. Chinese VTE space Figure 9. Japanese VTE space National differences in fabric hand feel 345 South Korean individuals differed regarding their strength of preference for fabri Moreover, both Japanese and Chinese participants associated likeability ratings w qualities of “soft,” “wet,” and “comfortable,” and the opposite was true for “dry” (Table Additionally, the associations of “likeable” with “wet” and “dry” were stronger among t Japanese participants. This may suggest that Chinese participants preferred soft fabri South Korean individuals differed regarding their strength of preference for fabrics. Moreover, both Japanese and Chinese participants associated likeability ratings with qualities of “soft,” “wet,” and “comfortable,” and the opposite was true for “dry” (Table 3). Additionally, the associations of “likeable” with “wet” and “dry” were stronger among the Japanese participants. This may suggest that Chinese participants preferred soft fabrics, Figure 10. Chinese VTE space Japanese VTE space South Korean individuals differed regarding their strength of preference for fabrics. Moreover, both Japanese and Chinese participants associated likeability ratings with qualities of “soft,” “wet,” and “comfortable,” and the opposite was true for “dry” (Table 3). Additionally, the associations of “likeable” with “wet” and “dry” were stronger among the Japanese participants. This may suggest that Chinese participants preferred soft fabrics, while Japanese participants favored both soft and moist fabrics. This is consistent with Suehiro et al. Intergroup differences in strength of preference (2014), who reported that Japanese participants displayed a stronger Fiber Weaving knitting Thickness of yarn Japanese Chinese X:Flexible preference factor Y:Thermal sensation factor X:Flexible preference factor Y:Thermal sensation factor Cotton PW F 0.30 1.32 0.11 0.87 PW M 1.81 0.42 1.05 0.02 PW T 1.39 0.61 0.56 1.00 T F 1.17 0.10 0.78 0.28 T M 0.46 0.78 0.53 0.39 T T 0.78 1.09 0.29 0.94 S F 0.17 0.28 0.11 0.05 S M 0.40 2.01 0.06 1.76 PS F 1.16 0.20 1.15 0.14 PS M 1.25 0.42 1.25 0.05 R F 1.80 0.11 1.99 0.12 R M 0.73 1.83 1.07 1.74 Hemp PW M 0.76 1.78 0.05 1.21 PW T 0.50 0.85 0.32 0.10 T F 0.47 1.41 0.32 0.99 Wool PW M 0.73 1.07 0.27 0.49 PW T 0.48 1.30 0.11 0.80 T M 0.81 0.11 0.43 0.45 T T 0.84 1.76 0.40 1.38 S M 0.44 1.46 0.27 0.90 PS M 0.99 1.74 0.67 1.48 PS T 0.38 1.08 0.00 1.00 R M 1.35 1.82 1.25 1.36 Silk PW F 0.81 1.30 0.79 1.58 T F 0.13 0.88 0.28 0.43 S F 0.98 0.28 0.66 1.13 Cupro PW F 0.43 1.31 0.60 1.29 T F 0.86 1.08 0.72 1.14 S F 0.13 0.08 0.07 0.84 Nylon PW F 1.67 0.66 1.33 0.37 PW M 2.51 0.21 2.35 0.03 T F 1.96 0.05 1.71 0.06 S F 1.78 0.94 1.96 1.14 Polyester PW F 0.22 1.85 0.28 1.57 T F 0.44 0.08 0.21 0.35 T M 1.62 0.06 0.83 0.42 S F 0.02 0.06 0.09 0.61 PS F 1.28 0.36 1.21 0.39 R F 1.27 0.42 0.74 0.29 Table 7. Factor scores for factor 1 and factor 2 for each fabric IJCST 35,3 346 while Japanese participants favored both soft and moist fabrics. This is consistent with Suehiro et al. (2014), who reported that Japanese participants displayed a stronger preference for moist fabrics than Chinese participants did. Intergroup differences in fabric hand spaces The fabric hand space for Japanese participants (Table 4) presented a 3-factor structure, while that of Chinese participants (Table 5) presented a 2-factor structure. This is in line with Ishikawa et al. (2012), who analyzed Japanese and Chinese perceptions of high dynamic range (HDR) images and found that Japanese perceptions had a more complex factor structure. Perceptual and linguistic differences with “crisp” and “stretchy” Perceptual and linguistic differences with “crisp” and “stretchy” p g ff p y This experiment was conducted under strict control, with the Chinese fabric texture evaluation words carefully translated by a native Chinese textile expert to preserve the meaning. The descriptors “crisp” and “stretchy” were negatively correlated among Japanese participants, but positively correlated among Chinese participants (see Figure 8). This implies that Japanese participants differentiated between crisp and stretchy textures, while Chinese participants perceived crispness similarlytostretchiness. Thismaybeduetosensory andlinguisticdifferences in evaluation. Regarding the sensory perspective, research indicates that the sensations generated by presentation stimuli are influenced by cultural aspects, such as growth environment and lifestyle(Ishiietal.,2009;Matsunagaetal.,2018;Rhodeetal.,2016).Chineseparticipantshavefewer opportunities and ways to distinguish between “crisp” and “stretchy” evaluations of fabrics than Japanese participants, and the sense of distinction was not cultivated. Therefore, “crisp” and “stretchy” were evaluated similarly. From the linguistic perspective, hari-no-aru (“crisp”) is a unique Japanese expression regarding fabric. It is related to the texture described in Kawabata (1980) and was defined as a representative word for expressing complex sensations (Ishikawa et al., 2015). Thus, it may have been evaluated as a synonym for “stretchy” because there was no direct translation in Chinese for hari-no-aru. The issue of semantic differences was highlighted by Kim and Winakor (1996), who asked American and South Korean participants to rate fabric stimuli using English and Hangul unipolar adjectives. Future research should address this issue. This finding represents an important discovery for designing subject-adaptive online shopping according to the relationship between the texture evaluation words of fabrics and their actual evaluations. This experiment was conducted under strict control, with the Chinese fabric texture evaluation words carefully translated by a native Chinese textile expert to preserve the meaning. The descriptors “crisp” and “stretchy” were negatively correlated among Japanese participants, but positively correlated among Chinese participants (see Figure 8). This implies that Japanese participants differentiated between crisp and stretchy textures, while Chinese participants perceived crispness similarlytostretchiness. Thismaybeduetosensory andlinguisticdifferences in evaluation. Regarding the sensory perspective, research indicates that the sensations generated by presentation stimuli are influenced by cultural aspects, such as growth environment and lifestyle(Ishiietal.,2009;Matsunagaetal.,2018;Rhodeetal.,2016).Chineseparticipantshavefewer lifestyle(Ishiietal.,2009;Matsunagaetal.,2018;Rhodeetal.,2016).Chineseparticipantshavefewer opportunities and ways to distinguish between “crisp” and “stretchy” evaluations of fabrics than Japanese participants, and the sense of distinction was not cultivated. Therefore, “crisp” and “stretchy” were evaluated similarly. From the linguistic perspective, hari-no-aru (“crisp”) is a unique Japanese expression regarding fabric. Perceptual and linguistic differences with “crisp” and “stretchy” It is related to the texture described in Kawabata (1980) and was defined as a representative word for expressing complex sensations (Ishikawa et al., 2015). Thus, it may have been evaluated as a synonym for “stretchy” because there was no direct translation in Chinese for hari-no-aru. The issue of semantic differences was highlighted by Kim and Winakor (1996), who asked American and South Korean participants to rate fabric stimuli using English and Hangul unipolar adjectives. Future research should address this issue. This finding represents an important discovery for designing subject-adaptive online shopping according to the relationship between the texture evaluation words of fabrics and their actual evaluations. Intergroup differences in strength of preference Notwithstanding the differences between the two studies, including the difference in stimuli, the findings suggest that Intergroup differences in fabric hand spaces The fabric hand space for Japanese participants (Table 4) presented a 3-factor structure, while that of Chinese participants (Table 5) presented a 2-factor structure. This is in line with Ishikawa et al. (2012), who analyzed Japanese and Chinese perceptions of high dynamic range (HDR) images and found that Japanese perceptions had a more complex factor structure. Notwithstanding the differences between the two studies, including the difference in stimuli, the findings suggest that VTE space Name Japanese Chinese Width difference Area Difference First Flexible 2.107 1.838 0.268 0.822 Factor Preference Second Thermal 1.796 1.498 0.298 Factor Sensation First Flexible 2.107 1.838 0.268 0.45 Factor Preference Third Surface 1.677 1.355 0.323 Factor Factor Second Thermal 1.796 1.498 0.298 0.117 Factor Sensation Third Surface 1.677 1.355 0.323 Factor Factor Table 8. Difference in width of each fiber space and area (VTE space) National differences in fabric hand feel 347 Japanese people perceive stimulus material in a more precise and nuanced manner than Chinese peopledo.Moreover, our resultsindicated thatthefibers’ factor width and areainVTEspaceswas greater among the Japanese participants. Given that each fabric had different attributes (fiber, weaving/knitting and yarn’s thickness and density), the factor width and space area can be regarded as indicative ofhow keenly the participants differentiated between theseattributes when evaluating the fabrics. Thus, Japanese responses exhibited greater factor width and space area, implying that Japanese participants perceived these differences more acutely. Note 1. Available at: https://english.keskato.co.jp/ 1. Available at: https://english.keskato.co.jp/ The influence of expertise compared with that of nationality The influence of expertise compared with that of nationality f f p p f y Tsunetou et al. (2018) examined how differences in sartorial knowledge affected fabric hand among Japanese individuals. They found that sartorially aware participants used their knowledge to discern differences in fabric attributes. Moreover, with the addition of visual information, the less knowledgeable participants struggled to discern the shear property as the visual information affected their tactile perceptions. Thus, differences in expertise may explain differences in fabric hand evaluations. The participants in our study were all students in clothing-related disciplines, but their nationality differed. Our results indicated that nationality explained numerous differences, including differences in fabric hand descriptors and the number of factors extracted in the factor analysis. Thus, the evidence suggests that differences in nationality affect perceived fabric hand more than differences in expertise do. The findings obtained in this paper are considered to provide design guidelines for realizing online shopping adapted to each subject group. 348 Conclusions Thi d i This study aimed to identify differences in the fabric hand perceptions between Japanese and Chinese participants. Fabric stimuli were tested on Japanese and Chinese participants in a visuotactile task. Significant intergroup differences in the ratings of “crisp,” “wet,” “cool,” “likeable” and “comfortable” were found. The ratings for these descriptors were significantly influenced by all three fabric attribute variables (fiber, weaving/knitting and yarn thickness and density). Theresults for “crisp” suggested that sensory orlinguisticinterpretationsdiffered among the groups and required further analysis. The results for “wet” revealed that Japanese participants rated the wetness of fabrics higher than their Chinese counterparts did although the reverse was true for the hemp fabrics. The results for the likeability descriptors (“likeable” and “comfortable”) revealed that Japanese participants showed a stronger preference for moist fabrics. A factor analysis suggested that the Japanese fabric hand evaluations were more precise, nuanced and varied. This was indicated by the larger number of factors extracted, as well as the larger factor widths and fiber space areas for the Japanese cohort. Finally, we would like to implement online shopping that is adapted to more consumers. Our study had three main limitations. First, all participants were in their 20s, which may result in finger sensitivity bias related to differences based on age and may have thus affected the fabric hand test results (Musa et al., 2019). Second, the results may have been affected by cross-cultural differences (both linguistic and perceptual) regarding the fabric hand descriptors. When individuals with different native languages select or substitute descriptors, they must strive for perceptual and linguistic equivalence. They must discuss ways to maximize both equivalences while considering multiple perspectives. Finally, the number of participants was small, affecting the generalizability of our results. Accordingly, future studies must focus on conducting these experiments using a larger sample size to verify the reliability of the results obtained in the present study. Inoue, H. (2002), “Quantitative study on qualitative words for descriptive scales in sensory analysis”, Japanese Journal of Sensory Evaluation, Vol. 6 No. 1, pp. 20-27, doi: 10.9763/jjsse.6.20. Ishii, K., Tsukasaki, T. and Kitayama, S. (2009), “Culture and visual perception: does perceptual inference depend on culture?”, Japanese Psychological Research, Vol. 51 No. 2, pp. 103-109, doi: 10.1111/j.1468-5884.2009.00393.x. IJCST 35,3 1. Available at: https://english.keskato.co.jp/ National differences in fabric hand feel References Aston, J., Vipond, O., Virgin, K. and Youssouf, O. (2020), “Retail e-commerce and COVID-19: how online shopping opened doors while many were closing”, Statcan COVID-19, Data to Insight for a Better Canada, Catalogue No. 45280001, Canada. Inoue, H. (2002), “Quantitative study on qualitative words for descriptive scales in sensory analysis”, Japanese Journal of Sensory Evaluation, Vol. 6 No. 1, pp. 20-27, doi: 10.9763/jjsse.6.20. Ishii, K., Tsukasaki, T. and Kitayama, S. (2009), “Culture and visual perception: does perceptual inference depend on culture?”, Japanese Psychological Research, Vol. 51 No. 2, pp. 103-109, doi: 10.1111/j.1468-5884.2009.00393.x. Ishikawa, T., Guan, Y., Chen, Y.C., Oguro, H., Kasuga, M. and Ayama, M. (2012), “Effect of tone curve and size on Kansei evaluation of high dynamic range images – comparison of Japanese and Chinese observers”, Kansei Engineering International Journal, Vol. 11 No. 2, pp. 67-79, doi: 10.5057/kei.11.67. Ishikawa, T., Nakamori, S., Sasaki, K., Miyatake, K. and Ayama, M. (2015), “Identification of common words for the evaluation of clothes’ appearance and tactile sensation in online shopping – an indicator for producing images that express clothes’ textures –”, International Journal of Affective Engineering: Special Issue on KEER 2014, Vol. 14 No. 3, pp. 143-149. 349 Kawabata, S. (1980), “The standardization and analysis of hand evaluation”, The Hand Evaluation and Standardization Committee, 2nd ed., The Textile Machinery Society of Japan, Osaka. Kim, H. and Winakor, G. (1996), “Fabric hand as perceived by U.S. and Korean males and females”, Clothing and Textiles Research Journal, Vol. 14 No. 2, pp. 133-144, doi: 10.1177/0887302X9601400204. Kobayashi, S. (1972), “Nuno no huuai imege pattern no bunseki”, Journal of the Japan Research Association for Textile End–Uses, Vol. 13 No. 1, pp. 4-11. Koch, J., Frommeyer, B. and Schewe, G. (2020), “Online shopping motives during the COVID-19 pandemic—lessons from the crisis”, Sustainability, Vol. 12 No. 24, 10247, doi: 10.3390/su122410247. Matsunaga, R., Yasuda, T., Johnson-Motoyama, M., Hartono, P., Yokosawa, K. and Abe, J. (2018), “A cross-cultural comparison of tonality perception in Japanese, Chinese, Vietnamese, Indonesian, and American listeners”, Psychomusicology: Music, Mind, and Brain, Vol. 28 No. 3, pp. 178-188, doi: 10.1037/pmu0000219. Musa, A.B.H., Malengier, B., Vasile, S. and Van Langenhove, L. (2019), “A comprehensive approach for human hand evaluation of split or large set of fabrics”, Textile Research Journal, Vol. 89 Nos 19-20, pp. 4239-4252, doi: 10.1177/0040517519832834. Nishimatsu, T. and Sakai, T. References (1987), “Significance of the influence of the sense of sight on the hand evaluation of pile fabrics”, Sen’i Gakkaishi, Vol. 43 No. 4, pp. 211-217, doi: 10.2115/fiber.43.4_211. Niwa, M. (1990), “Fabric hand and mechanical properties—objective evaluation of fabric hard and its application–”, Sen’i Gakkaishi, Vol. 46 No. 6, pp. 245-252. Rhode, A.K., Voyer, B.G. and Gleibs, I.H. (2016), “Does language matter? Exploring Chinese–Korean differences in holistic perception”, Frontiers in Psychology, Vol. 7, p. 1508, doi: 10.3389/fpsyg.2016.01508. Saito, M. and Harada, T. (1987), “Differences in texture judgment methods for various types of cloth”, Journal of Textile Engineering, Vol. 40 No. 6, pp. 239-245. Stean, A.E., D’Arcy, R.L., Postle, R. and Mahar, T.J. (1988), “A statistical analysis of subjective and objective methods of evaluating fabric handle. Part 1: men’s summer suiting fabrics”, Sen’i Kikai Gakkaishi (Journal of Textile Machinery Society of Japan), Vol. 40 No. 8, pp. 75-80. Suehiro, Y., Sakamoto, Y. and Sukigara, S. (2014), “Comparison of ‘Shittori’ characteristic between Japanese and Chinese for interlock knitted fabric of ultra-fine fibers with different stitch density”, Journal of Textile Engineering, Vol. 60 No. 2, pp. 35-40, doi: 10.4188/jte.60.35. Tsunetou, J., Ishikawa, T., Yanaka, M., Yanagida, Y., Sasaki, K. and Ayama, M. (2018), “Texture evaluation of various cloth attributes – influence of the differences between the subjective knowledge and sensory modality”, Journal of Textile Engineering, Vol. 64 No. 5, pp. 117-126, doi: 10.4188/jte.64.117. Yashima, K., Yamazaki, M., Miyatake, K., Kim, K. and Takatera, M. (2017), “Development of textile proposal system (TPS)-verification of validity for luster evaluation in textile retrieval system”, The 12th Japan Society of Kansei Engineering Spring Competition, 29 March–30, No. March, Osaka. Corresponding author Tomoharu Ishikawa can be contacted at: ishikawa@is.utsunomiya-u.ac.jp p g Tomoharu Ishikawa can be contacted at: ishikawa@is.utsunomiya-u.ac.jp For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com
https://openalex.org/W3106157995	https://hal.science/hal-02439660/document	English	null	Synthesis of Data Word Transducers	arXiv (Cornell University)	2,019	cc-by	11,311	To cite this version: Léo Exibard, Emmanuel Filiot, Pierre-Alain Reynier. Synthesis of Data Word Transducers. CONCUR 2019, Aug 2019, Amsterdam, Netherlands. 10.4230/LIPIcs.CONCUR.2019.24. hal-02439660 Pierre-Alain Reynier Pierre-Alain Reynier Aix Marseille Univ, Universit´e de Toulon, CNRS, LIS, Marseille, Franc Aix Marseille Univ, Universit´e de Toulon, CNRS, LIS, Marseille, France Abstract In reactive synthesis, the goal is to automatically generate an implementation from a speciﬁcation of the reactive and non-terminating input/output behaviours of a system. Speciﬁcations are usually modelled as logical formulae or automata over inﬁnite sequences of signals (ω-words), while implementations are represented as transducers. In the classical setting, the set of signals is assumed to be ﬁnite. In this paper, we consider data ω-words instead, i.e., words over an inﬁnite alphabet. In this context, we study speciﬁcations and implementations respectively given as automata and transducers extended with a ﬁnite set of registers. We consider diﬀerent instances, depending on whether the speciﬁcation is nondeterministic, universal or deterministic, and depending on whether the number of registers of the implementation is given or not. In the unbounded setting, we show undecidability for both universal and non-deterministic speciﬁcations, while decidability is recovered in the deterministic case. In the bounded setting, undecidability still holds for non-deterministic speciﬁcations, but can be recovered by disallowing tests over input data. The generic technique we use to show the latter result allows us to reprove some known result, namely decidability of bounded synthesis for universal speciﬁcations. 2012 ACM Subject Classiﬁcation Theory of computation →Logic and veriﬁcation; Theory of computation →Automata over inﬁnite objects; Theory of computation →Transducers Keywords and phrases Register Automata, Synthesis, Data words, Transducers Keywords and phrases Register Automata, Synthesis, Data words, Transducers Digital Object Identiﬁer 10.4230/LIPIcs.CONCUR.2019.24 Related Version An extended version can be found at https://arxiv.org/abs/1905.03538. Funding L´eo Exibard: Funded by a FRIA fellowship from the F.R.S.-FNRS. Funding Leo Exibard: Funded by a FRIA fellowship from the F.R.S. FNRS. Emmanuel Filiot: Research associate of F.R.S.-FNRS. He is supported by the ARC Project Transform F´ed´eration Wallonie-Bruxelles and the FNRS CDR J013116F and MIS F451019F projects. Pierre-Alain Reynier: Partly funded by the DeLTA project (ANR-16-CE40-0007). Acknowledgements We would like to thank Ayrat Khalimov for his remarks and suggestions, which helped improve the quality of the paper. © L´eo Exibard, Emmanuel Filiot, and Pierre-Alain Reynier; licensed under Creative Commons License CC-BY 30th International Conference on Concurrency Theory (CONCUR 2019). Editors: Wan Fokkink and Rob van Glabbeek; Article No. 24; pp. 24:1–24:15 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum f¨ur Informatik, Dagstuhl Publishing, Germany Synthesis of Data Word Transducers L´eo Exibard Aix Marseille Univ, Universit´e de Toulon, CNRS, LIS, Marseille, France Universit´e libre de Bruxelles, Brussels, Belgium Emmanuel Filiot Universit´e libre de Bruxelles, Brussels, Belgium HAL Id: hal-02439660 https://hal.science/hal-02439660v1 Submitted on 14 Jan 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Synthesis of Data Word Transducers systems are modelled as (sequential) transducers. Transducers are simple ﬁnite-state machines with transitions of type States × In →States × Out, which, at any state, can process any input signal and deterministically produce some output signal, while possibly moving, again deterministically, to a new state. A speciﬁcation is then a language S ⊆(In.Out)ω telling which are the acceptable behaviours of the system. It is also classically represented as an automaton, or as a logical formula then converted into an automaton. Some regular speciﬁcations may not be realisable by any transducer, and the realisability problem asks, given a regular speciﬁcation S, whether there exists a transducer T whose behaviours satisfy S (are included in S). The synthesis problem asks to construct T if it exists. A typical example of reactive system is that of a server granting requests from a ﬁnite set of clients C. Requests are represented as the set of input signals In = {(r, i) \| i ∈C} ∪{idle} (client i requests the ressource) and grants by the set of output signals Out = {(g, i) \| i ∈ C} ∪{idle} (server grants client i’s request). A typical constraint to be imposed on such a system is that every request is eventually granted, which can be represented by the LTL formula V i∈C G((r, i) →F(g, i)). The latter speciﬁcation is realisable for instance by the transducer which outputs (g, i) whenever it reads (r, i) and idle whenever it reads idle. It is well-known that the realisability problem is decidable for ω-regular speciﬁcations, ExpTime-c when represented by parity automata [9] and 2ExpTime-c for LTL speciﬁca- tions [16]. Such positive results have triggered a recent and very active research interest in eﬃcient symbolic methods and tools for reactive synthesis (see e.g. [1]). Extensions of this classical setting have been proposed to capture more realistic scenarios [1]. However, only a few works have considered inﬁnite sets of input and output signals. In the previous example, the number of clients is assumed to be ﬁnite, and small. To the best of our knowledge, existing synthesis tools do not handle large alphabets, and it is more realistic to consider an unbounded (inﬁnite) set of client identiﬁers, e.g. C = N. The goal of this paper is to investigate how reactive synthesis can be extended to handle inﬁnite sets of signals. Data words are inﬁnite sequences x1x2 . . . 1 Introduction 1 Reactive synthesis is an active research domain whose goal is to design algorithmic methods able to construct a reactive system from a speciﬁcation of its admissible behaviours. Such systems are notoriously diﬃcult to design correctly, and the main appealing idea of synthesis is to automatically generate systems correct by construction. Reactive systems are non- terminating systems that continuously interact with the environment in which they are executed, through input and output signals. At each time step, the system receives an input signal from a set In and produces an output signal from a set Out. An execution is then modelled as an inﬁnite sequence alternating between input and output signals, i.e., an ω-word in (In.Out)ω. Classically, the sets In and Out are assumed to be ﬁnite and reactive © L´eo Exibard, Emmanuel Filiot, and Pierre-Alain Reynier; licensed under Creative Commons License CC-BY 30th International Conference on Concurrency Theory (CONCUR 2019). Editors: Wan Fokkink and Rob van Glabbeek; Article No. 24; pp. 24:1–24:15 Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum f¨ur Informatik, Dagstuhl Publishing, Germany 24:2 Synthesis of Data Word Transducers L. Exibard, E. Filiot, and P.-A. Reynier “all input data but one are copied on the output, the missing one being replaced by some data which occurred before it”, modelled as the set of data sequences d1d1d2d2 . . . didjdi+1di+1 . . . for all i ≥0 and j < i (ﬁnite labels are irrelevant and not represented). S0 is not deﬁnable by any URA (it would require to guess j, which can be arbitrarily smaller than i), but it is expressible by some NRA making this guess. However, we show (unsurprisingly) that the realisability problem by register transducers of speciﬁcations deﬁned by NRA is undecidable. The same negative result also holds for URA, solving an open question raised in [12]. On the positive side, we show that decidability is recovered for deterministic (parity) register automata (DRA). One of the diﬃculties of register transducer synthesis is that the number of registers needed to realise the speciﬁcation is, a priori, unbounded with regards to the number of registers of the speciﬁcation. We show it is in fact not the case for DRA: any speciﬁcation expressed as a DRA with r registers is realisable by a register transducer iﬀit is realisable by a transducer with r registers. A way to obtain decidability is to ﬁx a bound k and to target register transducers with at most k registers. This setting is called bounded synthesis in [12], which establishes that bounded synthesis is decidable in 2ExpTime for URA. We show that unfortunately, bounded synthesis is still undecidable for NRA speciﬁcations. To recover decidability for NRA, we disallow equality tests on the input data and add a syntactic requirement which entails that on any accepted word, each output data is the content of some register which has been assigned an input data occurring before. This deﬁnes a subclass of NRA that we call (input) test-free NRA (NRAtf). NRAtf can express how output data can be obtained from input data (by copying, moving or duplicating them), although they do not have the whole power of register automata on the input nor the output side. Note that the speciﬁcation S0 given before is NRAtf-deﬁnable. To show that bounded synthesis is decidable for NRAtf, we establish a generic transfer property characterising realisable data word speciﬁcations in terms of realisability of corresponding speciﬁcations over a ﬁnite alphabet, thus reducing to the well-known synthesis problem over a ﬁnite alphabet. Synthesis of Data Word Transducers of labelled data, i.e., pairs (σ, d) with σ a label from a ﬁnite alphabet, and d ∈N. They can naturally model executions of reactive systems over an inﬁnite set of signals. Among other models, register automata are one of the main extensions of automata recognising languages of data words [10, 18]. They can use a ﬁnite set of registers in which to store data that are read, and to compare the current data with the content of some of the registers (in this paper, we allow comparison of equality). Likewise, transducers can be extended to register transducers as a model of reactive systems over data words: a register transducer is equipped with a set of registers, and when reading an input labelled data (σ, d), it can test d for equality with the content of some of its registers, and depending on the result of this test, deterministically assign some of its registers to d and output a ﬁnite label β together with the content of one of its registers. Its executions are then data words alternating between input and output labelled data, so register automata can be used to represent speciﬁcations, as languages of such data words. Contributions. We consider two classical semantics for register automata, non-deterministic and universal, both with a parity acceptance condition, which give two classes of register automata respectively denoted NRA and URA. Since NRA are not closed under complement (already over ﬁnite data words), NRA and URA deﬁne incomparable classes of speciﬁcations. The request-grant speciﬁcation given previously with an inﬁnite number of clients is expressible by an URA [12]: whenever a request is made by client i (labelled data (r, i)), universally trigger a run which stores i in some register and veriﬁes that the labelled data (g, i) eventually occurs in the data word; but no NRA can deﬁne it. On the contrary, consider the speciﬁcation S0: 24:3 Synthesis of Data Word Transducers Finally, classical reactive synthesis has strong connections with game theory on ﬁnite graphs. Some extension of games to inﬁnite graphs whose vertices are valuations of variables in an inﬁnite data domain have been considered in [8]. Such games are shown to be undecidable and a decidable restriction is proposed, which however does not seem to match our context. L. Exibard, E. Filiot, and P.-A. Reynier Such property also allows us to reprove the result of [12], with a rather short proof based on standard results from the theory of register automata, indicating that it might allow to establish decidability for other classes of data speciﬁcations. Our results are summarised in Table 1. Table 1 Decidability status of the problems studied. Table 1 Decidability status of the problems studied. Table 1 Decidability status of the problems studied. DRA NRA URA NRAtf Bounded Synthesis ExpTime Undecidable (k ≥1) 2ExpTime 2ExpTime (Thm. 13) (Thm. 3) ([12] and Thm. 12) (Thm. 16) General Case ExpTime Undecidable Undecidable Open (Thm. 6) (Thm. 2) (Thm. 4) Table 1 Decidability status of the problems studied. Related Work. As already mentioned, bounded synthesis of register transducers is consid- ered in [12] where it is shown to be decidable for URA. We reprove this result in a shorter way. Our proof bears some similarities with that of [12], but it seems that our formulation beneﬁts more from the use of existing results. The technique is also more generic and we instantiate it to NRAtf. NRAtf correspond to the one-way, non-deterministic version of the expressive transducer model of [5], which however does not consider the synthesis problem. [ ] The synthesis problem over inﬁnite alphabets is also considered in [6], in which data rep- resent identiﬁers and speciﬁcations (given as particular automata close to register automata) can depend on equality between identiﬁers. However, the class of implementations is very expressive: it allows for unbounded memory through a queue data structure. The synthesis problem is shown to be undecidable and a sound but incomplete algorithm is given. CONCUR 2019 24:4 1 In the terminology of [13], it corresponds to multiple-assignment register automata with registers initially ﬁlled, i.e. the class MF, with the additional requirement that all are ﬁlled with the same data. 2 Those sets are identical but have distinct semantics. 1 In the terminology of [13], it corresponds to multiple-assignment register automata with registers initially ﬁlled, i.e. the class MF, with the additional requirement that all are ﬁlled with the same data. 2 Th id i l b h di i i 2 Data Words and Register Automata For a (possibly inﬁnite) set S, we denote by Sω the set of inﬁnite words over this alphabet. For 1 ≤i ≤j, we let u[i:j] = uiui+1 . . . uj and u[i] = u[i:i] the ith letter of u. For u, v ∈Sω, we deﬁne their interleaving ⟨u, v⟩= u[1]v[1]u[2]v[2] . . . Data Words. Let Σ be a ﬁnite alphabet and D a countably inﬁnite set, denoting, all over this paper, a set of elements called data. We also distinguish an (arbitrary) data value d0 ∈D. Given a set R, let τ R 0 be the constant function deﬁned by τ R 0 (r) = d0 for all r ∈R. A labelled data (or l-data for short) is a pair x = (σ, d) ∈Σ × D, where σ is the label and d the data. We deﬁne the projections lab(x) = σ and dt(x) = d. A data word over Σ and D is an inﬁnite sequence of labelled data, i.e. a word w ∈(Σ × D)ω. We extend the projections lab and dt to data words naturally, i.e. lab(w) ∈Σω and dt(w) ∈Dω. We denote the set of data words over Σ and D by DW(Σ, D) (DW when clear from the context). A data word language is a subset L ⊆DW(Σ, D). Note that in this paper, data words are inﬁnite, otherwise they are called ﬁnite data words, and we denote by DWf(Σ, D) the set of ﬁnite data words. Register Automata. Register automata are automata recognising data word languages. They were ﬁrst introduced in [10] as ﬁnite-memory automata. Here, we deﬁne them in a spirit close to [18], but over inﬁnite words 1, with a parity acceptance condition. A register automaton (RA) is a tuple A = (Σ, D, Q, q0, δ, R, c), where: A register automaton (RA) is a tuple A = (Σ, D, Q, q0, δ, R, c), where: Σ is a ﬁnite alphabet of labels, D is an inﬁnite alphabet of data Q is a ﬁnite set of states and q0 ∈Q is the initial state R is a ﬁnite set of registers. We denote TstR = 2R and AsgnR = 2R. 2 R c : Q →{1, . . . 2 Those sets are identical but have distinct semantics. L. Exibard, E. Filiot, and P.-A. Reynier Conﬁgurations and Runs. A conﬁguration is a pair (q, τ) ∈Q×(R →D). Given tst ∈TstR and d ∈D, we say that τ, d satisﬁes tst, denoted τ, d \|= tst if τ −1(d) = tst. Given a transition t = p σ,tst,asgn −−−−−−→p′, we say that (q, τ) enables t on reading (σ′, d) if q = p, σ′ = σ and τ, d \|= tst. Let next(τ, asgn, d) be the conﬁguration τ ′ deﬁned by τ ′(i) = d if i ∈asgn, and τ ′(i) = τ(i) otherwise. We extend this notation to conﬁgurations as follows: if γ = (q, τ) enables t on input (σ, d), the successor conﬁguration of (q, τ) by t on input (σ, d) is next(γ, asgn, d) = (p′, next(τ, asgn, d)). We also write next(γ, t, σ, d) to denote the successor of (q, τ) by transition t when (q, τ) enables t on input (σ, d). The initial conﬁguration is (q0, τ R 0 ). Then, a run over a data word (σ1, d1)(σ2, d2) . . . is an inﬁnite sequence of transitions t0t1 . . . such that there exists a sequence of conﬁgurations γ0γ1 · · · = (q0, τ0)(q1, τ1) . . . such that γ0 is initial and for all i ≥0, γi+1 = next(γi, ti, σi, di). To a run ρ, we associate its sequence of states states(ρ) = q0q1 . . . Languages Deﬁned by RA. Given a run ρ, we denote, by a slight abuse of notation, c(ρ) = max{j \| c(ql) = j for inﬁnitely many ql ∈states(ρ)} the maximum color that occurs inﬁnitely often in ρ. Then, in the parity acceptance condition, ρ is accepting whenever c(ρ) is even. We consider two dual semantics for RA: non-deterministic (N) and universal (U). Given an RA A, depending on whether it is considered nondeterministic or universal, it recognises LN(A) = {w \| there exists an accepting run ρ on w} or LU(A) = {w \| all runs ρ on w are accepting}. We denote by NRA (resp. URA) the class of register automata interpreted with a non- deterministic (resp. universal) parity acceptance condition, and given A ∈NRA (resp. A ∈URA), we write L(A) instead of LN(A) (resp. LU(A)). We also denote by DRA the class of deterministic parity register automata. 2 Data Words and Register Automata , d}, where d ∈N is the number of priorities, is the colouring function, used to deﬁne the acceptance condition δ ⊆Q × Σ × TstR × AsgnR × Q is a set of transitions. δ ⊆Q × Σ × TstR × AsgnR × Q is a set of transitions. A transition (q, σ, tst, asgn, q′) is also written q σ,tst,asgn −−−−−−→ A q′. We may omit A in the latter notation. Intuitively such transition means that on input (σ, d) in state q the automaton: it ﬁrst checks that tst is exactly the set of registers containing d: for all r ∈tst, d is the current content of register r and for all r /∈tst, d is not in register r, then it assigns d to all the registers in asgn (asgn might be empty), and ﬁnally transitions to state q′. ⊆Q R g R Q A transition (q, σ, tst, asgn, q′) is also written q σ,tst,asgn −−−−−−→ A q′. We may omit A in the latter notation. Intuitively such transition means that on input (σ, d) in state q the automaton: it ﬁrst checks that tst is exactly the set of registers containing d: for all r ∈tst, d is the current content of register r and for all r /∈tst, d is not in register r, then it assigns d to all the registers in asgn (asgn might be empty), and ﬁnally transitions to state q′. A is said to be deterministic (resp. complete) when, given any state, any label and any possible test, at most (resp. at least) one transition can be taken: ∀q ∈Q, ∀σ ∈Σ, ∀tst ∈ TstR, ∃≤1asgn ∈Asgn, ∃≤1q′ ∈Q such that q σ,tst,asgn −−−−−−→q′ (resp. ∀q, ∀σ, ∀tst, ∃≥1asgn, ∃≥1q′ s.t. q σ,tst,asgn −−−−−−→q′). Since tests are mutually exclusive, this syntactically ensures that for any state and in any register conﬁguration, the transition to take is determined by the input l-data (respectively that a transition can always be taken, regardless the input l-data). The class of deterministic register automata will be denoted DRA. 24:5 3 Synthesis of Register Transducers NRA, URA) the class of speciﬁcations deﬁned by deterministic (resp. non-deterministic, universal) parity register automata. Register Transducers As Implementations. We consider implementations represented as transducers processing data words. A register transducer is a tuple T = (Σi, Σo, Q, q0, δ, R) where Q is a ﬁnite set of states with initial state q0, R is a ﬁnite set of registers, and δ : Q × Σi × TstR →AsgnR × Σo × R × Q is the (total) transition function (as before, TstR = AsgnR = 2R). When processing an l-data (σi, d), T compares d with the content of some of its registers, and depending on the result, moves to another state, stores d in some registers, and outputs some label in Σo along with the content of some register r ∈R. Let us formally deﬁne the semantics of a register transducer T, as an implementation IT . First, for a ﬁnite input data word w = (σ1 i , d1 i ) . . . (σn i , dn i ) in (Σi × D)∗, we denote by (qi, τi) the ith conﬁguration reached by T on w, where (q0, τ0) is initial and for all 0 < i < n, (qi, τi) is the unique conﬁguration such that there exists a transition δ(qi−1, σi i, tst) = (asgn, σo, r, qi) such that τi−1, di i \|= tst and τi = next(τi−1, di i, asgn). We let (σi o, di o) = (σo, τi(r)) and IT (w) = (σn o, dn o). Then, we denote fT = fIT and L(T) = L(IT ). Note that if T is interpreted as a DRA with exactly one transition per output state and whose states are all accepting (i.e. have even maximal parity 2), then L(IT ) is indeed the language of such register automaton. We denote by RT[k] the class of implementations deﬁned by register transducers with at most k registers, and by RT = S k≥0 RT[k] the class of implementations deﬁned by register transducers. Synthesis from Data-Free Speciﬁcations. If in the latter deﬁnitions of the problem, one considers speciﬁcations deﬁned by RA with no registers, and implementations deﬁned by RT with no registers, then the data in data-words can be ignored and we are in the classical reactive synthesis setting, for which important results are known: ▶Theorem 1 ([9]). 3 Synthesis of Register Transducers Given a (data-free) speciﬁcation S deﬁned by some (register-free) universal or non-deterministic parity automaton, the realisability problem of S by (register- free) transducers is ExpTime-c. 3 Synthesis of Register Transducers 3 Speciﬁcations, Implementations and the Realisability Problem. Let Σi and Σo be two ﬁnite alphabets of labels, and D a countable set of data. A relational data word is an element of w ∈[(Σi × D).(Σo × D)]ω. Such a word is called relational as it deﬁnes a pair of data words in DW(Σi, D) × DW(Σo, D) through the following projections. If w = x1 ix1 ox2 ix2 o . . . , we let inp(w) = x1 i x2 i . . . and out(w) = x1 ox2 o . . . We denote by RW(Σi, Σo, D) (just RW when clear from the context) the set of relational data words. A speciﬁcation is simply a language S ⊆RW(Σi, Σo, D). An implementation is a total function I : (Σi × D)∗→Σo × D. We associate to I another function fI : DW(Σi, D) →DW(Σo, D) which, to an input data word wi = x1 ix2 i · · · ∈Σi × D, associates the output data word fI(wi) = x1 ox2 o . . . such that ∀i ≥1, xi o = I(x1 i . . . xi−1 i ). I also deﬁnes a language of relational data words L(I) = {⟨wi, fI(wi)⟩\| wi ∈DW(Σi, D)}. We say that I realises S when L(I) ⊆S, and that S is realisable if there exists an implementation realising it. The realisability problem consists, given a (ﬁnite representation of a) speciﬁcation S, in checking whether S is realisable. In general, we parameterise this problem by classes of speciﬁcations S and of implementations I, deﬁning the (S, I)- realisability problem, denoted Real(S, I). Given a speciﬁcation S ∈S, it asks whether S is realisable by some implementation I ∈I. We now introduce the classes S and I we consider. Speciﬁcation Register Automata. In this paper, we consider speciﬁcations deﬁned from register automata alternately reading input and output l-data. We assume that the set of states is partitioned into Qi (called input states, reading only labels in Σi) and Qo (called output states, reading only labels in Σo), where q0 ∈Qi and F ⊆Qi, and such that the CONCUR 2019 24:6 Synthesis of Data Word Transducers transition relation δ alternates between these two sets, i.e. δ ⊆∪α=i,o(Qα × Σα × TstR × AsgnR × Qα), where i = o (resp. o = i). We denote by DRA (resp. L. Exibard, E. Filiot, and P.-A. Reynier Indeed, URA are closed under union, by the same product construction as for the intersection of NRA [10], and each part is URA-recognisable: S1 is, as described above, S2 is by simulating A on the output to check w ∈L(A) then looping over (a, d0), and T simply checks a regular property. Now, if L(A) ̸= ∅, let w ∈L(A), and k its number of distinct data. Then S is realisable by a k-register transducer realising S1 when the number of data in u is lower than or equal to k, and, when it is greater than k, by outputting u# bw#(a, d0)ω where bw is a membership- preserving renaming of w using k distinct data of u (this can always be done thanks to the so called “indistinguishability property” stated in [10]) Conversely if L(A) ∅then S is Now, if L(A) ̸= ∅, let w ∈L(A), and k its number of distinct data. Then S is realisable by a k-register transducer realising S1 when the number of data in u is lower than or equal to k, and, when it is greater than k, by outputting u# bw#(a, d0)ω where bw is a membership- preserving renaming of w using k distinct data of u (this can always be done thanks to the so-called “indistinguishability property” stated in [10]). Conversely, if L(A) = ∅, then S is not realisable. If it were, S ∩DWf#DW = S1 would be too, as a regular domain restriction, but we have seen above that this is not the case. Thus, S is realisable iﬀL(A) = ∅. ◀ However, we show that restricting to DRA allows to recover the decidability, modulo one additional assumption, namely that no output transition of the speciﬁcation is such that tst = ∅(the output data is diﬀerent from all register contents). We denote by DRA∅ this class of DRA. Such assumption rules out pathological, and to our opinion uninteresting and technical cases stemming from the asymmetry between the class of speciﬁcations and implementations. E.g., consider the single-register DRA in Fig. 1a (ﬁnite labels are arbitrary and not depicted). It starts by reading one input data d and stores it in r, asks that the corresponding output data is diﬀerent from the content d of r (with tst = ∅depicted here ̸=r), then accepts any output over any input (transitions ⊤are always takeable). 4 Unbounded Synthesis In this section, we consider the unbounded synthesis problem Real(RA, RT). Thus, we do not ﬁx a priori the number of registers of the implementation. Let us ﬁrst consider the case of NRA and URA, which are, in our setting, the most natural devices to express data word speciﬁcations. By reducing the universality of NRA over ﬁnite words (which is undecidable [14]) to our synthesis problems, we show: ▶Theorem 2. Real(NRA, RT) is undecidable. ▶Theorem 2. Real(NRA, RT) is undecidable. ▶Theorem 3. For all k ≥1, Real(NRA, RT[k]) is undecidable. ▶Theorem 3. For all k ≥1, Real(NRA, RT[k]) is undecidable. Now, we can show that the unbounded synthesis problem is also undecidable for URA, answering a question left open in [12]. ▶Theorem 4. Real(URA, RT) is undecidable. ▶Theorem 4. Real(URA, RT) is undecidable. 24:7 L. Exibard, E. Filiot, and P.-A. Reynier Proof. We present a reduction from the emptiness problem of URA over ﬁnite words (which is undecidable by a direct reduction from the universality problem of NRA, undecidable by [14]) to our synthesis problem. Proof. We present a reduction from the emptiness problem of URA over ﬁnite words (which is undecidable by a direct reduction from the universality problem of NRA, undecidable by [14]) to our synthesis problem. First, consider the relation S1 = {(u#v, u#w) \| u ∈DWf, v ∈DW, each data of u appears inﬁnitely often in w}. S1 is recognised by a 1-register URA which, upon reading a data d in u, stores it in its register and checks that it appears inﬁnitely often in w by visiting a state with maximal parity 2 every time it sees d (all other states have parity 1). Note that for all k ≥1, S1 ∩{(u#v, u#w) \| u ∈DWf, v, w ∈DW and \|dt(u)\| ≤k} is realisable by a k-register transducer: on reading u, store each distinct data in one register, and after the # outputs them in turn in a round-robin fashion. However, S1 is not realisable: on reading the # separator, any implementation must have all the data of dt(u) in its registers, but the size of dt(u) is not bounded (u can have pairwise distinct data and be of arbitary length). ( ) ( ) Then, let A be a URA over ﬁnite data words. Consider the speciﬁcation S = S1 ∪S2 ∪T, where S2 = {(u#v, u#w#(a, d0)ω) \| u ∈DWf, v ∈DW, w ∈L(A)} and T = {(u, w) \| u /∈ DWf#DW, w ∈DW}. S has total domain, and is recognisable by a URA. Indeed, URA are closed under union, by the same product construction as for the intersection of NRA [10], and each part is URA-recognisable: S1 is, as described above, S2 is by simulating A on the output to check w ∈L(A) then looping over (a, d0), and T simply checks a regular property. Then, let A be a URA over ﬁnite data words. Consider the speciﬁcation S = S1 ∪S2 ∪T, where S2 = {(u#v, u#w#(a, d0)ω) \| u ∈DWf, v ∈DW, w ∈L(A)} and T = {(u, w) \| u /∈ DWf#DW, w ∈DW}. S has total domain, and is recognisable by a URA. Synthesis of Data Word Transducers To check for realisability of DRA∅-speciﬁcations with r-registers, we show that it suﬃces to target transducers with r registers only. ▶Proposition 5. Let S be a speciﬁcation deﬁned by a DRA∅with r registers. If S is realisable by a register transducer, then it is realisable by a transducer with r registers. Proof. Let S = (Σi, Σo, D, QS, qS 0 , δS, RS, c) be a DRA∅speciﬁcation realisable by a register transducer I = (Σi, Σo, D, QI, qI 0, δI, RI). Without loss of generality, we assume that S does not conduct assignments on the output and that S is complete. Now, from S, we extract a transducer I′ realising S with RS registers, using I as a guide to make choices for the output. To this end, we simulate I synchronously with S. However, we cannot properly simulate I, since we only have RS registers, which are used to simulate S. Instead, we keep constraints in memory. Constraints. A constraint represents the equality relations between the registers in RS and those in RI (note that such idea is pervasive in the study of registers automata, e.g. to recognise the projection over ﬁnite labels). Thus, a constraint is a subset C ⊆RS ×RI, which is intended to be a set of equalities between the content of registers in RS and in RI. Then, knowing tests tstS, tstI and assignments asgnS, asgnI performed by S and I respectively allows to update the constraints: we deﬁne next(C, tstS, asgnS, tstI, asgnI) = C\((asgnS × RI) ∪(RS × asgnI)) ∪((tstS ∪asgnS) × (tstI ∪asgnI)) next(C, tstS, asgnS, tstI, asgnI) = C\((asgnS × RI) ∪(RS × asgnI)) ∪((tstS ∪asgnS) × (tstI ∪asgnI)) For instance, assume RI = {r1, r2} and RS = {s1, s2}, and at some point in a run, we have3 C = {(s2, r1), (s2, r2)}, i.e. s2 = r1 = r2 and s1 ̸= r1 (inequalities are implicit, since C is an exhaustive list of equalities). Now, S reads some data d which satisﬁes the tests tstS = {s1} in S and tstI = ∅in I (such tests are consistent because s1 ̸= r1, r2), and conducts assignments asgnS = ∅and asgnI = {r2}. Then, on the one hand, s1 = r2 (both contain d), and on the other hand s2 = r1 (since the content of those registers did not change). 3 For readability, we confuse a register with its content. L. Exibard, E. Filiot, and P.-A. Reynier It is not realisable because transducers necessarily output the content of some register (hence producing a data which already appeared). On the other hand, having tests tst = ∅does not imply unrealisability, as shown by the DRA of Fig. 1b: it starts by reading one data d1, asks to copy it on the output, then reads another data d2, and requires that the output is either distinct from d1 or equal to it, depending on whether d2 ̸= d1. It happens that such speciﬁcation is realisable by the identity. 1 2 3 5 4 6 7 ⊤, ↓r ↑r ̸=r, ↓r =r, ↓r ̸=r =r ⊤ ⊤ (b) A similar DRA, suprisingly realisable. 1 2 3 5 4 6 7 ⊤, ↓r ↑r ̸=r, ↓r =r, ↓r ̸=r =r ⊤ ⊤ (b) A similar DRA, suprisingly realisable. 1 2 3 4 ⊤, ↓r ̸= r ⊤ ⊤ (a) An unrealisable DRA. 1 2 3 5 4 6 7 ⊤, ↓r ↑r ̸=r, ↓r =r, ↓r ̸=r =r ⊤ ⊤ (b) A similar DRA, suprisingly realisable. Figure 1 Pathological DRA speciﬁcations. (b) A similar DRA, suprisingly realisable. Figure 1 Pathological DRA speciﬁcations. CONCUR 2019 24:8 L. Exibard, E. Filiot, and P.-A. Reynier Item (i) ensures with the help of constraints that in any reachable conﬁguration of I′, there exists at least one input data which satisﬁes both tstS i and tstI, which allows I′ to synchronise S with I. Note that this does not means that I′ is the synchronous product of S and I on any input: since I′ only has the registers of S, it cannot discriminate data as subtly as I, and might thus adopt a diﬀerent behaviour. For instance, it can be that upon reading some input data word, at some point, I would store some input data d in some register r that S would not, and use it later on in a test tstI = {r} to take diﬀerent actions, while neither I′ nor S could discriminate between those choices: on reading d, I′ simulates S with tstS i = ∅and synchronously simulates in I the transition with input test tstI = ∅. Nevertheless, we show the existence of some relational data word common to I and I′ for each run of I′ (which is also a run of S). This is suﬃcient to conclude that I′ realises S, because then each run of I′, interpreted as a run of S, is accepting. Then, items (iii) and (iv) ensures the same property as item (i) does, but this time on output positions. We shall see that for a transition t such that (qS, qI, C) is accessible on some ﬁnite input data word, tS i , tS o and tI exist and are unique. So, for a run ρ = t1t2 . . . of I′, we deﬁne ρS = tS i1tS o1tS i2tS o2 . . . and ρI = tI 1tI 2 . . . Proof of Correctness. Let us show that I′ is indeed a transducer realising S: we show that for all ui ∈DW(Σi, D), there exists a unique sequence of transitions ρ in I′, a unique output data word uo ∈DW(Σo, D) (we denote u = ⟨ui, uo⟩) and a w ∈L(I) such that: 3. ρ is the run of I′ over u 4. ρS is the run of S over u 1. ρI is the run of I over w S 1. ρI is the run of I over w S 2. L. Exibard, E. Filiot, and P.-A. Reynier ρS is the run of S over w Note that the above properties imply lab(u) = lab(w), but it can be that u ̸= w, which is consistent with the observations we made. Let us show that they entail the result we need: let ui ∈DW(Σi, D) be some input data word. By property 3, fI′(ui) exists and is unique, so I′ has total domain. Now, by denoting ρS the run of S over u = ⟨ui, fI′(ui)⟩, we know by property 2 that there exists w ∈L(I) such that ρS is the run of S over w. Then, ρS is accepting because I realises S so w ∈L(S), hence u ∈L(S). Thus, I′ realises S. The proof of properties 1-4 is rather technical, and can be found in [7]. Such result allows us to reduce unbounded synthesis to bounded synthesis for DRA∅. Bounded synthesis is in ExpTime for DRA (Thm. 13) and is the topic of the next section. ▶Theorem 6. Real(DRA∅, RT) is decidable in ExpTime. Synthesis of Data Word Transducers Moreover, r1 ̸= r2 since r2 has been reassigned and s1 ̸= r1 still holds. This is represented by the set of constraints C′ = {(s1, r2), (s2, r1)}, and indeed, next(C, {s1}, ∅, ∅, {r2}) = C′. Abstracting the behaviour of I modiﬁes its language (it somehow simpliﬁes it) but we Abstracting the behaviour of I modiﬁes its language (it somehow simpliﬁes it), but we will see that what we build is still an implementation. Deﬁnition of I′. We build I′ = (Σi, Σo, D, Q, q0, δ, RS), where Q = QS ×QI ×2RS×RI and q0 = (qS 0 , qI 0, RS × RI); we now deﬁne δ. For each state (qS i , qI, C) ∈Q, for each input test (σi, tstS i ) ∈Σi×TstRS, we construct a transition t = (qS i , qI, C) σi,tstS i ,asgnS,σo,so −−−−−−−−−−−−→ I′ (q′S i , q′I, C′) whenever there exist the following transitions of S and I: tS i = qS i σi,tstS i ,asgnS −−−−−−−−→ S qS o tS o = qS o σo,tstS o −−−−→ S q′S i tI = qI σi,tstI,asgnI,σo,ro −−−−−−−−−−−→ I q′I such that, for some ﬁxed arbitrary order on RS, we have: (i) tstI = {r ∈RI \| ∃s ∈tstS i , (s, r) ∈C} (ii) C′ = next(C, tstS i , asgnS, tstI, asgnI) (iii) tstS o = {s ∈RS \| (s, ro) ∈C′} (iv) so = min tstS o 24:9 Synthesis of Data Word Transducers Hence, since T ′ realises WS,k, we get Comp(a) ⊆S, so w ∈S, concluding the proof. ◀ Conversely, suppose that WS,k is realisable by some ﬁnite transducer T ′ over the input (output) alphabets Ak i (Ak o). Again, the transducer T can be seen as a transducer with k registers over data words. We show that T realises S, i.e., L(T) ⊆S. Let w ∈L(T). The run of T over w induces a sequence of actions a in (Ak i Ak o)ω which, by deﬁnition of compatibility, satisﬁes w ∈Comp(a). Moreover, a ∈L(T ′). Hence, since T ′ realises WS,k, we get Comp(a) ⊆S, so w ∈S, concluding the proof. ◀ Synthesis of Data Word Transducers ﬁnite input actions are Ak i = Σi × Tstk which corresponds to picking a label and a test over the k registers, and the output actions are Ak o = Σo × Asgnk × Rk, corresponding to picking some output symbol, some assignment and some register whose content is to be output. An alternating sequence of actions a = (σ1 i , test1)(σ1 o, asgn1, r1) · · · ∈(Ak i Ak o)ω abstracts a set of relational data words of the form w = (σ1 i , d1 i)(σ1 o, d1 o) · · · ∈RW(Σi, Σo, D) via a compatibility relation that we now deﬁne. We say that w is compatible with a if there exists a sequence of register conﬁgurations τ0τ1 · · · ∈(Rk →D)ω such that τ0 = τ Rk 0 and for all i ≥1, τi, di i \|= testi, di o = τi(ri) and τi+1 = next(τi, di i, asgni). Note that this sequence is unique if it exists. We denote by Comp(a) the set of relational data words compatible with a. Given a speciﬁcation S, we let WS,k = {a \| Comp(a) ⊆S}. The set WS,k can be seen as a speciﬁcation over the ﬁnite input (resp. output) alphabets Ak i (resp. Ak o). ▶Theorem 7 (Transfer). Let S be a data word speciﬁcation. The following are equivalent: 1. S is realisable by a transducer with k registers. ▶Theorem 7 (Transfer). Let S be a data word speciﬁcation. The following are equivalent: 1. S is realisable by a transducer with k registers. 2. The (data-free) word speciﬁcation WS,k is realisable by a (register-free) ﬁnite transducer. Proof. Let T be a transducer with k registers realising S. The transducer T can be seen as a ﬁnite transducer T ′ over input alphabet Ak i and output alphabet Ak o. Indeed, since the transition function of T is total, it is also the case of T ′ (this is required by the deﬁnition of transducer deﬁning implementations). Proof. Let T be a transducer with k registers realising S. The transducer T can be seen as a ﬁnite transducer T ′ over input alphabet Ak i and output alphabet Ak o. Indeed, since the transition function of T is total, it is also the case of T ′ (this is required by the deﬁnition of transducer deﬁning implementations). 5 Bounded Synthesis: A Generic Approach In this section, we study the setting where target implementations are register transducers in the class RT[k], for some k ≥0 that we now ﬁx for the whole section. For the complexity analysis, we assume k is given as input, in unary. Indeed, describing a k-register automaton in general requires O(k) bits, and not O(log k) bits. We prove the decidable cases of the ﬁrst line of Table 1 (page 3), by reducing the problems to realisability problems for data-free speciﬁcations. Abstract Actions. We reduce the problem to a ﬁnite alphabet problem. Since we synthesise k-register transducers, we take the input and output actions of the transducers as symbols of our ﬁnite input and output alphabets. Let Rk = {1, . . . , k} and Tstk = Asgnk = 2Rk. The CONCUR 2019 24:10 Synthesis of Data Word Transducers Synthesis of Data Word Transducers Let us show that WS,k is realisable by T ′, i.e. L(T ′) ⊆WS,k. Take a sequence a = a1e1a2e2 · · · ∈L(T ′). We show that Comp(a) ⊆S. Let w ∈Comp(a). Then, there exists a run q0q1q2 . . . of T ′ on a since a ∈L(T ′). By deﬁnition of compatibility for w, there exists a sequence of register conﬁgurations τ0τ1 · · · ∈(Rk →D)ω satisfying the conditions in the deﬁnition of compatibility. From this we can deduce that (q0, τ0)(q1, τ1) . . . is an initial sequence of conﬁgurations of T over w, so w ∈L(T). Finally, L(T) ⊆S, since T realises S. Conversely, suppose that WS,k is realisable by some ﬁnite transducer T ′ over the input (output) alphabets Ak i (Ak o). Again, the transducer T can be seen as a transducer with k registers over data words. We show that T realises S, i.e., L(T) ⊆S. Let w ∈L(T). Let us show that WS,k is realisable by T ′, i.e. L(T ′) ⊆WS,k. Take a sequence a = a1e1a2e2 · · · ∈L(T ′). We show that Comp(a) ⊆S. Let w ∈Comp(a). Then, there exists a run q0q1q2 . . . of T ′ on a since a ∈L(T ′). By deﬁnition of compatibility for w, there exists a sequence of register conﬁgurations τ0τ1 · · · ∈(Rk →D)ω satisfying the conditions in the deﬁnition of compatibility. From this we can deduce that (q0, τ0)(q1, τ1) . . . is an initial sequence of conﬁgurations of T over w, so w ∈L(T). Finally, L(T) ⊆S, since T realises S. ( ) a sequence of register conﬁgurations τ0τ1 · · · ∈(Rk →D)ω satisfying the conditions in the deﬁnition of compatibility. From this we can deduce that (q0, τ0)(q1, τ1) . . . is an initial sequence of conﬁgurations of T over w, so w ∈L(T). Finally, L(T) ⊆S, since T realises S. Conversely, suppose that WS,k is realisable by some ﬁnite transducer T ′ over the input (output) alphabets Ak i (Ak o). Again, the transducer T can be seen as a transducer with k registers over data words. We show that T realises S, i.e., L(T) ⊆S. Let w ∈L(T). The run of T over w induces a sequence of actions a in (Ak i Ak o)ω which, by deﬁnition of compatibility, satisﬁes w ∈Comp(a). Moreover, a ∈L(T ′). L. Exibard, E. Filiot, and P.-A. Reynier the test tst and the assignment asgn and goes to state asgn. In any state asgn ∈AsgnR, it is only allowed to read labelled data of the form (σo, asgn, r, d), for which it tests whether d is equal to the content of r. It does no assignment and moves back to state q. All states are accepting (i.e. have maximal even parity 2). Such NRA has size O(2k2). ◀ Let S be a speciﬁcation deﬁned by some URA AS with set of states Q. The following subset of Lk is deﬁnable by some NRA, where S denotes the complement of S: ▶Lemma 9. The language LS,k = {w ⊗a \| w ∈Comp(a) ∩S} is deﬁnable by some NRA. ▶Lemma 9. The language LS,k = {w ⊗a \| w ∈Comp(a) ∩S} is deﬁnable by some NRA. Proof. Since S is deﬁnable by the URA AS, S is NRA-deﬁnable with the same automaton, denoted now AS, interpreted as an NRA. Let B be some NRA deﬁning Lk (it exists by Lemma 8). It now suﬃces to take a product of AS and B to get an NRA deﬁning LS,k. ◀ Given a data word language L, we denote by lab(L) = {lab(w) \| w ∈L} its projection on labels. The language WS,k is obtained as the complement of the label projection of LS,k: ▶Lemma 10. WS,k = lab(LS,k). ▶Lemma 10. WS,k = lab(LS,k). ▶Lemma 10. WS,k = lab(LS,k). Proof. Let a ∈(Ak i Ak o)ω. Then, a /∈WS,k ⇔Comp(a) ̸⊆S ⇔∃w ∈RW, w ∈Comp(a)∩S ⇔ ∃w ∈RW, w ⊗a ∈LS,k ⇔a ∈lab(LS,k). ◀ We are now able to show regularity of WS,k. ▶Lemma 11. Let S be a data word speciﬁcation, k ≥0. If S is deﬁnable by some URA with n states and r registers, then WS,k is eﬀectively ω-regular, deﬁnable some deterministic parity automaton with O(2n216(r+k)2 ) states and O(n.4(r+k)2) priorities. Proof. First, LS,k is deﬁnable by some NRA with O(2k2n) states and O(r + k) registers by Lemma 10, obtained as product between the NRA AS and the automaton obtained in Lemma 8, of size O(2k2). It is known that the projection on the alphabet of labels of a language of data words recognised by some NRA is eﬀectively regular [10]. 5.1 The case of URA speciﬁcations In this section, we show that for any S a data word speciﬁcation given as some URA, the language WS,k is eﬀectively ω-regular, entailing the decidability of Real(URA, RT[k]), by Theorem 7 and the decidability of (data-free) synthesis. Let us ﬁrst prove a series of intermediate lemmas. We deﬁne an operation ⊗between relational data words w ∈RW(Σi, Σo, D) and sequences of actions a ∈(Ak i Ak o)ω as follows: w ⊗a ∈RW(Ak i , Ak o, D) is deﬁned only if for all i ≥1, lab(w[i]) = lab(a[i]) where lab(a[i]) is the ﬁrst component of a[i] (a label in Σi ∪Σo), by (w ⊗a)[i] = (a[i], dt(w[i])). ▶Lemma 8. The language Lk = {w ⊗a \| w ∈Comp(a)} is deﬁnable by some NRA. ▶Lemma 8. The language Lk = {w ⊗a \| w ∈Comp(a)} is deﬁnable by some NRA. Proof. We deﬁne an NRA with k registers which roughly follows the actions it reads on its input. Its set of states is {q}∪AsgnR, with initial state q. In state q, it is only allowed to read labelled data in Ak i × D. On reading (σi, tst, d), it guesses some assignment asgn, performs 24:11 ▶Theorem 13. Real(DRA, RT[k]) is in ExpTime. 5.2 The case of test-free NRA speciﬁcations Unfortunately, by Theorem 3, the synthesis problem for speciﬁcations expressed as NRA is undecidable, even when the number of registers of the implementation is bounded. And indeed, if we mimic the reasoning of the previous section, Lemma 10 does not allow to conclude because LS,k is deﬁnable by a URA but the string projection of a URA is not ω-regular in general. E.g., consider L = {(r, d1) . . . (r, dn)(g, d′ 1) . . . (g, d′ m) \| ∀i ̸= j, di ̸= dj ∧∀1 ≤i ≤n, ∃j, d′ j = di}, which consists in a word w ∈rn with pairwise distinct data followed by a word w ∈gm which contains at least all the data of w (it is over ﬁnite words for simplicity but can be extended to ω-words). L is recognised by the URA which, on reading (r, di), universally triggers a run checking three properties: ﬁrstly, once a label g is read, only g’s are read, secondly, (r, di) does not appear again, and thirdly, (g, di) appears at least once. Now, it is readily seen that lab(L) = {rngm \| m ≥n}, which is not regular. Here, we deﬁned L over ﬁnite words for simplicity but it is easily extended to an ω-language which is not ω-regular. Thus, in this section, we consider a restriction on NRA which do not perform tests on input data. A test-free register automaton is a tuple A = (Σi, Σo, D, Q, q0, δ, R, c) such that δ ⊆Q × Σi × AsgnR × Σo × R × Q. Such a register automaton reads two labelled data at once. In a conﬁguration (q, τ), when reading (σi, di)(σo, do), it can ﬁre any transition of the form (q, σi, asgn, r, σo, q′) ∈δ such that τ(r) = do and move to conﬁguration (q′, τ ′) where τ ′ = next(τ, asgn, di). It is easily seen that a test-free register automaton can be converted into a proper register automaton, justifying its name. Such automata will be interpreted by a non-deterministic parity acceptance condition; we denote the class NRAtf4. L. Exibard, E. Filiot, and P.-A. Reynier The same construction, which is based on extending the state space with register equality types, carries over to ω-words, and one obtains a non-deterministic parity automaton with O(n.4(r+k)2) states and d priorities recognising lab(LS,k). It can be complemented into a deterministic parity automaton with O(2n2.16(r+k)2 ) states and O(n.4(r+k)2) priorities using standard constructions [15]. ◀ We are now able to reprove the following result, known from [12]: ▶Theorem 12. For all k ≥0, Real(URA, RT[k]) is in 2ExpTime. Proof. By Lemma 11, we construct a deterministic parity automaton PS,k for WS,k. Then, according to Theorem 7, it suﬃces to check whether it is realisable by a (register-free) transducer. This is decidable by Theorem 1. The way to decide it is to see PS,k as a two-player parity game and check whether the protagonist has a solution. Parity games can be solved in time O(mlog d) [3] where m is the number of states of the game and d the number of priorities. Overall, solving it requires doubly exponential time, more precisely in O(2n316(r+k)2 ). ◀ In [11], it is shown that the complexity of the problem is actually only singly exponential in k, and such analysis extends to our construction. Similarly, when the speciﬁcation automaton is deterministic, we can show that: ▶Theorem 13. Real(DRA, RT[k]) is in ExpTime. CONCUR 2019 24:12 Synthesis of Data Word Transducers 4 The bounded synthesis of URA is already decidable, so we do not consider their test-free restriction. ▶Proposition 14. Let S be a NRAtf speciﬁcation. The following are equivalent: (i) S is realisable 5.2 The case of test-free NRA speciﬁcations It is not clear whether WS,k is regular for such speciﬁcations, but we show that it suﬃces to consider another set denoted W tf S,k which is easier to analyse (and can be proven regular), and based on the behaviour of S over input with pairwise distinct data. The intuition behind restricting to such case is that NRAtf cannot conduct test on input data, so they behave the same on an input word whose data are all distinct, and such choice ensures that two equal input data will not ease the task of the implementation. An interesting side-product of this approach is that it implies that we can restrict to test-free implementations. A test-free transducer is a transducer whose transitions do not depend on tests over input data; formally δ : Q × Σi →AsgnR × Σo × R × Q. In the following, we let AllDiff denote the set of relational data words whose input data are pairwise distinct: AllDiff = {w = (σ1 i , d1 i)(σ1 o, d1 o) · · · ∈RW \| ∀0 ≤i < i′, di i ̸= di′ i }; by convention d0 i = d0. ▶Proposition 14. Let S be a NRAtf speciﬁcation. The following are equivalent: (i) S is realisable (ii) W tf S,k = {a ∈(A∅ i Ak o)ω \| Comp(a) ∩S ∩AllDiff ̸= ∅}, where A∅ i = Σi × {∅}, has domain (A∅ i )ω and is realisable (by a register-free transducer) (iii) S is realisable by a test-free transducer Proof. (i) ⇒(ii): If S is realisable, then by Theorem 7 WS,k has total domain and is realisable by some transducer I. Now, since transducers are closed under regular domain restriction, W ∅ S,k = WS,k ∩(A∅ i Ak o)ω has domain (A∅ i )ω and is realisable by I ∩(A∅ i Ak o)ω. Moreover, W ∅ S,k ⊆W tf S,k. Indeed, if Comp(a) ⊆S, then, since S has total domain and a ∈(A∅ i Ak o)ω, Comp(a) ∩S ∩AllDiff ̸= ∅. Thus, W tf S,k also has domain (A∅ i )ω and is realisable by any transducer realising W ∅ S,k. 24:13 L. Exibard, E. Filiot, and P.-A. Reynier (iii) ⇒(i): is trivial. (ii) ⇒(iii): Intuitively, NRAtf can only rearrange input data (duplicate, erase, copy) regardless of the actual data values (as there are no tests), so its behaviour on AllDiff determines its behaviour on the entire domain. This can be formalised precisely through a notion of data origins given a run (this notion is also made explicit in [5]). To a run ρ = q0 σ1 i ,asgn1,r1,σ1 o −−−−−−−−−→q1 σ2 i ,asgn2,r2,σ2 o −−−−−−−−−→q2 . . . corresponds the origin function oρ : j 7→max{i ≤j \| rj ∈asgni}, with the convention max ∅= 0. To a run ρ = q0 σ1 i ,asgn1,r1,σ1 o −−−−−−−−−→q1 σ2 i ,asgn2,r2,σ2 o −−−−−−−−−→q2 . . . corresponds the origin function oρ : j 7→max{i ≤j \| rj ∈asgni}, with the convention max ∅= 0. Now, for an origin function o : N\{0} →N and for a relational data word w ∈RW, we say w is compatible with the origin function o, denoted w \|= o, whenever for all j ≥1, dt(out(w)[j]) = dt(inp(w)[o(j)]), with the convention dt(inp(w)[0]) = d0. The following lemma shows that actual data values in a word w do not matter with respect to membership in some NRAtf, only the compatibility with origin functions does: ▶Lemma 15. Let w ∈RW and ρ a sequence of transitions of some NRAtf. Then, ▶Lemma 15. Let w ∈RW and ρ a sequence of transitions of some NRAtf. Then, (i) If ρ is a run over w, then w \|= oρ. (i) If ρ is a run over w, then w \|= oρ. (ii) If ρ is a run over w and w ∈AllDiff, then for all o : N\{0} →N, w \|= o ⇔o = oρ. (ii) If ρ is a run over w and w ∈AllDiff, then for all o : N\{0} →N, w \|= o ⇔o = oρ. (iii) If w and ρ have the same ﬁnite labels and if w \|= oρ, then ρ is a run over w. (iii) If w and ρ have the same ﬁnite labels and if w \|= oρ, then ρ is a run over w. Proof. (i) and (iii) follow from the semantics of NRAtf, which do not conduct any test on the input data. The ⇐direction of (ii) is exactly (i). Now, assume w ∈AllDiff admits ρ as a run, and let o such that w \|= o. L. Exibard, E. Filiot, and P.-A. Reynier Then, let j ≥1 be such that dt(out(w)[j]) = dt(inp(w)[o(j)]). By (i) we know that dt(out(w)[j]) = dt(inp(w)[oρ(j)]), so dt(inp(w)[o(j)]) = dt(inp(w)[oρ(j)]). Since w ∈AllDiff, this implies o(j) = oρ(j), so, overall, o = oρ. ◀ Now, assume W tf S,k is realisable by some transducer I. We show that I, when ignoring the ∅input tests, is actually an implementation of S. Thus, let I′ be the same transducer as I except that all input transitions (σi, ∅) are now simply labelled σi. Note that I′, interpreted as a register transducer, is test-free. Let w ∈DW, and ai = lab(w) × ∅ω be the input action in A∅ i with same ﬁnite labels as w. Let a = I(ai), and let w′ ∈Comp(a)∩S ∩AllDiff (such w′ exists because W tf S,k has domain (A∅ i )ω and I realises W tf S,k). Then, since lab(w) = lab(w′), they admit the same run ρI in I, so w, w′ \|= oρI. Then, w′ ∈S, so it admits an accepting run ρS in S, which implies w′ \|= oρS. Moreover, w′ ∈AllDiff so, by Lemma 15 ii, we get oρI = oρS. Therefore, w \|= oρS, so, by iii, w admits ρS as a run, i.e. w ∈S. Overall, L(I) ⊆S meaning that I is a (test-free) implementation of S. End of proof of Prop. 14 ◀ Finally, W tf S,k = {a ∈(A∅ i Ak o)ω \| Comp(a) ∩S ∩AllDiff ̸= ∅} is regular. Indeed, W tf S,k = {a ∈(A∅ i Ak o)ω \| Comp(a) ∩S∅̸= ∅}, where S∅is the same automaton as S except that all transitions q σi,asgn,r,σo −−−−−−−→q′ have been replaced with q σi,∅,asgn,r,σo −−−−−−−−−→q′, because, for all a ∈(A∅ i Ak o)ω, Comp(a) ∩S ∩AllDiff ̸= ∅⇔Comp(a) ∩S∅̸= ∅(the ⇒direction is trivial, and the ⇐stems from the fact that an AllDiff input only takes tst = ∅transitions). Finally, W tf S,k = {a ∈(A∅ i Ak o)ω \| Comp(a) ∩S ∩AllDiff ̸= ∅} is regular. Indeed, W tf S,k = {a ∈(A∅ i Ak o)ω \| Comp(a) ∩S∅̸= ∅}, where S∅is the same automaton as S except that all transitions q σi,asgn,r,σo −−−−−−−→q′ have been replaced with q σi,∅,asgn,r,σo −−−−−−−−−→q′, because, for all a ∈(A∅ i Ak o)ω, Comp(a) ∩S ∩AllDiff ̸= ∅⇔Comp(a) ∩S∅̸= ∅(the ⇒direction is trivial, and the ⇐stems from the fact that an AllDiff input only takes tst = ∅transitions). L. Exibard, E. Filiot, and P.-A. Reynier Then, Ltf S,k = {w ⊗a ∈RW ⊗(A∅ i Ak o)ω \| w ∈Comp(a) ∩S∅} is NRA-deﬁnable. Indeed, S is NRAtf-deﬁnable, so S∅is NRA-deﬁnable, and by Lemma 8, Lk = {w ⊗a \| w ∈Comp(a)} is NRA-deﬁnable, so their product recognises Ltf S,k. Finally, W tf S,k = lab(Ltf S,k), and the projection of a NRA over some ﬁnite alphabet is regular [10]. Overall, by Theorems 1 and 7, we get (the complexity analysis is the same as for URA): ▶Theorem 16. For all k ≥0, Real(NRAtf, RT[k]) is decidable and in 2ExpTime. CONCUR 2019 24:14 Synthesis of Data Word Transducers 6 Synthesis and Uniformisation For all k ≥1, Unif(URA, RT[k]) is undecidable. ▶Theorem 18. For all k ≥1, Unif(URA, RT[k]) is undecidable. If the domain is DRA-recognisable, it is possible to reduce the uniformisation problem to realisability, by allowing any behaviour on the complement on the domain (which is DRA- recognisable). However, such property is undecidable as a direct corollary of Theorem 17. 6 Synthesis and Uniformisation In this section, we discuss the relation between realisability and uniformisation of relations: in this paper, if S is realisable by a register transducer, then, in particular, it has universal domain, i.e. inp(S) = DW(Σi, D), otherwise it cannot be that L(T) ⊆S for T a register transducer, since by deﬁnition inp(T) = DW(Σi, D). However, when deﬁning a speciﬁcation, the user might be interested only in a subset of behaviours (for instance, s/he knows that all input data will be pairwise distinct). In the ﬁnite alphabet setting, since the formalisms used to express speciﬁcations are closed under complement (whether it is LTL or ω-automata), it suﬃces to complete the speciﬁcation by allowing any behaviour on the input not considered. However, since register automata are not closed under complement, such approach is not suﬃcient here. Thus, it is relevant to generalise the realisability problem to the case where the domain of the speciﬁcation is not universal. This can be done by equipping register transducers with an acceptance condition. It is also necessary to adapt the notion of realisability; otherwise, any transducer accepting no words realises any speciﬁcation. A natural way is to consider synthesis as an instance of the uniformisation problem. An (implementation) function f : I →O is said to uniformise a (speciﬁcation) relation R ⊆I × O whenever dom(f) = dom(R) and for all i ∈dom(f), (i, f(i)) ∈R. In the context of reactive synthesis, where f = fI is deﬁned from an implementation I and R is given as a language of relational words, it can be rephrased as inp(L(I)) = inp(R) and for all wi ∈inp(L(I)), ⟨wi, fI(wi)⟩∈R. Note that such deﬁnition coincides with the one of realisability when the class of implementations has universal domain. In the following, we denote by Unif(S, I) the uniformisation problem from speciﬁcations in S to implementations in I. Unfortunately, this setting is actually much harder, as shown by the next two theorems: ▶Theorem 17. Given S a speciﬁcation represented by a DRA, checking whether inp(S) = DW(Σi, D) is undecidable. ▶Theorem 17. Given S a speciﬁcation represented by a DRA, checking whether inp(S) = DW(Σi, D) is undecidable. While the uniformisation setting obviously preserves the undecidability results from the synthesis setting, the above result allows to show that the somehow more general uniformisation problem is undecidable. For instance, we can prove: ▶Theorem 18. References 1 Roderick Bloem, Krishnendu Chatterjee, and Barbara Jobstmann. Graph Games and Reactive Synthesis, pages 921–962. Springer International Publishing, Cham, 2018. 2 Miko laj Boja´nczyk, Anca Muscholl, Thomas Schwentick, Luc Segouﬁn, and Claire David. Two-Variable Logic on Words with Data. In Proceedings of the 21th IEEE Symposium on Logic in Computer Science (LICS 2006), pages 7–16. ACM, 2006. doi:10.1109/LICS.2006.51. 3 Cristian S. Calude, Sanjay Jain, Bakhadyr Khoussainov, Wei Li, and Frank Stephan. Deciding Parity Games in Quasipolynomial Time. In Proceedings of the 49th Annual ACM SIGACT Symposium on Theory of Computing (STOC 2017), pages 252–263. ACM, 2017. doi:10.1145/ 3055399.3055409. 4 Luc Dartois, Emmanuel Filiot, and Nathan Lhote. Logics for Word Transductions with Synthesis. In Proceedings of the 33rd Annual ACM/IEEE Symposium on Logic in Computer Science (LICS 2018), pages 295–304. ACM, 2018. doi:10.1145/3209108.3209181. 5 Antoine Durand-Gasselin and Peter Habermehl. Regular Transformations of Data Words Through Origin Information. In Proceedings of the 19th International Conference on Foun- dations of Software Science and Computation Structures (FOSSACS 2016), volume 9634 of Lecture Notes in Computer Science, pages 285–300. Springer, 2016. doi:10.1007/ 978-3-662-49630-5_17. 6 R¨udiger Ehlers, Sanjit A. Seshia, and Hadas Kress-Gazit. Synthesis with Identiﬁers. In Proceedings of the 15th International Conference on Veriﬁcation, Model Checking, and Abstract Interpretation (VMCAI 2014), volume 8318 of Lecture Notes in Computer Science, pages 415–433. Springer, 2014. doi:10.1007/978-3-642-54013-4_23. 7 L´eo Exibard, Emmanuel Filiot, and Pierre-Alain Reynier. Synthesis of Data Word Transducers. CoRR, abs/1905.03538, 2019. arXiv:1905.03538. 8 Diego Figueira and M. Praveen. Playing with Repetitions in Data Words Using Energy Games. In Proceedings of the 33rd Annual ACM/IEEE Symposium on Logic in Computer Science (LICS 2018), pages 404–413. ACM, 2018. doi:10.1145/3209108.3209154. 9 J.R. B¨uchi and L.H. Landweber. Solving Sequential Conditions by Finite-State Strategies. Transactions of the American Mathematical Society, 138:295–311, 1969. 10 Michael Kaminski and Nissim Francez. Finite-memory Automata. Theor. Comput. Sci., 134(2):329–363, 1994. doi:10.1016/0304-3975(94)90242-9. 11 Ayrat Khalimov and Orna Kupferman. Register Bounded Synthesis. In Proceedings of the 30th International Conference on Concurrency Theory (CONCUR 2019), 2019. To appear. 12 Ayrat Khalimov, Benedikt Maderbacher, and Roderick Bloem. Bounded Synthesis of Register Transducers. In Proceedings of the 16th International Symposium on Automated Technology for Veriﬁcation and Analysis (ATVA 2018), volume 11138 of Lecture Notes in Computer Science, pages 494–510. Springer, 2018. 13 Andrzej S. Murawski, Steven J. Ramsay, and Nikos Tzevelekos. Bisimilarity in Fresh-Register Automata. In 30th Annual ACM/IEEE Symposium on Logic in Computer Science (LICS 2015), pages 156–167. 7 Conclusion In this paper, we have given a picture of the decidability landscape of the synthesis of register transducers from register automata speciﬁcations. We studied the parity acceptance condition because of its generality, but our results allow to reduce the synthesis problem for register automata speciﬁcations to the one for ﬁnite automata while preserving the acceptance condition. We have also introduced and studied test-free NRA, which do not have the ability to test their input, but still have the power of duplicating, removing or copying the input data to form the output. We have shown that they allow to recover decidability in presence of non-determinism, in the bounded case. We leave open the unbounded case, which we conjecture to be decidable. As future work, we want to study synthesis problems for speciﬁcations given by logical formulae, for decidable data words logics such as two-variable fragments of FO [2, 17, 4]. 24:15 L. Exibard, E. Filiot, and P.-A. Reynier L. Exibard, E. Filiot, and P.-A. Reynier References IEEE Computer Society, 2015. doi:10.1109/LICS.2015.24. 14 Frank Neven, Thomas Schwentick, and Victor Vianu. Finite State Machines for Strings over Inﬁnite Alphabets. ACM Trans. Comput. Logic, 5(3):403–435, 2004. doi:10.1145/1013560. 1013562. 15 Nir Piterman. From Nondeterministic B¨uchi and Streett Automata to Deterministic Parity Automata. Logical Methods in Computer Science, 3(3), 2007. 16 A. Pnueli and R. Rosner. On the synthesis of a reactive module. In ACM Symposium on Principles of Programming Languages, POPL. ACM, 1989. 17 Thomas Schwentick and Thomas Zeume. Two-Variable Logic with Two Order Relations. Logical Methods in Computer Science, 8(1), 2012. doi:10.2168/LMCS-8(1:15)2012. 18 Luc Segouﬁn. Automata and Logics for Words and Trees over an Inﬁnite Alphabet. In Proceedings of the 15th Annual Conference of the EACSL on Computer Science Logic (CSL 2006), volume 4207 of Lecture Notes in Computer Science, pages 41–57. Springer, 2006. doi:10.1007/11874683_3. CONCUR 2019
https://openalex.org/W1449031827	https://www.scienceopen.com/document_file/5a3426a7-026f-4918-a49a-1c2297764b60/ScienceOpen/001_Zwolinski.pdf	English	null	A Simulation Approach to the Design and Evaluation of Auditory Interfaces in a High Speed Train Driving Cab.	Electronic workshops in computing	1,998	cc-by	5,268	1 Introduction In the future, trains will travel at 350 km/h or more and will run on a European network of high speed tracks. In order to attain such performance, the design of high speed train driving cabs (control panels, informational devices and control devices) needs special attention, as mentioned by Foret [5]. Among the various informational devices, auditory signals play an important role in attracting attention and giving information to the train driver. There are many situations in which a train driver will use sounds e.g. when changing speed, to diagnose malfunctions, to receive a call from another operator, etc. These sounds are used because of the important visual load induced by the demands of the exterior environment (the railway and track signalling). But the extension of the train network throughout Europe, and the evolution of technologies have led to a growing number of different systems in trains and consequently of the different sounds generated. So, particular attention should be given to designing a coherent set of auditory signals for the future taking into account problems of redundant signals and distracting factors which could lead to signal vigilance errors, as demonstrated by Haga [6]. The aim of this paper is to illustrate a pragmatic methodology used to create a more efficient auditory system for the French high speed train driving cab. This is a first step in a study which will eventually include all the sounds used in all European trains. The intent of the paper is not to discuss the technical aspects of sound in high speed trains but to present a user-centred approach using simulation to create auditory interfaces for the future high speed train driving cab. The first section of this paper concentrates on existing sounds in the driving cab. It is the first stage in the simulation approach because we can build an accurate model of the present situation of auditory signals to determine the model of the future situation. In the second section we will show how to design a system in a co-operative approach using scenarios and by analysing the functions of each sound. In the final part of the paper, we will show how the twelve train drivers who have participated in the entire project have validated on a simulator the set of auditory signals they finally chose. Abstract As operators are spending more and more of their time monitoring and using control systems, auditory displays are becoming increasingly useful along with other control devices and control panels. This paper presents a method, elaborated with the help of engineers, ergonomists and train drivers, for designing and evaluating auditory interfaces for use in high speed train driving cabs. During this design project, ergonomists studied the usefulness and the usability of auditory signals, in relation to visual displays and future driving cab control devices. To take into account every aspect of auditory signals, members of the project have chosen to use a simulation approach benefiting from co-operation between ergonomists, engineers and volunteer train drivers who took part in the entire project for the design of the future driving cab. A Simulation Approach to the Design and Evaluation of Auditory Interfaces in a High Speed Train Driving Cab. Peggy Zwolinski Institut Polytechnique de Sévenans. L’Université de Technologie de Belfort-Montbéliard 90010 BELFORT cedex (FRANCE) Phone : 03-84-58-31-61 - Fax : 03-84-58-31-41 - E-mail : Peggy.zwolinski@utbm.fr Jean-Claude Sagot Institut Polytechnique de Sévenans. L’Université de Technologie de Belfort-Montbéliard 90010 BELFORT cedex (FRANCE) Phone : 03-84-58-30-13 - Fax : 03-84-58-31-41 Jean-Claude Sagot 2 Analysis of Existing Sounds ICAD’98 The first step in this study was to make an ergonomic evaluation of existing sounds during the driving activity. Indeed, this analysis of the driving activity is very important to identify factors for improving or for transforming existing working conditions and thus design a man-machine interface. In the present study concerning auditory interfaces, we first of all identified and recorded the 23 sounds being used at present in French train driving cabs. Then we evaluated, according to Momtahan et al. [10], the different sounds. A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. Twelve train drivers had to answer a questionnaire and then were tested for their detection and their comprehension of the various sounds. Twelve train drivers had to answer a questionnaire and then were tested for their detection and their comprehension of the various sounds. Next, with reference to Patterson [12], we examined the overall level, the temporal characteristics, the spectral characteristic and ergonomic aspects of each sound. It appears that the main problems of current auditory interfaces are : the sounds are too loud, the temporal patterns are not sufficiently distinctive and the ergonomics of existing auditory systems are deplorable. For example, let us examine the sounds which trigger an emergency stop. These sounds are defined as follows in the manufacturer’s specifications : radio alarm : continuous buzzer - 1400 Hz - 1800 Hz (sound 1). radio alarm : continuous buzzer - 1400 Hz - 1800 Hz (sound 1). false start :repeated bell rings (sound 2). false start :repeated bell rings (sound 2). passenger alarm : buzzer - 1000 Hz - generated 5 * [0.25 second ON and 0.75 second OFF] (sound 3) As no data was provided in the manual concerning the level of each sound and no recommendation was given concerning a general level, we took various readings inside several driving cabs. We were confronted with a wide variety of results (level, frequency, time,...) for one alarm, depending on which cab we were taking the measures in. It was clear that each generation of locomotive has its own sound generator and that even when the sounds seemed the same, there were differences between each cab. 2 Analysis of Existing Sounds Concerning the actions to be taken when the sound is played, we noted that drivers must make an emergency stop if they hear a radio alarm, a passenger alarm when starting in a station, or there is a false start from the master controller. But if they receive the passenger alarm in case of SAI (Interphone Alarm System) during the trip (sound 2) they must continue driving until they have more information concerning the incident. So, for the ergonomics of existing auditory systems, most of the problems derive from the fact that new control systems have been installed without a global ergonomic view of the whole system. For example, one sound may require the driver to take three different actions and one action may be required by different sounds. There are no priorities between sounds, so the driver could be called for nothing at any moment even if there is a sudden emergency. To maximise the effect of ergonomic principles, it’s important to review the whole warning system to prevent warnings from proliferating. One way to do this is to establish a warning philosophy and define categories of action urgency [2]. So, we have defined with train drivers the following sound classifications: * Class 1 : Sounds indicating information to the driver. * Class 1 : Sounds indicating information to the driver. * Class 2 : Sounds indicating changes in signalling to the d river. * Class 3 : Sounds to warn the driver and which require immediate action. * Class 4 : Sounds to warn the driver to make an emergency stop (danger or an accident). All these considerations have led us to determine, during the preliminary study, what we have called, according to Sagot et al. [14], "the field of desirable future activities " concerning auditory interfaces, desirable in terms of safety, comfort and efficiency. 3.1 Definition of the Field of Desirable Future Activities To define the field of desirable future activities, we have worked with twelve train drivers in order to determine what happens when a sound is made. In principle, acoustic signals have to meet 3 criteria to be effective. The recipient has to detect the signal, to interpret the signal and to take an appropriate action [8]. So, we have defined scenarios about what can happen before, after and at the moment when the sound is played and suggest a general scenario. On the basis of the general scenario (Figure 1), we have chosen to design and evaluate sounds keeping in mind these 3 classes of criteria : detection, comprehension, utility. ICAD’98 2 2 A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. Sound anticipated coherent detected explained detected explained detected repeated there is a redundant information The sound concerns the security of the train yes yes yes yes yes yes yes yes yes yes Emergency stop by the train no no no no no Unknown consequences which can lead to an accident Emergency stop by the security system no no no no no Action for correction Action for validation Warm up Memorisation Search for the solution Transmission of the information Figure 1 : General scenario concerning what happens when a sound is generated. Sound no Action for correction Action for validation Warm up Memorisation Search for the solution Transmission of the information Unknown consequences which can lead to an accident Figure 1 : General scenario concerning what happens when a sound is generated. 3.3 Design and First Evaluation of Sounds Concept Based on functional analysis, we have built different sounds for the 10 classes of auditory signals. As a wide variety of acoustic parameters have clear and consistent effects on the perceived urgency [7], we have developed 5 or more different non verbal propositions for each sound. All these propositions meet the criteria defined during the functional analysis. To create them, we simply chose sounds with dominant frequencies (340, 400, 425, 440, 500, 554, 750, 900, 1000, 1100, 1260, 1400, 1520, 1800, 2000, 2500, 3000, 3250 Hz). We then associated these different sounds with the PC to generate a large number of propositions. We have also used different sounds like buzzers, bells, etc., that we found in sound libraries. But, as we have noticed, increasing the number of sounds could require an inordinate amount of training and retraining to maintain a perfect identification by all the drivers. So we have suggested that voice warnings should be introduced to reduce the number of sounds required on the train and we have also proposed 2 or 3 messages for each class of auditory signals. Next, we developed a testing bench to test auditory signals in the noise environment of a cab which we have simulated with real recorded sounds. We generated three environmental sounds (we did not include the sounds from auxiliary equipment which are not continuous and are not very loud.) : Rail rolling sounds - Aerodynamic sounds - Electric motor sounds. To generate the different alarms and the environment, we used : * A DX 2/50 PC computer with a GRAVIS ULTRASOUND Max card, to generate the interactive sound of the environment and to play the alarm. * An EXPELEC MIX 003, for mixing the different sounds and to easily adjust the different of sound le * A MARANTZ PM-43 amplifier, with 2 JPW loudspeakers for the environment sound and one for the As mentioned by Antin et al. [1], who are working in the automotive sector, it is necessary to use a testing bench to test auditory signals in the environment noise of a cab, to test the appropriateness of the sound level in the environment. According to Fidell [4], it is one of the most efficient ways to validate whether or not the user is able to detect the signals. So, drivers have listen to the different propositions in the sound environment. Table 1 : Principles retained on solutions by the 12 drivers concerning each sound. 3.2 Functional Specifications of Sounds In order to take into account every aspect of the auditory signal in relation to the environment, we have analysed, according to the NF X 50-151 [11], the function of each sound as shown Figure 2. Driver in the cab Sound Reason for the sound Rules and standard specification of the French national railway Environment Control devices Visual displays Other sounds FC1 FC5 FP1 FC2 FC3 FC4 Figure 2 : Example of the functional diagram (FP : principal function - FC : constraint function) We have identified all the relations between a sound and the elements of its environment. For each relation we can define a function that the sound had to realise and for each function we have made a list of criteria to etail the level of satisfaction concerning each function. With this analysis, we have validated the usefulness f 10 different sounds of the present 23 sounds and given a list of criteria taking into account every aspect of ach sound. Reason for the sound Sound Rules and standard specification of the French national railway Environment Control devices Visual displays Figure 2 : Example of the functional diagram (FP : principal function - FC : constraint function We have identified all the relations between a sound and the elements of its environment. For each relation we can define a function that the sound had to realise and for each function we have made a list of criteria to detail the level of satisfaction concerning each function. With this analysis, we have validated the usefulness of 10 different sounds of the present 23 sounds and given a list of criteria taking into account every aspect of each sound. 3 ICAD’98 A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving ca A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. 3.3 Design and First Evaluation of Sounds Concept Then they have to chose between a verbal or non verbal auditory signal and to select the sound they prefer. During their selection, they made a large number of suggestions for optimising the different propositions concerning each sound. In Table 1 are shown the principles of the solutions retained by the majority of the drivers and validated by all the participants in the project. For each of the final 10 sounds retained, we have determined the type of auditory information (sound or vocal) and if the driver needed a redundant visual information. Auditory information concerned Class Type Visual information Message radio alarm 4 sound yes Emergency stop passenger alarm (at start) 4 sound yes Emergency stop false start 4 sound yes Emergency stop excess speed alarm 3 sound yes 1. Speed exceeded 2. You may be stopped passenger alarm (travelling) 3 Vocal yes "Passenger alarm" start signal 2 Vocal yes "Start " signals for b KVB deleted 2 sound + Vocal yes Speed 160 signals for anomaly 1 Vocal yes "consult GDI" radio call 1 sound yes Call on radio signalling changes 2 sound no VACMA sound 3 sound no radio alarm signal 1 Vocal yes "Radio alarm signal" A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. 4 Evaluation of Auditory Interfaces Using a Simulator On the basis of the first evaluation, we have selected and optimised auditory signals for their evaluation in a simulated context. Each volunteer driver has to select a set of sounds before the simulation to evaluate his own choice during a scenario on a simulator ( Figure 3). Figure 3 : The simulator Figure 3 : The simulator 4.2 The Scenario We have chosen a special scenario to test the set of auditory signals chosen by each train driver in a simulated context, as recommended by Maline [9]. During this scenario the train drivers had to: * stop at 3 stations * stop after a false order to start * react to 4 radio calls * obey sounds concerning changes in signalling * notice 2 problems concerning engine malfunction * react to a passenger warning * perform 2 emergency stops. 4.1 The Train Simulator The advantage of the simulator is that it provides an experimental site for reproducing the driving cab with its principle components [15] as well as the main environmental parameters which might influence the driving activity such as the forward movement of the rails, ambient sounds, etc. The variable configuration of this simulator also integrates all the new control and informational devices in relation with the new set of auditory signals chosen by the train drivers. 4.3 Evaluation For the three other classes, we have analysed the type of reaction induced by the sound and measured the time to realise the appropriate action and the time to see the visual information. For technical reason, one of the 12 subjects subject was forced to stop before the end of the experiments. 4.3 Evaluation During the realisation of the scenario, 2 cameras recorded the different actions of the driver and his eye movements. With the simulator we have recorded pertinent indicators, selected in agreement with the work group, such as the real speed, the indicated speed, the number of actions, the action realised with the main command to regulate the speed, etc. You can see for example the speed imposed, the speed realised and the moment when the sound was played for one driver in Figure 4. 5 ICAD’98 ICAD’98 ICAD’98 A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. Speed imposed and realised during the sound scenario 0 50 100 150 200 0 200 400 600 800 1000 1200 Time (seconds) Speed (km/h) Speed realised Speed imposed Sound Figure 4 : Speeds realised and speeds imposed for one driver during the scenario with sounds r a class 1 alarm, which does not necessarily require an action or a visual consultation, we have simply ified that they were indeed detected and that there were no consequences on the driving task. r the three other classes, we have analysed the type of reaction induced by the sound and measured the time realise the appropriate action and the time to see the visual information. For technical reason, one of the 12 Speed imposed and realised during the sound scenario Speed imposed and realised during the sound scenario Figure 4 : Speeds realised and speeds imposed for one driver during the scenario with sounds For a class 1 alarm, which does not necessarily require an action or a visual consultation, we have simply verified that they were indeed detected and that there were no consequences on the driving task. For the three other classes, we have analysed the type of reaction induced by the sound and measured the time to realise the appropriate action and the time to see the visual information. For technical reason, one of the 12 subjects subject was forced to stop before the end of the experiments. For a class 1 alarm, which does not necessarily require an action or a visual consultation, we have simply verified that they were indeed detected and that there were no consequences on the driving task. 4.4 Results Time to see the visual information (seconds) Time to see the visual information (seconds) Sound class Sound class Figure 6 : Mean times to see the visual information (± SD) concerning the different classes of sounds, for the 11 drivers. ANOVA (* :p<0.05). Figure 6 : Mean times to see the visual information (± SD) concerning the different classes of sounds, for the 11 drivers. ANOVA (* :p<0.05). Concerning the difference between the time to see the visual information and the time to realise the Concerning the difference between the time to see the visual information and the time to realise the appropriate action (Figure 7), the data analyses show an high significant difference between class 2 and class 4 sounds (*** : p<0.001), and a significant difference between class 2 and the two other classes (* : p<0.05). There is no significant effect concerning the different subjects(NS). Concerning the difference between the time to see the visual information and the time to realise the appropriate action (Figure 7), the data analyses show an high significant difference between class 2 and class 4 sounds (*** : p<0.001), and a significant difference between class 2 and the two other classes (* : p<0.05). There is no significant effect concerning the different subjects(NS). Concerning the difference between the time to see the visual information and the time to realise the appropriate action (Figure 7), the data analyses show an high significant difference between class 2 and class 4 sounds (*** : p<0.001), and a significant difference between class 2 and the two other classes (* : p<0.05). There is no significant effect concerning the different subjects(NS). -1 0 1 2 3 4 5 6 2 3 4 Sound class Difference between the time to see the visual information and the time to realise the action (seconds) *** * Figure 7 : Means differences between the time to see the visual information and the time to realise the appropriate action (± SD), concerning the different sound classes for the 12 drivers. ANOVA (*** : p<0.001 ; * :p<0.05). 4.4 Results A set of variance analyses (ANOVA) was conducted to test the effect of the sound class and of the subjects on alarm response measures (time to realise the action, time to see the visual information, and the difference between the time to see the visual information and the time to realise the action). Concerning the time to realise the appropriate action (Figure 5), the data analyses show an high significant difference (*** : p<0.001) between class 2 sounds (33 measures) and class 4 sounds (33 measures) and a significant effect between class 3 sounds (11 measures) and class 4 sounds (* : p<0.05). 0 1 2 3 4 5 6 7 8 9 2 3 4 Sound class Time to realise the action (seconds) *** * Figure 5 : Mean times to realise the appropriate action ( ± SD) concerning the different classes of sounds, for the 11 drivers. ANOVA (*** : p<0.001 ; * :p<0.05). Time to realise the action (seconds) Figure 5 : Mean times to realise the appropriate action ( ± SD) concerning the different classes of sounds, for the 11 drivers. ANOVA (*** : p<0.001 ; * :p<0.05). ICAD’98 6 A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. Concerning the time to see the visual information (Figure 6), the data analyses show a significant difference between class 2 sounds and class 4 sounds (* : p<0.05). There is no significant effect concerning the different subjects(NS). Concerning the time to see the visual information (Figure 6), the data analyses show a significant difference between class 2 sounds and class 4 sounds (* : p<0.05). There is no significant effect concerning the different subjects(NS). 0 1 2 3 4 5 6 7 2 3 4 Sound class Time to see the visual information (seconds) * Figure 6 : Mean times to see the visual information (± SD) concerning the different classes of sounds, for the 11 drivers. ANOVA (* :p<0.05). Concerning the difference between the time to see the visual information and the time to realise the appropriate action (Figure 7), the data analyses show an high significant difference between class 2 and class 4 sounds (*** : p<0.001), and a significant difference between class 2 and the two other classes (* : p<0.05). There is no significant effect concerning the different subjects(NS). 4.4 Results Difference between the time to see the visual information and the time to realise the action (seconds) Difference between the time to see the visual information and the time to realise the action (seconds) Figure 7 : Means differences between the time to see the visual information and the time to realise the appropriate action (± SD), concerning the different sound classes for the 12 drivers. ANOVA (*** : p<0.001 ; * :p<0.05). 7 ICAD’98 A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. These different analyses seem to verify that the differences between 3 classes of new sounds is significant in relation with the appropriate actions induced (action or visual consultation). Moreover, it would verify that these actions are coherent in relation with the driving activity. Indeed : Class 4 sounds are characterised by the shorter time to realise the action and to see the visual information. These two types of responses induced by the sound are generally simultaneous. It is very coherent because this class concerns sounds which indicate a danger or an accident and the situation needs a very quick and appropriate action. For class 3 sounds, the time to see the visual information is shorter, but the time to realise the action is longer than for class 4. In this case, the train driver is informed that he has done something wrong which he has to correct. So he has to identify and to verify the causes of sounds and to find the most effective things to do, to correct the mistake. Another wrong action could cause security system to make an emergency stop which could have consequences on his career if it is repeated. For class 2 sounds, the time to realise the action and the time to see the visual information is greater than for class 4. These two types of responses induced by the sound are generally simultaneous. In this case, there is an action to do because of changes in the signalling. It is important to execute it, but it is not necessary to react quickly because there is a short delay to do it. 5 Conclusion The design methodology, described in this paper, presents our experience with a simulation approach defined to develop auditory interfaces in the driving cab of the future high speed train. This method had the merit of associating several partners : designers, ergonomists and operators taking into account different opinions to define the final concept concerning auditory interfaces. The notion of "field of desirable future activities " defined by Sagot et al. [14] was central to our work. On this basis, we have determined and simulated some conditions of the activity in order to optimise and validate the new auditory interfaces. The simulation approach allows representative end-users provide input into the design, testing and modification sessions that are essential to determine an acceptable solution. There is now an increasing number of work environments for which auditory displays have to be designed and implemented but ergonomic aspects of sounds must not be neglected. The simulation approach, illustrated in this paper, constitutes a useful solution to have a user centred approach and a co-operative design which takes into account the ergonomic aspects of sounds. 4.5 Discussion These results enable us to verify : * the validity of the functional principle of the auditory system, * the validity of the functional principle of the auditory system, * the appropriateness between adopted design logic and the driver stereotypes, * the appropriateness between adopted design logic and the driver stereotypes, * comprehension simplification as a result of the grouping and organisation of sounds. * comprehension simplification as a result of the grouping and organisation of sounds Thus, the simulation approach has allowed us to make an initial validation of the new set of sounds for the future high speed train. On the basis of our results and according to Edworthy and Stanton [3] we were able to validate with all the members of the project the fact that we have developed auditory warnings that "sound like what they mean" to the appropriate group of users. The technical aspects of sound in high speed trains will be taken into account later when the same study will integrate sounds from other countries crossed by the high speed train. Then, when all the technical aspects concerning sounds will be defined as recommended by Robinson and Eberts [13], another evaluation will be necessary. Indeed, as mentioned by Haga [6], we may be confronted with other types of errors, depending on the automatic nature of the actions the drivers do which could result in neglecting a signal or which could influence the driver in his choice by masking the main information. 6 Acknowledgements ICAD’98 The authors extend their thanks to the SNCF for providing support for this study, in particular M. Lorinquer, M. Raimond and all the French drivers for their participation to the experiment, and all the members of the ERCO team who have actively participated in the realisation of the experiment. 8 A simulation approach to the design and evaluation of auditory interfaces in the high speed train driving cab. 15. Zwolinski P., Sagot JC., Gouin V. La simulation de l’activité comme outil d’aide à la conception et à l’innovation : Application à la conception de la nouvelle commande de régulation de la vitesse des TGV futurs. Actes des 2ème Journées "recherche et ergonomie ", les 9, 10 et 11 Février 1998; Université de Toulouse le Mirail, 71-75 7 References 1. Antin J.F., Lauretta D.J., Wolf L.D. The intensity of auditory warning tones in the automobile environment : detection and preference evaluations. Applied ergonomics, 1991, 22.1, 13-19. 2. Edworthy J. The design and implementation of non-verbal auditory warnings. Applied ergonomics 1994; 25(4):202-210 3. Edworthy J., Stanton N. A user centred approach to the design and evaluation of auditory warning signals : 1. Methodology. Ergonomics 1995; 38(11):2262-2280 4. Fidell S. Effectivness of audible warning signals for emergency vehicles. Human factors, 1978, 20(1), 19-26 5. Foret J. Conditions de travail des conducteurs de train : revue des études physiologiques et psychophysiologiques. Le travail humain, 1987, tome 50, N° 1, p 17-34. 6. Haga S. An experimental study of signal vigilance errors in train driving. Ergonomics, 1984, vol. 27, n°7, 755-765. 7. Hellier E., Edworthy J., Dennis I. Improving auditory warning design : Quantifying and predicting the effect of different warning parameters on perceived urgency. Human factors 1993; 35(4):693-706 8. Lazarus H., Höge H. Industrial safety : acoustic signals for danger situations in factories. Applied ergonomics 1986; 17(1):41-46. 9. Maline J. Simuler le travail, une aide à la conduite de projet. Editions de l’ANACT, Montrouge ; 1994, 156 p. 10. Momtahan K., Hétu R., Tansley B. Audibility and identification of auditory alarms in the operating room and intensive care unit. Ergonomics 1993; 36(10):1159-1176. 11. NF X 50-151. Analyse de la valeur, analyse fonctionnelle. Expression fonctionnelle du besoin et cahier des charges fonctionnel. Aux éditions AFNOR, Décembre 1991. 12. Patterson R.D. Guidelines for Auditory Warning Systems on Civil Aircraft. Civil Aviation Authority paper 82017, CAA London, 1983 13. Robinson C., Eberts R. Comparison of speech and pictorial displays in a cockpit environment Human factors, 1987, 29(1), 31-44. 14. Sagot J.C., Gouin V., Lorinquer J.P., Chappet P. The high speed train : an ergonomic approach for the driving cab design. In :World Congress on Railway Research. 16-19 November 1997, Florence (Italy), vol. A, 843-851. 15. Zwolinski P., Sagot JC., Gouin V. La simulation de l’activité comme outil d’aide à la conception et à l’innovation : Application à la conception de la nouvelle commande de régulation de la vitesse des TGV futurs. Actes des 2ème Journées "recherche et ergonomie ", les 9, 10 et 11 Février 1998; Université de Toulouse le Mirail, 71-75 ICAD’98 9
https://openalex.org/W2898280826	http://eprints.whiterose.ac.uk/141053/1/s41598-018-34327-5.pdf	English	null	Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry	Scientific reports	2,018	cc-by	8,244	Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 Received: 30 April 2018 Accepted: 17 October 2018 Published: xx xx xxxx Received: 30 April 2018 Accepted: 17 October 2018 Published: xx xx xxxx Runoff from high-elevation debris-covered glaciers represents a crucial water supply for millions of people in the Hindu Kush-Himalaya region, where peak water has already passed in places. Knowledge of glacier thermal regime is essential for predicting dynamic and geometric responses to mass balance change and determining subsurface drainage pathways, which ultimately influence proglacial discharge and hence downstream water availability. Yet, deep internal ice temperatures of these glaciers are unknown, making projections of their future response to climate change highly uncertain. Here, we show that the lower part of the ablation area of Khumbu Glacier, a high-elevation debris-covered glacier in Nepal, may contain ~56% temperate ice, with much of the colder shallow ice near to the melting- point temperature (within 0.8 °C). From boreholes drilled in the glacier’s ablation area, we measured a minimum ice temperature of −3.3 °C, and even the coldest ice we measured was 2 °C warmer than the mean annual air temperature. Our results indicate that high-elevation Himalayan glaciers are vulnerable to even minor atmospheric warming. A glacier’s thermal regime exerts a strong influence on its dynamics, mass balance, and thus its response to cli- matic change – a particular concern with rising atmospheric temperatures1,2. Temperate glacier ice (defined as ice at the melting-point temperature, Tm) yields greater ice velocities than cold ice (below Tm), both through the more rapid deformation of warmer ice under a given stress, and through basal motion, which is facilitated by the presence of meltwater at the ice-bed interface and within subglacial sediments, if present3,4. Temperate ice will also exhibit enhanced ablation rates and yield greater proglacial discharge than cold ice, aided by the increased importance of a subglacial drainage system to evacuate meltwater5. Millions of people in the foothills of the Hindu Kush-Himalaya region depend on glacier melt as part of their water resources6, yet measurements of the internal characteristics and dynamics of mountain glaciers, particularly their internal temperature field, are scarce. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 30 April 2018 Accepted: 17 October 2018 Published: xx xx xxxx SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 Inset (b) shows an image from Site 3 to demonstrate the glacier surface in the upper part of the ablation area (person drilling shown for scale, image taken by K.M.); inset (c) shows the location of the field site (red circle) within Nepal, including the glacierised area across Nepal from the Randolph Glacier Inventory51. Figure 1. Location map of Khumbu Glacier (a) showing the drill sites (blue diamonds) and relevant information about each site from field observations (debris thickness ranges are estimated from field observations). The approximate position of the 1974 Mae drill site18 is also marked (green triangle), with villages and Mt. Everest shown for reference (yellow stars). The background image is a RapidEye scene49 obtained on 23.02.2017. Contours were created from the 2015 SETSM DEM50 and are at 20 m intervals; each 100 m contour within the ablation area is shown in bold from 5,000–5,900 m a.s.l. Inset (b) shows an image from Site 3 to demonstrate the glacier surface in the upper part of the ablation area (person drilling shown for scale, image taken by K.M.); inset (c) shows the location of the field site (red circle) within Nepal, including the glacierised area across Nepal from the Randolph Glacier Inventory51. example, a single temperature measurement of −5.3 °C was made at 2.7 m depth on Khumbu Glacier in 197418; drilling reached 20.3 m depth where the borehole froze shut, which was interpreted to indicate a perennially cold shallow ice zone19. A shallow borehole drilled on Rongbuk Glacier (located north of Mt. Everest) in 1966 gave an ice temperature of −4 °C at 3 m depth and −2.1 °C at 10 m depth; this gradient was used to infer temperate ice at depth for other glaciers south and east of Mt. Everest that are at slightly lower elevations20. Ice temperatures have been modelled for East Rongbuk Glacier and matched to empirical measurements from three boreholes21: one from ice core analysis high in the accumulation area22, the other two from shallow boreholes in the ablation area, but no other methodological data were provided21. More recent work on four high-elevation Himalayan glaciers found that ice temperature on the Gyabrag Glacier (north-west of Mt. Everest) was −8.0 °C at a depth of 10 m, ~3–4 °C warmer than the mean annual air temperature (MAAT)23. Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 At higher elevations, rising surface temperatures may cause peak meltwater to elapse in the next 30 years, leading to a long-term reduction in the glacial contribution to downstream water resources1,2,7. It is therefore increasingly important to determine glacier thermal regimes to better forecast 21st Century glacier retreat and meltwater production.i p Most spatially-distributed numerical models of Himalayan glacier motion include only an unrefined rep- resentation of glacier dynamics8 while, to our knowledge, none includes an empirically-constrained thermal regime1,9,10. The only higher-order dynamic model that has been applied to a debris-covered Himalayan glacier11 calculated englacial and subglacial temperatures by solving for thermal fluxes12 that were estimated in the absence of field data. Consequently, predictions of future mass loss vary and contain large uncertainties; for example, pro- jections of glacier mass loss in the Everest region by 2100 range between 10% and 99%9–11.f Debris-covered glaciers have a more complex surface topography and differing mass loss processes compared to clean-ice glaciers13,14, complicating direct measurement of internal ice temperature. Seasonal variations in subglacial hydrology inferred from satellite-derived surface velocities suggest the presence of temperate ice at the base of high-elevation debris-covered glaciers15,16. Limited field measurements of ice temperatures have been made, but only reached shallow depths (<~20 m) where ice temperature is influenced by seasonal variations in air temperature17. Measurements in this zone do not therefore reflect longer-term and deeper ice temperatures. For 1Centre for Glaciology, Department of Geography and Earth Sciences, Aberystwyth University, Aberystwyth, UK. 2School of Geography, University of Leeds, Leeds, UK. 3Department of Environmental Science and Engineering, Kathmandu University, Kathmandu, Nepal. 4Department of Geography, University of Sheffield, Sheffield, UK. Correspondence and requests for materials should be addressed to K.E.M. (email: kam64@aber.ac.uk) SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 1 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Location map of Khumbu Glacier (a) showing the drill sites (blue diamonds) and relevant information about each site from field observations (debris thickness ranges are estimated from field observations). The approximate position of the 1974 Mae drill site18 is also marked (green triangle), with villages and Mt. Everest shown for reference (yellow stars). The background image is a RapidEye scene49 obtained on 23.02.2017. Contours were created from the 2015 SETSM DEM50 and are at 20 m intervals; each 100 m contour within the ablation area is shown in bold from 5,000–5,900 m a.s.l. Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 Star symbols are used to show data in panel c because malfunctioning equi i i i d (i l d i d d li ) h ll f i Figure 2. Time series of temperatures measured by each borehole thermistor string for: (a) Site 1; (b) Site 2; and (c) Site 3. Star symbols are used to show data in panel c because malfunctioning equipment resulted in some missing data (interpolated using dotted lines); however, all freezing curves were captured. Note the different axis limits on each panel. Thermistors are colour-coded by depth. The small diurnal signal recorded by the thermistors at Site 2 (b; <±0.06 °C variation), and to a lesser extent the other two sites (a,c; <±0.03 °C variation), results from battery voltage noise from the solar regulator operating during daylight hours. Our thermistor naming convention (e.g. S1_5.0) has two parts: ‘S’ refers to the site at which the borehole was drilled; the suffix denotes the depth (in metres) of each thermistor below the surface. thermistor into the borehole as the heat injected during drilling is dissipated26. Beneath this curve, thermistors settle towards the undisturbed temperature of the surrounding ice26,27. Ice temperatures range from −0.47 °C (S1_5.0) to −3.3 °C (S3_24.6). Ice is warmer at Site 1 than Site 3, and in general becomes warmer with depth along each borehole. The uppermost thermistors at Sites 2 and 3 (S2_2.6 and S3_4.7) record increasing temperatures between June and mid-October. Undisturbed ice temperatures were calculated for each thermistor that showed a freezing curve, and are pre- sented in Fig. 3 with the expected Tm (see Methods). Ice temperatures are generally colder near to the glacier sur- face and increase approximately linearly with depth (Site 2 R2 = 0.9206; Site 3 R2 = 0.9996). All ice temperatures at Site 3 (Fig. 3c) are colder than the coldest measured at Site 2 (Fig. 3b), which are very close in temperature to Tm (a conservative estimate; see Methods). These temperatures are plotted along Khumbu Glacier from the icefall to the terminus (Fig. 4), illustrating the general increase in the temperature field both with depth and towards the terminus. The estimated cold-temperate transition surface (CTS; see Methods)28 is included to show the extent of cold and temperate ice within the ablation area. Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 However, temperatures remain unknown below the shallow seasonally-influenced layer, particularly at depths where the thermal conditions would be most relevant for modelling ice flow.i gl Here, we present ice temperature profiles measured along Khumbu Glacier, which originates high on the Nepali side of Mt. Everest and currently terminates at ~4,850 m a.s.l. (Fig. 1). Boreholes were drilled in May 2017 at three locations along the glacier’s ablation area; the deepest at each site was instrumented with a thermistor string (see Methods). At Site 1, the 45.5 m deep borehole was instrumented with nine thermistors; at Site 2, the 22.6 m deep borehole was instrumented with five thermistors; at Site 3, the 132 m deep borehole was instru- mented with eleven thermistors. Data were retrieved in October 2017 having recorded englacial ice temperatures during the monsoon and post-monsoon periods24,25. Our thermistor naming convention (e.g. S1_5.0) has two parts: ‘S’ refers to the site; the suffix denotes the depth (in metres) of each thermistor below the surface. pfi p Borehole temperature time series (Fig. 2) show an initial decrease in temperature measured by all thermis- tors at Sites 2 and 3, and the uppermost thermistor at Site 1 (S1_5.0), which we interpret as the freezing of each SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 2 www.nature.com/scientificreports/ tificreports/ Figure 2. Time series of temperatures measured by each borehole thermistor string for: (a) Site 1; (b) Site 2; and (c) Site 3. Star symbols are used to show data in panel c because malfunctioning equipment resulted in some missing data (interpolated using dotted lines); however, all freezing curves were captured. Note the different axis limits on each panel. Thermistors are colour-coded by depth. The small diurnal signal recorded by the thermistors at Site 2 (b; <±0.06 °C variation), and to a lesser extent the other two sites (a,c; <±0.03 °C variation), results from battery voltage noise from the solar regulator operating during daylight hours. Our thermistor naming convention (e.g. S1_5.0) has two parts: ‘S*’ refers to the site at which the borehole was drilled; the suffix denotes the depth (in metres) of each thermistor below the surface. Figure 2. Time series of temperatures measured by each borehole thermistor string for: (a 2; and (c) Site 3. Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 At Sites 2 and 3, freezing curves initiate within 3 days of thermistor installation due to the rapid dissipation of heat within cold ice (Fig. 2). In contrast, thermistors between 15 and 45.3 m depth at Site 1 show no freezing curves over the 17 days of data available (Fig. 2a) before the cable ruptured due to debris movement, and are inter- preted to have recorded only water temperature during this period. We can, however, infer that the ice between SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 3 www.nature.com/scientificreports/ Figure 3. Vertical ice temperature profiles for each borehole thermistor string at: (a) Site 1; (b) Site 2; and (c) Site 3. Note the different axis limits on each panel. Error bars indicate estimated uncertainty in thermistor temperatures (horizontal), which are accurate to ±0.05 °C at 0 °C, and depths (vertical; see Methods). Panel (a) shows additional error bars in red to indicate the potential ice temperature range of the thermistors that did not freeze in and only recorded the borehole water temperature (as they did not freeze in before S1_5.0, the ice is inferred not to be colder than the ice around this thermistor; see Text). A dashed line indicates the melting- point temperature (Tm; see Methods). Thermistor naming convention is outlined in Fig. 2, and the thermistor colour-coding by depth matches that in Fig. 2. The grey bands mark the 10 m shallow ice layer that is expected to be influenced by seasonal variations in air temperature17. Figure 3. Vertical ice temperature profiles for each borehole thermistor string at: (a) Site 1; (b) Site 2; and (c) Site 3. Note the different axis limits on each panel. Error bars indicate estimated uncertainty in thermistor temperatures (horizontal), which are accurate to ±0.05 °C at 0 °C, and depths (vertical; see Methods). Panel (a) shows additional error bars in red to indicate the potential ice temperature range of the thermistors that did not freeze in and only recorded the borehole water temperature (as they did not freeze in before S1_5.0, the ice is inferred not to be colder than the ice around this thermistor; see Text). A dashed line indicates the melting- point temperature (Tm; see Methods). Thermistor naming convention is outlined in Fig. 2, and the thermistor colour-coding by depth matches that in Fig. 2. www.nature.com/scientificreports/ The temperature reversal at the base of the Site 2 borehole (Fig. 3b) may contradict the latter point if the temperature continues to decrease beyond our borehole depth. Indeed, similar minor reversals have been reported elsewhere, but with no explanation27. If this overturning is real, the mechanism remains to be explained, but is most likely to be related to the advection of a relatively cold ice layer. y y Assuming that the ice does remain temperate to the bed, we estimate that the CTS is at 20 m depth (or shallower) at Site 1, 31 m depth at Site 2, and 255 m at Site 3. This suggests a substantial layer of temperate ice, similar to results from lower-elevation valley glaciers elsewhere in the world17,27,29. The temperate layer on Khumbu Glacier would thus comprise ~56% of the ablation area ice volume (Site 3 to terminus). The accumulation area of Khumbu Glacier is above 6,200 m a.s.l. in the Western Cwm of Mt. Everest where the MAAT is below −9 °C31. Thus, ice is expected to form at or below −9 °C, as on the northeast side of Mt. Everest22. As this cold ice is advected downglacier, it will be warmed from below by geothermal heating, from above by warm air at lower elevations (the Khumbu ablation area is between ~5,300 and 4,850 m a.s.l.), and from within by deformation, refreezing and meltwater flow17,32,33. The emergence of ice in the lower part of the ablation area removes colder near-surface ice17. These processes explain the generally increasing ice temperatures downglacier and at depth (Fig. 3). h p p g y g p g p g Our near-surface thermistor located at Site 3 (S3_4.7) may be compared with the single nearby measurement of −5.3 °C recorded at 2.7 m depth and at a similar time of year to our measurements (late November, with bore- hole drilled in August) in 197418. As this measurement was taken in the shallow ice layer affected by seasonal tem- perature variations, a temperature recorded in November (shortly following the warm season24) likely represents close to the warmest temperature the ice reaches during the year. Our equivalent thermistor in this layer (S3_4.7) recorded a minimum temperature of −3.1 °C in late June and a maximum of −2.3 °C in late October at the end of the warm season (Fig. 2c). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Illustrative long profile of Khumbu Glacier showing the ice temperature values recorded by each thermistor within the three boreholes. The ice surface from the 2015 SETSM DEM50 was plotted along the approximate centreline of the glacier (blue line), with the modelled surface debris layer11 indicated by a grey line. A blue dashed line has been used to indicate the local borehole elevations in May 2017. It should be noted that the Site 1 borehole was drilled off the centreline, between two supraglacial ponds in a large surface depression. The bed depth was estimated using the SETSM DEM and modelled ice thicknesses11 and is plotted with a brown dotted line; the proglacial extent was interpolated between two measured points. It is not known whether the Site 1 borehole reached the bed. The blue dashed MAAT isotherms were calculated from the 1994– 2013 lapse rate up the Khumbu Valley31. The CTS, estimated from the thermistor data, is plotted as a red dotted line with cold ice above and temperate ice below. Figure 4. Illustrative long profile of Khumbu Glacier showing the ice temperature values recorded by each thermistor within the three boreholes. The ice surface from the 2015 SETSM DEM50 was plotted along the approximate centreline of the glacier (blue line), with the modelled surface debris layer11 indicated by a grey line. A blue dashed line has been used to indicate the local borehole elevations in May 2017. It should be noted that the Site 1 borehole was drilled off the centreline, between two supraglacial ponds in a large surface depression. The bed depth was estimated using the SETSM DEM and modelled ice thicknesses11 and is plotted with a brown dotted line; the proglacial extent was interpolated between two measured points. It is not known whether the Site 1 borehole reached the bed. The blue dashed MAAT isotherms were calculated from the 1994– 2013 lapse rate up the Khumbu Valley31. The CTS, estimated from the thermistor data, is plotted as a red dotted line with cold ice above and temperate ice below. Our ice temperature measurements reveal that Khumbu Glacier is polythermal; with cold ice in the upper part of the ablation area and temperate ice at depth in the lower part of the ablation area (Fig. 4). The coldest ice tem- peratures were measured near the surface, but beneath a seasonally influenced upper layer of ~10 m depth. Polythermal structure of a Himalayan debris-covered glacier revealed by borehole thermometry Katie E. Miles 1, Bryn Hubbard 1, Duncan J. Quincey 2, Evan S. Miles 2, Tenzing C. Sherpa3, Ann V. Rowan 4 & Samuel H. Doyle1 The grey bands mark the 10 m shallow ice layer that is expected to be influenced by seasonal variations in air temperature17. thermistors S1_15.0 and S1_45.3 is warmer than that around the uppermost thermistor at Site 1 (S1_5.0); if the deeper ice were as cold, these thermistors would have frozen in over a similar time frame. The potential temper- ature ranges for ice at the depths of these thermistors is indicated by the red range bars in Fig. 3a. All are warmer than −0.46 °C (S1_5.0), and we thus expect this ice to be very close to, or at, Tm 29.h The ice surrounding the uppermost thermistors at Sites 2 and 3 (S2_2.6, S3_4.7, and S2_12.6 to a much smaller degree) is influenced by seasonal air temperature variations, responding to rising air temperatures through the monsoon (the greater depth of S2_12.6 results in a lag, with warming only beginning in September). Therefore, despite the debris layer, at least the uppermost 10 m of the ablation area is notably influenced by seasonal surface temperatures17, similar to clean-ice glaciers23,30. The uppermost thermistor at Site 2 (S2_2.6) is most strongly sea- sonally influenced, reflecting its shallow location and thinner overlying debris layer (Fig. 1), showing a tempera- ture increase of ~1 °C from June to October (Fig. 2b). The sharp rise and more gradual fall in temperature towards the end of July is likely a result of water breaking into the borehole near this thermistor, possibly through a crack, and subsequently either seeping out the borehole or, more likely, cooling and freezing within it. SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 4 Methods ll Data collection - boreholes. Thirteen boreholes were drilled into Khumbu Glacier in May 2017 at three sites in the ablation area (Fig. 1) using a pressurised hot-water drilling system41 adapted for operation at high ele- vations. Sites were selected based on proximity to a water supply (a supraglacial pond) and a thin (<0.5 m) local debris layer that could be cleared prior to drilling. At Sites 1 (2 boreholes) and 2 (ten boreholes), drilling ceased due to the presence of debris in the borehole. At Site 1, we believe that the drill may have reached the bed, but this is difficult to confirm with the available observations. At Site 2, ten boreholes were drilled to 12–22 m depth at locations with surface elevation varying by ~10 m, suggesting a spatially extensive and possibly continuous debris layer beneath the surface. The borehole at Site 3 was drilled to the maximum length achievable (~155 m) with our equipment at 5,200 m elevation. Borehole inclinometry revealed that it was drilled off-vertical (reading a maxi- mum of 30° at the base). Thus, although the borehole length was 155 m, the depth of the borehole base was 132 m beneath the glacier surface. The sensor depths presented here have been corrected to reflect the true depth (rather than borehole length). Inclinometer data were not available for the boreholes at Sites 1 and 2, but the deviation at Site 3 is lower towards the surface so no thermistor at Sites 1 or 2 is likely to be more than ~1 m in error. Data collection - thermistors. The longest borehole at each site was selected to be instrumented with strings of thermistor sensors. The thermistor string contained negative temperature coefficient thermistors (Honeywell UNI-curve 192-502-LET-AOI) connected by a multicore cable, spaced more closely at depth. Higher up the cable, thermistors were spaced more evenly (typically 10–20 m apart) according to the expected length of the borehole which was based on measured42 and modelled11 ice thicknesses. Thermistor resistance was meas- ured every 10 minutes with Campbell Scientific CR1000 data loggers, using a half-bridge relative to a precision reference resistor with a low temperature coefficient (15 ppm/°C). Resistance was converted to temperature using a Steinhart and Hart43 polynomial fitted to the manufacturer’s calibration curve, with a further correction using a freezing-point offset for each thermistor obtained from an ice-bath calibration. www.nature.com/scientificreports/ penetrates crevasses and refreezes34, this might partially explain the presence of warmer ice at depth farther downglacier. Alternatively, amplified climate warming at high elevations1,2 may be penetrating deeper into the glacier. Since ice located above the CTS at Site 2 was within 0.8 °C of Tm, and at Site 1 all measurements were within 0.5 °C of Tm (Fig. 3), it is highly likely that towards the terminus the ice temperature is near, if not already at, Tm. This is despite the supraglacial debris layer acting to insulate shallow ice temperatures from atmospheric warming. However, the presence of supraglacial ponds35,36 hosting bare-ice cliffs, which are subject to thermal erosion37, appears to at least counteract the insulating effect of the surface debris layer at Khumbu Glacier. pp gf y Our measurements of the thermal regime of Khumbu Glacier have important implications for the future of Himalayan glaciers. Temperate shallow ice located near the terminus, where there are already increasingly large areas of supraglacial ponds36, could contribute to more rapid pond expansion29,38, increased ice mass loss and water storage within the supraglacial hydrological system39,40. If high melt rates continue, the CTS may become shallower as the volume of temperate ice expands, resulting in more energy absorption contributing directly to melt rather than warming cold ice. A layer of warm ice at depth, particularly if it extends to the bed, would allow a widespread englacial and/or subglacial drainage system to persist, potentially enhancing glacier velocity, ablation and water storage, all of which would influence downstream water delivery. gl y Our analysis of borehole-based ice temperatures within Khumbu Glacier indicates a polythermal regime with ~56% of the ice column being temperate in the lower 8 km of the ablation area. Even in the upper part of the ablation area, ice temperatures are no more than 3.5 °C colder than Tm, are up to 2 °C warmer than the MAAT and may be ~2 to 3 °C warmer than ~40 years ago. These data are the first of their kind for this region and for any debris-covered glacier, and will improve predictions of glacier response to climate change and their contribution to downstream water resources. The prevalence of temperate and warming ice at high elevations, even beneath thick supraglacial debris, indicates that these glaciers are highly vulnerable to 21st Century climate warming. www.nature.com/scientificreports/ Assuming similar seasonal surface heat transfer to ~5 m depth in both 1974 and 2017, comparison of these values could indicate a warming of the ice by ~2–3 °C in this area of the glacier in the 43 years between the observations. However, the difference could also be a result of differences in ice advection pathways between the sites, or measurement uncertainty (which is not presented for the Mae data).l Immediately below the uppermost layer, where ice is no longer influenced by seasonal surface temperatures, the undisturbed ice temperatures are warmer than the MAAT by as much as 2 °C (Fig. 4) suggesting additional warming of englacial ice beyond atmospheric heating inputs. Little is known about the thermodynamics of ice transport through icefalls, but if the primary stratification is deformed or latent energy released as meltwater SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 5 www.nature.com/scientificreports/ Methods ll The undisturbed ice temperature of this thermistor was calculated by using the following equation26,27 fitted to the raw thermistor data shown in Fig. 2a: π =    −   + T t Q k t s T ( ) 4 ( ) 0 where T is the borehole ice temperature at time t, Q is the heat released by drilling per unit length of the borehole; k is the thermal conductivity of pure ice at 0 °C (2.1 W m−1 K−1), T0 is the undisturbed ice temperature, and s is the time in seconds until the start of the freezing curve. Melting-point temperatures. The pressure-dependent melting-point temperature, Tm, was calculated at the depth of each thermistor using the Clausius-Clapeyron equation of melting-point depression27,46: γ ρ ρ = − − T T ( ) m tp tr where Ttp and ρtr are the triple point temperature (273.16 K) and the pressure of water (611.73 Pa) respectively, γ is the Clausius-Clapeyron constant and ρ is the ice overburden pressure, which can be approximated as: ρ ρ = g h i where ρi is the density of ice (900 kg m−3), g is the gravitational acceleration (9.81 m s−2) and h is the height of the overlying ice column given here by sensor depth (m). Values of the Clausius-Clapeyron constant range from that of pure (air-free) water/ice (0.0742 K MPa−1)30, a small content of soluble impurities and air within ice (0.079 K MPa−1)47 to that for pure ice and air-saturated water (0.098 K MPa−1)48. These values were all tested for the best match of each Tm to the freezing-point of each thermistor27: the constant for pure ice and air-saturated water provided the closest fit and was used in the analysis here. However, the theoretical Tm values still differed from the true freezing temperature of the thermistors by 0.04 to 1.1 °C, suggesting that a further factor depressed the freezing-point. One possible explanation is the presence of solutes and impurities within the ice, the concen- trations of which are currently unknown within Khumbu Glacier. Cold-temperate transition surface. The values of Tm were used along with the undisturbed ice temper- atures to estimate the CTS depth27,28,46. At Sites 2 and 3, a line of best fit through all the undisturbed thermistor values (as presented in Fig. 3) beneath the surface 10 m was extended until it intersected Tm. Methods ll Goodness-of-fit was calculated to be R2 = 0.9206 for Site 2, and R2 = 0.9996 for Site 3. The CTS is interpreted to occur at this inter- section of the extended best-fit line with Tm, and occurred at a depth of 255 m below the surface at Site 3, and 31 m below the surface at Site 2. To estimate the CTS depth at Site 1, a line of the same gradient as for Site 2 was extrapolated from the mid-point of the error bar for the first thermistor below the seasonally-affected shallow ice layer (S1_15.0). This line intersected Tm at 20 m depth. For reference, when a line of the same gradient as Site 3 was used, it intersected Tm at 30 m depth. a Availability datasets presented in this study are available for download from: https://doi.org/10.6084/m9.figshare.7165531.v1. The datasets presented in this study are available for download from: https://doi.org/10.6084/m9.figshare.7165531.v1. References 1. Kraaijenbrink, P. D. A., Bierkens, M. F. P., Lutz, A. F. & Immerzeel, W. W. Impact of a global temperature rise of 1.5 degrees Celsiu on Asia’s glaciers. Nature 549, 257–260 (2017). g 2. Pepin, N. et al. Elevation-dependent warming in mountain regions of the world. Nat. Clim. Chang. 5, 424–430 (2015) 2. Pepin, N. et al. Elevation-dependent warming in mountain regions of the world. Nat. Clim. Chang. 5, 424–430 (2015).h 3. Glen, J. The Creep of Polycrystalline Ice. Proc. R. Soc. A Math. Phys. Eng. Sci. 228, 519–538 (1955). h eertman, J. On the sliding of glaciers. J. Glaciol. 3, 33–38 (1957). g g 5. Irvine-Fynn, T. D. L., Hodson, A. J., Moorman, B. J., Vatne, G. & Hubbard, A. L. Polythermal Glacier Hydrology: A Review. Rev. Geophys. 49, 1–37 (2011).f p y . Immerzeel, W. W., van Beek, L. P. H. & Bierkens, M. F. P. Climate chan 6. Immerzeel, W. W., van Beek, L. P. H. & Bierkens, M. F. P. Climate change will affect the Asian water towers. Science. 328, 1382–1385 (2010) 7. Huss, M. & Hock, R. Global-scale hydrological response to future glacier mass loss. Nat. Clim. Chang. 8, 135–140 (2018).l f 7. Huss, M. & Hock, R. Global-scale hydrological response to future glacier mass loss. Nat. Clim. Chang. 8, 135–140 (2018). 8 R ttli S I l W W & P lli i tti F C t ti li t h i t i fl f hi h ltit d t h t i y g p g g 8. Ragettli, S., Immerzeel, W. W. & Pellicciotti, F. Contrasting climate change impact on river flows from high-altitude catchments in the Himalayan and Andes Mountains. Proc. Natl. Acad. Sci. USA 113, 9222–9227 (2016). y ( ) 9. Shea, J. M., Immerzeel, W. W., Wagnon, P., Vincent, C. & Bajracharya, S. Modelling glacier change in the Everest region, Nepa Himalaya. Cryosph. 9, 1105–1128 (2015).h y y p ( ) 0. Soncini, A. et al. Future hydrological regimes and glacier cover in the Everest region: The case study of the upper Dudh Koshi basin Sci. Total Environ. 1084–1101, https://doi.org/10.1016/j.scitotenv.2016.05.138 (2016).l 11. Rowan, A. V., Egholm, D. L., Quincey, D. J. & Glasser, N. F. Modelling the feedbacks between mass balance, ice flow and d transport to predict the response to climate change of debris-covered glaciers in the Himalaya. Earth Planet. Sci. Lett. 430, 427 (2015).l 12. Methods ll Previous studies using such ther- mistors44–46 suggest that with this secondary calibration, an accuracy of ±0.05 °C can be achieved. We therefore consider the thermistors to be accurate to ±0.05 °C at 0 °C, but accept that this value represents an indication of uncertainty rather than a maximum limit. y Uncertainty in vertical depth for each thermistor was estimated as the sum of error in the exact location of cable splicing (±0.2 m) and cable stretch upon lowering into each borehole (+0.5% of sensor depth). These are indicated in Fig. 3 as vertical error bars. The average depth uncertainty range was 0.66 m, with a maximum depth uncertainty range of 1.05 m (sensor S3_130.6). Undisturbed ice temperatures. The undisturbed ice temperature for each thermistor that froze in was estimated by taking the minimum of the running mean for one hour during the night (to avoid the slight noise-related influence from solar charging during the day), late in the time series (to ensure the settled tem- perature was as close to the true temperature as possible). Some thermistors, for example at Site 2 (Fig. 2b), still show a very slight cooling trend, but this is of the order of hundredths of a degree and is therefore not expected to significantly change the undisturbed ice temperatures we calculate. For the uppermost thermistors at these sites that were influenced by rising surface temperatures during the monsoon (S2_2.6 and S3_4.7), an hour during the night at the lowest point of the freezing curve was used, before the temperature began to rise. At Site 1, the CR1000 was detached from the thermistor string and removed from the site at the end of the May field season due to developing slope instability near the borehole, relating to pond expansion. During the return trip in October, an attempt was made to reconnect the strings to the CR1000, but the cable had been severed by debris and no further data were collected from Site 1. Only the surface-most thermistor at Site 1 (S1_5.0) froze into the borehole, but SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 6 www.nature.com/scientificreports/ the full settling curve was not captured before the CR1000 was removed. The undisturbed ice temperature of this thermistor was calculated by using the following equation26,27 fitted to the raw thermistor data shown in Fig. 2a: the full settling curve was not captured before the CR1000 was removed. www.nature.com/scientificreports/ Automated stereo-photogrammetric DEM generation at high latitudes: Surface Extraction from TIN-Based Search Minimization (SETSM) validation and demonstration over glaciated regions GIScience Remote Sens 52 198 217 (2015) 50. Noh, M. J. & Howat, I. M. Automated stereo-photogrammetric DEM generation at high latitudes: Surface Extraction from TIN-Based Search Minimization (SETSM) validation and demonstration over glaciated regions. GIScience Remote Sens. 52, 198–217 (2015). Search Minimization (SETSM) validation and demonstration over glaciated regions. GIScience Remote Sens. 52, 198 217 (2015). 51. RGI Consortium. Randolph Glacier Inventory - A Dataset of Global Glacier Outlines: Version 6.0: Technical Report, Global Land Ice Measurements from Space, Colorado, USA. Digit. Media, https://doi.org/10.7265/N5-RGI-60 (2017). ( ) g g R , ( ) 51. RGI Consortium. Randolph Glacier Inventory - A Dataset of Global Glacier Outlines: Version 6.0: Technical Report, Global Ice Measurements from Space, Colorado, USA. Digit. Media, https://doi.org/10.7265/N5-RGI-60 (2017). ( ) g g , ( ) 51. RGI Consortium. Randolph Glacier Inventory - A Dataset of Global Glacier Outlines: Version 6.0: Technical Report, Global Land Ice Measurements from Space, Colorado, USA. Digit. Media, https://doi.org/10.7265/N5-RGI-60 (2017). Acknowledgementsh g This research was supported by the ‘EverDrill’ Natural Environment Research Council Grant awarded to the Universities of Leeds and Sheffield (NE/P00265X) and Aberystwyth University (NE/P002021). K.M. is funded by an AberDoc PhD Scholarship (Aberystwyth University). The authors would like to thank Himalayan Research Expeditions for organising the logistics that supported fieldwork in Nepal during 2017, and in particular Mahesh Magar for guiding, navigation and general assistance with fieldwork, and Emily Potter for fieldwork support. We acknowledge the support of the Sagarmatha National Park and their assistance with permitting. www.nature.com/scientificreports/ Earth Sur 693–705 (2013). ( ) 28. Blatter, H. & Hutter, K. Polythermal conditions in Arctic glaciers. J. Glaciol. 37, 261–269 (1991). . Blatter, H. & Hutter, K. Polythermal conditions in Arctic glaciers. 9. Eisen, O., Bauder, A., Lüthi, M., Riesen, P. & Funk, M. Deducing the thermal structure in the tongue of Gornergletscher, Switzerland from radar surveys and borehole measurements. Ann. Glaciol. 50, 63–70 (2009).fh 29. Eisen, O., Bauder, A., Lüthi, M., Riesen, P. & Funk, M. Deducing the thermal structure in the t from radar surveys and borehole measurements. Ann. Glaciol. 50, 63–70 (2009).fh y . Cuffey, K. & Paterson, W. S. B. The Physics of Glaciers. (Butterwor y 30. Cuffey, K. & Paterson, W. S. B. The Physics of Glaciers. (Butterworth-Heinemann, 2010). f yh y f 31. Salerno, F. et al. Weak precipitation, warm winters and springs impact glaciers of south slop the last 2 decades (1994–2013). Cryosph. 9, 1229–1247 (2015). f yh y f 31. Salerno, F. et al. Weak precipitation, warm winters and springs impact glaciers of south slopes of Mt. Everest (central Himalay f yh y f alerno, F. et al. Weak precipitation, warm winters and springs impact glaciers of south slopes of Mt. Everest (central Himalaya) in e last 2 decades (1994 2013) Cryosph 9 1229 1247 (2015) 31. Salerno, F. et al. Weak precipitation, warm winters and springs im the last 2 decades (1994–2013). Cryosph. 9, 1229–1247 (2015). p p p g the last 2 decades (1994–2013). Cryosph. 9, 1229–1247 (2015). the last 2 decades (1994–2013). Cryosph. 9, 1229–1247 (2015). . Fisher, J. Internal temperatures of a cold glacier and conclusions t 32. Fisher, J. Internal temperatures of a cold glacier and conclusions therefrom. J. Glaciol. 18, 583–591 (1955).f 3. Phillips, T., Rajaram, H. & Steffen, K. Cryo-hydrologic warming: A potential mechanism for rapid thermal response of ice sheets Geophys. Res. Lett. 37, 1–5 (2010). 4. Lliboutry, B. L., Briat, M., Creseveur, M. & Pourchet, M. 15 m deep temperatures in the glaciers of Mont Blanc (French Alps). J Glaciol. 16, 197–203 (1976).h 5. Thompson, S. S., Benn, D. I., Dennis, K. & Luckman, A. A rapidly growing moraine-dammed glacial lake on Ngozumpa Glacier Nepal. Geomorphology 145–146, 1–11 (2012).h p p gy 6. Watson, C. S., Quincey, D. J., Carrivick, J. L. & Smith, M. W. The dynamics of supraglacial ponds in the Everest region, centra Himalaya. Glob. Planet. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 7. Blatter, H. On the thermal regime of an Arctic valley glacier: A study of White Glacier, Axel Heiberg Island, N.W.T., Canada. J Glaciol. 33, 200–211 (1987). Glaciol. 33, 200–211 (1987). 18 Mae S Wushiki H Ageta Y & Higuchi K Thermal Drilling and Temperature Measurements in Khumbu Glacier Nepal Glaciol. 33, 200 211 (1987). 18. Mae, S., Wushiki, H., Ageta, Y. & Higuchi, K. Thermal Drilling and Temperature Measurements in Khumbu Glacier, Nepal Measurements Himalayas J Japanese Soc Snow Ice 37 161–169 (1975) 18. Mae, S., Wushiki, H., Ageta, Y. & Higuchi, K. Thermal Drilling and Temperature Measurements in Khumbu Glacier, N Measurements Himalayas. J. Japanese Soc. Snow Ice 37, 161–169 (1975). y p 19. Mae, S. Ice Temperature of Khumbu Glacier. J. Japanese Soc. Snow Ice 38, 37–38 (1976). 0. Academia Sinica. Basic features of the glaciers of Mt. Jolmo Lungma Region, Southern part of the Tibet Autonomous Region, China Sci. Sin. 18, 106–130 (1975).l 1. Zhang, T. et al. Observed and modelled ice temperature and velocity along the main flowline of East Rongbuk Glacier, Qomolangma (Mount Everest), Himalaya. J. Glaciol. 59, 438–448 (2013). y 2. Hou, S. et al. Summer temperature trend over the past two millennia using air content in Himalayan ice. Clim. Past, Eur. Geosci Union 3, 89–95 (2007). 23. Liu, Y., Hou, S., Wang, Y. & Song, L. Distribution of borehole temperature at four high-altitude alpine glaciers in Central Asia. J. Mt. Sci. 6, 221–227 (2009). 4. Bollasina, M., Bertolani, L. & Tartari, G. Meteorological observations at high altitude in the Khumbu Valley, Nepal Himalayas 1994–1999. Bull. Glaciol. Res. 19, 1–11 (2002). 5. Shea, J. M. et al. A comparative high-altitude meteorological analysis from three catchments in the Nepalese Himalaya. Int. J. Wate Resour. Dev. 31, 174–200 (2015). ( ) 26. Humphrey, N. & Echelmeyer, K. Hot-water drilling and bore-hole closure in cold ice. J. Glaciol. 36, 287–298 (1990). 26. Humphrey, N. & Echelmeyer, K. Hot-water drilling and bore-h 26. Humphrey, N. & Echelmeyer, K. Hot-water drilling and bore-hole closure in cold ice. J. Glaciol. 36, 287–298 (1990). 27. Ryser, C. et al. Cold ice in the ablation zone: Its relation to glacier hydrology and ice water content. J. Geophys. Res. Earth S p y y g 27. Ryser, C. et al. Cold ice in the ablation zone: Its relation to glacier hydrology and ice water content. J. Geophys. Res. www.nature.com/scientificreports/ Change 142, 14–27 (2016).i 37. Miles, E. S. et al. Refined energy-balance modelling of a supraglacial pond, Langtang Khola, Nepal. Ann. Glaciol. 57, 29–40 (2016). 38 S k C R P i V Al ik A G S D & Sh h d M R l i i h C M i 37. Miles, E. S. et al. Refined energy-balance modelling of a supraglacial pond, Langtang Khola, Nepal. Ann. Glaciol. 57, 29–40 (2016). 38. Stokes, C. R., Popovnin, V., Aleynikov, A., Gurney, S. D. & Shahgedanova, M. Recent glacier retreat in the Caucasus Mountains, Russia and associated increase in supraglacial debris cover and supra /proglacial lake development Ann Glaciol 46 195 203 37. Miles, E. S. et al. Refined energy-balance modelling of a supraglacial pond, Langtang Khola, Nepal. Ann. Glaciol. 57, 29–40 (2016). 38. Stokes, C. R., Popovnin, V., Aleynikov, A., Gurney, S. D. & Shahgedanova, M. Recent glacier retreat in the Caucasus Mountains, Russia, and associated increase in supraglacial debris cover and supra-/proglacial lake development. Ann. Glaciol. 46, 195–203 (2007) i gy g p g p g g p 38. Stokes, C. R., Popovnin, V., Aleynikov, A., Gurney, S. D. & Shahgedanova, M. Recent glacier retreat in the Caucasus Mountains, Russia, and associated increase in supraglacial debris cover and supra-/proglacial lake development. Ann. Glaciol. 46, 195–203 (2007). 39. Miles, E. S. et al. Pond dynamics and supraglacial-englacial connectivity on debris-covered Lirung Glacier. Front. Earth Sci., ht doi.org/10.3389/feart.2017.00069 (2017).f g 40. Irvine-Fynn, T. D. L. et al. Supraglacial Ponds Regulate Runoff From Himalayan Debris-Covered Glaciers. Geophys. Res. Lett. 44 894–11,904 (2017). . Hubbard, B. & Glasser, N. Field Techniques in Glaciology and Glac q gy p gy y 42. Gades, A., Conway, H., Nereson, N., Naito, N. & Kadota, T. In Debris-Covered Glaciers (eds Nakawo, M., Raymond, C. F. & Foun 42. Gades, A., Conway, H., Nereson, N., Naito, N. & Kadota, T. In Debris-Covered Glaciers ( A.) 264, 13–22 (International Association of HydrologicalSciences, 2000). 42. Gades, A., Conway, H., Nereson, N., Naito, N. & Kadota, T. In Debris-Covered Glaciers (eds Nakawo, M., Raymond, C. F. & Fountain, A.) 264, 13–22 (International Association of HydrologicalSciences, 2000). 42. Gades, A., Conway, H., Nereson, N., Naito, N. & Kadota, T. In Debris Covered Glaciers (eds Nakawo, M., Raymond, C. F. & Fou A.) 264, 13–22 (International Association of HydrologicalSciences, 2000). Steinhart, J. S. & Hart, S. R. Calibration curves for thermistors. D.Q. conceived of and led the EverDrill project. B.H. and A.R. co-led the project. B.H. and S.D. designed, and K.M. and E.M. assisted in building the borehole sensors. B.H. led the hot-water drilling. K.M., B.H., D.Q., E.M. and T.S. carried out the data collection in the field. K.M. processed the data and wrote the manuscript. All authors contributed to the data analysis and editing of the manuscript. References Egholm, D. L., Pedersen, V. K., Knudsen, M. F. & Larsen, N. K. Coupling the flow of ice, water, and sediment in a glacial landscape evolution model. Geomorphology 141–142, 47–66 (2012).h p gy ( ) 13. Østrem, G. Ice Melting under a Thin Layer of Moraine, and the Existence of Ice Cores in Moraine Ridges. Geogr. Ann. 41, 228–230 (1959).h 4. Thompson, S., Benn, D. I., Mertes, J. & Luckman, A. Stagnation and mass loss on a Himalayan debris-covered glacier: Processes patterns and rates. J. Glaciol. 62, 467–485 (2016).i 15. Quincey, D. J., Luckman, A. & Benn, D. Quantification of Everest region glacier velocities between 1992 and 2002, using satellite radar interferometry and feature tracking. J. Glaciol. 55, 596–606 (2009). y g 16. Benn, D. I. et al. Structure and evolution of the drainage system of a Himalayan debris-covered glacier, and its relationship with patterns of mass loss. Cryosph. 11, 2247–2264 (2017). SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 7 www.nature.com/scientificreports/ Deep J p g ( ) 44. Iken, A., Echelmeyer, K., Harrison, W. & Funk, M. Mechanisms of fast flow in Jakobshavns Isbrae, West Greenla M t f t t d t l l i d b h l J Gl i l 39 (1993) 44. Iken, A., Echelmeyer, K., Harrison, W. & Funk, M. Mechanisms of fast flow in Jako Measurements of temperature and water level in deep boreholes. J. Glaciol. 39 (1993).l , , y , , , , J Measurements of temperature and water level in deep boreholes. J. Glaciol. 39 (1993).l R. Temperature of a ‘temperate’ alpine glacier: Glacier de Tsanfleuro 45. Bayley, O. D. R. Temperature of a ‘temperate’ alpine glacier: Gla lh 46. Doyle, S. H. et al. Physical Conditions of Fast Glacier Flow: 1. Measurements From Boreholes Drilled to the Bed of Store Glacier, West Greenland. J. Geophys. Res. Earth Surf. 123 (2018).fl lh 46. Doyle, S. H. et al. Physical Conditions of Fast Glacier Flow: 1. Measurements From Boreholes Drilled to the Bed of S West Greenland. J. Geophys. Res. Earth Surf. 123 (2018).fl West Greenland. J. Geophys. Res. Earth Surf. 123 (2018). p y f 47. Lüthi, M., Funk, M., Iken, A., Gogineni, S. & Truffer, M. Mechanisms of fast flow in Jakobshavn Isbræ, West Greenland: Part III. Measurements of ice deformation, temperature and cross-borehole conductivity in boreholes to the bedrock. J. Glaciol. 48, 369–385 (2002). 47. Lüthi, M., Funk, M., Iken, A., Gogineni, S. & Truffer, M. Mechanisms of fast flow in Jakobshavn Isbræ, West Greenland: Part III. Measurements of ice deformation, temperature and cross-borehole conductivity in boreholes to the bedrock. J. Glaciol. 48, 369–385 (2002). ( ) 48. Harrison, W. D. Temperature of a temperate glacier. J. Glaciol. 11, 15–29 (1972). rrison, W. D. Temperature of a temperate glacier. J. Glaciol. 11, 15– 49. Planet Team Planet Application Program Interface: In Space for Life on Earth. San Francisco, CA, https://api.planet.com (2017 50 N h M J & H I M A d h i DEM i hi h l i d S f E i f TIN B 49. Planet Team Planet Application Program Interface: In Space for Life on Earth. San Francisco, CA, h 49. Planet Team Planet Application Program Interface: In Space for Life on Earth. San Francisco, CA, https://api.planet.com (2017). 50. Noh, M. J. & Howat, I. M. Author Contributions D.Q. conceived of and led the EverDrill project. B.H. and A.R. co-led the project. B.H. and S.D. designed, and K.M. and E.M. assisted in building the borehole sensors. B.H. led the hot-water drilling. K.M., B.H., D.Q., E.M. and T.S. carried out the data collection in the field. K.M. processed the data and wrote the manuscript. All authors contributed to the data analysis and editing of the manuscript. 8 SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 www.nature.com/scientificreports/ Additional Informationh Competing Interests: The authors declare no competing interests. SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCiENtifiC REPOrTS \| (2018) 8:16825 \| DOI:10.1038/s41598-018-34327-5 9
https://openalex.org/W3157534524	https://www.frontiersin.org/articles/10.3389/fphar.2021.651790/pdf	English	null	Current Bioequivalence Study Designs in South Korea: A Comprehensive Analysis of Bioequivalence Study Reports Between 2013 and 2019	Frontiers in pharmacology	2,021	cc-by	5,404	Keywords: bioequivalence, regulations, generic drug, Intrasubject coefﬁcient of variation, biopharmaceutics classiﬁcation system Current Bioequivalence Study Designs in South Korea: A Comprehensive Analysis of Bioequivalence Study Reports Between 2013 and 2019 Ki Young Huh 1, Eunwoo Kim 1, Soyoung Lee 1, Hyounggyoon Yoo 2, Seonghae Yoon 3, 1Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Hospital, Seoul, South Korea, 2Department of Clinical Pharmacology and Therapeutics, CHA University Bundang Medical Center, CHA University School of Medicine, Gyeonggi-do, South Korea, 3Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine and Bundang Hospital, Gyeonggi-do, South Korea Demonstration of bioequivalence (BE) is mandatory while developing generic drugs. The scientiﬁc concept of BE applies equally to different regulatory agencies. However, the application of the concept may differ for each agency, which can affect the design of BE studies. To evaluate the study practices in terms of the BE concept in South Korea, we retrospectively analyzed BE study reports available from Ministry of Food and Drug Safety between 2013 and 2019. Statistical estimation of the pharmacokinetic parameters, including peak concentration and area under the concentration–time curve to the last measurable concentration, as well as study design, number of subjects in a study, study duration, fasting status, and formulation of speciﬁc drugs were obtained. The drugs were classiﬁed per World Health Organization Anatomical Therapeutic Chemical Classiﬁcation and Biopharmaceutics Classiﬁcation System. Post-hoc intrasubject coefﬁcient of variation and corresponding sample sizes were calculated from the 90% conﬁdence intervals of pharmacokinetic parameters. A total of 143 generic drugs in 588 BE studies were analyzed. The largest number of studies were performed in the area of Cardiovascular system (172 studies), followed by Nervous system (143 studies) and Alimentary tract and metabolism (92 studies). Overall, BE studies in South Korea were conducted in accordance with the global guideline despite the differences in details. BE studies were focused on the several therapeutic areas and conducted in a similar manner. The number of subjects was generally larger than that estimated with 90% power. Edited by: Sandor Kerpel-Fronius, Semmelweis University, Hungary y g y Reviewed by: Anna Bucsics, Federal Administrative Court of Austria, Austria Jordi Ocaña, University of Barcelona, Spain Correspondence: Jae-Yong Chung mekka@snu.ac.kr jychung@snubh.org Reviewed by: Anna Bucsics, Federal Administrative Court of Austria, Austria Jordi Ocaña, University of Barcelona, Spain Correspondence: Jae-Yong Chung mekka@snu.ac.kr jychung@snubh.org Specialty section: This article was submitted to Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Received: 11 January 2021 Accepted: 31 March 2021 Published: 04 May 2021 Specialty section: This article was submitted to Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Pharmaceutical Medicine and Outcomes Research, a section of the journal Frontiers in Pharmacology Received: 11 January 2021 Accepted: 31 March 2021 Published: 04 May 2021 Edited by: Edited by: Sandor Kerpel-Fronius, Semmelweis University, Hungary ORIGINAL RESEARCH published: 04 May 2021 doi: 10.3389/fphar.2021.651790 Data Collection BE study reports from March 2013 to November 2019 were collected from the public database of Ministry of Food and Drug Safety and analyzed retrospectively. In South Korea, the BE study reports are provided only for generic drugs that have demonstrated BE. All studies were conducted in accordance with Standard on Pharmaceutical Equivalence Study and were approved by Ministry of Food and Drug Safety. As one of the ICH members, South Korea follows global standards for BE (Davit et al., 2013). BE studies in South Korea are regulated by Ministry of Food and Drug Safety under Standard on Pharmaceutical Equivalence Test (Ministry of Food and Drug Safety, 2014). Currently, the Ministry of Food and Drug Safety only accepts domestic BE study results (Ministry of Food and Drug Safety, 2018b). Furthermore, generic products approved in other countries are required to submit BE study results from the authorized study centers in South Korea for approval (Ministry of Food and Drug Safety, 2018b). Following statistics for PK parameters were obtained from the BE study reports for test and reference products: Following statistics for PK parameters were obtained from the BE study reports for test and reference products: • Maximum plasma concentration (Cmax): mean and standard deviation, test-to-reference geometric mean ratio (GMR), and 90% conﬁdence interval (CI) The standard for BE studies in South Korea has been comprehensively reformed since 2006 to be at par with the global standards (Ryu and Kim, 2017). However, speciﬁc regulatory requirements are either absent or different from those of other countries. Several important criteria for evaluation of BE in South Korea are as follow (Ministry of Food and Drug Safety, 2018a): • Area under the concentration–time curve from zero to the last measurable point (AUClast): mean and standard deviation, test-to-reference GMR, and 90% CI • Elimination half-life (t1/2): mean and standard deviation • Time to reach Cmax (Tmax): median, minimum, and maximum • When blood samples are used, the comparative evaluation parameters include AUCt and Cmax in a single dose study, and AUCτ and Css,max in a multiple-dose study. [Article 17 (Lee et al., 2016)] The study information obtained from the reports included design (e.g., 2 × 2, 2 × 2 × 4), fasting status, study duration (duration up to the last PK sampling point), and the number of subjects analyzed. Brand names for test and reference drugs were obtained and standardized to generic names. INTRODUCTION Huh KY, Kim E, Lee S, Yoo H, Yoon S, Yu K-S and Chung J-Y (2021) Current Bioequivalence Study Designs in South Korea: A Comprehensive Analysis of Bioequivalence Study Reports Between 2013 and 2019. Front. Pharmacol. 12:651790. doi: 10.3389/fphar.2021.651790 Huh KY, Kim E, Lee S, Yoo H, Yoon S, Yu K-S and Chung J-Y (2021) Current Bioequivalence Study Designs in South Korea: A Comprehensive Analysis of Bioequivalence Study Reports Between 2013 and 2019. Front. Pharmacol. 12:651790. doi: 10.3389/fphar.2021.651790 Development of generic drugs is one of the effective strategies to increase patient access to therapeutic drugs. Regulatory agencies have adopted an abbreviated approval process for generic drugs (Sravika et al., 2017). Demonstration of bioequivalence (BE) is required for approval of generic drugs, instead of repeating clinical trials on safety and efﬁcacy (Lee et al., 2016). As a result of this abbreviated approval process, generic drugs can be supplied at lower cost. For instance, generic drug Development of generic drugs is one of the effective strategies to increase patient access to therapeutic drugs. Regulatory agencies have adopted an abbreviated approval process for generic drugs (Sravika et al., 2017). Demonstration of bioequivalence (BE) is required for approval of generic drugs, instead of repeating clinical trials on safety and efﬁcacy (Lee et al., 2016). As a result of this abbreviated approval process, generic drugs can be supplied at lower cost. For instance, generic drug May 2021 \| Volume 12 \| Article 651790 Frontiers in Pharmacology \| www.frontiersin.org Huh et al. Bioequivalence Study Designs in Korea accounted for only 27% of total prescription costs in the United States despite a large proportion (89%) in the total prescription cost (Howard et al., 2018). • Blood collection shall be conducted with sufﬁcient time period of more than 3 times the elimination half-life or AUC0-t to reach at least 80% of AUC∞. [Article 15 (Lee et al., 2016)] • Blood collection shall be conducted with sufﬁcient time period of more than 3 times the elimination half-life or AUC0-t to reach at least 80% of AUC∞. [Article 15 (Lee et al., 2016)] The scientiﬁc concept of BE is deﬁned uniformly across various regulatory agencies (Chen et al., 2018). INTRODUCTION BE is achieved when the bioavailabilities of two drugs “lie within acceptable predeﬁned limits” to ensure “similarity in terms of safety and efﬁcacy” (European Medicines Agency, 2010), thus demonstrating “the absence of signiﬁcant difference in the rate and extent of absorption under similar experimental conditions” (U.S. Food and Drug Administration, 2013; Cristofoletti et al., 2018). BE can be demonstrated in vivo and in vitro (Chow, 2014), although in vitro assessment has limited acceptance, i.e., only for drugs with high solubility and permeability (Cristofoletti et al., 2013). A standard approach for demonstrating BE is a two-way crossover (2 × 2) clinical trial conducted in healthy subjects (Chow, 2014). • The number of subjects shall be based on an appropriate sample size calculation and may be added or subtracted depending on types and characteristics of active ingredients. The number, in principle, shall be at least 12 or more. (Article 13) • The number of subjects shall be based on an appropriate sample size calculation and may be added or subtracted depending on types and characteristics of active ingredients. The number, in principle, shall be at least 12 or more. (Article 13) (Abbreviations: AUC∞, area under the drug concentration in blood-time curve from zero to inﬁnity; AUCt, area under the drug concentration in blood-time curve from zero to the ﬁnal sampling time t; AUCτ, area under the drug concentration in blood-time curve over one dose interval at steady-state; Cmax, the maximum drug concentration in blood; Css,max, The maximum drug concentration in blood at steady state; Tmax, Time to the maximum drug concentration in blood.) Although the concept of BE is accepted globally, regulatory requirements and standards for BE are not consistent among countries (Davit et al., 2013; Kaushal et al., 2016; Chen et al., 2018). The difference is observed even among the members of International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) (Davit et al., 2013; Kuribayashi et al., 2016). The difference lies in terms of recommended study design, method for pharmacokinetic (PK) parameter estimation, and modiﬁcation of BE criteria for highly variable drugs (Davit et al., 2013). In the light of the global concept of BE, we evaluated the BE studies conducted in South Korea by retrospectively analyzing the BE study reports available from Ministry of Food and Drug Safety between 2013 and 2019. RESULTS MSE 2 · ⎛ ⎜ ⎜ ⎜ ⎝ Δ 1 n1 + 1 n2 · t1−2α,n1+n2−2 ⎞⎟⎟⎟⎠ 2 MSE 2 · ⎛ ⎜ ⎜ ⎜ ⎝ Δ 1 n1 + 1 n2 · t1−2α,n1+n2−2 ⎞⎟⎟⎟⎠ 2 Data Collection BCS classiﬁcation was deﬁned as below (Löbenberg and Amidon, 2000): Intrasubject CV (%) 100 · eMSE −1 √ (t: t-values of the student t-distribution, α: probability of type I error (assumed 0.05), n1, n2: sample sizes of each group). The second step was the pooling and conversion of the estimated intrasubject CVs to the corresponding sample sizes. The intrasubject CV was pooled for drugs with a different formulation or fasting status (e.g., metformin hydrochloride, metformin hydrochloride ER, and metformin hydrochloride ER (fed) were calculated separately) using a method described previously (Chung et al., 2018). Estimation of the sample sizes was performed with the following criteria: • BCS class I: high solubility, high permeability • BCS class II: low solubility, high permeability • BCS class III: high solubility, low permeability • BCS class IV: low solubility, low permeability • Signiﬁcance level (α): 0.05 Data Collection Additionally, information was collected regarding the dose strength, ﬁxed-dose combination, modiﬁed-release dosage form [e.g., extended-release (ER), controlled-release (CR) form], and route of administration other than oral administration (e.g., patch). • When log transformation and statistical evaluation on comparative parameters of the reference and test product except Tmax are performed, the 90% conﬁdence intervals for the difference in mean values between the test and reference should be within log 0.8 to log 1.25. [Article 17 (Howard et al., 2018)] May 2021 \| Volume 12 \| Article 651790 Frontiers in Pharmacology \| www.frontiersin.org 2 Huh et al. Bioequivalence Study Designs in Korea FIGURE 1 \| The number of subjects compared with the estimated sample size. (A) Distribution of the actual and number of subjects with post-hoc estimation with 80 and 90% powers. (B) Maximum estimated post-hoc coefﬁcient of variation (CV) vs. the actual number of subjects. Dots represent the actual number of subjects. The estimated numbers of subjects are presented as solid lines. (Notes: The studies in which the estimated number of subjects was >120, the number was reduced to 120 for visualization. For ﬁxed-dose combination, the active pharmaceutical ingredient with the highest maximum CV was selected for analysis. Only 2 × 2 bioequivalence (BE) trials were analyzed.). FIGURE 1 \| The number of subjects compared with the estimated sample size. (A) Distribution of the actual and number of subjects with post-hoc estimation with 80 and 90% powers. (B) Maximum estimated post-hoc coefﬁcient of variation (CV) vs. the actual number of subjects. Dots represent the actual number of subjects. The estimated numbers of subjects are presented as solid lines. (Notes: The studies in which the estimated number of subjects was >120, the number was reduced to 120 for visualization. For ﬁxed-dose combination, the active pharmaceutical ingredient with the highest maximum CV was selected for analysis. Only 2 × 2 bioequivalence (BE) trials were analyzed ) Intrasubject CV (%) 100 · eMSE −1 √ Each generic drug corresponded to one BE study. Each drug was classiﬁed per World Health Organization Anatomical Therapeutic Chemical Classiﬁcation system. Active pharmaceutical ingredients for ﬁxed-dose combination products were classiﬁed separately for components. In addition, drugs were classiﬁed by Biopharmaceutics Classiﬁcation System (BCS) based on solubility and permeability reported in previous literature. Statistical Analysis • Power (1 −β): 80 and 90% (calculated separately) • Treatment to reference ratio: 1.05 (based on the convention) (Ring et al., 2019) Statistical analysis of the BE results was conducted in two steps. First, post-hoc estimation of the intrasubject coefﬁcient of variation (CV) for Cmax and AUClast was conducted for the number of subjects, and 90% conﬁdence intervals were determined for each study. The point estimate (PE) of each PK parameter was calculated as the geometric mean of the lower limit (CIlower) and upper limit (CIupper) of the conﬁdence interval ( CIlower · CIupper ) considering the log- transformation recommended in BE analysis. Margin of error on a log scale was calculated as the difference of the natural logarithm of PE and the lower limit of the CI [Δ ln(PE) −ln(CIlower)]. Mean squared error (MSE) and intrasubject CV were calculated using the following equations (Chung et al., 2018): • Intrasubject CV: maximum of pooled intrasubject CV of Cmax or AUClast R version 3.6.3. (R Core Team, Vienna, Austria) and statistical package “PowerTOST” were used (Labes et al., 2020) to estimate the post-hoc intrasubject CV of the Cmax and AUClast and to estimate the sample size with the intrasubject CV. Overall Characteristics A total of 143 generic drugs evaluated in 588 BE studies were included in the analysis (Supplementary Table S1). Considering May 2021 \| Volume 12 \| Article 651790 Frontiers in Pharmacology \| www.frontiersin.org 3 Huh et al. Huh et al. Bioequivalence Study Designs in Korea FIGURE 2 \| Interstudy variability of the number of subjects. Dots represent the median number of subjects in each drug, and horizontal lines represent the minimum and maximum number of subjects. The difference between the minimum and maximum number of subjects [the number of bioequivalence (BE) studies] is presented in the text. ATC classiﬁcation for each drug is provided with colors (Notes: Drugs with more than three BE studies were selected. Fixed-dose combination drugs were excluded from the analysis. Only 2 × 2 BE trials are analyzed.). FIGURE 2 \| Interstudy variability of the number of subjects. Dots represent the median number of subjects in each drug, and horizontal lines represent the minimum and maximum number of subjects. The difference between the minimum and maximum number of subjects [the number of bioequivalence (BE) studies] is presented in the text. ATC classiﬁcation for each drug is provided with colors (Notes: Drugs with more than three BE studies were selected. Fixed-dose combination drugs were excluded from the analysis. Only 2 × 2 BE trials are analyzed.). FIGURE 2 \| Interstudy variability of the number of subjects. Dots represent the median number of subjects in each drug, and horizontal lines represent the minimum and maximum number of subjects. The difference between the minimum and maximum number of subjects [the number of bioequivalence (BE) studies] is presented in the text. ATC classiﬁcation for each drug is provided with colors (Notes: Drugs with more than three BE studies were selected. Fixed-dose combination drugs were excluded from the analysis. Only 2 × 2 BE trials are analyzed.). May 2021 \| Volume 12 \| Article 651790 Frontiers in Pharmacology \| www.frontiersin.org 4 Bioequivalence Study Designs in Korea Huh et al. Huh et al. FIGURE 3 \| Ratio of study duration to half-life versus grouped half-life. Dotted lines represent recommended minimum ratio of study duration to half-life in South Korea. (Note: Ratios > 32 were reduced to 32 for visualization. For ﬁxed dose combination, drug with the highest maximum coefﬁcient of variation was selected.) studies). Most of the BE studies were conducted in the fasted state (565 studies in fasted status vs. Number of Subjects The distribution of actual number of subjects differed from the estimated post-hoc sample sizes (Figure 1A). The actual number of subjects centered between 24 and 40. Most studies were conducted with more subjects than the sample sizes estimated with 90% power (Figure 1B). For several drugs, the number of subjects was variable among studies (Figure 2). The studies on ezetimibe (n 4) exhibited the highest difference of 39 subjects, whereas the studies on apixaban (n 3) had the same number of subjects. Therapeutic area with the most types of drugs was the Nervous system (17 drugs), followed by Alimentary tract and metabolism (7 drugs) and the Cardiovascular system (5 drugs). BE studies with 2 × 2 × 4 design were separately analyzed (Supplementary Table S2 and Supplementary Figure S1) and ﬁxed-dose combination drugs were excluded for clarity. Intrasubject CVs for Cmax of studies with 2 × 2 × 4 design were larger than 30% except for two studies. FIGURE 3 \| Ratio of study duration to half-life versus grouped half-life. Dotted lines represent recommended minimum ratio of study duration to half-life in South Korea. (Note: Ratios > 32 were reduced to 32 for visualization. For ﬁxed dose combination, drug with the highest maximum coefﬁcient of variation was selected.) the formulation and fasting status, in total 171 combinations were present, of which the intrasubject CVs were calculated for both the Cmax and AUClast. Therapeutic area with the most generic drugs was the Nervous system (39 drugs), followed by the Alimentary tract and metabolism (22 drugs) and the Cardiovascular system (21 drugs). Therapeutic area with the largest number of studies was the Cardiovascular system (172 studies), followed by the Nervous system (143 studies) and Alimentary tract and metabolism (92 Overall Characteristics 23 studies in fed status). Fixed-dose combination accounted for one fourth of the total studies (155 studies) for 24 generic drugs. The formulation with the greatest number of generic drugs was the immediate-release formulation (131 drugs). Other formulations included ER (12 drugs), orodispersible (4 drugs), patch (2 drugs), power (2 drugs), CR (1 drug) and sublingual formulation (1 drug). All studies were conducted with a single- dose administration. Most studies were conducted in 2 × 2 design (139 drugs, 568 studies) and the others were conducted in 2 × 2 × 4 design (7 drugs, 20 studies). Study Duration Relative to Half-Life of the Reference Drug Most of the drugs exhibited terminal half-lives of less than 24 h. Study duration ranged up to 144 h and exhibited discrete distribution with an interval of 6 or 12 h. Study FIGURE 4 \| Coefﬁcient of variation (CV) by Biopharmaceutics Classiﬁcation System (BCS) classiﬁcation. (A) Scatter plot and (B) box plot for pooled CV of Cmax and AUClast by BCS classiﬁcation. Black solid line represents line of unity, while dotted line represents CV criterion of the highly variable drugs. FIGURE 4 \| Coefﬁcient of variation (CV) by Biopharmaceutics Classiﬁcation System (BCS) classiﬁcation. (A) Scatter plot and (B) box plot for pooled CV of Cmax and AUClast by BCS classiﬁcation. Black solid line represents line of unity, while dotted line represents CV criterion of the highly variable drugs. May 2021 \| Volume 12 \| Article 651790 5 Frontiers in Pharmacology \| www.frontiersin.org Bioequivalence Study Designs in Korea Huh et al. duration for drugs with short terminal half-lives (<24 h) was longer than three-fold of half-lives of the reference drugs. Study duration for drugs with long terminal half-lives was two- or three-fold of the half-lives of the reference drugs (Figure 3). Additionally, study duration was affected by regulatory requirements. As per the current guideline, AUClast should account for at least 80% of AUC extrapolated to inﬁnity. In addition, study duration can be reduced to 72 h when a drug has a long half-life and intrasubject variability of clearance is low (Ministry of Food and Drug Safety, 2018a). In several studies, study duration was shorter than three-fold of terminal half-life. Most cases pertained to the reduced duration of 72 h, when the drugs had a long half-life. However, eight cases that did not have a drug witlong half- life had a shorter study duration. The variability of reported terminal half-life of the reference drug or effective half-life might be the possible cause (Boxenbaum and Battle, 1995). CONCLUSION BE studies in South Korea were conducted in accordance with the global guideline despite the differences in details. BE studies were focused on the several therapeutic areas and conducted in similar manner. The number of subjects was generally larger than that estimated with 90% power. We observed that conducting BE studies in South Korea is affected by speciﬁc regulatory requirements. The current BE guidelines in South Korea mandate that the minimum number of subjects in a BE study should be >12 (Ministry of Food and Drug Safety, 2018a), which was amended in September 2014 from the previous requirement of the minimum number of 12 subjects in each sequence (Ministry of Food and Drug Safety, 2014). This could affect the number of subjects for the drugs with low intrasubject CV, such as amlodipine besylate (mean CV for Cmax: 10.4%; AUClast: 9.4%); this drug reported the lowest number of subjects (15 subjects). DISCUSSION In South Korea, the evaluated BE studies were performed in a similar manner. Most of the reference drugs were immediate- release oral formulations and administered in the fasted state. Therapeutic areas were focused on the Cardiovascular system, Nervous system, and Alimentary tract and metabolism, which collectively accounted for more than half of the total studies. The standard two-way crossover (2 × 2) design was adopted except for few drugs (azathioprine, carbidopa, entacapone, eperisone, naftopidil, R-thioctic acid tromethamine, and telmisartan), which adopted replicated crossover (2 × 2 × 4) design. Parallel design or partially replicated design (2 × 3 × 3) were not found. However, the intrasubject CV for most drugs was comparable with those reported in earlier studies. This can be supported by the fact that formulation-by-formulation effect was relatively lower than intrasubject variability (Yu et al., 2016). Furthermore, correlation between BCS class and intrasubject CV was comparable with that reported from 113 generic drugs (Sugihara et al., 2015). This comparability would support the extrapolation of intrasubject CVs reported in our study to other jurisdictions. Nonetheless, the ethnic sensitivity of BE results also needs to be investigated for extrapolation (Ozdin et al., 2020). Our study had some limitations. The BE study reports provided by Ministry of Food and Drug Safety only included drugs that demonstrated BE, which can be a possible source of publication bias. In addition, the study reports did not include the detailed features of the study design including sampling points and safety assessments. This limited further analysis of the BE studies. Thus, further investigations on the source of interstudy variability are warranted. The number of subjects mostly ranged between 24 and 40. More than half of the studies enrolled larger number of subjects than that estimated retrospectively with 90% power. In other words, the number of subjects was determined conservatively. Sample size is determined by intrasubject CV and the estimates of CV are usually referred from the previous studies. The determination of a conservative number of subjects might be attributable to higher estimated intrasubject CV. Furthermore, highly set dropout rates, which are empirically determined by the investigators, may contribute to the larger number of subjects. Coefﬁcient of Variation by Biopharmaceutics Classiﬁcation System Classiﬁcation Pooled intrasubject CV calculated from Cmax was larger than that from AUClast (Figure 4A). BCS class I drugs (high solubility, high permeability) exhibited the least intrasubject CV and Cmax exhibited a larger intrasubject CV than AUClast in most cases except for oseltamivir (mean intrasubject CV for Cmax 37.0%), hydroxychloroquine sulfate (Cmax 42.6%, AUClast 37.6%), and sildenaﬁl citrate (Cmax 33.3%). BCS class II and IV drugs (low solubility) accounted for most of the highly variable drugs (intrasubject CV for Cmax > 30%) (Figure 4B). Intrasubject CV exhibited some variability among studies. Besides the intrinsic randomness of the intrasubject CVs, different assay methods or distributions of baseline characteristics, such as demographics or genetic polymorphisms in drug metabolizing enzymes could be considered as sources of variability (Bebia et al., 2004). Several drugs exhibited intrasubject CV different than that reported in earlier studies. For example, contrary to the reported high variability of levothyroxine in a previous study (Meredith, 2003), intrasubject CV of levothyroxine was signiﬁcantly low (7.1–11.5%) in our data. This might be attributable to the factors other than the randomness of the intrasubject CVs. Frontiers in Pharmacology \| www.frontiersin.org REFERENCES Perception and attitude of Korean physicians towards generic drugs. BMC Health Serv. Res. 17, 610. doi:10.1186/s12913-017-2555-y Sravika, S., Bhavana, R., Sharmila, V., Anusha, S., Mounica, N. V. N., Nagarjuna Reddy, D., et al. (2017). A comprehensive study on regulatory requirements for development and ﬁling of generic drugs globally. Pharma Innovation 6, 153–158. doi:10.4103/2230-973X.104392 Davit, B., Braddy, A. C., Conner, D. P., and Yu, L. X. (2013). International guidelines for bioequivalenceofsystemicallyavailableorallyadministeredgenericdrugproducts:asurvey of similarities and differences. AAPS J. 15, 974–990. doi:10.1208/s12248-013-9499-x European Medicines Agency (2010). Guideline on the investigation of bioequivalence [Internet]. Available at: https://www.ema.europa.eu/en/ investigation-bioequivalence (Accessed August 01, 2010). Sugihara, M., Takeuchi, S., Sugita, M., Higaki, K., Kataoka, M., and Yamashita, S. (2015). Analysis of intra- and intersubject variability in oral drug absorption in human bioequivalence studies of 113 generic products. Mol. Pharm. 12, 4405–4413. doi:10.1021/acs.molpharmaceut.5b00602 Howard, J. N., Harris, I., Frank, G., Kiptanui, Z., Qian, J., and Hansen, R. (2018). Inﬂuencers of generic drug utilization: a systematic review. Res. Soc. Administrative Pharm. 14, 619–627. doi:10.1016/j.sapharm.2017.08.001 U.S. Food and Drug Administration (2013). Bioequivalence studies with pharmacokinetic endpoints for drugs submitted under an abbreviated new drug application [internet]. Available at: https://www.fda.gov/regulatory-information/ search-fda-guidance-documents/bioequivalence-studies-pharmacokinetic-endpoints- drugs-submitted-under-abbreviated-new-drug (Accessed December 05, 2013). Kaushal, N., Singh, S., Gulati, M., Vaidya, Y., and Kaushik, M. (2016). Study of regulatory requirements for the conduct of bioequivalence studies in US, Europe, Canada, India, ASEAN and SADC countries: impact on generic drug substitution. J. App Pharm. Sci. 6, 206–222. doi:10.7324/japs.2016.60430 Yu, Y., Teerenstra, S., Neef, C., Burger, D., and Maliepaard, M. (2016). A comparison of the intrasubject variation in drug exposure between generic and brand-name drugs: a retrospective analysis of replicate design trials. Br. J. Clin. Pharmacol. 81, 667–678. doi:10.1111/bcp.12828 Kuribayashi, R., Takishita, T., and Mikami, K. (2016). Regulatory considerations of bioequivalence studies for oral solid dosage forms in Japan. J. Pharm. Sci. 105, 2270–2277. doi:10.1016/j.xphs.2016.05.026 j Labes, D., Schutz, H., and Lang, B. (2020). PowerTOST: power and sample size for (Bio) Equivalence studies R package version 1.5-0. [Internet]. Available at: https://CRAN. R-project.org/packagePowerTOST (Accessed January 18, 2021). Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. REFERENCES Ministry of Food and Drug Safety (2014). Standard on pharmaceutical equivalence test, notice No. 2014-55, partially amended and enforced on feb. 2 [internet]. Available at: https://www.law.go.kr/LSW//admRulLsInfoP. do?chrClsCd&admRulSeq2100000000924 (Accessed February 12, 2014). Bebia, Z., Buch, S., Wilson, J., Frye, R., Romkes, M., Cecchetti, A., et al. (2004). Bioequivalence revisited: inﬂuence of age and sex on CYP enzymes. Clin. Pharmacol. Ther. 76, 618–627. doi:10.1016/j.clpt.2004.08.021 Ministry of Food and Drug Safety (2018a). Standard on pharmaceutical equivalence test, notice No. 2018-29, partially amended and enforced on apr. 18 [internet]. Available at: https://www.mfds.go.kr/eng/brd/m_18/view. do?seq71450&srchFr&srchTo&srchWordStandard+on+Pharmaceutical+ Equivalence+Study&srchTp7&itm_seq_10&itm_seq_20&multi_itm_seq 0&company_cd&company_nm&page1 (Accessed April 18, 2018). Boxenbaum, H., and Battle, M. (1995). Effective half-life in clinical Boxenbaum, H., and Battle, M. (1995). Effective half-life in clinical Pharmacology. J. Clin. Pharmacol. 35, 763–766. doi:10.1002/j.1552-4604.1995.tb04117.x J. Clin. Pharmacol. 35, 763–766. doi:10.1002/j.1552-4604.1995 Chen, M.-L., Blume, H., Beuerle, G., Davit, B., Mehta, M., Potthast, H., et al. (2018). The global bioequivalence harmonization initiative: summary report for EUFEPS international conference. Eur. J. Pharm. Sci. 111, 153–157. doi:10.1016/j.ejps.2017.09.047 Ministry of Food and Drug Safety (2018b). Frequently asked questions on the bioequivalence trials, [Internet]. Available at: https://www.nifds.go.kr/brd/m_ 197/view.do?seq12&srchFr&srchTo&srchWord&srchTp&itm_ seq_10&itm_seq_20&multi_itm_seq0&company_cd&company_ nm&page1 (Accessed August 14, 2018). Chow, S.-C. (2014). Bioavailability and bioequivalence in drug development. Wiley Interdiscip. Rev. Comput. Stat. 6. 304–312. doi:10.1002/wics.1310 Chung, I., Oh, J., Lee, S., Jang, I.-J., Lee, Y., and Chung, J.-Y. (2018). A post hoc analysis of intra-subject coefﬁcients of variation in pharmacokinetic measures to calculate optimal sample sizes for bioequivalence studies. Transl Clin. Pharmacol. 26, 6–9. doi:10.12793/tcp.2018.26.1.6 Ozdin, D., Ducharme, M. P., Varin, F., Fuglsang, A., and Al-Numani, D. (2020). Inﬂuence of different populations on pharmacokinetic bioequivalence results: can we extrapolate bioequivalence results from one population to another? J. Pharm. Pharm. Sci. 23, 357–388. doi:10.4103/2230-973X.104392 Cristofoletti, R., Chiann, C., Dressman, J. B., and Storpirtis, S. (2013). A comparative analysis of biopharmaceutics classiﬁcation system and biopharmaceutics drug disposition classiﬁcation system: a cross-sectional survey with 500 bioequivalence studies. J. Pharm. Sci. 102, 3136–3144. doi:10.1002/jps.23515 Ring, A., Lang, B., Kazaroho, C., Labes, D., Schall, R., and Schütz, H. (2019). Sample size determination in bioequivalence studies using statistical assurance. Br. J. Clin. Pharmacol. 85, 2369–2377. doi:10.1111/bcp.14055 Cristofoletti, R., Rowland, M., Lesko, L. J., Blume, H., Rostami-Hodjegan, A., and Dressman, J. B. (2018). Past, present, and future of bioequivalence: improving assessment and extrapolation of therapeutic equivalence for oral drug products. J. Pharm. Sci. 107, 2519–2530. doi:10.1016/j.xphs.2018.06.013 Ryu, M., and Kim, J. (2017). SUPPLEMENTARY MATERIAL Ministry of Food and Drug Safety open database for approved drugs, https://nedrug.mfds.go.kr/pbp/CCBAC02/getList? totalPages11&page1&limit50&sort&sortOrder&searchYn &targetGb1&title%EC%A0%9C%EB%84%A4%EB%A6%AD. Ministry of Food and Drug Safety open database for approved drugs, https://nedrug.mfds.go.kr/pbp/CCBAC02/getList? The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.651790/ full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.651790/ full#supplementary-material totalPages11&page1&limit50&sort&sortOrder&searchYn &targetGb1&title%EC%A0%9C%EB%84%A4%EB%A6%AD. SUPPLEMENTARY FIGURE 1 \| Interstudy variability of the number of subjects in 2 × 2 × 4 bioequivalence (BE) trials. Dots represent the median number of subjects in each drug and horizontal lines represent minimum and maximum number of subjects. Difference between the minimum and maximum number of subjects [the number of BE studies] are presented in the text. ATC classiﬁcation for each drug was provided with colors. (Notes: Drugs with more than three BE studies were selected. Fixed-dose combination drugs were excluded from the analysis.) DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/ repositories and accession number(s) can be found below: May 2021 \| Volume 12 \| Article 651790 Frontiers in Pharmacology \| www.frontiersin.org 6 Huh et al. Bioequivalence Study Designs in Korea AUTHOR CONTRIBUTIONS KH, EK, SL, HY, SY, K-SY and J-YC wrote the manuscript. J-YC designed research. KH, EK, SL, HY, SY and J-YC performed research. KH and J-YC analyzed data. REFERENCES Lee, C.-Y., Chen, X., Romanelli, R. J., and Segal, J. B. (2016). Forces inﬂuencing generic drug development in the United States: a narrative review. J. Pharm. Policy Pract. 9, 1–6. doi:10.1186/s40545-016-0079-1 Copyright © 2021 Huh, Kim, Lee, Yoo, Yoon, Yu and Chung. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Löbenberg, R., and Amidon, G. L. (2000). Modern bioavailability, bioequivalence and biopharmaceutics classiﬁcation system. New scientiﬁc approaches to international regulatory standards. Eur. J. Pharm. Biopharm. 50, 3–12. doi:10.1016/s0939-6411(00)00091-6 Meredith, P. (2003). Bioequivalence and other unresolved issues in generic drug substitution. Clin. Ther. 25, 2875–2890. doi:10.1016/s0149-2918(03)80340-5 May 2021 \| Volume 12 \| Article 651790 Frontiers in Pharmacology \| www.frontiersin.org 7
https://openalex.org/W4213037940	https://www.aanda.org/10.1051/0004-6361/202038553/pdf	English	null	The New Generation Planetary Population Synthesis (NGPPS)	Astronomy & astrophysics	2,021	cc-by	48,328	ABSTRACT Context. The explosion of observational data on exoplanets gives many constraints on theoretical models of planet formation and evolution. Observational data probe very large areas of the parameter space and many different planet properties. Ai Context. The explosion of observational data on exoplanets gives many constraints on theoretical models of planet formation and evolution. Observational data probe very large areas of the parameter space and many different planet properties. Aims. Comparing theoretical models with observations allows one to take a key step forward towards understanding planetary sys- tems. It however requires a model able to (i) predict all the necessary observable quantities (not only masses and orbits, but also radii, luminosities, magnitudes, or evaporation rates) and (ii) address the large range in relevant planetary masses (from Mars mass to super- Jupiters) and distances (from stellar-grazing to wide orbits). Aims. Comparing theoretical models with observations allows one to take a key step forward towards understanding planetary sys- tems. It however requires a model able to (i) predict all the necessary observable quantities (not only masses and orbits, but also radii, luminosities, magnitudes, or evaporation rates) and (ii) address the large range in relevant planetary masses (from Mars mass to super- Jupiters) and distances (from stellar-grazing to wide orbits). Methods. We have developed a combined global end-to-end planetary formation and evolution model, the Generation III Bern model, based on the core accretion paradigm. This model solves as directly as possible the underlying differential equations for the structure and evolution of the gas disc, the dynamical state of the planetesimals, the internal structure of the planets yielding their planetesi- mal and gas accretion rates, disc-driven orbital migration, and the gravitational interaction of concurrently forming planets via a full N-body calculation. Importantly, the model also follows the long-term evolution of the planets on gigayear timescales after formation including the effects of cooling and contraction, atmospheric escape, bloating, and stellar tides. Results. To test the model, we compared it with classical scenarios of Solar System formation. For the terrestrial planets, we ﬁnd that we obtain a giant impact phase of protoplanet-protoplanet collisions provided enough embryos (∼100) are initially emplaced in the disc. For the giant planets, we ﬁnd that Jupiter-mass planets must accrete their core shortly before the dispersal of the gas disc to prevent strong inward migration that would bring them to the inner edge of the disc. Astronomy & Astrophysics Astronomy & Astrophysics Astronomy & Astrophysics A&A 656, A69 (2021) https://doi.org/10.1051/0004-6361/202038553 © A. Emsenhuber et al. 2021 A&A 656, A69 (2021) https://doi.org/10.1051/0004-6361/202038553 © A. Emsenhuber et al. 2021 ABSTRACT Regarding the emergence of entire planetary sys- tems, many aspects can be understood with the comparison of the timescales of growth and migration, the capture into resonances, and the consequences of large-scale dynamical instabilities caused by the gravitational interactions of protoplanets, including the situation when a second core starts runaway gas accretion. Conclusions. The Generation III Bern model provides one of the most comprehensive global end-to-end models of planetary sys- tem formation and evolution developed so far, linking a multitude of crucial physical processes self-consistently. The model can form planetary systems with a wide range of properties. We ﬁnd that systems with only terrestrial planets are often well-ordered (in period, mass, and radius), while giant-planet bearing systems show no such similarity. In a series of papers, the model will be used to per- form extensive planetary population syntheses, putting the current theoretical understanding of planet formation and evolution to the observational test. Key words. planets and satellites: formation – planets and satellites: interiors – planet-disk interactions – protoplanetary disks – methods: numerical ds. planets and satellites: formation – planets and satellites: interiors – planet-disk interactions – protoplanetary disks numerical Alexandre Emsenhuber1,2,3 , Christoph Mordasini2 , Remo Burn2,4 , Yann Alibert2, Willy Benz2, and Erik Asphaug1 1 Lunar and Planetary Laboratory, University of Arizona, 1629 E. University Blvd., Tucson, AZ 85721, USA 2 Physikalisches Institut, Universität Bern, Gesellschaftsstrasse 6, 3012 Bern, Switzerland 3 Universitäts-Sternwarte München, Ludwig-Maximilians-Universität München, Scheinerstraße 1, 81679 München, Germany e-mail: emsenhuber@usm.lmu.de 4 Max-Planck-Institut für Astronomie, Königstuhl 17, 69117 Heidelberg, Germany 4 Max-Planck-Institut für Astronomie, Königstuhl 17, 69117 Heidelberg, Germany Max-Planck-Institut für Astronomie, Königstuhl 17, 69117 H Received 1 June 2020 / Accepted 6 July 2021 Received 1 June 2020 / Accepted 6 July 2021 Received 1 June 2020 / Accepted 6 July 2021 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction of days, which is well within the orbit of Mercury (Mayor et al. 2011; Fabrycky et al. 2014); others were detected at large sepa- rations using the direct imaging technique (Marois et al. 2008; Lagrange et al. 2010; Rameau et al. 2013; Macintosh et al. 2015; Chauvin et al. 2017; Keppler et al. 2018). Since the discovery of the ﬁrst exoplanet detected around a main sequence star (Mayor & Queloz 1995), the number of known exoplanets has greatly increased. These planets span a wide range of masses and sizes, and they were detected using vari- ous techniques, such as radial velocity, transit, direct imaging, and microlensing. Despite all these observational constraints, the exact formation pathways are not yet certain. To highlight this, we ﬁrst discuss possible formation mechanisms for different planet kinds. pp Most giant planets are thought to form via the core accre- tion mechanism as gravitational instability (Boss 1997, 2003) is found to work only at large separation (several tens of astronom- ical units, Raﬁkov 2005; Schib et al. 2021), though the clumps could migrate after formation (Nayakshin 2010), and for bodies above about 5 MX (Schlaufman 2018) or even the deuterium- burning limit (Kratter et al. 2010). On the other extreme, for very close-in giant planets, core accretion (Perri & Cameron 1974; Giant planets have been found orbiting their host star over a wide range of periods. Some are of the order of days or tens A69, page 1 of 44 A69, page 1 of 44 A&A 656, A69 (2021) for instance, atmospheric escape (e.g. Jin et al. 2014) or giant impacts (Schlichting & Mukhopadhyay 2018). Mizuno 1980) requires for in situ formation a very strong pile- up of solids. While this has been proposed (Boley et al. 2016; Batygin et al. 2016; Bailey & Batygin 2018), the possibility remains heavily debated. A scenario where these planets formed further out and were subsequently moved to their ﬁnal location (Lin et al. 1996) is usually considered more likely. Multi-planetary systems provide additional information. Many super-Earth systems have similar mass and spacing (Millholland et al. 2017; Weiss et al. 2018), though this is debated (Zhu 2020; Weiss & Petigura 2020). However, most of the planet pairs are out of mean-motion resonances (Fabrycky et al. 2014). The low number of planets in mean-motion resonances (MMR) may be surprising, as gas-driven migration is efﬁcient at captur- ing the planets in MMRs. 1. Introduction The ﬁnal mass and location of the planet depends thus on the interplay between growth and migration, not to mention the interactions with the other planets forming in the same system. Observations show that the giant planets are divided into two sub-groups depending on the host-star metallicity (Dawson & Murray-Clay 2013; Buchhave et al. 2018). Hot-Jupiters around metal-poor stars exhibit lower stellar obliquity and eccentric- ity than the ones around metal-rich stars. The usual con- cept of inward migration due to interaction with the gas disc (Goldreich & Tremaine 1979; Ward 1997; Tanaka et al. 2002) cannot account for the obliquity of these planets, which more likely were brought there by few-body interactions combined with tidal circularisation (Dawson & Johnson 2018). As the model has many parameters, a large number of planets with different properties are required to constrain their possible values. The model must then be able to predict all the necessary observable quantities for the different observational techniques, not only masses and distances, but also radii (for transits), lumi- nosities, magnitudes (for direct imaging), and evaporation rates. To leverage the enormous amount of statistical observational data on exoplanets, the models should also be able to make quan- titative predictions which can be compared statistically with the actual planetary population. For this, planetary population syn- thesis (Ida & Lin 2004a; Mordasini et al. 2009a) is a frequently used approach. For the distant giant planets, core accretion is still favoured (Wagner et al. 2019). A possible formation pathway for some of these distant planets is accretion in the inner region of the disc followed by close encounters and scattering (Marleau et al. 2019a). This pathway is supported by evidence that it is able to reproduce the distribution of eccentricities of giant planets (Chatterjee et al. 2008; Juri´c & Tremaine 2008; Ford & Rasio 2008; Raymond et al. 2010; Sotiriadis et al. 2017), and that most giant planet-harbouring system are multiple (Knutson et al. 2014; Bryan et al. 2016; Wagner et al. 2019). pp In this work, we introduce a strongly improved and extended version the Bern global end-to-end model of planetary formation and evolution for multi-planetary systems. The model com- bines the work of Alibert et al. (2013, hereafter A13) (inclusion of N-body interactions) and the internal structure calculations and long-term thermodynamical evolution model of Mordasini et al. (2012c,b). 1. Introduction But it is possible for the resonances to be broken during the retreat of the gas disc (Liu et al. 2017) or after the dispersal by dynamical instabilities (Inamdar & Schlichting 2016; Izidoro et al. 2017, 2021). The mutual inclina- tions remain relatively low (Lissauer et al. 2011; Fang & Margot 2012) and they exhibit low-to-moderate eccentricities (Xie et al. 2016; Mills et al. 2019). In the standard view, giant planets form from embryos located beyond the ice line, where solids are abundant owing to the volatiles being present in the solid form. This allows the embryo to form rapidly enough before the dispersal of the gas disc, which occurs in a time frame of several million years (Haisch et al. 2001; Fedele et al. 2010; Richert et al. 2018). Embryos initially accrete solids and a small quantity of gas. The further growth results in the accretion of gas, which is gov- erned by the ability of the planet to radiate away the accretion energy (Pollack et al. 1996; Lee & Chiang 2015). The cooling process becomes more efﬁcient as the mass increase, so that when the planet reaches a mass of several times that of the Earth, the amount of solids and gas are equal (the critical mass, Stevenson 1982). Once the accretion rate becomes greater than what the disc is able to supply, the envelope can no longer remain in equilibrium with the surrounding nebula and it contracts. ) Many models have been developed to capture the above- mentioned effects. We may cite Pollack et al. (1996), Ida & Lin (2004a,b), Alibert et al. (2004, 2005a), Miguel et al. (2011), Coleman & Nelson (2014), Cridland et al. (2016, 2017), Liu et al. (2017), Ormel et al. (2017), Ronco et al. (2017), Ndugu et al. (2018), Chambers (2018), Alessi & Pudritz (2018), Bitsch et al. (2019), Johansen et al. (2019), Booth & Ilee (2019), or Alessi et al. (2020a,b), to mention only a few. To capture all of the above effects, models of planetary formation must include many phys- ical processes occurring during the formation of the systems, which lead to feedbacks and non-linearities. Then, comparing the outcomes of global end-to-end models with observations is a key step for the understanding of the origins and evolution of planets systems. This process is further complicated by the implications of planetary migration (Baruteau et al. 2014, 2016). A69, page 2 of 44 2.2.2. Evolution phase p g The original model was introduced in Alibert et al. (2004, 2005a) for individual planets, then used in Mordasini et al. (2009a,b) for entire planetary populations. We refer to it as Gen- eration I, which computed the formation on a single planet until the gas disc disperses. The model subsequently diverged into two different branches: one with the aim to follow the long- term evolution of the formed planet (Generation Ib; Mordasini et al. 2012c,b) while the other obtained the ability to form multi-planetary systems with an improved description of the planetesimals disc (Generation II; Alibert et al. 2013; Fortier et al. 2013). In this work we bring these two variants of the model back together so that we can follow the formation and the long- term evolution of multi-planetary systems. At the same time, we extend the model with new elements, which are shown in italic on Fig. 1. The long-term evolution of the planets (20 Myr–10 Gyr) is calcu- lated by solving, like already in the formation phase, the standard spherically symmetric internal structure equations, but with dif- ferent boundary conditions, and taking into account different physical effects like atmospheric escape, or radius inﬂation. In this phase, the planets evolve individually; N-body interactions and the accretion of planetesimals are no more considered. The orbits and masses of the planets may however still evolve because of effects like tides and atmospheric escape. As described in Mordasini et al. (2012c), the coupling between the formation and evolution phases is made self- consistently, that is both the compositional information as well as the gravothermal heat content given by the formation model are given to the evolution model as initial conditions. Previous versions of the model have been extensively described in referenced papers (see also Benz et al. 2014; Mordasini et al. 2015; Mordasini 2018 for reviews and the inter- actions between the different mechanisms involved in planet population syntheses). We nevertheless describe this new ver- sion in the remainder of this section. Regarding the temporal evolution, we now also take the ther- mal energy content of the planet’s core into account for a planet’s luminosity, as described in Linder et al. (2019). This is impor- tant for core-dominated low-mass planets (e.g. Lopez & Fortney 2014). 1. Introduction Here, we track the planets with full N-body interactions, in contrast to Ida & Lin (2010) for instance, who introduced a semi-analytical approach, an improvement over pre- vious works, such as Ida & Lin (2004a), to follow planet-planet interactions. The model follows the formation of many embryos, as it usually obtained from the end stage of the runaway growth of solids (Kokubo & Ida 1998), so that both terrestrial as well as giant planetary systems can be obtained. Exoplanets include planets unknown in the solar system, those between the Earth and Neptune (Mayor et al. 2011; Youdin 2011; Howard et al. 2012). The density of these planets vary more than one order of magnitude (Hatzes & Rauer 2015; Otegi et al. 2020). Sub-Neptunes exhibit a low bulk density, indicating the pres- ence of a gaseous envelope (Weiss & Marcy 2014; Rogers 2015). This implies that they mostly formed in a time scale comparable with the lifetime of the protoplanetary disc. However, whether they formed early (in the same way as the core of giant planets) or towards the end of the disc (Lee & Chiang 2016) is not yet settled. The structure of this work is as follows: in the ﬁrst part, we describe our global model. In Sect. 2, we introduce the new ver- sion of our model with a general overview of its conception, along with its relationship to previous work. Detailed description are provided in Sect. 3 for the stellar and nebular components, in Sect. 4 for the planets, and in Sect. 5 for the migration and dynamical evolution. In a second part we perform some tests for different kind of planets and show possible resulting systems. In Super-Earths on the other hand are compatible with being gas-free. They are not constrained by the lifetime of the pro- toplanetary disc and can form over longer periods of time (Lambrechts et al. 2019; Ogihara et al. 2018). These could also have had a gaseous envelope in the past that was removed by, A69, page 2 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. phase, we follow the thermodynamical evolution of each planet individually to 10 Gyr. phase, we follow the thermodynamical evolution of each planet individually to 10 Gyr. Sect. 2.1. History The model presented in this work, the Generation III Bern model, combines the formation and evolution stage of planetary system. It is based on many contributions in the ﬁeld that aim to study different aspects of the physics of planetary formation and evolution. We thus start by a short history of the series of model, and its different branches that we couple together in this work. A graphical sketch of the different generations of the Bern model is provided in Fig. 1. 2.2.2. Evolution phase As in previous calculations, the other gravothermal energy sources are the cooling and contraction of the H/He envelope, the contraction of the core, and radiogenic heating due to the presence of long-lived radionuclides in the core. 1. Introduction 6, we aim at reproducing formation of terrestrial planets with our improved model to determine whether it is applicable to kind of planets. The formation of giant planets and the impli- cations for Jupiter are discussed in Sect. 7. Finally, in Sect. 8, we apply the presented model to speciﬁc systems to assess the interaction between the different mechanisms occurring during the formation and evolution of planetary systems. 2.2. General description We base our study on the Bern model of planetary formation and evolution. This global model self-consistently computes the evolution of the gas and planetesimals discs, the accretion of gas and solids by the protoplanets, their internal and atmospheric structure, as well as interactions between the protoplanets and between the gas disc and the protoplanets. We provide a diagram of the main components of the overall model as well as the most important exchanged quantities in Fig. 2. 2.2.1. Formation phase During the formation stage (0–20 Myr), the model follows the evolution of a gaseous protoplanetary disc and the dynamical state of planetesimals (Sect. 3). These serve as sources for the accretion of the protoplanets (Sect. 4). The lifetime of the gas disc is shorter than the simulated formation stage, so that solids accretion in a gas-free environment can also take place. The gas disc also leads to planetary migration, and interactions (scatter- ing, collisions) between the concurrently growing proptoplanets are tracked via a N-body integrator (Sect. 5). This work is the ﬁrst of a series of several. In a compan- ion paper, Emsenhuber et al. (2021, hereafter referred to as Paper II), we will use this model to compute synthetic popu- lations of planetary systems and perform statistical analysis. In subsequent articles, we will perform more detailed comparison with observations, and analyse various parameters that we have in the present model. The formation of planets is based on the core accretion paradigm (Mizuno 1980; Pollack et al. 1996): ﬁrst, a solid core is formed, and once it becomes massive enough, it starts to bind a signiﬁcant H/He envelope. Core growth results from the accre- tion of planetesimals. Gas accretion is initially governed by the ability of the planet to radiate away the energy released by the accretion of both solids and gas. Once the gas accretion rate of the envelope exceeds the limit from the disc, the envelope can no longer maintain equilibrium with the disc; hence it subsequently contracts and passes into the detached phase (Bodenheimer et al. 2000). Generation I (Alibert et al. 2005a): base model 2006), (7) disc-limited gas accretion rate, (8) later additions and improvements, (9) additional output relative to older generation, (10) population synthesis publications using this generation. In the bottom right panel, text in italic indicates new elements. Fig. 1. Overview of the physical mechanisms included in the Bern model. At the top, the processes and base assumptions made in all model generations are given. The four boxes below show the four model generations with the main paper introducing each generation. The further points are: (1) number of initial embryos per disc, N-body integrator type, initial embryo mass, (2) phases simulated, (3) planetesimal accretion mode and planetesimal size, (4) phases with calculation of the planets’ internal structure, (5) disc model characteristics, (6) orbital migration: type I, type II, transition criterion from type I to II (here thermal refers to a criterion only with the ratio between the Hill radius and the scale height of the disc, while ‘thermal and viscous’ refers to the full criterion of Crida et al. 2006), (7) disc-limited gas accretion rate, (8) later additions and improvements, (9) additional output relative to older generation, (10) population synthesis publications using this generation. In the bottom right panel, text in italic indicates new elements. Fig. 1. Overview of the physical mechanisms included in the Bern model. At the top, the processes and base assumptions made in all model generations are given. The four boxes below show the four model generations with the main paper introducing each generation. The further points are: (1) number of initial embryos per disc, N-body integrator type, initial embryo mass, (2) phases simulated, (3) planetesimal accretion mode and planetesimal size, (4) phases with calculation of the planets’ internal structure, (5) disc model characteristics, (6) orbital migration: type I, type II, transition criterion from type I to II (here thermal refers to a criterion only with the ratio between the Hill radius and the scale height of the disc, while ‘thermal and viscous’ refers to the full criterion of Crida et al. 2006), (7) disc-limited gas accretion rate, (8) later additions and improvements, (9) additional output relative to older generation, (10) population synthesis publications using this generation. In the bottom right panel, text in italic indicates new elements. Compared to some other 1D internal structures models in the literature (e.g. Vazan et al. 2013; Venturini et al. Generation I (Alibert et al. 2005a): base model Multiplicity, eccentricities, MMR 10. Pfyﬀer et al. (2015); Alibert & Benz (2017) Generation III (this work): long-term evolution and N-body evolution 1. Many embryos per disc (Mercury N-body integrator), 0.01 M⊕ 2. Formation (to 20 Myr) and (thermodynamic) evolution (to 10 Gyr) 3. Oligarchic planetesimals accretion, 300 m 4. Attached, detached, evolutionary (with D-burning, escape, bloating, Roche lobe overﬂow, core structure) 5. Vertically integrated, with stellar irradiation and stellar evo- lution 6. Non-isothermal type I, non-equilibrium type II, thermal and viscous transition criterion 7. Bondi-limited gas accretion 8. None 9. Combines output of Ib and II 10. This work (NGPPS series) Fig. 1. Overview of the physical mechanisms included in the Bern model. At the top, the processes and base assumptions made in all model generations are given. The four boxes below show the four model generations with the main paper introducing each generation. The further points are: (1) number of initial embryos per disc, N-body integrator type, initial embryo mass, (2) phases simulated, (3) planetesimal accretion mode and planetesimal size, (4) phases with calculation of the planets’ internal structure, (5) disc model characteristics, (6) orbital migration: type I, type II, transition criterion from type I to II (here thermal refers to a criterion only with the ratio between the Hill radius and the scale height of the disc, while ‘thermal and viscous’ refers to the full criterion of Crida et al. 2006), (7) disc-limited gas accretion rate, (8) later additions and improvements, (9) additional output relative to older generation, (10) population synthesis publications using this generation. In the bottom right panel, text in italic indicates new elements. Fig. 1. Overview of the physical mechanisms included in the Bern model. At the top, the processes and base assumptions made in all model generations are given. The four boxes below show the four model generations with the main paper introducing each generation. The further points are: (1) number of initial embryos per disc, N-body integrator type, initial embryo mass, (2) phases simulated, (3) planetesimal accretion mode and planetesimal size, (4) phases with calculation of the planets’ internal structure, (5) disc model characteristics, (6) orbital migration: type I, type II, transition criterion from type I to II (here thermal refers to a criterion only with the ratio between the Hill radius and the scale height of the disc, while ‘thermal and viscous’ refers to the full criterion of Crida et al. 2.2.3. Envelope structure Afterwards, in the evolutionary A69, page 3 of 44 A&A 656, A69 (2021) A proofs: manuscript no m Physical mechanisms and base assumptions included in all model generations – Formation paradigm: core accretion – Formation paradigm: core accretion – Formation paradigm: core accretion – Protoplanetary disc model: solution of 1D evolution equation for gas surface density in an axissymetric constant α-disc with photoevaporation – Solid accretion: rate equation (Safronov-type) from planetesimals of a single size; planetesimals are represented by a solid surface density with dynamical state – Gas accretion and planet interior structure: from solving 1D radially symmetric hydrostatic planet interior structure equations – Orbital migration: gas disc-driven, types I and II Formation paradigm: core accretion – Protoplanetary disc model: solution of 1D evolution equation for gas surface density in an axissymetric constant α-disc with photoevaporation – Solid accretion: rate equation (Safronov-type) from planetesimals of a single size; planetesimals are represented by a solid surface density with dynamical state Formation paradigm: core accretion – Protoplanetary disc model: solution of 1D evolution equation for gas surface density in an axissymetric constant α-disc with photoevaporation – Solid accretion: rate equation (Safronov-type) from planetesimals of a single size; planetesimals are represented by a solid surface density with dynamical state – Gas accretion and planet interior structure: from solving 1D radially symmetric hydrostatic planet interior structure equations – Orbital migration: gas disc-driven, types I and II with dynamical state – Gas accretion and planet interior structure: from solving 1D radially symmetric hydrostatic planet interior structure equations – Orbital migration: gas disc-driven, types I and II y ccretion and planet interior structure: from solving 1D radially symmetric hydrostatic planet interior structure equations al migration: gas disc-driven, types I and II Evolution of physical mechanisms considered in various model generations Evolution of physical mechanisms considered in various model generations s Generation Ib (Mordasini et al. 2012c,b): inclusion of long- term evolution 1. 1 embryo per disc (no N-body), 0.6 M⊕ 2. Formation (to tdisc) and (thermodynamic) evolution (to 10 Gyr) 3. Runaway planetesimals accretion, 100 km 4. Attached, detached and evolutionary, with core structure 5. Vertical disc structure, with stellar irradiation, no stellar evo- lution 6. According to Dittkrist et al. (2014): Non-isothermal type I, non-equilibrium type II, thermal and viscous transition crite- rion 7. Non-equilibrium gas ﬂux in disc 8. D-burning, atmospheric escape 9. Radii, luminosities, envelope evaporation rates 10. Mordasini et al. Generation I (Alibert et al. 2005a): base model 2016; Valletta & Helled 2020), the model is simpliﬁed ﬁrst by assuming that the gaseous envelope consists of pure H/He, while accreted solids sink to the core. In this sense, the model is similar to the original Pollack et al. (1996) model. We neglect thus the consequences of heavy element enrichment and compositional gradients in the envelope. These eﬀects will be added in future work. One should note that also other modern models make use of the simpliﬁca- tion of pure H/He envelopes (D’Angelo et al. 2021). Including enrichment would generally speed up gas giant formation (Ven- turini et al. 2016). Second, the eﬀects of hydrodynamic ﬂows Compared to some other 1D internal structures models in the literature (e.g. Vazan et al. 2013; Venturini et al. 2016; Valletta & Helled 2020), the model is simpliﬁed ﬁrst by assuming that the gaseous envelope consists of pure H/He, while accreted solids sink to the core. In this sense, the model is similar to the original Pollack et al. (1996) model. We neglect thus the consequences of heavy element enrichment and compositional gradients in the envelope. These effects will be added in future work. One should note that also other modern models make use of the simpliﬁ- cation of pure H/He envelopes (D’Angelo et al. 2021). Includ- ing enrichment would generally speed up gas giant formation (Venturini et al. 2016). Second, the effects of hydrodynamic ﬂows affecting the (upper) envelope structure and cooling p g p y, y gaseous envelope made of pure H/He. In contrast to earlier syn- theses predicting planetary radii (Mordasini et al. 2012c, 2014), we now use self-consistently the iron mass fraction as given by the disc compositional model (according to Thiabaud et al. 2014, Section 3.3.3), instead of assuming a ﬁxed 2:1 silicate:iron mass ratio. Physical eﬀects that are included in the model besides the spherically symmetric structure with an iron core, a silicate man- tle, and depending of a planet’s accretion history, a water ice layer and a gaseous envelope made of pure H/He. In contrast to earlier syntheses predicting planetary radii (Mordasini et al. 2012c, 2014), we now use self-consistently the iron mass fraction as given by the disc compositional model (according to Thi- abaud et al. 2014, Sect. 3.3.3), instead of assuming a ﬁxed 2:1 silicate:iron mass ratio. p g p y, y gaseous envelope made of pure H/He. Generation I (Alibert et al. 2005a): base model Generation I (Alibert et al. 2005a): base model 1. 1 embryo per disc (no N-body), 0.6 M⊕ 9. Radii, luminosities, envelope evaporation rates 10. Mordasini et al. (2014, 2017); Jin & Mordasini (2018) Generation III (this work): long-term evolution and N-body evolution 1. Many embryos per disc (Mercury N-body integrator), 0.01 M⊕ 2. Formation (to 20 Myr) and (thermodynamic) evolution (to 10 Gyr) 3. Oligarchic planetesimals accretion, 300 m 4. Attached, detached, evolutionary (with D-burning, escape, bloating, Roche lobe overﬂow, core structure) 5. Vertically integrated, with stellar irradiation and stellar evo- lution 6. Non-isothermal type I, non-equilibrium type II, thermal and viscous transition criterion 7. Bondi-limited gas accretion 8. None 9. Combines output of Ib and II 10. This work (NGPPS series) Generation II (Alibert et al. 2013): inclusion of N-body inter- action 1. Several embryos per disc (EMPS N-body integrator), 0.01 M⊕ 2. Formation only (to tdisc) 3. Oligarchic planetesimals accretion, 300 m (Fortier et al. 2013) 4. Attached phase only 5. Vertical disc structure, no stellar irradiation, no stellar evolu- tion 6. According to Dittkrist et al. (2014): Non-isothermal type I, non-equilibrium type II, thermal and viscous transition crite- rion 7. Non-equilibrium gas ﬂux in disc 8. Composition tracking (Thiabaud et al. 2015) 9. Multiplicity, eccentricities, MMR 10. Pfyﬀer et al. (2015); Alibert & Benz (2017) Generation II (Alibert et al. 2013): inclusion of N-body inter- action 1. Several embryos per disc (EMPS N-body integrator), 0.01 M⊕ 2. Formation only (to tdisc) 3. Oligarchic planetesimals accretion, 300 m (Fortier et al. 2013) 4. Attached phase only 5. Vertical disc structure, no stellar irradiation, no stellar evolu- tion 6. According to Dittkrist et al. (2014): Non-isothermal type I, non-equilibrium type II, thermal and viscous transition crite- rion 7. Non-equilibrium gas ﬂux in disc 8. Composition tracking (Thiabaud et al. 2015) 9. Multiplicity, eccentricities, MMR 10. Pfyﬀer et al. (2015); Alibert & Benz (2017) Generation II (Alibert et al. 2013): inclusion of N-body inter- action 1. Several embryos per disc (EMPS N-body integrator), 0.01 M⊕ 2. Formation only (to tdisc) 3. Oligarchic planetesimals accretion, 300 m (Fortier et al. 2013) 4. Attached phase only 5. Vertical disc structure, no stellar irradiation, no stellar evolu- tion 6. According to Dittkrist et al. (2014): Non-isothermal type I, non-equilibrium type II, thermal and viscous transition crite- rion 7. Non-equilibrium gas ﬂux in disc 8. Composition tracking (Thiabaud et al. 2015) 9. 2.2.3. Envelope structure (2014, 2017); Jin & Mordasini (2018) Generation I (Alibert et al. 2005a): base model 1. 1 embryo per disc (no N-body), 0.6 M⊕ 2. Formation only (to tdisc) 3. Runaway planetesimals accretion, 100 km 4. Attached phase only 5. Vertical disc structure, no stellar irradiation, no stellar evolu- tion 6. Isothermal type I, equilibrium type II, thermal only transition criterion 7. Equilibrium gas ﬂux in disc 8. Stellar irradiation of the disc (Fouchet et al. 2012) 9. Masses, orbital distances, bulk composition 10. Mordasini et al. (2009a,b); Alibert et al. (2011); Mordasini et al. (2012a) 2.2.3. Envelope structure The calculation of the internal structure of all planets (Sect. 4) during their entire formation and evolution is a crucial aspect of the Bern Model, as visible from its central position in Fig. 2. It not only yields the planetary gas accretion rate in the attached phase but is also key for the accretion of planetesimals via the drag enhanced capture radius. It also yields the radii and lumi- nosities that on one hand enter multiple other sub-modules, and on the other hand are key observable quantities. The inter- nal structure model assumes that planets have an onion-like In our coupled formation and evolution model, we ﬁrst model the planets’ main formation phase for a ﬁxed time interval (set to 20 Myr, see the related discussion in Sect. 6 regarding the impact of this speciﬁc choice). A69, page 4 of 44 Generation I (Alibert et al. 2005a): base model In contrast to earlier syn- theses predicting planetary radii (Mordasini et al. 2012c, 2014), we now use self-consistently the iron mass fraction as given by the disc compositional model (according to Thiabaud et al. 2014, Section 3.3.3), instead of assuming a ﬁxed 2:1 silicate:iron mass ratio. Physical eﬀects that are included in the model besides the spherically symmetric structure with an iron core, a silicate man- tle, and depending of a planet’s accretion history, a water ice layer and a gaseous envelope made of pure H/He. In contrast to earlier syntheses predicting planetary radii (Mordasini et al. 2012c, 2014), we now use self-consistently the iron mass fraction as given by the disc compositional model (according to Thi- abaud et al. 2014, Sect. 3.3.3), instead of assuming a ﬁxed 2:1 silicate:iron mass ratio. usual cooling and contraction are XUV-driven atmospheric es- cape (Jin et al. 2014), D-burning (Mollière & Mordasini 2012), Roche-lobe overﬂow, and bloating of the close-in planets (Sarkis et al. 2021). Article number page 4 of 45 Physical effects that are included in the model besides the usual cooling and contraction are XUV-driven atmospheric escape (Jin et al. 2014), D-burning (Mollière & Mordasini 2012), Roche-lobe overﬂow, and bloating of the close-in planets (Sarkis et al. 2021). A69, page 4 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. y p y ( ) Fig. 2. Sub-modules and most important exchanged quantities of the Generation III Bern model. The colours denote the stages at which processes are considered. Blue indicates processes active in the formation stage, but only before the dispersal of the gas disc. Green processes are considered during the entire formation stage, even after the dispersal of the gas disc. Processes in red are only considered during the evolution stage. The processes in black are included in all stages. Fig. 2. Sub-modules and most important exchanged quantities of the Generation III Bern model. The colours denote the stages at which processes are considered. Blue indicates processes active in the formation stage, but only before the dispersal of the gas disc. Green processes are considered during the entire formation stage, even after the dispersal of the gas disc. Processes in red are only considered during the evolution stage. The processes in black are included in all stages. behaviour are currently also neglected (Ali-Dib et al. 2020; Moldenhauer et al. 2021). Generation I (Alibert et al. 2005a): base model to ‘see’ the stellar XUV irradiation. This could lead to a cer- tain under-estimation of atmospheric escape. The consequences should, however, be small, since escape continues to be impor- tant for at least the ﬁrst 100 Myr when stars are in the saturated phase of high XUV emission, and not only for the ﬁrst 20 Myr. The effect that atmospheric escape can destabilise resonant chains for sufﬁciently high mass loss (Matsumoto & Ogihara 2020) is thus not included. On the other hand, we include during the entire formation phase (also after gas disc dissipation) the accretion of planetesimals, which also changes planet masses. On the other hand, our internal structure model allows to model the entire ‘life’ of planets from t = 0 to 10 Gyr, modelling and coupling self-consistently all phases (attached, detached, evolutionary), for both the gaseous envelope and the solid core. Importantly, the model is capable of calculating the internal structure and temporal evolution of planets ranging in mass from 10−2 M⊕to the lithium-burning limit (about 63 Jovian masses, Burrows et al. 2001). It includes besides the standard aspects (accretion, cooling, contraction) also atmospheric escape, bloat- ing, Roche-lobe overﬂow, and deuterium burning. In particular, this makes it possible to model planets that reside very close to their host star. This quite unique general applicability to very different planet types reﬂects the needs arising from a population synthesis calculation. In the following sections, we describe in detail all the sub- modules visible in Fig. 2. 3.1. Stellar model As shown in Fig. 2, atmospheric escape is only included in the evolution phase starting at 20 Myr. In reality, it would start immediately once the gas disc has dissipated and the planets start Instead of assuming a ﬁxed 1 L⊙stellar luminosity for a 1 M⊙star as in previous model generations, stellar evolution is now consid- A69, page 5 of 44 A&A 656, A69 (2021) It is clear that this treatment of the opacities is simpliﬁed: in reality, the evolution of the dust via coagulation, fragmentation, and drift inﬂuences via the resulting grain opacity the thermal and density structure of the disc. This structure in turn feeds back onto the dust evolution, meaning that the processes must be treated together in a self-consistently coupled way (Gorti et al. 2015; Savvidou et al. 2020). ered by incorporating the stellar evolution tracks from Baraffe et al. (2015). These provide the radius R⋆, luminosity L⋆and temperature T⋆for a given stellar mass M⋆at any moment. Stellar temperature and radius are used for the outer bound- ary conditions of the gas disc; stellar radius is also used in the N-body integrator to detect collisions with the star and to cal- culate the stellar tidal migration. Finally, the stellar irradiation enters into the calculation of the outer (atmospheric) tempera- ture (at τ = 2/3) of the planets’ interior structure as described in Mordasini et al. (2012c) and radius bloating (Sect. 4.2.2). Such a more realistic coupled model affects for example the disc lifetime, the local dust-to-gas ratio (Gorti et al. 2015), or – in the context of planet formation – the locations of the outward migration zones (Savvidou et al. 2020, see Sect. 5.1.3). They also show that the ratio of Planck and Rosseland opacity is in real- ity not simply a constant as currently assumed. In Voelkel et al. (2020) we have recently coupled the Birnstiel et al. (2012) dust- pebble evolution model to the Bern Model. Based on this, future version of the Bern Model will include also a more physically realistic grain opacity and therefore disc structure model. This will in particular also include the dependency of the disc opac- ity on the stellar metallicity, which is currently not taken into account. 3.2. Gas disc The protoplanetary gas disc is modelled with a 1D radial axisy- metric structure. The evolution is given by solving the viscous diffusion equation as function of the time t and orbital distance r (Lüst 1952; Lynden-Bell & Pringle 1974), ∂Σg ∂t = 1 r ∂ ∂r " 3r1/2 ∂ ∂r r1/2νΣg # −˙Σg,photo −˙Σg,planet, (1) (1) (1) In equilibrium, the radiative ﬂux is identical as the viscous dissipation rate, which is given by where Σg = R ∞ −∞ρdz is the surface density of gas, and ˙Σg,photo and ˙Σg,planet are the sink terms related to photo-evaporation (Sect. 3.2.2) and accretion by the planets respectively. The vis- cosity is parametrised, following Shakura & Sunyaev (1973), with where Σg = R ∞ −∞ρdz is the surface density of gas, and ˙Σg,photo and ˙Σg,planet are the sink terms related to photo-evaporation (Sect. 3.2.2) and accretion by the planets respectively. The vis- cosity is parametrised, following Shakura & Sunyaev (1973), with ˙E = Σν r∂Ω ∂r !2 = 9 4ΣνΩ2 (4) (4) with Ωbeing the Keplerian angular frequency at distance r from the star. The second equality holds only if purely the mass of the central star is accounted for in the Keplerian frequency, that is Ω= p GM⋆/r3, G being the gravitational constant. The self- gravity of the disc has been neglected. ν = αcsH (2) (2) ν = αcsH This equation is solved on a grid spaced regularly in log with 3400 points that extends from the inner location of the disc rin (an initial condition) to rmax = 1000 au. At these two locations, the surface density is ﬁxed to zero. The disc’s outer temperature due to irradiation is given by T 4 s = T 4 ⋆ " 2 3π R⋆ r 3 + 1 2 R⋆ r 2 H r ∂ln H ∂ln r −1 !# + T 4 irr + T 4 cd (5) (5) 3.2.1. Vertical structure The disc’s vertical structure is computed at each step of the evo- lution following the approach of Nakamoto & Nakagawa (1994). This change is necessary to accommodate the new stellar model with variable quantities. With this approach, the link between the outer and midplane temperatures is given by following Hueso & Guillot (2005), but also accounting for the direct irradiation through the disc’s mid plane. The ﬁrst term inside the bracket is the irradiation of the star onto a ﬂat disc. The second term in the square brackets accounts for the ﬂaring of the disc at large separation. In our case, we do not compute this factor explicitly and instead adopt ∂ln H/∂ln r = 9/7 (Chiang & Goldreich 1997). following Hueso & Guillot (2005), but also accounting for the direct irradiation through the disc’s mid plane. The ﬁrst term inside the bracket is the irradiation of the star onto a ﬂat disc. The second term in the square brackets accounts for the ﬂaring of the disc at large separation. In our case, we do not compute this factor explicitly and instead adopt ∂ln H/∂ln r = 9/7 (Chiang & Goldreich 1997). σSBT 4 mid = 1 2 3 8τR + 1 2τP ! ˙E + σSBT 4 s (3) (3) The Tirr term accounts for the direct irradiation through the disc midplane. It is computed as with Tmid the disc mid-plane temperature, Ts the temperature due to irradiation (see below), σSB the Stefan-Boltzmann constant, τR and τP are the Rosseland and Planck mean optical depths respectively, and ˙E is the viscous dissipation rate. This formula yields the mid-plane temperature both in the optically-thick (the term with τR) and optically-thin (the term with τP) regimes. The Rosseland optical depth is given by τR = κdisc(ρmid, Tmid)Σ where ρmid = Σ/( √ 2πH) is the central density, H = cs/Ωthe disc’s vertical scale height, cs = p kBTmid/(µmH) the isothermal sound speed, µ = 2.24 the mean molecular weight of the gas, and mH the mass of an hydrogen atom. The opacity κdisc is given by the maximum of the opacities computed according to Bell & Lin (1994) (which accounts for micrometre size with a ﬁxed interstel- lar dust-to-gas ratio of 1%, independently of the dust-to-gas ratio chosen for the solids disc) and Freedman et al. (2014) (which gives molecular opacities for a grain-free gas). A69, page 6 of 44 3.2.2. Disc photoevaporation ˙Σg,photo,ext(r) =  0 for r < βIrg,I ˙Mwind π(r2max−β2 I r2 g,I) otherwise (9) (9) The temporal evolution shows overall a decrease in the sur- face density. A hole forms inside roughly 2 au by about 4.7 Myr. The change in the temperature proﬁle initially between 1.5 and 3 au and that moves inwards is due to a maximum in the opacity (top right panel). This different behaviour is reﬂected in the sur- face density as the temperature affects the sound speed, hence the viscosity. with ˙Mwind a parameter that provides the total mass loss rate if the disc would extend to rmax = 1000 au. In practice however, the actual mass loss rate due to external photoevaporation is clearly smaller than that parameter, as the disc does not extend up to rmax, but to a dynamically obtained radius which results from the interplay of viscous spreading (increasing the outer radius) and external photoevaporation (decreasing the outer radius). For the midplane temperature, the direct irradiation term is only important in the innermost region (within about 0.2 au) until a few hundred thousand years before the dispersal of the gas disc. The last proﬁle before dispersal shows an increase of temperature within 0.2 au due to this contribution; otherwise the midplane temperature remains below the equilibrium temperature, apart from the inner region (≪3 au) at early times. For the internal photoevaporation, we follow Clarke et al. (2001), which in turn is based on ‘weak stellar wind’ case of (Hollenbach et al. 1994). Here, extreme-ultraviolet (EUV; > 13.6 eV) creates a layer of ionised hydrogen whose temperature is TII ≈104 K and with a mean molecular weight µII = 0.68. The sound speed and gravitational radius are computed in analogy with Eqs. (7) and (8). The scaling radius r14 = βIIrg,II/1014 cm follows Clarke et al. (2001) while we select again βII = 0.14 fol- lowing Alexander & Pascucci (2012). With this, we can estimate the base density with We compared the results obtained here with prescriptions from other works, as for instance Bitsch et al. (2015a). We ﬁnd that in general, for a given stellar accretion rate (which is the parametrisation of the Bitsch et al. 2015a prescription) we obtain lower surface density proﬁles by 30% coupled with larger temperature by 20–40%. 3.2.2. Disc photoevaporation ˙Σg,photo = ˙Σg,photo,ext + ˙Σg,photo,int. (12) Photoevaporation in the protoplanetary discs is the principal means of controlling their lifetimes. For the prescription, we follow Mordasini et al. (2012b). In this scheme, we include contributions from both internal (due the host start itself) and external (due to nearby massive stars in the birthplace of the system) sources. (12) 3.2.3. Initial gas surface density proﬁle and example 3.2.3. Initial gas surface density proﬁle and example We initialise the gas surface density proﬁle with (Veras & Armitage 2004) For the external photo-evaporation, we use the far-ultraviolet (FUV) description of Matsuyama et al. (2003). FUV radia- tion (6–13.6 eV) creates a neutral layer of dissociated hydrogen whose temperature is TI ≈103 K. The corresponding sound speed is then Σg(t = 0) = Σg,0 r r0 !−βg exp − r rcut,g !2−βg 1 − r rin r ! (13) (13) where r0 = 5.2 au is the reference distance, βg = 0.9 the power- law index (Andrews et al. 2010), rcut,g the characteristic radius for the exponential decay and rin the inner edge of the disc. c2 s,I = kBTI µImH , (7) c2 s,I = kBTI µImH , ( ) ,g for the exponential decay and rin the inner edge of the disc. (7) The conversion between the total mass and the normalisation surface density Σg,0 at r0 is obtained with where the mean molecular weight µI = 1.35 for the dissociated gas. It corresponds to the gravitational radius (where the sound speed equals the escape velocity) of Mg = 2πΣg,0 2 −βg (r0)βg rcut,g 2−βg . (14) (14) rg,I = GM⋆ c2 s,I . (8) It should be noted that this formula neglects the lack of gas within rin, but since the total mass is dominated by the outer disc as we have a shallow power-law, there has in practice very limited effect. rg,I = GM⋆ c2 s,I . (8) We assume that mass is removed uniformly outside of βIrg,I with βI = 0.14 (similar to Alexander & Pascucci 2012), so that the rate is given by An example of evolution of such as disc, without any planets (i.e. ˙Σg,planet = 0), is provided in Fig. 3. The initial conditions and parameter are provided in Table 1 (note that the table also contains planetesimals disc properties that are not used here). The lifetime of that disc is nearly 5.3 ×106 yr. 3.2.1. Vertical structure The distance-dependent base density can then be calculated as which is the ionising photon luminosity in the units of 1041 s−1. The distance-dependent base density can then be calculated as in which a star and its planetary system are born (Krumholz 2006; Ndugu et al. 2018). On the other hand, different cluster environments and thus different levels of the interstellar FUV ﬁeld (Fatuzzo & Adams 2008) are taken into account by varying in the population syntheses (see Paper II) the magnitude of the external photoevaporation rate ˙Mwind. External photoevaporation is likely the most important environment-related factor for discs (Winter et al. 2020). n0(r) = n0(r14) r rg,II !−5 2 . (11) (11) We further follow Clarke et al. (2001) to get ˙Σg,photo,int = 2cs,IIn0mH outside of βIIrg,II. The ﬁnal photoevaporation rate is given by the sum of the effects of host star + nearby massive stars with 3.2.1. Vertical structure For the Planck optical depth, we follow further Nakamoto & Nakagawa (1994) and set τP = 2.4τR. with Tmid the disc mid-plane temperature, Ts the temperature due to irradiation (see below), σSB the Stefan-Boltzmann constant, τR and τP are the Rosseland and Planck mean optical depths respectively, and ˙E is the viscous dissipation rate. This formula yields the mid-plane temperature both in the optically-thick (the term with τR) and optically-thin (the term with τP) regimes. The Rosseland optical depth is given by τR = κdisc(ρmid, Tmid)Σ where ρmid = Σ/( √ 2πH) is the central density, H = cs/Ωthe disc’s vertical scale height, cs = p kBTmid/(µmH) the isothermal sound speed, µ = 2.24 the mean molecular weight of the gas, and mH the mass of an hydrogen atom. The opacity κdisc is given by the maximum of the opacities computed according to Bell & Lin (1994) (which accounts for micrometre size with a ﬁxed interstel- lar dust-to-gas ratio of 1%, independently of the dust-to-gas ratio chosen for the solids disc) and Freedman et al. (2014) (which gives molecular opacities for a grain-free gas). For the Planck optical depth, we follow further Nakamoto & Nakagawa (1994) and set τP = 2.4τR. T 4 irr = L⋆ 16πr2σSB e−τmid, (6) (6) which is the black-body equilibrium temperature accounting for the optical depth through the midplane of the disc τmid = R which is the black-body equilibrium temperature accounting for the optical depth through the midplane of the disc τmid = R ρmidκ(ρmid, Tmid)dr. This contribution is usually important only at the very end of the disc lifetime while it clears; otherwise, the optical depth conﬁnes the contribution to the very innermost region. However, taking this contribution in account is necessary to provide a smooth transition of the temperature at the surface of the planets (see Sect. 4.1) from the time when they are embed- ded in the nebula to the time when they are exposed to the direct stellar irradiation. The last term accounts for the heating by the surround- ing environment (molecular cloud), which we set constant to Tcd = 10 K. We thus neglect possible variations of this back- ground temperature depending on the stellar cluster environment A69, page 6 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. which is the ionising photon luminosity in the units of 1041 s−1. 3.2.2. Disc photoevaporation The two models cannot be very well compared directly due to the different underlying assumptions, like constant radial ﬂow rate in Bitsch et al. (2015a). Our mod- els accounts for the full evolutionary equation for the surface density including photoevaporation and gas accretion by the pro- toplanets, which means we have the radial ﬂow rate varying with n0(r14) = kHolΦ1/2 41 r−3/2 14 , (10) (10) n0(r14) = kHolΦ1/2 41 r−3/2 14 , where we set kHol = 5.7 × 104 following the hydrodynamical simulations of Hollenbach et al. (1994) and Φ41 = 0.1 √M⋆/M⊙, where we set kHol = 5.7 × 104 following the hydrodynamical simulations of Hollenbach et al. (1994) and Φ41 = 0.1 √M⋆/M⊙, A69, page 7 of 44 A69, page 7 of 44 A69, page 7 of 44 A&A 656, A69 (2021) 10−1 100 101 102 103 Distance [AU] 10−3 10−2 10−1 100 101 102 103 104 Gas surface density [g/cm2] 10−1 100 101 102 103 Distance [AU] 10−4 10−3 10−2 10−1 100 Opacity [cm2/g] 10−1 100 101 102 103 Distance [AU] 101 102 103 Midplane temperature [K] 10−1 100 101 102 103 Distance [AU] −10−7 −10−8 −10−9 −10−10 0 10−10 10−9 10−8 10−7 Radial gas ﬂow [M/yr] 0 1 2 3 4 5 Time [106 yr] Fig. 3. Time evolution of the surface density (top left), opacity κ (top right), midplane temperature (bottom left), and radial ﬂow rate (bottom right) of a protoplanetary disc. The lines represent each one snapshot the state, and are spaced by about 2 × 105 yr. The blue line in both panels shows the nitial proﬁle, which has not yet been evolved at all, and is therefore not in equilibrium. The green line in the temperature proﬁle shows the proﬁle at disc’s dispersal, which is given by the equilibrium temperature with the host star’s luminosity. 10−1 100 101 102 103 10−3 10−2 10−1 100 101 102 103 104 Gas surface density [g/cm2] Radial gas ﬂow [M/yr] Radial gas ﬂow [M Radial g Fig. 3. Time evolution of the surface density (top left), opacity κ (top right), midplane temperature (bottom left), and radial ﬂow rate (bottom right) of a protoplanetary disc. The lines represent each one snapshot the state, and are spaced by about 2 × 105 yr. 3.3.1. Dynamical state distance (bottom right panel of Fig. 3, where the radially con- stant inﬂow in the inner disc, and the viscous spreading (outﬂow) in the outer disc can be seen). There are other model assump- tions that result in the differences between the surface density and temperature in the two models: (1) the stellar luminosity, which in our case it starts with roughly 3 L⊙as predicted by the Baraffe et al. (2015) tracks whereas Bitsch et al. (2015a) begins with 1.5 L⊙following Baraffe et al. (1998), (2) the opacity which affects the relation between midplane and disc photo- spheric temperature, and (3) the different approach of including stellar irradiation (vertically integrated assuming an equilibrium for the ﬂaring angle versus an explicit 1D vertical structure with radiative transfer). For the time evolution of the dynamical state, we use the approach of Fortier et al. (2013) and explicitly solve the dif- ferential equations describing the change of eccentricity and inclination. In this framework, these are stirred by both the pro- toplanets, and to a lesser extent the other planetesimals, and damped by drag from the gas disc. The equations for the root mean square (RMS) of the planetesimals’ eccentricity eplan and inclination iplan read as ˙e2 plan = ˙e2 plan drag + ˙e2 plan VS,M + ˙e2 plan VS,plan (15) ˙i2 plan = ˙i2 plan drag + ˙i2 plan VS,M + ˙i2 plan VS,plan . (16) (15) (16) The contributions from the aerodynamical drag, stirring by the protoplanets and the planetesimals are denoted by ‘drag’, ‘VS,M’ and ‘VS,plan’ respectively. The dynamical state is followed dur- ing the entire formation stage. The drag term is only evaluated while the gas disc is still present. After the dissipation of the gas disc, the term is set to 0. 3.2.2. Disc photoevaporation The blue line in both panels shows the initial proﬁle, which has not yet been evolved at all, and is therefore not in equilibrium. The green line in the temperature proﬁle shows the proﬁle at disc’s dispersal, which is given by the equilibrium temperature with the host star’s luminosity. A69, page 8 of 44 3.3. Planetesimal disc Planetesimals are represented by a ﬂuid-like description, that is they are modelled not as individual particles but on a grid as a surface density (Σs) with eccentricity (eplan) and inclination (iplan) as dynamical state. A69, page 8 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. is the gas drag time scale and CD = 1. In the Stokes regimes the drag expressions are is the gas drag time scale and CD = 1. In the Stokes regimes the drag expressions are Table 1. Initial conditions and parameters for the example system. Table 1. Initial conditions and parameters for the example system. Quantity Value Stellar mass M⋆ 1 M⊙ Reference surface density Σg,0 at 5.2 au 145 g cm−2 Initial gas disc mass Mg 3.90 × 10−2 M⊙ Inner edge of the gas disc rin 0.091 au (10 d) Characteristic radius of the gas disc rcut,g 66.5 au Disc viscosity parameter α 2 × 10−3 External photoevaporation rate ˙Mwind 6.42 × 10−7 M⊙yr−1 Power law index of the gas disc βg 0.9 Dust-to-gas ratio 3.4% Planetesimal disc mass 348 M⊕ Power law index of the solids disc βs 1.5 Characteristic radius of the solids disc rcut,s rcut,g/2 Planetesimal radius 300 m Planetesimal density (rocky) 3.2 g cm−3 Planetesimal density (icy) 1 g cm−3 Embryo mass Memb,0 1 × 10−2 M⊕ Opacity reduction factor fopa 3 × 10−3 Notes. The upper part contains the gas disc properties, the middle part the planetesimals disc properties, and the bottom part show planetary embryos properties. ˙e2 plan drag = −3 2 λρmide2 plan ρplanR2 plan (22) ˙i2 plan drag = −3 4 λρmidi2 plan ρplanR2 plan , (23) (22) (23) while in the Epstein regime they read as while in the Epstein regime they read as ˙e2 plan drag = −e2 plan csρmid ρplanRplan (24) ˙i2 plan drag = − i2 plan 2 csρmid ρplanRplan (25) (24) (25) (Adachi et al. 1976; Raﬁkov 2004; Fortier et al. 2013). We also i h d d l h f i f i (Adachi et al. 1976; Raﬁkov 2004; Fortier et al. 2013). We also want to point out that we do not model the formation of gap in the gas disc by giant planets. This means that drag in the vicinity of such planets might be overestimated, resulting in lower eccen- tricities and inclination. 3.3. Planetesimal disc As consequence, the accretion rate of planetesimals would be overestimated in this stage, which affects the heavy element contents of the planets. Notes. The upper part contains the gas disc properties, the middle part the planetesimals disc properties, and the bottom part show planetary embryos properties. As in Fortier et al. (2013), the stirring by the protoplanets follows the approach of Guilera et al. (2010), where the stirring of Ohtsuki et al. (2002) is modulated with the separation from the protoplanets. The contribution reads as The form of the drag term depends on the regime: Epstein, Stokes (laminar) or quadratic (turbulent). The distinc- tion between those regimes is made using the criterion proposed by Raﬁkov (2004) using the molecular Reynolds number Remol = vrelRplan/νmol, where νmol = λcs/3 is the molecular viscosity, λ = (nH2σH2)−1 the gas molecules’ mean free path, nH2 the number density assuming all of the gaseous molecules having hydrogen mass, σH2 their collisional cross-section, Rplan the planetesimals’ radius, ˙e2 plan VS,M = n X =1 f∆,  ΩMplanet,  6πbM⋆ ! PVS (26) ˙i2 plan VS,M = n X =1 f∆,  ΩMplanet,  6πbM⋆ ! QVS (27) (26) (27) where the sum is over all the protoplanets present in the system, (17) f −1 ∆, = 1 + \|r −aplanet, \| bRH !5 (28) vrel = vK q η2 + 5/8e2 plan + 1/2i2 plan (17) (28) (17) their relative velocity, is the modulation due to separation so that the perturbation is effectively restricted to the planet’s feeding zone, η = − 1 2Ωrρmid ∂p ∂r (18) (18) RH = aplanet,  3 s Mplanet,  3M⋆ (29) = 2Ωrρmid ∂r (29) the deviation between the gas and Keplerian velocities due the support of the gas by the radial pressure gradient, ρmid the mid- plane gas density, and vK = Ωr the Keplerian velocity. When Rplan < λ, the gas drag is assumed to be in the Epstein regime. Otherwise, if Remol > 20, the gas drag is taken to be in the quadratic (or turbulent) regime and in the Stokes regime if not. the planet’s Hills radius, and b = 5 is the half-width of the feed- ing zone (see Sect. 4.3.3). The terms PVS and QVS are given by (Fortier et al. 3.3. Planetesimal disc Including in the Bern Model an explicit model for the evolution of the solid building blocks across the entire size range (dust-pebble-planetesimals) is thus subject of ongoing research. A ﬁrst important step was recently made in Voelkel et al. (2020) where we have coupled the dust-and-pebble model of Birnstiel et al. (2012) and the planetes- imal formation model of Lenz et al. (2019) to our global model. These effects are, however, not yet included in the Generation III Model presented here. hplan = 3 s 2Mplan 3M⋆ , (36) hplan = 3 s 2Mplan 3M⋆ , (36) and Mplan = 4/3πR3 planρplan, the mass of a planetesimal. and Mplan = 4/3πR3 planρplan, the mass of a planetesimal. p To set the initial dynamical state, we assume that the disc is initially in a cold state, that is only the self-stirring of the planetesimals contributes to their eccentricities and inclinations. In other words, this assumes that the embryos appear instantly at the beginning of the simulation. The equilibrium values can be derived by equating the contributions of self-stirring and damping (Thommes et al. 2003; Chambers 2006), which results in eplan = 2.31 M4/15 plan r1/5ρ2/15 plan Σ1/5 g C1/5 D ρ1/5M2/5 ⋆ (37) and iplan = 1 2eplan. (38) eplan = 2.31 M4/15 plan r1/5ρ2/15 plan Σ1/5 g C1/5 D ρ1/5M2/5 ⋆ (37) (37) and iplan = 1 2eplan. (38) (38) We also compared our prescription for the dynamical state with gamma-stirring from, for instance, Ida et al. (2008) and Okuzumi & Ormel (2013). Although this is not straightforward due to the differences in the sources, we ﬁnd that, generally, the eccentricities resulting from γ-stirring are larger than the self- stirring from the planetesimals, but lower than the stirring by the forming protoplanets. Thus, accounting for the stirring of plan- etesimals by turbulent diffusion in the disc would increase their eccentricities at locations far away from growing protoplanets. Close to the growing protoplanets however, where the planetes- imals’ eccentricities are important for the solids accretion rate, neglecting this effect does not signiﬁcantly affect planetesimals’ eccentricities. 3.3. Planetesimal disc 2009), and the theoretical predictions of planetesimal formation models (e.g. Klahr & Schreiber 2020) rather point at ∼100 km plan- etesimals. The ﬁrst two points can, however, also be explained with other effects (Zheng et al. 2017; Wei et al. 2018, although the former work makes no determination about the initial size frequency distribution of planetesimals). Λ = ˜ıplan(˜e2 plan + ˜ı2 plan)/12, β = iplan/eplan, while for IPVS and IQVS we use the approximations obtained by Chambers (2006): Λ = ˜ıplan(˜e2 plan + ˜ı2 plan)/12, β = iplan/eplan, while for IPVS and IQVS we use the approximations obtained by Chambers (2006): As already in the Generation II Model (Alibert et al. 2013), we assume a constant planetesimals radius of 300 m through- out the disc, which is a strong assumption and simpliﬁcation. IPVS(β) ≃ β −0.36251 0.061547 + 0.16112β + 0.054473β2 , (32) IQVS(β) ≃ 0.71946 −β 0.21239 + 0.49764β + 0.14369β2 . (33) (33) The stirring by the other planetesimals is given by, following Ohtsuki et al. (2002), ˙e2 plan VS,plan = 1 6 s Gr M⋆ ΣshplanPVS (34) ˙i2 plan VS,plan = 1 6 s Gr M⋆ ΣshplanQVS (35) (34) (35) with q y p ) In the more speciﬁc context of the simulations presented here, this choice was made for the following reasons: (1) small planetesimals undergo sufﬁcient eccentricity and inclination damping by the disc gas to sustain a planetesimal accretion rate in the oligarchic growth regime that is high enough to build giant planet cores during typical disc lifetimes (Fortier et al. 2013). We note that the Generation I and Ib Bern Models assumed in contrast runaway planetesimal accretion as Pollack et al. (1996). In the runaway regime, the eccentricities and inclinations of the planetesimals are assumed to remain low even without damping by the disc gas. Therefore, fast core growth occurred in these models also with 100 km planetesimals, which was the assumed size in these early model generations. (2) Their drift time scales are longer than typical lifetimes of gas discs (Burn et al. 2019) and (3) this size was shown to be able to reproduce several of the known exoplanet properties across a wide range of masses (Fortier et al. 2013). In any case, the constant planetesimal size is an important limitation of the model. 3.3. Planetesimal disc 2013), PVS =  73˜e2 plan 10Λ2 ln 1 + 10Λ2/˜e2 plan + "72IPVS(β) π˜eplan˜ıplan # ln 1 + Λ2 (30) QVS =  4˜ı2 plan + 0.2˜ıplan˜e3 plan 10Λ2˜eplan ln 1 + 10Λ2˜e2 plan + "72IQVS(β) π˜eplan˜ıplan # ln 1 + Λ2 . (31) The expressions for the drag in the quadratic regimes are (Adachi et al. 1976; Chambers 2006), ˙e2 plan drag = − e2 plan τdrag r η2 + 5 8e2 plan + 1 2i2 plan (19) ˙i2 plan drag = − i2 plan 2τdrag r η2 + 5 8e2 plan + 1 2i2 plan, (20) where + "72IPVS(β) π˜eplan˜ıplan # ln 1 + Λ2 (30) QVS =  4˜ı2 plan + 0.2˜ıplan˜e3 plan 10Λ2˜eplan ln 1 + 10Λ2˜e2 plan + "72IQVS(β) π˜eplan˜ıplan # ln 1 + Λ2 . (31) ˙e2 plan drag = − e2 plan τdrag r η2 + 5 8e2 plan + 1 2i2 plan (19) ˙i2 plan drag = − i2 plan 2τdrag r η2 + 5 8e2 plan + 1 2i2 plan, (20) where QV (30) (19) (31) τdrag = 8ρplanRplan 3CDρmidvK τdrag = 8ρplanRplan 3CDρmidvK (21) Here, ˜eplan = replan/RH and ˜ıplan = riplan/RH are respec- tively the reduced planetesimals’ eccentricity and inclination, A69 9 f 44 τdrag = 8ρplanRplan 3CDρmidvK (21) Here, ˜eplan = replan/RH and ˜ıplan = riplan/RH are respec- tively the reduced planetesimals’ eccentricity and inclination, (21) Here, ˜eplan = replan/RH and ˜ıplan = riplan/RH are respec- tively the reduced planetesimals’ eccentricity and inclination, A69, page 9 of 44 A&A 656, A69 (2021) As already in the Generation II Model (Alibert et al. 2013), we assume a constant planetesimals radius of 300 m through- out the disc, which is a strong assumption and simpliﬁcation. There is an ongoing discussion about the characteristic primor- dial planetesimal size in the literature. Observations of extrasolar debris belts (Krivov & Wyatt 2021), the presence of hyper- volatile ices in comets that can only be preserved in impacts involving small bodies (Golabek & Jutzi 2021), direct size deter- minations by stellar occultations (Arimatsu et al. 2019) and some theoretical studies (Fraser 2009; Schlichting et al. 2013) suggest small (∼1 km) characteristic planetesimals sizes. On the other hand, the absence of small craters on Pluto (Singer et al. 2019), the size distribution in the asteroid belt (Morbidelli et al. 3.3. Planetesimal disc To set the initial surface density proﬁle of planetesimals, we thus use a slightly different description than for the gas, that is, Σs(t = 0) = Σs,0 fs(r) r r0 !−βs exp − r rcut,s !2 (39) (39) with the power-law exponent is set to βs = 1.5, as in the MMSN, and rcut,s = rcut,g/2 is the exponential cutoff radius of the solids, set half the value of the gas disc following Ansdell et al. (2018). This formula also enables us to model relatively sharp outer edges of the solids disc (Birnstiel & Andrews 2014). 3.3.2. Size, initial surface density proﬁle, and evolution The reference surface density value Σs,0 is adjusted so that the bulk solids-to-gas ratio remains to the prescribed value (e.g. 1%). To roughly take into account the observational (e.g. Ansdell et al. 2018) and theoretical (e.g. Birnstiel & Andrews 2014) ﬁnding that solids have a more concentrated distribution than the gas, the initial surface density proﬁle of planetesimals now follows a steeper slope than the one of the gas disc (Lenz et al. 2019; Voelkel et al. 2020). This leads to a higher concentration of solids in the inner part of the disc. The surface density of planetesimals is reduced by accretion onto and ejection by the protoplanets to ensure mass conser- vation (see Sect. 4.3), or removed entirely if e2 plan > 0.95. Our model only includes ejection (Sect. 4.3.2) and not scattering by the forming planets. Thus, we do not have redistribution of A69, page 10 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. except for the giant planets where 65% of planetesimals were ejected and 35% accreted. material to other regions of the disc by planets, as obtained by Raymond & Izidoro (2017) for instance. Finally, the planetesi- mals disc remains after the dispersal of the gas disc; the only difference is that the damping terms for eccentricity ˙e2 plan drag and The stirring by the protoplanets heats the planetesimals in the surrounding region. This effect is heavily dependent on the protoplanet’s masses; the most massive one is the second outer- most one (close to 10 au), which reaches a mass of about 5.4 MX at the end of the formation stage. That planet has a core mass of 47 M⊕, which corresponds (for a pure H/He envelope) to a metal- licity slightly lower than that of the star (2.8% versus 3.0%). This is below the relationship found by Thorngren et al. (2016) for the planet’s mass. This, however, is not unexpected for the idealised setup used here: ﬁrst, planets that form in the in-situ case tend to have lower core masses than planets that migrated (e.g. Alibert et al. 2005b). Second, with N-body interactions switched off here, giant impacts otherwise increasing the heavy element content are not possible. In the more realistic example in Sect. 4.1. Envelope structure In the Bern model, the internal structure of the planets (and thus their gas accretion rate, radius, luminosity, and interior struc- ture) are found at all stages (attached, detached, evolution) by directly solving the 1D structure equations. In contrast, many other global models use in contrast approximations and ﬁts to ﬁnd for example the gas accretion rate (see Alibert & Venturini 2019). While the 1D hydrostatic picture is also not the ﬁnal word for low-mass planets because of multidimensional hydro- dynamic effects (e.g. Ormel et al. 2015; Lambrechts & Lega 2017; Cimerman et al. 2017; Moldenhauer et al. 2021), the ﬁts (except the deep neural networks) often fail grossly to reproduce the result of 1D structure equations that they should in principle recover (Alibert & Venturini 2019). Many ﬁts also neglect the inﬂuence of the luminosity on the gas accretion rate (e.g. Ida & Lin 2004a; Bitsch et al. 2015b). In reality, there is an important interplay between solid accretion which is dominant for the lumi- nosity at early stages, and gas accretion. This leads to important feedbacks that can only be captured when solving the internal structure equations (Dittkrist et al. 2014). 3.3.2. Size, initial surface density proﬁle, and evolution 8.1.2, where these effects are included, giant impacts strongly increase the solid content of the giant planets by a fac- tor 2–3 relative to value at the moment gas runaway begins. The impacts are themselves triggered by the fast mass growth, destabilising neighbouring lower mass protoplanets. inclination ˙i2 plan drag vanish. inclination ˙i2 plan drag vanish. 3.3.3. Compositional model The Bern model includes the simple condensation model of Thiabaud et al. (2014) and Marboeuf et al. (2014a). The initial abundance of volatile and refractory species is identical to the one given in Marboeuf et al. (2014b). Volatile species are com- posed of H, O, C, and S atoms whose abundance reﬂect solar composition (Lodders 2003). The relative abundances of the molecules are set according to interstellar medium. Then at each location in the disc at t = 0, we check whether each molecule is the solid or gas phase assuming local thermodynamical equilib- rium. This yields the fraction of heavy elements that is locally condensed and thus contributes to the solid surface density (the ice line locations), and the chemical composition. This com- position is tracked into the protoplanets when a propotoplanet accretes planetesimals, and in giant impacts between protoplan- ets. This yields in particular the ﬁnal iron to silicate ratio and the volatile mass fraction of all the planets. As noted by Fortier et al. (2013), the usual assumption that β = iplan/eplan ≈1/2 does not hold. We ﬁnd that the stirring of eccentricities takes place over larger separation to the protoplan- ets than for the inclinations. This can be seen for instance in the region affected by the most massive planet. The factor fs(r) in Eq. (39) for the initial planetesimal surface density accounts for the mass fraction of all elements that are in the solid phase at a given location. To compute its value, we use the aforementioned condensation model. Only the contribution of molecules in the solid phase are accounted for the resulting solid surface density. Thus, the value of fs in the inner locations is the mass fraction of condensed to total solids and this value increases by small jumps each time an ice line is crossed until it becomes unity at large separation. Further, the effect of the planet is not only limited to the sur- rounding area because of the following effect: the massive planet is able to signiﬁcantly reduce the inward gas ﬂow such that the region inside its orbit becomes gas-poor. This greatly reduces the damping of the planetesimals dynamical state to a such point that their eccentricity becomes close to unity. 4. Planet properties For the density of planetesimals, we assume that in the region where only refractory materials contributes to the solid phase ρplan = 3.2 gcm−3 while when volatiles are in the solid phase we take ρplan = 1 gcm−3. This transition corresponds to the H2O-ice line in all discs, which induces the largest surface density jump because H2O makes up ∼60% of all ices in mass (Marboeuf et al. 2014a). 3.3.4. Example The blue line in both panels denote the initial proﬁle. The dashed vertical lines represent the location of the embryos, which is ﬁxed in this ase. N-body interactions were also disabled. The lifetime of the gas disc is shorter than the case presented in Fig. 3 due to the accretion by the protoplanets. 10−1 100 101 102 103 10−5 10−4 10−3 10−2 10−1 100 RMS of planetesimals eccentricity 10−1 100 101 102 103 10−5 10−4 10−3 10−2 10−1 100 RMS of planetesimals inclination Location [AU] Location [AU] Location [AU] 10−1 100 101 102 103 Location [AU] 10−3 10−2 10−1 100 101 102 103 Planetsimals surface density [g/cm2] 0.12M⊕ 0.48M⊕ 0.56M⊕ 1.0M⊕ 1.4M⊕ 2.5M⊕ 4.4M⊕ 6.9M⊕ 1709M⊕ 0.02M⊕ Fig. 4. Time evolution of the RMS of planetesimals’ eccentricity (top left), inclination (top right), and surface density (bottom left) of a circum- stellar disc that also contains 10 embryos. The lines represent temporal snapshots of the three quantities, and are spaced by about 2 × 105 yr. The blue line in both panels denote the initial proﬁle. The dashed vertical lines represent the location of the embryos, which is ﬁxed in this case. N-body interactions were also disabled. The lifetime of the gas disc is shorter than the case presented in Fig. 3 due to the accretion by the protoplanets. just with methods measuring quantities depending on the plan- ets’ mass (like RV, astrometry or microlensing), but also transit and direct imaging surveys. rate is governed by the ability to radiate the gravitational energy liberated by the accretion of solids and gas, and the envelope’s contraction. For the forming giant planets, this phase generally lasts until the planets reach a total mass in the range of 30– 100 M⊕where envelope contraction becomes fast, depending on the conditions. There is no ﬁxed mass boundary; the transi- tion occurs when the gas accretion rate obtained from solving the internal structure equations (that is the envelope’s Kelvin- Helmholtz contraction) becomes larger than the disc-limited rate (Sect. 4.1.2. For low-mass planets which have very low gas accre- tion rates (very long Kelvin-Helmholtz timescales), the attached phase lasts (almost) until the gas disc dissipates. The downside is that solving the internal structure for bod- ies ranging in mass from 10−2 M⊕to beyond the deuterium limit requires an internal structure model that is very versatile and numerically stable in all stages of planetary formation and evo- lution. 3.3.4. Example An example of the dynamical state of planetesimals is provided in Fig. 4. The initial conditions and parameters are provided in Table 1. This is the same initial disc as shown in Fig. 3, except than ten embryos were added to the disc, at the locations shown by the dashed vertical lines. In addition, both migration and N- body interactions were artiﬁcially disabled so that the embryos remain at the same location throughout the simulation. The different jumps in the initial surface density proﬁle are due to the crossing of the different ice lines; the most con- sequential one at about 3 au is due to the water-ice line. The surface density of planetesimals is equalised inside the feeding zone of each planet. It should be noted that besides this effect, we do not include planetesimals redistribution, as was found by, for instance, Levison et al. (2010). In total, the planets accreted 61 M⊕of planetesimals (47 M⊕of which by the giant planet) while 89 M⊕were ejected (according to the prescription detailed in Sect. 4.3.2; virtually all of them by the giant planet). The feed- ing zones are all nearly depleted by the planets due to accretion, Also, from the point of view of guiding and interpreting astronomical observations, it is crucial to solve the internal struc- ture equations, as this gives self-consistently at each moment in time the planet’s radius and luminosity and associated magni- tudes. These are the observable quantities for transit and direct imaging surveys. By predicting them self-consistently, the output of the Bern model can be compared in population syntheses not A69, page 11 of 44 A69, page 11 of 44 A&A 656, A69 (2021) A&A 656, A69 (2021) 10−1 100 101 102 103 Location [AU] 10−5 10−4 10−3 10−2 10−1 100 RMS of planetesimals eccentricity 10−1 100 101 102 103 Location [AU] 10−5 10−4 10−3 10−2 10−1 100 RMS of planetesimals inclination 10−1 100 101 102 103 Location [AU] 10−3 10−2 10−1 100 101 102 103 Planetsimals surface density [g/cm2] 0.12M⊕ 0.48M⊕ 0.56M⊕ 1.0M⊕ 1.4M⊕ 2.5M⊕ 4.4M⊕ 6.9M⊕ 1709M⊕ 0.02M⊕ 0 1 2 3 4 Time [106 yr] Fig. 4. Time evolution of the RMS of planetesimals’ eccentricity (top left), inclination (top right), and surface density (bottom left) of a circum- tellar disc that also contains 10 embryos. The lines represent temporal snapshots of the three quantities, and are spaced by about 2 × 105 yr. 3.3.4. Example Solving the internal structure also comes with signiﬁcant computational cost. A69, page 12 of 44 4.1.2. Maximum gas accretion rate with L being the luminosity. with L being the luminosity. with L being the luminosity. The opacity in the envelope κ is obtained in similar way as for the gas disc, but following Mordasini et al. (2014), the interstellar medium (ISM) grain opacity contribution in Bell & Lin (1994) is multiplied by a factor fopa = 0.003. This value was found in Mordasini et al. (2014) to ﬁt best the detailed simulations by Movshovitz & Podolak (2008) and Movshovitz et al. (2010) of the grain dynamics in protoplanetary atmospheres (growth, set- tling) and the resulting dust opacities. Using one global reduction factor of the ISM opacity can of course not reproduce the full complex behaviour of the grain opacity which depends on plan- etary properties like the core or envelope mass as found in grain dynamics models (Movshovitz & Podolak 2008). But as shown in Mordasini et al. (2014), it still provides a useful ﬁrst approxi- mation. The value is not increased when a planetary system with higher metallicity is simulated. The reason is that a higher dust input in the outermost layer (as possibly associated with a high metallicity system) does not lead to a strong increase of the opac- ity. This was found numerically in Movshovitz & Podolak (2008) and explained analytically in Mordasini (2014): a higher dust input leads to a higher dust-to-gas mass ratio (which increases the opacity), but also larger grains (which decreases the opacity). These effects cancel each other out in the dominating growth regime of differential settling. In the initial stages, the gas accretion is limited by the planet’s ability to radiate away the potential energy provided of the accre- tion material, that is the Kelvin-Helmholtz process. The rate at which gas can be accreted is set by the Kelvin-Helmholtz time scale, τKH = GM2 tot RtotLtot . (49) τKH = GM2 tot RtotLtot . (49) However, as the planet’s core reaches a mass of about 10 M⊕, the value of τKH becomes so low that the planet undergo runaway gas accretion. In this phase, the amount of gas that the planet can accrete is constrained by the supply from the gas disc. There- fore, we compute the quantity ˙Menv,max, which is used to limit the value of ˙Menv found by solving the internal structure equations. Our approach to compute the maximum rate is similar to Mordasini et al. 4.1.2. Maximum gas accretion rate (2012c) but using only the ‘local reservoir’ com- ponent. This a major difference from the previous versions of the Bern model, where gas accretion was constrained from the radial ﬂow of the gas. Following D’Angelo & Lubow (2008) and Zhou & Lin (2007), we adopt a Bond- or Hill-like accretion in a region of size Rgc around the planet. For simplicity, we compute Rgc according to Eq. (44). Depending on the value of Rgc with respect to H, the local disc’s scale height, two different regimes occur. In the case where Rgc < H, the planet will not accrete from the full vertical extent of the disc, and so the gas ﬂow through the gas capture cross section σcross = πR2 gc is given by The boundary conditions for the integration are taken as follows: the outer radius is given by, following Lissauer et al. (2009), 1 Rtot = 1 k1Racc + 1 k2RH , (44) (44) (50) ˙Menv,max,3D = ρσcrossvrel (50) with ρ ≈Σ/H the approximate density of the gas and vrel = max (ΩRtot, cs) the relative velocity between the gas and the planet. where Racc = GMtot c2s (45) Racc = GMtot c2s (45) p On the other hand, in the case Rgc > H, the planet will accrete from the whole gas column and the approximation of constant gas density breaks down. In this situation, the gas ﬂow through the planet’s capture radius is provided only by the radial extension of the gas capture area, hence we have is the Bondi radius, RH is the Hill’s radius (Eq. (29)), k1 = 1 and k2 = 1/4. The pressure and temperature are derived from the local properties of the disc with is the Bondi radius, RH is the Hill’s radius (Eq. (29)), k1 = 1 and k2 = 1/4. The pressure and temperature are derived from the local properties of the disc with ˙Menv,max,2D = 2RgcΣvrel. 4.1.1. Attached phase Once Mtot is found, the envelope mass can be retrieved by Menv = Mtot −Mcore, and the gas accretion rate by taking the difference of the envelope mass between two successive steps of the envelope structure calculation ˙Menv = (Menv(t) −Menv(t −∆t))/∆t. ∂P/∂R = −GMρ/R2 (41) (42) ∂T/∂R = ∂P/∂R min (∇ad, ∇rad), with M the mass enclosed in the radius R, P the pressure, T the temperature, ρ = ρ(P, T) the density, computed using the SCvH equation of state (Saumon et al. 1995), and ∇ad and ∇rad the adi- abatic and radiative gradients respectively. The minimum of the two indexes is the Schwarzschild criterion (e.g. Kippenhahn & Weigert 1994), and is used to ensure stability against convection. The adiabatic gradient comes from the equation of state, while the radiative gradient is given by with M the mass enclosed in the radius R, P the pressure, T the temperature, ρ = ρ(P, T) the density, computed using the SCvH equation of state (Saumon et al. 1995), and ∇ad and ∇rad the adi- abatic and radiative gradients respectively. The minimum of the two indexes is the Schwarzschild criterion (e.g. Kippenhahn & Weigert 1994), and is used to ensure stability against convection. The adiabatic gradient comes from the equation of state, while the radiative gradient is given by ∇rad = 3κL 64πσSBGMT 3 , (43) (43) 4.1.1. Attached phase Gas accretion is calculated by solving the classical 1D radi- ally symmetric internal structure equations (Bodenheimer & Pollack 1986), In the initial phase, known as the attached phase, the envelope is in equilibrium with the gas disc and the gas density smoothly transitions from the value in the protoplanetary envelope to the one in the background nebula. The planets do not yet have a well-deﬁned outer radius. During this phase, the gas accretion (40) ∂M/∂R = 4πR2ρ ∂M/∂R = 4πR2ρ ∂M/∂R = 4πR2ρ A69, page 12 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. ∂P/∂R = −GMρ/R2 (41) ∂T/∂R = ∂P/∂R min (∇ad, ∇rad), (42) ∂P/∂R = −GMρ/R2 (41) being the optical depth at the surface of the planet (Mordasini et al. 2012c), using the reduced opacities for the grains. The more complex parts come from the luminosity and the mass. The cal- culation of the outer luminosity L(Rtot) is described in Sect. 4.2. In the case of the mass, what is known is the core mass, that is M(Rcore) = Mcore, while M(Rtot) = Mtot is the quantity that is being searched for. We thus use an iterative method by guess- ing Mtot, which is then used to integrate the internal structure equations until the boundary condition at the inner boundary is fulﬁlled, that is M(Rcore) = Mcore. Once Mtot is found, the envelope mass can be retrieved by Menv = Mtot −Mcore, and the gas accretion rate by taking the difference of the envelope mass between two successive steps of the envelope structure calculation ˙Menv = (Menv(t) −Menv(t −∆t))/∆t. being the optical depth at the surface of the planet (Mordasini et al. 2012c), using the reduced opacities for the grains. The more complex parts come from the luminosity and the mass. The cal- culation of the outer luminosity L(Rtot) is described in Sect. 4.2. In the case of the mass, what is known is the core mass, that is M(Rcore) = Mcore, while M(Rtot) = Mtot is the quantity that is being searched for. We thus use an iterative method by guess- ing Mtot, which is then used to integrate the internal structure equations until the boundary condition at the inner boundary is fulﬁlled, that is M(Rcore) = Mcore. 4.1.2. Maximum gas accretion rate 2001; Kley & Dirksen 2006) allows the planets ( p y ) p to efﬁciently access disc material even after a gap has formed. For circular orbits, gap formation would in contrast strongly reduce the gas accretion rate (Lubow et al. 1999; Bryden et al. 1999), and limit planetary masses to ∼5−10 MX. X The radial extent of the feeding zone is set by rfeed,inf = aplanet (1 −e) 1 −ffeed r Mtot 3M⋆  (53) rfeed,sup = aplanet (1 + e) 1 + ffeed r Mtot 3M⋆  (54) (53) (54) with ffeed = 0.5 so that the overall extent is a half a Hill radius larger than the radial excursion of the planet’s orbit. This radial extent provides the location over which the disc properties (Σ, H, etc.) are averaged for the calculation of the maximum rate and the removal of the accreted gas, with [ y ] 0.0 0.5 1.0 1.5 2.0 2.5 Time [Myr] 0 500 1000 1500 2000 2500 Enveloppe mass [M⊕] Radial ﬂow Bondi rate ˙Σg,planet = ˙Menv π r2 feed,sup −r2 feed,inf . (55) (55) The planet’s eccentricity consequently does not directly affect the maximum gas accretion rate, but only indirectly through the size of the feeding zone. The self-limitation of gas accretion by removal of local disc gas by the planet, which then needs to be replenished by the inﬂow from more distant disc regions (i.e. mass conservation) is fully taken into account in our scheme via the ˙Σg,planet term entering the evolutionary equation of the disc gas surface density. We also take into account that for planets of any mass growing in multi-planet systems, the eccentricity can be increased via gravitational planet-planet interactions, which then affects the feeding zone width and thus indirectly the gas accretion rate. On the other hand, we currently do not take into account that the eccentric instability (i.e. the increase of a sin- gle giant’s eccentricity because of gravitational interaction with the gas disc) in reality only acts for sufﬁciently massive plan- ets (Papaloizou et al. 2001; Kley & Dirksen 2006). This could lead to an overestimation of gas accretion at lower to intermedi- ate giant planet masses. This could potentially explain why our current model of disc-limited gas accretion seems to too strongly reduce the stellar gas accretion rate (Manara et al. 2019; Bergez- Casalou et al. 2020). 4.1.2. Maximum gas accretion rate Gap formation would reduce this effect, but could potentially lead to another issue: observationally, the giant planet mass function seems to extend smoothly to about 30 MX (Sahlmann et al. 2011; Adibekyan 2019) (though Santos et al. 2017 and Schlaufman 2018 found a change in the metallicity dependency at about 4 to 5 MX and concluded that the plan- ets above that threshold formed predominantly by gravitational instability). Reaching such high masses could be difﬁcult given the expected reduction of gas accretion because of gap formation in the circular case. Fig. 5. Comparison of two prescriptions for the maximum (i.e. disc lim- ited) gas accretion rate, the one presented in this work (labelled ‘Bondi rate’) with that of Mordasini et al. (2012c) (labelled ‘Flow rate’). These are two different simulations (one for each prescription) whose initial conditions represent the second outermost planet in Fig. 4. Top panel: maximum value that can be supplied by the gas disc (labelled ‘Max.’) and effective accretion rates (labelled ‘Eff.’), which is given by intrin- sic cooling in the initial attached phase and the maximum rate in the detached phase. Bottom panel: corresponding enveloppe mass (i.e. total gas accreted). et al. (2012c), with a limit of 0.9 of the radial ﬂow to allow some gas to ﬂow through the gap (Lubow & D’Angelo 2006). The results show that using the Bondi rate, as we presented here, gives a somewhat stronger limitation of gas accretion by the forming planet, especially during the onset of the runaway gas accretion. As a result, the ﬁnal planet’s mass is a bit lower when using the Bondi rate. The reduction of gas inﬂow into the inner disc because of an accreting giant planet can result in the clearing of the inner region of the protoplanetary disc by photoevaporation (Rosotti et al. 2013). This effect is also automatically taken into account by our model. A69, page 14 of 44 4.1.2. Maximum gas accretion rate (51) (51) P(Rtot) = Pneb(aplanet) and (46) T 4(Rtot) = T 4 neb(aplanet) + 3τoutL(Rtot) 8πσSBR2 tot , (47) (46) To distinguish between the two regimes, we use the lower rate of the two, that is (47) (47) ˙Menv,max = min ˙Menv,max,2D, ˙Menv,max,3D (52) ˙Menv,max = min ˙Menv,max,2D, ˙Menv,max,3D (52) and and Finally, to ensure that no more gas than available in the feeding zone Mfeed is accreted during one time step, we fur- ther constrain ˙Menv,max < Mfeed/∆t. We consider the limiting τout = max κ(ρneb, Tneb)ρnebRtot, 2 3 ! (48) (48) A69, page 13 of 44 A69, page 13 of 44 A&A 656, A69 (2021) 0.0 0.5 1.0 1.5 2.0 2.5 Time [Myr] 10−5 10−4 10−3 10−2 10−1 100 Accretion rate [M⊕/yr] Max. Flow Eﬀ. Flow Max. Bondi Eﬀ. Bondi 0.0 0.5 1.0 1.5 2.0 2.5 Time [Myr] 0 500 1000 1500 2000 2500 Enveloppe mass [M⊕] Radial ﬂow Bondi rate Fig. 5. Comparison of two prescriptions for the maximum (i.e. disc lim- ited) gas accretion rate, the one presented in this work (labelled ‘Bondi rate’) with that of Mordasini et al. (2012c) (labelled ‘Flow rate’). These are two different simulations (one for each prescription) whose initial conditions represent the second outermost planet in Fig. 4. Top panel: maximum value that can be supplied by the gas disc (labelled ‘Max.’) and effective accretion rates (labelled ‘Eff.’), which is given by intrin- sic cooling in the initial attached phase and the maximum rate in the detached phase. Bottom panel: corresponding enveloppe mass (i.e. total gas accreted). 0.0 0.5 1.0 1.5 2.0 2.5 Time [Myr] 10−5 10−4 10−3 10−2 10−1 100 Accretion rate [M⊕/yr] Max. Flow Eﬀ. Flow Max. Bondi Eﬀ. Bondi case to be that gap formation does not reduce the planetary gas accretion rate. Such a situation arises if the eccentric instability (Papaloizou et al. 2001; Kley & Dirksen 2006) allows the planets to efﬁciently access disc material even after a gap has formed. For circular orbits, gap formation would in contrast strongly reduce the gas accretion rate (Lubow et al. 1999; Bryden et al. 1999), and limit planetary masses to ∼5−10 MX. case to be that gap formation does not reduce the planetary gas accretion rate. Such a situation arises if the eccentric instability (Papaloizou et al. 4.1.4. Evolutionary phase The value ˙Mtot in the attached phase and of ˙Rtot in the detached phase are determined from the guess for the mass or radius during the iterations. The same is not true for ˙ξtot. To circumvent this problem, we estimate the luminosity with Lint ≈C ξGMtot Rtot ˙Mtot −ξGM2 tot 2R2 tot ˙Rtot ! . (62) (62) The correction factor C corrects for neglecting the ˙ξtot term. The value of C can be calculated a posteriori by determining the actual total energy of the new planet, with C = −(Etot(t)−Etot(t− ∆t))/(Lint · ∆t). The value of C is then used for the next time step. Marleau et al. (2017, 2019b) conducted 1D radiation- hydrodynamic simulations of the planetary gas accretion shock, a feature that is seen in various 3D radiation-hydrodynamic sim- ulations of accreting protoplanets of sufﬁciently high mass (e.g. Szulágyi & Mordasini 2017; Schulik et al. 2020). High postshock entropies were found, suggesting that warm or hot gas accretion is more plausible than cold accretion (see also Berardo et al. 2017; Berardo & Cumming 2017). We therefore assume in our model that gas accretion in the detached phase is hot, which means that we do not subtract the accretion shock luminosity from Lint (see Mordasini et al. 2012c). The correction factor C corrects for neglecting the ˙ξtot term. The value of C can be calculated a posteriori by determining the actual total energy of the new planet, with C = −(Etot(t)−Etot(t− ∆t))/(Lint · ∆t). The value of C is then used for the next time step. 4.1.4. Evolutionary phase with u being the speciﬁc internal energy of the gas, as obtained from the equation of state. The gravitational binding energy term includes the contribution from the core. For simplicity, we assume that it has a constant density, so its contribution is taken as −3/5GM2 core/Rcore. It should be noted that this is not strictly self-consistent with our model to determine its density or radius, which assumes differentiation (Mordasini et al. 2012b); however, the difference remains small (Linder et al. 2019). The param- eter ξ in Eq. (60) represents as in polytropic models the mass distribution and additionally the thermal energy content. It is retrieved from Eq. (60). The internal luminosity resulting from the accretion, cooling, and contraction Lint can then be obtained as For the evolutionary phase (after the dispersal of the gas disc), the outer boundary conditions are set to P(Rtot) = Pedd + Prad (58) T 4(Rtot) = T 4 int + (1 −A)T 4 eq (59) (58) (59) where T 4 int = Ltot/(4πσSBR2 tot) is the intrinsic temperature, Teq = T⋆∗pR⋆/(2 ∗aplanet), and A = 0.343 is the albedo, which is taken be the same as Jupiter (Guillot 2005). This value was selected for simplicity, although hot-Jupiter planets may have lower values (e.g. Mallonn et al. 2019). We thus use an Eddington grey boundary condition taking the stellar irradiation into account, as described in Mordasini et al. (2012c). During evolution, we assume a solar-composition condensate-free gas for the opacities, using the opacity tables of Freedman et al. (2014). Nebular grain opacity is neglected, at they are found to rain out quickly once gas accretion stops (Movshovitz & Podolak 2008). The identical envelope and atmo- spheric composition (pure H/He, solar composition opacities) in all planets means that for planets with identical bulk properties (orbital distance, core and envelope mass), the predicted radii will exhibit an artiﬁcially reduced spread. In reality, planets have different enrichment levels of heavy elements in the envelope (e.g. Fortney et al. 2013). This affects the equation of state and opacity, resulting in particular in a larger spread of the radii (e.g. Burrows et al. 2011; Müller et al. 2020). as Lint = ˙Etot = ξGMtot Rtot ˙Mtot −ξGM2 tot 2R2 tot ˙Rtot + GM2 tot 2Rtot ˙ξtot, (61) (61) with ˙Mtot = ˙Mcore + ˙Menv being the total accretion rate of the planet (solids and gas). 4.2.1. Accretion and contraction The luminosity calculation suffers from the same problem as the total mass in the attached phase, or the outer radius in the detached phase; that is that the new structure needs to be known to retrieve its energy, hence the luminosity. This means that the total energy of the new structure needs to be estimated for a luminosity to be obtained. Pram = ˙Menv 4πR2 tot vﬀ; v2 ﬀ= 2GMtot 1 Rtot −1 RH ! (57) (57) The model uses the approach from Mordasini et al. (2012c). The total energy is given as being the ram pressure due to the accretion shock and the free- fall velocity at the surface of the planet. Etot = −G Z Mtot 0 M R dM + Z Mtot Mcore udM = −ξGM2 tot 2Rtot (60) (60) 4.2. Luminosity with Pneb(aplanet) being the pressure at the midplane of the gas disc, Pedd = (2g)/(3κ) the Eddington expression for the pho- tospheric pressure due to the material residing above the τ = 2/3 surface, Prad = (2σSBT 4(Rtot))/(3c) the radiation pressure, c being the speed of light in vaccum, and 4.1.3. Detached phase Once the gas accretion rate exceeds the maximum that can be provided by the disc – which includes the planet no longer being in a region where gas is present – the accretion regimes changes to the detached phase (Bodenheimer et al. 2000). In the detached phase, the solid and gas accretion rate are known (for the gas, it is given by the disc-limited rate), but not the planet’s radius. The radius is determined following the approach of Mordasini et al. (2012c,b), that is by using the same internal structure equations To compare the prescription presented here with previous work, we provide in Fig. 5 the comparison of the gas accre- tion rate for the second outermost planet from the case shown in Fig. 4. The previous methodology, using the radial gas ﬂow and taking into account the geometry was described in Mordasini A69, page 14 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. as in the attached, but iterating on the radius until convergence is reached. structure at the beginning of the transition from the attached to detached phase (the time marked with a dashed vertical line in the insert in Fig. 7). The pressure outer boundary conditions are modiﬁed to take into account that the disc and the envelope are no longer con- nected, and that the gas free-falls onto the surface of the planet Note that the planet is still accreting during the initial stages of the detached phase. P(Rtot) = Pneb(aplanet) + Pedd + Pram + Prad (56) (56) 4.1.5. Example To illustrate the calculation of the internal structure, we provide snapshots of envelope structures in Fig. 6 and the time evolution of the radius and luminosity in Fig. 7. These are taken from the second outermost planet of the system shown in Fig. 4, which is a giant planet whose ﬁnal mass is 6.4 MX. Due to the differ- ent scales involved in the attached, detached, and evolutionary phases, they are shown in different panels. During the attached phase, the structure extends to the Bondi radius (Eq. (45)), which is much larger than the core radius. Therefore, the structure spans a wider range of pressure. The upper part of the envelope is radiative while the lower part is convective, with several tran- sitions in the mid region. The red proﬁle shows the internal Marleau et al. (2017, 2019b) conducted 1D radiation- hydrodynamic simulations of the planetary gas accretion shock, a feature that is seen in various 3D radiation-hydrodynamic sim- ulations of accreting protoplanets of sufﬁciently high mass (e.g. Szulágyi & Mordasini 2017; Schulik et al. 2020). High postshock entropies were found, suggesting that warm or hot gas accretion is more plausible than cold accretion (see also Berardo et al. 2017; Berardo & Cumming 2017). We therefore assume in our model that gas accretion in the detached phase is hot, which means that we do not subtract the accretion shock luminosity from Lint (see Mordasini et al. 2012c). A69, page 15 of 44 A69, page 15 of 44 A69, page 15 of 44 A&A 656, A69 (2021) A&A 656, A69 (2021) A&A 656, A69 (2021) 102 103 104 Temperature [K] 10−7 10−5 10−3 10−1 101 103 105 107 Pressure [bar] Attached 102 103 104 105 Temperature [K] 10−7 10−4 10−1 102 105 108 Pressure [bar] Transition 103 104 105 Temperature [K] 10−2 100 102 104 106 108 1010 Pressure [bar] Detached 103 104 105 Temperature [K] 10−2 100 102 104 106 108 1010 Pressure [bar] Evolution 105 106 107 108 109 1010 Time [yr] Fig. 6. Snapshots of the internal structure of the second outermost planet of Fig. 4. The structures are split according to the phases, with attached (top left), transition (the initial stage of the detached phase; top right), detached (bottom left) and evolutionary (bottom right). The red line shows the ﬁrst proﬁle of the detached phase and is shown of both panels. 4.1.5. Example Lbloat = ϵF⋆e−τmidπR2 tot (63) Lbloat = ϵF⋆e−τmidπR2 tot (63) with ϵ = 2.37 exp − log (F⋆/109erg cm−1 s−1) −0.14 2 2 · 0.372 , (64) (64) F⋆= L⋆/(4πa2 planet) the total stellar ﬂux at the planet’s location, F⋆= L⋆/(4πa2 planet) the total stellar ﬂux at the planet’s location, and τmid is the optical depth from the star to the planet location through the mid-plane of the disc, as in Eq. (6). We only apply bloating if the stellar ﬂux F⋆(in the evolutionary phase) or the stellar ﬂux multiplied by the optical depth F⋆exp (−τmid) (before the dispersal of the gas disc) is greater than 2 × 108 erg cm−2 s−1 (Demory & Seager 2011). F⋆= L⋆/(4πa2 planet) the total stellar ﬂux at the planet’s location, d i h i l d h f h h l l i and τmid is the optical depth from the star to the planet location through the mid-plane of the disc, as in Eq. (6). We only apply bloating if the stellar ﬂux F⋆(in the evolutionary phase) or the stellar ﬂux multiplied by the optical depth F⋆exp (−τmid) (before the dispersal of the gas disc) is greater than 2 × 108 erg cm−2 s−1 (Demory & Seager 2011). 4.1.5. Example The green and blue proﬁles lie at the transition between two stages and are shown of two panels each. In each proﬁle, thin lines show the part where energy transport is radiative and thick lines for convective. 102 103 104 T [K] 10−7 10−5 10−3 10−1 101 103 105 107 Pressure [bar] Attached 102 103 104 105 T t [K] 10−7 10−4 10−1 102 105 108 Pressure [bar] Transition 108 109 1010 [yr] Pressure [bar] Pressure [bar] Time [yr] Temperature [K] p [ ] 103 104 105 Temperature [K] 10−2 100 102 104 106 108 1010 Pressure [bar] Detached Detached Pressure [bar] Pressure [bar] Temperature [K] Fig. 6. Snapshots of the internal structure of the second outermost planet of Fig. 4. The structures are split according to the phases, with attached (top left), transition (the initial stage of the detached phase; top right), detached (bottom left) and evolutionary (bottom right). The red line shows the ﬁrst proﬁle of the detached phase and is shown of both panels. The green and blue proﬁles lie at the transition between two stages and are shown of two panels each. In each proﬁle, thin lines show the part where energy transport is radiative and thick lines for convective. which are in the detached and evolutionary phase and directly irradiated by the host star, we include an additional luminos- ity contribution that is based on the best empirical ﬁt formula obtained by Thorngren & Fortney (2018): In addition to the accretion and contraction luminosity, we include the luminosity from radioactive decay, bloating for close-in planets, and, in the case of brown-dwarfs, deuterium fusion. The radiogenic luminosity Lradio includes contributions from the three most important long-lived radionucleides 40K, 238U and 232Th (Wasserburg et al. 1964). To compute the lumi- nosity contributions, we follow the procedure of Mordasini et al. (2012b): we assume the mantle of the protoplanets has a chron- dritic composition and the energy production rate are retrieved from meteoritic values of William (2007). The initial radio- genic contribution is Q0 ≈5×−7 erg g−1 s−1 of mantle material (all elements besides iron). 4.2.2. Bloating of close-in planets Massive, close-in planets exhibit anomalously large radii (Laughlin et al. 2011). To reproduce this effect, we include a bloating mechanism based on Sarkis et al. (2021). For planets A69, page 16 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 1.05 1.10 Time [Myr] 101 102 Radius [R] 105 106 107 108 109 1010 Time [yr] 100 101 102 Radius [R] 105 106 107 108 109 1010 Time [yr] 100 101 102 103 104 105 106 107 Luminosity [L] Fig. 7. Time evolution of the planet’s radius and luminosity of the second outermost planet of Fig. 4; the same as in Fig. 6. The insert on the left panel shows the contraction at the transition between attached and detached phase. The exact time where the model switches between the two phases is shown with the vertical dashed line. 1.05 1.10 Time [Myr] 101 102 Radius [R] 105 106 107 108 109 1010 Time [yr] 100 101 102 Radius [R] 105 106 107 108 109 1010 Time [yr] 100 101 102 103 104 105 106 107 Luminosity [L] Fig. 7. Time evolution of the planet’s radius and luminosity of the second outermost planet of Fig. 4; the same as in Fig. 6. The insert on the left panel shows the contraction at the transition between attached and detached phase. The exact time where the model switches between the two phases is shown with the vertical dashed line. (Berardo et al. 2017; Berardo & Cumming 2017) and we do not account for the decrease of the luminosity with depth. This will be addressed in future work. (Berardo et al. 2017; Berardo & Cumming 2017) and we do not account for the decrease of the luminosity with depth. This will be addressed in future work. 4.2.3. Deuterium-burning For the calculation of the luminosity due to deuterium fusion, we follow the procedure of Mollière & Mordasini (2012). In this framework, the energy generation rate (per unit mass and time) is given by Kippenhahn & Weigert (1994), with the assump- tion that nuclei are fully ionised and non-degenerate. The energy released in each process is computed according to Fowler et al. (1967). The speciﬁc deuterium burning luminosity of a planet depends on the conditions in the planet’s gaseous envelope, most notably the density, temperature, and the remaining deuterium nuclei. This implies that there is no universal mass at which deuterium burning starts, but as already found in Mollière & Mordasini (2012) (see also Bodenheimer et al. 2013), the mass where burning becomes important clusters around about 13 MX. The presence of a solid core does thus not signiﬁcantly alter the mass where burning starts relative to (coreless) brown dwarfs (Chabrier & Baraffe 2000). We use an initial deuterium number fraction [D/H] = 2 × 10−5, which is the primordial value. 4.3. Accretion of solids The growth of the astrophysical core of the planets can occur via three channels: (1) the accretion of planetesimals (e.g. Greenzweig & Lissauer 1992; Thommes et al. 2003), (2) the accretion of pebbles (e.g. Ormel & Klahr 2010; Johansen & Lacerda 2010; Lambrechts & Johansen 2012), and (3) by the collision with other embryos (which we call giant impacts). In the Generation III model, we consider accretion by planetesimals and giant impacts; the inclusion of pebble accretion is subject of ongoing work (Voelkel et al. 2020). g g For planetesimals accretion, core growth is given by the probability of collisions with planetesimals in the oligarchic regime (Ida & Makino 1993), as described in Fortier et al. (2013). This is a major difference to the ﬁrst generation of the Bern model which followed Pollack et al. (1996) for the planetesimal accretion rate. According to Chambers (2006), the core growth can be computed assuming a particle-in-a-box approximation is Our model also includes the enhancing of the reaction rate by screening, that is the shielding of the positive charges by the surrounding electron. In turn, screening is affected by the elec- tron degeneracy, as we are dealing with objects of high central densities. This procedure follows the work of Dewitt et al. (1973) and Graboske et al. (1973). (66) ˙Mcore = Ω¯ΣsR2 Hpcoll, (66) ˙Mcore = Ω¯ΣsR2 Hpcoll, with ¯Σs the mean surface density of planetesimals in the planet’s feeding zone and pcoll the collision probability with planetes- imals. As Ida & Lin (2008), we use the same prescription to calculate the planetesimal accretion rate independently of a pro- toplanet’s orbital migration rate. In addition, we address the possible impact that orbital migration could have in the con- text of the shepherd/predator regimes proposed by Tanaka & Ida (1999): in the idealised situation studied by Tanaka & Ida (1999) (single protoplanet per disc, no local reservoir of planetesimals, no growth via collisions with other protoplanets), shepherding was found to signiﬁcantly reduce the planetesimal accretion rate for protoplanets migrating sufﬁciently slowly. However, in the 4.2.4. Total luminosity The ﬁnal luminosity is then given by (65) L(Rtot) = Ltot = Lint + Lradio + Lbloat + LD−burn. (65) L(Rtot) = Ltot = Lint + Lradio + Lbloat + LD−burn. We assume that at a given time, the luminosity does not change within the envelope, that is ∂L/∂r = 0. This approximation is ﬁne under most circumstances because energy transport is due to convection and the luminosity enters only in the radiative gra- dient. During rapid gas accretion in the detached phase, under the effect of hot accretion, the interior may become radiative We assume that at a given time, the luminosity does not change within the envelope, that is ∂L/∂r = 0. This approximation is ﬁne under most circumstances because energy transport is due to convection and the luminosity enters only in the radiative gra- dient. During rapid gas accretion in the detached phase, under the effect of hot accretion, the interior may become radiative A69, page 17 of 44 A&A 656, A69 (2021) 102 103 104 105 106 107 Time [yr] 10−2 10−1 100 101 102 Radius [R] Total radius Capture radius Core radius 10−2 10−1 100 101 102 Core mass [M⊕] 10−2 10−1 100 101 102 Radius [R] 105 yr 106 yr Total radius Capture radius Core radius Fig. 8. Evolution of the planet’s core, planetesimals capture, and total radii as function of time (left panel) and core mass (right panel) for the second outermost planet of Fig. 4. In the right panel, we also include two time indicators at 105 and 106 yr with dashed vertical lines. The upper-right portion of the plot for the total radius in the left panel is also shown on the left panel of Fig. 7. 102 103 104 105 106 107 Time [yr] 10−2 10−1 100 101 102 Radius [R] Total radius Capture radius Core radius 10−2 10−1 100 101 102 Core mass [M⊕] 10−2 10−1 100 101 102 Radius [R] 105 yr 106 yr Total radius Capture radius Core radius Radius [R] Fig. 8. Evolution of the planet’s core, planetesimals capture, and total radii as function of time (left panel) and core mass (right panel) for the second outermost planet of Fig. 4. In the right panel, we also include two time indicators at 105 and 106 yr with dashed vertical lines. 4.2.4. Total luminosity The upper-right portion of the plot for the total radius in the left panel is also shown on the left panel of Fig. 7. IG(β) ≃ 1 + 0.39960β β 0.0369 + 0.048333β + 0.006874β2. (71) more realistic N-body simulations by Daisaka et al. (2006) where multiple protoplanets (oligarchs) form and grow concurrently as expected in the oligarchic regime (Kokubo & Ida 1998), the trap- ping of planetesimals by the protoplanets is only tentative and does not signiﬁcantly reduce their accretion rates. We similarly ﬁnd that in the more realistic situation we consider here with many embryos per disc, the existence of a local reservoir of planetesimals in a protoplanet’s initial feeding zone accessible without migration and a time sequence of a solid accretion domi- nated initially by planetesimals and later on collisions with other protoplanets (Sect. 8.1), shepherding should only be of limited importance. We discuss these points further in Appendix A. (71) The ﬁnal collision is then given by pcoll = min pmed, p−2 high + p−2 low −1/2 . (72) (72) In the initial stage, the capture radius Rcap is the physical radius of the core Rcore. Once the planet has sufﬁciently mas- sive H/He envelope, it will enhance the capture cross-section of planetesimals. As in Fortier et al. (2013), the capture radius is obtained following Inaba & Ikoma (2003) by solving the implicit equation 4.3.1. Capture probability Rplan = 3 2 ρ(Rcap)Rcap ρplan  v2 rel + 2GM(Rcap)/Rcap v2 rel + 2GM(Rcap)/RH . (73) (73) We distinguish three different accretion regimes depending on the random velocities: low-, mid- and high-velocity. The dis- tinction is based on the reduced planetesimals’ eccentricity ˜eplan = replan/RH and inclination ˜ıplan = riplan/RH (r is the helio- centric distance): the high-velocity regime for ˜eplan, ˜ıplan ≳2, mid-velocity for 2 ≳˜eplan, ˜ıplan ≳0.2 and low-velocity for 0.2 ≳ ˜eplan, ˜ıplan. According to Inaba et al. (2001), each regime has a different expression for the collision probability, The enhancement of the capture radius over the physical radius is very important for increasing the planet’s planetesimals accretion rate (Podolak et al. 1988; Venturini & Helled 2020). We highlight this in Fig. 8, which compares the planetesimals capture radius to that of the core for the same planet we high- lighted in Fig. 7. The calculation of the envelope structure begins at about 104 yr, before that, the capture radius is equal to that of the core. At that moment, the core mass is 9 × 10−2 M⊕. By the time the core reaches 1 M⊕at 4.8 × 105 yr, the capture radius is 9 times the core radius. Therefore, for small roughly km-sized planetesimals as in our case, the enhancement of the capture radius is already important for low-mass bodies (starting about ×10−1 M⊕), and the calculation of gaseous envelopes cannot be omitted at any stage. Besides the factor that the eccentricity and inclination damping by nebular gas drag is more efﬁcient for smaller planetesimals which leads to a larger gravitational cross section (a larger Safronov factor), the larger envelope drag enhancing the planet capture radius further is the second effect making the accretion of small planetesimal more efﬁcient. This reﬂects that the accretion of km-sized planetesimals is not a pure gravitational process. phigh = (Rcap + Rplan)2 2πRH IF(β) + 6RHIG(β) (Rcap + Rplan)˜e2 plan  (67) pmid = (Rcap + Rplan)2 4πRH˜ıplan 17.3 + 232RH Rcap + Rplan ! (68) plow = 11.3 Rcap + Rplan RH ! 4.3.1. Capture probability , (69) (67) (68) (69) where Rcap is the planetesimal capture radius of the planet, β = iplan/eplan and the IF and IG functions can be approximated as, following Chambers (2006): where Rcap is the planetesimal capture radius of the planet, β = iplan/eplan and the IF and IG functions can be approximated as, following Chambers (2006): IF(β) ≃1 + 0.95925β + 0.77251β2 β (0.13142 + 0.12295β) (70) (70) A69, page 18 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. Region 1 Region 2 Before separation After separation Central star Fig. 9. Illustration of the procedure to separate planetesimals’ feeding zones when zones would otherwise overlap. The horizontal axis rep- resents the separation to the central star and four planets are shown. The light colour areas below the horizontal line show the initial feeding zones while the ones above show the ﬁnal zones. amid2,3 and amid3,4 are the edges of the new feeding zone. Region 1 Region 2 Before separation After separation Central star to rapo + Rfeed, with rperi and rapo being the peri- and apocentre of the planet’s orbit respectively. When multiple planets are present in the same disc, their feeding zones may overlap. To avoid problems with two plan- ets accreting from the same location, such as mass-conservation issues, we separate the feeding zones so that there is at most one planet accreting at any location the disc. A graphical represen- tation of the following procedure is provided in Fig. 9. First, we compute regions in the disc from where planets accrete. In the case a region contains a single planet, then the feeding zone is the same as in the single planet case (as in Region 1 on Fig. 9). If there are multiple planets in one region (as in Region 2 on that ﬁgure), the inner edge of the innermost planet and the outer edge of the outermost planet are set to the edges of the region. For the other edges, we sort planets by distance, and for each pair, we compute the location of the limit between their feeding zones with Fig. 9. Illustration of the procedure to separate planetesimals’ feeding zones when zones would otherwise overlap. The horizontal axis rep- resents the separation to the central star and four planets are shown. 4.3.4. Core radius To obtain the radius of the core (and its density), we applied a methodology similar to Mordasini et al. (2012b). This model also accounts for the composition of the core and the pressure burden exerted by the envelope. ˙Mcore ˙Mejec = vesc vsurf !4 (74) ˙Mcore ˙Mejec = vesc vsurf !4 The principle is to solve similar structure equations as for the envelope, that is Eqs. (40) and (41), but with an equation of state that takes the form of a modiﬁed polytrope from Seager et al. (2007), which reads (74) with vsurf = pGMtot/Rcap the characteristic surface velocity. The rate at which planetesimals are removed from the disc is then with vsurf = pGMtot/Rcap the characteristic surface velocity. The rate at which planetesimals are removed from the disc is then (78) ρ(P) = ρ0 + cPn. (78) ρ(P) = ρ0 + cPn. ˙Mplan = ˙Mcore + ˙Mejec. (75) ˙Mplan = ˙Mcore + ˙Mejec. (75) We include three different materials: iron, silicates (perovskite, MgSiO3) and ice, whose parameters ρ0, c and n are taken from Seager et al. (2007). Because of the small thermal expansion coefﬁcient of these materials compared to H/He, we neglect via the temperature-independent modiﬁed polytropic EOS a possi- ble temperature dependency of the radius of the core. It should, in any case, be small (Grasset et al. 2009). We include three different materials: iron, silicates (perovskite, MgSiO3) and ice, whose parameters ρ0, c and n are taken from Seager et al. (2007). Because of the small thermal expansion coefﬁcient of these materials compared to H/He, we neglect via the temperature-independent modiﬁed polytropic EOS a possi- ble temperature dependency of the radius of the core. It should, in any case, be small (Grasset et al. 2009). It should be noted that our model does not include the redis- tribution of planetesimals by scattering (Raymond & Izidoro 2017). 4.3.2. Ejection of planetesimals Planets not only accrete material; they also induce gravitational perturbations on the planetesimals that come close-by but are not accreted. These planetesimals, if they receive a sufﬁcient velocity kick from a close approach by a planet, can be ejected from the system. To estimate this effect, we follow a procedure similar to Ida & Lin (2004a). The planetesimals that receive a velocity kick greater than the escape velocity from the pri- mary, vesc = p2GM⋆/aplanet, will likely be ejected from the system. Thus, we have that the fraction of accreted-to-ejected planetesimals is (Ida & Lin 2004a) 4.3.1. Capture probability The light colour areas below the horizontal line show the initial feeding zones while the ones above show the ﬁnal zones. amid2,3 and amid3,4 are the edges of the new feeding zone. amid = aout √Min + ain √Mout √Min + √Mout (77) (77) where the subscripts indicate the inner (in) and outer (out) plan- ets of the pair. We scale with the square root of the planet masses because the area of the feeding zone scales with the square of the distance. This scaling keeps the area of the feeding zones related to the planet masses. We tested alternative prescriptions, like using the cubic root of the mass (as in the Hill sphere) or the mid- point between the two planets and found that the prescription does not signiﬁcantly affect the outcomes of the simulations. 4.5. Initial conditions The simulation begin with a predetermined number of embryos whose initial mass is Memb,0 = 10−2 M⊕(approximately the mass of our Moon). They are randomly placed with an uniform prob- ability in log a, where a is the semi-major axis, between rin and 40 au. The starting location zone is slightly more extended that in the previous studies, where the upper boundary was set to 20 au. Also, two embryos cannot be placed within 10 Hill radii from each other. It should be noted that for the simulations with largest initial number of embryos, 100, this represents an average spacing of 28 Hill radii. The evaporation model is based on Jin et al. (2014). It takes into account contributions from X-ray and extreme-ultraviolet (XUV) irradiation. At the early stages, the evaporation is typ- ically X-ray driven. We describe this regime using the energy- limited rate from Jackson et al. (2012) using the ﬂux in the 1 to 20 Å range from Ribas et al. (2005) and assuming an efﬁciency factor ϵ = 0.1. The presence of a number of embryos right at the beginning of the simulations is a strong assumption we made because the model does not track the formation of the embryos themselves. This shortcoming of the model will be addressed in future evo- lutions of the model (Voelkel et al. 2020), where the evolution of the dust, pebbles and planetesimal and embryo formation is followed. At later stages, the evaporation from EUV takes over. We also use the work of Ribas et al. (2005) to obtain the time-dependent EUV stellar luminosity for a Sun-like star. EUV evaporation can be divided into two sub-regimes (Murray-Clay et al. 2009). At low EUV ﬂuxes, the same energy-limited approximation as for the X-ray ﬂux is used. In this case, the escape ﬂux is given by 4.3.3. Feeding zone For gas giant planets, where envelopes can reach masses of thousands of Earth masses, this can cause a signiﬁcant com- pression of the core (Baraffe et al. 2008). Thus, the pressure on the core’s surface is taken as boundary condition of the calcula- tion to include this effect. Core compression can be observed in Fig. 8, where the core radius shrinks after the envelope contracts at 1.06 Myr. To obtain the mean surface density of planetesimals in the feed- ing zone, we must determine its extent. The half-width of the feeding zone (centred at the planet’s location) is usually given in terms of the Hill radius with Rfeed = bRH. (76) (76) Rfeed = bRH. For a planet on a circular orbit, conservation of the Jacobi energy implies that b = q 12 + 4/3(˜e2 plan + ˜ı2 plan) (e.g. Hayashi et al. 1977). So, in a quiescent disc with ˜eplan, ˜ıplan ≪1, b = 2 √ 3 ≈ 3.5. For numerical stability reasons, however, we assume b = 5, as in Fortier et al. (2013). For a planet on a circular orbit, conservation of the Jacobi energy implies that b = q 12 + 4/3(˜e2 plan + ˜ı2 plan) (e.g. Hayashi et al. 1977). So, in a quiescent disc with ˜eplan, ˜ıplan ≪1, b = 2 √ 3 ≈ 3.5. For numerical stability reasons, however, we assume b = 5, as in Fortier et al. (2013). The core composition is retrieved from the accreted planetes- imals described in Sect. 3.3.3 and other embryos in case of giant impacts. The chemical composition is used to obtain the fraction of the different elements to compute the core radius. While in the chemistry model includes 32 (Thiabaud et al. 2014) refrac- tory and 8 volatile (Marboeuf et al. 2014b) chemical species, the core radius calculation groups them into only three types: iron, silicates, and water ice. Thus, we map all ice species to water ice In the general case, to account for a non-circular orbit of the planet, we take the feeding zone to span from rperi −Rfeed A69, page 19 of 44 A&A 656, A69 (2021) with n0,base the density of neutrals at the base, hν0 = 20 eV, σν0 = 6 × 10−18 cm2(hν0/13.6 eV)−3, αrec = 2.7 × 10−13, and ρbase = n+,basemH. when calculating the core structure and all refractories except iron to the silicate mantle. 4.4. Atmospheric escape During the evolutionary phase, that is after the dissipation of the gaseous disc, planets at small distances of their host star (∼0.1 au) receive intense XUV stellar irradiation, which will drive atmospheric escape. This effect is especially important for the low-mass planets, that can loose the whole of their gaseous envelope due to their low gravitational binding energy (e.g. Lammer et al. 2009; Lopez et al. 2012; Owen & Jackson 2012; Jin et al. 2014; Jin & Mordasini 2018). The stripping of the whole envelope has a signiﬁcant effect on the planets radius. Due to the low density of gas, the presence of an envelope will result a signiﬁcant increase of the planets’ sizes even if the envelope mass is only on a percent level of the total planet mass. Bare cores are thus clearly separated from object that retain a gaseous envelope, and a gap is observed in the distribution of planetary radii (Owen & Wu 2013; Lopez & Fortney 2013; Jin et al. 2014; Chen & Rogers 2016; Fulton & Petigura 2018). 4.3.3. Feeding zone The reason for this is that ﬁrst, equa- tions of state are only available for a limited number of species. Second, the differences between different types of, for instance, silicates is not very large (Seager et al. 2007). n+,basemH. , The model also includes the effect of Roche lobe overﬂow. When solving the internal structure equations, there are some- times solutions found in the detached and evolutionary phase where the radius is larger than the Hill sphere. This occurs in two situations: ﬁrst, for close-in low-mass planets with a high envelope mass fraction. At the moment when the nebula dissi- pates (and thus the ambient pressure vanishes), and when the star starts to irradiate the planets directly (resulting in an increase of the temperature, see Fig. 3), these planets bloat. Second, giant planets that get very close to the star because of tidal spiral in (see Sect. 5.3) can also overﬂow their Roche lobe. In this case, we remove at each time step the part of the H/He envelope that is outside of the Hill sphere. 5.1. Planetary migration at the sonic point Rs, which is calculated the same way as Racc. Here cs = √kBT/(mH/2) is the isothermal sound speed of ionised gas with T = 104 K. The density can be related to the one at the ionisation base, where τ = 1, with at the sonic point Rs, which is calculated the same way as Racc. Here cs = √kBT/(mH/2) is the isothermal sound speed of ionised gas with T = 104 K. The density can be related to the one at the ionisation base, where τ = 1, with We include two types of migration, Type I for low mass planets embedded in the gas disc and Type II for planets massive enough to open a gap in the disc. ρs ∼ρbase exp "GMtot Rtotc2s Rtot Rs −1 !# . (81) (80) (80) (79) (79) As the planet mass increases, it will generate a stronger perturba- tion in the density of the gas around the planet. This perturbation will cause the nebula to no longer be axis-symmetric, and as a consequence produces a torque back on the planet, leading to planetary migration. At the same time, convergent migration can result in capture in mean-motion resonances or orbital desta- bilisation. Hence migration and dynamical evolution must be performed together to capture all the effects. where FEUV is the EUV ﬂux, Rbase the radius of the photoionisa- tion base, calculated as in Murray-Clay et al. (2009), and ϵ = 0.3 is the heating efﬁciency, taken as in Murray-Clay et al. (2009). On the other hand, energy-limited evaporation is not suitable when the EUV ﬂux is high (>104erg cm−2 s−1), as a substantial part of the heating is lost in cooling radiation. In this regime, we adopt the radiation-recombination-limited approximation of Murray-Clay et al. (2009). The mass loss rate is given by wind due to escape ˙Menv,rr ∼4πρscsR2 s (80) ˙Menv,rr ∼4πρscsR2 s 5. Dynamical evolution: orbital migration, N-body interaction, and tides ˙Menv,e = ϵ πFEUVR3 base GMtot (79) 5.1.2. Type II migration The total Type I torque on a planet, following Eqs. (50)–(53) of Paardekooper et al. (2011) and (15) of Coleman & Nelson (2014), is given by The criterion to detect gap opening and switch migration to Type II is from Crida et al. (2006), Γ1 = FLΓL + FeFi Γc,baro + Γc,ent , (83) with Type II is from Crida et al. (2006), 3H 4RH + 50νM⋆ Mplaneta2 planetΩ≤1, Γ1 = FLΓL + FeFi Γc,baro + Γc,ent , (83) yp 3H 50νM (83) 3H 4RH + 50νM⋆ Mplaneta2 planetΩ≤1, (93) (93) with with Γc,baro = Γhs,baroF(pν)G(pν) + Γc,lin,baro(1 −K(pν)) Γc,ent = Γhs,entF(pν)F(pχ) q G(pν)G(pχ) +Γc,lin,baro q (1 −K(pν))(1 −K(pχ)), (84) (84) with ν is the viscosity from Eq. (2). with ν is the viscosity from Eq. (2). y q Type II orbital migration follows the non-equilibrium approach from Dittkrist et al. (2014). Here, the planet follows the radial velocity of the gas, (85) vrad = 1 Σg √r ∂ ∂r νΣg √r (94) (94) where ΓL, Γhs,baro, Γhs,ent, Γc,lin,baro and Γc,lin,baro are the Linblad torque, barotropic and entropy part of the horseshoe drag and lin- ear corotation torque respectively. They are given by Eqs. (3)–(7) of Paardekooper et al. (2011). The function F governs saturation, while G and K provide the cutoff at high viscosity, and are given by Eqs. (22), (30) and (31) of Paardekooper et al. (2011). (Pringle 1981), but is limited if the planet’s mass is much larger than the local disc mass (the fully suppressed case, see Alexander & Armitage 2009). The radial velocity of the planet vplanet is given by The other factors in Eq. (83) account for the shape of the orbit. FL provides the reduction of the Lindblad torque for eccen- tric or inclined orbits following Cresswell & Nelson (2008), with vplanet vrad = min 1, 2Σga2 planet Mplanet . (95) (95) F−1 L = Pe + Pe \|Pe\| ! 0.07ˆı + 0.085ˆı4 −0.08ˆeˆı2 (86) For the larger planet masses, when the migration rate is con- strained by the disc-to-planet mass ratio, this expression result in a similar behaviour as the formula obtained by Kanagawa et al. (2018), although it does not take into account the aspect ratio of the disc h. (86) and and Pe = 1 + ˆe 2.25 1/2 + ˆe 2.84 6 1 − ˆe 2.02 4 . 5.1.2. Type II migration (87) For our migration scheme, we convert the radial velocity into a torque according to (87) Γ2 = 1 2 MplanetΩaplanetvplanet. (96) (96) Here, ˆe = e/h = e/(H/r) and ˆı = i/h = i/(H/r) are the planet’s orbital eccentricity and inclination scaled by the disc’s aspect ratio h = H/r. Fe and Fi provide the reduction of the corotation torques due to eccentricity and inclination (Bitsch & Kley 2010). We use This prescription allows in principle planets in Type II to migrate outwards if the disc is decreting (Veras & Armitage 2004). How- ever, in practice this mechanism is limited by the restriction to planets that are already at large distances or during the ﬁnal moments of the disc, and limited by the small surface density (Dittkrist et al. 2014). Fe = exp − e h/2 + 0.01 ! (88) (88) During type II migration, the eccentricity and inclination damping time scales are set to as suggested by Fendyke & Nelson (2014) for the reduction due to eccentricity and τe = τi = 1 10\|τa\| = 1 10 aplanet \|vplanet\|. (97) Fi = 1 −tan h(ˆı) (89) Fi = 1 −tan h(ˆı) Fi = 1 −tan h(ˆı) (97) (89) for the reduction due to inclination (Coleman & Nelson 2014). This relationship was selected because hydrodynamical simu- lations of migrating planets in this regime have shown that eccentricity and inclination damping act on time scales that are shorter than migration (Kley et al. 2004; Kley 2019). Eccentricity and inclination damping time scales follow Cresswell & Nelson (2008), with τe = twave 0.78 1 −0.14ˆe2 + 0.06ˆe3 + 0.18ˆeˆı2 (90) (90) (90) ) 5.1.1. Type I migration (81) For Type I migration, our model follows the approach of Coleman & Nelson (2014). This includes the torques formulation from Paardekooper et al. (2011), modiﬁed to consider that orbital eccentricity and inclinations attenuate the co-rotation torques (Bitsch & Kley 2010, 2011). For Type I migration, our model follows the approach of Coleman & Nelson (2014). This includes the torques formulation from Paardekooper et al. (2011), modiﬁed to consider that orbital eccentricity and inclinations attenuate the co-rotation torques (Bitsch & Kley 2010, 2011). The photoionisation base is located where there is equilibrium between photoionisations and recombination: FUV σ FUV hν0 σν0n0,base ∼n2 +,baseαrec FUV hν0 σν0n0,base ∼n2 +,baseαrec (82) (82) A69, page 20 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 5.1.3. Migration map and An example of the outcome of the whole migration scheme for one disc proﬁle is provided in Fig. 10. The disc is the same as the example shown in Fig. 3 at 1 Myr; at this time the disc mass is 1.46 × 10−02 M⊙. Its outer radius is 123 au, so we cut the ﬁgure at 200 au since there is no migration outside this distance. τi = twave 0.544 1 −0.3ˆı2 + 0.24ˆı3 + 0.14ˆe2ˆı , (91) τi = twave 0.544 1 −0.3ˆı2 + 0.24ˆı3 + 0.14ˆe2ˆı , (91) where twave = M⋆ Mplanet !  M⋆ Σa2 planet h4Ω−1 (92) 5.2. N-body integration Radial surface density proﬁle (top panel), temperature proﬁle (middle panel), and migration map (bottom) as function of the planet mass (assuming zero eccentricity and inclination), for the same disc pre- sented in Fig. 3 at t = 1 Myr. The value plotted in the bottom panel is the relative migration rate 1/τa = −vplanet/aplanet; blue regions indicate inward migration, red regions outward migration. For both directions, the locations in bright colours are where migration is inefﬁcient while dark tones indicate efﬁcient migration. The dashed black line shows the boundary between type I (below) and type II (above) migration regimes. Fig. 10. Radial surface density proﬁle (top panel), temperature proﬁle (middle panel), and migration map (bottom) as function of the planet mass (assuming zero eccentricity and inclination), for the same disc pre- sented in Fig. 3 at t = 1 Myr. The value plotted in the bottom panel is the relative migration rate 1/τa = −vplanet/aplanet; blue regions indicate inward migration, red regions outward migration. For both directions, the locations in bright colours are where migration is inefﬁcient while dark tones indicate efﬁcient migration. The dashed black line shows the boundary between type I (below) and type II (above) migration regimes. The evolution of such a system by splitting is done using a second-order method, HI τ 2 HS τ 2 HK (τ) HS τ 2 HI τ 2 , (103) (103) also note two convergence zones for low- to mid-mass plan- ets. These are due to opacity transitions (Lyra et al. 2010) or structures in the gas disc (Kretke & Lin 2012) such as the increase of the surface density close to the inner edge of the disc (Masset et al. 2006). These are the locations where, for a given planet mass, outward migration happens on the inner side and inward migration on the outer side. Hence, at this moment of evolution, planets with masses less than ≈8 M⊕cannot reach the inner edge of the disc by migration only. However, as time goes and gas becomes scarcer, the zones of outward migration (hence where the notation H...(τ) is used to represent the evolution under the given Hamiltonian for a step τ. For HI, this means that the planets receive a kick in velocity due to the interactions with the other bodies (except the central star). (91) (91) (91) where where twave = M⋆ Mplanet !  M⋆ Σa2 planet h4Ω−1 (92) Migration is most efﬁcient for intermediate mass planets, above about 10 M⊕up to the transition to Type II migration (shown with the dashed black line on the migration map). The outward migration at large separation for the type II migration regime is due to the outward spreading of the gas disc. We (92) is the characteristic time of evolution of density waves (Tanaka & Ward 2004). A69, page 21 of 44 A&A 656, A69 (2021) 10−1 100 101 102 Distance [AU] 10−1 100 101 102 103 Planet mass [M⊕] −10−5−10−6−10−7−10−8 0 10−8 10−7 10−6 10−5 Migration rate [yr−1] 102 103 Temperature [K] 10−3 10−1 101 Surf. dens. [g/cm2] Fig. 10. Radial surface density proﬁle (top panel), temperature proﬁle ( ddl l) d i i (b ) f i f h l the convergence zones) shift towards lower planetary masses. Thus, by the end of the gas disc, planet with masses down to ≈2 M⊕could reach the inner edge of the disc. 5.2. N-body integration Gravitational interactions between the protoplanets are now modelled with the mercury N-body code (Chambers 1999) using the hybrid method. Unlike the direct resolution of the equation of motion (as performed in A13), this use a symplectic integration scheme (see e.g. Sanz-Serna 1992, for a review). The basic principle is to use the solution of Hamilton’s equations, ˙xi = ∂H ∂pi , ˙pi = −∂H ∂xi (98) (98) where x denotes the position coordinates, p the momentum coordinates, and H = N X i=0 p2 i 2Mi −G N X i=0 Mi N X j=i+1 M j ∆xi j (99) (99) Planet mass [M⊕] is the Hamiltonian of the system, with ∆xi j = \|xi −xj\|. Here, the index i = 0 refers to the central star and M0 = M⋆while the subsequent are the planet with Mi = Mplanet,i so that N is the number of planets in the system. However, while H has no analytical solution for N > 1, it is possible to split the Hamiltonian into several pieces, solving the simpler problems to ﬁnally combine them back so that a solution close to that of the original system. The Hamiltonian is divided into three components, so that H = HK + HS + HI, and 10−1 100 101 102 Distance [AU] 10−1 −10−5−10−6−10−7−10−8 0 10−8 10−7 10−6 10−5 Migration rate [yr−1] HK = N X i=1  p2 i 2Mi −G M⋆Mi ∆xi0  (100) HS = 1 2M⋆  N X i=1 pi  2 (101) HI = −G N X i=1 N X j=i+1 MiM j ∆xi j . (102) HK = N X i=1  p2 i 2Mi −G M⋆Mi ∆xi0  (100) HS = 1 2M⋆  N X i=1 pi  2 (101) HI = −G N X i=1 N X j=i+1 MiM j ∆xi j . (102) (100) (101) (102) Here, HK represents the unperturbed Keplerian orbits of the planets about the central star, HS the kinetic energy of the star and HI the interactions between the planets. The separation into three different Hamiltonians (rather than two) is required because the scheme uses mixed-centre coordinates (also called ‘democratic heliocentric’): heliocentric positions and barycen- tric velocities. These coordinates are chosen so that HK ≫ HS, HI, unless two planets come close together. Fig. 10. 5.2.2. Collision detection ˙Mcore,supp = Mcore,2 τimpact √ 2π exp −1 2 t −timpact τimpact −3 !2 (109) Lcore,supp = Eimpact τimpact √ 2π exp −1 2 t −timpact τimpact −3 !2 (110) Collisions are detected when two planets come closer than a pre- determined distance, which is the sum of their radii. When the closet approach is found inside to be during one of the substeps of the N-body, the minimum distance is retrieved by ﬁtting a third-degree polynomial equation whose condition are set by the relative separation and their radial velocity at the beginning and end of the substep (similar to A13). (109) (110) where timpact is the time of the impact, τimpact = 104 yr is the time scale of release taken as in Broeg & Benz (2012). These two terms are added to the core accretion rate due to planetesimal accretion, and to the luminosity (Sect. 4.2) used in the internal structure calculation, respectively. For planets with a signiﬁcant and extended envelope (like during the attached phase), the assumption that planets have a unique radius which decides whether a collisions occurs or not is no very accurate, as the outcome is determined by gas dynamics inside the merging envelopes. As we do not have the full enve- lope structure in the N-body, we nevertheless remaining with a unique radius approach. In the attached phase, the envelope transitions smoothly to surrounding nebula. The outer radius, as provided by Eq. (44), is unsuitable for the detection of collisions, as it corresponds to very low gas densities. Thus, the radius used to detect collisions is computed assuming that the whole planet mass has the same density as its core. This is an approxima- tion, but reﬂects that the gas density in the envelope is much higher close to the (solid) core surface. In the detached phase, we use the planetesimals’ capture radius Rcap; this is normally an This impact model was tailored for the most common col- lisions that we ﬁnd in our simulations. We highlight this by showing cumulative distrubutions of impactor-to-target mass ratio γ for different ranges of target masses in Fig. 11. At low masses, most target/impactor pairs are of similar masses, thus this source of growth cannot be neglected. In contrast, most col- lisions involving giant planets are with much smaller impactors (the red curve in Fig. 11). 5.2.3. Collision treatment When a collision is detected, the following procedure is applied: the cores merge, the eventual envelope of the less massive body is deemed to be ejected, and the impact energy is added as a addi- tional contribution to the luminosity for the structure calculation of the new body. The symplectic integration scheme has a huge advantage in terms of computational requirements compared to a standard Bulirsch-Stoer method, as the interaction between the plan- ets, the one part that is O(N2), is only computed once per step. The merger of the cores will make that a part of the impact energy will already be taken into account consistently with the luminosity calculation described in Sect. 4.2; so the additional energy is calculated using We do not use the N-body when there is only one protoplanet in a system as the solution is analytical. This happens either for populations with one embryo per system or in the unlikely case that only one planet survives in a planetary system with initially multiple embryos per system. Eimpact = max 1 2µv2 imp −Eacc,core, 0 ! (107) (107) 5.2.1. Additional forces where µ = Mtot,1Mtot,2/(Mtot,1 + Mtot,2) is the reduced mass, and the indexes 1 and 2 refer to the quantities of the larger and smaller body respectively. vimp is the relative velocity at time of contact. Here Migration and damping are included as additional forces in the N-body. The contributions from migration and eccentricity damping apply in the orbital plane and are split into tangential (θ) and radial (r) components, while the inclination damping acts on the vertical component (z), resulting in Eacc,core = G Mtot,1Mcore,2 Rcore,1 + Rcore,2 (108) (108) aθ = −vθ −vK 2τe + Γ Mplanet (104) ar = −vr τe (105) az = −2vz τi (106) (104) is the centre-of-mass impact energy of two bodies with the total mass of the target and the core mass of the impactor colliding at their mutual escape velocity. Also, we restrict the supplementary energy to positive values. Negative value can arise if the bodies are colliding at below the mutual escape velocity, which is pos- sible due to the drag by the gas disc or in the case of speciﬁc conﬁguration, such as co-orbitals. However, the impact velocity is never quite lower than the mutual escape velocity, so that the error remains small. (105) (106) with a denoting the additional accelerations, v the planet’s velocity along each direction. Here, vK = Ωr is the Kelperian velocity. The addition of the core mass and luminosity is performed via via 5.2. N-body integration In our case, HI is extended to include additional forces representing the effect of the gas disc, see Sect. 5.2.1. The evolution under HS results in a shift τ/(2M⋆) P pi while the evolution under HK is a Keplerian motion around the central star for a period τ. A69, page 22 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. As we noted, the assumption that HI is small compared to HK is no longer valid when two bodies become close together. In that situation, the idea is to bring the interaction between the two close-by bodies into HK so that the interaction Hamiltonian remains small. This implies that HK is no longer analytically integrable during that period, but only for the orbits of the involved bodies. In practice, the orbits of the two close-by bod- ies are integrated with a conventional Bulirsch-Stoer method (Stoer & Bulirsch 1980) for the duration of the encounter. That algorithm is described in detail in Chambers (1999). overestimation of the effective collision radius, larger bodies needing to penetrate deeper down in the envelope to be captured. However, in this phase the envelope scale height is small com- pared to the radius except for the very short time directly after detachment, so the actual error is small. 6.1. Setup For this test cases, we performed a few modiﬁcations to our main model to mimic earlier work like Chambers (2001) and Raymond et al. (2005). Orbital migration has been disabled; as for the envelope structure calculation and the evolution phase, here, all planets are treated as purely rocky. We adopt an ini- tial surface density proﬁle close the minimum-mass solar nebula (MMSN; Weidenschilling 1977; Hayashi 1981), with a reference surface density of Σ0,s = 7.1 g cm−2 at r0 = 1 au, but truncated at 2 au, as we are primarily interested in the inner planets. This also helps to determine more precisely which fraction of the planetes- imal disc has been accreted by the terrestrial planets during their formation. This gives a solids mass of 3.67 M⊕. The initial num- ber of embryos is selected to have a similar spacing as the two populations presented Emsenhuber et al. (2021, Paper II) with most embryos per system, which means that we have initially 23 (correspond to 50 in Paper II) and 46 (corresponding to 100) lunar-mass (0.01 M⊕) embryos. In addition to that, we perform one run with 9 embryos initially in Sect. 6.3, which corresponds to 20 embryos in Paper II. Fig. 11. Cumulative distribution of the impactor-to-target mass ratio γ = Mtot,2/Mtot,1 for different target mass ranges (as provided in the legend). Data come from the 100-embryos population presented in Paper II. 5.3. Tidal evolution During the evolution phase we include the inward migration of planets due to tides they raise onto the central star. In addition to planets that are pushed inwards due to capture in mean-motion resonances, this gives another channel to obtain planets that are within the inner boundary of the gas disc. For the tidal migration rate, we compute the rate according to It should be noted that the model lacks the ‘dynamical fric- tion’ obtained in N-body simulations with a large number of small bodies (O’Brien et al. 2006; Raymond et al. 2006) because we do not include the effect of the damping of eccentricities and inclinations of the embryos by the planetesimals. However, after all material has been accreted onto the planets, the remainder of the formation process is similar to pure N-body simulations of terrestrial planet accretion, as all the mass is now contained in bodies that are directly followed by the N-body. ∂aplanet ∂t = −9 2 s G M⋆ R5 ⋆Mplanet Q⋆ a−11/2 planet (111) (111) (Ferraz-Mello et al. 2008; Jackson et al. 2009; Benítez-Llambay et al. 2011), where Q⋆= 106 is the stellar dissipation parameter. It is clear that this model for the tidal spiralling-in is strongly simpliﬁed. It will be improved in future work along the lines of, for example, Bolmont & Mathis (2016). y y y For some simulations, we include Jupiter and Saturn to deter- mine the effects they have on the formation of the inner planets. In that case, Jupiter and Saturn are on their present-day orbits, but they are rotated so that their invariant plane coincides with that of the disc (as in Chambers 2001, 2013; Emsenhuber et al. 2020). We do not model the formation of these planets, because they form over a period that is much shorter than the terrestrial planets. 5.2.2. Collision detection Our models neglect the envelope of the impactor, but there are only few collisions where this could provide signiﬁcant source of mass. A69, page 23 of 44 A&A 656, A69 (2021) 0.0 0.2 0.4 0.6 0.8 1.0 γ = Mtot,2/Mtot,1 0.0 0.2 0.4 0.6 0.8 1.0 Cumulative fraction < 0.1 M⊕ 0.1 −1 M⊕ 1 −10 M⊕ 10 −100 M⊕ > 100 M⊕ Fig. 11. Cumulative distribution of the impactor-to-target mass ratio γ = Mtot,2/Mtot,1 for different target mass ranges (as provided in the legend). Data come from the 100-embryos population presented in Paper II. 0.0 0.2 0.4 0.6 0.8 1.0 γ = Mtot,2/Mtot,1 0.0 0.2 0.4 0.6 0.8 1.0 Cumulative fraction < 0.1 M⊕ 0.1 −1 M⊕ 1 −10 M⊕ 10 −100 M⊕ > 100 M⊕ tens Myr (Yin et al. 2002; Kleine et al. 2002) to roughly 100 Myr (Touboul et al. 2007; Allègre et al. 2008; Kleine et al. 2009). This is longer than the expected lifetime of the solar system’s nebula of 4 Myr (Wang et al. 2017) by about an order of magnitude or more. Hence the modelling of formation of planetary systems with terrestrial planets needs to span a longer time period for dynamical effects (i.e. the ‘late stage’) than for gas-dominated planets. tens Myr (Yin et al. 2002; Kleine et al. 2002) to roughly 100 Myr (Touboul et al. 2007; Allègre et al. 2008; Kleine et al. 2009). This is longer than the expected lifetime of the solar system’s nebula of 4 Myr (Wang et al. 2017) by about an order of magnitude or more. Hence the modelling of formation of planetary systems with terrestrial planets needs to span a longer time period for dynamical effects (i.e. the ‘late stage’) than for gas-dominated planets. A69, page 24 of 44 6. Terrestrial planet formation We begin by studying whether the new generation of the Bern model with a higher initial number of embryos, but which still includes a statistical description of planetesimals, is capable of reproducing models of terrestrial planets that use purely N-body (e.g. Chambers 2001), that is where the planetesimals are repre- sented as individual (test) particles. This test is crucial to assess whether we can reach our goal of having a formation model which is able to simulate the growth of planets with a very large mass range from about that of Mars, to brown dwarfs. This is in contrast with earlier generations of the Bern Model, where mainly more massive planets were at the focus (or more speciﬁcally, planets for which the giant impact phase after disc dissipation is not very important). To obtain a better overview of the inﬂuence of the parame- ters we are studying, and to reduce (and better understand) the stochastic effects of N-body interactions, we perform 10 sim- ulations for each combination of parameters (initial number of embryos and presence of the outer planets). The only differences between the 10 simulations are the initial position of the ter- restrial planet embryos. For the 10 simulations, we consider the average outcomes as being representative (e.g. Fig. 12). The simulations starts with a gas disc, which lives for roughly 4.4 Myr. Its only effect however is to damp the eccentricities and inclinations of the planetesimals. Planetesimals accretion con- tinues after the dispersal of the gas disc. As the planets do not have envelopes, we perform only the formation stage of the cal- culation. However, the duration of that stage has been extended to 400 Myr to account for the much longer time needed for the solar system’s terrestrial planets to converge. The formation of terrestrial planets does not have the same time constraint as for gas giants. In the case of planets with a signiﬁcant H/He envelope, a sufﬁciently massive core must be formed before the dispersal of the gas disc, but this does not apply to terrestrial planets. Indeed, in the case of the Earth, cos- mochemical evidences point to a formation time between a few A69, page 24 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 6. Terrestrial planet formation Middle panels: mass versus time; sudden increases in mass are due to embryo-embryo collisions. Bottom panels: semi-major axis versus time. 10−1 100 10−2 10−1 100 Mass [M⊕] 9 embryos 10−1 100 Distance [AU] 23 embryos 10−1 100 46 embryos 105 107 10−2 10−1 100 Mass [M⊕] 105 107 Time [yr] 105 107 105 107 10−1 100 Distance [AU] 105 107 Time [yr] 105 107 Fi 13 C i f f i k f i h MMSN lik 0 1 2 3 4 Mass [M⊕] 103 104 105 106 107 108 Time [yr] 101 Number of embryos 46 emb, no J/S 46 emb, with J/S 23 emb, no J/S 23 emb, with J/S Distance [AU] Number of embryos Time [yr] 105 107 10−1 100 Distance [AU] 105 107 Time [yr] 105 107 Fig. 12. Average over the 10 simulations for each set of parameters. The top panels show the masses of solids (excepting the outer giant planets in the relevant cases) in the protoplanetary disc, that is still in planetes- imals (solid lines), accreted by the embryos (dashed lines) and ejected (dotted lines). The dashed black line denotes the total mass of solids in each simulation. The bottom panel shows the number of embryos that remain. The ‘J/S’ in the legend refers to Jupiter and Saturn. Fig. 13. Comparison of formation tracks for a system with a MMSN-like surface density of planetesimals, and with 9 embryos (left), 23 (cen- tre), and 46 (right), and no outer giant planets. Each line represents one embryo. Top panels: mass versus semi-major axis; embryos start at the bottom and move upwards as they grow. The ﬁnal positions of the remaining planets are shown by dots. The dashed black line denotes the isolation mass (Lissauer 1987). Middle panels: mass versus time; sudden increases in mass are due to embryo-embryo collisions. Bottom panels: semi-major axis versus time. 6.2. Gravitational interactions If the embryos remain at their initial locations during the whole formation process, then they grow to their isolation mass (Lissauer 1987). In our model, we obtain this behaviour if we artiﬁcially remove the N-body interactions, unless the feeding zones of two adjacent embryos overlap at some point, in which case the masses become slightly lower. When using this mode, the runs starting with 46 embryos have accreted roughly half of the disc’s mass onto the embryos by about 4 Myr (the time at which the gas disc disperses) and accrete very slowly thereafter. For the runs starting with 23 embryos, only a quarter of the mass ends in the embryos by 4 Myr. redistributed to locations outside of the embryos’ feeding zone rather than be accreted. However, when they add the mecha- nism that embryos can reside in all parts of the disc (which is more realistic, Levison et al. 2010) no gap in the planetesimal disc opens, as embryos mutually scatter planetesimal into their vicinity and accrete them eventually. This leads to an efﬁcient formation of massive planets. Thus, feeding zones overlap in these simulations, therefore the effect of planetesimal redistri- bution should play little role in our case as there are very few locations in the disc where planetesimals would not be accreted by the local embryo and/or scattered back into the feeding zone of other embryos. For the other parameter sets (all with gravitational interac- tions), Fig. 12 provides the averaged results over 10 simulations, for the masses of solids and the number of embryos. The story is quite different when N-body interactions are included. We see for instance that in the case with 46 embryos and no outer giant planets, nearly all the planetesimals have been accreted onto the embryos. For the case with 23 embryos initially and no outer giant planets, more than half of the planetesimals end up accreted. 6. Terrestrial planet formation 0 1 2 3 4 Mass [M⊕] 103 104 105 106 107 108 Time [yr] 101 Number of embryos 46 emb, no J/S 46 emb, with J/S 23 emb, no J/S 23 emb, with J/S Fig. 12. Average over the 10 simulations for each set of parameters. The top panels show the masses of solids (excepting the outer giant planets in the relevant cases) in the protoplanetary disc, that is still in planetes- imals (solid lines), accreted by the embryos (dashed lines) and ejected (dotted lines). The dashed black line denotes the total mass of solids in each simulation. The bottom panel shows the number of embryos that remain. The ‘J/S’ in the legend refers to Jupiter and Saturn. 6.2. Gravitational interactions If the embryos remain at their initial locations during the 10−1 100 10−2 10−1 100 Mass [M⊕] 9 embryos 10−1 100 Distance [AU] 23 embryos 10−1 100 46 embryos 105 107 10−2 10−1 100 Mass [M⊕] 105 107 Time [yr] 105 107 105 107 10−1 100 Distance [AU] 105 107 Time [yr] 105 107 Fig. 13. Comparison of formation tracks for a system with a MMSN-like surface density of planetesimals, and with 9 embryos (left), 23 (cen- tre), and 46 (right), and no outer giant planets. Each line represents one embryo. Top panels: mass versus semi-major axis; embryos start at the bottom and move upwards as they grow. The ﬁnal positions of the remaining planets are shown by dots. The dashed black line denotes the isolation mass (Lissauer 1987). Middle panels: mass versus time; sudden increases in mass are due to embryo-embryo collisions. Bottom panels: semi major axis versus time 10−1 100 10−2 10−1 100 Mass [M⊕] 9 embryos 10−1 100 Distance [AU] 23 embryos 10−1 100 46 embryos 105 107 10−2 10−1 100 Mass [M⊕] 105 107 Time [yr] 105 107 105 107 10−1 100 Distance [AU] 105 107 Time [yr] 105 107 Fig. 13. Comparison of formation tracks for a system with a MMSN-like surface density of planetesimals, and with 9 embryos (left), 23 (cen- tre), and 46 (right), and no outer giant planets. Each line represents one embryo. Top panels: mass versus semi-major axis; embryos start at the bottom and move upwards as they grow. The ﬁnal positions of the remaining planets are shown by dots. The dashed black line denotes the isolation mass (Lissauer 1987). 6.3. Interactions lead to more massive planets To understand how the embryo-embryo interactions lead to a quasi-complete accretion of the planetesimals disc, we show the formation tracks for one particular system with a varying number of embryos in Fig. 13. There are two aspects we point out here. First, in the ﬁg- ure, the planetesimal mass accreted by embryos that have been later ejected is accounted as accreted. Second, our planetesimal model does not include redistribution of material by interac- tions with the embryos. For instance, in their less realistic setup where embryos only populate a limited orbital distance range in the disc, Levison et al. (2010) found that planetesimals can be We can easily observe that the larger the number of embryos, the more and the sooner they start to move around. In the system with only 9 embryos, they basically remain where they started and grow slightly above their isolation mass. For the other two simulations, however, the local isolation mass is sufﬁcient to A69, page 25 of 44 A&A 656, A69 (2021) 0.0 0.2 0.4 Ecc. With J/S 0.2 Myr No J/S 0.0 0.2 0.4 Ecc. 2 Myr 0.0 0.2 0.4 Ecc. 20 Myr 10−1 100 Distance [AU] 0.0 0.2 0.4 Ecc. 10−1 100 Distance [AU] 200 Myr Fig. 14. Stacked eccentricity versus distance snapshots of 10 simula- tions with each 46 embryos initially. The left column shows the runs with outer giant planets whereas the right column has no outer giant planets. In each column, the 10 systems are represented with a different colour for each one. The bodies are shown by points whose sizes are proportional to their physical ones. Black crosses show the solar system planets. 0.0 0.2 0.4 Ecc. With J/S 0.2 Myr No J/S 0.0 0.2 0.4 Ecc. 2 Myr 0.0 0.2 0.4 Ecc. 20 Myr 10−1 100 Distance [AU] 0.0 0.2 0.4 Ecc. 10−1 100 Distance [AU] 200 Myr trigger signiﬁcant embryo-embryo interactions that will change their positions in the disc. This in turn enables them to accrete from regions that would otherwise inaccessible, which creates a positive feedback since more massive planets will result in yet more interactions. This feedback only ends when nearly all planetesimals have been accreted onto the embryos. Thus, closer packed embryos lead to enhanced stirring of their eccentricities, which has two consequences: the increase of the feeding zone size because of radial excursion for eccen- tric orbits, and collisions between embryos. 6.4. Time needed for formation We ﬁnd a similar pattern for the timing at which interactions start in the two simulations with the higher number of embryos of Fig. 13 (23 and 46 embryos). In the early phase (a few 105 yr), no dynamical interactions occur, because the embryos need to reach a certain mass before the eccentricities can be signiﬁcantly excited. Then, the ﬁrst embryos to show an increased eccentric- ity are located at ∼0.3 au, and then this propagates both inwards and outwards. In the inner part of the system, collisions happen rather rapidly so that the system has essentially obtained its ﬁnal conﬁguration by several Myr. Fig. 14. Stacked eccentricity versus distance snapshots of 10 simula- tions with each 46 embryos initially. The left column shows the runs with outer giant planets whereas the right column has no outer giant planets. In each column, the 10 systems are represented with a different colour for each one. The bodies are shown by points whose sizes are proportional to their physical ones. Black crosses show the solar system planets. g y y On the other hand, in the outer region we observe that embryos remain on eccentric orbits for a certain amount of time before suffering from collisions. It takes more than 10 Myr for the planets located at about 1au to reach their ﬁnal mass. In the even more distant regions, it takes even longer, and we see the phase with several embryos on eccentric orbits remaining for more than 100 Myr. Such a growth wave travelling from the inside to the outside is expected, as the growth process scales with the local Keplerian frequency. To provide a better comparison point between the two cases, we provide in Figs. 14 and 15 several snapshots of the simulations. One general consequence at earlier times is that there is slower growth for the embryos beyond 1.5 au. We see in the two top rows of Fig. 14 that the outermost embryos remain smaller in the runs with outer giant planets. Also, their eccentricities have already increased in the ﬁrst snapshot, while this is not the case at all for the runs without giant planets. The underlying cause is stirring of planetesimal’s eccentricity and inclination by the giant planets; this heavily reduces the collision probability with the low-mass protoplanets (Inaba et al. 2001) and hence the accretion rate. 6.3. Interactions lead to more massive planets Embryos having a greater eccentricity can sample a broader region of the disc, thus grow to a larger mass before depleting the disc. Collisions with other embryos are capable to bring material from more dis- tant regions of the disc that would otherwise not be accessible to one embryo. At the end, we arrive at a result that is maybe counter-intuitive at ﬁrst: the larger the number of embryos, the less planets remain. We observe this for instance in the bottom panel of Fig. 12. 6.4. Time needed for formation p q y Therefore, our choice of the integration time dictates the location where and how accurately the model can follow the formation of the terrestrial planets. With our choice of an inte- gration time limited to 20 Myr for the formation phase, the model can only track most of the giant impact stage inside of roughly 1 au for systems that have a MMSN-like surface density of solids. Even within 1 au, the giant impact stage is not entirely ﬁnished within our set time frame, as it can be see in the innermost planet by about 300 Myr in the bottom right panel of Fig. 13. Neverthe- less, these events remain rare. Locations further away or systems with a lower amount of solids (as formation is slower for less massive systems, Kokubo et al. 2006; Dawson et al. 2015) will, however, not have reached a ﬁnal state by end of the formation stage at 20 Myr. A consequence of the longer timescales of accretion in the outer part of the disc is the state at the moment of the dispersal of the gas disc. In the runs with giant planets, a larger percent- age of the planetesimals remains unaccreted at the moment the gas disperses. In addition, after that point, there is no longer gas present to counterbalance the effects of the stirring by the giant planets. This means that after a short moment, the planetesimals will reach eccentricities of the order of unity and will be ejected. This can be observed in Fig. 12, where we see that up to a quar- ter of the original initial mass is ejected from the planetesimals disc. The ﬁnal eccentricities of the terrestrial bodies are similar in both cases (Fig. 14), as the inner region is subject to the self- stirring while in the outer region, excitation by the outer planets makes up from a weaker self-stirring as the masses are lower. 6.5. With outer giant planets As the ﬁnal stage of terrestrial planet formation (the giant impact stage) takes longer than formation of the giant planets, we also want to consider the effects of their presence on terrestrial planet formation. Here we perform the same simulations again, each time with the addition of two outer giant planets that represent Jupiter and Saturn. A69, page 26 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 10−1 100 Mass [M⊕] With J/S 20 Myr No J/S 10−1 100 Distance [AU] 10−1 100 Mass [M⊕] 10−1 100 Distance [AU] 200 Myr Fig. 15. Same as Fig. 14, but showing mass versus distance stacked diagram of 10 simulations with each 46 embryos initially. As before, the black crosses show the solar system planets. of the gas disc leads for example to very closely packed sys- tems of massive planets, instabilities will often occur shortly after disc dispersal. On the other hand, if at gas disc dispersal only low-mass, widely-space planets are present, they will ﬁrst have to grow further via accretion of remaining planetesimals and embryos – which can take a very long time – to eventually (or also never) become unstable. For larger planet masses, grav- itational interactions can extend further: even on distant orbits, massive planets can destabilise the system as noted by Bitsch et al. (2020) and Matsumura et al. (2021). This could explain why Izidoro et al. (2021) ﬁnd that by 20 Myr only a fraction of the instabilities between the planets have happened in their setup, whereas Mulders et al. (2020) on the contrary ﬁnd that increas- ing the integration time from 10 Myr to 100 Myr only leads to minor further evolution in their simulations. For systems lack- ing outer planets, Izidoro et al. (2021) also found a convergence after ∼30 Myr (their Model III). This is in better agreement to the analysis for planets with a < 1au done in Mulders et al. (2020). The purpose of the model here is to obtain planetary sys- tems that can be compared with observations at the population level. For this, it is important to see that the region where long-term growth will be most important (distant low-mass plan- ets) represents at the same time the parameter space currently not accessible to most detection techniques of extrasolar plan- ets (radial velocity, transits, and direct imaging). 7. Giant planets The formation of giant planets is quite different. Cores must form before the dispersal of the gas disc so that they can undergo run- away gas accretion, and since we have massive cores in a gas disc, migration is efﬁcient. To gain an understanding of the inter- play of accretion and migration, we here show some illustrative cases with a single embryo per disc. For this case, we use the model without modiﬁcations, but the N-body is not used. The following examples are taken from the single-embryo population of Paper II. Simulations parameters are the same as provided in Table 1, except for disc masses and surface density (both gas and planetesimals), inner edge, characteristic radius, and external photoevaporation rate of the gas disc. In the following simula- tions, the inner radius has negligible effect on the ﬁnal outcome, as we do not study close-in planets, and so we do not mention it. The characteristic radius rcut,g of the gas disc is set as p An integration period (for the formation stage) longer than the lifetime of the protoplanetary disc is necessary to follow the giant impact phase. The time required for the bodies to obtain their ﬁnal characteristics increase with distance (as shown here) and with decreasing initial amount of solids (e.g. Kokubo et al. 2006; Dawson et al. 2015). The limitation of the formation stage to 20 Myr (Sect. 2.2) permits to capture all the accretion of plan- etesimals (provided there are enough embryos initially) and most of the dynamical interactions of Earth-mass and larger planets forming via giant impacts up to roughly 1 au, and sub-Earths planets in the ﬁrst few tenths of an au (corresponding to periods of roughly 100 days). For the population syntheses in Paper II, we estimate from tracking major changes of the planets’ orbits, that for orbital distances of ≲1 au around 90% of the major insta- bilities should have been captured when integrating the systems for 20 Myr. Mg 2 × 10−3 M⊙ = rcut,g 10 au 1.6 . (112) (112) The integration time needed to capture most instabilities within a given orbital distance range is a function of the archi- tecture of the planetary systems that results from the previous growth stages. If the growth and migration during the presence (see Paper II for the motivation). 6.6. Summary for terrestrial planets To summarise, we have just seen that as long as the separation between the embryos is sufﬁciently small that dynamical interac- tions are triggered before the embryos reach their local isolation mass, the model is capable of reproducing the main features of the formation of terrestrial planets in good agreement with pure N-body models. This is due to embryo-embryo interactions being able to increase the eccentricities, so that the embryos can move out of their original locations, and almost entirely depletes the planetesimals. 6.5. With outer giant planets This should minimise the impact of this limitation. We acknowledge, how- ever, that generally speaking, not all dynamical interactions will have taken place by the end of the integration time of 20 Myr in the model. While the later evolution should not be substan- tial enough to strongly affect the statistical results in the inner systems, this limitations must be critically kept in mind when comparing for example to microlensing surveys (e.g. Suzuki et al. 2018) that probe more affected regions. Fig. 15. Same as Fig. 14, but showing mass versus distance stacked diagram of 10 simulations with each 46 embryos initially. As before, the black crosses show the solar system planets. Thus, the outer giant planets will limit and delay growth of the terrestrial planets in the outer region. The number of objects is a bit higher than the one obtained by pure N-body simula- tions of terrestrial planet formation, but we are using a somewhat smaller initial surface density proﬁle compared to, for example, Raymond et al. (2006), which prevents the accretion into a lower number of higher mass bodies (Kokubo et al. 2006). Nevertheless, we conclude that the new generation of syn- theses can be used to describe in a much more comprehensive way planetary sub-populations ranging from sub-Earths to super- Jupiters. 7.1. Formation and evolution of Jupiter-mass planets to type II migration) happen in the same period, not always in the same order. In one case (the inner most planet shown in red), the change of the migration regime occurs ﬁrst, while in the three other cases it is the reverse. Once the migration regime changes to type II, the rate slows down (bottom centre panel of Fig. 17) but the accretion remains mostly constant. Thus, accretion dom- inates at the onset of this stage, but this reverses at the end. In contrast, Mordasini et al. (2009a) used the equilibrium values of the radial gas ﬂow for both gas accretion and migration. Thus, the slope of detached planet migrating with the planet-dominated case of the Type II regime exhibited a common slope in the mass- distance diagram. It should also be noted that for planets inside roughly 1 au, it happens that the criterion limiting the gas accre- tion rate changes to the mass in the feeding zone which leads to a reduction of the rate at the end of the formation. It can also be noted that our model allows for the growth of embryos at large separation (up to about 30 au, unlike the work of Johansen & Bitsch (2019). The difference is mainly related to the plan- etesimals size. Smaller planetesimals have lower eccentricities and inclinations because of more efﬁcient damping by the disc We show in Fig. 16 the formation tracks of a few synthetic giant planets whose masses are in the 100–500 M⊕range and have a wide range of ﬁnal positions. Due the inclusion of migration in the model, we observe that the ﬁnal position of these planets is closer-in that the initial location of the embryo: all the embryos start beyond 10 au, with one close to 30 au, while all the planets end up inside 10 au. During the initial stage, both accretion and migration are slow, but accretion is still faster. As the planets grow, migration becomes more efﬁcient; we observe that most of the migration occurs while the planets are close to the transition to gas giants, with masses between 20 and 50 M⊕. The innermost planet shows a strong inward migration at this stage, but this is due to lim- ited accretion while migration remains at the same rate. 7. Giant planets We provide the remaining two parameters, the initial masses of the gas and planetesimals discs in the following. A69, page 27 of 44 A&A 656, A69 (2021) 10−1 100 101 102 Distance [AU] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 104 105 106 107 Time [yr] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 0.115M 728M⊕ 0.030M 177M⊕ 0.032M 334M⊕ 0.027M 201M⊕ 104 106 108 1010 Time [yr] 10−2 101 104 107 Luminosity [L] 104 106 108 1010 Time [yr] 10−1 100 101 102 Distance [AU] 104 106 108 1010 Time [yr] 101 102 103 Radius [R⊕] Fig. 16. Formation and evolution tracks of four giant planets with ﬁnal masses in the 1/3 to 2 MX range in discs with a single embryo. Top panels: formation tracks with total mass Mtot versus distance (time goes towards the top) and total mass Mtot (solid lines) and core mass Mcore (dashed lines) versus time. Three panels on the bottom row: time dependence of the outer luminosity Ltot (bottom left), the distance (bottom centre) and the total radius Rtot (bottom right). For all panels except for the mass versus time (top right), the line styles denote the phase: dashed lines for the attached phase, solid line for the detached phase during formation and dash-dotted lines for the evolution stage. Line widths denote the migration regime, with tick lines for Type I and think lines for Type II. The legend in the top right panel shows the gas (in Solar masses) and planetesimals (in Earth mass) disc masses. 10−1 100 101 102 Distance [AU] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 104 105 106 107 Time [yr] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 0.115M 728M⊕ 0.030M 177M⊕ 0.032M 334M⊕ 0.027M 201M⊕ Mass [M⊕] Time [yr] 104 106 108 1010 Time [yr] 101 102 103 Radius [R⊕] AU] 0 104 106 108 1010 Time [yr] 10−1 100 101 102 Distance [AU] [ 104 106 108 1010 Time [yr] 10−2 101 104 107 Luminosity [L] Luminosity [L] Distance [AU] Radius [R⊕] Distance [ Fig. 16. Formation and evolution tracks of four giant planets with ﬁnal masses in the 1/3 to 2 MX range in discs with a single embryo. Top panels: formation tracks with total mass Mtot versus distance (time goes towards the top) and total mass Mtot (solid lines) and core mass Mcore (dashed lines) versus time. 7. Giant planets Three panels on the bottom row: time dependence of the outer luminosity Ltot (bottom left), the distance (bottom centre) and the total radius Rtot (bottom right). For all panels except for the mass versus time (top right), the line styles denote the phase: dashed lines for the attached phase, solid line for the detached phase during formation and dash-dotted lines for the evolution stage. Line widths denote the migration regime, with tick lines for Type I and think lines for Type II. The legend in the top right panel shows the gas (in Solar masses) and planetesimals (in Earth mass) disc masses. 7.1. Formation and evolution of Jupiter-mass planets Once the planets undergo the runaway accretion of gas and switch to type II migration, accretion is strong, and they experience lim- ited migration. This leads again to near-vertical tracks. The two changes (from an attached to a detached envelope and from type I A69, page 28 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 10−1 100 101 102 Distance [AU] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 0.090M 1038M⊕ 0.099M 558M⊕ 0.112M 737M⊕ 0.077M 332M⊕ 104 105 106 107 Time [yr] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 104 106 108 1010 Time [yr] 10−2 101 104 107 Luminosity [L] 104 106 108 1010 Time [yr] 10−1 100 101 102 Distance [AU] 104 106 108 1010 Time [yr] 101 102 103 Radius [R⊕] Fig. 17. Formation and evolution tracks of two groups of four giant planets with ﬁnal masses between 2 and 10 MX in discs with a single embryo. Panel and line descriptions are the same as Fig. 16. 10−1 100 101 102 Di t [AU] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 0.090M 1038M⊕ 0.099M 558M⊕ 0.112M 737M⊕ 0.077M 332M⊕ 104 105 106 107 Ti [ ] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 10−1 100 101 102 Distance [AU] 10−2 10−1 100 101 102 103 104 Mass [M⊕] 0.090M 1038M⊕ 0.099M 558M⊕ 0.112M 737M⊕ 0.077M 332M⊕ Mass [M⊕] Time [yr] 104 106 108 1010 Time [yr] 101 102 103 Radius [R⊕] U] 104 106 108 1010 Time [yr] 10−1 100 101 102 Distance [AU] 104 106 108 1010 Time [yr] 10−2 101 104 107 Luminosity [L] Di [AU] Distance [AU] Radius [R⊕] Fig. 17. Formation and evolution tracks of two groups of four giant planets with ﬁnal masses between 2 and 10 MX in discs with a single embryo. Panel and line descriptions are the same as Fig. 16. Fig. 17. Formation and evolution tracks of two groups of four giant planets with ﬁnal masses between 2 and 10 MX Panel and line descriptions are the same as Fig. 16. The simulations presented here show a situation with fast migration where no intermediate stage is possible, because the planets would otherwise end up at the inner edge of the gas without the opportunity to undergo runaway gas accretion. 7.1. Formation and evolution of Jupiter-mass planets This means that the cores must form just at the time to undergo runaway gas accretion. The usual picture of the formation of Jupiter-mass planets in our model is then more similar that what was found by Alibert et al. (2005a), with an almost nonexistent intermediate phase (left panel on the second row of Fig. 17). As the accretion time scale are longer at large separation, the embryos will accrete their mass over a longer period. At the same time, the inward migration experienced by the protoplan- ets means that their feeding zone is not depleted as in the in situ formation scenario. gas, and in addition, a larger capture probability by the planets for a given surface density because of the more strongly drag- enhanced capture radius for small planetesimals. This results in a larger accretion rate of solids, which enables planets to sufﬁciently grow to undergo runaway gas accretion before the dispersal of the gas disc also in the outer parts of the disc. The formation of Jupiter-like planets with migration and planetesimals accretion follows a different pattern in the one- planet-per-disc approximation studied here than what was found by some other models using the in situ (and one-embryo-per- disc) approximation. For the latter, the favoured scenario is that a core between 10 and 20 M⊕forms early (less than 105 yr) and undergoes runaway gas accretion only close to the disper- sal of the gas disc (Pollack et al. 1996; Alibert et al. 2018). The slow accretion of planetesimals, resulting in a steady lumi- nosity, is able to prevent runaway gas accretion during the intermediate stage. This intermediate stage is the problematic part when migration is included; the reason being that migration is most efﬁcient for planets that are between 10 and 50 M⊕(see Sect. 5.1.3 and Fig. 10). Hybrid pebble-planetesimals (Alibert et al. 2018) or pure pebble (Bitsch et al. 2019) accretion mod- els can account for the migration during the intermediate phase, as the cores are able to form at larger separation, provided that far out, bodies emerge early and massive enough to be able to efﬁciently capture pebbles. In contrast, for the multi-embryos simulations that we present in Paper II (see also Sect. 8.1), migration can be signif- icantly altered by mean-motion resonances chains. 7.1. Formation and evolution of Jupiter-mass planets In that case, the torque acting on one planet must be spread over all the bod- ies, meaning that the planet with the largest speciﬁc torque will migrate slower than it would were it not in a resonance chain. This provides a way to obtain an intermediate stage and less overall efﬁciency of migration, as we show in that work. This effect leaves open the possibility to have an intermediate stage for the formation of giant planets, as obtained in Alibert et al. (2018). Thus, once multiplicity is included, the simulations here A69, page 29 of 44 A&A 656, A69 (2021) 10−2 10−1 100 101 Distance [AU] 10−2 10−1 100 101 102 103 Mass [M⊕] 104 105 106 107 108 109 1010 Time [yr] 10−2 10−1 100 101 102 103 Mass [M⊕] 104 106 108 1010 Time [yr] 100 102 104 106 Luminosity [L] 104 106 108 1010 Time [yr] 10−2 10−1 100 101 Distance [AU] 104 106 108 1010 Time [yr] 101 102 103 Radius [R⊕] Fig. 18. Formation and evolution tracks of one giant planet that ends up being accreted by the star during the evolution stage due to tidal migration. Top left: total mass Mtot versus distance; top right: total mass Mtot (black) and core mass Mcore (red) versus time; bottom left: total luminosity Ltot (black) and the bloating contribution Lbloat (red) versus time; bottom centre: distance versus time; bottom right: total radius Rtot (black) and outer (attached phase) or Hill (detached phase) radius (red) versus time. 10−2 10−1 100 101 Distance [AU] 10−2 10−1 100 101 102 103 Mass [M⊕] 104 105 106 107 108 109 1010 Time [yr] 10−2 10−1 100 101 102 103 Mass [M⊕] 104 105 106 107 108 109 1010 Ti [ ] 10−2 10−1 100 101 102 103 Mass [M⊕] 10−2 10−1 100 101 Distance [AU] 10−2 10−1 100 101 102 103 Mass [M⊕] Mass [M⊕] Time [yr] 104 106 108 1010 Time [yr] 101 102 103 Radius [R⊕] Luminosity [L] Distance [AU] Radius [R⊕] Fig. 18. Formation and evolution tracks of one giant planet that ends up being accreted by the star during the evolution stage due to tidal migration. 7.2. More massive planets Figure 17 shows the formation tracks of planets that are in the 2 to 10 MX range. Compared to the planets previously discussed, these ones show a greater range of initial locations (from 6 to 40 au) and overall effect of migration. The planet shown in orange is the quickest to accrete a massive core and undergo run- away gas accretion, due to both the more massive disc and the inner location. The latter is made possible due to the disc’s mass. Figure 17 shows the formation tracks of planets that are in the 2 to 10 MX range. Compared to the planets previously discussed, these ones show a greater range of initial locations (from 6 to 40 au) and overall effect of migration. The planet shown in orange is the quickest to accrete a massive core and undergo run- away gas accretion, due to both the more massive disc and the inner location. The latter is made possible due to the disc’s mass. This is also the one to migrate the least before reaching 10 M⊕ because (1) the fast formation limits the effect of migration and (2) enters a convergence zone (see Fig. 10 and the discussion in Sect. 5.1.3). As the boundary of convergence zone moves inward (Lyra et al. 2010; Dittkrist et al. 2014) and to lower planetary masses over time, the planet shown in red will encounter the convergence zone at a different location, which will not affect the planet as much. become more similar to Jupiter formation models like Alibert et al. (2018). phase. This is seen on the top right panel of Fig. 17, where the tracks become dashed and thin during a brief section. The slope break that was discussed in for the Jupiter-mass is stronger for the two innermost planets. Comparing the time evolution of the two, it can be noted that the migration rate remains mostly constant while in the type II regime, while the accretion rate decreases. Concerning the radius and luminosity, we observe that all the planets show a similar behaviour even with the difference in the ﬁnal location. A69, page 30 of 44 7.1. Formation and evolution of Jupiter-mass planets Top left: total mass Mtot versus distance; top right: total mass Mtot (black) and core mass Mcore (red) versus time; bottom left: total luminosity Ltot (black) and the bloating contribution Lbloat (red) versus time; bottom centre: distance versus time; bottom right: total radius Rtot (black) and outer (attached phase) or Hill (detached phase) radius (red) versus time. become more similar to Jupiter formation models like Alibert et al. (2018). 8.1.1. Low initial solid content same time (as it can be seen in the lower right panel of Fig. 18). As the planet migrates inward, the Hill radius shrinks. Once the detached phase begins, the Hill radius continues th shrink as further inward migration continues. Many aspects of the emergence of the planetary systems can be understood with the comparison of the timescales of growth and migration, and the consequences of (large-scale) dynamical instabilities caused by the gravitational interactions of protoplan- ets. Therefore we colour code in Fig. 19 showing the temporal evolution of the system in the a −M plane the tracks of the planets by the ratio \|τmig/τgrow\| = \|d ln m/d ln a\|. Regarding the timescales, it is of fundamental importance that the oligarchic planetesimal accretion timescale increases with increasing planet mass (e.g. Thommes et al. 2003), whereas the orbital migration timescale in the Type I regime decreases with planet mass (e.g. Ward 1989). As this planet is close to the star (0.04 au), the evolution stage is different from the case shown previously. The luminos- ity increases over time time, the envelope gradually expands and looses mass due to atmospheric escape and the planet migrates further inward due to the tides raised onto the star. The migra- tion rate increases over time due to its strong dependence on the distance between the planet and the star (see Eq. (111)). To determine the reason for the luminosity increase, we print along- side the total value, the contribution from bloating (Eq. (63)). We see that from late in the formation stage until the end, this contributes to nearly all the planet’s luminosity. And as it goes with the stellar ﬂux, it increases at late times due to tidal migra- tion. The luminosity increase in turns leads to an expansion of the envelope, which increases the loss rate by atmospheric escape. But rather than this being the main cause of gas loss, we see that the bulk of the envelope is removed because it over- ﬂows the Hill sphere. This occurs suddenly at the end of the planet’s life, once the outer radius gets larger than the Hill sphere. Only a bare core remains, which get accreted by the star shortly thereafter. ) At the beginning (105 yr, top left panel of Fig. 19), the quasi in-situ accretion of planetesimals present in the initial)feeding zone of the embryos is the dominating process. 8. Individual systems After discussing formation pathways of terrestrial under ide- alised conditions, and of single giant planets, we ﬁnally show results obtained with the full model. Using many embryos per system, the model is able to produce a very large variety of plan- etary systems. These range from terrestrial planets (as we saw in the previous section) to giant planets. We ﬁrst provide two examples of the temporal emergence of planetary systems and then show the variety of the ﬁnal architecture of 23 systems. At the very beginning, all protoplanets grow as if they were the only bodies in the disc, not feeling the inﬂuence of the other protoplanets. With increasing mass, the interaction (either directly via N-body interactions) or indirectly via resonant migration, become important. By ×105 yr, the ﬁrst dynamical interactions have started among some of the more massive pro- toplanets, which is visible as a ‘jitter’ in some tracks, and two collisions, which are shown by two open grey circles. At 1 Myr (top right panel in Fig. 19), inside of the iceline, the character of growth has changed from planetesimal-dominated, to some ﬁrst growth via giant impacts (embryo-embryo colli- sions) for some protoplanets or stalled growth for others. As can be seen in panel a of Fig. 20 which shows the semi-major axis of the (proto)planets as a function of time colour coding the mass, about 10 further giant impacts have occurred. This has allowed the protoplanets in the inner disc to grow beyond the local isolation mass. As visible in panel b of Fig. 19, at 1 Myr, the planetesimal disc is now depleted out to about 1.3 au, and as time proceeds, the depletion moves even further out. We thus see a growth wave moving outwards (Thommes et al. 2003). All solid mass has been transferred into the embryos in this part, and 8.1.1. Low initial solid content Migration occurs at these very low masses on a much longer timescale, leading to nearly vertical upward tracks. We note that the model does not include any artiﬁcial reduction factors of Type I migration. The speciﬁc distance dependency of the mass to which the proto- planets have grown by 105 yr is given by the following interplay of growth timescale as a function of orbital distance and the local availability of solids: from the innermost embryo at about 0.03 au to the one at about 0.6 au, the protoplanets have already grown to the local planetesimal isolation mass (Lissauer 1987). Given the planetesimal surface density scaling with r−3/2, the isolation mass increases with orbital distance. As can be seen in panel b of Fig. 20 which shows the mean planetesimal surface density in the feeding zone of the planets, at 105 yr, the surface density is already strongly depleted in the inner parts of the disc. Between the local maximum at 0.7 au and the water iceline at 2.7 au, the mass is in contrast decreasing with distance because protoplanets further out grow slower. The next feature is a sharp increase of the protoplanets’ mass by about a factor 2 across the water iceline because of the increase of the solid surface density. One protoplanets grows in the transition zone, giving it an inter- mediate mass. Outside of the iceline, the masses decrease again with distance because of the longer growth timescales. For the protoplanets in the inner part that have already reached the isola- tion mass, the growth is temporally stalled. Because of the very low (isolation) masses of these protoplanets, orbital migration is nevertheless negligible. 7.4. Summary for giant planets The formation and evolution of giant planets involves multiple concurrent processes. Migration being most efﬁcient during the onset of the gas runaway accretion, this phase must occur in a relatively short time for the planets to not end up at the inner edge of the disc, in the absence of another planet to prevent migration. This also means that the cores must form late (i.e. shortly before the dispersal of the disc) to prevent a massive envelope from being accreted. Close-in planets will experience addition effects during their evolution, such as atmospheric escape and inward tidal migration that can lead to accretion by the star. In the latter case, it is possible for Hill sphere overﬂow to cause the loss of most of the envelope. 7.3. Giant planets ending in the star by tidal migration As an illustration how close-in planets are affected by the newly added physical processes during evolution, we ﬁnally discuss the formation and evolution of a close-in giant planet. These will raise tides onto the star, which will result in tidal migration. The consequence is that the planet can be accreted by the star at some point during its evolution. We show such a case in Fig. 18. The formation stage looks quite similar to the previous example, with the difference that the planet ends at a close-in location, 0.04 au. The radius shrinks already before the planet goes to the detached phase, because it experiences a strong inward migration at the Unlike the Jupiter-mass planets, all the ones of this group ﬁrst switch to type II migration before going to the detached A69, page 30 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 8.1. Two examples of the temporal evolution of the emergence of planetary systems Figures 19 and 20 show the formation of a planetary system in a protoplanetary disc with a low initial content of solids (System 30 in NG76, see Paper II). The initial disc gas mass is 0.023 M⊙ while [Fe/H] is −0.13, corresponding to a dust to gas ratio of 0.011. This results in a low initial solid content of 65.1 M⊕in the disc of planetesimals. The disc is seeded with 100 lunar mass embryos at t = 0, distributed uniformly in the logarithm of the semi-major axis inside of 40 au (see Paper II for more details on the initial conditions). A69, page 31 of 44 A&A 656, A69 (2021) Fig. 19. Example of the formation of a planetary system from initially 100 lunar-mass embryos in low gas mass initial mass 0.023 M⊙), low metallicity ([Fe/H] = −0.13) disc. The initial mass of planetesimals is 65.1 M⊕. Four moments in time (in years) are shown. Lines shows the growth tracks in the semi-major axis-mass plane. Black points show (proto)planets existing at a given epoch. Grey open circles show the last position of protoplanets that were accreted by another more massive body in a giant impact. The colours of the lines are \|τmig/τgrow\| = \|d ln m/d ln a\|. Fig. 19. Example of the formation of a planetary system from initially 100 lunar-mass embryos in low gas mass initial mass 0.023 M⊙), low metallicity ([Fe/H] = −0.13) disc. The initial mass of planetesimals is 65.1 M⊕. Four moments in time (in years) are shown. Lines shows the growth tracks in the semi-major axis-mass plane. Black points show (proto)planets existing at a given epoch. Grey open circles show the last position o protoplanets that were accreted by another more massive body in a giant impact. The colours of the lines are \|τmig/τgrow\| = \|d ln m/d ln a\|. Fig. 19. Example of the formation of a planetary system from initially 100 lunar-mass embryos in low gas mass initial mass 0.023 M⊙), low metallicity ([Fe/H] = −0.13) disc. The initial mass of planetesimals is 65.1 M⊕. Four moments in time (in years) are shown. Lines shows the growth tracks in the semi-major axis-mass plane. Black points show (proto)planets existing at a given epoch. Grey open circles show the last position of protoplanets that were accreted by another more massive body in a giant impact. 8.1. Two examples of the temporal evolution of the emergence of planetary systems The colours of the lines are \|τmig/τgrow\| = \|d ln m/d ln a\|. are now present. In the outer disc, beyond the iceline, the afore- mentioned group of the about 10 most massive protoplanets has grown further now reaching a maximum mass of 3 M⊕, and has also migrated further inward. As these planets migrate into zones that have been previously depleted by inner planets (in particular inside of the iceline), planetesimal accretion is quickly stalled. This means that planetesimal accretion for migrating planets is usually limited in low-mass multiple systems like the one present here. This means that a possible shepherding effect (Tanaka & Ida 1999) that we do not include in the model should not affect the outcome very much, except for a transition phase where τmig ≈τacc for some planets. This phase can be seen for the outer group from the cyan line colours. As can be seen in panel a of Fig. 20, the planets capture each other in very large resonant convoys and migrate together (e.g. Cresswell & Nelson 2008; Alibert et al. 2013). In this conﬁguration, outer more massive planets push inner smaller planets. their mutual interaction (giant impacts) governs the further mass growth. This implies that the accretion of planetesimals is only important at the early phases when the planets grow mostly in- situ. In the outer disc beyond the iceline, growth in contrast still proceeds mainly via planetesimal accretion, as there is a larger mass reservoir available. Between 2 and 4 au, a group of about 10 protoplantes with a mass of about 1 M⊕has formed, meaning that the most massive planets are now found further out than before. These protoplanets originate from (just) beyond the iceline. The colours of the lines in Fig. 19 show that migration is still much slower than accretion for these planets at 1 Myr, but some slight inward migration is now occurring, causing the tracks to bend inwards. This applies also to the inner disc, where horizontal tracks are visible. They result from the depletion of the planetes- imal disc, and the fact that the cores are of such a low mass that virtually no gas accretion is possible. At 3 Myr (bottom left panel of Fig. 19), in the inner disc, the dominant effect is further growth via giant impacts. A69, page 32 of 44 8.1. Two examples of the temporal evolution of the emergence of planetary systems About 25 protoplanets with masses between the one of Mars and Earth As visible in Fig. 20 by the small black circles, many giant impacts seem to occur in groups (i.e. at similar moments in time A69, page 32 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. Fig. 20. Same system as in Fig. 19, but now showing the semi-major axes a of the planets as a function of time, colour coding in panel a the planets’ mass, in (b) the planetesimal surface density in the planets’ feeding zone, and in (c) the local gas surface density. Here, the vertical line indicates the moment of gas disc dissipation. Panel d: mass as a function of time, colour coding the semi-major axis. Small black circles indicate giant impacts, by showing the position or mass of the target (the more massive collision partner) at the moment of the impact. Fig. 20. Same system as in Fig. 19, but now showing the semi-major axes a of the planets as a function of time, colour coding in panel a the planets’ mass, in (b) the planetesimal surface density in the planets’ feeding zone, and in (c) the local gas surface density. Here, the vertical line indicates the moment of gas disc dissipation. Panel d: mass as a function of time, colour coding the semi-major axis. Small black circles indicate giant impacts, by showing the position or mass of the target (the more massive collision partner) at the moment of the impact. One also sees that more massive bodies tend to be less eccentric, likely a consequence of energy equipartition. in fast sequence): a ﬁrst group occurs at about 3 Myr, a next one at 4 Myr, and again one at the moment when the disc inside of about 2 au becomes free of gas. This is visible in panel c of Fig. 20, which colour codes the gas surface density at the planets’ position. This moment corresponds to the opening of the inner hole in the gas disc because of internal photoevaporation (cf. Fig. 3). At this moment, the damping effect of the gas vanishes, allowing orbit crossings and collisions (e.g. Ida & Lin 2010). The outer gas disc dissipates a bit later, at 5.1 Myr, shown by the ver- tical line in panel c of Fig. 20. 8.1. Two examples of the temporal evolution of the emergence of planetary systems However, shortly after 0.5 Myr, a second core, located about 0.5 au outside of the ﬁrst giant, also starts runaway gas accretion. The embryo of this planets started at about 5.3 au, and was for some time in a resonant conﬁguration with the ﬁrst giant- to-be. It will eventually become the most massive giant planet in the system (about 2100 M⊕) at 1.2 au. Fig. 21. Temporal evolution of the eccentricities of the planets of the system emerging in the low-mass disc shown in Fig. 19. Colours indi- cate the planet mass. For better visibility, only planets more massive than 0.1 M⊕are shown. The curves are running averages such that one sees more clearly the mean values instead of rapid variations of the eccentricities. The thick black line is the mass of the gas disc relative to the value at 105 yr, which is in turn very similar to the initial value. The increase of the eccentricities at around 5 Myr when the gas disc dissipates is visible. The bottom right panel of Fig. 19 shows the system at 20 Myr, which corresponds to the time where we stop the N- body integration and planetesimal accretion. Between 3 and 20 Myr, numerous giant impacts have reduced the number of planets and destroyed the mean motion resonances (see also Fig. 20). The inner system now contains 8 roughly Earth-mass planets, exhibiting a certain inter-system similarity of the mass scale (Millholland et al. 2017) with an increase towards the exte- rior (Weiss et al. 2018). At 0.7 au, there is a sudden increase in the typical mass, corresponding to the transition from volatile-poor planets that have formed inside of the iceline, to very volatile- rich planets originating from beyond the iceline. Compared to the original location of the iceline at 2.7 au, there was thus an inward shift in this transition by about 2 au because of orbital migration. y ⊕ The growth of this second giant planet has important system- wide consequences, as can be seen in the panel at 1 Myr. It not only destabilises several Neptunian planets in the vicinity of the forming giants, but it also sends a protoplanet of about 3 M⊕from about 0.9 au into the inner system (close to 0.1 au). The orbit of this planet is eccentric, and triggers numerous giant impacts among the protoplanets in the inner system (see panel d of Fig. 8.1. Two examples of the temporal evolution of the emergence of planetary systems 0.0001 0.001 0.01 0.1 1 105 106 107 Eccentricity or gas disc mass fraction Time [yr] 0.1 1 M [M⊕] Fig. 21. Temporal evolution of the eccentricities of the planets of the system emerging in the low-mass disc shown in Fig. 19. Colours indi- cate the planet mass. For better visibility, only planets more massive than 0.1 M⊕are shown. The curves are running averages such that one sees more clearly the mean values instead of rapid variations of the eccentricities. The thick black line is the mass of the gas disc relative to the value at 105 yr, which is in turn very similar to the initial value. The increase of the eccentricities at around 5 Myr when the gas disc dissipates is visible. 0.0001 0.001 0.01 0.1 1 105 106 107 Eccentricity or gas disc mass fraction Time [yr] 0.1 1 M [M⊕] In the top left panel of Fig. 22 we see that at 105 yr, the basic picture regarding the (relative) mass of the protoplanets as a function of orbital distances is analogous to the one in the low-mass disc at the same time. In absolute terms, the planet masses are, however, about one order of magnitude larger. As can be seen in panel b of Fig. 23, the planetesimal disc is already strongly depleted out to about 1 au. Some giant impacts have also already occurred in the inner disc. This fast development away from the initial conditions is a sign that the early phase of solid growth (from dust to embryos) should be treated more explicitly (e.g. Voelkel et al. 2021). ( g ) The situation at 0.5 Myr is already quite different, as a ﬁrst core has undergone runaway gas accretion, at about 0.35 Myr. By 0.5 Myr, its mass has already grown to about 350 M⊕. In the end, it will have a mass close to 750 M⊕and be the innermost giant planet. The starting position of this embryo was 4.5 au. The water iceline in this system is for comparison found at about 3.4 au. The formation of this ﬁrst giant planet does not yet strongly affect the rest of the system, at least at this moment. In the inner system, we in particular see a similar development as in the low-mass disc: the formation of very large resonant convoys and some giant impacts. 8.1. Two examples of the temporal evolution of the emergence of planetary systems 23). These orbit crossings and impacts are facilitated because the runaway gas accretion by the two forming giants strongly reduces the gas surface density in the inner disc temporarily, reducing eccentric damping (panel c of Fig. 23). In the end, only the intruder from the exterior remains, the mass of which has increased to about 13 M⊕by accreting the local protoplan- ets. The formation of the second giant also scatters an initially low-mass protoplanet (0.7 M⊕) from about 2 au onto a very eccentric orbit with a semi-major axis of about 15 au. This proto- planet grows then out there (potentially in a monarchical growth mode, Weidenschilling 2005), reaching a mass of about 3 M⊕by 1 Myr. In the end, the planetesimal disc is depleted out to about 5 au. Outside, about 35 M⊕remain in the form of planetesimals. This corresponds to a fraction of about 46% of the initial planetesimal mass that was converted into planets. Since we follow the accre- tion for only 20 Myr, this remaining planetesimal mass must be considered an upper limit for the actual mass of remaining planetesimals, as over longer timescales, the distant protoplan- ets would continue to accrete. However, since the accretion timescales at several 10 au in the absence of eccentricity damping (corresponding to orderly growth) become extremely long (Ida & Lin 2004a), at least some part of these planetesimals could remain to eventually form a debris disc, in analogy to the Kuiper belt beyond the orbit of Neptune in the Solar System. y By about 1.4 Myr, its mass has increased to 8 M⊕, and a phase of rapid inward migration sets in. It then runs from out- side into a group of 7 protoplanets at about 2 to 4 au that are captured in MMRs with the giant planet that had formed sec- ond (see panel a of Fig. 23. A series of giant impacts occur, and at 1.8 Myr, the protoplanet coming from the outside starts run- away gas accretion. It eventually becomes the third giant planet in the system with a mass of about 630 M⊕at 2.5 au. Interest- ingly enough, this implies that giant planets in a system need not to be strictly coeval, which could be of importance for exam- ple for direct imaging observations. 8.1. Two examples of the temporal evolution of the emergence of planetary systems After the dissipation of the disc, only 3 more giant impacts occur in this system to 20 Myr. In our model, dynamical friction by residual planetesimals is neglected. This would reduce the eccentricities and inclination of the protoplanets. This implies that our model tends to overes- timate the eccentricities and inclinations of lower mass planets for which dynamical friction by planetesimals would play a role. The general sequence of solid growth that is ﬁrst dominated by the near in-situ accretion of planetesimals followed by the sec- ond phase of growth via giant impacts is well visible in panel d of Fig. 20. It shows the mass of the protoplanets as a function of time. The line colours show the semi-major axis. We note how the transition between the two regimes occurs the later the more distant a planet is. At the largest orbital distances where embryos were inserted into the disc (maximum starting distance is 40 au), nearly no growth at all has occurred during the simulated period. As described in Sect. 5.2.3, numerically speaking, we add the mass of the impactor in a giant impact over a timescale of 104 yr to the target. This is the reason why the vertical steps in the curves corresponding to giant impacts (indicated with the black circles) are not strictly vertical. This is visible particularly at the early ages. The temporal evolution of the eccentricities is shown in Fig. 21. The colours show the planet mass. For clarity, only plan- ets with a mass of at least 0.1 M⊕were included. One can clearly see the increase of the typical values of the eccentricities near the time the gas disc dissipates at around 5 Myr. Before, typical values of the eccentricities are of the order of 10−3 to a few 10−2. After disc dissipation, they increase to values between about 0.02 to 0.2. Such values are expected from the increase of the velocity dispersion of the orbits until they are comparable to the escape velocity from their surfaces resulting from close encoun- ters, once the damping by the gas is gone (Goldreich et al. 2004). A69, page 33 of 44 A&A 656, A69 (2021) an orbital distance of 40 au. The evolution in the a −M plane is shown in Fig. 22. The semi-major axis and mass as function of time is shown in Fig. 23. 8.1. Two examples of the temporal evolution of the emergence of planetary systems Here, the outermost giant is nearly 1.5 Myr younger, and starts runaway accretion only when the inner two planets have already reached nearly their ﬁnal mass. Actually, the fact that this third outer planet forms strongly reduces the gas accretion rate of the middle giant, by 8.1.2. High initial solid content So, more precisely speaking, the formation of this third giant actually sets the ﬁnal mass of the giant planet inside of it. A comparable, transient depletion of the inner gas disc is already also seen when the inner two giants form, as mentioned. It should be noted that the degree of depletion of the inner disc because of gas accreting giant planets might be overestimated in our model (Manara et al. 2019; Nayakshin et al. 2019; Bergez- Casalou et al. 2020). Then, this indirect interactions via the disc would be reduced. The lifetime of the gas disc is in this exam- ple about 3.4 Myr. This is less than the lifetime of the low mass system studied in the previous section, despite the higher start- ing mass. The difference is mainly a consequence of the higher external photoevaporation by nearly a factor 5 (it is an indepen- dent initial condition, see Paper II). The gas accretion of the giant planets also contributes to the dispersal by them containing in the end about 0.01 M⊙of gas (out of the initial disc mass of 0.066 M⊙). reducing the gas surface density in the inner system (see panel c of Fig. 23). So, more precisely speaking, the formation of this third giant actually sets the ﬁnal mass of the giant planet inside of it. A comparable, transient depletion of the inner gas disc is already also seen when the inner two giants form, as mentioned. It should be noted that the degree of depletion of the inner disc because of gas accreting giant planets might be overestimated in our model (Manara et al. 2019; Nayakshin et al. 2019; Bergez- Casalou et al. 2020). Then, this indirect interactions via the disc would be reduced. The lifetime of the gas disc is in this exam- ple about 3.4 Myr. This is less than the lifetime of the low mass system studied in the previous section, despite the higher start- ing mass. The difference is mainly a consequence of the higher external photoevaporation by nearly a factor 5 (it is an indepen- dent initial condition, see Paper II). The gas accretion of the giant planets also contributes to the dispersal by them containing in the end about 0.01 M⊙of gas (out of the initial disc mass of 0.066 M⊙). their gas envelopes, they get hit by several lower-mass proto- planets that they destabilise. 8.1.2. High initial solid content The second system we consider is System 852 in NG76 (Paper II). The initial conditions are here a disc mass of 0.066 M⊙and a metallicity of [Fe/H] = 0.23. This leads to an initial planetesimal mass of 432 M⊕, 6.6 times as much as in the ﬁrst example. As in the previous case, 100 lunar mass embryos are put into the disc at the beginning, uniform in log of the semi-major axis out to A69, page 34 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. Fig. 22. Example of the formation of a planetary system from initially 100 lunar-mass embryos in a high gas mass (initial mass 0.066 M⊙), h metallicity ([Fe/H] = 0.23) disc. The initial mass of planetesimals is 432 M⊕. The plot is analogous to Fig. 19, but the y-axis now extends to m higher masses, and the moments in time that are shown are different. At the end of the simulation at 20 Myr, this system contains one clos sub-Neptunian planet, three giant planets, and a group of outer very low-mass planets. Fig. 22. Example of the formation of a planetary system from initially 100 lunar-mass embryos in a high gas mass (initial mass 0.066 M⊙), high metallicity ([Fe/H] = 0.23) disc. The initial mass of planetesimals is 432 M⊕. The plot is analogous to Fig. 19, but the y-axis now extends to much higher masses, and the moments in time that are shown are different. At the end of the simulation at 20 Myr, this system contains one close-in sub-Neptunian planet, three giant planets, and a group of outer very low-mass planets. Fig. 22. Example of the formation of a planetary system from initially 100 lunar-mass embryos in a high gas mass (initial mass 0.066 M⊙), high metallicity ([Fe/H] = 0.23) disc. The initial mass of planetesimals is 432 M⊕. The plot is analogous to Fig. 19, but the y-axis now extends to much higher masses, and the moments in time that are shown are different. At the end of the simulation at 20 Myr, this system contains one close-in sub-Neptunian planet, three giant planets, and a group of outer very low-mass planets. reducing the gas surface density in the inner system (see panel c of Fig. 23). 8.1.2. High initial solid content It is an example of an inner sub-Neptunian planet in a system with outer giant planets (see Paper III). Finally, it is worth mentioning that systems with three giant planets are statistically a very rare outcome in the population synthesis (Paper II): there are only ﬁve such systems among the 1000 synthesised in the nominal population NG76. Systems with one or two giants are in comparison much more common (each about 100 systems). In the system at hand, orbital stability is provided by the giant planets residing in the 3:1 mean motion resonance for both pairs of planets. This allows them to remain stable (Alves et al. 2016) despite their relative proximity to each other, corresponding for both pairs to about 6 to 7 mutual Hill radii, and their signiﬁcant eccentricities (about 0.08, 0.18, and 0.40 for the inner, middle, and outer planet). We have further tested the stability of this system by extending the orbital inte- gration (including all bodies in the system) from 20 to 100 Myr. At least on this timescale, the system remained stable without secular growth of the eccentricities. It is important to point out a potential complication concern- ing the formation of the outer two giant planets in the system shown in the panel q of Fig. 24. These planets accreted their cores and started undergoing runaway gas accretion in the inner region of the system. They were subsequently moved to the outer region of the disc where they continue to accrete gas. However, the reason for their ﬁnal distant locations are not planet-planet scatterings. The presence of a inner massive giant planet (in this case, the one at 0.2 au with nearly 30 MX, which corresponds to 3% of the mass of the central star, for an initial disc mass of 9% of the stellar mass) results here in the outer planets obtain- ing large eccentricities. This, in turn, causes the prescription for the modulation of the torque (Eqs. (86) and (87)) to reverse its sign. Via the additional forces added to the N-body integrator (Sect. 5.2.1), this if found to lead to outward migration in the present case. Generally speaking, a positive torque means that the angu- lar momentum of a planet has to grow. For an eccentric planet, this can occur via two ways (Cresswell et al. 2007): by eccen- tricity reduction (circularisation) or outward migration (increase of the semi-major axis). 8.1.2. High initial solid content This increases the core mass of the three giants from about 24, 14 and 10 M⊕at the onset of gas runaway accretion to clearly higher ﬁnales values of 64, 26, and 21 M⊕, respectively. Such giants impacts thus strongly inﬂuence the ﬁnal heavy element content (Thorngren & Fortney 2018), and could potentially lead to the existence of diluted cores as found in Jupiter (Liu et al. 2019). p At the end of the simulation at 20 Myr, the system contains four planets more massive than one Earth mass. During the emer- gence of the system, eight protoplanets have collided with the host star and four were ejected. About 244 M⊕of planetesimals remain out of the starting value of 432 M⊕, corresponding to a difference of 188 M⊕. However, the planets actually existing at the end contain only 123 M⊕, meaning that about 65 M⊕of plan- etesimals were ‘lost’ because they were either directly ejected or contained in planets that were themselves ejected or fell into the star. This correspond to a solid conversion efﬁciency of plan- etesimals into planets of about 28%. Over gigayear timescales, atmospheric escape reduces the mass of the close-in planet at 0.08 au from 13.2 to 11.6 M⊕, but it does retain a remaining H/He envelope. Under the effect of bloating, the planet therefore has a The temporal evolution of this system shows how the growth of multiple giant planets strongly affects the overall system architecture. This also has important consequences for the giant planets themselves (see panel d of Fig. 23): while they accrete A69, page 35 of 44 A&A 656, A69 (2021) ig. 23. Temporal evolution of the system shown in Fig. 22. The four panels have the same meaning as in Fig. 20. A&A 656 A&A 656, A69 (2021) A&A 656, A69 (2021) Fig. 23. Temporal evolution of the system shown in Fig. 22. The four panels have the same meaning as in Fig. 20. Fig. 23. Temporal evolution of the system shown in Fig. 22. The four panels have the same meaning as in Fig. 20. individual systems without taking into account the likelihood of such systems in populations. Hereafter we give an overview of some major correlations that we ﬁnd. For quantitative results, we refer to the next papers of the series. relatively large radius of 5.3 R⊕at 5 Gyr. 8.1.2. High initial solid content The different approaches how to trans- late the positive torque found in hydrodynamical simulations into the additional N-body forces have been inconsistent with one A69, page 36 of 44 8.2. Overview of the diversity of system architectures Figures 24 and 25 show the mass-distance and radius-distance of 23 synthetic systems. The solar system is shown in the top- left panel for comparison. All these systems are again taken from the nominal synthetic population NG76 for 1 M⊙star that will be presented in Paper II. However, here we study these as A69, page 36 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 10−2 100 102 104 Mass [M⊕] 100 102 104 106 Period [days] a 0.41 1261 M⊕ 100 102 104 106 Period [days] b 0.39 338 M⊕ 100 102 104 106 Period [days] c 0.35 625 M⊕ 100 102 104 106 Period [days] 10−4 10−2 100 102 Mass [MJup] 10−2 100 102 104 Mass [M⊕] d 0.24 155 M⊕ e 0.23 285 M⊕ f 0.22 184 M⊕ g 0.17 37 M⊕ 10−4 10−2 100 102 Mass [MJup] 10−2 100 102 104 Mass [M⊕] h 0.17 104 M⊕ i 0.15 402 M⊕ j 0.12 92 M⊕ k 0.04 700 M⊕ 10−4 10−2 100 102 Mass [MJup] 10−2 100 102 104 Mass [M⊕] l 0.03 103 M⊕ m 0.02 419 M⊕ n 0.00 118 M⊕ o 0.00 398 M⊕ 10−4 10−2 100 102 Mass [MJup] 10−2 100 102 104 Mass [M⊕] p −0.01 350 M⊕ q −0.01 377 M⊕ r −0.13 65 M⊕ s −0.13 39 M⊕ 10−4 10−2 100 102 Mass [MJup] 10−2 100 102 Distance [AU] 10−2 100 102 104 Mass [M⊕] t −0.21 72 M⊕ 10−2 100 102 Distance [AU] u −0.31 221 M⊕ 10−2 100 102 Distance [AU] v −0.36 25 M⊕ 10−2 100 102 Distance [AU] w −0.41 47 M⊕ 10−4 10−2 100 102 Mass [MJup] g. 24. Mass-distance diagrams of speciﬁc systems with 100 embryos initially (panels a–w), which are taken from the nominal populatio edicted for a 1 M⊙star (NG76). Symbols are as follows: red points show gas-rich planets where Menv/Mcore > 1. Blue symbols are planets th ve accreted some volatile material (ices) outside of the ice line(s). Green symbols are planets that have only accreted refractory solids. Ope een and blue circles have 0.1 ≤Menv/Mcore ≤1 while ﬁlled green points and blue crosses have Menv/Mcore ≤0.1. For all these bodies, the gr rizontal bars go from a −e to a + e. 8.2. Overview of the diversity of system architectures The top left panel with black crosses shows the solar system. Bodies lost because of collisions or ejectio e shown in light grey. Planets accreted by the central star are show in the very left of each panel, the ejected ones on the very right and plane at collided with another (more massive) planet are shown at their last position on the diagram. The dotted vertical line in each system shows th cation of the ice line. The number after each panel name is the metallicity [M/H] of the system expressed in dex, while the value on the top rig the initial mass of the planetesimals disc. other in the literature in the past in this regard (Ida et al 2020) is becoming ill deﬁned The setups used to derive the eccen Fig. 24. Mass-distance diagrams of speciﬁc systems with 100 embryos initially (panels a–w), which are taken from the nominal population predicted for a 1 M⊙star (NG76). Symbols are as follows: red points show gas-rich planets where Menv/Mcore > 1. Blue symbols are planets that have accreted some volatile material (ices) outside of the ice line(s). Green symbols are planets that have only accreted refractory solids. Open green and blue circles have 0.1 ≤Menv/Mcore ≤1 while ﬁlled green points and blue crosses have Menv/Mcore ≤0.1. For all these bodies, the grey horizontal bars go from a −e to a + e. The top left panel with black crosses shows the solar system. Bodies lost because of collisions or ejections are shown in light grey. Planets accreted by the central star are show in the very left of each panel, the ejected ones on the very right and planets that collided with another (more massive) planet are shown at their last position on the diagram. The dotted vertical line in each system shows the location of the ice line. The number after each panel name is the metallicity [M/H] of the system expressed in dex, while the value on the top right is the initial mass of the planetesimals disc. another in the literature in the past in this regard (Ida et al. 2020). A re-assessment was recently made in Ida et al. (2020), but is not yet included in the simulations shown here. is becoming ill-deﬁned. The setups used to derive the eccen- tricity and inclination damping expressions and their translation into additional N-body forces (e.g. 8.2. Overview of the diversity of system architectures Papaloizou & Larwood 2000; Cresswell & Nelson 2008; Bitsch et al. 2013; Ida et al. 2020) assume that the disc orbits on a nearly circular orbit centred on the star. However, in the case here, the planet and outer disc will The problem we encounter in the special setup here (the pres- ence of an inner very massive giant planet) is likely that the eccentricity state towards which eccentricity damping is acting A69, page 37 of 44 A&A 656, A69 (2021) A&A 656, A69 (2021) A&A 656, A69 (2021) 10−1 100 101 100 102 104 106 Period [days] d 0.24 155 M⊕ 100 102 104 106 Period [days] h 0.17 104 M⊕ 100 102 104 106 Period [days] l 0.03 103 M⊕ 100 102 104 106 Period [days] 10−2 10−1 100 10−1 100 101 Radius [R⊕] m 0.02 419 M⊕ n 0.00 118 M⊕ r −0.13 65 M⊕ s −0.13 39 M⊕ 10−2 10−1 100 Radius [R] 10−2 100 102 Distance [AU] 10−1 100 101 t −0.21 72 M⊕ 10−2 100 102 Distance [AU] u −0.31 221 M⊕ 10−2 100 102 Distance [AU] v −0.36 25 M⊕ 10−2 100 102 Distance [AU] w −0.41 47 M⊕ 10−2 10−1 100 Fig. 25. Radius-distance diagrams for a subset of the systems shown in Fig. 24. Here, we focus on systems with multiple low-mass planets; panel letters correspond to the same system in Fig. 24. In contrast to Fig. 24, lost planets are not shown for clarity. Fig. 25. Radius-distance diagrams for a subset of the systems shown in Fig. 24. Here, we focus on systems with multiple low-mass planets; panel letters correspond to the same system in Fig. 24. In contrast to Fig. 24, lost planets are not shown for clarity. (1) the gas accretion rate in the disc-limited regime could be too high and (2) the simple Type II migration model we employ in this work leads to too much inward migration. tend to orbit the barycenter of the star-inner giant pair, which means that the eccentricity can likely not be stabilised near zero. Figuring out the consequences for the orbital evolution of the different involved planets would likely require dedicated hydro- dynamical simulations. This shows a limitation of our N-body approach with additional forces instead of direct hydrodynami- cal simulations. This implies that the model results for distant giant planets with an inner massive planet must be taken with caution. 8.2.2. Similarity in the low-mass systems Systems where only terrestrial planets are present have planets with similar properties. It can be seen in panels d, g, h, l, n, r, s, t, v, and w. This is result consistent with observational results about masses and spacing (Millholland et al. 2017). To provide a comparison point with the similarity of planet radii (Weiss et al. 2018), we provide a radius-distance diagram in Fig. 25. For the rocky planets, both masses and radii show the same similarity. The transition from rocky to icy planets affects the radii only slightly. More important is the presence of (remaining) H/He envelopes that were not removed by photoevaporation. 8.2.1. Mass and ﬁnal number of planets The number of planets that remain past the formation stage is anti-correlated to the mass of the formed planets. Systems form- ing giant planets loose more embryos than the ones forming low-mass planets only. We obtained some systems where only one giant planets remains, for instance in panels a and c (includ- ing a single one in the latter case), where all the other embryos were removed during the formation stage. When this occurs, at least one of the ﬁnal planets remains on a wide orbit, as it needs to clear the outer embryos. If this is not the case, then we observe that some embryos with low masses remain in the outer region (e.g. panels e and i). We observe a general slight increase of mass with distance, at least in the inner region. This is most likely linked to the surface density proﬁle of solids. The isolation mass Miso ∝r(1.5(2−βs)) (Lissauer 1987), and so since we have βs = 1.5, the value increases with distance. This increase stops at locations usu- ally slightly outside of 1 au, which could be due to our limited integration time, as we discussed in the previous section. g p Systems that still form giant planets, but of lower masses, are able to retain more bodies. We have a few examples that have an architecture in the fashion the solar system, with terrestrial planets inside of giants, such as in panels e, i, m and p. However, those are not comparable to the solar system for several reasons. First, the giant planets are quite more massive than in the solar system; it is not uncommon to ﬁnd masses of the order of 5– 10 MX. Likewise, the terrestrial planets are many Earth masses. Further, the location of the giants is much closer in that Jupiter, with distances that are around 1 au. These ﬁndings indicate that 8.2. Overview of the diversity of system architectures Finally, systems that form low-mass planets only remain with the largest number of bodies. This is seen for instance in panels l, n, r, t, v and w, where many ice-free bodies (shown in green circles) are present at the end. A69, page 38 of 44 8.2.3. Composition of the close-in planets We ﬁnd that close-in terrestrial planets are likely to be rocky, which is in agreement with inferences from observations (Jin & Mordasini 2018). This is especially the case for systems where A69, page 38 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. allows for example to study the luminosities at any time (Mordasini et al. 2017), and enables the comparison with directly-imaged exoplanets (e.g. Vigan et al. 2017). no planets grow to more than a few Earth masses. We observe in all systems that icy planets are found inside the location of the ice line (the dotted vertical line). Nevertheless, the innermost planets only accrete from the inner region of the disc where the planetesimals are rocky. This indicates that these planets neither migrate from beyond the ice line to their current position, nor get moved to other locations by mean of dynamical instability. It should be noted that in our simulations, planetesimals composi- tion is set from the initial temperature and pressure proﬁle of the gas disc (Sect. 3.3.3). The model now includes a multitude of physical processes (see Fig. 2). The following are included during both the forma- tion and evolution phase: – A solution of 1D radially symmetric internal structure equa- tions (Bodenheimer & Pollack 1986) is used to calculate the internal structure of the H/He envelope and thus the gas accretion rate (during the attached phase), radius and luminosity, which includes Deuterium burning (Mollière & Mordasini 2012) and bloating of close-in planets. Nevertheless, there are systems without giant planet that con- sist of only ice-bearing bodies; these are shown in panels d, g and u. These systems form planets that are more massive than the previous ones, with most of them having at least one planet above 10 M⊕. The Type I migration timescale decreases with increas- ing mass; therefore these more massive planets can migrate from outside of the water ice line to their current position, increas- ing the compositional diversity of the systems (Raymond et al. 2018). y g ( Mordasini 2012) and bloating of close-in planets. – The solution of the 1D internal core structure is used to obtain the radius of the solid core with a modiﬁed polytropic EOS (Seager et al. 2007). g – An atmospheric model yields the outer boundary conditions during the attached, detached, and evolutionary phase. 8.2.3. Composition of the close-in planets For the detached phase, we assume hot gas accretion. For the evolutionary phase, we use a simple grey atmosphere. – The host star properties are retrieved from tabulated stellar evolution tracks (Baraffe et al. 2015). Systems with giant planets exhibit different behaviours. Some have only ice-bearing planets (panels b, f and q) while others have also terrestrial planets. In the latter case, the giant planets do not necessarily separate rocky bodies from icy ones. Panels e and o show systems where rocky and icy planets are separated by giants, while in panels i, m and p icy planets are present both inside and outside of the gas giants. This points at a high diversity of the composition of planets in systems con- taining both giant and low-mass planets. Correlations between the occurrences of giant planets and others in planetary systems will be investigated in more details in Paper II. Schlecker et al. (2021a, hereafter Paper III) will look thoroughly at correlations between close-in Super-Earth planets and long-period giants. During formation, the following processes are included: – The radial structure of the protoplanetary gas disc is com- puted with a 1D radial (axis-symmetric) constant α-disc model. The effects of internal and external photoevaporation are included. – The vertical structure of the disc is modelled by building on radiative equilibrium (Nakamoto & Nakagawa 1994), includ- ing viscous heating and stellar irradiation (Fouchet et al. 2012). Irradiation now also includes the direct irradiation in the disc midlplane important when the disc becomes optically thin. – Planetesimals are presented by a 1D radial (axis-symmetric) disc, with a surface density and a dynamical state (eccentricity, inclination). The temporal evolution of e and i are explicitly followed, including the dynamic excitation by protoplanets and planetesimals, and damping from gas drag (Fortier et al. 2013). The composition of the planetesimal and the position of ice lines is found from an equilibrium condensation model (Thiabaud et al. 2015). 9. Summary and conclusions In this work, we presented the Generation III version of the Bern global model of planetary formation and evolution. In this gen- eration, the following two main aspects were improved. First is the ability to simulate planets with a mass range from Mars to deuterium-burning planets. Older generations of the Bern model could not address terrestrial planets, as they we lack- ing the giant-impact stage. To reach this goal, we improved the N-body integrator so that per disc, hundreds of concur- rently forming embryos can now be included. This is crucial for the formation of low-mass planets in general and the Solar System. We also added several new physical processes to take into account the consequences of stellar proximity, allowing us to simulate with the new model planets that cover the widest range of orbital separations, from star-grazing to distant and even rogue planets. Second, the ability to predict self-consistently for multi-planet systems as many directly observable quantities as possible: not only masses and orbital elements as in the past, but also other key observables like luminosities, magnitudes, transit radii, or evaporation rates. To achieve this, we coupled our planet formation model (to 20 Myr) to our planet evolution model (20 Myr–10 Gyr). Thanks to this, we can now self-consistently and statistically compare the same population to all important observational techniques, as will be done in the series of NGPPS papers. This is crucial, as different methods probe distinct plane- tary sub-populations. This combined comparison puts extremely compelling and powerful constraints on any theoretical model. In this work, we presented the Generation III version of the Bern global model of planetary formation and evolution. In this gen- eration, the following two main aspects were improved. First is the ability to simulate planets with a mass range from Mars to deuterium-burning planets. Older generations of the Bern model could not address terrestrial planets, as they we lack- ing the giant-impact stage. To reach this goal, we improved the N-body integrator so that per disc, hundreds of concur- rently forming embryos can now be included. This is crucial for the formation of low-mass planets in general and the Solar System. We also added several new physical processes to take into account the consequences of stellar proximity, allowing us to simulate with the new model planets that cover the widest range of orbital separations, from star-grazing to distant and even rogue planets. References 2013, A&A, 558, A109 Alibert, Y., Venturini, J., Helled, R., et al. 2018, Nat. Astron., 2, 873 Ali-Dib, M., Cumming, A., & Lin, D. N. C. 2020, MNRAS, 494, 2440 Allègre, C. J., Manhès, G., & Göpel, C. 2008, Earth Planet. Sci. Let Alves, A. J., Michtchenko, T. A., & Tadeu dos Santos, M. 2016, Celest. Mech. Dyn. Astron., 124, 311 Andrews, S. M., Wilner, D. J., Hughes, A. M., Qi, C., & Dullemond, C. P. 2010, ApJ, 723, 1241 Ansdell, M., Williams, J. P., Trapman, L., et al. 2018, ApJ, 859, 21 Arimatsu, K., Tsumura, K., Usui, F., et al. 2019, Nat. Astro Bailey, E., & Batygin, K. 2018, ApJ, 866, L2 Baraffe, I., Chabrier, G., Allard, F., & Hauschildt, P. H. 1998, A&A, 337, 403 Baraffe, I., Chabrier, G., & Barman, T. 2008, A&A, 482, 315 Baraffe, I., Homeier, D., Allard, F., & Chabrier, G. 2015, A&A, 577, A42 Baruteau C Crida A Paardekooper S J et al 2014 in Protostars and P Baruteau, C., Crida, A., Paardekooper, S. J., et al. 2014, in Protostars and Planets We also give a short overview of the diversity of planetary systems that were obtained using the model. We ﬁnd that systems containing giant planets can have a great diversity of conﬁgu- rations, while for systems forming only low-mass (Earth-like) planets exhibit arranged planets with similar masses. p VI, eds. H. Beuther, R. S. Klessen, C. P. Dullemond, & T. Henning (Tucson: University of Arizona Press) 667 VI, eds. H. Beuther, R. S. Klessen, C. P. Dullemond, & T. Henning (Tucson: University of Arizona Press), 667 VI, eds. H. Beuther, R. S. Klessen, C. P. Dullemond, & T. Henning (Tucson: University of Arizona Press), 667 Baruteau, C., Bai, X., Mordasini, C., & Mollière, P. 2016, Space Sci. Rev., 205, Baruteau, C., Bai, X., Mordasini, C., & Mollière, P. 2016, Space Sci. Rev., 205, 77 77 Batygin, K., Bodenheimer, P. H., & Laughlin, G. P. 2016, ApJ, 829, 114 Bell, K. R., & Lin, D. N. C. 1994, ApJ, 427, 987 Benítez-Llambay, P., Masset, F., & Beaugé, C. 2011, A&A, 528, A2 Batygin, K., Bodenheimer, P. H., & Laughlin, G. P. 2016, ApJ, 829, 114 Batygin, K., Bodenheimer, P. H., & Laughlin, G. P. 2016, ApJ, 829, 114 Bell, K. R., & Lin, D. N. C. 1994, ApJ, 427, 987 Bell, K. R., & Lin, D. N. C. 9. Summary and conclusions The authors thank David Swoboda, Natacha Brügger, Martin Schlecker, and the members of the Theoretical Astrophysics and Plan- etary Science (TAPS) group at the University of Bern for fruitful discussions. We also thank the anonymous reviewer, whose remarks and suggestions helped improve the quality of this manuscript. A.E. and E.A. acknowledge the support from The University of Arizona. A.E. and C.M. acknowledge the support from the Swiss National Science Foundation under grant BSSGI0_155816 ‘PlanetsIn- Time’. Parts of this work have been carried out within the frame of the National Center for Competence in Research PlanetS supported by the SNSF. The plots shown in this work were generated using matplotlib (Hunter 2007). Acknowledgements. The authors thank David Swoboda, Natacha Brügger, Martin Schlecker, and the members of the Theoretical Astrophysics and Plan- etary Science (TAPS) group at the University of Bern for fruitful discussions. We also thank the anonymous reviewer, whose remarks and suggestions helped improve the quality of this manuscript. A.E. and E.A. acknowledge the support from The University of Arizona. A.E. and C.M. acknowledge the support from the Swiss National Science Foundation under grant BSSGI0_155816 ‘PlanetsIn- Time’. Parts of this work have been carried out within the frame of the National Center for Competence in Research PlanetS supported by the SNSF. The plots shown in this work were generated using matplotlib (Hunter 2007). – tidal spiral-in because of stellar tides (Benítez-Llambay et al. 2011), along with Roche-lobe overﬂow. We show in Sect. 6 where we study the formation of terres- trial planets that provided there are initially enough embryo in each disc, mutual gravitational interactions will stir their eccen- tricities. Due to the radial excursions, embryos will have access to more material until all the planetesimals are accreted. After- wards, a phase of giant impacts sets in. Thus, despite the use a ﬂuid-like description for the planetesimals, the model is able to reproduce the giant impact phase of terrestrial planet forma- tion. Due to the limitation of the integration time (20 Myr), this is only completely modelled within a distance of roughly 1 au. Giant planets, in contrast, are not affected by the integration time limitation as they must anyway form before the dispersal of the gas disc. The model is then able to track the formation of all planets in the inner part of planetary systems. A69, page 40 of 44 9. Summary and conclusions Second, the ability to predict self-consistently for multi-planet systems as many directly observable quantities as possible: not only masses and orbital elements as in the past, but also other key observables like luminosities, magnitudes, transit radii, or evaporation rates. To achieve this, we coupled our planet formation model (to 20 Myr) to our planet evolution model (20 Myr–10 Gyr). Thanks to this, we can now self-consistently and statistically compare the same population to all important observational techniques, as will be done in the series of NGPPS papers. This is crucial, as different methods probe distinct plane- tary sub-populations. This combined comparison puts extremely compelling and powerful constraints on any theoretical model. – The equation for the planetesimal accretion rate of the protoplanet is computed assuming the oligarchic regime (Chambers 2006). The enhancement of the planetesimal capture radius because of the planetary H/He envelope is included (Inaba & Ikoma 2003). – A prescription based on Bondi- and Hill-type gas accretion in the 2D and 3D cases limits the planetary gas accretion rate in the disc-limited regime. – Gas-driven Type I and Type II orbital migration are com- puted including the effects of non-isothermality and of the planet’s eccentricity and inclination (Paardekooper et al. 2011; Coleman & Nelson 2014; Dittkrist et al. 2014). – Full N-body interaction between all the embryos forming concurrently in one disc are tracked using the mercury inte- grator (Chambers 1999). Orbital migration and the damping of eccentricity and inclination are input in the integrator via additional forces. In case of a collision, the impact energy is added as an additionally luminosity term (Broeg & Benz 2012) to the internal structure model. This can lead to the loss of the H/He envelope. During the evolutionary phase we include: g y p – XUV-driven atmospheric photoevaporation in the energy and radiation-recombination-limited approximation (Jin et al. 2014), The formation and evolution model follows the envelope structure of the giant planets during they entire lifetime. This A69, page 39 of 44 A&A 656, A69 (2021) – for close-in planets, the addition of a bloating luminos- ity modelled with the empirical relation of Thorngren & Fortney (2018), and – for close-in planets, the addition of a bloating luminos- ity modelled with the empirical relation of Thorngren & Fortney (2018), and Acknowledgements. References Adachi, I., Hayashi, C., & Nakazawa, K. 1976, Prog. Theor. Phys., 56, 1756 Adibekyan, V. 2019, Geosciences, 9, 105 Alessi, M., & Pudritz, R. E. 2018, MNRAS, 478, 2599 Alessi, M., Pudritz, R. E., & Cridland, A. J. 2020a, MNRAS, 493, 1013 Alessi, M., Inglis, J., & Pudritz, R. E. 2020b, MNRAS, 497, 4814 Alexander, R. D., & Armitage, P. J. 2009, ApJ, 704, 989 Alexander, R. D., & Pascucci, I. 2012, MNRAS, 422, L82 Alib Y & B W 2017 A&A 598 L5 Adachi, I., Hayashi, C., & Nakazawa, K. 1976, Prog. Theor. Phys., 56, 1756 Adibekyan, V. 2019, Geosciences, 9, 105 Alessi, M., & Pudritz, R. E. 2018, MNRAS, 478, 2599 Alessi, M., Pudritz, R. E., & Cridland, A. J. 2020a, MNRAS, 493, 1013 Alessi, M., Inglis, J., & Pudritz, R. E. 2020b, MNRAS, 497, 4814 Alexander, R. D., & Armitage, P. J. 2009, ApJ, 704, 989 Alexander, R. D., & Pascucci, I. 2012, MNRAS, 422, L82 Alibert, Y., & Benz, W. 2017, A&A, 598, L5 Alibert, Y., & Venturini, J. 2019, A&A, 626, A21 After the description of the model, we study how the many different sub-models included in the Bern Generation III Model interact in the full end-to-end model by simulating the formation of two planetary systems. To understand the results, it is helpful to compare the timescales of growth and migration, reveal- ing which process is dominant. It is also helpful to study the planetesimal surface density, revealing the solid accretion mode (planetesimal accretion versus growth by giant impacts). Other key processes occurring during the emergence of the planetary systems include the capture of many protoplanets into large res- onant convoys, and the consequences of dynamical instabilities caused by the gravitational interactions between the protoplan- ets. This includes the destabilisation of other protoplanets at the moment a giant planet (especially a second one in the system) starts runaway gas accretion as well as series of giant impacts at the moment the gas disc dissipates. Alibert, Y., & Venturini, J. 2019, A&A, 626, A21 Alibert, Y., Mordasini, C., & Benz, W. 2004, A&A, 417, L25 Y., Mordasini, C., Benz, W., & Winisdoerffer, C. 2005a, A&A, 434 Alibert, Y., Mousis, O., Mordasini, C., & Benz, W. 2005b, ApJ, 626, L57 Alibert, Y., Mordasini, C., & Benz, W. 2011, A&A, 526, A63 Alibert, Y., Carron, F., Fortier, A., et al. g A&A, 643, A133 Birnstiel, T., & Andrews, S. M. 2014, ApJ, 780, 153 g – Paper III will look for correlations between of the occurrence of inner low-mass and outer giant planets. p Birnstiel, T., Klahr, H., & Ercolano, B. 2012, A&A, 539, A148 Bitsch, B., & Kley, W. 2010, A&A, 523, A30 – Paper IV (Burn et al. 2021) will extend the population synthesis to lower-mass stars (down to late M-dwarfs) and analyse the effects of the stellar mass. Bitsch, B., & Kley, W. 2011, A&A, 530, A41 Bitsch, B., Crida, A., Libert, A. S., & Lega, E. 2013, A&A, 555, A124 Bitsch, B., Crida, A., Libert, A. S., & Lega, E. 2013, A&A, Bitsch, B., Johansen, A., Lambrechts, M., & Morbidelli, A. 2015a, A&A, 575, A28 References 1994, ApJ, 427, 987 This work is the ﬁrst of a series. Here we present the outline of the series: Benítez-Llambay, P., Masset, F., & Beaugé, C. 2011, A&A, 528, A2 B W Id S Alib Y Li D & M d i i C 2014 i P Benítez-Llambay, P., Masset, F., & Beaugé, C. 2011, A&A, 528, A2 Benz, W., Ida, S., Alibert, Y., Lin, D., & Mordasini, C. 2014, in Proto Planets VI, eds. H. Beuther, R. S. Klessen, C. P. Dullemond, & T. Henning – Paper II will introduce the methods to calculate population syntheses. Several populations for Solar-mass stars with dif- ferent numbers of initial embryos per system are computed. The effects of this parameter at the population level will be investigated. (Tucson: University of Arizona Press), 691 Berardo, D., & Cumming, A. 2017, ApJ, 846, L17 Berardo, D., Cumming, A., & Marleau, G.-D. 2017, ApJ, 834, 149 Berardo, D., Cumming, A., & Marleau, G.-D. 2017, Bergez-Casalou, C., Bitsch, B., Pierens, A., Crida, A., & Raymond, S. N. 2020, A&A, 643, A133 Bergez-Casalou, C., Bitsch, B., Pierens, A., Crida, A g A&A, 643, A133 Krumholz, M. R. 2006, ApJ, 641, L45 Krumholz, M. R. 2006, ApJ, 641, L45 Dittkrist, K. M., Mordasini, C., Klahr, H., Alibert, Y., & Henning, T. 2014, A&A, 567, A121 p Lagrange, A. M., Bonnefoy, M., Chauvin, G., et al. 2010, Science, 329, 57 Lagrange, A. M., Bonnefoy, M., Chauvin, G., et al. 2010 Emsenhuber, A., Cambioni, S., Asphaug, E., et al. 2020, ApJ, 891, 6 Lambrechts, M., & Johansen, A. 2012, A&A, 544, A32 Emsenhuber, A., Mordasini, C., Burn, R., et al. 2021, A&A, 656, A70 (Paper II) Lambrechts, M., & Lega, E. 2017, A&A, 606, A146 Fabrycky, D. C., Lissauer, J. J., Ragozzine, D., et al. 2014, ApJ, 790, 146 Lambrechts, M., Morbidelli, A., Jacobson, S. A., et al. 2019, A&A, 627, A83 Lambrechts, M., Morbidelli, A., Jacobson, S. A., et al Fang, J., & Margot, J.-L. 2012, ApJ, 761, 92 Lammer, H., Odert, P., Leitzinger, M., et al. 2009, A&A, 506, 399 g Laughlin, G., Crismani, M., & Adams, F. C. 2011, ApJ, 729, L7 Fatuzzo, M., & Adams, F. C. 2008, ApJ, 675, 1361 Fatuzzo, M., & Adams, F. C. 2008, ApJ, 675, 1361 Lee, E. J., & Chiang, E. 2015, ApJ, 811, 41 Fedele, D., van den Ancker, M. E., Henning, T., Jayawardhana, R., & Oliveira, J. M. 2010, A&A, 510, A72 Fedele, D., van den Ancker, M. E., Henning, T., Jayaw Lee, E. J., & Chiang, E. 2016, ApJ, 817, 90 Fendyke, S. M., & Nelson, R. P. 2014, MNRAS, 437, 96 Lenz, C. T., Klahr, H., & Birnstiel, T. 2019, ApJ, 874, 36 Fendyke, S. M., & Nelson, R. P. 2014, MNRAS, 437, 96 Levison, H. F., Thommes, E., & Duncan, M. J. 2010, AJ, 139, 1297 Ferraz-Mello, S., Rodríguez, A., & Hussmann, H. 2008, Celest. Mech. Dyn. Astron., 101, 171 Lin, D. N. C., Bodenheimer, P., & Richardson, D. C. 1996, Nature, 380, 606 Ford, E. B., & Rasio, F. A. 2008, ApJ, 686, 621 Linder, E. F., Mordasini, C., Mollière, P., et al. 2019, A&A, 623, A Ford, E. B., & Rasio, F. A. 2008, ApJ, 686, 621 Lissauer, J. J. 1987, Icarus, 69, 249 Fortier, A., Alibert, Y., Carron, F., Benz, W., & Dittkrist, K. M. 2013, A&A, 549, A44 Lissauer, J. J., Hubickyj, O., D’Angelo, G., & Bodenheimer, P. 2009, Icarus, 199, 338 Fortney, J. J., Mordasini, C., Nettelmann, N., et al. 2013, ApJ, 775, 80 Lissauer, J. J., Ragozzine, D., Fabrycky, D. C., et al. A28 – Paper V (Schlecker et al. 2021b) will study the mapping of disc initial conditions to planet properties with machine learning. Bitsch, B., Lambrechts, M., & Johansen, A. 2015b, A&A, 582, A1 Bitsch, B., Izidoro, A., Johansen, A., et al. 2019, A&A, 623, A88 Bitsch, B., Trifonov, T., & Izidoro, A. 2020, A&A, 643, A66 Bodenheimer, P., & Pollack, J. B. 1986, Icarus, 67, 391 – Paper VI (Mishra et al. 2021) will look for the diversity between planets in each system compared to diversity of the overall population (Weiss et al. 2018). Bodenheimer, P., & Pollack, J. B. 1986, Icarus, 67, 391 Bodenheimer, P., Hubickyj, O., & Lissauer, J. J. 2000, Icarus, 143, 2 Bodenheimer, P., D’Angelo, G., Lissauer, J. J., Fortney, J. J., & Saumon, D. 2013, ApJ, 770, 120 – There are then three papers on the quantitative comparison with various observational techniques: radial velocity with HARPS and CARMENES, and transits with Kepler. Boley, A. C., Granados Contreras, A. P., & Gladman, B. 2016, ApJ, 817, L17 ont, E., & Mathis, S. 2016, Celest. Mech. Dyn. Astron., 126, 275 Bolmont, E., & Mathis, S. 2016, Celest. Mech. Dyn. Ast Booth, R. A., & Ilee, J. D. 2019, MNRAS, 487, 3998 Boss, A. P. 1997, Science, 276, 1836 The discrepancies uncovered in these comprehensive and multi- aspect comparisons with observations will be helpful to improve the understanding of planet formation and evolution. Boss, A. P. 2003, ApJ, 599, 577 p Broeg, C. H., & Benz, W. 2012, A&A, 538, A90 Bryan, M. L., Knutson, H. A., Howard, A. W., et al. Bryan, M. L., Knutson, H. A., Howard, A. W., et al. 2016, ApJ, 821, 89 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. 2008, ApJ, 686, 580 Chauvin, G., Desidera, S., Lagrange, A. M., et al. 2017, A&A, 605, L9 Johansen, A., & Lacerda, P. 2010, MNRAS, 404, 475 Chauvin, G., Desidera, S., Lagrange, A. M., et al. 2017, A&A, 6 Johansen, A., Ida, S., & Brasser, R. 2019, A&A, 622, A202 Chen, H., & Rogers, L. A. 2016, ApJ, 831, 180 Juri´c, M., & Tremaine, S. 2008, ApJ, 686, 603 g p Chiang, E. I., & Goldreich, P. 1997, ApJ, 490, 368 g p Chiang, E. I., & Goldreich, P. 1997, ApJ, 490, 368 Juri´c, M., & Tremaine, S. 2008, ApJ, 686, 603 Kanagawa, K. D., Tanaka, H., & Szuszkiewicz, E. 2018, ApJ, 861, 140 Cimerman, N. P., Kuiper, R., & Ormel, C. W. 2017, MNRAS, 471, 4662 Cimerman, N. P., Kuiper, R., & Ormel, C. W. 2017, Keppler, M., Benisty, M., Müller, A., et al. 2018, A&A, 617, A44 Clarke, C. J., Gendrin, A., & Sotomayor, M. 2001, MNRAS, 328, 485 y Coleman, G. A. L., & Nelson, R. P. 2014, MNRAS, 445, 479 Kippenhahn, R., & Weiagert, A. 1994, Stellar Structure and Evolution (Berlin: Springer), 192 Cresswell, P., & Nelson, R. P. 2008, A&A, 482, 677 Klahr, H., & Schreiber, A. 2020, ApJ, 901, 54 Cresswell, P., Dirksen, G., Kley, W., & Nelson, R. P. 2007, A&A Klahr, H., & Schreiber, A. 2020, ApJ, 901, 54 Crida, A., Morbidelli, A., & Masset, F. 2006, Icarus, 181, 587 Kleine, T., Münker, C., Mezger, K., & Palme, H. 2002, Nature, 418, 952 Kleine, T., Münker, C., Mezger, K., & Palme, H Cridland, A. J., Pudritz, R. E., & Alessi, M. 2016, MNRAS, 461, 3274 Kleine, T., Touboul, M., Bourdon, B., et al. 2009, Geochim. Cosmochim. Acta, 73, 5150 Cridland, A. J., Pudritz, R. E., Birnstiel, T., Cleeves, L. I., & Bergin, E. A. 2017, MNRAS, 469, 3910 Cridland, A. J., Pudritz, R. E., Birnstiel, T., Cleeves, L. I., & Bergin, E. A. 2017, MNRAS, 469, 3910 Kley, W. 2019, Planet Formation and Disk-Planet Interactions (Berlin, Heidel- berg: Springer Berlin Heidelberg), 151 berg: Springer Berlin Heidelberg), 151 Kley, W., & Dirksen, G. 2006, A&A, 447, 369 D’Angelo, G., & Lubow, S. H. 2008, ApJ, 685, 560 D’Angelo, G., Weidenschilling, S. J., Lissauer, J. J., & Bodenheimer, P. 2021, I 355 114087 D’Angelo, G., Weidenschilling, S. J., Lissauer, J. J., & Bodenheimer, P. 2021, Icarus, 355, 114087 Kley, W., Peitz, J., & Bryden, G. 2004, A&A, 414, 735 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. Ida, S., & Lin, D. N. C. 2010, ApJ, 719, 810 Bryden, G., Chen, X., Lin, D. N. C., Nelson, R. P., & Papaloizou, J. C. B. 1999, ApJ, 514, 344 Ida, S., & Makino, J. 1993, Icarus, 106, 210 Buchhave, L. A., Bitsch, B., Johansen, A., et al. 2018, ApJ, 856, 37 Ida, S., Guillot, T., & Morbidelli, A. 2008, ApJ, 686, 1292 Ida, S., Muto, T., Matsumura, S., & Brasser, R. 2020, MNRA urn, R., Marboeuf, U., Alibert, Y., & Benz, W. 2019, A&A, 629, A Burn, R., Schlecker, M., Mordasini, C., et al. 2021, A&A, 656, A72 (Paper IV) Inaba, S., & Ikoma, M. 2003, A&A, 410, 711 Inaba, S., Tanaka, H., Nakazawa, K., Wetherill, G. W., & Kokubo, E. 2001, Inaba, S., Tanaka, H., Nakazawa, K., Wetherill, G. W., & Kokubo, E. 2001, Icarus 149 235 Inaba, S., Tanaka, H., Nakazawa, K., Wetherill, G. W., & Inaba, S., Tanaka, H., Nakazawa, K., Wethe Burrows, A., Hubbard, W. B., Lunine, J. I., & Liebert, J. 2001, Rev. Mod. Phys., 73, 719 Icarus, 149, 235 Inamdar, N. K., & Schlichting, H. E. 2016, ApJ, 817, L13 Burrows, A., Heng, K., & Nampaisarn, T. 2011, ApJ, 736, 47 Chabrier, G., & Baraffe, I. 2000, ARA&A, 38, 337 Izidoro, A., Ogihara, M., Raymond, S. N., et al. 2017, MNRAS, 470, 1750 Chambers, J. E. 1999, MNRAS, 304, 793 zidoro, A., Bitsch, B., Raymond, S. N., et al. 2021, A&A, 650, A15 Chambers, J. E. 2001, Icarus, 152, 205 Jackson, B., Barnes, R., & Greenberg, R. 2009, ApJ, 698, 1357 g p Jackson, A. P., Davis, T. A., & Wheatley, P. J. 2012, MNRAS, 422, 2024 Ji S & M d i i C 2018 A J 853 163 Chambers, J. 2006, Icarus, 180, 496 Jackson, A. P., Davis, T. A., & Wheatley, P. J. 2012, MNRAS, 42 , , , , y, , , Jin, S., & Mordasini, C. 2018, ApJ, 853, 163 Jin, S., & Mordasini, C. 2018, ApJ, 853, 163 Chambers, J. E. 2013, Icarus, 224, 43 p Jin, S., Mordasini, C., Parmentier, V., et al. 2014, ApJ, 795, 65 Jin, S., Mordasini, C., Parmentier, V., et al. 201 Chambers, J. 2018, ApJ, 865, 30 p Johansen, A., & Bitsch, B. 2019, A&A, 631, A70 Johansen, A., & Bitsch, B. 2019, A&A, 631, A70 Chatterjee, S., Ford, E. B., Matsumura, S., & Rasio, F. A. g Icarus, 355, 114087 Knutson, H. A., Fulton, B. J., Montet, B. T., et al. 2014, ApJ, 785, 126 Knutson, H. A., Fulton, B. J., Montet, B. T., et al. 20 Dawson, R. I., & Johnson, J. A. 2018, ARA&A, 56, 175 Kokubo, E., & Ida, S. 1998, Icarus, 131, 171 Kokubo, E., Kominami, J., & Ida, S. 2006, ApJ, 642, 1131 Dawson, R. I., & Murray-Clay, R. A. 2013, ApJ, 767, L24 Dawson, R. I., Chiang, E., & Lee, E. J. 2015, MNRAS, 453, 1471 Kratter, K. M., Murray-Clay, R. A., & Youdin, A. N. 2010, ApJ, 710, 1375 Demory, B.-O., & Seager, S. 2011, ApJS, 197, 12 Kretke, K. A., & Lin, D. N. C. 2012, ApJ, 755, 74 Dewitt, H. E., Graboske, H. C., & Cooper, M. S. 1973, ApJ, 181, 439 Krivov, A. V., & Wyatt, M. C. 2021, MNRAS, 500, 718 Krumholz, M. R. 2006, ApJ, 641, L45 Krumholz, M. R. 2006, ApJ, 641, L45 2011, ApJS, 197, 8 Fouchet, L., Alibert, Y., Mordasini, C., & Benz, W. 2012, A&A, 540, A107 Liu, B., Ormel, C. W., & Lin, D. N. C. 2017, A&A, 601, A15 Fowler, W. A., Caughlan, G. R., & Zimmerman, B. A. 1967, ARA&A, 5, 525 Fraser, W. C. 2009, ApJ, 706, 119 Liu, S.-F., Hori, Y., Müller, S., et al. 2019, Nature, 572, 355 Fraser, W. C. 2009, ApJ, 706, 119 Lodders, K. 2003, ApJ, 591, 1220 Freedman, R. S., Lustig-Yaeger, J., Fortney, J. J., et al. 2014, ApJS, 214, 25 Lopez, E. D., & Fortney, J. J. 2013, ApJ, 776, 2 Fulton, B. J., & Petigura, E. A. 2018, AJ, 156, 264 Golabek, G. J., & Jutzi, M. 2021, Icarus, 363, 114437 Lopez, E. D., & Fortney, J. J. 2014, ApJ, 792, 1 Goldreich, P., & Tremaine, S. 1979, ApJ, 233, 857 Lopez, E. D., Fortney, J. J., & Miller, N. 2012, ApJ, 761, 59 Goldreich, P., Lithwick, Y., & Sari, R. 2004, ApJ, 614, 497 Lubow, S. H., & D’Angelo, G. 2006, ApJ, 641, 526 Gorti, U., Hollenbach, D., & Dullemond, C. P. 2015, ApJ, 804, 29 Lubow, S. H., Seibert, M., & Artymowicz, P. 1999, ApJ, 526, 1001 Lüst, R. 1952, Zeitschrift Naturforschung Teil A, 7, 87 Graboske, H. C., Dewitt, H. E., Grossman, A. S., & Cooper, M. S. 1973, ApJ, 181, 457 Lynden-Bell, D., & Pringle, J. E. 1974, MNRAS, 168, 603 Lyra, W., Paardekooper, S.-J., & Mac Low, M.-M. 2010, ApJ, 715, L68 Grasset, O., Schneider, J., & Sotin, C. 2009, ApJ, 693, 722 Grasset, O., Schneider, J., & Sotin, C. 2009, ApJ, 693, 722 Greenzweig, Y., & Lissauer, J. J. 1992, Icarus, 100, 440 y p p Macintosh, B., Graham, J. R., Barman, T., et al. 2015, Science, 350, 64 Greenzweig, Y., & Lissauer, J. J. 1992, Icarus, 100, 44 Guilera, O. M., Brunini, A., & Benvenuto, O. G. 2010, A&A, 521, A50 Guillot T 2005 AREPS 33 493 Guilera, O. M., Brunini, A., & Benvenuto, O. G. 2010, A&A, 521, A50 Mallonn, M., Köhler, J., Alexoudi, X., et al. 2019, A&A, 624, A62 Guilera, O. M., Brunini, A., & Benvenuto, O. G. 2010, Guilera, O. M., Brunini, A., & Benvenuto, O. G. 2010, A&A, 521, A50 Guillot, T. 2005, AREPS, 33, 493 Guillot, T. 2005, AREPS, 33, 493 Guillot, T. 2005, AREPS, 33, 493 Manara, C. F., Mordasini, C., Testi, L., et al. 2019, A&A, 631, L2 Haisch, Karl E., J., Lada, E. A., & Lada, C. J. 2001, ApJ, 553, L15 Haisch, Karl E., J., Lada, E. A., & Lada, C Marboeuf, U., Thiabaud, A., Alibert, Y., Cabral, N., & Benz, W. 2014a, A&A, 570, A36 MNRAS, 469, 3910 Daisaka, J. K., Tanaka, H., & Ida, S. 2006, Icarus, 185, 492 570, A36 Hatzes, A. P., & Rauer, H. 2015, ApJ, 810, L25 Marboeuf, U., Thiabaud, A., Alibert, Y., Cabral, N., & Benz, W. 2014b, A&A, 570, A35 Hayashi, C. 1981, Prog. Theor. Phys. Suppl., 70, 35 y g y pp Hayashi, C., Nakazawa, K., & Adachi, I. 1977, PASJ, 29, 163 Hollenbach, D., Johnstone, D., Lizano, S., & Shu, F. 1994, ApJ, 428, 654 Marleau, G.-D., Klahr, H., Kuiper, R., & Mordasini, C. 2017, ApJ, 836, 221 Howard, A. W., Marcy, G. W., Bryson, S. T., et al. 2012, ApJS, 20 Marleau, G.-D., Coleman, G. A. L., Leleu, A., & Mordasini, C. 2019a, A&A, 624, A20 y y Hueso, R., & Guillot, T. 2005, A&A, 442, 703 Hunter, J. D. 2007, Comput. Sci. Eng., 9, 90 Marleau, G.-D., Mordasini, C., & Kuiper, R. 2019b, ApJ, 881, 144 p g Ida, S., & Lin, D. N. C. 2004a, ApJ, 604, 388 Marois, C., Macintosh, B., Barman, T., et al. 2008, Science, 322, 13 p Ida, S., & Lin, D. N. C. 2004b, ApJ, 616, 567 Masset, F. S., Morbidelli, A., Crida, A., & Ferreira, J. 2006, ApJ, 642, 478 Masset, F. S., Morbidelli, A., Crida, A., & Ferreira, J p Ida, S., & Lin, D. N. C. 2008, ApJ, 673, 487 Matsumoto, Y., & Ogihara, M. 2020, ApJ, 893, 43 A69, page 41 of 44 A69, page 41 of 44 A&A 656, A69 (2021) Matsumura, S., Brasser, R., & Ida, S. 2021, A&A, 650, A116 Matsuyama, I., Johnstone, D., & Hartmann, L. 2003, ApJ, 582, 893 Rosotti, G. P., Ercolano, B., Owen, J. E., & Armitage, P. J. 2013, MNRAS, 430, 1392 y p Mayor, M., & Queloz, D. 1995, Nature, 378, 355 y Mayor, M., & Queloz, D. 1995, Nature, 378, 355 hlmann, J., Ségransan, D., Queloz, D., et al. 2011, A&A, 525, A95 Mayor, M., Marmier, M., Lovis, C., et al. 2011, ArXiv e-prints [arXiv:1109.2497] Sahlmann, J., Ségransan, D., Queloz, D., et al. 2011, A&A, 525, A95 Santos, N. C., Adibekyan, V., Figueira, P., et al. 2017, A&A, 603, A30 Sahlmann, J., Ségransan, D., Queloz, D., et al. 2011, A&A, 525, A95 Santos, N. C., Adibekyan, V., Figueira, P., et al. 2017, A&A, 603, A30 Miguel, Y., Guilera, O. M., & Brunini, A. 2011, MNRAS, 417, 314 Sanz-Serna, J. M. 1992, Acta Numerica, 1, 243 Millholland, S., Wang, S., & Laughlin, G. , , 642, A75 Owen, J. E., & Jackson, A. P. 2012, MNRAS, 425, 2931 Voelkel, O., Deienno, R., Kretke, K., & Klahr, H. 2021, A&A, 645, Owen, J. E., & Wu, Y. 2013, ApJ, 775, 105 p Paardekooper, S. J., Baruteau, C., & Kley, W. 2011, MNRAS, 410, 293 Wagner, K., Apai, D., & Kratter, K. M. 2019, ApJ, 877, 46 p y Papaloizou, J. C. B., & Larwood, J. D. 2000, MNRAS, 315, 823 Wang, H., Weiss, B. P., Bai, X.-N., et al. 2017, Science, 355, 623 Papaloizou, J. C. B., Nelson, R. P., & Masset, F. 2001, A&A, 366, Ward, W. R. 1986, Icarus, 67, 164 Ward, W. R. 1986, Icarus, 67, 164 Perri, F., & Cameron, A. G. W. 1974, Icarus, 22, 416 Ward, W. R. 1989, ApJ, 345, L99 Pfyffer, S., Alibert, Y., Benz, W., & Swoboda, D. 2015, A&A, 579, A37 Ward, W. R. 1997, Icarus, 126, 261 Wasserburg, G. J., MacDonald, G. J. F., Hoyle, F., & Fo Podolak, M., Pollack, J. B., & Reynolds, R. T. 1988, Icarus, 73, 1 Wasserburg, G. J., MacDonald, G. J. Wasserburg, G. J., MacDonald, G. J. F., Hoyle, F., & Fowler, W. A. 1964, Science, 143, 465 645, A43 Mollière, P., & Mordasini, C. 2012, A&A, 547, A105 Mollière, P., & Mordasini, C. 2012, A&A, 547, A105 Schlaufman, K. C. 2018, ApJ, 853, 37 Schlecker, M., Mordasini, C., Emsenhuber, A., et al. 2021a, A&A, 656, A71 (Paper III) Morbidelli, A., Bottke, W. F., Nesvorný, D., & Levison, H. F. 2009, Icarus, 204, 558 ( p ) Schlecker, M., Pham, D., Burn, R., et al. 2021b, A&A, 656, A73 (Paper V) Mordasini, C. 2014, A&A, 572, A118 Mordasini, C. 2018, in Handbook of Exoplanets, eds. H. J. Deeg, & J. A. Belmonte (Berlin: Springer Living Reference Work), 143 Schlichting, H. E., & Mukhopadhyay, S. 2018, Space Sci. Rev., 214, 34 Schlichting, H. E., Fuentes, C. I., & Trilling, D. E. 2013, AJ, 146, 36 p g g Mordasini, C., Alibert, Y., & Benz, W. 2009a, A&A, 501, 1139 Schulik, M., Johansen, A., Bitsch, B., Lega, E., & Lambrechts, M. 2020, A&A, 642, A187 Mordasini, C., Alibert, Y., Benz, W., & Naef, D. 2009b, A&A, 501, 116 Seager, S., Kuchner, M., Hier-Majumder, C. A., & Militzer, B. 2007, ApJ, 669, 1279 Mordasini, C., Alibert, Y., Benz, W., Klahr, H., & Henning, T. 2012a, A&A, 541, A97 Shakura, N. I., & Sunyaev, R. A. 1973, A&A, 500, 33 Mordasini, C., Alibert, Y., Georgy, C., et al. 2012b, A&A, 547, A112 Shakura, N. I., & Sunyaev, R. A. 1973, A&A, 500, 33 Mordasini, C., Alibert, Y., Georgy, C., et al. 2012b, A&A, 547, A112 Mordasini, C., Alibert, Y., Klahr, H., & Henning, T. 2012c, A&A, 547, A111 Mordasini, C., Alibert, Y., Klahr, H., & Henning, T. 2012c, A&A, 547, A111 Singer, K. N., McKinnon, W. B., Gladman, B., et al. 2019, Science, 363, 955 Singer, K. N., McKinnon, W. B., Gladman, B., et al. 201 Sotiriadis, S., Libert, A.-S., Bitsch, B., & Crida, A. 2017, A&A, 598, A70 Mordasini, C., Klahr, H., Alibert, Y., Miller, N., & Henning, T. 2014, A&A, 566, A141 Stevenson, D. J. 1982, Planet. Space Sci., 30, 755 Mordasini, C., Mollière, P., Dittkrist, K. M., Jin, S., & Alibert, Y. 2015, Int. J. Astrobiol., 14, 201 Stoer, J., & Bulirsch, R. 1980, Introduction to Numerical Analysis (New York: Springer-Verlag) Mordasini, C., Marleau, G. D., & Mollière, P. 2017, A&A, 608, A72 p g g Suzuki, D., Bennett, D. P., Ida, S., et al. 2018, ApJ, 869, L34 Movshovitz, N., & Podolak, M. yj Pringle, J. E. 1981, ARA&A, 19, 137 Wei, Q., Hu, Y., Liu, Y., et al. 2018, ApJ, 856, L14 g Raﬁkov, R. R. 2004, AJ, 128, 1348 Weidenschilling, S. J. 1977, Ap&SS, 51, 153 Weidenschilling, S. J. 2005, Space Sci. Rev., 116, 53 Raﬁkov, R. R. 2005, ApJ, 621, L69 g Science, 143, 465 y Pollack, J. B., Hubickyj, O., Bodenheimer, P., et al. 1996, Icarus, 124, 62 yj Pringle, J. E. 1981, ARA&A, 19, 137 Raﬁkov, R. R. 2005, ApJ, 621, L69 Rameau, J., Chauvin, G., Lagrange, A. M., et al. 2013, ApJ, 772, L15 Weiss, L. M., & Marcy, G. W. 2014, ApJ, 783, L6 Weiss, L. M., & Petigura, E. A. 2020, ApJ, 893, L1 Raymond, S. N., & Izidoro, A. 2017, Icarus, 297, 134 Weiss, L. M., Marcy, G. W., Petigura, E. A., et al. 2018, AJ, 155, 48 Raymond, S. N., Quinn, T., & Lunine, J. I. 2005, ApJ, 632, 670 Raymond, S. N., Quinn, T., & Lunine, J. I. 2006, Icarus, 183, 265 William, L. 2007, Fundamentals of Geophysics (Cambridge: Cambridge University Press) Raymond, S. N., Armitage, P. J., & Gorelick, N. 2010, ApJ, 711, 772 570, A36 2017, ApJ, 849, L33 Sarkis, P., Mordasini, C., Henning, T., Marleau, G. D., & Mollière, P. 2021, A&A, 645, A79 Mills, S. M., Howard, A. W., Petigura, E. A., et al. 2019, AJ, 157, 198 Saumon, D., Chabrier, G., & van Horn H. M. 1995, ApJS, 99, 713 Mishra, L., Alibert, Y., Leleu, A., et al. 2021, A&A, 656, A74 (Paper VI) Mizuno, H. 1980, Prog. Theor. Phys., 64, 544 Savvidou, S., Bitsch, B., & Lambrechts, M. 2020, A&A, 640, A6 Moldenhauer, T. W., Kuiper, R., Kley, W., & Ormel, C. W. 2021, A&A, 646, L11 Schib, O., Mordasini, C., Wenger, N., Marleau, G. D., & Helled, R. 2021, A&A, 645, A43 645, A43 2008, Icarus, 194, 368 Szulágyi, J., & Mordasini, C. 2017, MNRAS, 465, L64 Tanaka, H., & Ida, S. 1999, Icarus, 139, 350 Movshovitz, N., Bodenheimer, P., Podolak, M., & Lissauer, J. J. 2010, Icarus, 209, 616 Tanaka, H., & Ward, W. R. 2004, ApJ, 602, 388 Mulders, G. D., O’Brien, D. P., Ciesla, F. J., Apai, D., & Pascucci, I. 2020, ApJ, 897, 72 Tanaka, H., Takeuchi, T., & Ward, W. R. 2002, ApJ, 565, 1257 Tanaka, H., Takeuchi, T., & Ward, W. R. 2002, A Thiabaud, A., Marboeuf, U., Alibert, Y., et al. 2014, A&A, 562, A27 Müller, S., Ben-Yami, M., & Helled, R. 2020, ApJ, 903, 147 Thiabaud, A., Marboeuf, U., Alibert, Y., Leya, I., & Mezger, K. 2015, A&A, 574, A138 Murray-Clay, R. A., Chiang, E. I., & Murray, N. 2009, ApJ, 693, 23 A138 Thommes, E. W., Duncan, M. J., & Levison, H. F. 2003, Icarus, 161, 431 Thommes, E. W., Duncan, M. J., & Levison, H. F. 2003, Icaru Nakamoto, T., & Nakagawa, Y. 1994, ApJ, 421, 640 Nayakshin, S. 2010, MNRAS, 408, L36 Thorngren, D. P., & Fortney, J. J. 2018, AJ, 155, 214 Nayakshin, S., Dipierro, G., & Szulágyi, J. 2019, MNRAS, 488, L12 Thorngren, D. P., Fortney, J. J., Murray-Clay, R. A., & Lopez, E. D. 2016, ApJ, 831, 64 Naya s , S., p e o, G., & S u ágy , J. 0 9, N S, 88, Ndugu, N., Bitsch, B., & Jurua, E. 2018, MNRAS, 474, 886 Ndugu, N., Bitsch, B., & Jurua, E. 2018, MNRAS, 474, 886 g O’Brien, D. P., Morbidelli, A., & Levison, H. F. 2006, Icarus, 184, 39 Touboul, M., Kleine, T., Bourdon, B., Palme, H., & Wieler, R. 2007, Nature, 450, 1206 Ogihara, M., Kokubo, E., Suzuki, T. K., & Morbidelli, A. 2018, A&A, 615, A63 Valletta, C., & Helled, R. 2020, ApJ, 900, 133 Valletta, C., & Helled, R. 2020, ApJ, 900, 133 Ohtsuki, K., Stewart, G. R., & Ida, S. 2002, Icarus, 155, 436 Vazan, A., Kovetz, A., Podolak, M., & Helled, R. 2013, MNR Vazan, A., Kovetz, A., Podolak, M., & Helled, R. 2013, MNRAS, 434, 3283 Okuzumi, S., & Ormel, C. W. 2013, ApJ, 771, 43 Venturini, J., & Helled, R. 2020, A&A, 634, A31 p Ormel, C. W., & Klahr, H. H. 2010, A&A, 520, A43 Venturini, J., Alibert, Y., & Benz, W. 2016, A&A, 596, A90 Ormel, C. W., Shi, J.-M., & Kuiper, R. 2015, MNRAS, 447, 3512 Veras, D., & Armitage, P. J. 2004, MNRAS, 347, 613 Vigan, A., Bonavita, M., Biller, B., et al. 2017, A&A, 603, A Ormel, C. W., Liu, B., & Schoonenberg, D. 2017, A&A, 604, A1 Otegi, J. F., Bouchy, F., & Helled, R. 2020, A&A, 634, A43 Voelkel, O., Klahr, H., Mordasini, C., Emsenhuber, A., & Lenz, C. 2020, A&A, 642, A75 A.1. General considerations A.1. General considerations Second, according to the Tanaka & Ida (1999) timescale cri- terion one ﬁnds (at least formally) that whenever the migration timescale is longer than the critical value, no planetesimal accre- tion occurs. This includes in particular the case that a planet does not migrate at all. However, as shown by many works (Kokubo & Ida 1998; Levison et al. 2010; Alibert et al. 2013), multiple protoplanets forming concurrently can grow in-situ by planetesi- mals that are already in their feeding zones. When a protoplanet grows, its feeding zone which is proportional to the Hill sphere and thus the mass of the protoplanet, also expands, bringing new planetesimals in reach of the protoplanet in a process that is unaf- fected by shepherding. The protoplanet’s mass growth can occur via the accretion of solids, but also via the accretion of gas. In fact, the interplay of gas accretion leading to an extension of the solid feeding zone which in turn increases the core mass, and then again the gas accretion rate is the underlying process for Phase II in Pollack et al. (1996). Because of the absence of a gap in planetesimal disc around the planet if many protoplan- ets grow concurrently thanks to scattering (Levison et al. 2010; Alibert et al. 2013), each protoplanet has a local reservoir of planetesimals from which it grows. Thus, when judging whether shepherding could affect growth, we do not need to consider planets that have not (yet) migrated outside of the planetesi- mal feeding zone around their starting location, since they still accrete their local reservoir. Ward (1986) and Ward (1989) were the ﬁrst to suggested that the orbital migration of a protoplanet could strongly accelerate its planetesimal accretion if the planet is able to catch most plan- etesimals through which it is sweeping during its migration. In the terminology of Ward, the protoplanet would then act as a predator. On the other hand, Tanaka & Ida (1999) found that in the case of a slow migration timescale τmig, the protoplanet rather carves a gap in the planetesimals disc around its orbit and shepherds the planetesimals, which stalls the accretion of the protoplanet. They derived a criterion in terms of a critical migration timescale τmig,crit only below which the protoplanets can act as predator, τmig ≤τmig,crit. Otherwise, the protoplanets acts as shepherd. Alibert et al. University Press) Winter, A. J., Kruijssen, J. M. D., Chevance, M., Keller, B. W., & Longmore, S. N. 2020, MNRAS, 491, 903 Raymond, S. N., Boulet, T., Izidoro, A., Esteves, L., & Bitsch, B. 2018, MNRAS, 479, L81 Xie, J.-W., Dong, S., Zhu, Z., et al. 2016, Proc. Natl. Acad. Sci. U.S.A., 113, 11431 Yin, Q., Jacobsen, S. B., Yamashita, K., et al. 2002, Nature, 418, 949 Xie, J.-W., Dong, S., Zhu, Z., et al. 2016, Proc. Natl. Acad. Sci. U.S.A., 113, 11431 Ribas, I., Guinan, E. F., Güdel, M., & Audard, M. 2005, ApJ, 622, 680 Ri h t A J W G t K V F i l E D t l 2018 MNRAS 477 Ribas, I., Guinan, E. F., Güdel, M., & Audard, M. 2005, ApJ, 622, 680 Ribas, I., Guinan, E. F., Güdel, M., & Audard, M. 2005, ApJ, 622, 680 Richert, A. J. W., Getman, K. V., Feigelson, E. D., et al. 2018, MNRAS, 477, 5191 g Yin, Q., Jacobsen, S. B., Yamashita, K., et al. 2002, Nature, 418, 949 Youdin, A. N. 2011, ApJ, 742, 38 Youdin, A. N. 2011, ApJ, 742, 38 Rogers, L. A. 2015, ApJ, 801, 41 Rogers, L. A. 2015, ApJ, 801, 41 Zheng, X., Lin, D. N. C., & Kouwenhoven, M. B. N. 2017, ApJ, 836, 207 Zheng, X., Lin, D. N. C., & Kouwenhoven, M. B. N. 2017, ApJ, 836, 207 Zhou, J.-L., & Lin, D. N. C. 2007, ApJ, 666, 447 A69, page 42 of 44 A. Emsenhuber et al.: The New Generation Planetary Population Synthesis (NGPPS). I. A.2. Applicability of Tanaka & Ida (1999) Coming to the general applicability of the work of Tanaka & Ida (1999), one should note that they studied a highly spe- cial setup. In what follows, we discuss several important effects they neglected. When considering them, it becomes much less clear whether the shepherding effect can at all represent a major impediment to planetesimal accretion. We mention these points also in view of the speciﬁc results discussed in Sect. A.3 where we try to identify planets for which shepherding could potentaially be relevant. First, the predator-shepherd mechanism of Tanaka & Ida (1999) was found for a single core growing alone in a disc of planetesimals. This seems a highly unlikely setup given that many protoplanets (oligarchs) emerge concurrently from the pre-dating runaway planetesimal accretion phase and that sub- sequently grow further in lockstep (e.g. Kokubo & Ida 1998). As Tanaka & Ida (1999) state actually themselves, in the case of such multiple protoplanets, ‘the protoplanets push planetesimals into the feeding zones of others and they can grow.’ Finally, we also do not need to consider protoplanets where the gas disc has already dissipated. In this case, no gas disc- driven orbital migration occurs and thus no shepherding. A.1. General considerations (2005a) studied this effect with the Generation I Bern Model and found that the migration rates were gener- ally high enough for the protoplanets to be predators, and thus ignored the shepherding effect. Since the Generation 3 Model differs in numerous aspects (like the oligarchic growth mode, the disc model, the planetesimal size, the migration model, or the multiplicity of forming planets) from the model of 2005, we here re-assess this question. We do this based on existing simula- tions and published criteria. It is clear that for a more deﬁnitive assessment, direct N-body simulations of thousands of concur- rently growing and migrating oligarchs accreting planetesimals and gas in the setup that we consider here would be needed. In these simulations, the planetesimals would have to be included as individual fully interacting bodies in the N-body. This differs from our current approach of representing planetesimals statisti- cally as a surface density with a dynamic state (eccentricity and inclination, see Sect. 3.3). Third, the temporal sequence of how solid accretion pro- ceeds in forming multi-planet systems itself reduces the impact of shepherding. Here it is important to consider that solid growth of protoplanets occurs via two channels: the accretion of plan- etesimals, but also via protoplanet-protoplanet collisions (giant impacts). As can be seen in the growth tracks in Figures 19 and 22, at the lower masses, when the planetesimal accretion timescale is less than the migration timescale, the protoplanets grow nearly in-situ by accreting planetesimals from their local feeding zone. As explained before, in this case shepherding is not important. In the subsequent phase, when the planets have grown to a mass where they start migrating, they also start accreting other protoplanets via giant impacts, which is also unaffected by shepherding. They also often migrate into parts of the disc which were previously depleted in planetesimals by another pro- toplanet that has formed further in. Here, shepherding would not have an effect. This insight also further justiﬁes why using the in-situ prescription for the planetesimal accretion rate is appro- priate. This also implies that when identifying planets that could be affected by shepherding, we do not need to consider plan- ets that accrete in any case only very slowly planetesimals even when neglecting shepherding. Appendix A: Possible impact of migration on the accretion of planetesimals reduce their accretion rates. This was already noted by Ida & Lin (2008), motivating them to not modify the core accretion rate for migrating protoplanets. A.3. Assessment of the population-wide importance of shepherding Indeed, the more recent and realistic work of Daisaka et al. (2006) who run N-body simulation including type-I migration and tidal damping of eccentricity and inclination, starting from 7000–14’000 equal-mass self-gravitating planetesimals whose size is roughly 1000 km showed a different picture than the sin- gle protoplanet simulations of Tanaka & Ida (1999): namely that in the multiple protoplanet situation, the trapping of plan- etesimals by cores is only tentative and does not signiﬁcantly Except for the ﬁrst point (the effect of protoplanet multiplicity) which questions the existence of shepherding in a fundamental way, the considerations from the previous section can - in an approximate way - be cast into a set of conditions where shep- herding could be important. This allows us to identify these protoplanets which might at least in principle be affected by shepherding. A69, page 43 of 44 A&A 656, A69 (2021) Fig. A.1. Mass-distance diagram of the nominal synthetic population NG76 of solar-like starts which stars with initially 100 moon-mass embryos per disc (see Paper II). The epochs of 0.1, 0.5, and 1 Myr are shown. Coloured points show protoplanets that can no longer accrete planetesimals of the initial local reservoir, which have a planetesimal accretion timescale of less than 3 Myr, and which are still embedded in the parent gaseous disc. When ˜τmig ≳˜τmig,c, the planetesimal accretion of these planets could in principle be affected by shepherding if they would be the only protoplanets growing in the disc. Fig. A.1. Mass-distance diagram of the nominal synthetic population NG76 of solar-like starts which stars with initially 100 moon-mass embryos per disc (see Paper II). The epochs of 0.1, 0.5, and 1 Myr are shown. Coloured points show protoplanets that can no longer accrete planetesimals of the initial local reservoir, which have a planetesimal accretion timescale of less than 3 Myr, and which are still embedded in the parent gaseous disc. When ˜τmig ≳˜τmig,c, the planetesimal accretion of these planets could in principle be affected by shepherding if they would be the only protoplanets growing in the disc. Figure A.1 shows the mass-distance diagram of the nomi- nal synthetic population NG76 from Paper II. Three moments in time are shown where planetesimal accretion is in gen- eral important. A69, page 44 of 44 A.3. Assessment of the population-wide importance of shepherding We colour code the absolute value of the ratio of the normalised migration timescale of a planet ˜τmig to the normalised critical migration timescale ˜τmig,c which both are cal- culated as in Tanaka & Ida (1999). When this ratio is larger than approximately unity, shepherding would occur for a single pro- toplanet migrating alone through a disc of planetesimals. Only protoplanets which could in principle be affected by shepherd- ing by fulﬁlling the following criteria are colour coded: First, the distance a planet has migrated away from its starting location is larger than ﬁve times the size of its Hill sphere. This means that it can no longer accrete from its initial local reservoir of plan- etesimals. Second, the planetesimal accretion timescale is less than three million years (the typical disc lifetime), meaning that planetesimal accretion (as opposed to growth via giant impacts) is still relevant. Third, the gas disc has not yet dissipated. Other protoplanets should in any case not be signiﬁcantly affected by shepherding and are shown in grey. The plot ﬁrst shows that the large majority of protoplanets are grey, meaning that shepherding should not be important for them in any case. Then, more speciﬁcally, at 0.1 Myr, there is a group of Mars- to Earth-mass protoplanets inside of the ice line where ˜τmig ≫˜τmig,c. At 0.5 Myr, there is a radial interval from about 1 to 4 au where ˜τmig is longer than ˜τmig,c, however in most cases by less than one order of magnitude. These are regions where usu- ally groups of tens of protoplanets form together (see Sect. 8.1), so that it is not clear if shepherding would occur at all. Planets where the ratio is clearly larger, and thus where the effect could in principle be particularly strong, are rare. At 1 Myr, a similar pattern is seen, but the potentially affected region is reduced. It is clear that this simple a posteriori analysis cannot be seen as a ﬁnal result - for this, simulations where planetesimal are included directly in the N-body would be necessary. Never- theless, together with the ﬁnding of Daisaka et al. (2006) that shepherding is by principle not important when several proto- planets form concurrently, they indicate that shepherding can only affect a relatively limited part of all growing protoplanets. A69, page 44 of 44
https://openalex.org/W2896559048	https://parasitesandvectors.biomedcentral.com/track/pdf/10.1186/s13071-018-3096-x	English	null	Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference	Parasites & vectors	2,018	cc-by	7,784	Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference Magalla Bastian Chalitha Lakmal Somarathne1, Yasanthi Illika Nilmini Silva Gunawardene2, Naduvilath Vishvanath Chandrasekharan1, Arjuna Nisantha Bandara Ellepola3 and Ranil Samantha Dassanayake1* Abstract Background: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). Results: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non- treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. Conclusion: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study. Keywords: Setaria digitata, SDNP, RNAi, Culex quinquefasciatus, Gene knockdown 40 million of them [1–3]. Additionally, plant-parasitic nematodes destroy approximately 12.3% of the annual crop yield, and animal parasitic nematodes kill millions of livestock animals. This contributes to a loss of billions of US dollars annually and necessitates the allocation of additional billions of US dollars for anthelminthic drugs for livestock [4, 5]. Strategies for controlling this prob- lem have mainly focused on chemical treatments [5]. However, due to the limited efficiency and high toxicity of the available drugs, adverse effects on the environment, and emergence of drug resistance in parasites, these chemical treatment methods have become somewhat inef- fective [6–8]. Hence, global interest has recently arisen in © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Somarathne et al. Parasites & Vectors (2018) 11:541 https://doi.org/10.1186/s13071-018-3096-x Somarathne et al. Parasites & Vectors (2018) 11:541 https://doi.org/10.1186/s13071-018-3096-x Background Nematodes are the most abundant metazoans, capable of inhabiting both terrestrial and aquatic habitats. This facilitates their ability to cause both human and animal health problems, as well as to impair crop production worldwide. Gastrointestinal nematodes infect 3.5 billion humans annually worldwide, and cause around 125,000 deaths per year, whereas filarial nematodes infect 120 million people in the globe annually, disabling around * Correspondence: rsdassanayake@chem.cmb.ac.lk 1Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03 00300, Sri Lanka Full list of author information is available at the end of the article * Correspondence: rsdassanayake@chem.cmb.ac.lk 1Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03 00300, Sri Lanka Full list of author information is available at the end of the article Page 2 of 11 Page 2 of 11 Somarathne et al. Parasites & Vectors (2018) 11:541 several shortcomings such as difficulty in maintaining the worms’ viability in the culture, poor RNAi trigger delivery, and compatibility issues of the culture medium with different life stages of the worm [25]. To overcome these drawbacks of in vitro protocols, in our study an in vivo approach was developed, where S. digitata larval stages were cultured in its intermediate host, Culex quinquefasciatus, and the RNAi trigger (siRNA) was dir- ectly injected into the mosquito [19], and the possibility of using the mosquito as a better siRNA delivery plat- form examined. Furthermore, we focused on developing an effective RNAi protocol to specifically shut down the gene of interest to study its function and explore the gene’s potential as a likely drug target. developing more effective controlling strategies for para- sitic nematodes. In this context, screening of the nema- tode genome will enable us to gain a comprehensive knowledge of the molecular mechanisms associated with the development of the nematode, which may facilitate the control of parasitic nematodes [9, 10]. RNA mediated gene silencing has become a promising and unbiased tool in genetic screening [11]. RNA interfer- ence (RNAi) is a double-stranded RNA (dsRNA) induced post-transcriptional, homology-dependent gene silencing mechanism, naturally occurring in organisms, assumed to be involved in defence against virus attacks, heterochromati- nisation and retrotransposon silencing [11–13]. RNAi was initially utilised in the genetic screening of Caenorhabditis elegans, and after that, this technique was expanded to other invertebrates (nematodes, insects, molluscs), vertebrates (fish, mice, humans), plants, fungi and algae [14–17]. Background RNAi allows the temporary shut-down of gene expression more specifically and rapidly and gives an insight into gene func- tion [18, 19]. Mosquito infection with S. digitata Adult female worms obtained from a cattle slaughter- house in Welisara, Ragama, Sri Lanka were cut into small pieces after washing three times with mosquito physiological saline (MPS) [28] to release microfilariae (MF) into a tube containing 300 μl MPS. This was vor- texed and centrifuged at 1500× rpm for 30 s, and the supernatant recovered was then re-centrifuged at 1500× rpm for 3 min to obtain a pellet containing MF, which was then re-suspended in MPS to achieve a microfilariae (MF) concentration of 40 MF/μl. Unveiling the biology of the parasitic nematodes genes, which are specific and functionally crucial for their sur- vival, is crucial in identifying targets to control the nematode population [16]. SDNP is such a gene found in S. digitata. Intriguingly, this gene has demonstrated high sequence similarities with putative proteins of W. bancrofti (79%), Brugia malayi (77%) and Loa loa (81%). Moreover, bioinformatics studies of SDNP revealed that this protein contains a nuclear localisation signal, an RNAP_Rpb7_N_like domain, and an inter-domain linker of muscle-specific twitchin kinase similar to that of C. elegans [24]. Considering the background, our study fo- cused on the functional characterisation of SDNP. The thorax of anaesthetized adult mosquitoes (aged 3 days) on ice [29] was injected with a volume of 0.5 μl of microfilariae suspension using a pre-pulled borosilicate glass capillary attached to a manual injector [29–31]. The injected mosquitoes were kept in paper cups cov- ered with a net while providing them with cotton pads soaked with the sucrose solution. Mosquito colony establishment Culex quinquefasciatus were collected from a cattle trap and maintained at 27 °C with 80% relative humidity, with cycles of 16 h of light and 8 h of darkness, in 12 × 12 × 12-inch cages [26]. The eggs obtained from these mosquitoes were hatched in cups containing pre-boiled water, and the larvae which emerged from the eggs were fed with a larval diet (brewer yeast, bovine liver powder, tuna meal) until pupae appeared. The pupae were then separated from the larvae and placed in containers with fresh water in separate cages for 36 h until adult mos- quitoes emerged. These were then fed with 10% sucrose solution [27]. They were used as an in vivo culturing medium for S. digitata microfilariae. Setaria digitata is a parasitic nematode that naturally resides in the peritoneum and mesentery of cattle, zebu and water buffalo. Under normal circumstances, the nematode does not show any pathogenicity towards the natural hosts. The microfilariae produced by the adult female worm travel into the bloodstream of the cattle and are transmitted into new hosts via mosquito bites. However, when the larvae are transfected into non-permissive hosts such as goats, sheep and horses, they invade the brain, spinal cord, and eyes, causing cere- brospinal setariosis, conjunctivitis, and filariasis oculi, re- spectively [20]. Intriguingly, studies on S. digitata have revealed that it has a high resemblance to the human parasitic nematode Wuchereria bancrofti with regard to its morphology, antigenicity and histology [21, 22]. Hence S. digitata has been recommended as a model organism for studying W. bancrofti and Brugia malayi [23]. siRNA synthesis, Cy3 labelling and RNAi treatment Commonly, RNAi protocols are practised by isolation of the nematodes from their natural hosts and maintain- ing them in artificial liquid cultures (in vitro). These in vitro protocols reduce the efficiency of RNAi due to The entire coding sequence of SDNP was divided into four overlapping regions, and each of them was ampli- fied by separate PCR reactions using four separate Page 3 of 11 Somarathne et al. Parasites & Vectors (2018) 11:541 Page 3 of 11 H2O, 2.0 μl 10× buffer, 0.5 μl Kpn I (15 U/μl), 10 μl DNA (200 ng/μl); incubated 1 h at 37 °C] and Xho I [10 μl nu- clease free H2O, 3.0 μl 10× buffer, 0.3 μl BSA, 0.5 μl Xho I (20 U/μl), 16.2 μl DNA (200 ng/μl); incubated 3 h at 37 ° C] separately. The Kpn I digested vector was in vitro tran- scribed by T7 RNA polymerase using the same protocol indicated above. The Xho I digested vector was in-vitro transcribed by Sp6 RNA polymerase using the following reaction; 60 μl of nuclease-free H2O, 8.0 μl of 10x Sp6 buf- fer, 2 μg (10 μl) of pCR II-TOPO vector, 8 μl of rNTP mix (80 mM), 2 μl of Sp6 RNA polymerase (20 U/μl), incu- bated at 40°C for 4 h. The separately in vitro transcribed RNA was mixed and incubated at 75 °C for 5 min and cooled down to room temperature for the complementary strands of synthesised RNA to anneal to make dsRNA. primer sets containing T7 promoter sequence at 5' ends (Table 1) to enrich the total yield of the template and PCR products of overlapping regions were used to synthesize siRNA. The PCR conditions used for the amplification were initial denaturation at 94 °C for 2 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for the primer sets SDNP-1-F/R, SDNP-2-F/R and SDNP-3-F/R and 45 °C for the primer set SDNP-4-F/R, and extension at 72 °C for 30 s with a final extension of 10 min at 72 °C (Table 1). The resulting PCR fragments were transcribed separ- ately in vitro by T7 RNA polymerase (New England Bio- labs, Hitchin, UK) using slightly modified manufacturer’s protocols, to yield a high amount of dsRNA. siRNA synthesis, Cy3 labelling and RNAi treatment Briefly, the in vitro transcription reaction was carried out overnight at 37 °C in a 120 μl reaction mix containing 81.5 μl of nuclease-free H2O, 12.0 μl of 10× T7 buffer, 3.0 μl of DTT (100 mM), 10 μl (2 μg) of PCR product, 12 μl of rNTP mix (80 mM), 1.5 μl of T7 RNA polymerase (18 U/μl). The RNA products of in vitro transcribed DNA were first treated with DNase 1 (New England BioLabs) to remove DNA templates and then incubated at 75 °C for 5 min be- fore cooling them down to room temperature for the complementary strands of synthesized RNA to anneal to make dsRNA. The dsRNA (assessed by gel electrophor- esis) was digested with RNase III (New England Biolabs) according to the manufacturer’s protocol to produce 21– 23 mer siRNA. They were analyzed by polyacrylamide gel electrophoresis before labelling with Cy3 fluorescent dye using a Silencer® siRNA Labeling Kit - Cy™3 dye (Ambion, Life Technologies, California, USA) according to the man- ufacturer’s instructions [32]. The S. digitata infected mosquitoes were initially injected with 150 ng of Cy3 labelled SDNP siRNA mix into their thorax at 12 h post infection (hpi) to monitor the uptake of siRNA by larvae. To target the L2 to L3 larvae transformation, non-labelled siRNA mix was injected at eight days post infection (dpi). The GFP siRNA (150 ng) was injected as a negative control. Abbreviation: T temperature Mosquito dissection The mosquitoes infected with S. digitata and injected with SDNP siRNA, non-injected, GFP siRNA-injected and buffer-injected were dissected at different time in- tervals (24 hpi, 8–14 dpi) under a dissection microscope. In this process, the bodies of cold anaesthetized mosqui- toes were separated into the thorax, midgut, and head, and each of these body parts was further dissected to re- lease the larvae on to glass slides. The MF obtained from the smeared content on the slides at 24 hpi were visual- ized under the fluorescent microscope using Cy3 filter set (excitation: 550 nm, emission: 570 nm) to detect the uptake of siRNA. The transformation of L2 larvae to L3 was recorded from the mosquitoes dissected at 8 to 14 dpi, and the following data were recorded only from the mosquitoes dissected at 14 dpi: (i) larvae length and The ds-RNA synthesized from the gene encoding for the green fluorescent protein (GFP) of Aequorea victoria was also used as the non-specific siRNA control as this gene neither has any homologous sequences in S. digitata nor mosquito. pCRII TOPO vector (pCRII TOPO-GFP) containing GFP was used in the synthesis of GFP dsRNA. The vector was digested with Kpn I [7.5 μl nuclease free Table 1 Primer sets used for the synthesis of dsRNA. The positions of the primers with respect to SDNP sequence (GenBank: GU222920), sequence corresponding to T7 promoter region of primers (underlined), the size of the PCR products, and the optimize annealing temperatures of primer sets are given annealing temperatures of primer sets are given Primer Position Sequence (5'-3') Product size (bp) Annealing T (°C) SDNP-1-F 1 TAATACGACTCACTATAGGGCGACGAGGGTTCCATTGAGTGA 154 55 SDNP-1-R 134 TAATACGACTCACTATAGGGGCTTCCTGACAAGCCAAACAT SDNP-2-F 155 TAATACGACTCACTATAGGGCGACGAGGGTTCCATTGAGTGA 150 55 SDNP-2-R 282 TAATACGACTCACTATAGGGTTGGAATATAAACATGCGGTATA SDNP-3-F 305 TAATACGACTCACTATAGGGCTACCGGTATCAGAACTCAGAA 157 55 SDNP-3-R 462 TAATACGACTCACTATAGGGTAGCGCTGGACCGAATTCTTTT SDNP-4-F 463 TAATACGACTCACTATAGGGGGAAATGGAACTTGTGAAATAA 155 45 SDNP-4-R 618 TAATACGACTCACTATAGGGTCAGTAATTAATCAAATT Abbreviation: T temperature Somarathne et al. Parasites & Vectors (2018) 11:541 Page 4 of 11 Table 2 Primers used for qPCR. The primer sequences of the target (SDNP) and internal control (Actin) are given Primer Sequence (5'-3') Position Product size (bp) Annealing T (°C) SDNP amplification SDNP-RTPCR-FP AAGACTTGCAACTCCATCTCGAT 219 170 60 SDNP-RTPCR-RP ATTTTCTCGCTGACCACCACAACT 366 S. digitata actin amplification (reference gene) SDACT-RTPCR-FP CGCTCGAGAAATCTTACGAATTGC 707 147 55 SDACT-RTPCR-RP TCCTTTCTGATATCGATATCACATT 854 Abbreviation: T temperature used pre-fashioned 5-point scheme based on Song et al. [19]). Mosquito dissection Changes of SDNP specific transcript levels were analysed by qPCR, and the 2-ΔΔCT method was used in evaluating the results. For the reference gene, S. digitata actin gene was used. GFP was used as the non-specific control. SDNP transcript level was presented as relative to that of non-treated worms. Each experiment was performed in triplicate. Bars display mean ± SD. Statistical analysis was performed using Student’s t- test (P < 0.05). Significant differences are represented by different lowercase letters Fig. 2 Relative mRNA expression changes owing to the RNAi. a Relative mRNA levels of SDNP at two dpi. b Relative mRNA levels of SDNP at 14 dpi. Changes of SDNP specific transcript levels were analysed by qPCR, and the 2-ΔΔCT method was used in evaluating the results. For the reference gene, S. digitata actin gene was used. GFP was used as the non-specific control. SDNP transcript level was presented as relative to that of non-treated worms. Each experiment was performed in triplicate. Bars display mean ± SD. Statistical analysis was performed using Student’s t- test (P < 0.05). Significant differences are represented by different lowercase letters Fig. 2 Relative mRNA expression changes owing to the RNAi. a Relative mRNA levels of SDNP at two dpi. b Relative mRNA levels of SDNP at 14 dpi. Changes of SDNP specific transcript levels were analysed by qPCR, and the 2-ΔΔCT method was used in evaluating the results. For the reference gene, S. digitata actin gene was used. GFP was used as the non-specific control. SDNP transcript level was presented as relative to that of non-treated worms. Each experiment was performed in triplicate. Bars display mean ± SD. Statistical analysis was performed using Student’s t- test (P < 0.05). Significant differences are represented by different lowercase letters binding dye. qPCR conditions were initial denatur- ation at 95 °C for 2 min, followed by 40 cycles of de- naturation at 95 °C for 30 s annealing at 60 °C for SDNP primers and 55 °C for actin primers for 45 s (Table 2), and with an extension at 72 °C for 30 s. In qPCR, the actin gene (GenBank: AF079359.1) was used as the internal amplification control in a separ- ate tube for each qPCR reaction. In calculating the relative mRNA expression level, the 2-ΔΔCT method was used as mentioned in qPCR Application Guide (BioRad). Mosquito dissection The scoring scheme was as follows: 1, immobile, dead; 2, compromised motility; 3, partial movement of the body; 4, slightly reduced motion in the body; and 5, all parts of the body are vigorously twisting. Values in width (Image J software was utilized in obtaining the measurements); (ii) larvae location in the mosquito (lar- vae in the head, thorax and abdomen of the mosquito; (iii) larvae motility (motility of the RNAi treated and control worms were compared according to a previously Fig. 1 Uptake and distribution of Cy3 labelled SDNP siRNA by microfilariae at 24 hpi. a Fluorescent image of microfilariae from siRNA non- injected mosquitoes (position indicated by a red square). b Bright field image of microfilariae from siRNA non-injected mosquitoes. c Fluorescent image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes (arrow indicates the dispersion of Cy3 labelled siRNA). d Bright field image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes. Fluorescence was visualised using Olympus BX53 using Cy3 filter. Magnification 400×. Scale-bars: a-d, 20 μm Fig. 1 Uptake and distribution of Cy3 labelled SDNP siRNA by microfilariae at 24 hpi. a Fluorescent image of microfilariae from siRNA non- injected mosquitoes (position indicated by a red square). b Bright field image of microfilariae from siRNA non-injected mosquitoes. c Fluorescent image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes (arrow indicates the dispersion of Cy3 labelled siRNA). d Bright field image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes. Fluorescence was visualised using Olympus BX53 using Cy3 filter. Magnification 400×. Scale-bars: a-d, 20 μm e at 24 hpi a Fluorescent image of microfilariae from siRNA non Fig. 1 Uptake and distribution of Cy3 labelled SDNP siRNA by microfilariae at 24 hpi. a Fluorescent image of microfilariae from siRNA non- injected mosquitoes (position indicated by a red square). b Bright field image of microfilariae from siRNA non-injected mosquitoes. c Fluorescent image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes (arrow indicates the dispersion of Cy3 labelled siRNA). d Bright field image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes. Fluorescence was visualised using Olympus BX53 using Cy3 filter. Magnification 400×. Scale-bars: a-d, 20 μm Somarathne et al. Parasites & Vectors (2018) 11:541 Page 5 of 11 Fig. 2 Relative mRNA expression changes owing to the RNAi. a Relative mRNA levels of SDNP at two dpi. b Relative mRNA levels of SDNP at 14 dpi. Relative quantification of SDNP mRNA level Total RNA was extracted at two dpi and 14 dpi from non-injected, GFP siRNA injected, SDNP siRNA-injected and buffer-injected mosquitoes infected with S. digitata using a GenoSpin RTM Total RNA Extraction Kit (CEY- GEN Biotech, Colombo, Sri Lanka) according to the manu- facturer’s instructions. Five mosquitoes were used from each batch. DNA contamination in extracted RNA was re- moved by treating with DNase I and heat inactivating the DNase I at 75 °C for 5 min. cDNA synthesis was carried out using RNA (2 μg) purified using an RNeasy Plus Mini Kit (Qiagen, California, USA) in a 20 μl reaction mix con- taining 5.0 μl of M-MLV transcription buffer, 1.0 μl of oligo dT primers (5 nM), 1.25 μl of dNTP mix (10 nM), 1.0 μl RNase inhibitor (20 U), 1.0 μl M-MLV reverse transcriptase (200 U, Promega, California, USA), RNA (2 μg), and RNase free water according to the vendor’s protocol. Mosquito dissection The data obtained were presented as the mean ± standard deviation (SD). A P value < 0.05 was considered statistically significant. between 5 and 1 in the scale were assigned according to the decreasing motility blindly by an independent evalu- ator; (iv) at 14 dpi, mosquito survival rate was also mea- sured by counting the live mosquitoes. host A previous study demonstrated the possibility of in vivo suppression of the cathepsin L-like cysteine protease gene of B. malayi using RNAi in Aedes aegypti [19]. Therefore, in this study, the possibility of using C. quinquefasciatus as an in vivo siRNA delivery platform for developing S. digitata larvae was tested, where the trans- formation of MF to L3 larvae of the latter organism has been observed. Since the previously stated study revealed Relative quantification of SDNP mRNA levels was car- ried out by quantitative real-time PCR [CFX96 RealTime PCR System (Bio-Rad)] using SYBR green as the DNA Somarathne et al. Parasites & Vectors (2018) 11:541 Page 6 of 11 Fig. 3 Larvae development in C. quinquefasciatus mosquitoes at 14 dpi. a Larvae in siRNA non-injected mosquito. b Larvae in SDNP siRNA- injected mosquito. c Larvae in GFP siRNA-injected mosquito. d Larvae in buffer-injected mosquito (magnification 400×). e Mean length and width of larvae form SDNP siRNA-injected, non-injected, buffer-injected and GFP siRNA-injected mosquitoes. To target L2 to L3, siRNA injections were made at 8 dpi. Triplicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are represented by different lowercase letters. Scale-bars: a-d, 400 μm Fig. 3 Larvae development in C. quinquefasciatus mosquitoes at 14 dpi. a Larvae in siRNA non-injected mosquito. b Larvae in SDNP siRNA- injected mosquito. c Larvae in GFP siRNA-injected mosquito. d Larvae in buffer-injected mosquito (magnification 400×). e Mean length and width of larvae form SDNP siRNA-injected, non-injected, buffer-injected and GFP siRNA-injected mosquitoes. To target L2 to L3, siRNA injections were made at 8 dpi. Triplicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are represented by different lowercase letters. Scale-bars: a-d, 400 μm the dissemination of siRNA to the MF of B. malayi through the mosquito, in the current study the uptake of the Cy3 labeled RNAi trigger by MF was investigated by, comparing the internal fluorescence of the MF with the mosquito physiological saline (MPS) injected con- trol under a fluorescent microscope (Fig. 1a). A dis- persion of the fluorescent signal was observed throughout the body of the tested MF at 24 hpi (Fig. 1c) indicating that uptake of Cy3 labelled siRNA by MF is occurring in the mosquitoes. host Although the mechanism of siRNA uptake is unclear at this point, it is possible that this would occur via feeding or through the cuticle of MF. Therefore, here, it was possible to recreate the in vitro siRNA soaking condi- tions for S. digitata larvae in vivo, and the possibility of utilizing the mosquito as a siRNA delivering plat- form for parasitic nematodes hosted by the mosquito was affirmed. The effect of siRNA on SDNP mRNA level of MF Several recent studies involving RNAi on nematodes showed that genes could be specifically suppressed by soaking the nematode in in vitro culture media consist- ing of RNAi triggers [25, 32–35]. In this study, instead Somarathne et al. Parasites & Vectors (2018) 11:541 Page 7 of 11 Fig. 4 Percentage of larval development to L3 stage out of total larvae inside the mosquito. S. digitata larvae from non-injected, SDNP siRNA- injected, GFP siRNA-injected and buffer-injected C. quinquefasciatus are represented by four different patterns in the graph. To target L2 to L3, siRNA injections were made at 8 dpi. Triplicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are indicated by * Fig. 4 Percentage of larval development to L3 stage out of total larvae inside the mosquito. S. digitata larvae from non-injected, SDNP siRNA- injected, GFP siRNA-injected and buffer-injected C. quinquefasciatus are represented by four different patterns in the graph. To target L2 to L3, siRNA injections were made at 8 dpi. Triplicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are indicated by * Fig. 4 Percentage of larval development to L3 stage out of total larvae inside the mosquito. S. digitata larvae from non-injected, SDNP siRNA- injected, GFP siRNA-injected and buffer-injected C. quinquefasciatus are represented by four different patterns in the graph. To target L2 to L3, siRNA injections were made at 8 dpi. Triplicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are indicated by * t-test, t44 = 15.2220, P = 0.05) in the worms from SDNP siRNA-injected mosquitoes, compared to the non-injected at 2 dpi and 14 dpi, respectively, compared to worms from non-injected mosquitoes (Fig. 2a, b). Worms from the buffer-injected, and GFP siRNA-injected mosquitoes displayed no significant changes in the SDNP transcript levels (Fig. 2a, b). The qPCR results obtained proved the worms’ susceptibility to specific RNAi while showing the effectiveness of the new siRNA delivery platform developed in this study and the capability of siRNA to knockdown the targeted gene specifically in this parasitic nematode. of using the in vitro culture conditions, we recreated the soaking conditions in vivo by injecting the mosquitoes harbouring nematode larvae with siRNA. The effect of siRNA on SDNP mRNA level of MF The RNAi ef- fect on SDNP transcript level was assessed two dpi, and 14 dpi of siRNA using qPCR and the knockdown of the gene was determined by comparing the transcript levels of the worms derived from non-injected, buffer-injected and GFP siRNA-injected mosquitoes. A significant sup- pression of the target gene expression was observed in this study, and the transcript abundance of the SDNP was reduced by 93% (unpaired two-tailed t-test, t44 = 15.8716, P = 0.05) and 87.4 % (unpaired two-tailed Fig. 5 Motility comparison of larvae at 14 dpi in, a non-injected, b SDNP siRNA-injected, c GFP siRNA-injected, d buffer-injected mosquitoes (Magnification 40×) Scale-bars: a-d, 400 μm Fig. 5 Motility comparison of larvae at 14 dpi in, a non-injected, b SDNP siRNA-injected, c GFP siRNA-injected, d buffer-injected mosquitoes (Magnification 40×) Scale-bars: a-d, 400 μm Somarathne et al. Parasites & Vectors (2018) 11:541 Page 8 of 11 Fig. 6 Frequency distribution for motility of S. digitata larvae at 14 dpi in, a siRNA non-injected, b SDNP siRNA-injected, c GFP siRNA-injected and d buffer-injected mosquitoes. The scoring scheme was: 1, immobile or dead; 2, compromised motility; 3, partial movement of the body; 4, slightly reduced motion in the body; 5, all parts of the body are vigorously twisting. Values in between 5 and 1 in the scale were assigned according to the decreasing motility blindly by an independent evaluator. Five replicates were performed for each experiment. Bars represent mean ± SD Fig. 6 Frequency distribution for motility of S. digitata larvae at 14 dpi in, a siRNA non-injected, b SDNP siRNA-injected, c GFP siRNA-injected and d buffer-injected mosquitoes. The scoring scheme was: 1, immobile or dead; 2, compromised motility; 3, partial movement of the body; 4, slightly reduced motion in the body; 5, all parts of the body are vigorously twisting. Values in between 5 and 1 in the scale were assigned according to the decreasing motility blindly by an independent evaluator. Five replicates were performed for each experiment. Bars represent mean ± SD Fig. 7 Frequency distribution of S. digitata larvae in C. quinquefasciatus head, thorax, or abdomen. Migration of the larvae was monitored at 14 dpi in non-injected, SDNP siRNA-injected, GFP siRNA-injected and buffer-injected mosquitoes. Mosquitoes were dissected into head, thorax and abdomen and larvae in each part was counted. Five replicates were performed for each experiment. Bars represent mean ± SD. Changes in developmental phenotypes and motility of larvae because of RNAi the involvement of SDNP in growth and development of nematodes. In the motility changes monitored at 14 dpi of the control worms and worms from SDNP siRNA-injected mosquitoes, the former displayed a vigorously convolut- ing body movement (Fig. 5a, c, d) of 4 and 5 in the mo- tility scale (Fig. 6a, c, d) while the SDNP siRNA-treated showed a significantly depressed body movement, feeble body twitching, and total body paralysis (Figs. 5b, 6b). Also, the worms from SDNP siRNA-injected worms and monitored at 14 dpi displayed irregular migration pat- terns in the mosquitoes compared to the controls (Fig. 7). Worms from the control mosquitoes were predomin- antly found in the head (non-injected: ~91%; GFP siRNA-injected: ~85%; buffer-injected: ~87%) of the mosquito, while SDNP-suppressed worms were mainly found either in the thorax (~85%) or abdomen (~10%) indicating that motility irregularities of larvae are associ- ated with the migration within the mosquito. It has been previously revealed that SDNP had homology with the inter-domain linkers of the muscle-specific twitchin kin- ase of C. elegans and was expressed in the longitudinal muscles of the adult worm [24]. Hence, the downregula- tion of the SDNP could affect the muscle movement of the developing worm, which in turn reduces the migra- tion of the parasite in the mosquito. The increased mos- quito survival rate was seen in the SDNP siRNA-injected mosquito group compared to the control groups (Fig. 8). This observation suggests that the compromised motility and development of the larvae induced by the SDNP siRNA treatment could reduce the parasitic burden on the mosquito, thereby increasing its survival. To study the phenotypic changes, if any, associated with the transformation of L2 to L3 in the SDNP siRNA-injected group in comparison to the control mos- quito groups, the infected mosquitoes were dissected to recover the larval stages at eight dpi to 14 dpi following the RNAi trigger. A significant reduction in body length and width together with deformities in the body struc- ture was observed in the larvae from SDNP siRNA-injected mosquitoes at 14 dpi (Fig. 3b, e) com- pared to the non- injected, buffer-injected and GFP siRNA-injected counterparts (Fig. 3a, c-e). The effect of siRNA on SDNP mRNA level of MF Statistical analysis was performed using Student’s t-test (P < 0.05). Significant differences at P < 0.05 are indicated by * Fig. 7 Frequency distribution of S. digitata larvae in C. quinquefasciatus head, thorax, or abdomen. Migration of the larvae was monitored at 14 dpi in non-injected, SDNP siRNA-injected, GFP siRNA-injected and buffer-injected mosquitoes. Mosquitoes were dissected into head, thorax and abdomen and larvae in each part was counted. Five replicates were performed for each experiment. Bars represent mean ± SD. Statistical analysis was performed using Student’s t-test (P < 0.05). Significant differences at P < 0.05 are indicated by * Fig. 7 Frequency distribution of S. digitata larvae in C. quinquefasciatus head, thorax, or abdomen. Migration of the larvae was monitored at 14 dpi in non-injected, SDNP siRNA-injected, GFP siRNA-injected and buffer-injected mosquitoes. Mosquitoes were dissected into head, thorax and abdomen and larvae in each part was counted. Five replicates were performed for each experiment. Bars represent mean ± SD. Statistical analysis was performed using Student’s t-test (P < 0.05). Significant differences at P < 0.05 are indicated by * Somarathne et al. Parasites & Vectors (2018) 11:541 Page 9 of 11 Fig. 8 Mosquito survival rate. Survivability of the non-injected, SDNP siRNA-injected, GFP siRNA-injected and buffer-injected mosquitoes at 14 dpi was measured. Five replicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are indicated with different lowercase letters Fig. 8 Mosquito survival rate. Survivability of the non-injected, SDNP siRNA-injected, GFP siRNA-injected and buffer-injected mosquitoes at 14 dpi was measured. Five replicates were performed for each experiment. Bars represent mean ± SD. Significant differences at P < 0.05 are indicated with different lowercase letters Changes in developmental phenotypes and motility of larvae because of RNAi In addition, aberrations, with larval lethality was also observed in SDNP siRNA treated larvae compared to its control groups, and with ~96% of L2 to L3 transformation in non-treated, ~95% in buffer-treated and ~93% in GFP siRNA treated compared to ~18% of such transform- ation in treated at 14 dpi (Fig. 4). The L2 and L3 lar- vae were differentiated based on their length of the body. No significant developmental changes were ob- served in the worms from GFP siRNA-injected mos- quitoes suggesting the phenotypic changes are specific to SDNP siRNA and not due to the injection of non-specific siRNA. Therefore, it is reasonable to as- sume these phenotypic changes are most likely due to the downregulation of SDNP transcripts, which in turn affects the larval development. The previous finding reported by us, i.e., the ubiquitous expression of SDNP in all life stages of S. digitata and also its predominant expression in longitudinal muscle, repro- ductive systems, and developing embryos [24, 36], to- gether with the findings of the present study, i.e. association of SDNP downregulation with irregular- ities in the larvae development, transformation arrest, changes in phenotypes, etc. in larvae can be used to further strengthen our previous suggestion regarding Competing interests Competing interests The authors declare that they have no competing interests. 23. Hawking F & World Health Organization. Diethylcarbamazine: a review of the literature with special reference to its pharmacodynamics, toxicity, and use in the therapy of onchocerciasis and other filarial infections. Geneva: World Health Organization; 1978. http://www.who.int/iris/handle/10665/70735 Funding Th The research was funded by a grant (AP/3/2011/CG/03) awarded to Professor R.S. Dassanayake by the University of Colombo, Sri Lanka, to promote collaborative research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 14. Behm CA, Bendig MM, McCarter JP, Sluder AE. RNAi-based discovery and validation of new drug targets in filarial nematodes. Trends Parasitol. 2005; 21:97–100. 15. Fire A, Albertson D, Harrison SW, Moerman DG. Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development. 1991;113:503–14. Availability of data and materials The data supporting the conclusions of this study are included within the article. Clones and raw data are available from the authors upon request. 16. Grant WN, Behm CA. Target identification and validation for anthelmintic discovery. Expert Opin Drug Discov. 2007;2:S91–8. 17. Pfarr K, Heider U, Hoerauf A. RNAi mediated silencing of actin expression in adult Litomosoides sigmodontis is specific, persistent and results in a phenotype. Int J Parasitol. 2006;36:661–9. Abbreviations R R f and genetics. Int J Parasitol. 2006;36:1227 39. 7. Kaplan RM. Drug resistance in nematodes of veterinary importance: a statu report. Trends Parasitol. 2004;20:477–81. 8. Richard-Lenoble D, Chandenier J, Gaxotte P. Ivermectin and filariasis. Fundam Clin Pharmacol. 2003;17:199–203. 9. Berriman M, Lustigman S, McCarter JP. Genomics and emerging drug bb e at o s RNAi: RNA interference; SDNP: Setaria digitata novel protein; qPCR: Quantitative real-time polymerase chain reaction; siRNA: Small interfering RNA; dsRNA: Double stranded RNA; MPS: Mosquito physiological saline; MF: Microfilariae; hpi: Hour post infection; dpi: Days post-infection; GFP: Green fluorescent protein. 7. Kaplan RM. Drug resistance in nematodes of veterinary importance: a statu report. Trends Parasitol. 2004;20:477–81. 8. Richard-Lenoble D, Chandenier J, Gaxotte P. Ivermectin and filariasis. Fundam Clin Pharmacol. 2003;17:199–203. 9. Berriman M, Lustigman S, McCarter JP. Genomics and emerging drug 7. Kaplan RM. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol. 2004;20:477–81. 8. Richard-Lenoble D, Chandenier J, Gaxotte P. Ivermectin and filariasis. Fundam Clin Pharmacol. 2003;17:199–203. 9. Berriman M, Lustigman S, McCarter JP. Genomics and emerging drug discovery technologies. Expert Opin Drug Discov. 2007;2:S83–9. 9. Berriman M, Lustigman S, McCarter JP. Genomics and emerging drug discovery technologies. Expert Opin Drug Discov. 2007;2:S83–9. Conclusions In this study, we demonstrated the successful usage of the RNAi approach to turn off the expression of SDNP in the larval stages of S. digitata in their natural milieu, Page 10 of 11 Page 10 of 11 Page 10 of 11 Somarathne et al. Parasites & Vectors (2018) 11:541 Somarathne et al. Parasites & Vectors (2018) 11:541 Somarathne et al. Parasites & Vectors (2018) 11:541 C. quinquefasciatus, and the possibility of using this host as an in vivo culture platform in the absence of proper culture conditions to study the biological functions of the genes that the S. digitata genome encodes. Further, we showed that the specific silencing of SDNP is associ- ated with developmental deformities and motility changes in the developing larvae of S. digitata, in addition to the reduction of migration of the larvae to the head of the mosquito. Therefore, by taking the out- comes of present and our previous studies into consider- ation, it can be concluded that SDNP plays a vital role in the movement and development of larvae. Hence, it can be suggested that the SDNP may be utilised as a candi- date anthelminthic drug target. Received: 10 May 2018 Accepted: 4 September 2018 Received: 10 May 2018 Accepted: 4 September 2018 Acknowledgement 10. Foster JM, Zhang Y, Kumar S, Carlow CKS. Mining nematode genome data for novel drug targets. Trends Parasitol. 2005;21:101–4. 10. Foster JM, Zhang Y, Kumar S, Carlow CKS. Mining nematode genome da for novel drug targets. Trends Parasitol. 2005;21:101–4. The corresponding author wishes to acknowledge Professor Mark Taylor and his team for providing him training in RNA interference at his laboratories in Liverpool School of Tropical Medicine, Liverpool under the Commonwealth Fellowship Programme in 2012. 11. Kuwabara PE, Coulson A. RNAi - prospects for a general technique for determining gene function. Parasitol Today. 2000;16:347–9. 11. Kuwabara PE, Coulson A. RNAi - prospects for a general technique for determining gene function. Parasitol Today. 2000;16:347–9. 12. Allshire R. Retraction. Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science. 2005;310:49. 12. Allshire R. Retraction. Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing. Science. 2005;310:49. 13. Hall IM, Shankaranarayana GD, Noma K-I, Ayoub N, Cohen A, Grewal SIS. Establishment and maintenance of a heterochromatin domain. Science. 2002;297:2232–7. References 1. Bhadury P, Austen MC, Bilton DT, Lambshead PJD, Rogers AD, Smerdon GR. Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes. Mar Ecol Prog Ser. 2006;320:1–9. 2. Stepek G, Buttle DJ, Duce IR, Behnke JM. Human gastrointestinal nematode infections: are new control methods required? Int J Exp Pathol. 2006;87:325–41. 3. Global Programme to Eliminate Lymphatic Filariasis. Progress report on mass drug administration 2010. Switzerland: World Health Organization; 2011. 4. Singh S, Singh B, Singh AP. Nematodes: a threat to sustainability of agriculture. Procedia Environ Sci. 2015;29:215–6. 1. Bhadury P, Austen MC, Bilton DT, Lambshead PJD, Rogers AD, Smerdon GR. Development and evaluation of a DNA-barcoding approach for the rapid identification of nematodes. Mar Ecol Prog Ser. 2006;320:1–9. 5. Roeber F, Jex AR, Gasser RB. Impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance - an Australian perspective. Parasit Vectors. 2013;6:153. 6. Gilleard JS. Understanding anthelmintic resistance: the need for genomics and genetics. Int J Parasitol. 2006;36:1227–39. Publisher’s Note 24. Rodrigo WWP, Dassanayake RS, Voronin D. Novel parasitic nematode-specific protein of Setaria digitata largely localized in longitudinal muscles, reproductive systems and developing embryos. Exp Parasitol. 2014;141:12–20. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Consent for publication 22. Decruse SW, Raj RK. Histological studies on female Setaria digitata (von Linstow 1906), a filaria of bovine, Bos indicus. Proc Anim Sci. 1990;99:103–12. Authors’ contributions MBCLS carried out research work, collected samples, analysed data, and wrote the manuscript. YINSG supervised research work, analysed research data and wrote the manuscript. NVC supervised research work data and corrected the manuscript. ANBE analysed research data and corrected the manuscript. RSD designed and supervised the study and corrected the manuscript. All authors read and approved the final manuscript. 18. Kamath RS, Ahringer J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods. 2003;30:313–21. elegans. Methods. 2003;30:313–21. 19. Song C, Gallup JM, Day TA, Bartholomay LC, Kimber MJ. Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi. PLoS Pathog. 2010;6:e1001239. 20. Jinkichi F, Soichi I, Toshio I, Tetsuo N, Ken T, Tadashi T, et al. Heterotopic parasitism of Setaria digitata (Linstow, 1906) in the heart of a cattle. Japanese J Vet Sci. 1985;47:999–1002. Ethics approval and consent to participate Not applicable. 21. Bal M, Das MK. Antigenicity of a filarial protease from Setaria digitata in Wuchereria bancrofti infection. Ann Trop Med Parasitol. 1999;93:279–88. Somarathne et al. Parasites & Vectors (2018) 11:541 Somarathne et al. Parasites & Vectors (2018) 11:541 27. Gerberg EJ. Manual for mosquito rearing and experimental techniques. Bull Am Mosq Control Assoc. 1970;5:1–109. 28. Hayes RO. Determination of a physiological saline solution for Aedes aegypti (L.). J Econ Entomol. 1953;46:624–7. 29. Benedict M, Howell P. 3.8 Mosquito Anesthesia. In: Methods in Anopheles Research. 2nd ed. Atlanta: bei RESOURCES; 2010. p. 1–4. 30. Garver L, Dimopoulos G. Protocol for RNAi assays in adult mosquitoes (A. gambiae). J Vis Exp. 2007;5:230. 31. Luna BM, Juhn J, James AA. Injection of dsRNA into female A. aegypti mosquitoes. J Vis Exp. 2007;215:224. 32. Ford L, Zhang J, Liu J, Hashmi S, Fuhrman JA, Oksov Y, et al. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference. PLoS Negl Trop Dis. 2009;3:e377. 33. Ma HB, Lu Q, Liang J, Zhang XY. Functional analysis of the cellulase gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference. Genet Mol Res. 2011;10:1931–41. 34. Aboobaker AA, Blaxter ML. Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi. Mol Biochem Parasitol. 2003;129:41–51. 35. Lustigman S, Zhang J, Liu J, Oksov Y, Hashmi S. RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting. Mol Biochem Parasitol. 2004;138:165–70. 36. Dassanayake RS, Rodrigo WWP, Karunanayake EH, Weerasena OVDSJ, Chandrasekharan NV. A putative nuclear growth factor-like globular nematode-specific protein. Bioinformation. 2009;3:370–4. 27. Gerberg EJ. Manual for mosquito rearing and experimental techniques. Bull Am Mosq Control Assoc. 1970;5:1–109. 27. Gerberg EJ. Manual for mosquito rearing and experimental techniques. Bull Am Mosq Control Assoc. 1970;5:1–109. 28. Hayes RO. Determination of a physiological saline solution for Aedes aegypti (L.). J Econ Entomol. 1953;46:624–7. 28. Hayes RO. Determination of a physiological saline solution for Aedes aegypti (L.). J Econ Entomol. 1953;46:624–7. 29. Benedict M, Howell P. 3.8 Mosquito Anesthesia. In: Methods in Anopheles Research. 2nd ed. Atlanta: bei RESOURCES; 2010. p. 1–4. 29. Benedict M, Howell P. 3.8 Mosquito Anesthesia. In: Methods in Anopheles Research. 2nd ed. Atlanta: bei RESOURCES; 2010. p. 1–4. 30. Garver L, Dimopoulos G. Protocol for RNAi assays in adult mosquitoes (A. gambiae). J Vis Exp. 2007;5:230. 30. Garver L, Dimopoulos G. Protocol for RNAi assays in adult mosquitoes (A. gambiae). J Vis Exp. 2007;5:230. 31. Luna BM, Juhn J, James AA. Injection of dsRNA into female A. aegypti mosquitoes. J Vis Exp. 2007;215:224. 31. 27. Gerberg EJ. Manual for mosquito rearing and experimental techniques. Bull Am Mosq Control Assoc. 1970;5:1–109. 28. Hayes RO. Determination of a physiological saline solution for Aedes aegypti (L.). J Econ Entomol. 1953;46:624–7. 29. Benedict M, Howell P. 3.8 Mosquito Anesthesia. In: Methods in Anopheles Research. 2nd ed. Atlanta: bei RESOURCES; 2010. p. 1–4. 30. Garver L, Dimopoulos G. Protocol for RNAi assays in adult mosquitoes (A. gambiae). J Vis Exp. 2007;5:230. 31. Luna BM, Juhn J, James AA. Injection of dsRNA into female A. aegypti mosquitoes. J Vis Exp. 2007;215:224. 32. Ford L, Zhang J, Liu J, Hashmi S, Fuhrman JA, Oksov Y, et al. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference. PLoS Negl Trop Dis. 2009;3:e377. 33. Ma HB, Lu Q, Liang J, Zhang XY. Functional analysis of the cellulase gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference. Genet Mol Res. 2011;10:1931–41. 34. Aboobaker AA, Blaxter ML. Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi. Mol Biochem Parasitol. 2003;129:41–51. 35. Lustigman S, Zhang J, Liu J, Oksov Y, Hashmi S. RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting. Mol Biochem Parasitol. 2004;138:165–70. 36. Dassanayake RS, Rodrigo WWP, Karunanayake EH, Weerasena OVDSJ, Chandrasekharan NV. A putative nuclear growth factor-like globular nematode-specific protein. Bioinformation. 2009;3:370–4. Author details 1 25. Lendner M, Doligalska M, Lucius R, Hartmann S. Attempts to establish RNA interference in the parasitic nematode Heligmosomoides polygyrus. Mol Biochem Parasitol. 2008;161:21–31. 1Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03 00300, Sri Lanka. 2Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Ragama 11010, Sri Lanka. 3Department of Bioclinical Sciences, Faculty of Dentistry, Health Sciences Center, University of Kuwait, Kuwait City, Kuwait. 26. Tung KC, Cheng FP, Lai CH, Wang KS, Wang JS, Lee WM. Demonstration of vector competence of Culex quinquefasciatus (Diptera: Culicidae) for Setaria digitata. Vet Parasitol. 2004;123:279–84. Page 11 of 11 Somarathne et al. Parasites & Vectors (2018) 11:541 Luna BM, Juhn J, James AA. Injection of dsRNA into female A. aegypti mosquitoes. J Vis Exp. 2007;215:224. 32. Ford L, Zhang J, Liu J, Hashmi S, Fuhrman JA, Oksov Y, et al. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference. PLoS Negl Trop Dis. 2009;3:e377. 32. Ford L, Zhang J, Liu J, Hashmi S, Fuhrman JA, Oksov Y, et al. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference. PLoS Negl Trop Dis. 2009;3:e377. 33. Ma HB, Lu Q, Liang J, Zhang XY. Functional analysis of the cellulase gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference. Genet Mol Res. 2011;10:1931–41. 33. Ma HB, Lu Q, Liang J, Zhang XY. Functional analysis of the cellulase gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference. Genet Mol Res. 2011;10:1931–41. 34. Aboobaker AA, Blaxter ML. Use of RNA interference to investigate gene function in the human filarial nematode parasite Brugia malayi. Mol Biochem Parasitol. 2003;129:41–51. 35. Lustigman S, Zhang J, Liu J, Oksov Y, Hashmi S. RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting. Mol Biochem Parasitol. 2004;138:165–70. 36. Dassanayake RS, Rodrigo WWP, Karunanayake EH, Weerasena OVDSJ, Chandrasekharan NV. A putative nuclear growth factor-like globular nematode-specific protein. Bioinformation. 2009;3:370–4.
https://openalex.org/W3080384475	https://pure.au.dk/ws/files/198818569/Fehr_et_al._2020_Diversity_Distributions.pdf	English	null	Non‐native palms (Arecaceae) as generators of novel ecosystems: A global assessment	Diversity and distributions	2,020	cc-by	14,983	B I O D I V E R S I T Y R E V I E W B I O D I V E R S I T Y R E V I E W Non-native palms (Arecaceae) as generators of novel ecosystems: A global assessment ent Fehr1,2 \| Robert Buitenwerf1,2 \| Jens-Christian Svenning1,2 1Center for Biodiversity Dynamics in a Changing World (BIOCHANGE), Department of Biology, Aarhus University, Aarhus C, Denmark 2Section for Ecoinformatics and Biodiversity, Department of Biology, Aarhus University, Aarhus C, Denmark Correspondence Vincent Fehr, Center for Biodiversity Dynamics in a Changing World (BIOCHANGE), Department of Biology, Aarhus University, Ny Munkegade 114, DK- 8000 Aarhus C, Denmark. Email: vincent.fehr@bios.au.dk Funding information Villum Fonden, Grant/Award Number: 16549 1Center for Biodiversity Dynamics in a Changing World (BIOCHANGE), Department of Biology, Aarhus University, Aarhus C, Denmark 2Section for Ecoinformatics and Biodiversity, Department of Biology, Aarhus University, Aarhus C, Denmark Correspondence Vincent Fehr, Center for Biodiversity Dynamics in a Changing World (BIOCHANGE), Department of Biology, Aarhus University, Ny Munkegade 114, DK- 8000 Aarhus C, Denmark. Email: vincent.fehr@bios.au.dk Funding information Villum Fonden, Grant/Award Number: 16549 1Center for Biodiversity Dynamics in a Changing World (BIOCHANGE), Department of Biology, Aarhus University, Aarhus C, Denmark Diversity and Distributions. 2020;26:1523–1538. Received: 27 March 2020 \| Revised: 29 June 2020 \| Accepted: 17 July 2020 Received: 27 March 2020 \| Revised: 29 June 2020 \| Accepted: 17 July 2020 DOI: 10.1111/ddi.13150 1524 \| 1 \| INTRODUCTION FEHR et al. 1524 FEHR et al. (Galetti, Keuroghlian, Hanada, & Morato, 2001; Henderson, 2002; Sica, Bravo, & Giombini, 2014). We therefore expect introduced palms to have disproportionate effects on ecological communities and ecosystem functioning. Palms have been widely introduced be- yond their native range for ornamental purposes, food and other uses (Byg & Balslev, 2001, 2006; Campos & Ehringhaus, 2003; Martins, Filgueiras, & Albuquerque, 2014; Sosnowska & Balslev, 2009), result- ing in the spread of many palm species (Figure 1). For example, the coconut palm (Cocos nucifera) has a pantropical distribution due to long-distance dispersal by humans (Gunn, Baudouin, & Olsen, 2011). The spread of palms to new environments is expected to accelerate due to ongoing global warming, which will allow palms to colonize higher latitudes and altitudes (Reichgelt, West, & Greenwood, 2018; Walther et al., 2007). Palms are also a practical model for studying novel ecosystems as they are one of the best known tropical plant families (Baker & Dransfield, 2016; Eiserhardt, Svenning, Kissling, & Balslev, 2011; Henderson, 2002; Kissling et al., 2019). This body of knowledge is key to move beyond descriptive work, and identify and quantify the mechanisms by which invasive species assemble novel communities and ecosystem processes. Human agency is an increasingly dominant force in the assembly of ecological communities. An important component is the trans- location of species to regions outside their native range (Seebens et al., 2017), with introductions sometimes resulting in species as- semblages markedly without historical precedent (Hobbs, Higgs, & Harris, 2009). Such ecosystems have been termed “novel ecosys- tems” if they are self-sustaining without ongoing human interven- tion (Hobbs et al., 2009; Morse et al., 2014). Species invasions that trigger the emergence of novel ecosystems have far-reaching eco- logical—and in some cases also socio and economic—effects, as eco- system functioning and services may be strongly affected (Hobbs et al., 2009). The generation of novel ecosystems with non-native plants as the main drivers has been well documented within a wide range of habitats around the globe (Lugo, 2004; Rogers & Chown, 2014; Yu, Okin, Ravi, & D’Odorico, 2016). Despite awareness of the various mechanisms through which non-native plants can alter ecosystem composition and processes (Hejda, Pyšek, & Jarošík, 2009; Vilà et al., 2011), the degree of novelty that different taxonomic groups or functional types generate within an ecosystem remains unclear. Abstract Abstract Aims: Novel ecosystems are self-maintaining ecosystems that support species assem- blages without historical precedent. Despite much interest and controversy around novel ecosystems, it remains poorly understood how they are generated, what their capacity to support biodiversity is and what the implications for society are. Here, we address these issues through a global synthesis of non-native palms, since palms are likely generators of novel ecosystems because they are introduced widely beyond their native range and have the capacity to act as ecosystem engineers. Location: Global. Methods: We gathered data on non-native palms from peer-reviewed literature/pa- pers, grey literature and online databases. We extracted data on the biogeographic context of palm invasions, plant functional traits and anthropogenic drivers to quan- tify their effects on biodiversity, ecosystem functioning and ecosystem services. Results: Of the 2,557 palm species, 3.4% (86 species) were recorded as naturalized and 1.1% (28 species) as invasive, which exceeds the average invasion success across all woody plants. Naturalized palms are present in most tropical and subtropical re- gions around the world, often in urban areas, reflecting the use of palms in horti- culture. Many naturalized palms were taller and more likely to originate from open habitats or dry forest than non-naturalized palms. These features likely represent the naturalized palms’ competitive ability, high fecundity and dispersal ability along with ecological matching to human-disturbed environments. Overall, literature on ecological effects of palm invasions was sparse, but we found multiple cases in which palm invasions resulted in strong ecosystem changes or even biome shifts. Main Conclusions: We found strong evidence that palm invasions can generate novel ecosystems. Although there are substantial knowledge gaps on the ecological effects of palm invasion, anthropogenic drivers like urbanization and ongoing global warming will continue to expand palm ranges and promote non-native palms as generators of novel ecosystems. Diversity and Distributions. 2020;26:1523–1538. \| 1523 wileyonlinelibrary.com/journal/ddi This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Diversity and Distributions published by John Wiley & Sons Ltd. K E Y W O R D S alien invasive species, Arecaceae, biological invasions, ecosystem effects, global change, invasion biology, non-native species, novel ecosystems, palms, urban ecology \| 152 wileyonlinelibrary.com/journal/ddi 1524 \| 1 \| INTRODUCTION This inability to generalize hampers the ability to make future projec- tions of community assembly, structure and functioning and to fore- cast social and economic implications. Furthermore, the ecological dynamics of novel ecosystems, their ability to sustain biodiversity, and how this can be improved through conservation and restoration actions remain poorly understood (Svenning, 2018). Here, we provide the first global synthesis on palms as potential generators of novel ecosystems, drawing on peer-reviewed literature, as well as non-peer-reviewed reports and verifiable anecdotal evi- dence in order to overcome data limitations. The study addresses three broad objectives. First, we assess the biogeographic and environmen- tal context of palm invasions and quantify the role of functional traits and human-mediated dispersal. Second, we assess evidence that palm invasions generate novel ecosystems as well as effects more generally on other taxonomic groups, ecosystem functioning and human soci- ety. Finally, we discuss the future of palm-generated novel ecosystems under scenarios of societal and climate change. Tools to predict future ranges under different scenarios of global change exist, notably environmental niche models (Guisan & Zimmermann, 2000; Thuiller, 2003). However, these models typically ignore species interactions, which are crucial in species invasions. Even joint species distribution models (Blois, Zarnetske, Fitzpatrick, & Finnegan, 2013; Svenning et al., 2014), in which species interac- tions are considered through co-occurrence patterns (Ovaskainen et al., 2017), suffer from an intractability of the ecological processes through which species interact and typically focus on a single taxo- nomic or trophic level (Zurell, Pollock, & Thuiller, 2018). Evidence of the effects of species introductions across entire taxonomic groups may thus lead to new insights and generalizations that can be used to predict the degree to which invasions will create novel ecosystems. 2.1 \| Data compilation and classification of non- native palm records We gathered information on non-native palms from several sources including alien species databases, peer-reviewed publications, books and grey literature like inventory lists of alien species or factsheets from local authorities (see Appendix S1: Table S1.1 and S1.2 and Appendix S3) and examined the references from the retrieved sources for further records. A total of 302 records were gathered from the World Checklist of Arecaceae by Govaerts, Dransfield, Hodel, and Henderson (2015). We standardized the taxonomic names according to the nomenclature of Govaerts et al. (2015) and classified the species into three naturalization stages according to Richardson et al. (2000): Palms (Arecaceae) are potential generators of novel ecosystems as they (a) have the capacity to act as ecosystem engineers, and (b) have been widely introduced elsewhere for agri- and horticulture. Many palms have the capacity to act as ecosystem engineers within their native range, primarily because of their unique physiognomy and functional traits compared to other woody plants (Dransfield & Uhl, 2008; Tomlinson, Horn, & Fisher, 2011), combined with a capac- ity to become hyper-dominant (Steege et al., 2013). An example of a unique palm functional trait is the big, heavy and slow-decomposing leaves that modify the habitat in multiple ways (Farris-Lopez, Denslow, Moser, & Passmore, 2004). Many palms are considered as keystone species in their native range due to their important role for frugivores • Casual species: Seedlings, juveniles or reproductive individuals that spread locally and close to cultivated parent plants, but do not form self-sustaining populations (including species reported • Casual species: Seedlings, juveniles or reproductive individuals that spread locally and close to cultivated parent plants, but do not form self-sustaining populations (including species reported FEHR et al. 1525 \| \| (a) (b) (c) (d) (e) (f) FI G U R E 1 Examples of naturalized palms dominating non-native ecosystems, with potentially large ecological impacts. (a) Archontophoenix alexandrae in Hawaii, United States (Photo: Bo-Göran Lundkvist); (b) Washingtonia robusta around San Diego, California, United States (Photo: Vincent Fehr); (c) Phoenix dactylifera in central Baja California, Mexico (Photo: V. Fehr); (d) Trachycarpus fortunei in Ticino, Southern Switzerland, invading a temperate, deciduous forest (Photo: V. Fehr); (e) Archontophoenix cunninghamiana in Southern Brazil (Photo: V. Fehr); (f) Phoenix canariensis around San Diego, California, United States (Photo: V. Fehr) (a) (b) (c) (d) (e) (f) FI G U R E 1 Examples of naturalized palms dominating non-native ecosystems, with potentially large ecological impacts. 2.1 \| Data compilation and classification of non- native palm records (a) Archontophoenix alexandrae in Hawaii, United States (Photo: Bo-Göran Lundkvist); (b) Washingtonia robusta around San Diego, California, United States (Photo: Vincent Fehr); (c) Phoenix dactylifera in central Baja California, Mexico (Photo: V. Fehr); (d) Trachycarpus fortunei in Ticino, Southern Switzerland, invading a temperate, deciduous forest (Photo: V. Fehr); (e) Archontophoenix cunninghamiana in Southern Brazil (Photo: V. Fehr); (f) Phoenix canariensis around San Diego, California, United States (Photo: V. Fehr) (b) (a) (d) (c) (c) (f) (e) (e) (e) (f) as “casual alien species”, “not established”, “escaped”, “locally nat- uralized”, “semi-naturalized” or “introduced”). introduced range (e.g. Phoenix dactylifera in Austria and Hungary; DAISIE, 2018). To ensure a high-quality data set, we checked each report for plausibility, verified the original source in doubtful cases and removed incorrect or implausible reports. In all the following analyses, we included only records of naturalized and invasive palms (i.e. excluding casual species records). All analyses were conducted using the R software (version 3.6.1; R Core Team, 2019). • Naturalized species: Fertile individuals that exist over considerable distance from parent plants and form self-sustaining populations (including species reported as “naturalised” or “established”). All invasive species were also included in this category. • Invasive species: Naturalized species that produce reproductive offspring, often in very large numbers, at considerable distances from parent plants and for this reason could potentially have un- desirable ecological, social or economic effects. This stage of nat- uralization was only assigned to non-native species if they were classified as such in a peer-reviewed publication or by an accepted authority (including species reported as “weeds”). for 1,052 species (52% of all non-climbing palms). For species with- out recorded maximum stem height (n = 3) and habitat type (n = 25) in Henderson (2002) or Kissling et al. (2019), we substituted values using carefully selected records from (online) palm literature (Craft, Riffle, & Zona, 2014; Palmpedia, 2019; Palmweb, 2019). Pearson cor- relation tests were computed to assess how maximum stem height is related to maximum stem diameter, maximum blade length and average fruit length (Kissling et al., 2019). (Randall, 2007) or Cuba (Oviedo et al., 2012), or publications specifi- cally focusing on non-native palms (e.g. for Panama [Svenning, 2002] or for tropical islands [Meyer, Lavergne, & Hodel, 2008]). In contrast, many regions seemed underexplored (e.g. Philippines and some countries in the tropics of Africa and South America). Data on the native distribution of the palm family were derived from Govaerts et al. (2015). We performed a Spearman correlation test to analyse the relation between the number of native species on a continent and the number of naturalized species originating from that continent. To test whether palms with a conspicuous fruit colour are more naturalized (e.g. due to increased dispersal by birds), we compared the proportion of species with conspicuous fruit colour and species with cryptic fruit colour in naturalized and non-naturalized palm species using Fisher's exact test. Data on fruit conspicuousness were available for 1,799 species (Kissling et al., 2019). We filled in missing values on fruits conspicuousness of naturalized species (n = 2) using Palmpedia (2019). To examine the relationship between the environment and non-native palm establishment, we assigned each occurrence record to “tropical” and “extratropical” climates based on the Köppen cli- mate classification (Peel, Finlayson, & McMahon, 2007) and to eight habitat classes (IUCN, 2019), which were combined and renamed (see Table S1.3 in Appendix S1). Habitat classes were only applied to non-native palm records that specifically state the habitat type in the original source. Species may occur in multiple climatic regions and habitats. The climate and habitat information for many records were not detailed enough to assign species to biomes. 2.4 \| Ecological, social and economic effects of palm invasions To estimate the ability of palms to generate novel ecosystems, we compiled information on their effects on different taxonomic groups, ecosystem functions and processes from our records (see Appendix 3). We collected information on their effects on aquatic fauna, birds, invertebrates, mammals, other plants and reptiles and ecosystem variables: fire regime, physical vegetation structure, soil moisture, soil nutrients, temperature regime and water regime. We classified these into desirable and undesirable effects based on the evaluation of the authors of the original source. For every reported case, we indicated if information was based on strong (experimental) or on weak (anecdotal or observational) evidence. It was not pos- sible to evaluate the severity of these effects because many of the reported effects were not comparable among the different sources. Therefore, an “undesirable effect on native plants” can indicate an ecosystem-level threat or the suppression of a single native species. To evaluate whether palm invasions are more or less likely to occur on islands, we compared the number of naturalized palm spe- cies on mainlands and islands. We defined islands as landmasses that are surrounded by water and that were not connected to a continental landmass (i.e. Australia or larger) during the Last Glacial Maximum, when global sea levels were ~120 m below current levels (Rabineau et al., 2006). 2.3 \| Functional traits of non-native palms Height is a core trait in determining naturalization success as it re- lates to the plants’ ability to compete for light, their fecundity and their dispersal ability (Jelbert, Stott, McDonald, & Hodgson, 2015; van Kleunen, Weber, & Fischer, 2010; Thomson, Moles, Auld, & Kingsford, 2011). We also expected that palms originating from open environments are more likely to be pre-adapted for tolerat- ing or benefitting from human disturbances and associated open- or semi-open anthropogenic environments. To test whether natural- ized palms are on average taller than non-naturalized palms and are primed for anthropogenically disturbed habitats, we used a logistic regression. We excluded all climbing palms (i.e. 546 species, of which eight species were naturalized) because of their different functional attributes compared to non-climbing palms. Data on habitat types were derived from Henderson (2002) and were available for 1,069 species (53% of the non-climbing species [n = 2,011] of the palm family). To simplify the habitat types used by Henderson (2002), we created the following habitat classes: (a) moist forest (= lowland moist forest + montane forest), (b) dry forest, and (c) open habitat (= grassland, shrubland and savanna + desert and xeric shrubland). Data on maximum stem height were taken from Kissling et al. (2019) and were available for 1,741 species (87% of all non-climbing palms). Data on both maximum stem height and habitat type were available 2.2 \| Inventory, distribution and biogeography of non-native palms To assess whether palm species from certain subfamilies are more likely to naturalize outside their native range, we compared the proportion of naturalized species (excluding casual species) and non-naturalized species in each subfamily with species from the re- maining subfamilies using Fisher's exact test. If we found contradicting information regarding the naturaliza- tion stage of a species in consulted peer-reviewed and grey litera- ture, we adopted peer-reviewed records over grey literature. When equally reliable sources reported different naturalization stages, we assigned the most advanced stage of naturalization. To analyse the geographical distribution of non-native palms, we used the World Geographical Scheme for Recording Plant Distributions of the International Working Group on Taxonomic Databases (TDWG, Brummitt, 2001). Specifically, we used botanical continents (level 1), botanical regions (level 2) and botanical countries (level 3), which are units based on a combination of political bound- aries and botanical traditions. For some parts of the world, com- prehensive inventories of non-native biota were available including non-native palms, for example for Florida (FLEPPC, 2017), Australia The criteria and the use of terms for categorizing non-native palm species varied considerably between different sources. We no- ticed several cases where the naturalization stage of a species was incorrectly adopted from the original source into a publication of a peer-reviewed journal or an online database. We also found entries in online databases where the climatic requirements of the non-na- tive palm species did not comply with the climatic conditions in the FEHR et al. FEHR et al. 1526 We hypothesized that the level of urbanization in a region will be a major determinant of palm invasions because propagule pressure of non-native palms is usually high in urban settings due to the presence of ornamental palm plantings. To test this hypothesis, we used the human footprint index (HFI) from Venter et al. (2016) as a proxy vari- able for urbanization. For each TDWG level-3 country, we calculated the human footprint index by taking the 90th percentile of all 1 × 1 km HFI raster cells within the country. We used the 90th percentile in- stead of the mean, as we expect that the presence of highly modified areas (i.e. a high HFI value) better predicts palm invasions in a country. We selected the HFI of 1993 instead of the more current version from 2009 to take into account that the majority of currently naturalized palms are the result of introductions prior to 2009. We performed a logistic regression to test the effect of the HFI on the proportion of naturalized palms from the total number of palms (i.e. native + natu- ralized). We then calculated Nagelkerke's R2 to quantify the predictive power of the HFI for the proportion of naturalized palms. naturalized species (2.2%, p = .074). The only species of the mono- typic subfamily Nypoideae, Nypa fruticans, was also reported to be in- vasive (100%, p = .034). The subfamily Ceroxyloideae (0%, p = .404) contained no naturalized or invasive species, but one casual species. 3.2 \| Distribution and biogeography of non- native palms We found 86 species of naturalized palms that were spread over 185 TDWG level-3 countries, resulting in 567 unique species-country combinations. Non-native palm establishment was concentrated in the tropics and subtropics, where most countries had naturalized palm species (Figure 2a). However, the number of naturalized palms varied between continents. South America (including the Caribbean Islands) was the recipient of the most naturalized species (46 spe- cies), followed by tropical Asia (31), the Pacific islands (29), Africa (22), North America (22), temperate Asia (19), Australasia (14) and Europe (6). Of the 86 naturalized palm species, 64 species were nat- uralized outside their native continent (Table 1). Globally, the most widespread naturalized palms were commercially important species, that is coconut palm (C. nucifera; 97 botanical countries), date palm (P. dactylifera; 57 bot. countries), African oil palm (Elaeis guineensis; 38 bot. countries) and the betel nut palm (Areca catechu; 37 bot. countries). 3.1 \| Inventory of non-native palms We found 96 palm species and one hybrid (Phoenix sp.) that were in- troduced outside their native ranges. Of those 96 casual species (of 52 genera), 86 species (of 48 genera) were naturalized in their new range, with 28 species (of 21 genera) also classified as invasive (Table 1). Of the total number of palm species (n = 2,557, Govaerts et al., 2015), 3.4% were naturalized in the new range and 1.1% were invasive. The majority of naturalized species originated from tropical Asia (33 species) and South America (18 species). These two regions are also natural palm diversity “hotspots” (Figure 2b), and for this reason, we expected more naturalized species to come from these continents relative to other regions with fewer native palms. Indeed, we found a strong positive correlation between the number of na- tive species on a continent and the number of naturalized species originating from that continent (Spearman's r = .78, n = 8, p = .023, Figure 3). Naturalized palm species originating from tropical Asia were the most widespread and were present on every continent except Europe (Figure 4). Most naturalized species in tropical Asian countries originated from other parts of tropical Asia. The conti- nental region receiving the largest number of naturalized palms was South America including the Caribbean, with most species originat- ing from tropical Asia and from areas elsewhere in South America (Figure 4). In contrast to the records of species originating from trop- ical Asia and South America, the large number of naturalized palm records of African and temperate Asian species (Figure 4) stemmed from only a few widespread species (i.e. Livistona chinensis, P. dac- tylifera and Trachycarpus fortunei from temperate Asia; Dypsis lute- scens, E. guineensis and Phoenix canariensis from Africa). All five subfamilies included at least one casual species. The sub- families Arecoidea, Coryphoideae and Nypoideae included signifi- cantly more naturalized species compared to the average relative frequency of naturalized species across the remaining subfamilies. 2.5 \| Anthropogenic drivers of non-native palms To determine the potential role of human-driven spread of non-na- tive palm species, we assigned four human use categories to each naturalized palm species: (a) ornamental, (b) food, (c) rattan (i.e. the flexible stem of climbing palms used for furniture, wickerwork etc.), and (d) other uses (e.g. fuel, building material, handcrafts, erosion protection and medicine). We only included human uses that are likely to have resulted in cultivation of palm species outside their native range. We thus ignored local uses of palms, as they generally do not contribute to an inter-continental range expansion of spe- cies outside their native habitat. The attribution of uses was mainly based on peer-reviewed papers. However, only few studies focused on the use of palms as ornamentals (e.g. Broschat, Elliott, & Hodel, 2014). To fill in these gaps, we relied on grey literature, personal ob- servations and discussions with palm experts. FEHR et al. FEHR et al. 1527 3.3 \| Functional traits of non-native palms FI G U native species Number of native palm species that became naturalised outside its native range Naturalized palm species were significantly taller (M = 16.94 m) than non-naturalized species (M = 8.43 m) (see Appendix S2: Tables S2.1 and S2.2 for the logistic regression coefficients). The proportion of species becoming naturalized was higher in palms originating in open habitats (20.8%) and in dry forests (20%) compared to palms origi- nating in moist forests (5.4%; Figure 6a). When grouping palms ac- cording to their native habitat type, naturalizing species were taller than non-naturalizing species in each habitat (Figure 6b). Stem height was positively correlated with stem diameter (Pearson's r = .67, n = 1,546, p < .001), blade length (Pearson's r = .38, n = 1,500, p < .001) and fruit length (Pearson's r = .27, n = 1,586, p < .001). The proportion of palm species with conspicuous fruit colour was not significantly different between naturalized (39.5%) and non-natural- ized palm species (41.1%; p = .822). 3.1 \| Inventory of non-native palms Species richness per region is shown using a log10 scale 5 10 15 20 Species richness (a) Naturalised palm distribution 1 10 100 Species richness (b) Native palm distribution FI G U R E 3 Strong positive correlation between the number of native palm species on a continent and the number of naturalized species originating from that continent (Spearman's r = .78, n = 8, p = .023) R = 0.78 , p = 0.023 0 10 20 30 0 400 800 1200 Number of native palm species Number of native palm species that became naturalised outside its native range 1528 \| F d (B s c a t o in G G p s 5 10 15 20 Species richness (a) Naturalised palm distribution 1 10 100 Species richness (b) Native palm distribution FEHR et al. 1528 1528 \| 5 10 15 20 Species richness (a) Naturalised palm distribution FEHR et al. FI G U R E 2 Naturalized and native palm distribution per TDWG level-3 countries (Brummitt, 2001). Dots represent small islands (based on TDWG level-3 countries). We used Mollweide equal- area projection. (a) Global distribution of the 86 naturalized palm species based on our collected records (including invasive species, excluding casuals). (b) Global native palm distribution based on Govaerts et al. (2015). Species richness per region is shown using a log10 scale (a) Naturalised palm distribution (b) Native palm distribution 1 10 100 Species richness (b) Native palm distribution FI G U R E 3 Strong positive correlation between the number of native palm species on a continent and the number of naturalized species originating from that continent (Spearman's r = .78, n = 8, p = .023) R = 0.78 , p = 0.023 0 10 20 30 0 400 800 1200 Number of native palm species Number of native palm species that became naturalised outside its native range R = 0.78 , p = 0.023 0 10 20 30 0 400 800 1200 Number of native palm species Number of native palm species that became naturalised outside its native range 3.1 \| Inventory of non-native palms The subfamily Coryphoideae with 37 species (7.3%, p < .001) con- tained the most naturalized species, followed by Arecoideae with 34 naturalized species (2.5%, p = .008) and Calamoideae with 14 TA B LE 1 Number of non-native palm species and percentages of all species in the palm family (n = 2,557, Govaerts et al., 2015) belonging to the three naturalization stages considering different distances to their native habitat Totala Outside native botanical continentb [n] [%] [n] [%] Casual species 96 3.8 69 2.7 Naturalized species 86 3.4 64 2.5 Invasive species 28 1.1 24 0.9 The tropics contained the majority of naturalized palms (80 species) while the extratropics (including the humid subtropics, Mediterranean climate regions and deserts) were acceptors of only 31 species, reflecting the native distribution of the palm family per climate region (Figure 5a). However, the proportion of naturalized palm species was on average higher in the extratropics than in the tropics, and variation of this proportion was stronger in extratropical aIncluding all reports of species reported to be spreading inside and/or outside the botanical countries and/or continent where the species is native. bIncluding only reports of species spreading outside the native botanical continent (TDWG level-1). bIncluding only reports of species spreading outside the native botanical continent (TDWG level-1). 1528 \| FEHR et al. FI G U R E 2 Naturalized and native palm distribution per TDWG level-3 countries (Brummitt, 2001). Dots represent small islands (based on TDWG level-3 countries). We used Mollweide equal- area projection. (a) Global distribution of the 86 naturalized palm species based on our collected records (including invasive species, excluding casuals). (b) Global native palm distribution based on Govaerts et al. (2015). Species richness per region is shown using a log10 scale 5 10 15 20 Species richness (a) Naturalised palm distribution 1 10 100 Species richness (b) Native palm distribution 1528 \| FEHR et al regions (Figure 5b). Naturalized palms were reported in forests (63 records), wetlands (35), anthropogenic areas (25), coastal areas (21), deserts (13), shrubland (6), grassland and savannas (3) and rocky areas (3). We found 248 and 319 records of naturalized palms on 3.3 \| Functional traits of non-native palms Naturalized palm species were significantly taller (M = 16.94 m) than non-naturalized species (M = 8.43 m) (see Appendix S2: Tables S2.1 and S2.2 for the logistic regression coefficients). FI G U R E 2 Naturalized and native palm distribution per TDWG level-3 countries (Brummitt, 2001). Dots represent small islands (based on TDWG level-3 countries). We used Mollweide equal- area projection. (a) Global distribution of the 86 naturalized palm species based on our collected records (including invasive species, excluding casuals). (b) Global native palm distribution based on Govaerts et al. (2015). Species richness per region is shown using a log10 scale 3.1 \| Inventory of non-native palms The proportion o species becoming naturalized was higher in palms originating in open habitats (20.8%) and in dry forests (20%) compared to palms origi nating in moist forests (5.4%; Figure 6a). When grouping palms ac cording to their native habitat type, naturalizing species were talle than non-naturalizing species in each habitat (Figure 6b). Stem heigh was positively correlated with stem diameter (Pearson's r = .67 n = 1,546, p < .001), blade length (Pearson's r = .38, n = 1,500 p < .001) and fruit length (Pearson's r = .27, n = 1,586, p < .001). The proportion of palm species with conspicuous fruit colour was no significantly different between naturalized (39.5%) and non-natural ized palm species (41.1%; p = .822). 3.4 \| Ecological, social and economic effects of palm invasions Effects of naturalized palms on receiving ecosystems were reported for 18 of 28 invasive species. For the remaining 10 species, effect were either not mentioned or unclear. In total, we found 73 cases (a case is defined as a unique combination of an invasive palm species, an affected taxonomic group or an ecosystem function, a botanical re gion [TDWG-level-2] and the desirability of the effect) where an inva sive palm was reported to affect a taxonomic group or an ecosystem FI G U R E 2 Naturalized and native palm distribution per TDWG level-3 countries (Brummitt, 2001). Dots represent small islands (based on TDWG level-3 countries). We used Mollweide equal- area projection. (a) Global distribution of the 86 naturalized palm species based on our collected records (including invasive species, excluding casuals). (b) Global native palm distribution based on Govaerts et al. (2015). 3.4 \| Ecological, social and economic effects of palm invasions For species with a native range that extends to more than one botanical continent (n = 8 spp.), the region in which the greatest proportion of the species' distribution range is located was Tropical Asia Temperate Asia Southern America Pacific Northern America Europe Australia Africa Tropical Asia Temperate Asia Southern America Pacific Northern America Europe Australasia Africa Region of origin Region of naturalisation Pacific Southern America Southern America Tropical Asia Tropical Asia Region of origin Region of naturalisation FI G U R E 4 Exchange of naturalized palm species between botanical continents (TDWG level-1 from Brummitt, 2001). Single species are counted multiple times in their region of origin if they have naturalized in multiple continents. For species with a native range that extends to more than one botanical continent (n = 8 spp.), the region in which the greatest proportion of the species' distribution range is located was selected as the region of origin ecosystem service provisioning, down- or upgrading the aesthetic landscape value, cost causation due to naturalized palm removal and posing a risk to human health. an invasive palm affected a taxonomic group or an ecosystem func- tion in an undesirable way (evaluated as such in the original source). Approximately half of the undesirable cases were based on strong ev- idence. Reported undesirable effects of palm invasions ranged from effects on a single species to effects across several trophic levels. Competition with the native flora and alteration of the physical struc- ture of the ecosystem were the most frequently reported effects. We found much fewer reports on effects on vertebrates, invertebrates and other ecosystem functions. However, our results illustrate that invasive palms were not only able to affect single species, but also can reduce the richness of an entire taxonomic group such as arthropods or birds and affect ecosystem functioning. In nine cases, naturalized palms were reported to have desirable ecological effects. an invasive palm affected a taxonomic group or an ecosystem func- tion in an undesirable way (evaluated as such in the original source). Approximately half of the undesirable cases were based on strong ev- idence. Reported undesirable effects of palm invasions ranged from effects on a single species to effects across several trophic levels. 3.4 \| Ecological, social and economic effects of palm invasions FI G U R E 3 Strong positive correlation between the number of native palm species on a continent and the number of naturalized species originating from that continent (Spearman's r = .78, n = 8, p = .023) FI G U R E 3 Strong positive correlation between the number of native palm species on a continent and the number of naturalized species originating from that continent (Spearman's r = .78, n = 8, p = .023) Effects of naturalized palms on receiving ecosystems were reported for 18 of 28 invasive species. For the remaining 10 species, effects were either not mentioned or unclear. In total, we found 73 cases (a case is defined as a unique combination of an invasive palm species, an affected taxonomic group or an ecosystem function, a botanical re- gion [TDWG-level-2] and the desirability of the effect) where an inva- sive palm was reported to affect a taxonomic group or an ecosystem function (Figure 7). The majority of the 73 cases were reported where regions (Figure 5b). Naturalized palms were reported in forests (63 records), wetlands (35), anthropogenic areas (25), coastal areas (21), deserts (13), shrubland (6), grassland and savannas (3) and rocky areas (3). We found 248 and 319 records of naturalized palms on islands and continental landmasses, respectively. FEHR et al. 1529 \| 1529 FEHR et al. FI G U R E 4 Exchange of naturalized palm species between botanical continents (TDWG level-1 from Brummitt, 2001). Single species are counted multiple times in their region of origin if they have naturalized in multiple continents. For species with a native range that extends to more than one botanical continent (n = 8 spp.), the region in which the greatest proportion of the species' distribution range is located was selected as the region of origin Tropical Asia Temperate Asia Southern America Pacific Northern America Europe Australia Africa Tropical Asia Temperate Asia Southern America Pacific Northern America Europe Australasia Africa Region of origin Region of naturalisation FI G U R E 4 Exchange of naturalized palm species between botanical continents (TDWG level-1 from Brummitt, 2001). Single species are counted multiple times in their region of origin if they have naturalized in multiple continents. 3.5 \| Anthropogenic drivers of non-native palms b) Proportion of naturalized palm FI G U R E 5 (a) Number of native palm species in tropical and extratropical TDWG level-3 countries. b) Pro FI G U R E 5 (a) Number of native palm species in tropical and extratropical TDWG level-3 countries. b) Proportion of naturalized palm species of the total number of palm species (native + naturalized) in tropical and extratropical TDWG level-3 countries. (c) Proportion of naturalized palm species against the total number of palm species (native + naturalized) in TDWG level-3 countries as a function of the human footprint index (HFI) from 1993 from Venter et al. (2016). Low HFI values represent a low level of urbanization; high values represent a high level of urbanization. The black line shows the logistic regression curve (for logistic regression coefficients, see Table S2.4 in Appendix S2). Only TDWG level-3 countries with native palms (black points) were included in the logistic regression analysis FI G U R E 6 (a) Proportion of naturalized and non-naturalized species for each native habitat type. (b) Kernel density estimate of the stem height distribution of naturalized and non-naturalized species for three native habitat types 0 25 50 75 100 Open habitat (n = 72) Dry forest (n = 120) Moist forest (n = 860) Habitat type of the native range Proportion of species [%] Non−naturalised Naturalised Open habitat (n = 72) Dry forest (n = 120) Moist forest (n = 860) 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0.000 0.025 0.050 0.075 0.100 Stem height [m] Density (a) (b) Open habitat (n = 72) Dry forest (n = 120) Moist forest (n = 860) 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0.000 0.025 0.050 0.075 0.100 Stem height [m] Density (b) 0 25 50 75 100 Open habitat (n = 72) Dry forest (n = 120) Moist forest (n = 860) Habitat type of the native range Proportion of species [%] Non−naturalised Naturalised (a) (b) Moist forest (n = 860) Density Stem height [m] FI G U R E 6 (a) Proportion of naturalized and non-naturalized species for each native habitat type. (b) Kernel density estimate of the stem height distribution of naturalized and non-naturalized species for three native habitat types 3.5 \| Anthropogenic drivers of non-native palms The most common human use of naturalized palms was cultivation as ornamental plants (61 species). A smaller number of naturalized palms are or have been cultivated for food (20), rattan (8) or other uses (13). For nine species, the reason for their introduction outside their native range was unclear, as no uses are known. The uses of all the naturalized palms are presented in Appendix S2: Table S2.3. Reported social and economic effects of naturalized palms ap- peared limited as were only reported for six species and included We found a positive relationship between the proportion of naturalized palm species relative to the total pool of palms 1530 \| FEHR et al. FI G U R E 5 (a) Number of native palm species in tropical and extratropical TDWG level-3 countries. b) Proportion of naturalized palm species of the total number of palm species (native + naturalized) in tropical and extratropical TDWG level-3 countries. (c) Proportion of naturalized palm species against the total number of palm species (native + naturalized) in TDWG level-3 countries as a function of the human footprint index (HFI) from 1993 from Venter et al. (2016). Low HFI values represent a low level of urbanization; high values represent a high level of urbanization. The black line shows the logistic regression curve (for logistic regression coefficients, see Table S2.4 in Appendix S2). Only TDWG level-3 countries with native palms (black points) were included in the logistic regression analysis 1 10 100 extratropical tropical Number of native palm species 0.00 0.25 0.50 0.75 1.00 extratropical tropical Prop. of naturalised palm species 0.00 0.25 0.50 0.75 1.00 10 20 30 40 Human Footprint Index Prop. of naturalised palm species Countries with native palms Countries without native palms (a) (b) (c) FEHR et al. 0.00 0.25 0.50 0.75 1.00 extratropical tropical Prop. of naturalised palm species (b) \| 1 10 100 extratropical tropical Number of native palm species (a) 0.00 0.25 0.50 0.75 1.00 10 20 30 40 Human Footprint Index Prop. of naturalised palm species Countries with native palms Countries without native palms (c) Number of native palm specie Prop. of naturalised palm s Human Footprint Index Countries with native palms Countries without native palms r of native palm species in tropical and extratropical TDWG level-3 countries. Desirability and evidence FI G U R E 7 Effects of palm invasions on taxonomic groups and ecosystem functions. The y-axis shows the number of cases where an invasive palm species was reported to affect a taxonomic group or an ecosystem function within a botanical region (TDWG-level-2). Effects are classified as desirable or undesirable based on the evaluation in the original source. For every effect, the level of evidence is indicated (strong [experimental] or weak [anecdotal or observational]) FI G U R E 7 Effects of palm invasions on taxonomic groups and ecosystem functions. The y-axis shows the number of cases where an invasive palm species was reported to affect a taxonomic group or an ecosystem function within a botanical region (TDWG-level-2). Effects are classified as desirable or undesirable based on the evaluation in the original source. For every effect, the level of evidence is indicated (strong [experimental] or weak [anecdotal or observational]) urbanization and climate change will likely promote novel ecosys- tems generated by palm invasions in the future. naturalized palm species was higher in the extratropics than in the tropics because the number of native species in the extratropics is on average very low but the horticultural demand for having a variety of different species is similar in both climatic regions. Thus, the num- ber of species that were introduced by humans is not proportional to the number of native species in the tropics and the extratropics. Naturalized palms were reported in a wide variety of habitats in- cluding forests, wetlands, anthropogenic habitats, coastal areas and deserts, indicating that many regions of the world could potentially be colonized by non-native palms. Likely more habitats per species would be suitable for naturalized palms than were reported. Islands can be considered major receivers of naturalized palms, with 42.6% of the naturalized palm records coming from islands, especially when taking into account their smaller area compared to mainlands. Islands are known to be more prone to plant invasions than mainlands likely due to their open niche space, increased ecological naiveté of na- tive biota or simply higher numbers of introductions on islands (van Kleunen et al., 2015; Moser et al., 2018). 4.1 \| Distribution and biogeography of non- native palms We found that the palm family has a higher proportion of invasive species (1.1%, 28 species) than the global pool of trees and shrubs (0.5%–0.7%, Richardson & Rejmánek, 2011), suggesting that the palm family is a major source of invasive woody plants within their climatic range (Rejmánek, 2014). South America (including the Caribbean Islands), tropical Asia and the Pacific islands were the major receivers of naturalized palms, likely because the climate of these continents matches the climatic requirements of many palm species. The number of naturalized species in Africa was lower, even though large regions of Africa are climatically suitable for palms, possibly reflecting lower propagule pressure of non-native palms due to a lower degree of ur- banization. However, historical and cultural factors (e.g. colonization history) are known to strongly influence the distribution of non-na- tive plants and data availability varies considerably between regions (Richardson & Rejmánek, 2011), making it difficult to interpret the geographical distribution of non-native palms. The largest number of naturalized species originated from tropical Asia and South America, reflecting the high number of native species on these continents. Overall, the number of naturalized species originating from a conti- nent was proportional to the continents’ number of native species, suggesting that there are likely no continent-specific characteristics of palms promoting or preventing their naturalization. 4 \| DISCUSSION (native + naturalized species) and the human footprint index (HFI) on a TDWG-level-3 country level (Figure 5c, for logistic regression coefficients, see Table S2.4 in Appendix S2). Nagelkerke's R2 showed that HFI explains 25% of the proportion of naturalized palms within TDWG level-3 country. The association between harbouring natu- ralized palm species and human footprint is further illustrated by the fact that every TDWG level-3 country with a HFI > 20 had at least one naturalized palm species (Figure 5c). These findings strongly support our hypothesis that urbanization positively influences palm invasions, if the environmental conditions of a region match with the requirements of palms. Our extensive literature review revealed that a relatively large pro- portion of the palm family has naturalized (3.4%, 86 species) or become invasive (1.1%, 28 species) in a wide variety of habitats in the tropics, subtropics and warm temperate regions. We found that naturalized palms were taller than non-naturalized palms and were more likely to originate from open habitats or dry forest than non- naturalized palms. The number of reported effects of naturalized palms was limited, but we found strong evidence that non-native palms can generate novel ecosystems in some contexts. Increased \| 1531 FEHR et al. FI G U R E 7 Effects of palm invasions on taxonomic groups and ecosystem functions. The y-axis shows the number of cases where an invasive palm species was reported to affect a taxonomic group or an ecosystem function within a botanical region (TDWG-level-2). Effects are classified as desirable or undesirable based on the evaluation in the original source. For every effect, the level of evidence is indicated (strong [experimental] or weak [anecdotal or observational]) Taxonomic group Ecosystem function Plants MammalsInvertebrats Birds Aquatic fauna Reptiles Physical structure Water regime Soil nutrients Soil moisture Temperature regime Fire regime 0 5 10 15 20 25 Number of cases of effects of invasive palms Desirability and evidence Desirable, weak evidence Desirable, strong evidence Undesirable, weak evidence Undesirable, strong evidence 1531 FEHR et al. Taxonomic group Ecosystem function Plants MammalsInvertebrats Birds Aquatic fauna Reptiles Physical structure Water regime Soil nutrients Soil moisture Temperature regime Fire regime 0 5 10 15 20 25 Number of cases of effects of invasive palms Taxonomic group Ecosystem function Aquatic fauna Physical structure Soil moisture Soil nutrients Desirability and evidence 4.3 \| Do non-native palm invasions generate novel ecosystems? We found support for the hypothesis that non-native palms generate novel ecosystems. For example, the coconut palm (C. nucifera) inva- sion of a Pacific island triggered a trophic cascade (Young, Miller-ter Kuile, McCauley, & Dirzo, 2017). Because birds did not nest on the non-native coconut palms, reduced nutrient input by guano altered plant community composition, which in turn affected herbivorous invertebrate communities. In another example of ecosystem ef- fects, the Chinese windmill palm (T. fortunei) has transformed the understory of deciduous forests in Southern Switzerland into ev- ergreen, palm-dominated thickets, which may classify as a biome shift (Figure 1d, Fehr & Burga, 2016). Further evidence for the abil- ity of palms to generate novel ecosystems includes the modification of habitats by changing the physical habitat structure (e.g. Talley, Nguyen, & Nguyen, 2012; Young, McCauley, Pollock, & Dirzo, 2014), by altering key ecosystem functioning like the water regime (e.g. flow reduction of desert springs [Furler & Willing, 2006] or reduction of plant-available water [Young et al., 2017]), by inducing nutrient de- pletion (Young, McCauley, Dunbar, & Dirzo, 2010) or by triggering wildfires (DiTomaso & Kyser, 2013; Ortiz-Uribe, Salomón-Torres, & Krueger, 2019). Such habitat modifications can lead to cascading com- munity effects and result in a strong ecosystem change (Buma, 2015). We found that palm species associated with open habitats and dry forests are more likely to naturalize than palms occurring in moist forest. Palms from these habitats may have traits that favour rapid establishment within a broad range of environmental conditions (e.g. physiological traits promoting fast growth and drought tolerance) and would be pre-adapted to tolerate the high light levels and often rela- tively dry conditions in urban or other anthropogenic environments. Some palms have been reported to flourish in their native range as a result of human-induced disturbances (Smith, 2015). The expansion of anthropogenically disturbed habitats in the tropics due to ongoing deforestation would be expected to open up niche space for non-na- tive palms adapted to open or disturbed habitats. Two traits, long-lived seeds and remote germination may help palms of the Coryphoideae subfamily to establish in anthropogenic dry habitats, explaining why this subfamily is particularly over-represented among naturalizing palms. The longer-lived seeds in coryphoid palms relative to other subfamilies (Jones, 1995) are likely an adaption to climate seasonality (Casas et al., 2017) and might facilitate establishment in disturbed habi- tats, creating seedbanks persisting several months. This may be because other dis- persal agents and mechanism override the effect of bird dispersal, for example because some palm fruits are too big for extant birds (Furler & Willing, 2006; NSW Department of Planning, Industry, & Environment, 2019). Fruit size was indeed weakly positively corre- lated with stem height, indicating that naturalized palms have rather larger fruits than non-naturalized palms. environments and climates, is coupled to selection criteria for or- namental palms. We expect this to have a strong influence on nat- uralization patterns, as it is well known that traits that make plants attractive for horticulture also promote naturalization (van Kleunen et al., 2018), and, as we found here, the majority of naturalized palms were introduced for ornamental purposes. Notably, a complemen- tary explanation for the height effect on naturalization could be that tall palms are more likely to be introduced outside their native range, as they are used in higher numbers in horticulture than short palms due to the popularity of large palms in landscaping (V. Fehr & J.-C. Svenning, personal observation). Overall, we expect an increase in the number of naturalized palm species as new species are being introduced to horticulture on a frequent basis. Knowing which traits are over-represented in naturalized palms will be crucial for predict- ing the naturalization potential of new species in cultivation. competitors for light and strong dispersers. We found that leaf size is positively correlated with height in palms likely resulting in a compet- itive advantage, with naturalized palms frequently reported to shade out native plants due to their large leaves (Ishii, Ichinose, Ohsugi, & Iwasaki, 2016; Starr, Starr, & Loope, 2003; Williams, 2008). We suspect that the capacity of tall palms to produce more propagules than smaller palms is likely favouring tall palms to naturalize more frequently as it is well known that tall plants usually exhibit a higher fecundity leading to increased propagule pressure, likely result- ing in a competitive advantage (Jelbert et al., 2015; van Kleunen et al., 2010). Further, naturalization also depends on the dispersal ability of the species. Greater height increases the dispersal ability of plants as tall plants yield greater fecundity, resulting in an increased dispersal probability (Jelbert et al., 2015) and because greater height promotes greater dispersal distance (Thomson et al., 2011). Small fruit size and conspicuous fruit colour are known to favour the short-distance dispersal by birds (Levey, 1987; Schmidt, Schaefer, & Winkler, 2004). Although many naturalized palm species are dis- persed by birds (e.g. Ishii et al., 2016; Mengardo & Pivello, 2012; Meyer et al., 2008; Spennemann, 2019), we did not find evidence that fruits with conspicuous colours are more frequent among natu- ralized palm species as expected. 4.2 \| Functional traits of non-native palms To become naturalized, a species has to overcome certain barriers, including survival, reproduction, dispersal and environmental barri- ers (Blackburn et al., 2011). The capacity of a species to overcome these barriers depends strongly on plant functional traits such as size and fecundity (Jelbert et al., 2015; van Kleunen et al., 2010). Our data show clearly that naturalized palms are on average taller than non-naturalized palms. This finding is consistent with the pattern found for plants in general that invasive plant species are larger than their non-invasive relatives (Jelbert et al., 2015). Height can promote naturalization in various ways. Tall palms were expected to be strong The tropics contained a larger number of naturalized palm species (80 species) than the extratropics (31 species) reflecting the native distribution of palms per climate region. However, the proportion of 1532 FEHR et al. FEHR et al. 4.5 \| Future of palm-generated novel ecosystems We expect that increased urbanization (United Nations, Department of Economic and Social Affairs, Population Division, 2017) and as- sociated horticultural trade will promote future palm invasions and thus the emergence of novel ecosystems, especially in regions with- out (many) native palms. On a country-level scale, we found strong evidence that most palm invasions occur within or close to urbanized regions, that is every country with native palms and areas with a high human footprint index (HFI > 20) had at least one naturalized palm species (Figure 5c). This is most likely due to the high density of gardens and parks that host a large number of cultivated, orna- mental palms in urbanized areas and therefore propagule pressure is high. This is consistent with the results of van Kleunen et al. (2018), who showed that between 75% and 93% of the global naturalized alien flora is grown in domestic and botanical gardens, which are typically located in urbanized regions. Given the long generation time of palms, long lags between the first introduction and subse- quent spread are expected. The four most widespread naturalized palm species (coconut palm, date palm, African oil palm and betel nut palm) are all agricultural crops that have been cultivated for cen- turies (Harries, 1978; Mathew et al., 2015) compared to many or- namental palm species that have been disseminated more recently, again illustrating the importance of time but also propagule pressure in the process of naturalization of palms. Overall, we expect a strong increase in the reports of palm invasions in the near future, as many regions of the world may still be within this lag phase and propagule pressure will likely increase due to increased urbanization. Besides the reported ecological effects of naturalized palms, there is evidence of palms acting as ecosystem engineers within their native range through various ways. For example, palm leaves can re- duce the light availability (Farris-Lopez et al., 2004), senescent falling palm leaves can cause disturbances in the forest understory (Peters, Pauw, Silman, & Terborgh, 2004) and the slow decomposition of palm leaves can result in thick litter layers hindering the establishment of small-seeded (tree-) species (Farris-Lopez et al., 2004). Schlaepfer, 2020; Warren, King, Tarsa, Haas, & Henderson, 2017). This biased view emerged as invasion biologists traditionally focus on non-native species perceived to pose a threat to ecosystems or society to inform policy, in order to guide potential management actions (Davis, 2009). The focus of invasion biologists on undesir- able effects might also explain the paucity of reported desirable ecosystem effects of naturalized palms. However, we found lim- ited evidence of non-native palms benefiting the invaded ecosys- tem by creating habitats and providing food for the native biota and contributing to a higher biodiversity. For example, in Brazil's Atlantic rain forest, the long-term survival of endangered primates depends on the non-native oil palm (E. guineensis) (Canale, Kierulff, & Chivers, 2013; de Jesus Rodrigues Malta & Pontes, 2013). Overall, we expect that desirable ecosystem effects of natural- ized palms are more common, especially cases where naturalized palms benefit native frugivores, as many palms are part of mutu- alistic relationships with frugivores in their native range (Galetti et al., 2001; Henderson, 2002; Sica et al., 2014). of P. canariensis can cause injuries that often have a protracted course and multiple surgical procedures may be required to remove all of the foreign material (Adams, Timms, & Hanlon, 2000). Further, the pollen of the same species has been reported to cause asthma and rhinoconjunctivitis (Blanco et al., 1995). The only substantial economic costs of non-native palms that we found reported were costs for controlling or eradicating non-native palms. 4.5 \| Future of palm-generated novel ecosystems Palms are widely hyper-dominant members of their ecosystems, for example in the Amazonian rain forest, where 227 tree species (1.4%) account for 50% of all stems, with 15 of these species being palms, nearly five times as many hyper-dominant species for the family than expected by chance (Steege et al., 2013). It is likely that naturalized palms have the ability to affect the invaded ecosystem through similar pathways. In summary, considering the ecological key role of palms within their native ecosystem and the few, but strong, examples of non-na- tive palms as ecosystem engineers, we expect that ecosystem effects of palm invasions are far more diverse and that many mecha- nisms through which they can affect native biota or ecosystem func- tioning are still unknown (Svenning, 2018). From a biogeographic perspective, we expect that palm invasions will increase around the globe in tropical, subtropical and warm tem- perate regions. Areas with exceptionally high diversity of natural- ized palms might become more frequent in the tropics in the future (Svenning, 2002). In the subtropics and temperate regions, the nat- uralized palm richness is unlikely to increase strongly, as the pool of species adapted to cooler, more seasonal climate is relatively small. 4.3 \| Do non-native palm invasions generate novel ecosystems? In remote germina- tion, the radicle is pushed down deep before sending up the first leaves (Broschat et al., 2014). As a result, the meristem and the sensitive zone where the roots emerge are located deeper in the soil, an adaption that is likely advantageous in drought-prone settings (Pinheiro, 2001). However, direct measurements of ecosystem effects of non-native palms were scarce, and for more than one third of the 28 invasive palm species, no ecological effects were reported. This is surprising, as invasive species by definition should have nega- tive effects on ecosystems and native species. The lack of data on ecosystem effects may partly stem from logistical challenges in generating such data for long-lived species (Stricker, Hagan, & Flory, 2015). However, we suspect that several naturalizing palm species are incorrectly classified as invasive, for example, be- cause palms are highly noticeable and there is a tendency among biologists to regard non-native biota in general as negative for the native biodiversity (Davis et al., 2011; Gbedomon, Salako, & The abundance of certain traits in naturalizing palms, such as size, fast growth, ease of propagation and good performance in different FEHR et al. 1533 4.6 \| Management of non-native palms Where non-native palms are perceived as negative, various manage- ment approaches have been implemented. However, information on control techniques for invasive palms is scarce (Dyer et al., 2019; Enloe, Langeland, Ferrell, Sellers, & MacDonald, 2018) and only scattered cases of local attempts to control invasive palms exist. In Southern California, Washingtonia robusta is removed at signifi- cant costs (removal with helicopter; Anderson & Castellanos, 2014; DiTomaso & Kyser, 2013), despite no strong evidence for negative bio- diversity effects or additional economic costs (National Park Service & U.S. Department of the Interior, 2015). The economic investment into eradicating W. robusta is even more surprising as it is native only 350 km further south, and the closely related Washingtonia filifera occurs only 80 km to the east (Minnich, Franco-Vizcaíno, & Salazar- Ceseña, 2011). In Indonesia, the invasive arenga palm (Arenga obtusi- folia) is controlled by both cutting the palm down and using herbicide in an attempt to restore the habitat for the Javan rhino (Rhinoceros sondaicus) (Konstant, 2014). In outhern Switzerland, the Chinese windmill palm (T. fortunei) is cut down regularly in certain areas as it is suspected to negatively affect the native flora and the protective forest to prevent erosion (Info Flora, 2014). We suggest that manage- ment should be based on careful assessments of the extent to which Novel ecosystems are on the rise, in large part driven by in- troductions of non-native species, but also promoted by ongoing global warming, increased atmospheric carbon dioxide (CO2) and land use change (Feng et al., 2014; Hobbs et al., 2009; Perring & Ellis, 2013). The new species assemblages and ecosystem func- tions of these novel ecosystems are likely to alter ecosystem ser- vices (Collier, 2014; Evers et al., 2018; Hobbs et al., 2009; Millenium Ecosystem Assessment, 2005) may threaten native species (Bellard, Genovesi, & Jeschke, 2016; Pyšek et al., 2012) but could also gen- erate new habitats for native species (Padovani, Salisbury, Bostock, Roy, & Thomas, 2020). An improved understanding of the func- tioning of novel ecosystems to these increasingly widespread nat- ural environments should clearly be an important research priority (e.g. Svenning, 2018). Further, to restore these novel ecosystems to a historical baseline would in many cases be an unfeasible task (Hobbs et al., 2009), for example due to climate change (Ordonez & Svenning, 2016). 4.7 \| Knowledge gaps and the way forward Factors that have been hypothesized to regulate many plant in- vasions are diseases and pathogens. Invasive species are suggested to become less invasive over time as they accumulate pests and pathogens in their new locations (Flory & D’Antonio, 2015). We are aware of one example in Southern California, where a population of naturalized P. canariensis was infested and decimated by (non-native) palm weevils (Milosavljević et al., 2019; V.Fehr, personal observation, Figure 1f). Considering the large number of diseases and pests that can affect palms (Gitau, Gurr, Dewhurst, Fletcher, & Mitchell, 2009), cases where naturalized palms are infested by diseases and pests are likely to increase in the future and eventually benefit native plants. Hence, such ecological dynamics may increase the biodiversity of novel ecosystems in the long term (Svenning, 2018), similar to what has been achieved through the introduction of biological control agents in cases such as Opuntia spp. in Australia (Novoa et al., 2019). Our review revealed important knowledge gaps. First, data on non- native palms were geographically and taxonomically biased, suggest- ing potential under-reporting of non-native palms. Second, studies on ecological, social and economic effects of non-native palms were scarce and available studies focused on palm effects on specific taxa rather than on ecosystem-level effects. Based on the ecological im- portance of palms within their native range, we anticipated that de- sirable ecosystem effects, that is where non-native palms benefit the native biota and contribute to a higher biodiversity, could be common. However, we did not find many reported desirable effects, which could reflect that such outcomes of novel ecosystem formation may simply be underexplored due to a bias in non-native species research towards focusing on undesirable effects. Hence, there is a strong need for more field-based studies aiming to uncover if and how non-native palms modify habitats and affect key ecosystem functioning and bio- diversity in different climate and land use settings. Here, it will be im- portant to address the temporal dynamics and spatial scaling of the ecological effects, as effects that are locally negative for other species could be transient or lead to higher biodiversity at larger scales. the non-native species cause major net negative ecological or social effects and if control is likely to be effective in the long-term, nota- bly to not waste scarce resources for environmental management (Svenning, 2018). Given the over-representation of open habitat- associated palms among the naturalizing species, reducing human disturbance may be in many cases an effective long-term strategy. In some cases, expansions could also reflect defaunation, notably losses of large herbivores (Ripple et al., 2015) that could otherwise limit palm populations (Mandle & Ticktin, 2012; Watson, Cameron, & Iifo, 2020), and here trophic rewilding (Svenning et al., 2016) could be a long-term solution (cf. Guyton et al., 2020). Dransfield, Balslev, & Svenning, 2013; Kissling et al., 2012). If this is true, ongoing urbanization and associated horticultural activity should increase palm propagule pressure and thus lead to more palm invasions in Africa. Ongoing global warming will promote the spread of non-na- tive palm species in the subtropics, warm temperate regions of the Northern Hemisphere (>30°N) and higher elevations (Reichgelt et al., 2018). The expansion of the Chinese windmill palm (T. for- tunei) in Southern Switzerland (Figure 1d) is an example where global warming expanded palm niche space, increased urbanization increased propagule pressure and less intensive forest manage- ment allowed establishment (Conedera, Wohlgemuth, Tanadini, & Pezzatti, 2018; Fehr & Burga, 2016; Walther et al., 2007). 4.4 \| Social and economic effects of palm invasions We found six studies that provided information on the social and economic effects of naturalized palms, which is surprisingly scarce. Palms are valued for aesthetic reasons, as evidenced by a lively horticultural trade in palms (Broschat et al., 2014), but sometimes also by the appreciation of wild naturalized palms (Lindemann- Matthies, 2016). In contrast, naturalized palms can also be perceived as an undesirable, foreign landscape element (Williams, 2008). In one case, naturalized palms were suggested to provide ecosystem services including food, fibres and wood to local communities (de Grenade, 2013). One palm species was reported as posing a risk to human health (albeit likely a small one): The barbs on the leaf bases Some subtropical and warm temperate climates are currently able to support palms, but have no native palms, for example, due to past extinctions during ice ages or post-glacial dispersal limita- tion (Kissling et al., 2012), as is the case for the Southern European Alps where the Chinese windmill palm (T. fortunei) is spreading. This unoccupied niche space for palms might further facilitate palm inva- sions. Open niches for non-native palms might also be available in Africa where palm richness is low, potentially due to climate change during the Neogene and Quaternary (Blach-Overgaard, Kissling, 1534 FEHR et al. FEHR et al. REFERENCES Davis, M. A. (2009). Invasion biology. Oxford, UK: Oxford University Press. Adams, C. D., Timms, F. J., & Hanlon, M. (2000). Phoenix date palm injuries: A review of injuries from the Phoenix date palm treated at the Starship Children’s Hospital. The Australian and New Zealand Journal of Surgery, 70, 355–357. https://doi.org/10.1046/j.1440-1622.2000.01823.x Davis, M. A., Chew, M. K., Hobbs, R. J., Lugo, A. E., Ewel, J. J., Vermeij, G. J., … Briggs, J. C. (2011). Don’t judge species on their origins. Nature, 474, 153–154. https://doi.org/10.1038/474153a Anderson, E., & Castellanos, M. (2014). Helicopter Used To Remove 60 Palm Trees From Tierrasanta Canyon. Retrieved from Kpbs Website https://www.kpbs.org/news/2014/dec/29/environmental-rescu e-tierrasanta-canyon/ de Casas, R. R., Willis, C. G., Pearse, W. D., Baskin, C. C., Baskin, J. M., & Cavender-Bares, J. (2017). Global biogeography of seed dormancy is determined by seasonality and seed size: A case study in the le- gumes. New Phytologist, 214, 1527–1536. https://doi.org/10.1111/ nph.14498 Baker, W. J., & Dransfield, J. (2016). Beyond Genera Palmarum: Progress and prospects in palm systematics. Botanical Journal of the Linnean Society, 182, 207–233. de Grenade, R. (2013). Date palm as a keystone species in Baja California peninsula, Mexico oases. Journal of Arid Environments, 94, 59–67. https://doi.org/10.1016/j.jaridenv.2013.02.008 Bellard, C., Genovesi, P., & Jeschke, J. M. (2016). Global patterns in threats to vertebrates by biological invasions. Proceedings of the Royal Society B: Biological Sciences, 283, 20152454. de Jesus Rodrigues Malta, A., & Pontes, A. R. M. (2013). The simplified novel diet of the highly threatened blond capuchin in the vanishing Pernambuco Endemism Center. In L. K. Marsh, & C. A. Chapman (Eds.), Primates in fragments: Complexity and resilience (pp. 245–257). New York, NY: Springer. Blach-Overgaard, A., Kissling, W. D., Dransfield, J., Balslev, H., & Svenning, J.-C. (2013). Multimillion-year climatic effects on palm species diversity in Africa. Ecology, 94, 2426–2435. https://doi. org/10.1890/12-1577.1 DiTomaso, J. M., & Kyser, G. B. (2013). Weed control in natural areas in the western United State. Weed Research and Information Center, University of California. Blackburn, T. M., Pyšek, P., Bacher, S., Carlton, J. T., Duncan, R. P., Jarošík, V., … Richardson, D. M. (2011). A proposed unified framework for bi- ological invasions. Trends in Ecology & Evolution, 26, 333–339. https:// doi.org/10.1016/j.tree.2011.03.023 Dransfield, J., & Uhl, N. W. (2008). Genera Palmarum: The evolution and classification of palms, Kew, UK: Kew Pub. Blanco, C., Carrillo, T., Quiralte, J., Pascual, C., Martin Esteban, M., & Castillo, R. (1995). Byg, A., & Balslev, H. (2001). Diversity and use of palms in Zahamena, eastern Madagascar. Biodiversity & Conservation, 10, 951–970. PEER REVIEW The peer review history for this article is available at https://publo ns.com/publon/10.1111/ddi.13150. Canale, G. R., Kierulff, M. C. M., & Chivers, D. J. (2013). A critically endan- gered capuchin Monkey (Sapajus xanthosternos) living in a highly frag- mented hotspot. In L. K. Marsh, & C. A. Champman (Eds.), Primates in fragments: Complexity and resilience (pp. 299–311). New York, NY: Springer. 4.6 \| Management of non-native palms Hence, adapting conservation and restoration prac- tices to promote the biodiversity value of these novel ecosystems may be the most feasible approach in many cases (Svenning, 2018). FEHR et al. 1535 ORCID Vincent Fehr https://orcid.org/0000-0002-9609-6873 Robert Buitenwerf https://orcid.org/0000-0003-3356-2301 Jens-Christian Svenning https://orcid. org/0000-0002-3415-0862 Vincent Fehr https://orcid.org/0000-0002-9609-6873 Robert Buitenwerf https://orcid.org/0000-0003-3356-2301 Jens-Christian Svenning https://orcid. org/0000-0002-3415-0862 Craft, P., Riffle, R. L., & Zona, S. (2014). The encyclopedia of cultivated palms, Portland, OR: Timber Press. DAISIE (2018). DAISIE European invasive alien species gateway. Retrieved from http://www.europe-aliens.org/speciesFactsheet.do?speciesId=2699# ACKNOWLEDGEMENTS We thank VILLUM FONDEN for economic support through the VILLUM Investigator project “Biodiversity Dynamics in a Changing World” (grant 16549, to J.-C.S.). We thank VILLUM FONDEN for economic support through the VILLUM Investigator project “Biodiversity Dynamics in a Changing World” (grant 16549, to J.-C.S.). Byg, A., & Balslev, H. (2006). Palms in indigenous and settler communities in southeastern Ecuador: Farmers’ perceptions and cultivation prac- tices. Agroforestry Systems, 67, 147–158. https://doi.org/10.1007/ s10457-005-1704-1 Campos, M. T., & Ehringhaus, C. (2003). Plant virtues are in the eyes of the beholders: A comparison of known palm uses among indigenous and folk communities of Southwestern Amazonia. Economic Botany, 57, 324. DATA AVAILABILITY STATEMENT The literature-based dataset on non-native palms used in this study can be found in Appendix S3. All other data used are available pub- licly from the sources given in the manuscript. Collier, M. J. (2014). Novel ecosystems and the emergence of cultural ecosystem services. Ecosystem Services, 9, 166–169. https://doi. org/10.1016/j.ecoser.2014.06.002 ORCID Conedera, M., Wohlgemuth, T., Tanadini, M., & Pezzatti, G. B. (2018). Drivers of broadleaved evergreen species spread into deciduous for- ests in the southern Swiss Alps. Regional Environmental Change, 18, 425–436. https://doi.org/10.1007/s10113-017-1212-7 Frugivory and seed dispersal by the lowland tapir (Tapirus terrestris) in Southeast Brazil. Biotropica, 33, 723–726. Lugo, A. E. (2004). The outcome of alien tree invasions in Puerto Rico. Frontiers in Ecology and the Environment, 2, 265–273. Gbedomon, R. C., Salako, V. K., & Schlaepfer, M. A. (2020). Diverse views among scientists on non-native species. NeoBiota, 54, 49–69. https:// doi.org/10.3897/neobiota.54.38741 Mandle, L., & Ticktin, T. (2012). Interactions among fire, grazing, har- vest and abiotic conditions shape palm demographic responses to disturbance. Journal of Ecology, 100, 997–1008. https://doi. org/10.1111/j.1365-2745.2012.01982.x Gitau, C. W., Gurr, G. M., Dewhurst, C. F., Fletcher, M. J., & Mitchell, A. (2009). Insect pests and insect-vectored diseases of palms. Australian Journal of Entomology, 48, 328–342. https://doi. org/10.1111/j.1440-6055.2009.00724.x Martins, R. C., de Filgueiras, T., & Albuquerque, U. P. (2014). Use and diversity of palm (Arecaceae) resources in central western Brazil. The Scientific World Journal, 2014, 942043. https://doi. org/10.1155/2014/942043 Govaerts, R., Dransfield, J., Hodel, D. R., & Henderson, A. (2015). World checklist of Arecaceae. Facilitated by the Royal Botanic Gardens, Kew. Published on the Internet. Retrieved from http://wcsp.science.kew.org Mathew, L. S., Seidel, M. A., George, B., Mathew, S., Spannagl, M., Haberer, G., … Malek, J. A. (2015). A genome-wide survey of date palm cultivars supports two major subpopulations in Phoenix dactylif- era. G3: Genes, Genomes, Genetics, 5, 1429–1438. Guisan, A., & Zimmermann, N. E. (2000). Predictive habitat distribution models in ecology. Ecological Modelling, 135, 147–186. https://doi. org/10.1016/S0304-3800(00)00354-9 Gunn, B. F., Baudouin, L., & Olsen, K. M. (2011). Independent origins of cultivated coconut (Cocos nucifera L.) in the old world tropics. PLoS One, 6, e21143. Mengardo, A. L. T., & Pivello, V. R. (2012). Phenology and fruit traits of Archontophoenix cunninghamiana, an invasive palm tree in the Atlantic forest of Brazil. Ecotropica, 18, 45–54. Guyton, J. A., Pansu, J., Hutchinson, M. C., Kartzinel, T. R., Potter, A. B., Coverdale, T. C., … Pringle, R. M. (2020). Trophic rewilding revives biotic resistance to shrub invasion. Nature Ecology & Evolution, 4, 712–724. https://doi.org/10.1038/s41559-019-1068-y Meyer, J. Y., Lavergne, C., & Hodel, D. R. (2008). Time bombs in gar- dens: Invasive ornamental palms in tropical islands, with emphasis on French Polynesia (Pacific Ocean) and the Mascarenhas (Indian Ocean). Palms, 52, 71–83. Harries, H. C. (1978). The evolution, dissemination and classification of Cocos nucifera L. Botanical Review, 44, 265–319. https://doi. org/10.1007/BF02957852 Millenium Ecosystem Assessment (Ed.) (2005). Ecosystems and human well-being: Synthesis. Washington, DC: Island Press. Conservation, 13, e00362. https://doi.org/10.1016/j.gecco.2017. e00362 Conservation, 13, e00362. https://doi.org/10.1016/j.gecco.2017. e00362 IUCN. (2019). Habitats classification scheme (version 3.1). Retrieved from https://www.iucnredlist.org/resources/habitat-classification-scheme Jelbert, K., Stott, I., McDonald, R. A., & Hodgson, D. (2015). Invasiveness of plants is predicted by size and fecundity in the native range. Ecology and Evolution, 5, 1933–1943. https://doi.org/10.1002/ece3.1432 Farris-Lopez, K., Denslow, J. S., Moser, B., & Passmore, H. (2004). Influence of a common palm, Oenocarpus mapora, on seedling es- tablishment in a tropical moist forest in Panama. Journal of Tropical Ecology, 20, 429–438. Jones, D. L. (1995). Palms throughout the world, Washington, DC: Smithsonian Institution Press. Fehr, V., & Burga, C. A. (2016). Aspects and causes of earlier and current spread of Trachycarpus fortunei in the forests of southern Ticino and northern Lago Maggiore (Switzerland, Italy). Palms, 60, 125–136. Kissling, W. D., Balslev, H., Baker, W. J., Dransfield, J., Göldel, B., Lim, J. Y., … Svenning, J.-C. (2019). PalmTraits 1.0, a species-level functional trait database of palms worldwide. Scientific Data, 6, 178. Feng, S., Hu, Q., Huang, W., Ho, C.-H., Li, R., & Tang, Z. (2014). Projected climate regime shift under future global warming from multi-model, multi-scenario CMIP5 simulations. Global and Planetary Change, 112, 41–52. https://doi.org/10.1016/j.gloplacha.2013.11.002 Kissling, W. D., Eiserhardt, W. L., Baker, W. J., Borchsenius, F., Couvreur, T. L. P., Balslev, H., & Svenning, J.-C. (2012). Cenozoic imprints on the phylogenetic structure of palm species assemblages worldwide. Proceedings of the National Academy of Sciences of the United States of America, 109, 7379–7384. https://doi.org/10.1073/pnas.1120467109 FLEPPC. (2017). List of invasive plant species. Florida Exotic Pest Plant Council. Retrieved from http://www.fleppc.org/publications.htm Konstant, B. (2014). Indonesia: If you cut it, they will come. The Horn Spring, 12, 4. Flory, S. L., & D’Antonio, C. M. (2015). Taking the long view on the eco- logical effects of plant invasions. American Journal of Botany, 102, 817–818. https://doi.org/10.3732/ajb.1500105 Levey, D. J. (1987). Seed size and fruit-handling techniques of avian frugivores. The American Naturalist, 129, 471–485. https://doi. org/10.1086/284652 Furler, J., & Willing, R. (2006). Expedition Witjira: A biodiversity survey of the mound springs and surrounding area. Scientific Expedition Group (SEG) with National Parks and Wildlife (NPWS) and Department of Environment and Heritage (DEH). Lindemann-Matthies, P. (2016). Beasts or beauties? Laypersons’ per- ception of invasive alien plant species in Switzerland and atti- tudes towards their management. NeoBiota, 29, 15–33. https://doi. org/10.3897/neobiota.29.5786 Galetti, M., Keuroghlian, A., Hanada, L., & Morato, M. I. (2001). REFERENCES Occupational rhinoconjunctivitis and bronchial asthma due to Phoenix canariensis pollen allergy. Allergy, 50, 277–280. https://doi.org/10.1111/j.1398-9995.1995.tb01147.x Dyer, M. J. B., Keppel, G., Watling, D., Tuiwawa, M., Vido, S., & Boehmer, H. J. (2019). Using expert knowledge and field surveys to guide man- agement of an invasive alien palm in a Pacific Island lowland rainfor- est. Australian Journal of Botany, 66, 647–656. Blois, J. L., Zarnetske, P. L., Fitzpatrick, M. C., & Finnegan, S. (2013). Climate change and the past, present, and future of biotic interactions. Science, 341, 499–504. https://doi.org/10.1126/science.1237184 Eiserhardt, W. L., Svenning, J.-C., Kissling, W. D., & Balslev, H. (2011). Geographical ecology of the palms (Arecaceae): Determinants of di- versity and distributions across spatial scales. Annals of Botany, 108, 1391–1416. https://doi.org/10.1093/aob/mcr146 Broschat, T. K., Elliott, M. L., & Hodel, D. R. (2014). Ornamental palms: Biology and horticulture. In J. Janick (Ed.), Horticultural reviews: Volume 42 (pp. 1–120). Hoboken, NJ: John Wiley & Sons Ltd. Enloe, S. F., Langeland, K., Ferrell, J., Sellers, B., & MacDonald, G. (2018). Integrated management of non-native plants in natural areas of Florida. Gainesville, FL: Institute of Food and Agricultural Sciences, University of Florida, SP 242. Brummitt, R. K. (2001). World geographical scheme for recording plant distributions (2nd ed.). Pittsburgh, PA: Hunt Inst. for Botanical Documentation. Evers, C. R., Wardropper, C. B., Branoff, B., Granek, E. F., Hirsch, S. L., Link, T. E., … Wilson, C. (2018). The ecosystem services and biodi- versity of novel ecosystems: A literature review. Global Ecology and Buma, B. (2015). Disturbance interactions: Characterization, prediction, and the potential for cascading effects. Ecosphere, 6, art70. https:// doi.org/10.1890/ES15-00058.1 1536 FEHR et al. FEHR et al. Lista nacional de especies de plan- tas invasoras y potencialmente invasoras en la República de Cuba. Bissea, 6, 22–96. Sica, Y. V., Bravo, S. P., & Giombini, M. I. (2014). Spatial pattern of Pindó Palm (Syagrus romanzoffiana) recruitment in Argentinian Atlantic Forest: The importance of tapir and effects of defaunation. Biotropica, 46, 696–703. Padovani, R. J., Salisbury, A., Bostock, H., Roy, D. B., & Thomas, C. D. (2020). Introduced plants as novel Anthropocene habitats for in- sects. Global Change Biology, 26, 971–988. https://doi.org/10.1111/ gcb.14915 Smith, N. (2015). Acrocomia aculeata. In N. Smith (Ed.), Palms and people in the Amazon (pp. 9–15). Cham, Switzerland: Springer International Publishing. Palmpedia. (2019). Palmpedia. Retrieved from Palmpedia—Palm grower’s guide website: http://www.palmpedia.net Sosnowska, J., & Balslev, H. (2009). American palm ethnomedicine: A me- ta-analysis. Journal of Ethnobiology and Ethnomedicine, 5, 43. https:// doi.org/10.1186/1746-4269-5-43 Palmweb. (2019). Palmweb. Retrieved from Palmweb—Palms of the world online website: http://www.palmweb.org/ Spennemann, D. H. (2019). The contribution of the Canary Island date palm (Phoenix canariensis) to the winter diet of frugivores in novel ecosystems. European Journal of Ecology, 5, 27–37. https://doi. org/10.2478/eje-2019-0005 Peel, M. C., Finlayson, B. L., & McMahon, T. A. (2007). Updated world map of the Köppen-Geiger climate classification. Hydrology and Earth System Sciences, 11, 1633–1644. https://doi.org/10.5194/ hess-11-1633-2007 Perring, M. P., & Ellis, E. C. (2013). The extent of novel ecosystems: Long in time and broad in space. In R. J. Hobbs, E. S. Higgs, & C. M. Hall (Eds.), Novel Ecosystems (pp. 66–80). Hoboken, NJ: John Wiley & Sons Ltd. Starr, F., Starr, K., & Loope, L. (2003). Report: Archontophoenix alexandrae. United States Geological Survey - Biological Resources Division. Stricker, K. B., Hagan, D., & Flory, S. L. (2015). Improving methods to evaluate the impacts of plant invasions: Lessons from 40 years of research. AoB Plants, 7, plv028. https://doi.org/10.1093/aobpla/ plv028 Peters, H. A., Pauw, A., Silman, M. R., & Terborgh, J. W. (2004). Falling palm fronds structure Amazonian rainforest sapling communities. Proceedings of the Royal Society B: Biological Sciences, 271, S367–S369. Pinheiro, C. U. B. (2001). Germination strategies of palms: The case of Schippia concolor Burret in Belize. Brittonia, 53, 519–527. https://doi. org/10.1007/BF02809652 Svenning, J.-C. (2002). Non-native ornamental palms invade a secondary tropical forest in Panamá. Palms, 46, 81–86. Svenning, J.-C. (2018). Proactive conservation and restoration of botani- cal diversity in the Anthropocene’s “rambunctious garden”. American Journal of Botany, 105, 963–966. https://doi.org/10.1002/ajb2.1117 Pyšek, P., Jarošík, V., Hulme, P. National Park Service, U.S. Department of the Interior. (2015). Fan Palm oases. Retrieved from Joshua Tree National Park website: https:// www.nps.gov/jotr/learn/nature/oases.htm Rejmánek, M. (2014). Invasive trees and shrubs: Where do they come from and what we should expect in the future? Biological Invasions, 16, 483–498. https://doi.org/10.1007/s10530-013-0603-z Richardson, D. M., Pyšek, P., Rejmanek, M., Barbour, M. G., Panetta, F. D., & West, C. J. (2000). Naturalization and invasion of alien plants: Concepts and definitions. Diversity and Distributions, 6, 93–107. https://doi.org/10.1046/j.1472-4642.2000.00083.x Novoa, A., Brundu, G., Day, M. D., Deltoro, V., Essl, F., Foxcroft, L. C., … Wilson, J. R. U. (2019). Global actions for managing cactus invasions. Plants, 8, 421. https://doi.org/10.3390/plants8100421 Plants, 8, 421. https://doi.org/10.3390/plants8100421 NSW Department of Planning, Industry and Environment (2019). Cocos palms: A threat to flying-foxes. Retrieved from NSW Department of Planning, Industry and Environment website: https://www.envir onment.nsw.gov.au/topics/animals-and-plants/native-animals/nativ e-animal-facts/flying-foxes/cocos-palms-a-threat-to-flying-foxes Richardson, D. M., & Rejmánek, M. (2011). Trees and shrubs as in- vasive alien species – A global review: Global review of invasive trees & shrubs. Diversity and Distributions, 17, 788–809. https://doi. org/10.1111/j.1472-4642.2011.00782.x Ordonez, A., & Svenning, J.-C. (2016). Functional diversity of North American broad-leaved trees is codetermined by past and current en- vironmental factors. Ecosphere, 7, e01237. https://doi.org/10.1002/ ecs2.1237 Ripple, W. J., Newsome, T. M., Wolf, C., Dirzo, R., Everatt, K. T., Galetti, M., … Van Valkenburgh, B. (2015). Collapse of the world’s largest herbivores. Science Advances, 1, e1400103. https://doi.org/10.1126/ sciadv.1400103 Ortiz-Uribe, N., Salomón-Torres, R., & Krueger, R. (2019). Date palm status and perspective in Mexico. Agriculture, 9, 46. https://doi. org/10.3390/agriculture9030046 Rogers, A. M., & Chown, S. L. (2014). Novel ecosystems support sub- stantial avian assemblages: The case of invasive alien Acacia thickets. Diversity and Distributions, 20, 34–45. Schmidt, V., Schaefer, H. M., & Winkler, H. (2004). Conspicuousness, not colour as foraging cue in plant–animal signalling. Oikos, 106, 551–557. https://doi.org/10.1111/j.0030-1299.2004.12769.x Ovaskainen, O., Tikhonov, G., Norberg, A., Guillaume Blanchet, F., Duan, L., Dunson, D., … Abrego, N. (2017). How to make more out of community data? A conceptual framework and its implementation as models and software. Ecology Letters, 20, 561–576. https://doi. org/10.1111/ele.12757 Seebens, H., Blackburn, T. M., Dyer, E. E., Genovesi, P., Hulme, P. E., Jeschke, J. M., … Essl, F. (2017). No saturation in the accumulation of alien species worldwide. Nature Communications, 8, 14435. https:// doi.org/10.1038/ncomms14435 Oviedo, R., Herrera, P., Caluff, M. G., Regalado, L., Ventosa, I., Placencia, J. M., & González-Oliva, L. (2012). Milosavljević, I., El-Shafie, H. A. F., Faleiro, J. R., Hoddle, C. D., Lewis, M., & Hoddle, M. S. (2019). Palmageddon: The wasting of ornamental palms by invasive palm weevils, Rhynchophorus spp. Journal of Pest Science, 92, 143–156. https://doi.org/10.1007/s10340-018-1044-3 Hejda, M., Pyšek, P., & Jarošík, V. (2009). Impact of invasive plants on the species richness, diversity and composition of invaded communities. Journal of Ecology, 97, 393–403. https://doi. org/10.1111/j.1365-2745.2009.01480.x Minnich, R. A., Franco-Vizcaíno, E., & Salazar-Ceseña, M. (2011). Distribution and regional ecology of Californian palm oases interpreted from Google earth images. Aliso: A Journal of Systematic and Evolutionary Botany, 29, 1–12. https://doi.org/10.5642/aliso.20112901.02 Henderson, A. (2002). Evolution and ecology of palms. New York, NY: New York Botanical Garden Press. Hobbs, R. J., Higgs, E., & Harris, J. A. (2009). Novel ecosystems: Implications for conservation and restoration. Trends in Ecology & Evolution, 24, 599–605. https://doi.org/10.1016/j.tree.2009.05.012 Morse, N. B., Pellissier, P. A., Cianciola, E. N., Brereton, R. L., Sullivan, M. M., Shonka, N. K., … McDowell, W. H. (2014). Novel ecosys- tems in the Anthropocene: A revision of the novel ecosystem con- cept for pragmatic applications. Ecology and Society, 19, https://doi. org/10.5751/ES-06192-190212 Info Flora. (2014). Schwarze Liste und Watch-List der Schweiz. Retrieved from https://www.infoflora.ch/de/assets/content/documents/ neophyten/neophyten_diverses/SchwarzeListe_WatchListe_2014. pdf Moser, D., Lenzner, B., Weigelt, P., Dawson, W., Kreft, H., Pergl, J., … Essl, F. (2018). Remoteness promotes biological invasions on islands worldwide. Proceedings of the National Academy of Sciences of the United States of America, 115, 9270–9275. https://doi.org/10.1073/ pnas.1804179115 Ishii, H., Ichinose, G., Ohsugi, Y., & Iwasaki, A. (2016). Vegetation recov- ery after removal of invasive Trachycarpus fortunei in a fragmented urban shrine forest. Urban Forestry & Urban Greening, 15, 53–57. https://doi.org/10.1016/j.ufug.2015.11.008 FEHR et al. 1537 BIOSKETCH wrote the manuscript with contributions from R.B. and J.-C.S. Watson, L. H., Cameron, M. J., & Iifo, F. (2020). Elephant herbivory of knob-thorn (Senegalia nigrescens) and ivory palm (Hyphaene petersi- ana) in Bwabwata National Park, Caprivi, Namibia: The role of ivory palm as a biotic refuge. African Journal of Ecology, 58, 14–22. Williams, P. A. (2008). Biological success and weediness of some terrestrial weeds not presently in the Northland Regional Council’s RPMS. New Zealand: Landcare Research Contract Report: LC0708/079. BIOSKETCH Vincent Fehr is a PhD student at the Center for Biodiversity Dynamics in a Changing World (BIOCHANGE) at Aarhus University in Denmark. Beside his enthusiasm for the diversity and ecology of palms, he is interested in how non-native spe- cies are driving the emergence of novel ecosystems in a human- dominated world. van Kleunen, M., Dawson, W., Essl, F., Pergl, J., Winter, M., Weber, E., … Pyšek, P. (2015). Global exchange and accumulation of non-na- tive plants. Nature, 525, 100–103. https://doi.org/10.1038/natur e14910 van Kleunen, M., Essl, F., Pergl, J., Brundu, G., Carboni, M., Dullinger, S., … Dehnen-Schmutz, K. (2018). The changing role of ornamental hor- ticulture in alien plant invasions. Biological Reviews, 93, 1421–1437. https://doi.org/10.1111/brv.12402 Robert Buitenwerf is a broadly interested ecologist, with a re- search focus on the impacts of global change on ecosystem func- tioning and biodiversity. van Kleunen, M., Weber, E., & Fischer, M. (2010). A meta-anal- ysis of trait differences between invasive and non-inva- sive plant species. Ecology Letters, 13, 235–245. https://doi. org/10.1111/j.1461-0248.2009.01418.x Jens-Christian Svenning is an ecologist and biogeographer with strong interests in palms and novel ecosystems, among many other aspects of the biosphere. Venter, O., Sanderson, E. W., Magrach, A., Allan, J. R., Beher, J., Jones, K. R., … Watson, J. E. M. (2016). Global terrestrial Human Footprint maps for 1993 and 2009. Scientific Data, 3, 160067. https://doi. org/10.1038/sdata.2016.67 Vilà, M., Espinar, J. L., Hejda, M., Hulme, P. E., Jarošík, V., Maron, J. L., … Pyšek, P. (2011). Ecological impacts of invasive alien plants: A me- ta-analysis of their effects on species, communities and ecosystems: Ecological impacts of invasive alien plants. Ecology Letters, 14, 702– 708. https://doi.org/10.1111/j.1461-0248.2011.01628.x Robert Buitenwerf is a broadly interested ecologist, with a re- search focus on the impacts of global change on ecosystem func- tioning and biodiversity. Walther, G.-R., Gritti, E. S., Berger, S., Hickler, T., Tang, Z., & Sykes, M. T. (2007). Palms tracking climate change. Global Ecology and Biogeography, 16, 801–809. https://doi.org/10.1111/j.1466-8238.2007.00328.x Jens-Christian Svenning is an ecologist and biogeographer with strong interests in palms and novel ecosystems, among many other aspects of the biosphere. Warren, R. J. W. I., King, J. R., Tarsa, C., Haas, B., & Henderson, J. (2017). A systematic review of context bias in invasion biology. PLoS One, 12, e0182502. https://doi.org/10.1371/journal.pone.0182502 Author contributions: V.F., R.B., and J.-C.S. conceived the ideas. V.F. collected the data. V.F. analysed the data with help from R.B., and V.F. coconut palms (Cocos nucifera) in tropical islands. Canadian Journal of Zoology, 95, 139–148. the Amazonian tree flora. Science, 342, 1243092. https://doi. org/10.1126/science.1243092 Thomson, F. J., Moles, A. T., Auld, T. D., & Kingsford, R. T. (2011). Seed dispersal distance is more strongly correlated with plant height than with seed mass. Journal of Ecology, 99, 1299–1307. https://doi. org/10.1111/j.1365-2745.2011.01867.x Yu, K., Okin, G. S., Ravi, S., & D’Odorico, P. (2016). Potential of grass invasions in desert shrublands to create novel ecosystem states under variable climate. Ecohydrology, 9, 1496–1506. https://doi. org/10.1002/eco.1742 Thuiller, W. (2003). BIOMOD – Optimizing predictions of spe- cies distributions and projecting potential future shifts under global change. Global Change Biology, 9, 1353–1362. https://doi. org/10.1046/j.1365-2486.2003.00666.x Zurell, D., Pollock, L. J., & Thuiller, W. (2018). Do joint species distribu- tion models reliably detect interspecific interactions from co-occur- rence data in homogenous environments? Ecography, 41, 1812–1819. https://doi.org/10.1111/ecog.03315 Tomlinson, P. B., Horn, J. W., & Fisher, J. B. (2011). The anatomy of palm Arecaceae – Palmae. Oxford, UK: Oxford University Press. Tomlinson, P. B., Horn, J. W., & Fisher, J. B. (2011). The anatomy of Arecaceae – Palmae. Oxford, UK: Oxford University Press. United Nations, Department of Economic and Social Affairs, Population Division (2017). World Population Prospects: The 2017 Revision, Key Findings and Advance Tables. New York, NY: United Nations Population Division. E., Pergl, J., Hejda, M., Schaffner, U., & Vilà, M. (2012). A global assessment of invasive plant impacts on resident species, communities and ecosystems: The interaction of impact mea- sures, invading species’ traits and environment. Global Change Biology, 18, 1725–1737. https://doi.org/10.1111/j.1365-2486.2011.02636.x Svenning, J.-C., Gravel, D., Holt, R. D., Schurr, F. M., Thuiller, W., Münkemüller, T., … Normand, S. (2014). The influence of interspecific interactions on species range expansion rates. Ecography, 37, 1198– 1209. https://doi.org/10.1111/j.1600-0587.2013.00574.x R Core Team. (2019). R: A language and environment for statistical comput- ing. Vienna, Austria: R Foundation for Statistical Computing. Svenning, J.-C., Pedersen, P. B. M., Donlan, C. J., Ejrnæs, R., Faurby, S., Galetti, M., … Vera, F. W. M. (2016). Science for a wilder Anthropocene: Synthesis and future directions for trophic rewild- ing research. Proceedings of the National Academy of Sciences of the United States of America, 113, 898–906. https://doi.org/10.1073/ pnas.1502556112 Rabineau, M., Berné, S., Olivet, J.-L., Aslanian, D., Guillocheau, F., & Joseph, P. (2006). Paleo sea levels reconsidered from direct obser- vation of paleoshoreline position during Glacial Maxima (for the last 500,000 yr). Earth and Planetary Science Letters, 252, 119–137. https://doi.org/10.1016/j.epsl.2006.09.033 Talley, T. S., Nguyen, K.-C., & Nguyen, A. (2012). Testing the effects of an introduced palm on a riparian invertebrate community in south- ern California. PLoS One, 7, e42460. https://doi.org/10.1371/journ al.pone.0042460 Randall, R. P. (2007). The introduced flora of Australia and its weed sta- tus. Retrieved from https://www.cabdirect.org/cabdirect/abstr act/20093033838 Reichgelt, T., West, C. K., & Greenwood, D. R. (2018). The relation be- tween global palm distribution and climate. Scientific Reports, 8, 4721. https://doi.org/10.1038/s41598-018-23147-2 ter Steege, H., Pitman, N. C. A., Sabatier, D., Baraloto, C., Salomao, R. P., Guevara, J. E., … Silman, M. R. (2013). Hyperdominance in 1538 FEHR et al. SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. Young, H. S., McCauley, D. J., Dunbar, R. B., & Dirzo, R. (2010). Plants cause ecosystem nutrient depletion via the interruption of bird-de- rived spatial subsidies. Proceedings of the National Academy of Sciences of the United States of America, 107, 2072–2077. https://doi. org/10.1073/pnas.0914169107 Additional supporting information may be found online in the Supporting Information section. How to cite this article: Fehr V, Buitenwerf R, Svenning J-C. Non-native palms (Arecaceae) as generators of novel ecosystems: A global assessment. Divers Distrib. 2020;26: 1523–1538. https://doi.org/10.1111/ddi.13150 Young, H. S., McCauley, D. J., Pollock, A., & Dirzo, R. (2014). Differential plant damage due to litterfall in palm-dominated forest stands in a Central Pacific atoll. Journal of Tropical Ecology, 30, 231–236. https:// doi.org/10.1017/S026646741400008X Young, H. S., Miller-ter Kuile, A., McCauley, D. J., & Dirzo, R. (2017). Cascading community and ecosystem consequences of introduced
https://openalex.org/W3205620409	https://inria.hal.science/hal-03385317/document	Latin	null	COVID19 Disease Map, a computational knowledge repository of virus–host interaction mechanisms	Molecular systems biology	2,021	cc-by	24,082	To cite this version: Marek Ostaszewski, Anna Niarakis, Alexander Mazein, Inna Kuperstein, Robert Phair, et al.. COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms. Molecular Systems Biology, 2021, 17 (10), pp.e10387. 10.15252/msb.202110387. hal-03385317v2 COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms Marek Ostaszewski, Anna Niarakis, Alexander Mazein, Inna Kuperstein, Robert Phair, Aurelio Orta-Resendiz, Vidisha Singh, Sara Sadat Aghamiri, Marcio Luis Acencio, Enrico Glaab, et al. Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03385317 https://inria.hal.science/hal-03385317v2 Submitted on 19 Oct 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 1–82 The list of affiliations appears at the end of this article Corresponding author. Tel: +352 46 66 44 5604; E-mail: marek.ostaszewski@uni.lu †FAIRDOMHub: https://fairdomhub.org/projects/190 1–82 The list of affiliations appears at the end of this article Corresponding author. Tel: +352 46 66 44 5604; E-mail: marek.ostaszewski@uni.lu †FAIRDOMHub: https://fairdomhub.org/projects/190 1–82 The list of affiliations appears at the end of this article pp Corresponding author. Tel: +352 46 66 44 5604; E-mail: marek.ostaszewski@uni.lu †FAIRDOMHub: https://fairdomhub.org/projects/190 HAL Id: hal-03385317 https://inria.hal.science/hal-03385317v2 Submitted on 19 Oct 2021 Distributed under a Creative Commons Attribution 4.0 International License Article COVID19 Disease Map, a computational knowledge repository of virus–host interaction mechanisms Marek Ostaszewski1, , Anna Niarakis2,3 , Alexander Mazein1 , Inna Kuperstein4,5,6 , Robert Phair7 , Aurelio Orta-Resendiz8,9 , Vidisha Singh2 , Sara Sadat Aghamiri10 , Marcio Luis Acencio1 , Enrico Glaab1 , Andreas Ruepp11 , Gisela Fobo11, Corinna Montrone11, Barbara Brauner11, Goar Frishman11, Luis Cristobal Monraz Gomez4,5,6, Julia Somers12 , Matti Hoch13 , Shailendra Kumar Gupta13 , Julia Scheel13 , Hanna Borlinghaus14, Tobias Czauderna15 , Falk Schreiber14,15, Arnau Montagud16 , Miguel Ponce de Leon16 , Akira Funahashi17 , Yusuke Hiki17 , Noriko Hiroi18 , Takahiro G Yamada17 , Andreas Dr€ager19,20,21 , Alina Renz19,20 , Muhammad Naveez13,22 , Zsolt Bocskei23 , Francesco Messina24,25 , Daniela Börnigen26 , Liam Fergusson27, Marta Conti28, Marius Rameil28 , Vanessa Nakonecnij28, Jakob Vanhoefer28 , Leonard Schmiester28,29 , Muying Wang30, Emily E Ackerman30 , Jason E Shoemaker30,31 , Jeremy Zucker32 , Kristie Oxford32 , Jeremy Teuton32 , Ebru Kocakaya33 , Gökçe Yagmur Summak33 , Kristina Hanspers34 , Martina Kutmon35,36 , Susan Coort35 , Lars Eijssen35,37 , Friederike Ehrhart35,37 , Devasahayam Arokia Balaya Rex38 , Denise Slenter35 , Marvin Martens35 , Nhung Pham35 , Robin Haw39 , Bijay Jassal39 , Lisa Matthews40 , Marija Orlic-Milacic39 , Andrea Senff Ribeiro39,41 , Karen Rothfels39 , Veronica Shamovsky40, Ralf Stephan39 , Cristoffer Sevilla42 , Thawfeek Varusai42 , Jean-Marie Ravel43,44 , Rupsha Fraser45 , Vera Ortseifen46 , Silvia Marchesi47 , Piotr Gawron1,48 , Ewa Smula1 , Laurent Heirendt1 , Venkata Satagopam1 , Guanming Wu49 , Anders Riutta34 , Martin Golebiewski50 , Stuart Owen51 , Carole Goble51 , Xiaoming Hu50 , Rupert W Overall52,53,54 , Dieter Maier55, Angela Bauch55 , Benjamin M Gyori56 , John A Bachman56 , Carlos Vega1 , Valentin Groues1 , Miguel Vazquez16 , Pablo Porras42 , Luana Licata57 , Marta Iannuccelli57, Francesca Sacco57 , Anastasia Nesterova58 , Anton Yuryev58, Anita de Waard59 , Denes Turei60 , Augustin Luna61,62 , Ozgun Babur63 , Sylvain Soliman3 , Alberto Valdeolivas60 , Marina Esteban-Medina64,65 , Maria Pe~na-Chilet64,65,66 , Kinza Rian64,65 , Tomas Helikar67 , Bhanwar Lal Puniya67 , Dezso Modos68,69 , Agatha Treveil68,69 , Marton Olbei68,69 , Bertrand De Meulder70 , Stephane Ballereau71 , Aurelien Dugourd60,72 , Aurelien Naldi3 , Vincent No€el4,5,6 , Laurence Calzone4,5,6 , Chris Sander61,62 , Emek Demir12 , Tamas Korcsmaros68,69 , Tom C Freeman73 , Franck Auge23 , Jacques S Beckmann74 , Jan Hasenauer75,76 , Olaf Wolkenhauer13 , Egon L Wilighagen35 , Alexander R Pico34 , Chris T Evelo35,36 , Marc E Gillespie39,77 , Lincoln D Stein39,78 , Henning Hermjakob42 , Peter D’Eustachio40 , Julio Saez-Rodriguez60 , Joaquin Dopazo64,65,66,79 , Alfonso Valencia16,80 , Hiroaki Kitano81,82 , Emmanuel Barillot4,5,6 , Charles Auffray71 , Rudi Balling1 , Reinhard Schneider1 & the COVID-19 Disease Map Community,† 1–82 The list of affiliations appears at the end of this article COVID19 Disease Map, a computational knowledge repository of virus–host interaction mechanisms An interoperable repository of comprehensive and computable diagrams Keywords computable knowledge repository; large-scale biocuration; omics data analysis; open access community effort; systems biomedicine Subject Categories Computational Biology; Microbiology, Virology & Host Pathogen Interaction DOI 10.15252/msb.202110387 \| Received 7 April 2021 \| Revised 25 August 2021 \| Accepted 26 August 2021 Mol Syst Biol. (2021) 17: e10387 Keywords computable knowledge repository; large-scale biocuration; omics data analysis; open access community effort; systems biomedicine Subject Categories Computational Biology; Microbiology, Virology & Host Pathogen Interaction We constructed a comprehensive diagrammatic description of disease mechanisms in a way that is both human- and machine- readable, lowering communication barriers between experimental and computational biologists. To this end, we aligned the biocura- tion efforts of the Disease Maps Community (Mazein et al, 2018), Reactome (Jassal et al, 2020), and WikiPathways (Slenter et al, 2018) and developed guidelines for building and annotating these diagrams. In addition, we integrated relevant knowledge from public repositories (Licata et al, 2020; Perfetto et al, 2020; Rodchenkov et al, 2020; T€urei et al, 2021) and text mining resources to update and refine the contents of the C19DMap based on other knowledge-building efforts. This work resulted in a series of pathway diagrams constructed de novo, describing key events in the COVID-19 infectious cycle and host response. DOI 10.15252/msb.202110387 \| Received 7 April 2021 \| Revised 25 August 2021 \| Accepted 26 August 2021 DOI 10.15252/msb.202110387 \| Received 7 April 2021 \| Revised 25 August 2021 \| Accepted 26 August 2021 Mol Syst Biol. (2021) 17: e10387 COVID19 Disease Map, a computational knowledge repository of virus–host interaction mechanisms Marek Ostaszewski1,* , Anna Niarakis2,3 , Alexander Mazein1 , Inna Kuperstein4,5,6 , Robert Phair7 , Aurelio Orta-Resendiz8,9 , Vidisha Singh2 , Sara Sadat Aghamiri10 , Marcio Luis Acencio1 , Enrico Glaab1 , Andreas Ruepp11 , Gisela Fobo11, Corinna Montrone11, Barbara Brauner11, Goar Frishman11, Luis Cristobal Monraz Gomez4,5,6, Julia Somers12 , Matti Hoch13 , Shailendra Kumar Gupta13 , Julia Scheel13 , Hanna Borlinghaus14, Tobias Czauderna15 , Falk Schreiber14,15, Arnau Montagud16 , Miguel Ponce de Leon16 , Akira Funahashi17 , Yusuke Hiki17 , Noriko Hiroi18 , Takahiro G Yamada17 , Andreas Dr€ager19,20,21 , Alina Renz19,20 , Muhammad Naveez13,22 , Zsolt Bocskei23 , Francesco Messina24,25 , Daniela Börnigen26 , Liam Fergusson27, Marta Conti28, Marius Rameil28 , Vanessa Nakonecnij28, Jakob Vanhoefer28 , Leonard Schmiester28,29 , Molecular Systems Biology 17: e10387 \| 2021 1 of 22 ª 2021 The Authors. Published under the terms of the CC BY 4.0 license Molecular Systems Biology Molecular Systems Biology Marek Ostaszewski et al Marek Ostaszewski et al Abstract We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective. Keywords computable knowledge repository; large-scale biocuration; omics data analysis; open access community effort; systems biomedicine Subject Categories Computational Biology; Microbiology, Virology & Host Pathogen Interaction DOI 10.15252/msb.202110387 \| Received 7 April 2021 \| Revised 25 August 2021 \| Accepted 26 August 2021 Mol Syst Biol. (2021) 17: e10387 We need to effectively combine the knowledge from surging litera- ture with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive repre- sentation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines neces- sary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective. In the article, we explain the effort of our multidisciplinary community to construct the interoperable content of the resource, involving biocurators, domain experts and data analysts. We intro- duce the scope of the C19DMap and the insight it brings into the crosstalk and regulation of COVID-19-related molecular mechanisms. Next, we outline analytical workflows that can be used on the contents of the map, including the initial outcomes of two case stud- ies. We conclude with a discussion on the utility and perspectives of the C19DMap as a disease-relevant computational repository. 2 of 22 Molecular Systems Biology 17: e10387 \| 2021 Introduction The coronavirus disease 2019 (COVID-19) pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already resulted in the infection of over 250 million people worldwide, of whom almost 5 million have died (https://covid19.who.int, accessed on 05.10.2021). This global challenge motivated researchers world- wide to an unprecedented effort towards understanding the pathology to treat and prevent it. To date, over 170 thousand articles have been published in relation to COVID-19 (PubMed query “covid-19[Title/ Abstract] or sars-cov-2[Title/Abstract]”, accessed on 01.07.2021). The reported molecular pathophysiology that links SARS-CoV-2 infec- tion to the clinical manifestations and course of COVID-19 is complex and spans multiple biological pathways, cell types and organs (Gagliardi et al, 2020). Resources such as Protein Data Bank repository of viral protein structures (preprint: Lubin et al, 2020) or the IMEx coro- navirus interactome (Perfetto et al, 2020) offer detailed information about particular viral proteins and their direct binding partners. However, the scope of this information is limited. To gain insight into the large network of molecular mechanisms, knowledge from the vast body of scientific literature and bioinformatic databases needs to be inte- grated using systems biology standards. A repository of such compu- table knowledge will support data analysis and predictive modelling. The C19DMap project involved three main groups of partici- pants: the biocurators, the domain experts, and the analysts and modellers. Biocurators developed a collection of systems biology diagrams focused on the molecular mechanisms of SARS-CoV-2. Domain experts refined the contents of the diagrams using interac- tive visualisation and annotations. Analysts and modellers devel- oped computational workflows to generate hypotheses and predictions about the mechanisms encoded in the diagrams. Figure 1 illustrates the ecosystem of the C19DMap Community, highlighting the roles of the participants, available format conver- sions, interoperable tools and downstream uses. The community members and their contributions are listed on FAIRDOMHub (Wols- tencroft et al, 2017). Abstract analysts from 120 institutions in 30 countries to develop the COVID-19 Disease Map (C19DMap), an open access collection of curated computational diagrams and models of molecular mecha- nisms implicated in the disease (Ostaszewski et al, 2020). The C19DMap is a constantly evolving resource, refined and updated by ongoing biocuration, sharing and analysis efforts. Currently, it is a collection of 42 diagrams containing 1,836 interactions between 5,499 elements, supported by 617 publications and preprints. The summary of diagrams available in the C19DMap can be found online (https://covid.pages.uni.lu/map_contents) and in Table EV1. In the article, we explain the effort of our multidisciplinary community to construct the interoperable content of the resource, involving biocurators, domain experts and data analysts. We intro- duce the scope of the C19DMap and the insight it brings into the crosstalk and regulation of COVID-19-related molecular mechanisms. Next, we outline analytical workflows that can be used on the contents of the map, including the initial outcomes of two case stud- ies. We conclude with a discussion on the utility and perspectives of the C19DMap as a disease-relevant computational repository. analysts from 120 institutions in 30 countries to develop the COVID-19 Disease Map (C19DMap), an open access collection of curated computational diagrams and models of molecular mecha- nisms implicated in the disease (Ostaszewski et al, 2020). The C19DMap is a constantly evolving resource, refined and updated by ongoing biocuration, sharing and analysis efforts. Currently, it is a collection of 42 diagrams containing 1,836 interactions between 5,499 elements, supported by 617 publications and preprints. The summary of diagrams available in the C19DMap can be found online (https://covid.pages.uni.lu/map_contents) and in Table EV1. We need to effectively combine the knowledge from surging litera- ture with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive repre- sentation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines neces- sary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. Creating and accessing the diagrams The biocurators of the C19DMap diagrams followed the guidelines developed by the Community, WikiPathways (Slenter et al, 2018) and Reactome (Jassal et al, 2020) based on systems biology With this goal in mind, we initiated a collaborative effort involv- ing over 230 biocurators, domain experts, modellers and data ª 2021 The Authors Molecular Systems Biology Marek Ostaszewski et al Curators Domain experts Analysts Modellers GPML SBGNML CD SBML SBML qual CD SBML XGMML SBML SBGN GPML Graph PhysiBoSS 2.0 modelling SBML Boolean modelling Network analysis BSC Text mining resource Text mining modelling SBGN-ED CellDesigner WikiPathways Reactome Intera s XGMML Hipathia SIF Enrichment Analysis Curation SBML Squeezer User groups COVID-19 Disease Map use Newt Editor yED + ySGBGN SBML Format tr Input for pla rms or tools Input for users oups Legend: Carnival modelling Hipathia Domain experts Analysts Modellers User groups COVID-19 Disease Map use Format tr Input for pla rms or tools Input for users oups Legend: COVID-19 Disease Map use Format tr User groups COVID-19 Disease Map use Format tr Input for pla rms o Legend: Figure 1. Ecosystem of the COVID-19 Disease Map Community. The main groups of the C19DMap Community are biocurators, domain experts, and analysts and modellers; communicating to refine, interpret, and apply C19DMap diagrams. These diagrams are created and maintained by biocurators, following pathway database workflows or stand-alone diagram editors, and reviewed by domain experts. The content is shared via pathway databases or a GitLab repository; all can be enriched by integrated resources of text mining and interaction databases. The C19DMap diagrams are available in several layout-aware systems biology formats and integrated with external repositories, allowing a range of computational analyses, including network analysis and Boolean, kinetic or multiscale simulations. The main groups of the C19DMap Community are biocurators, domain experts, and analysts and modellers; communicating to refine, interpret, and apply C19DMap diagrams. These diagrams are created and maintained by biocurators, following pathway database workflows or stand-alone diagram editors, and reviewed by domain experts. The content is shared via pathway databases or a GitLab repository; all can be enriched by integrated resources of text mining and interaction databases. The C19DMap diagrams are available in several layout-aware systems biology formats and integrated with external repositories, allowing a range of computational analyses, including network analysis and Boolean, kinetic or multiscale simulations. Molecular Systems Biology 17: e10387 \| 2021 3 of 22 ª 2021 The Authors Creating and accessing the diagrams standards (Le Novere et al, 2009; Demir et al, 2010; Keating et al, 2020) and persistent identifiers (Wimalaratne et al, 2018). The diagrams are composed of biochemical reactions and interactions (altogether called interactions) between different molecular entities in various cellular compartments. As multiple teams worked on related topics, biocurators reviewed other diagrams, also across platforms (see also Materials and Methods). The diagrams are acces- sible online and can be explored using an intuitive user interface. Table 1 summarises information about the curated diagrams, and Table EV1 lists the diagrams and provides links to access them. biocuration needs to be supported by automated procedures to iden- tify and prioritise crucial articles, molecules and their interactions to be included in the C19DMap. Potential knowledge sources for such assisted biocuration are interaction and pathway databases, especially those with dedicated COVID-19 content (Licata et al, 2020; Perfetto et al, 2020). Their structured and annotated information on protein interactions or causal relationships was generated using separate biocuration guide- lines and formats. Nevertheless, their comparable identifiers and references to source publications make them plausible building blocks for constructing the C19DMap (see Materials and Methods). Enrichment using knowledge from databases and text mining Text mining approaches are another source of information that can direct the biocurators towards the most recent and relevant find- ings. They automatically extract and annotate biomolecule names and their interactions from abstracts, full-text documents or path- way figures (Bauch et al, 2020; Hanspers et al, 2020). Networks of molecule interactions constructed by text mining can carry substan- tially more noise than the contents of interaction databases but offer broader literature coverage. g g f g The knowledge of COVID-19 mechanisms is rapidly evolving, as shown by the growth of the COVID-19 Open Research Dataset (CORD-19), a source of manuscripts and metadata on COVID-19- related research (preprint: Lu Wang et al, 2020). CORD-19 currently contains almost 480,000 articles and preprints, over ten times more than when it was introduced more than a year ago (accessed on 05.10.2021). In such a quickly evolving environment, manual 3 of 22 Molecular Systems Biology 17: e10387 \| 2021 3 of 22 ª 2021 The Authors Molecular Systems Biology Molecular Systems Biology Molecular Systems Biology Marek Ostaszewski et al Table 1. COVID-19 Disease Map contents. Table 1. COVID-19 Disease Map contents. Source Individual diagrams Reactome WikiPathways Diagram contents 21 diagrams 1,334 interactions 4,272 molecular entities 397 publications 2 diagrams 101 interactions 489 molecular entities 227 publications 19 diagrams 401 interactions 738 molecular entities 61 publications Access GitLab gitlab.lcsb.uni.lu/covid/models SARS-CoV-1 and SARS-CoV-2 infections collection reactome.org/PathwayBrowser/#/ R-HSA-9679506 COVID pathway collection covid.wikipathways.org Exploration The MINERVA Platform (Gawron et al, 2016) covid19map.elixir-luxembourg.org Guide: covid.pages.uni.lu/minerva-guide Native web interface Guide: covid.pages.uni.lu/reactome-guide Native web interface Guide: covid.pages.uni.lu/wikipathways-guide Biocuration guidelines Communitya Platform-specificb Platform-specificc Diagram Editors CellDesigner (Matsuoka et al, 2014), Newtd, SBGN-ED (Czauderna et al, 2010), yEd+ySBGNe Reactome pathway editorb PathVisio (Kutmon et al, 2015) Formats CellDesigner SBML SBGNML (Bergmann et al, 2020) Internal, SBML and SBGNML compliant GPML (Kutmon et al, 2015) The table summarises biocuration resources and content of the C19DMap across three main parts of the repository. All diagrams are listed in Table EV1, and available online at: https://covid.pages.uni.lu/map_contents. ahttps://fairdomhub.org/documents/661 bhttps://reactome.org/community/training chttps://www.wikipathways.org/index.php/Help:Editing_Pathways dhttps://newteditor.org ehttps://github.com/sbgn/ySBGN Table 2 summarises open access interaction databases and text mining knowledge bases supporting the biocuration of the C19DMap. Molecular interactions from these sources have a broad coverage at the cost of depth of mechanistic representation. The biocurators used this content to build and update the map by manual exploration or by programmatic comparison. Molecular Systems Biology Marek Ostaszewski et al meaningful answers extracted from text mining platforms. Second, we developed routines for programmatic queries of the interaction databases, providing automated and reproducible exploration of the selected databases. This was realised using data endpoints: Applica- tion programming interfaces (API) for INDRA, AILANI or Pathway Studio, and SPARQL for BioKB. This automated exploration retrieved functions, interactions, pathways or drugs associated with submitted queries, e.g. gene lists. This way, otherwise time- consuming tasks such as an assessment of completeness of a given diagram or search for new literature evidence were automated. Section “Exploration of the networked knowledge” describes an application of such automated queries in crosstalk analysis. properties, supported by a common ontology (Courtot et al, 2011), allowed cross-format translation of the diagrams, which was essen- tial for harmonising the effort between biocuration platforms. The ecosystem of tools and resources supporting the C19DMap (see Fig 1) ensured interoperability between SBML, SBGNML and GPML via translation, preserving the diagram layout (Bohler et al, 2016; Balaur et al, 2020; Hoksza et al, 2020) for harmonised visuali- sation of diagrams. Additionally, these diagrams were transformed into inputs of computational pipelines and data repositories, allow- ing network analysis, pathway modelling and interoperability with molecular interaction repositories (Pillich et al, 2017) (see Materials and Methods). Interoperability of the diagrams and annotations The biocuration of the C19DMap diagrams was distributed across multiple teams, using varying tools and associated systems biology representations. This required a common approach to annotations of diagram elements and their interactions. Additionally, to compare and combine the diagrams in the C19DMap, interoperability of layout-aware formats was needed. The C19DMap was built bottom-up, exploiting a rich bioinformatics framework discussed in Section “An interoperable repository of comprehensive and computable diagrams” of the Results, based on knowledge from existing studies of other coronaviruses (Fung & Liu, 2019) and contextualised with data emerging from studies of SARS-CoV-2 (Gordon et al, 2020). The contents of the C19DMap are available online, summarised in a constantly updated overview at https://covid.pages.uni.lu/map_contents (see also Table EV1). Currently, the C19DMap focuses on molecular processes involved in SARS-CoV-2 entry and replication and host–virus interactions (see Fig 2). Emerging scientific evidence of host susceptibility, immune The diagrams were encoded in three layout-aware formats for standardised representation of molecular interactions: SBML, SBGNML and GPML. All three formats, centred around molecular interactions, provided a constrained vocabulary to encode element and interaction types, encode layout of corresponding diagrams and support stable identifiers for diagram components. These shared Innate immune and inflammatory response T-cells Adaptive immune response Antigen-presenting cell Natural killer Systemic circulation Respiratory Tract Antibody production Coagulation and thrombosis Cytokine release SARS-CoV-2 B cell Red blood cells Th1 Th2 Macrophages Monocytes Viral replication cycle Endosome and uncoating Genome replication Transcription and translation Virion Target Cell T-cell activation Specific T-cell response COVID-19 Disease Map Vascular endothelial cell Cellular metabolism Viral protein interactions CD8+ CD4+ PAMP Signalling Unfolded protein response Mitochondrial ETC Viral molecules (ssRNA, dsRNA) Apoptosis JNK Pathway Autophagy Endoplasmic Reticulum Stress Dendritic cells IFN-I Attachment and entry Integrative stress response Viral shedding (cardiovascular, renal, gastrointestinal, nervous) Other tissues/organs/systems Golgi Mitochondria Granulocytes E.R. IL-6 SARS-CoV-2 Bronchial epithelium Nasal mucosa Alveoli itocho S-CoV-2 tic cells d h Renin–angiotensin– aldosterone system (RAAS) AMP S anscri ACE2 TMPRSS2 ACE2 Figure 2. The structure and content of the COVID-19 Disease Map. An overview of the areas of focus of the C19DMap biocuration. Go to covid19map.elixir-luxembourg.org for an interactive version. Full list of diagrams and browsing instructions are available online at covid.pages.uni.lu/map_contents. Figure 2. The structure and content of the COVID-19 Disease Map. An overview of the areas of focus of the C19DMap biocuration. Go to covid19map.elixir-luxembourg.org for an interactive version. Enrichment using knowledge from databases and text mining First, the biocurators visually explored the contents of such networks using available search interfaces to identify interactions of interesting molecules and encoded them in the diagrams. This task was supported by a dedicated visualisation tool COVIDminer (https:// rupertoverall.net/covidminer). The biocurators also used assistant chatbots that respond to natural language queries and return Table 2. Resources supporting biocuration of the COVID-19 Disease Map. Resource Type Manually curated Directed Layout COVID-19 specific IMEx Consortium (Orchard et al, 2012) Interaction database Yes No No Yesa SIGNOR 2.0 (Licata et al, 2020) Yes Yes Yes Yesb OmniPath (T€urei et al, 2016) No Yes No No Elsevier Pathway Collectionc Pathway Yes Yes Yes Yesd INDRA (Gyori et al, 2017) Text mining Yes Yes No Yese BioKBf No Yes No Yes AILANI COVID-19g No Yes No Yes OpenNLP+GNormPlush No Yes No Yes They include (i) collections of COVID-19 interactions and pathways, (ii) interaction databases and (iii) text mining corpora. ahttps://www.ebi.ac.uk/intact/resources/datasets#coronavirus bhttps://signor.uniroma2.it/covid/ chttps://pathwaystudio.com dhttp://dx.doi.org/10.17632/d55xn2c8mw.1 ehttps://emmaa.indra.bio/dashboard/covid19 fhttps://biokb.lcsb.uni.lu ghttps://ailani.ai hhttps://gitlab.lcsb.uni.lu/covid/models/-/tree/master/Resources/Text%20mining Table 2. Resources supporting biocuration of the COVID-19 Disease Map. ª 2021 The Authors Molecular Systems Biology Interoperability of the diagrams and annotations For the literature on clinical manifestations, see Lauer et al, 2020; He et al, 2020; Huang et al, 2020; Bajema et al, 2020; preprint: Chen et al, 2020b; Wang et al, 2020a; Tong et al, 2020. response, cell and organ specificity will be incorporated into the next versions in accordance with our curation roadmap (https:// fairdomhub.org/documents/907). the map was proposed in Section “Case study: analysis of cell- specific mechanisms using single-cell expression data”. SARS-CoV-2 infection and COVID-19 progression are sequential events that start with viral attachment and entry (Fig 3). These events involve various dynamic processes and different timescales that are not captured in static representations of pathways. The correlation of symptoms and potential drugs suggested to date helps downstream data exploration and drug target interpretation in the context of therapeutic interventions. While the interactions of SARS-CoV-2 with various host cell types are vital determinants of COVID-19 pathology (Hui et al, 2020; Mason, 2020; Ziegler et al, 2020), the current C19DMap represents an infection of a generic host cell. Several pathways included in the map are shared between different cell types; for example, the IFN-1 pathway is active in dendritic and lung epithelial cells and in alveo- lar macrophages (Hadjadj et al, 2020; Lee & Shin, 2020; Sa Ribero et al, 2020). Continued annotations of emerging expression datasets (Delorey et al, 2021) and other sources of information will allow the construction of cell-specific versions of the C19DMap to provide an integrated view of the effects of SARS-CoV-2 on the human organ- ism. An example workflow to construct such a focused version of 6 of 22 Molecular Systems Biology 17: e10387 \| 2021 Interoperability of the diagrams and annotations Full list of diagrams and browsing instructions are available online at covid.pages.uni.lu/map_contents. An overview of the areas of focus of the C19DMap biocuration. Go to covid19map.elixir-luxembourg.org for an interactive versio instructions are available online at covid.pages.uni.lu/map_contents. ocus of the C19DMap biocuration. Go to covid19map.elixir-luxembourg.org for an interactive version. Full list of diagrams and browsing ine at covid.pages.uni.lu/map_contents. Molecular Systems Biology 17: e10387 \| 2021 5 of 22 ª 2021 The Authors Molecular Systems Biology Marek Ostaszewski et al A B C D E Figure 3. Overview of the C19DMap in the context of COVID-19 progression. The figure summarises the main sections and content of the C19DMap by illustrating the progressive but overlapping mechanisms at different levels and study features of the disease intended as quick references for the map. A Cellular level (light yellow), the immune response (blue) and other systemic responses (red) of the host following SARS-CoV-2 infection. B The progression of pathophysiology from tissue damage to organ damage and multiple organ dysfunction in severe cases. C Clinical manifestations, depending on the severity of the infection from asymptomatic to critical COVID-19. D Potential intervention strategies that may be suggested based on the analysis of the C19DMap before, during and after infection, depending on the type and target of the intervention. E Clinical outcomes of SARS-CoV-2 infection. ARDS, acute respiratory distress syndrome. RAAS, renin–angiotensin–aldosterone system. SIRS, systemic inflammatory response syndrome. For the literature on clinical manifestations, see Lauer et al, 2020; He et al, 2020; Huang et al, 2020; Bajema et al, 2020; preprint: Chen et al, 2020b; Wang et al, 2020a; Tong et al, 2020. Figure 3. Overview of the C19DMap in the context of COVID-19 progression. sections and content of the C19DMap by illustrating the progressive but overlapping mechanisms at different levels and study features references for the map. q p level (light yellow), the immune response (blue) and other systemic responses (red) of the host following SARS-CoV-2 infection. gression of pathophysiology from tissue damage to organ damage and multiple organ dysfunction in severe cases es that may be suggested based on the analysis of the C19DMap before, during and after infection, depending on the type and target E Clinical outcomes of SARS-CoV-2 infection. ARDS, acute respiratory distress syndrome. RAAS, renin–angiotensin–aldosterone system. SIRS, systemic inflammatory response syndrome. Viral subversion of host defence Viral subversion of host defence Endoplasmic reticulum (ER) stress results from the production of large amounts of viral proteins that create an overload of unfolded proteins (Kr€ahling et al, 2009; DeDiego et al, 2011; Fukushi et al, 2012). The mechanisms of the unfolded protein response (UPR) include the mitigation of the misfolded protein load by reduced protein synthesis and increased protein degradation (Sureda et al, 2020) through the ubiquitin–proteasome system (UPS) and autop- hagy (Choi et al, 2018; Bello-Perez et al, 2020). SARS-CoV-2 may perturb the process of UPS-based protein degradation via the inter- action of the viral Orf10 protein with the Cul2 ubiquitin ligase complex and its putative substrates (Gordon et al, 2020; Zhang et al, 2020). The involvement of SARS-CoV-2 in autophagy is less documented (Yang & Shen, 2020). Key metabolic pathways modulate the availability of nutrients and critical metabolites of the immune microenvironment (Rao et al, 2019). They are a target of infectious agents that reprogram host metabolism to create favourable conditions for their reproduc- tion (Kedia-Mehta & Finlay, 2019). The C19DMap encodes several immunometabolic pathways and provides detailed information about the way SARS-CoV-2 proteins interact with them. The meta- bolic pathways include haem catabolism (Batra et al, 2020) and its downstream target, the NLRP3 inflammasome (van den Berg & Te Velde, 2020), tryptophan-kynurenine metabolism governing the response to inflammatory cytokines (Murakami et al, 2013; preprint: Su et al, 2020), and nicotinamide and purine metabolism (Renz et al, 2020). Finally, we represent the pyrimidine synthesis pathway, tightly linked to purine metabolism, affecting viral DNA and RNA syntheses (Hayek et al, 2020; Xiong et al, 2020). The increased burden of misfolded proteins due to viral replica- tion and subversion of mitigation mechanisms may trigger programmed cell death (apoptosis). The C19DMap encodes major signalling pathways triggering this final form of cellular defence against viral replication (Diemer et al, 2010). Many viruses block or delay cell death by expressing anti-apoptotic proteins to maximise the production of viral progeny (Kanzawa et al, 2006; Liu et al, 2007) or induce it in selected cell types (Diemer et al, 2010; Chu et al, 2016; preprint: Chen et al, 2020b). These mechanisms are illustrated in the diagrams of the “Viral subversion of host defence” section in Table EV1: “ER stress and unfolded protein response”, “Autophagy and protein degradation” and “Apoptosis”. Viral subversion of host defence These mechanisms are illustrated in the diagrams of the “Innate Immune Response” section in Table EV1: “PAMP signalling”, “Induction of interferons and the cytokine storm” and “Altered host metabolism”. Host integrative stress response Severe acute respiratory syndrome coronavirus 2 infection damages the epithelium and the pulmonary capillary vascular endothelium (Bao et al, 2020), impairing respiration and leading to acute respira- tory distress syndrome (ARDS) in severe forms of COVID-19 (Huang et al, 2020). The release of pro-inflammatory cytokines and hyper- inflammation are known complications, causing further widespread damage (Chen et al, 2020a; Lucas et al, 2020). Coagulation distur- bances and thrombosis are associated with severe cases, but specific mechanisms have not been described yet (Iba et al, 2020; Klok et al, 2020). Nevertheless, it was shown that SARS-CoV-2 disrupts the coagulation cascade and causes renin–angiotensin system (RAS) imbalance (Magro et al, 2020; Urwyler et al, 2020). Contents of the COVID-19 Disease Map Virus replication cycle The virus replication cycle includes binding of the spike surface glycoprotein (S) to angiotensin-converting enzyme 2 (ACE2) facili- tated by TMPRSS2 (Hoffmann et al, 2020b; Letko et al, 2020) and 6 of 22 Molecular Systems Biology 17: e10387 \| 2021 6 of 22 ª 2021 The Authors Marek Ostaszewski et al Molecular Systems Biology other receptors (preprint: Amraei et al, 2020; preprint: Gao et al, 2020). Viral entry occurs either by direct fusion of the virion with the cell membranes or by endocytosis (Hoffmann et al, 2020a; Xia et al, 2020) of the virion membrane and the subsequent injection of the nucleocapsid into the cytoplasm. Within the host cell, the C19DMap depicts how SARS-CoV-2 hijacks the rough endoplasmic reticulum (RER)-linked host translational machinery for its replica- tion (Chen et al, 2010; Angelini et al, 2013; Nakagawa et al, 2016; V’kovski et al, 2019). The RER-attached translation machinery produces structural proteins, which, together with the newly gener- ated viral RNA, are assembled into new virions and released to the extracellular space via smooth-walled vesicles (Nakagawa et al, 2016) or hijacked lysosomes (Ghosh et al, 2020). Angiotensin-converting enzyme 2, used by SARS-CoV-2 for host cell entry, is a regulator of RAS and is widely expressed in the affected organs. The diagrams in the repository describe how ACE2- converted angiotensins trigger the counter-regulatory arms of RAS and the downstream signalling via AGTR1, regulating the coagula- tion cascade (Gheblawi et al, 2020; McFadyen et al, 2020). These mechanisms are illustrated in the diagrams of the “Integra- tive stress response” section in Table EV1: “Renin–angiotensin system” and “Coagulopathy”. Host immune response The innate immune system detects specific pathogen-associated molecular patterns, through pattern recognition receptors (PRRs) that recognise viral RNA in the endosome during endocytosis or in the cytoplasm during virus replication. The PRRs activate associated transcription factors promoting the production of antiviral proteins such as interferon-alpha, interferon-beta and interferon-lambda (Takeuchi & Akira, 2010; Berthelot & Liote, 2020; Blanco-Melo et al, 2020; Hadjadj et al, 2020; Park & Iwasaki, 2020). SARS-CoV-2 impairs this mechanism (Chu et al, 2020), but the exact components are yet to be elucidated (Liao et al, 2005; Devaraj et al, 2007; Frie- man et al, 2007; Li et al, 2016; Bastard et al, 2020). The C19DMap includes both the virus recognition process and the viral evasion mechanisms. It provides the connection between virus entry, its replication cycle, and the effector pathways of pro-inflammatory cytokines, especially of the interferon type I cascade (Wong et al, 2018; Mesev et al, 2019; Mantlo et al, 2020; Su & Jiang, 2020; Thoms et al, 2020; Ziegler et al, 2020). These mechanisms are illustrated in the diagrams of the “Virus repli- cation cycle” section in Table EV1: “Attachment and entry”, “Transcrip- tion, translation and replication” and “Assembly and release”. ª 2021 The Authors Molecular Systems Biology 17: e10387 \| 2021 7 of 22 Exploration of the networked knowledge The network structure of the diagrams and their interactions based on existing crosstalk (shared elements), candidate crosstalk, and candidate regulators. Colour code: green—pathways or pathway groups; blue—proteins with one or two neighbours; yellow—proteins with three or four neighbours; and red—proteins with five or more neighbours. Candidate molecular interactions are shown as directed edges. Candidate regulator elements are marked with a solid black border. See Materials and Methods for details. Exploration of the networked knowledge The diagrams of the C19DMap were curated in a distributed manner across various platforms and tools. In order to coordinate such an effort and get a systematic overview of the contents of the map, we programmatically analysed the content of the diagrams, benefiting from their standard encoding and annotation (see Materials and Methods). This allowed us to identify crosstalk and functional over- laps across pathways. Then, we linked the diagrams to interaction and text mining databases to fill the gaps in our understanding of COVID-19 mechanisms and generate new testable hypotheses. Exploration of the networked knowledge Molecular Systems Biology 17: e10387 \| 2021 7 of 22 ª 2021 The Authors Molecular Systems Biology Marek Ostaszewski et al JAK1 FOS TYK2 ITCH MINERVA: PAMP signalling RIPK1 WP5039: SARS-CoV-2 Innate Immunity Evasion and Cell-speciﬁc immune response MINERVA: NLRP3 inﬂammasome activation DDX58 TREML4 NFKB2 IL1B IFNAR2 MINERVA: Interferon lambda pathway IFNAR1 MINERVA: Kynurenine synthesis pathway CASP1 NFKB1 MAP3K7 MINERVA: Orf3a protein interactions WP4868: Type I Interferon Induction and Signaling During SARS-CoV-2 Infection CHUK WP4876: Activation of NLRP3 Inﬂammasome by SARS-CoV-2 IKBKB MINERVA: Pyrimidine deprivation WP4880: Host-pathogen interaction of human corona viruses - Interferon induction IKBKG IRF3 NLRP3 TBK1 MYD88 IFNA1 WP4912: SARS coronavirus and innate immunity HMOX1 TICAM1 IL6 IFNA2 RELA IFNA4 NFKBIA MAPK14 IFNA5 PYCARD EIF2AK2 IFNA6 TRAF3 CASP8 IFNA7 TLR7 FADD MAVS IFNA8 TLR8 MINERVA: Interferon 1 pathway IFNA10 TLR3 IFIH1 IFNA14 TRADD RNF135 IFNA16 TRIM25 IFNA17 STAT2 IFNA21 JUN STAT1 WP4961: STING pathway in Kawasaki-like disease and COVID-19 IRF9 STING1 MAPK8 OAS1 TRAF2 OAS2 OAS3 IKBKE IRF7 TRAF5 ACE2 TANK A C B Coagulopathy pathway Renin-angiotensin system Apoptosis pathway NFKBIA TNF AKT1 RHOA NLRP3 MAVS ADAM17 Induction of interferons and the cytokine storm Autophagy and protein degradation ER stress and unfolded protein response CASP1 CASP4 Altered host metabolism RELA PAMP signalling Virus replication cycle SRC EGFR PIN1 STAT3 ELAVL1 IRF3 Induction of interferons and the cytokine storm PTGS2 PRKRA TF GZMB USP7 TFRC CASP3 MDM2 Altered host metabolism HMGB1 AXL TLR2 TLR9 SOCS1 MYD88 IFNG AURKA Virus replication cycle IFNGR1 TNFAIP3 PAMP signalling STAT1 TLR5 IFNGR2 CD40 JAK1 SIRT1 TRAF3 IL6ST PIAS3 TP53BP2 JAK2 RCHY1 TP53 LEP CYLD ADAMTS13 SMAD7 PIAS1 VWF SMAD3 BIRC2 Coagulopathy pathway MAP3K14 BIRC3 IL22 CHUK XIAP CD14 MKNK1 CASP9 TLR4 EIF4E BIRC5 CDKN2A MMP9 CASP7 CFLAR CXCL5 NFKB2 CASP8 MYC MDM4 ER stress and unfolded protein response NFE2L2 SERPINA5 Autophagy and protein degradation HMOX1 F2 G3BP1 NPM1 EIF2AK2 ATM E2F1 TRAF6 BRAF APAF1 MAP2K1 ECE1 NR3C1 Apoptosis pathway EDN1 RELA Nodes with 1 or 2 neighbours 3 or 4 neighbours 5 or more neighbours Diagrams or diagram groups in the COVID-19 Disease Map Nodes suggested as novel regulators Interactions (undirected) connecting elements and diagrams or diagram groups in the COVID-19 Disease Map, in which they participate Novel interactions (directed) suggested by text mining and interaction databases Figure 4. Exploration of the existing and candidate crosstalk between the diagrams of the COVID-19 Disease Map. Exploration of the networked knowledge Exploration of the existing and candidate crosstalk between the diagrams of the COVID-19 Disease Map. Figure 4. Exploration of the existing and candidate crosstalk between the diagrams of the COVID-19 Disease Map. he existing and candidate crosstalk between the diagrams of the COVID-19 Disease Map. g p g g p The network structure of the diagrams and their interactions based on existing crosstalk (shared elements), candidate crosstalk, and candidate regulators. Colour code: green—pathways or pathway groups; blue—proteins with one or two neighbours; yellow—proteins with three or four neighbours; and red—proteins with five or more neighbours. Candidate molecular interactions are shown as directed edges. Candidate regulator elements are marked with a solid black border. See Materials and Methods for details. A Existing crosstalk between individual diagrams of IFN-I and RELA-related mechanisms. B Candidate crosstalk between pathway groups. C Candidate regulators of existing diagrams from text mining and interaction databases. The network structure of the diagrams and their interactions based on existing crosstalk (shared elements), candidate crosstalk, and candidate regulators. Colour code: green—pathways or pathway groups; blue—proteins with one or two neighbours; yellow—proteins with three or four neighbours; and red—proteins with five or more neighbours. Candidate molecular interactions are shown as directed edges. Candidate regulator elements are marked with a solid black border. See Materials and Methods for details. The network structure of the diagrams and their interactions based on existing crosstalk (shared elements), candidate crosstalk, and candidate regulators. Colour code: green—pathways or pathway groups; blue—proteins with one or two neighbours; yellow—proteins with three or four neighbours; and red—proteins with five or more neighbours. Candidate molecular interactions are shown as directed edges. Candidate regulator elements are marked with a solid black border. See Materials and M th d f d t il A Existing crosstalk between individual diagrams of IFN-I and RELA-related mechanisms. B Candidate crosstalk between pathway groups. C Candidate regulators of existing diagrams from text mining and interaction databases. C Candidate regulators of existing diagrams from text mining and interaction databases. Below, we discuss three specific examples of exploration of this networked knowledge (see Fig 4). Access to the complete content of the crosstalk diagrams can be found in Materials and Methods. (Mogensen, 2009). Downstream, IFN-1 activates Tyk2 and Jak1 protein tyrosine kinases, causing STAT1:STAT2:IRF9 (ISGF3) complex formation to promote the transcription of IFN-stimulated genes (ISGs). Exploration of the networked knowledge Importantly, TBK1 also phosphorylates IKBA, an NF- kB inhibitor, for proteasomal degradation in crosstalk with the UPS pathway, allowing free NF-kB and IRF3 to co-activate ISGs (Fang et al, 2017). Another TBK1 activator, STING, links IFN signalling with pyrimidine metabolism. 8 of 22 Molecular Systems Biology 17: e10387 \| 2021 Exploration of the networked knowledge A E i ti t lk b t i di id l di f IFN I d RELA l t d h i JAK1 FOS TYK2 ITCH MINERVA: PAMP signalling RIPK1 WP5039: SARS-CoV-2 Innate Immunity Evasion and Cell-speciﬁc immune response MINERVA: NLRP3 inﬂammasome activation DDX58 TREML4 NFKB2 IL1B IFNAR2 MINERVA: Interferon lambda pathway IFNAR1 MINERVA: Kynurenine synthesis pathway CASP1 NFKB1 MAP3K7 MINERVA: Orf3a protein interactions WP4868: Type I Interferon Induction and Signaling During SARS-CoV-2 Infection CHUK WP4876: Activation of NLRP3 Inﬂammasome by SARS-CoV-2 IKBKB MINERVA: Pyrimidine deprivation WP4880: Host-pathogen interaction of human corona viruses - Interferon induction IKBKG IRF3 NLRP3 TBK1 MYD88 IFNA1 WP4912: SARS coronavirus and innate immunity HMOX1 TICAM1 IL6 IFNA2 RELA IFNA4 NFKBIA MAPK14 IFNA5 PYCARD EIF2AK2 IFNA6 TRAF3 CASP8 IFNA7 TLR7 FADD MAVS IFNA8 TLR8 MINERVA: Interferon 1 pathway IFNA10 TLR3 IFIH1 IFNA14 TRADD RNF135 IFNA16 TRIM25 IFNA17 STAT2 IFNA21 JUN STAT1 WP4961: STING pathway in Kawasaki-like disease and COVID-19 IRF9 STING1 MAPK8 OAS1 TRAF2 OAS2 OAS3 IKBKE IRF7 TRAF5 ACE2 TANK A A B Coagulopathy pathway Renin-angiotensin system Apoptosis pathway NFKBIA TNF AKT1 RHOA NLRP3 MAVS ADAM17 Induction of interferons and the cytokine storm Autophagy and protein degradation ER stress and unfolded protein response CASP1 CASP4 Altered host metabolism RELA PAMP signalling Virus replication cycle C SRC EGFR PIN1 STAT3 ELAVL1 IRF3 Induction of interferons and the cytokine storm PTGS2 PRKRA TF GZMB USP7 TFRC CASP3 MDM2 Altered host metabolism HMGB1 AXL TLR2 TLR9 SOCS1 MYD88 IFNG AURKA Virus replication cycle IFNGR1 TNFAIP3 PAMP signalling STAT1 TLR5 IFNGR2 CD40 JAK1 SIRT1 TRAF3 IL6ST PIAS3 TP53BP2 JAK2 RCHY1 TP53 LEP CYLD ADAMTS13 SMAD7 PIAS1 VWF SMAD3 BIRC2 Coagulopathy pathway MAP3K14 BIRC3 IL22 CHUK XIAP CD14 MKNK1 CASP9 TLR4 EIF4E BIRC5 CDKN2A MMP9 CASP7 CFLAR CXCL5 NFKB2 CASP8 MYC MDM4 ER stress and unfolded protein response NFE2L2 SERPINA5 Autophagy and protein degradation HMOX1 F2 G3BP1 NPM1 EIF2AK2 ATM E2F1 TRAF6 BRAF APAF1 MAP2K1 ECE1 NR3C1 Apoptosis pathway EDN1 RELA s Interactions (undirected) connecting elements and diagrams or diagram groups in the COVID-19 Disease Map, in which they participate Novel interactions (directed) suggested by text mining and interaction databases C B Diagrams or diagram groups in the COVID-19 Disease Map Nodes suggested as novel regulators Interactions (undirected) connecting elements and diagrams or diagram groups in the COVID-19 Disease Map, in which they participate Nodes with 1 or 2 neighbours 3 or 4 neighbours 5 or more neighbours Interactions (undirected) connecting elements and diagrams or diagram groups in the COVID-19 Disease Map, in which they participate Interactions (undirected) connecting elements and diagrams or diagram groups in the COVID-19 Disease Map, in which they participate Novel interactions (directed) suggested by text mining and interaction databases in the COVID-19 Disease Map, in which they participate Novel interactions (directed) suggested by text mining and interaction databases Novel interactions (directed) suggested by text mining and interaction databases Figure 4. Novel regulators of protein activity Finally, we identified potential novel regulators of proteins in the C19DMap using interaction and text mining databases (see Fig 4C). These proteins take no part in the current version of the map but interact with molecules already represented in at least one of the diagrams. An example of such a novel regulator was NFE2L2, which controls the activity of HMOX1 in the context of viral infection (Kesic et al, 2011). In turn, HMOX1 controls immunomodulatory haem metabolism (Zhang et al, 2019), the mechanisms of viral replication, and is a target of SARS-CoV-2 Orf3a protein (Miao et al, 2020). The suggested NFE2L2-HMOX1 interaction is supported by the literature reports of NFE2L2 importance in COVID-19 cardiovas- cular complications due to crosstalk with the renin–angiotensin signalling pathway (Valencia et al, 2020) and potential interactions with viral entry mechanisms (Hassan et al, 2020). Interestingly, the modulation of the NFE2L2-HMOX1 axis was already proposed as a therapeutic measure for inflammatory diseases (Attucks et al, 2014), making it an appealing extension of the C19DMap. New crosstalk from interaction and text mining datasets New relationships emerging from associated interaction and text mining databases (see Section “Exploration of the networked knowledge” of the Results) suggested new pathway crosstalk (see Figs 4B and EV3). One of these was the interplay between ER stress and the immune pathways, as PPP1R15A regulates the expression of TNF and the translational inhibition of both IFN-1 and IL-6 (Smith, 2018). This finding coincided with the proposed interaction of pathways responsible for protein degradation and viral detection, as SQSTM1, an autophagy receptor and NFKB1 regulator, controls the activity of cGAS, a double-stranded DNA detector (Seo et al, 2018). Another association revealed by text mining data was ADAM17 and TNF release from the immune cells in response to ACE2-S protein interaction with SARS-CoV-1 (Haga et al, 2008), potentially increasing the risk of COVID-19 infection (Zipeto et al, 2020). This new interaction connected diagrams of the (i) “Viral replication cycle” via ACE2-S protein interactions, (ii) “Viral subver- sion of host defence mechanisms” via ER stress, (iii) “Host Existing crosstalk between COVID-19 Disease Map diagrams First, the existing pathways crosstalk emerged by matching entities between the diagrams (Figs 4A, EV1 and EV2). For instance, they link different pathways involved in type I IFN (IFN-1) signalling. Responses to RNA viruses and pathogen-associated molecular patterns (PAMPs) share common pathways, involving RIG-I/Mda-5, TBK1/IKKE and TLR signalling, leading to the production of IFN-1s, especially IFN-beta (H€acker & Karin, 2006) and IFN-alpha SARS-CoV-2 M protein affects these IFN responses by inhibiting the RIG-I:MAVS:TRAF3 complex and TBK1, preventing IRF3 phos- phorylation, nuclear translocation and activation (Zheng et al, 2020). In severe COVID-19 cases, elevated NF-kB activation 8 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors ª 2021 The Authors Marek Ostaszewski et al Marek Ostaszewski et al Molecular Systems Biology Molecular Systems Biology integrative stress response” via the renin–angiotensin system and (iv) “Host innate immune response” via pathways implicating TNF signalling. associated with impaired IFN-1 (Hadjadj et al, 2020) may be a host attempt to compensate for the lack of IFN-1 activation (Rubio et al, 2013), leading to NF-kB hyperactivation and release of pro- inflammatory cytokines. Also, SARS-CoV-1 viral papain-like proteases, contained within the nsp3 and nsp16 proteins, inhibit STING and its downstream IFN secretion (Chen et al, 2014). Perturbations in these pathways may impair the IFN response against SARS-CoV-2 and explain persistent blood viral load and an exacerbated inflammatory response in COVID-19 patients (Hadjadj et al, 2020). associated with impaired IFN-1 (Hadjadj et al, 2020) may be a host attempt to compensate for the lack of IFN-1 activation (Rubio et al, 2013), leading to NF-kB hyperactivation and release of pro- inflammatory cytokines. Also, SARS-CoV-1 viral papain-like proteases, contained within the nsp3 and nsp16 proteins, inhibit STING and its downstream IFN secretion (Chen et al, 2014). Perturbations in these pathways may impair the IFN response against SARS-CoV-2 and explain persistent blood viral load and an exacerbated inflammatory response in COVID-19 patients (Hadjadj et al, 2020). Case study: analysis of cell-specific mechanisms using single-cell expression data To investigate cell-specific mechanisms of COVID-19, we projected single-cell expression data onto the C19DMap. To this end, we calcu- lated differentially expressed genes (DEGs) for two datasets relevant to the disease. The first dataset describes non-infected bronchial secretory cells (Lukassen et al, 2020; Data ref: Lukassen et al, 2020), where we selected DEGs from three different subtypes of secretory cells dubbed (i) secretory1, (ii) secretory2 and (iii) secretory3 (tran- sient) cells. The second dataset describes SARS-CoV-2-infected intesti- nal organoids (Triana et al, 2021; Data ref: Triana et al, 2021), where we selected DEGs from (iv) infected and (v) bystander immature enterocytes from the intestinal organoids infected with SARS-CoV-2. DEGs (i), (ii) and (iii) can serve as an illustration of pathway activity across normal lung cells, while datasets (iv) and (v) demonstrate a comparison of molecular activity in cells of infected intestinal tissue. These selected datasets are available as overlays in the C19DMap and can be interactively explored, showing cell-type-specific dysregulation of particular diagrams (see Materials and Methods). Datasets projected on the C19DMap can create signatures of molecular regulation determined by the expression levels of the corre- sponding molecules. Together, multiple omics readouts and multiple measurements can increase the robustness of such signatures (De Meulder et al, 2018). This interpretation can be extended using avail- able SARS-CoV-2-related omics and interaction datasets (Bouhaddou et al, 2020) to infer which transcription factors, their target genes and signalling pathways are affected upon infection (Dugourd & Saez- Rodriguez, 2019). Combining regulatory interactions of the C19DMap with such data collections extends the scope of the analysis and may suggest new mechanisms to include in the map. Besides the visual exploration of omics datasets, the network structure of the C19DMap allows extended network analysis of viral–human protein–protein interactions (PPIs) (Gordon et al, 2020). It can be expanded by merging virus–host with human PPIs and proteomics data to discover clusters of interactions indicating human biological processes affected by the virus (Messina et al, 2020). These clusters can be interpreted by visualising them on the C19DMap diagrams to reveal additional pathways or interactions to add to the map. Visual exploration of the differential expression profiles in the C19DMap revealed that transient secretory cells specifically express molecules associated with the virus replication cycle (TMPRSS2). This suggests that these cells are more susceptible to viral entry than the other types of bronchial secretory cells. Computational analysis and modelling for hypothesis generation The standardised representation and programmatic access to the contents of the C19DMap support reproducible analytical and modelling workflows. Here, we discuss the range of possible Table 3. Examples of computational workflows using the COVID-19 Disease Map Workflow COVID-19 Disease Map contents User input Tools Output Data interpretation Online diagrams Transcriptomics, Proteomics, Metabolomics The MINERVA Platform (Gawron et al, 2016) PathVisio (Kutmon et al, 2015) Reactome (Jassal et al, 2020) Visualisation of SARS-CoV-2 mechanisms contextualised to dataa Diagrams in SIF format (via CasQ) Transcriptomics DoRothEA (Garcia-Alonso et al, 2019) Contextualised evaluation of transcription factors activity under SARS-CoV-2 infectionb Diagrams in SIF format (via CasQ) Interactome data Proteomics Network clustering (Messina et al, 2020) Identification of new SARS-CoV-2- relevant interactions Mechanistic modelling Diagrams in SIF format (via CasQ) TranscriptomicsMetabolomics HiPATHIA (Salavert et al, 2016) CARNIVAL (Liu et al, 2019) Endpoint predictionc Drug target effect prediction Discrete modelling Diagrams in SBML qual format (via CasQ) Perturbation hypothesis (loss/gain of function) CellCollective (Helikar et al, 2012) GINSim (Naldi et al, 2018a) BoolNet Perturbation outcomes:c - Real-time simulation - Attractor analysis Stochastic & Multiscale modelling Diagrams in SBML qual format (via CasQ) Perturbation hypothesis (loss/gain of function) PhysiBoSS (Letort et al, 2019): MaBoSS (Stoll et al, 2017) + PhysiCell (Ghaffarizadeh et al, 2018) Perturbation outcomes:d - Real-time simulation - Stochastic multiscale modelling Each workflow relies on the input from the C19DMap, either a direct diagram or its transformed contents, available in the GitLab repository. The workflow users may supply omics datasets to interpret them in the context of the map or test their hypotheses about how disease models will behave under specific perturbations. aExample: see Results, Case study—analysis of cell-specific mechanisms using single-cell expression data. bExample: see Results, Case study—RNA-Seq-based analysis of transcription factor activity. cExample: see Results, Case study—RNA-Seq-based analysis of pathway signalling. dhttps://colomoto.github.io/colomoto-docker/ Table 3. Examples of computational workflows using the COVID-19 Disease Map Molecular Systems Biology 17: e10387 \| 2021 9 of 22 ª 2021 The Authors Molecular Systems Biology Molecular Systems Biology Marek Ostaszewski et al approaches and demonstrate preliminary results, focusing on inter- operability, reproducibility and applicability of the methods and tools. Table 3 summarises selected computational workflows that can support data interpretation and hypothesis testing in COVID-19 research. behaviour of heterogeneous cell populations and their intercellular communications at different time scales, e.g. Data interpretation and network analysis The projection of omics data onto the C19DMap broadens and deep- ens our understanding of disease-specific mechanisms, in contrast to classical pathway enrichment analyses, which often produce lists of generic biological mechanisms. Visualisation of omics datasets on the map diagrams creates overlays, allowing interpretation of speci- fic conditions, such as disease severity or cell types (Satagopam et al, 2016). Mechanistic and dynamic computational modelling Diagrams from the C19DMap can be coupled with omics datasets to estimate their functional profiles and predict the effect of interven- tions, e.g. effects of drugs on their targets (Salavert et al, 2016). However, such an approach has a substantial computational complexity, limiting the size of the input diagrams. Large-scale mechanistic pathway modelling can address this challenge but requires transformation of diagrams into causal networks, which, combined with transcriptomics, (phospho-)proteomics or metabolo- mics data (Dugourd et al, 2021), contextualise the networks and hypotheses about intervention outcomes. Both approaches provide a set of coherent causal links connecting upstream drivers such as stimulations or pathogenic mutations to downstream changes in diagram endpoints or transcription factor activities. Dynamic modelling allows analysis of changes of molecular networks in time to understand their complexity under disease- related perturbations (Naldi et al, 2018b). C19DMap diagrams, trans- lated to SBML qual using CaSQ (see Materials and Methods), can be used in discrete modelling, using modelling software that supports SBML qual file import. Notably, multiscale processes involved in viral infection, from molecular interactions to multicellular behaviour, can be simulated using a dedicated computational architecture. In such a multiscale setup, single-cell models run in parallel to capture the Computational analysis and modelling for hypothesis generation diffusion, cell mechan- ics, cell cycle, or signal transduction (Osborne et al, 2017; preprint: Wang et al, 2020). Implementing detailed COVID-19 signalling models in the PhysiBoSS framework (Letort et al, 2019) may help better understand complex dynamics of interactions between immune system components and the host cell. 10 of 22 Molecular Systems Biology 17: e10387 \| 2021 Case study: analysis of cell-specific mechanisms using single-cell expression data Also, the interferon 1 signalling pathway was up-regulated in both secretory1 and tran- sient secretory cells. However, transient secretory cells showed up- regulation of elements up- and downstream of the pathway (IFNAR1-JAK1, and ISG15 or OAS1); in secretory1 cells, the up- regulated proteins were downstream (transcription factor AP-1). In the intestinal organoid dataset, the comparison of infected and bystander immature enterocytes confirmed the downregulation of the ACE2 receptor reported by the original article (Triana et al, 2021; Data ref: Triana et al, 2021), as visualised in the virus replica- tion cycle diagram. In addition, exploration of other affected path- ways may suggest the context of this observation – for instance, the C19DMap demonstrated the differential activity of the pyrimidine deprivation pathway, which could suggest a reduction of transcrip- tional activity as a host response to the viral infection. Enrichment analysis of diagrams indicated that mitochondrial dysfunction, apoptosis, and inflammasome activation were dysregulated in infected enterocytes. The enrichment analysis of the cell-type- specific overlays was obtained by the GSEA plugin of the C19DMap. These results can be replicated and examined directly by the users via the visual interface of the C19DMap (see https://covid.pages. uni.lu/minerva-guide/ and Materials and Methods). Case study: RNA-Seq-based analysis of pathway signalling reveals the transcription factors (TFs) related to SARS-CoV-2 infec- tion. To do so, we conducted differential expression analysis between SARS-CoV-2 infected Calu-3 human lung adenocarcinoma cell line and controls. Results were used to estimate TF activity deregulation upon viral infection. We mapped the outcomes of the TF activities to pathway diagrams of the C19DMap (see Materials and Methods). Case study: RNA-Seq-based analysis of pathway signalling Case study: RNA Seq based analysis of pathway signalling The diagrams of the C19DMap allow for a complex analysis of how the infection may affect signalling sequences in encoded pathways based on available omics data. To demonstrate this approach, we applied a mechanistic modelling algorithm that estimates the func- tional profiles of signalling circuits in the context of omics datasets. We used expression profiles from nasopharyngeal swabs of COVID- 19 patients and controls (Lieberman et al, 2020; Data ref: Lieberman et al, 2020) to calculate the differential expression profiles and derive the pathway signalling activities (see Materials and Meth- ods). The results for the interferon type I signalling diagram are shown in Fig 5. This pathway included some of the most active TFs after SARS-CoV-2 infection, such as STAT1, STAT2, IRF9 and NFKB1. These are well-known components of cytokine signalling and antiviral responses (Cheon et al, 2013; Fink & Grandvaux, 2013). Interestingly, these TFs were located downstream of various viral proteins (E, S, Nsp1, Orf7a and Orf3a) and members of the MAPK pathway (MAPK8, MAPK14 and MAP3K7). SARS-CoV-2 infection is known to promote MAPK activation, which mediates the cellular response to pathogenic infection and promotes the production of pro-inflammatory cytokines (Bouhaddou et al, 2020). Overall, these results highlighted that the molecular mecha- nisms of the response of the human cells to SARS-CoV-2 infection can be investigated by combining omics datasets with the diagrams of the C19DMap. To illustrate this approach, we focused on the results of the anal- ysis of the apoptosis pathway, also shown in Fig 6 and Table EV2. We observed an overall downregulation of both the CASP3 and CASP7 subpathways and an inhibition of the circuit ending in effector protein CASP3, possibly due to the downregulation of AKT1 and BAD and the downstream inhibition of BAX. Although the BAX downstream genes were up-regulated, the signal arriving at them was diminished by the effect of the previous nodes. Case study: RNA-Seq-based analysis of transcription factor activity As discussed above, the diagrams of the C19DMap can be coupled with omics datasets. Here, we highlight how the map systematically 10 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors Molecular Systems Biology Marek Ostaszewski et al Molecular Systems Biology Marek Ostaszewski et al Case study: RNA-Seq-based analysis of pathway signalling Although CASP8 was up-regulated, the cumulative effect of the individual node activ- ities resulted in the inhibition of CASP7. Indeed, inflammatory response via CASP8 has been described as a result of SARS-CoV-2 MYD88 NFKB_NFKBIA_complex STING1_ubiquitinated E RIPK1 FOS MAVS J U N Viral_replication_phenotype TRAF3_TANK_TBK1_IKKepsilon_complex AP-1_complex_cytoplasm RNF135 TRAF6_ubiquitinated T Y K 2 TRIM25 Nsp3 J A K 1 Orf9b S TRAF3 NFKB1_nucleus GRL0617_drug Nsp13 p50_p65_complex_nucleus Orf8_complex IRF7_cytoplasm AP-1_complex_nucleus IRF7_nucleus TRAF3_TBK1_IKBKE_complex_cytoplasm Azithromycin_drug ITCH Orf3a OAS3_rna PAMP_signalling_phenotype Orf7a OAS2_rna IRF3_nucleus S EIF2AK_rna IFNA1_cytoplasm Nsp1 OAS1_rna IFNB1_cytoplasm NFKBIA_cytoplasm MYD88_TCAM1_complex MAPK8 OAS2_cytoplasm TREML4 MAPK14 EIF2AK IRF3_cytoplasm MAP3K7 OAS3_cytoplasm 3 Orf6 IFNA1_rna OAS1_cytoplasm STAT2 IFNB1_rna Immune_Response_phenotype IRF9 Inflammation_phenotype OAS1_EIF2AK_complex STAT1 Nsp15_cytoplasm I K B K E ISRE_complex Viral_dsRNA_rna TBK1_IKBKE_complex STAT1/2_IRF9_complex IFIH1 T B K 1 IFNB1_Extracellular_Space DDX58_ubiquitinated N_cytoplasm IFNA1_Extracellular_Space RIG1_MDA5_complex IKK_complex IFNB1_IFNAR_complex pp1ab NFKB1_cytoplasm IFNAR_complex ypeM_cytoplasm RELA JAK1_TYK2_complex T A N K p50_p65_complex_cytoplasm IF IFNA1_IFNAR_complex TLR7 MAPK8/14_complex TCAM1 NLRP3_inflammasome_complex Figure 5. Interferon type I signalling pathway diagram of the COVID-19 Disease Map integrated with TF activity derived from transcriptomics data after SARS- CoV-2 infection. A zoom was applied in the area containing the most active TFs (red nodes) after infection. Node shapes: host genes (rectangles), host molecular complex (octagons), viral proteins (V shape), drugs (diamonds) and phenotypes (triangles). NFKB_NFKBIA_complex Figure 5. Interferon type I signalling pathway diagram of the COVID-19 Disease Map integrated with TF activity derived from transcriptomics data after SARS- CoV-2 infection. A oom as applied in the area containing the most acti e TFs (red nodes) after infection Node shapes host genes (rectangles) host molec lar comple (octagons) iral alling pathway diagram of the COVID-19 Disease Map integrated with TF activity derived from transcriptomics data after SARS Figure 5. Interferon type I signalling pathway diagram of the COVID-19 Disease Map integrated with TF activity derived fro CoV-2 infection. Figure 5. Interferon type I signalling pathway diagram of the COVID-19 Disease Map integrated with TF activity derived from transcriptomics data after SARS- CoV-2 infection. A zoom was applied in the area containing the most active TFs (red nodes) after infection. Node shapes: host genes (rectangles), host molecular complex (octagons), viral proteins (V shape), drugs (diamonds) and phenotypes (triangles). A zoom was applied in the area containing the most active TFs (red nodes) after infection. Node shapes: host genes (rectangles), hos proteins (V shape), drugs (diamonds) and phenotypes (triangles). ontaining the most active TFs (red nodes) after infection. Case study: RNA-Seq-based analysis of pathway signalling Node shapes: host genes (rectangles), host molecular complex (octagons), vira ds) and phenotypes (triangles). Molecular Systems Biology 17: e10387 \| 2021 11 of 22 ª 2021 The Authors Marek Ostaszewski et al Molecular Systems Biology Figure 6. Representation of the activation levels of apoptosis pathway in nasopharyngeal swabs from SARS-CoV-2-infected individuals. Activation levels were calculated using transcriptional data from GSE152075 and the Hipathia mechanistic pathway analysis algorithm. Each node represents a gene (ellipse), a human metabolite/viral protein (circle) or a function (rectangle). The pathway is composed of circuits from a receptor to an effector. Significant differential regulation of circuits in infected cells is highlighted by colour arrows (blue: inactive in infected cells). The colour of elements corresponds to the level of differential expression in SARS-CoV-2-infected human nasopharyngeal swabs versus non-infected nasopharyngeal human swabs. Blue: downregulated, red: up-regulated and white: no statistically significant differential expression. Figure 6. Representation of the activation levels of apoptosis pathway in nasopharyngeal swabs from SARS-CoV-2-infected individuals. Activation levels were calculated using transcriptional data from GSE152075 and the Hipathia mechanistic pathway analysis algorithm. Each node represents a gene (ellipse), a human metabolite/viral protein (circle) or a function (rectangle). The pathway is composed of circuits from a receptor to an effector. Significant differential regulation of circuits in infected cells is highlighted by colour arrows (blue: inactive in infected cells). The colour of elements corresponds to the level of differential expression in SARS-CoV-2-infected human nasopharyngeal swabs versus non-infected nasopharyngeal human swabs. Blue: downregulated, red: up-regulated and white: no statistically significant differential expression. presentation of the activation levels of apoptosis pathway in nasopharyngeal swabs from SARS-CoV-2-infected individuals. Figure 6. Representation of the activation levels of apoptosis pathway in nasopharyngeal swabs from SARS-CoV Activation levels were calculated using transcriptional data from GSE152075 and the Hipathia mechanistic pathway analysis algorithm. Each node represents a gene (ellipse), a human metabolite/viral protein (circle) or a function (rectangle). The pathway is composed of circuits from a receptor to an effector. Significant differential regulation of circuits in infected cells is highlighted by colour arrows (blue: inactive in infected cells). The colour of elements corresponds to the level of differential expression in SARS-CoV-2-infected human nasopharyngeal swabs versus non-infected nasopharyngeal human swabs. Blue: downregulated, red: up-regulated and white: no statistically significant differential expression. data interpretation. Case study: RNA-Seq-based analysis of pathway signalling The diagrams of the C19DMap describe molecu- lar mechanisms of COVID-19, grounded in the relevant published SARS-CoV-2 research, completed where necessary by mechanisms discovered in related beta-coronaviruses. infection (Li et al, 2020), and the role of caspase-induced apoptosis has been established, together with the ripoptosome/caspase-8 complex, as a pro-inflammatory checkpoint (Chauhan et al, 2018), which may be triggering up-regulation of such processes in other pathways. Overall, our findings recapitulate reported outcomes and provide explanations of the effects of interactions on pathway elements. We developed the contents of the C19DMap de novo in an unprecedented, community-driven effort involving independent biocurators, as well as WikiPathway and Reactome biocurators. Over forty diagrams with molecular resolution have been constructed since March 2020, shared across three platforms. In this work, we combined and harmonised expertise in biocuration across multiple teams, formulated clear guidelines and cross-reviewed the outcomes of our work with domain experts. Although the approach of community curation was applied in the past (Slayden et al, 2013; Naithani et al, 2019), we are not aware of any curation effort on a similar scale for a single human disease to date. 12 of 22 Molecular Systems Biology 17: e10387 \| 2021 Discussion Our knowledge of COVID-19 molecular mechanisms is growing at a great speed, fuelled by global research efforts to investigate the pathophysiology of SARS-CoV-2 infection. Keeping an overview of all the findings, many of which focus on individual molecules, is a great challenge just one year after the start of the pandemic. The C19DMap aggregates this knowledge into molecular interac- tion diagrams, making it available for visual exploration by life science and clinical researchers and analysis by computational biologists. In this work, we established a computational framework accom- panying the biocuration process, integrating interaction databases and text mining solutions to accelerate diagram building. This allowed us not only to enrich particular diagrams but also to explore crosstalk between them and prioritise key novel regulators of the encoded pathways. Thanks to the interoperability of dif- ferent systems biology formats, we performed this analysis for diagrams constructed in different biocuration environments, extending current advances in pathway interoperability (Bohler et al, 2016). The map complements and interfaces with other COVID-19 resources such as interaction databases (Licata et al, 2020; Perfetto et al, 2020), protein-centric resources (preprint: Lubin et al, 2020) and relevant omics data repositories (Delorey et al, 2021) by provid- ing a context to particular pieces of information and helping with 12 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors Marek Ostaszewski et al Molecular Systems Biology Moreover, by developing reproducible analysis pipelines for the contents of the C19DMap, we promoted early harmonisation of formats, support of standards and transparency in all steps. Prelimi- nary results of such efforts are illustrated in the case studies above. Notably, the biocurators and domain experts participated in the analysis helped to evaluate the outcomes and corrected the curated content if necessary. This way, we improve the quality of the analy- sis and increase the reliability of the models used to generate testable predictions. The project aims to provide the tools to deepen our understand- ing of the mechanisms driving the infection and help boost drug development supported by testable suggestions. It offers insights into the dynamic nature of the disease at the molecular level and its propagation at the systemic level. Thus, it provides a platform for a precise formulation of models, accurate data interpretation, the potential for disease mitigation and drug repurposing. Discussion In the longer run, the constantly growing C19DMap content will be used to facili- tate the finding of robust signatures related to SARS-CoV-2 infection predisposition, disease evolution or response to various treatments, along with the prioritisation of new potential drug targets or drug candidates. The C19DMap is an open access repository of diagrams and reproducible workflows for content conversion and analysis. We followed FAIR principles in making our content and code available to the entire research community (Wilkinson et al, 2016). Impor- tantly, FAIRDOMHub is an essential platform for disseminating all information about the project and linking contributors to their contributions. The C19DMap Community is open and expanding as more people with complementary expertise join forces. Using the FAIR approach for sharing the results of our work makes this effort more scalable. Recognising individual contributions and open access policy promote the distributed knowledge building and generation of research data. This approach to an emerging worldwide pandemic leveraged the capacity and expertise of an entire swath of the bioinformatics community, bringing them together to improve the way we build and share knowledge. By aligning our efforts, we strive to provide COVID-19-specific pathway models, synchronise content with simi- lar resources and encourage discussion and feedback at every stage of the curation process. Such an approach may help to deal with new waves of COVID-19 or similar pandemics in the long-term perspective. Materials and Methods Reagents and Tools table Layout-aware systems biology formats Individual diagrams were encoded in systems biology layout-aware formats (see below) by biocurators using CellDesigner (Matsuoka et al, 2014), Newt (https://newteditor.org), SBGN-ED (Czauderna et al, 2010) and ySBGN (https://github.com/sbgn/ySBGN). This community-based curation was coordinated by sharing curation topics, e.g. relevant pathways or particular SARS-CoV-2 proteins across the community to cover the available literature and identify synergies. Curation guidelines (https://fairdomhub.org/documents/ 661) were established to ensure proper representation and annota- tion of the key features of the diagrams. Curation guidelines for logi- cal models (Niarakis et al, 2020) were followed. Regular technical reviews of the diagrams were performed following a previously established checklist to harmonise their content. The diagrams are stored and versioned in a GitLab repository (https://gitlab.lcsb.uni. lu/covid/models). Individual diagrams are visualised in the MINERVA Platform (Gawron et al, 2016). The entry-level view is based on Fig 2. The diagrams are available in SBML format (Keating et al, 2020), allowing computational modelling of biological processes. SBML stores visual information about encoded elements and reactions using render (Bergmann et al, 2018) and layout (Gauges et al, 2015) packages. An early version of SBML adapted by CellDesigner allows storing layout and rendering information. Systems Biology Graphical Notation (SBGN) format is a graphical standard for visual encodings of molecular entities and their interactions, implemented using SBGNML (Bergmann et al, 2020) for encoding the layout of SBGN maps and their annotations. Finally, GPML (Kutmon et al, 2015) is a structured XML format for computable representation of biological knowledge used by the WikiPathways platform. Interactions and interacting entities are annotated following a uniform, persistent identification scheme, using either MIRIAM Registry or Identifiers.org (Juty et al, 2012) and the guidelines for annotations of computational models. Viral protein interactions are explicitly annotated with their taxonomy identifiers to highlight find- ings from strains other than SARS-CoV-2. Stable protein complexes from SARS-CoV-2 and SARS are annotated using the Complex Portal. Reactome (Jassal et al, 2020) biocuration efforts initially focused on SARS-CoV-1 and its proteins, and their functions are extensively documented in the experimental literature. Reactome curators were assigned a subpathway from the viral life cycle, a host pathway or potential therapeutics. Curators were supported by an editorial manager and a dedicated SARS literature triage process. The result- ing set of pathways for SARS-CoV-1 provided the basis for computa- tional inference of the corresponding SARS-CoV-2 pathways based on structural and functional homologies between the two viruses. Reagents and Tools table Reagent/Resource Reference or Source Identifier or Catalog Number Software CellDesigner v4.4.2 http://www.celldesigner.org (Matsuoka et al, 2014) Newt v3.0 https://newteditor.org SBGN-ED Czauderna et al (2010) ySBGN https://github.com/sbgn/ySBGN The MINERVA Platform v15.1.4 https://minerva-web.lcsb.uni.lu (Gawron et al, 2016) Reactome https://reactome.org (Jassal et al, 2020) WikiPathways https://www.wikipathways.org (Slenter et al, 2018) PathVisio v3.3.0 https://pathvisio.github.io (Kutmon et al, 2015) INDRA Gyori et al (2017) AILANI COVID-19 https://ailani.ai BioKB https://biokb.lcsb.uni.lu/topic/DOID:0080599 OpenNLP + GNormPlus https://opennlp.apache.org/ (Wei et al, 2015) COVIDminer https://rupertoverall.net/covidminer rWikipathways v 1.12 10.18129/B9.bioc.rWikiPathways OmniPathR https://github.com/saezlab/OmnipathR The MINERVA Conversion API v15.1 https://minerva.pages.uni.lu/doc/api/15.1/converter/ (Hoksza et al, 2020) cd2sbgml https://github.com/sbgn/cd2sbgnml (Balaur et al, 2020) rnef2sbgn https://github.com/golovatenkop/rnef2sbgn Seurat v4.0 https://satijalab.org/seurat/ (Hao et al, 2021) DESeq2 10.18129/B9.bioc.DESeq2 (Love et al, 2014) Viper v1.26.0 10.18129/B9.bioc.viper (Alvarez et al, 2016) DoRothEA v1.4.1 10.18129/B9.bioc.dorothea (Garcia-Alonso et al, 2019) CaSQ v0.9.11 Aghamiri et al (2020) Identifier or Catalog Number Marek Ostaszewski et al Molecular Systems Biology Reagents and Tools table (continued) Reagent/Resource Reference or Source Identifier or Catalog Number CoV-HiPathia Rian et al (2021) Datasets IMEx Consortium COVID-19 dataset Perfetto et al (2020) SIGNOR 2.0 COVID-19 dataset Licata et al (2020) OmniPath T€urei et al (2021) INDRA EMMAA Collection, accessed: 2020.12.01 https://emmaa.indra.bio/dashboard/covid19 RNA-Seq transcriptomic single-cell profiles https://eils-lung.cells.ucsc.edu https://doi.org/10.6084/m9.figshare.11981034.v1 (Lukassen et al, 2020) SARS-CoV-2-infected intestinal organoids Triana et al (2021) GSE156760 SARS-CoV-2-infected Calu-3 cells Blanco-Melo et al (2020) GSE147507 SARS-CoV-2 nasopharyngeal swabs Lieberman et al (2020) GSE152075 Reagents and Tools table (continued) cellular processes relevant to the virus–host interactions (https:// www.wikipathways.org/index.php/Portal:COVID-19). cellular processes relevant to the virus–host interactions (https:// www.wikipathways.org/index.php/Portal:COVID-19). Layout-aware systems biology formats The computationally inferred SARS-CoV-2 infection pathway events and entities were then reviewed and manually curated using published SARS-CoV-2 experimental data. Reactome diagrams are available via a dedicated pathway collection (https://reactome.org/ PathwayBrowser/#/R-HSA-9679506). 14 of 22 Molecular Systems Biology 17: e10387 \| 2021 Text and figure mining Text mining was performed on the CORD-19: COVID-19 Open Research Dataset dataset (preprint: Lu Wang et al, 2020). INDRA (Gyori et al, 2017), AILANI COVID-19 (https://ailani.ai) and BioKB processed CORD-19 dataset (https://biokb.lcsb.uni.lu/topic/DOID: 0080599), with their results available programmatically via REST API and SPARQL interfaces. An OpenNLP-based (https://opennlp. apache.org/) text mining workflow using GNormPlus (Wei et al, 2015) was applied to the CORD-19 dataset and the collection of MEDLINE abstracts associated with the genes in the SARS-CoV-2 PPI network (Gordon et al, 2020) using the Entrez GeneRIFs, https://www.ncbi.nlm.nih.gov/gene/about-generif. (https://gitlab. lcsb.uni.lu/covid/models/-/tree/master/Resources/Text%20mining). Also, we used data from 221 CORD-19 dataset figures using a dedi- cated Figure Mining Workflow (Hanspers et al, 2020), with results available at https://gladstone-bioinformatics.shinyapps.io/shiny-cov idpathways. Results of text mining were accessed by the curators in the form of molecular interactions with references to the articles and to sentences from which these interactions were derived. We systematically aligned the C19DMap with assembled INDRA State- ments, both to enrich and to extend the map (see “Crosstalk analysis” below). The content of INDRA and AILANI COVID-19 was accessible via interfaces that allow users to provide natural language queries, such as “What are COVID-19 risk factors?” or “What are the interac- tors of ACE2?”, facilitating extracting knowledge from the results of text mining workflows. The results of the INDRA workflow were visualised using the COVIDminer project (https://rupertoverall.net/ covidminer). Each extracted statement describes a directed interac- tion between two gene products, small molecules or biological processes. The causal network representing the COVIDminer data- base is browsable through a web interface. The results of the OpenNLP-based text mining workflow were imported into a BioKC biocuration platform for structured processing and SBML export. Molecular Systems Biology uniroma2.it/covid/). The Elsevier Pathway Collection (Daraselia et al, 2004; Nesterova et al, 2020) COVID-19 dataset comprises manually reconstructed and annotated pathway diagrams. State- ments about molecular interactions are extracted into a knowledge graph by a dedicated text mining technology adapted for extracting facts about viral proteins and viruses from the literature. These interactions were filtered for experimental evidence, used for path- way reconstruction and made openly available (http://dx.doi.org/ 10.17632/d55xn2c8mw.1). Information from OmniPath (T€urei et al, 2021) on existing interactions gathered from pathway and interac- tion databases was used in a programmatic way to suggest cell- specific interactions and cell–cell interactions specific to immune reactions. quality control of the text mining data were obtained from Omni- Path using the OmnipathR package (https://github.com/saezlab/ OmnipathR). We filtered text mining interactions of the EMMAA dataset for “belief” of 0.8 or higher and retained those matching the direction and interacting molecules to the OmniPath dataset. We call this filtered group of interactions “EMMAA-OP interactions”. g Crosstalk between C19DMap diagrams was calculated based on the HGNC identifiers of their elements. For simplification, all elements of the same diagram were considered to be interacting with each other. Three types of networks were constructed: existing cross- talk, new crosstalk and new regulators. Diagram groups followed the scheme in the list of C19DMap diagrams (Table EV1). The networks were visualised using Cytoscape (Shannon et al, 2003). The colour code is common for the networks: light green for nodes representing a diagram or a diagram group, light blue for nodes having one or two neighbours, yellow for nodes having three or four neighbours and red for nodes with five or more neighbours. Diagram nodes have pre- fixes indicating their provenance. Diagram groups have no prefixes, as they combine diagrams across platforms. Existing crosstalk between diagrams, or groups of diagrams, was calculated by identify- ing shared HGNC identifiers linking diagrams or groups of diagrams. To calculate new crosstalk between diagrams, we merged the EMMAA-OP interactions with the network of existing crosstalk and kept only those new interactions that link at least two upstream and two downstream diagrams or diagram groups. To calculate new upstream regulators of existing diagrams, we merged the EMMAA-OP interactions with the network of existing crosstalk. We kept interac- tions with source elements, not within existing diagrams, and target elements in at least one existing diagram or diagram group. Diagram interoperability and translation for computational modelling Bidirectional translation of curated diagrams between CellDesigner and SBGNML formats is supported by the MINERVA Conversion API https://minerva.pages.uni.lu/doc/api/15.1/converter/ (Hoksza et al, 2020), and cd2sbgml converter https://github.com/sbgn/ cd2sbgnml (Balaur et al, 2020). The MINERVA Conversion API supports bidirectional translation between CellDesigner, SBML and GPML. Unidirectional translation from Reactome format to GPML is supported by the Reactome-to-WikiPathways converter (Bohler et al, 2016). Diagrams in the RNEF format of Elsevier Pathway Studio were translated to SBGNML using a dedicated rnef2sbgn soft- ware (https://github.com/golovatenkop/rnef2sbgn). p g g p g The C19DMap diagrams (Table EV1) in CellDesigner format were translated using CaSQ (Aghamiri et al, 2020) into executable Boolean networks. Conversion rules and logical formulae were inferred according to the topology and the annotations of the diagrams. SBML-qual files (Chaouiya et al, 2013) generated with CaSQ (Aghamiri et al, 2020) retained their references, annotations and layout of the original CellDesigner file. They can be used for in silico simulations and analysis with CellCollective (Helikar et al, 2012), GINsim (Naldi et al, 2018a) or MaBoSS (Stoll et al, 2017). CaSQ was adapted to produce SIF files necessary for HiPATHIA (Hidalgo et al, 2017) and CARNIVAL (Liu et al, 2019) pipelines. The C19DMap GitLab repository (https://gitlab.lcsb.uni.lu/covid/ models) was configured to translate stable versions of diagrams into SBML qual and SIF files. The diagrams were translated to XGMML using Cytoscape and GINSim. Interaction databases The biocuration process was supported by interaction and pathway databases storing structured, annotated and curated information about COVID-19 virus–host interactions. The IMEx Consortium (Meldal et al, 2019) dataset (Perfetto et al, 2020) contains curated Coronaviridae-related interaction data from reviewed manuscripts and preprints, resulting in a dataset of roughly 7,300 interactions extracted from over 250 publications, including data from SARS- CoV-2, SARS, CoV, and other strains of Coronaviridae. The dataset is updated with every release of IMEx data and is open access (https://www.ebi.ac.uk/intact/resources/datasets#coronavirus). The SIGNOR 2.0 (Licata et al, 2020) dataset contains manually anno- tated and validated signalling interactions related to the host–virus interaction, including cellular pathways modulated during SARS- CoV-2 infection. The dataset was constructed from the literature on causal interactions between SARS-CoV-2, SARS-COV-1, MERS proteins and the human host and is openly available (https://signor. The WikiPathways (Slenter et al, 2018) diagrams were constructed using PathVisio (Kutmon et al, 2015), with annotation of pathway elements from the integrated BridgeDb identifier mapping framework (van Iersel et al, 2010). All pathways are stored in GPML format (Kutmon et al, 2015). The WikiPathways diagrams are available via a dedicated pathway portal, grouping pathway models specific to SARS-CoV-2, other coronaviruses and general 14 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors Molecular Systems Biology Marek Ostaszewski et al ª 2021 The Authors Crosstalk analysis Crosstalk analysis was performed for the list of C19DMap diagrams (Table EV1). The code is available at: https://gitlab.lcsb.uni.lu/ covid/models/-/tree/master/Resources/Crosstalks. Individual diagrams were accessed via the API of the MINERVA Platform, WikiPathway diagrams via the rWikipathways package (https://github.com/ wikipathways/rWikiPathways) and Reactome diagrams via the Reactome API. Text mining interactions are from the INDRA EMMAA Collection (https://emmaa.indra.bio/dashboard/covid19), dataset timestamp: 2020-12-01-21-05-54. Verified molecular interactions for Molecular Systems Biology 17: e10387 \| 2021 15 of 22 ª 2021 The Authors Molecular Systems Biology Molecular Systems Biology Marek Ostaszewski et al RNA-seq-based analysis of pathway signalling The CoV-HiPathia (Rian et al, 2021) web tool was used to calculate the level of activity of the subpathways of the apoptosis diagram (https://fairdomhub.org/models/712) from the C19DMap. RNA-Seq transcriptomic profiles come from a public dataset of nasopharyngeal swabs from 430 individuals with SARS-CoV-2 and 54 negative controls, Gene Expression Omnibus reference GSE152075 (Lieberman et al, 2020; Data ref: Lieberman et al, 2020). RNA-Seq gene expres- sion data with the trimmed mean of M-values (TMM) normalisation (Robinson et al, 2010) were rescaled to the range [0;1] for the calcu- lation of the signal and normalised using quantile normalisation (Bol- stad et al, 2003). Normalised gene expression values and the experimental design (case/control sample names files) were uploaded to CoV-Hipathia to calculate the level of activation of the signalling in the selected diagram. A case/control contrast with a Wilcoxon test was used to assess differences in signalling activity between the two conditions. To reproduce the results, files with normalised gene expression data and the experimental design can be generated using the code https://gitlab.lcsb.uni.lu/covid/models/-/tree/master/ Resources/Hipathia/data_preprocessing. These files can then be used in CoV-HiPathia at http://hipathia.babelomics.org/covid19/ under the “Differential signalling” tab. Diagrams from the C19DMap can be selected in the “Pathway source” section, under “Disease Maps Community curated pathways”. Data availability COVID-19 Disease Map diagrams are available via: -the GitLab repository (https://gitlab.lcsb.uni.lu/covid/models). -WikiPathways collection (http://covid.wikipathways.org). -Reactome collection (https://reactome.org/PathwayBrowser/#/R- HSA-9679506). Workflows, executable models and network models are available via: -the GitLab repository (https://gitlab.lcsb.uni.lu/covid/models). -FAIRDOMHub (https://fairdomhub.org/projects/190). Calculation and visualisation of single-cell RNA-Seq expression profiles results of this case study are available at https://github.com/ saezlab/Covid19. RNA-Seq transcriptomic single-cell profiles were calculated for (i) non-infected airway cells (Lukassen et al, 2020; Data ref: Lukassen et al, 2020) and (ii) SARS-CoV-2-infected intestinal organoids (Tri- ana et al, 2021; Data ref: Triana et al, 2021). The Seurat package (Hao et al, 2021) was used to calculate cell-specific transcriptional profiles. For dataset (i), differential expression was calculated using every cell type against remaining cell types and applying the FindAllMarkers function of the Seurat package with min pct 0.25 and log fold change threshold 0.25. For dataset (ii), the cells were classified into bystander or infected based on the absence or pres- ence of SARS-CoV-2 mRNA measured by scRNAseq (Triana et al, 2021; Data ref: Triana et al, 2021). Differential expression was calculated by contrasting the mock organoids with the bystander or infected cells after 12 h or 24 h of treatment. Expression profiles for the following cell types and conditions were selected for visualisa- tion and enrichment analysis: for dataset (i), three types of secretory cells; and for dataset (ii), infected and bystander immature entero- cytes 24 h post-infection versus mock. The datasets were selected to recapitulate the findings in the original papers and demonstrate the capability of the C19DMap for cell-specific data interpretation. Selected differentially expressed genes (DEGs) were prepared for visualisation in the MINERVA Platform as follows. Differential expression values were normalised to [1,1] range by dividing by three and setting the outliers to their respective border values. Expression values and their corresponding HGNC symbols were used to create visual overlays in the C19DMap in the MINERVA Platform (https://covid19map.elixir-luxembourg.org/ minerva/index.xhtml?id=covid19_map_17Jun21, “Overlays” tab). On-the-fly exploration and enrichment analyses using the GSEA plugin (Hoksza et al, 2019) are described in a dedicated guide (https://covid.pages.uni.lu/minerva-guide/). Complete expression analysis and transformation scripts are available in RMarkdown files at https://gitlab.lcsb.uni.lu/covid/models/-/tree/master/Reso urces/Omics%20analysis. 16 of 22 Molecular Systems Biology 17: e10387 \| 2021 RNA-Seq-based analysis of transcription factor activity q y f p f y RNA-Seq transcriptomic profiles of SARS-CoV-2 infection come from SARS-CoV-2-infected Calu-3 cells measured 24 h after infection, Gene Expression Omnibus reference GSE147507 (Blanco-Melo et al, 2020; Data ref: Blanco-Melo et al, 2020). Differential expression analysis of the transcript abundances between conditions was performed with DESeq2 (Love et al, 2014). The resulting t-values from the differential expression analysis were used to estimate the effect of SARS-CoV-2 at the transcription factor (TF) activity level. This analysis was performed using the software Viper (Alvarez et al, 2016) algorithm coupled with TF–target interactions from DoRothEA (Garcia-Alonso et al, 2019). DoRothEA TF–target interactions have a confidence level based on the reliability of their source, which ranges from A (most reliable) to E (least reliable). Here, interactions with confidence levels A, B and C were selected. Activities of TFs having at least five different targets were computed. The TFs normalised enrichment score from the Viper output was mapped on the “Interferon type I signalling pathway diagram” (https:// fairdomhub.org/models/713) of the C19DMap using the SIF files generated by CaSQ. The resulting network was visualised using Cytoscape (Shannon et al, 2003). Notebooks to reproduce the Expanded View for this article is available online. Expanded View for this article is available online. Conflict of interest A. Niarakis collaborates with SANOFI-AVENTIS R&D via a public–private part- nership grant (CIFRE contract, n° 2020/0766). D. Maier and A. Bauch are employed at Biomax Informatics AG and will be affected by any effect of this publication on the commercial version of the AILANI software. J.A. Bachman and B. Gyori received consulting fees from Two Six Labs, LLC. T. Helikar has served as a shareholder and/or has consulted for Discovery Collective, Inc. R. Balling and R. Schneider are founders and shareholders of MEGENO S.A. and ITTM S.A. J. Saez-Rodriguez receives funding from GSK and Sanofi and consul- tant fees from Travere Therapeutics. The remaining authors have declared that they have no Conflict of interest. Blanco-Melo D, Nilsson-Payant BE, Liu W-C, Uhl S, Hoagland D, Møller R, Jordan TX, Oishi K, Panis M, Sachs D et al (2020) imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell 181: 1036 – 1045 Blanco-Melo D, Nilsson-Payant BE, Liu W-C, Uhl S, Hoagland D, Møller R, Jordan TX, Oishi K, Panis M, Sachs D et al (2020) Gene Expression Omnibus GSE147507 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE147507). [DATASET] Bohler A, Wu G, Kutmon M, Pradhana LA, Coort SL, Hanspers K, Haw R, Pico AR, Evelo CT (2016) Reactome from a WikiPathways perspective. PLoS Comput Biol 12: e1004941 Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinforma Oxf Engl 19: 185 – 193 Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinforma Oxf Engl 19: 185 – 193 Bouhaddou M, Memon D, Meyer B, White KM, Rezelj VV, Correa Marrero M, Polacco BJ, Melnyk JE, Ulferts S, Kaake RM et al (2020) The global phosphorylation landscape of SARS-CoV-2 infection. Cell 182: 685 – 712 Molecular Systems Biology Marek Ostaszewski et al Governments (A. Renz); German Center for Infection Research (DZIF), grant no 8020708703 (A. Dr€ager); The Netherlands Organisation for Health Research and Development (ZonMw), grant no 10430012010015, (M. Kutmon, S. Coort, F. Ehrhart, N. Pham, E.L. Willighagen, C.T. Evelo); H2020 Marie Skłodowska-Curie Actions, grant number 765274 (J. Scheel); National Institutes of Health, USA (NIH), grant number U41 HG003751 (L.D. Stein). The development of Reactome is supported by grants from the US National Institutes of Health (U41 HG003751) and the European Molecular Biology Laboratory. Attucks OC, Jasmer KJ, Hannink M, Kassis J, Zhong Z, Gupta S, Victory SF, Guzel M, Polisetti DR, Andrews R et al (2014) Induction of heme oxygenase I (HMOX1) by HPP-4382: a novel modulator of Bach1 activity. PLoS One 9: e101044 Balaur I, Roy L, Mazein A, Karaca SG, Dogrusoz U, Barillot E, Zinovyev A (2020) cd2sbgnml: bidirectional conversion between cell designer and SBGN formats. Bioinform Oxf Engl 36: 2620 – 2622 Bao C, Liu X, Zhang H, Li Y, Liu J (2020) Coronavirus disease 2019 (COVID-19) CT findings: A Systematic Review and Meta-analysis. J Am Coll Radiol JACR 17: 701 – 709 Author contributions Biol Open 9: bio053934 Bauch A, Pellet J, Schleicher T, Yu X, Gelemanovic A, Cristella C, Fraaij PL, Polasek O, Auffray C, Maier D et al (2020) Informing epidemic (research) responses in a timely fashion by knowledge management - a Zika virus use case. Biol Open 9: bio053934 Bello-Perez M, Sola I, Novoa B, Klionsky DJ, Falco A (2020) Canonical and noncanonical autophagy as potential targets for COVID-19. Cells 9: 1619 Bello-Perez M, Sola I, Novoa B, Klionsky DJ, Falco A (2020) Canonical and noncanonical autophagy as potential targets for COVID-19. Cells 9: 1619 Bergmann FT, Czauderna T, Dogrusoz U, Rougny A, Dr€ager A, Toure V, Mazein A, Blinov ML, Luna A (2020) Systems biology graphical notation markup language (SBGNML) version 0.3. J Integr Bioinforma 17: 20200016 Bergmann FT, Keating SM, Gauges R, Sahle S, Wengler K (2018) SBML level 3 package: Render, Version 1, Release 1. J Integr Bioinforma 15: 20170078 Berthelot J-M, Liote F (2020) COVID-19 as a STING disorder with delayed over-secretion of interferon-beta. EBioMedicine 56: 102801 Bello-Perez M, Sola I, Novoa B, Klionsky DJ, Falco A (2020) Canonical and noncanonical autophagy as potential targets for COVID-19. Cells 9: 1619 Bergmann FT, Czauderna T, Dogrusoz U, Rougny A, Dr€ager A, Toure V, Mazein A, Blinov ML, Luna A (2020) Systems biology graphical notation markup language (SBGNML) version 0.3. J Integr Bioinforma 17: 20200016 Bergmann FT, Keating SM, Gauges R, Sahle S, Wengler K (2018) SBML level 3 package: Render, Version 1, Release 1. J Integr Bioinforma 15: 20170078 Berthelot J-M, Liote F (2020) COVID-19 as a STING disorder with delayed over-secretion of interferon-beta. EBioMedicine 56: 102801 Author contributions MO, AN, AM and IK planned and coordinated the project. RP, AO-R, J-MR, RF, VO and SM advised the project as domain experts. MO, AN, AM, IK, VS, SSA, MLA, EG, AR, GF, CM, BB, GF, LCMG, JS, MH, SG, JS, HB, TC, FS, AM, MPL, AF, YH, NH, TGY, AD, AR, MN, ZB, FM, DB, LF, MC, MR, VN, JV, LS, MW, EEA, JS, JZ, KO, JT, EK, GYS, KH, MK, SC, LE, FE, DABR, DS, MM, NP, RH, BJ, LM, MO-M, ASR, KR, VS, RS, CS and TV curated and reviewed the diagrams. MO, PG, ES, LH, VS, GW, AR, MG, SO, CG and XH designed, developed and implemented key elements of the data sharing and communication infrastructure. RW. Overall, DM, AB, BMG, JAB, CV, VG, MV, PP, LL, MI, FS, AN, AY and AW designed and developed the contents of interac- tion and pathway databases, and text mining platforms and their visualisation and interoperability functionalities. AN, DT, AL, OB, SS, AV, ME, MP, KR, TH, BLP, DM, AT, MO, BDM, SB, AD, AN, VN and LC developed format interoperability, analysis and modelling workflows. CS, ED, TK, TF, FA, JSB, JH, OW, ELW, ARP, CTE, MEG, LS, HH, PD’E, JS-R, JD, AV, HK, EB, CA, RB and RS defined the strategy and scope of the project and revised its progress. MO, AN, AM and IK wrote the manuscript. AO-R, IK and AM designed the overview figures. AW, PD’E, JSB and LDS revised and contributed significantly to the structure of the manuscript. All authors have revised, read and accepted the manuscript in its final form. Bastard P, Rosen LB, Zhang Q, Michailidis E, Hoffmann H-H, Zhang Y, Dorgham K, Philippot Q, Rosain J, Beziat V et al (2020) Auto-antibodies against type I IFNs in patients with life-threatening COVID-19. Science 370: eabd4585 Batra N, De Souza C, Batra J, Raetz AG, Yu A-M (2020) The HMOX1 pathway as a promising target for the treatment and prevention of SARS-CoV-2 of 2019 (COVID-19). Int J Mol Sci 21: 6412 Bauch A, Pellet J, Schleicher T, Yu X, Gelemanovic A, Cristella C, Fraaij PL, Polasek O, Auffray C, Maier D et al (2020) Informing epidemic (research) responses in a timely fashion by knowledge management - a Zika virus use case. Acknowledgements We would like to thank Andjela Tatarovic, architect, and Gina Crovetto, a researcher in the field of cancer, for their help with the design of the top-level view diagrams. We would like to acknowledge the Responsible and Reproducible Research (R3) team of the Luxembourg Centre for Systems Biomedicine for supporting the project and providing necessary communication and data shar- ing resources. The work presented in this paper was carried out using the ELIXIR Luxembourg tools and services. This study was supported by the Luxembourg National Research Fund (FNR) COVID-19 Fast-Track grant programme, grant COVID-19/2020-1/14715687/CovScreen (E. Glaab); European Commission, INFORE grant H2020-ICT-825070 (A. Montagud, M. Ponce de Leon, M. Vazques and A. Valencia); European Commission, PerMedCoE grant H2020-ICT-951773 (A. Montagud, M. Ponce de Leon, M. Vazques and A. Valencia) the Federal Ministry of Education and Research (BMBF, Germany) and the Baden-W€urttemberg Ministry of Science, the Excellence Strategy of the German Federal and State 16 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors Molecular Systems Biology Molecular Systems Biology Molecular Systems Biology Molecular Systems Biology Marek Ostaszewski et al multi-omics data with prior knowledge to generate mechanistic hypotheses. Mol Syst Biol 17: e9730 Dugourd A, Saez-Rodriguez J (2019) Footprint-based functional analysis of multiomic data. Curr Opin Syst Biol 15: 82 – 90 Fang R, Wang C, Jiang Q, Lv M, Gao P, Yu X, Mu P, Zhang R, Bi S, Feng J-M et al (2017) (2017) NEMO-IKKb are essential for IRF3 and NF-jB activation in the cGAS-STING pathway. J Immunol Baltim Md 199: 3222 – 3233 Fink K, Grandvaux N (2013) STAT2 and IRF9: Beyond ISGF3. JAK-STAT 2: e27521 Frieman M, Yount B, Heise M, Kopecky-Bromberg SA, Palese P, Baric RS (2007) Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane. J Virol 81: 9812 – 9824 Chen S, Jonas F, Shen C, Hilgenfeld R, Higenfeld R (2010) Liberation of SARS- CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode. Protein Cell 1: 59 – 74 multi-omics data with prior knowledge to generate mechanistic hypotheses. Mol Syst Biol 17: e9730 Dugourd A, Saez-Rodriguez J (2019) Footprint-based functional analysis of multiomic data. Curr Opin Syst Biol 15: 82 – 90 Chen X, Yang X, Zheng Y, Yang Y, Xing Y, Chen Z (2014) SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex. Protein Cell 5: 369 – 381 Fink K, Grandvaux N (2013) STAT2 and IRF9: Beyond ISGF3. JAK-STAT 2: e27521 Chen Y, Feng Z, Diao B, Wang R, Wang G, Wang C, Tan Y, Liu L, Wang C, Liu Y et al (2020b) The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly decimates human spleens and lymph nodes. medRxiv https://doi.org/10.1101/2020.03.27.20045427 [PREPRINT] Frieman M, Yount B, Heise M, Kopecky-Bromberg SA, Palese P, Baric RS (2007) Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane. J Virol 81: 9812 – 9824 Cheon H, Holvey-Bates EG, Schoggins JW, Forster S, Hertzog P, Imanaka N, Rice CM, Jackson MW, Junk DJ, Stark GR (2013) IFNb-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage. Molecular Systems Biology EMBO J 32: 2751 – 2763 Fukushi M, Yoshinaka Y, Matsuoka Y, Hatakeyama S, Ishizaka Y, Kirikae T, Sasazuki T, Miyoshi-Akiyama T (2012) Monitoring of S protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus. J Virol 86: 11745 – 11753 Choi Y, Bowman JW, Jung JU (2018) Autophagy during viral infection – a double-edged sword. Nat Rev Microbiol 16: 341 – 354 Fung TS, Liu DX (2019) Human coronavirus: host-pathogen interaction. Annu Rev Microbiol 73: 529 – 557 Chu H, Chan JF-W, Wang Y, Yuen TT-T, Chai Y, Hou Y, Shuai H, Yang D, Hu B, Huang X et al (2020) Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19. Clin Infect Dis Publ Infect Dis Soc Am 71: 1400 – 1409 Gagliardi I, Patella G, Michael A, Serra R, Provenzano M, Andreucci M (2020) COVID-19 and the kidney: from epidemiology to clinical practice. J Clin Med 9: 2506 Gao C, Zeng J, Jia N, Stavenhagen K, Matsumoto Y, Zhang H, Li J, Hume AJ, M€uhlberger E, van Die I et al (2020) SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors. BioRxiv https://doi.org/10.1101/ 2020.07.29.227462 [PREPRINT] Chu H, Zhou J, Wong BH-Y, Li C, Chan JF-W, Cheng Z-S, Yang D, Wang D, Lee AC-Y, Li C et al (2016) Middle east respiratory syndrome coronavirus efficiently infects human primary T lymphocytes and activates the extrinsic and intrinsic apoptosis pathways. J Infect Dis 213: 904 – 914 Garcia-Alonso L, Holland CH, Ibrahim MM, Turei D, Saez-Rodriguez J (2019) Benchmark and integration of resources for the estimation of human Courtot M, Juty N, Kn€upfer C, Waltemath D, Zhukova A, Dr€ager A, Dumontier Courtot M, Juty N, Kn€upfer C, Waltemath D, Zhukova A, Dr€ager A, Dumontier M, Finney A, Golebiewski M, Hastings J et al (2011) Controlled vocabularies and semantics in systems biology. Mol Syst Biol 7: 543 Czauderna T, Klukas C, Schreiber F (2010) Editing, validating and translating of SBGN maps Bioinforma Oxf Engl 26: 2340 – 2341 Courtot M, Juty N, Kn€upfer C, Waltemath D, Zhukova A, Dr€ager A, Dumontier M, Finney A, Golebiewski M, Hastings J et al (2011) Controlled vocabularies and semantics in systems biology. Mol Syst Biol 7: 543 transcription factor activities. Molecular Systems Biology Nature 583: 459 – 468 Gyori BM, Bachman JA, Subramanian K, Muhlich JL, Galescu L, Sorger PK (2017) From word models to executable models of signaling networks using automated assembly. Mol Syst Biol 13: 954 H€acker H, Karin M (2006) Regulation and function of IKK and IKK-related kinases. Sci STKE Signal Transduct Knowl Environ 2006: re13 Hadjadj J, Yatim N, Barnabei L, Corneau A, Boussier J, Smith N, Pere H, Charbit B, Bondet V, Chenevier-Gobeaux C et al (2020) Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369: 718 – 724 Ghosh S, Dellibovi-Ragheb TA, Kerviel A, Pak E, Qiu Q, Fisher M, Takvorian PM, Bleck C, Hsu VW, Fehr AR et al (2020) b-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell 183: 1520– 1535 Demir E, Cary MP, Paley S, Fukuda K, Lemer C, Vastrik I, Wu G, D’Eustachio P, Schaefer C, Luciano J et al (2010) The BioPAX community standard for pathway data sharing. Nat Biotechnol 28: 935 – 942 Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K, White KM, O’Meara MJ, Rezelj VV, Guo JZ, Swaney DL et al (2020) A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583: 459 – 468 Gyori BM, Bachman JA, Subramanian K, Muhlich JL, Galescu L, Sorger PK (2017) From word models to executable models of signaling networks using automated assembly. Mol Syst Biol 13: 954 H€acker H, Karin M (2006) Regulation and function of IKK and IKK-related kinases. Sci STKE Signal Transduct Knowl Environ 2006: re13 Hadjadj J, Yatim N, Barnabei L, Corneau A, Boussier J, Smith N, Pere H, Charbit B, Bondet V, Chenevier-Gobeaux C et al (2020) Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 369: 718 – 724 Gyori BM, Bachman JA, Subramanian K, Muhlich JL, Galescu L, Sorger PK (2017) From word models to executable models of signaling networks using automated assembly. Mol Syst Biol 13: 954 Devaraj SG, Wang N, Chen Z, Chen Z, Tseng M, Barretto N, Lin R, Peters CJ, Tseng C-TK, Baker SC et al (2007) Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus. J Biol Chem 282: 32208 – 32221 Diemer C, Schneider M, Sch€atzl HM, Gilch S (2010) Modulation of host cell death by SARS coronavirus proteins. Molecular Systems Biology Genome Res 29: 1363 – 1375 Gauges R, Rost U, Sahle S, Wengler K, Bergmann FT (2015) The Systems Biology Markup Language (SBML) Level 3 Package: Layout, Version 1 Core. J Integr Bioinforma 12: 267 Czauderna T, Klukas C, Schreiber F (2010) Editing, validating and translating of SBGN maps. Bioinforma Oxf Engl 26: 2340 – 2341 Gawron P, Ostaszewski M, Satagopam V, Gebel S, Mazein A, Kuzma M, Zorzan S, McGee F, Otjacques B, Balling R et al (2016) MINERVA-a platform for visualization and curation of molecular interaction networks. NPJ Syst Biol Appl 2: 16020 Daraselia N, Yuryev A, Egorov S, Novichkova S, Nikitin A, Mazo I (2004) Extracting human protein interactions from MEDLINE using a full- sentence parser. Bioinforma Oxf Engl 20: 604 – 611 De Meulder B, Lefaudeux D, Bansal AT, Mazein A, Chaiboonchoe A, Ahmed H, Balaur I, Saqi M, Pellet J, Ballereau S et al (2018) A computational framework for complex disease stratification from multiple large-scale datasets. BMC Syst Biol 12: 60 Ghaffarizadeh A, Heiland R, Friedman SH, Mumenthaler SM, Macklin P (2018) PhysiCell: an open source physics-based cell simulator for 3-D multicellular systems. PLoS Comput Biol 14: e1005991 DeDiego ML, Nieto-Torres JL, Jimenez-Guarde~no JM, Regla-Nava JA, Alvarez E, Oliveros JC, Zhao J, Fett C, Perlman S, Enjuanes L (2011) Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis. PLoS Pathog 7: e1002315 Gheblawi M, Wang K, Viveiros A, Nguyen Q, Zhong J-C, Turner AJ, Raizada MK, Grant MB, Oudit GY (2020) Angiotensin-Converting enzyme 2: SARS-CoV-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ACE2. Circ Res 126: 1456 – 1474 Delorey TM, Ziegler CGK, Heimberg G, Normand R, Yang Y, Segerstolpe A, Abbondanza D, Fleming SJ, Subramanian A, Montoro DT et al (2021) COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets. Nature 595: 107 – 113 Ghosh S, Dellibovi-Ragheb TA, Kerviel A, Pak E, Qiu Q, Fisher M, Takvorian PM, Bleck C, Hsu VW, Fehr AR et al (2020) b-Coronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell 183: 1520– 1535 Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K, White KM, O’Meara MJ, Rezelj VV, Guo JZ, Swaney DL et al (2020) A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. References Bouhaddou M, Memon D, Meyer B, White KM, Rezelj VV, Correa Marrero M, Polacco BJ, Melnyk JE, Ulferts S, Kaake RM et al (2020) The global phosphorylation landscape of SARS-CoV-2 infection. Cell 182: 685 – 712 Aghamiri SS, Singh V, Naldi A, Helikar T, Soliman S, Niarakis A (2020) Automated inference of Boolean models from molecular interaction maps using CaSQ. Bioinforma Oxf Engl 36: 4473 – 4482 Chaouiya C, Berenguier D, Keating SM, Naldi A, van Iersel MP, Rodriguez N, Dr€ager A, B€uchel F, Cokelaer T, Kowal B et al (2013) SBML qualitative models: a model representation format and infrastructure to foster interactions between qualitative modelling formalisms and tools. BMC Syst Biol 7: 135 Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH, Califano A (2016) Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nat Genet 48: 838 – 847 Amraei R, Yin W, Napoleon MA, Suder EL, Berrigan J, Zhao Q, Olejnik J, Chandler KB, Xia C, Feldman J et al (2020) CD209L/L-SIGN and CD209/DC- SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells. BioRxiv https://doi.org/10. 1101/2020.06.22.165803 [PREPRINT] Chauhan D, Bartok E, Gaidt MM, Bock FJ, Herrmann J, Seeger JM, Broz P, Beckmann R, Kashkar H, Tait SWG et al (2018) BAX/BAK-induced apoptosis results in caspase-8-dependent IL-1b maturation in macrophages. Cell Rep 25: 2354 – 2368.e5 Angelini MM, Akhlaghpour M, Neuman BW, Buchmeier MJ (2013) Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles. Mbio 4: e00524-13 Chen G, Wu DI, Guo W, Cao Y, Huang DA, Wang H, Wang T, Zhang X, Chen H, Yu H et al (2020a) Clinical and immunological features of severe and moderate coronavirus disease 2019. J Clin Invest 130: 2620 – 2629 Molecular Systems Biology 17: e10387 \| 2021 17 of 22 ª 2021 The Authors Molecular Systems Biology Molecular Systems Biology Med Arch Sarajevo Bosnia Herzeg 74: 134 – 138 Hayek S, Pietrancosta N, Hovhannisyan AA, Alves de Sousa R, Bekaddour N, Ermellino L, Tramontano E, Arnould S, Sardet C, Dairou J et al (2020) Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition. Eur cell data. Cell 184: 3573 – 3587 Hassan SM, Jawad MJ, Ahjel SW, Singh RB, Singh J, Awad SM, Hadi NR (2020) The Nrf2 activator (DMF) and covid-19: is there a possible role? Med Arch Sarajevo Bosnia Herzeg 74: 134 – 138 Hassan SM, Jawad MJ, Ahjel SW, Singh RB, Singh J, Awad SM, Hadi NR (2020) The Nrf2 activator (DMF) and covid-19: is there a possible role? Med Arch Sarajevo Bosnia Herzeg 74: 134 138 Kesic MJ, Simmons SO, Bauer R, Jaspers I (2011) Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells. Free Radic Biol Med 51: 444 – 453 Klok FA, Kruip M, van der Meer N, Arbous MS, Gommers D, Kant KM, Kaptein F, van Paassen J, Stals M, Huisman MV et al (2020) Incidence of thrombotic complications in critically ill ICU patients with COVID-19. Thromb Res 191: 145 – 147 Ermellino L, Tramontano E, Arnould S, Sardet C, Dairou J et al (2020) Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition. Eur J Med Chem 186: 111855 Kr€ahling V, Stein DA, Spiegel M, Weber F, M€uhlberger E (2009) Severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase R but is resistant to its antiviral activity. J Virol 83: 2298 – 2309 Helikar T, Kowal B, McClenathan S, Bruckner M, Rowley T, Madrahimov A, Wicks B, Shrestha M, Limbu K, Rogers JA (2012) The Cell Collective: Toward an open and collaborative approach to systems biology. BMC Syst Biol 6: 96 Kutmon M, van Iersel MP, Bohler A, Kelder T, Nunes N, Pico AR, Evelo CT (2015) PathVisio 3: an extendable pathway analysis toolbox. PLoS Comput Biol 11: e1004085 Hidalgo MR, Cubuk C, Amadoz A, Salavert F, Carbonell-Caballero J, Dopazo J (2017) High throughput estimation of functional cell activities reveals disease mechanisms and predicts relevant clinical outcomes. Oncotarget 8: 5160 – 5178 Lee JS, Shin E-C (2020) The type I interferon response in COVID-19: implications for treatment. Molecular Systems Biology In Molecular biology of the SARS-coronavirus, Lal SK (ed) pp 231 – 245. Berlin, Heidelberg: Springer Berlin Heidelberg Dugourd A, Kuppe C, Sciacovelli M, Gjerga E, Gabor A, Emdal KB, Vieira V, Bekker-Jensen DB, Kranz J, EricMJ B et al (2021) Causal integration of Dugourd A, Kuppe C, Sciacovelli M, Gjerga E, Gabor A, Emdal KB, Vieira V, Bekker-Jensen DB, Kranz J, EricMJ B et al (2021) Causal integration of 18 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors Marek Ostaszewski et al Molecular Systems Biology Kanzawa N, Nishigaki K, Hayashi T, Ishii Y, Furukawa S, Niiro A, Yasui F, Kohara M, Morita K, Matsushima K et al (2006) Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/ X4 proteins through NF-kappaB activation. FEBS Lett 580: 6807 – 6812 Haga S, Yamamoto N, Nakai-Murakami C, Osawa Y, Tokunaga K, Sata T, Yamamoto N, Sasazuki T, Ishizaka Y (2008) Modulation of TNF-alpha- converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry. Proc Natl Acad Sci USA 105: 7809 – 7814 Keating SM, Waltemath D, König M, Zhang F, Dr€ager A, Chaouiya C, Bergmann FT, Finney A, Gillespie CS, Helikar T et al (2020) SBML Level 3: an extensible format for the exchange and reuse of biological models. Mol Syst Biol 16: e9110 Hanspers K, Riutta A, Summer-Kutmon M, Pico AR (2020) Pathway information extracted from 25 years of pathway figures. Genome Biol 21:273 Hanspers K, Riutta A, Summer-Kutmon M, Pico AR (2020) Pathway information extracted from 25 years of pathway figures. Genome Biol 21:273 Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, Lee MJ, Wilk Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, Lee MJ, Wilk Kedia-Mehta N, Finlay DK (2019) Competition for nutrients and its role in controlling immune responses. Nat Commun 10: 2123 AJ, Darby C, Zager M et al (2021) Integrated analysis of multimodal single- cell data. Cell 184: 3573 – 3587 AJ, Darby C, Zager M et al (2021) Integrated analysis of multimodal single- cell data. Cell 184: 3573 – 3587 J y g ( ) g y g cell data. Cell 184: 3573 – 3587 Hassan SM, Jawad MJ, Ahjel SW, Singh RB, Singh J, Awad SM, Hadi NR (2020) The Nrf2 activator (DMF) and covid-19: is there a possible role? Molecular Systems Biology Nat Rev Immunol 20: 585 – 586 Letko M, Marzi A, Munster V (2020) Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5: 562 – 569 Hoffmann M, Kleine-Weber H, Pöhlmann S (2020a) A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells Mol Cell 78: 779 – 784 Letort G, Montagud A, Stoll G, Heiland R, Barillot E, Macklin P, Zinovyev A, Calzone L (2019) PhysiBoSS: a multi-scale agent-based modelling framework integrating physical dimension and cell signalling. Bioinforma Oxf Engl 35: 1188 – 1196 Hoffmann M, Kleine-Weber H, Schroeder S, Kr€uger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu N-H, Nitsche A et al (2020b) SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181: 271 – 280 Li S, Zhang Y, Guan Z, Li H, Ye M, Chen X, Shen J, Zhou Y, Shi Z-L, Zhou P et al (2020) SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation. Signal Transduct Target Ther 5: 235 Hoksza D, Gawron P, Ostaszewski M, Smula E, Schneider R (2019) MINERVA API and plugins: opening molecular network analysis and visualization to the community. Bioinforma Oxf Engl 35: 4496 – 4498 Li S-W, Wang C-Y, Jou Y-J, Huang S-H, Hsiao L-H, Wan L, Lin Y-J, Kung S-H, Lin C-W (2016) SARS coronavirus papain-like protease inhibits the TLR7 signaling pathway through removing Lys63-linked polyubiquitination of TRAF3 and TRAF6. Int J Mol Sci 17: 678 Hoksza D, Gawron P, Ostaszewski M, Hasenauer J, Schneider R (2020) Closing the gap between formats for storing layout information in systems biology. Brief Bioinform 21: 1249 – 1260 Liao Q-J, Ye L-B, Timani KA, Zeng Y-C, She Y-L, Ye L, Wu Z-H (2005) Activation of NF-kappaB by the full-length nucleocapsid protein of the SARS Huang C, Wang Y, Li X, Ren L, Zhao J, Hu YI, Zhang LI, Fan G, Xu J, Gu X et al (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet Lond Engl 395: 497 – 506 Huang C, Wang Y, Li X, Ren L, Zhao J, Hu YI, Zhang LI, Fan G, Xu J, Gu X et al (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet Lond Engl 395: 497 – 506 coronavirus. Molecular Systems Biology Acta Biochim Biophys Sin 37: 607 – 612 p y Licata L, Lo Surdo P, Iannuccelli M, Palma A, Micarelli E, Perfetto L, Peluso D, Calderone A, Castagnoli L, Cesareni G (2020) SIGNOR 2.0, the SIGnaling network open resource 2.0: 2019 update. Nucleic Acids Res 48: D504 – D510 Lieberman NAP, Peddu V, Xie H, Shrestha L, Huang M-L, Mears MC, Cajimat MN, Bente DA, Shi P-Y, Bovier F et al (2020) Gene Expression Omnibus GSE152075 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE152075). [DATASET] Licata L, Lo Surdo P, Iannuccelli M, Palma A, Micarelli E, Perfetto L, Peluso D, Calderone A, Castagnoli L, Cesareni G (2020) SIGNOR 2.0, the SIGnaling network open resource 2.0: 2019 update. Nucleic Acids Res 48: D504 – D510 Hui KPY, Cheung M-C, Perera RAPM, Ng K-C, Bui CHT, Ho JCW, Ng MMT, Kuok DIT, Shih KC, Tsao S-W et al (2020) Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures. Lancet Respir Med 8: 687 – 695 Lieberman NAP, Peddu V, Xie H, Shrestha L, Huang M-L, Mears MC, Cajimat MN, Bente DA, Shi P-Y, Bovier F et al (2020) Gene Expression Omnibus GSE152075 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE152075). [DATASET] Iba T, Levy JH, Connors JM, Warkentin TE, Thachil J, Levi M (2020) The unique characteristics of COVID-19 coagulopathy. Crit Care Lond Engl 24: 360 Iba T, Levy JH, Connors JM, Warkentin TE, Thachil J, Levi M (2020) The unique characteristics of COVID-19 coagulopathy. Crit Care Lond Engl 24: 360 GSE152075). [DATASET] [DATASET] Niarakis A, Kuiper M, Ostaszewski M, Malik Sheriff RS, Casals-Casas C, Thieffry D, Freeman TC, Thomas P, Toure V, No€el V et al (2020) Setting the basis of best practices and standards for curation and annotation of logical models in biology-highlights of the [BC]2 2019 CoLoMoTo/SysMod Workshop. Brief Bioinform 22: 1848 – 1859. Lukassen S, Chua RL, Trefzer T, Kahn NC, Schneider MA, Muley T, Winter H, Meister M, Veith C, Boots AW et al (2020) SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J 39: e105114 p f f Novere NL, Hucka M, Mi H, Moodie S, Schreiber F, Sorokin A, Demir E, Wegner K, Aladjem MI, Wimalaratne SM et al (2009) The systems biology graphical notation. Nat Biotechnol 27: 735 – 741 Orchard S, Kerrien S, Abbani S, Aranda B, Bhate J, Bidwell S, Bridge A, Briganti L, Brinkman FSL, Brinkman F et al (2012) Protein interaction data curation: the International Molecular Exchange (IMEx) consortium. Nat Methods 9: 345 – 350 Novere NL, Hucka M, Mi H, Moodie S, Schreiber F, Sorokin A, Demir E, Wegner K, Aladjem MI, Wimalaratne SM et al (2009) The systems biology graphical notation. Nat Biotechnol 27: 735 – 741 Magro C, Mulvey JJ, Berlin D, Nuovo G, Salvatore S, Harp J, Baxter-Stoltzfus A, Laurence J (2020) Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases. Transl Res J Lab Clin Med 220: 1 – 13 Orchard S, Kerrien S, Abbani S, Aranda B, Bhate J, Bidwell S, Bridge A, Briganti L, Brinkman FSL, Brinkman F et al (2012) Protein interaction data curation: the International Molecular Exchange (IMEx) consortium. Nat Methods 9: 345 – 350 five cases. Transl Res J Lab Clin Med 220: 1 – 13 Mantlo E, Bukreyeva N, Maruyama J, Paessler S, Huang C (2020) Antiviral activities of type I interferons to SARS-CoV-2 infection. Antiviral Res 179: 104811 Osborne JM, Fletcher AG, Pitt-Francis JM, Maini PK, Gavaghan DJ (2017) Comparing individual-based approaches to modelling the self- organization of multicellular tissues. PLoS Comput Biol 13: e1005387 Mason RJ (2020) Pathogenesis of COVID-19 from a cell biology perspective. Eur Respir J 55: 2000607 Matsuoka Y, Funahashi A, Ghosh S, Kitano H (2014) Modeling and simulation using Cell Designer. GSE152075). [DATASET] Methods Mol Biol Clifton NJ 1164: 121 – 145 Ostaszewski M, Mazein A, Gillespie ME, Kuperstein I, Niarakis A, Hermjakob H, Pico AR, Willighagen EL, Evelo CT, Hasenauer J et al (2020) COVID-19 Disease Map, building a computational repository of SARS-CoV-2 virus- host interaction mechanisms. Sci Data 7: 136 Mazein A, Ostaszewski M, Kuperstein I, Watterson S, Le Novere N, Lefaudeux D, De Meulder B, Pellet J, Balaur I, Saqi M et al (2018) Systems medicine disease maps: community-driven comprehensive representation of disease mechanisms. NPJ Syst Biol Appl 4: 21 Park A, Iwasaki A (2020) Type I and type III interferons-induction, signaling, evasion, and application to combat COVID-19. Cell Host Microbe 27: 870 – 878 McFadyen JD, Stevens H, Peter K (2020) The emerging threat of (Micro)Thrombosis in COVID-19 and Its therapeutic implications. Circ Res 127: 571–587 McFadyen JD, Stevens H, Peter K (2020) The emerging threat of (Micro)Thrombosis in COVID-19 and Its therapeutic implications. Circ Res 127: 571–587 Meldal BHM, Bye-A-Jee H, Gajdos L, Hammerova Z, Horackova A, Melicher F, Perfetto L, Pokorny D, Lopez MR, T€urkova A et al (2019) Complex Portal 2018: extended content and enhanced visualization tools for macromolecular complexes. Nucleic Acids Res 47: D550 – D558 Perfetto L, Pastrello C, Del-Toro N, Duesbury M, Iannuccelli M, Kotlyar M, Licata L, Meldal B, Panneerselvam K, Panni S et al (2020) The IMEx coronavirus interactome: an evolving map of Coronaviridae-host molecular interactions. Database (Oxford) 2020: baaa096 Meldal BHM, Bye-A-Jee H, Gajdos L, Hammerova Z, Horackova A, Melicher F, Meldal BHM, Bye-A-Jee H, Gajdos L, Hammerova Z, Horackova A, Melicher F, Perfetto L, Pokorny D, Lopez MR, T€urkova A et al (2019) Complex Portal 2018: extended content and enhanced visualization tools for macromolecular complexes. Nucleic Acids Res 47: D550 – D558 Pillich RT, Chen J, Rynkov V, Welker D, Pratt D (2017) NDEx: a community resource for sharing and publishing of biological networks. Methods Mol Biol Clifton NJ 1558: 271 – 301 Mesev EV, LeDesma RA, Ploss A (2019) Decoding type I and III interferon signalling during viral infection. Nat Microbiol 4: 914 – 924 Rao M, Dodoo E, Zumla A, Maeurer M (2019) Immunometabolism and pulmonary infections: implications for protective immune responses and host-directed therapies. GSE152075). [DATASET] FASEB J Publ Fed Am Soc Exp Biol 21: 1586 – 1596 Naithani S, Gupta P, Preece J, Garg P, Fraser V, Padgitt-Cobb LK, Martin M, Vining K, Jaiswal P (2019) Involving community in genes and pathway curation. Database 2019: bay146 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15: 550 dispersion for RNA seq data with DESeq2. Genome Biol 15: 550 Lu Wang L, Lo K, Chandrasekhar Y, Reas R, Yang J, Eide D, Funk K, Kinney R, Liu Z, Merrill W et al (2020) CORD-19: The Covid-19 Open Research Dataset. ArXiv https://arxiv.org/abs/2004.10706v2 [PREPRINT] Lubin JH, Zardecki C, Dolan EM, Lu C, Shen Z, Dutta S, Westbrook JD, Hudson BP, Goodsell DS, Williams JK et al (2020) Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic. BioRxiv https://doi.org/10.1101/2020.12.01.406637 [PREPRINT] Naldi A, Hernandez C, Abou-Jaoude W, Monteiro PT, Chaouiya C, Thieffry D (2018a) Logical modeling and analysis of cellular regulatory networks with GINsim 3.0. Front Physiol 9: 646 Lubin JH, Zardecki C, Dolan EM, Lu C, Shen Z, Dutta S, Westbrook JD, Hudson BP, Goodsell DS, Williams JK et al (2020) Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic. BioRxiv https://doi.org/10.1101/2020.12.01.406637 [PREPRINT] Naldi A, Hernandez C, Levy N, Stoll G, Monteiro PT, Chaouiya C, Helikar T, Zinovyev A, Calzone L, Cohen-Boulakia S et al (2018b) The CoLoMoTo interactive notebook: accessible and reproducible computational analyses for qualitative biological networks. Front Physiol 9: 680 Lucas C, Wong P, Klein J, Castro TBR, Silva J, Sundaram M, Ellingson MK, Mao T, Oh JE, Israelow B et al (2020) Longitudinal analyses reveal immunological misfiring in severe COVID-19. Nature 584: 463 – 469 Nesterova AP, Klimov EA, Zharkova M, Sozin S, Sobolev V, Ivanikova NV, Shkrob M, Yuryev A (2020) Guide and legend. In Disease Pathways, pp xxi – xxviii. Cambridge, MA: Elsevier Lukassen S, Chua RL, Trefzer T, Kahn NC, Schneider MA, Muley T, Winter H, Meister M, Veith C, Boots AW et al (2020) Figshare 11981034 (https://doi.org/https://doi.org/10.6084/m9.figshare.11981034.v1). GSE152075). [DATASET] van Iersel MP, Pico AR, Kelder T, Gao J, Ho I, Hanspers K, Conklin BR, Evelo CT (2010) The BridgeDb framework: standardized access to gene, protein and metabolite identifier mapping services. BMC Bioinformatics 11: 5 Lieberman NAP, Peddu V, Xie H, Shrestha L, Huang M-L, Mears MC, Cajimat MN, Bente DA, Shi P-Y, Bovier F et al (2020) In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age. PLoS Biol 18: e3000849 Jassal B, Matthews L, Viteri G, Gong C, Lorente P, Fabregat A, Sidiropoulos K, Cook J, Gillespie M, Haw R et al (2020) The Reactome Pathway Knowledgebase. Nucleic Acids Res 48: D498 – D503 Liu A, Trairatphisan P, Gjerga E, Didangelos A, Barratt J, Saez-Rodriguez J (2019) From expression footprints to causal pathways: contextualizing large signaling networks with CARNIVAL. NPJ Syst Biol Appl 5: 40 Liu A, Trairatphisan P, Gjerga E, Didangelos A, Barratt J, Saez-Rodriguez J (2019) From expression footprints to causal pathways: contextualizing large signaling networks with CARNIVAL. NPJ Syst Biol Appl 5: 40 Liu M, Yang Y, Gu C, Yue Y, Wu KK, Wu J, Zhu Y (2007) Spike protein of SARS- CoV stimulates cyclooxygenase-2 expression via both calcium-dependent Juty N, Le Novere N, Laibe C (2012) Identifiers.org and MIRIAM Registry: community resources to provide persistent identification. Nucleic Acids Res 40: D580 – 586 Liu M, Yang Y, Gu C, Yue Y, Wu KK, Wu J, Zhu Y (2007) Spike protein of SARS- CoV stimulates cyclooxygenase-2 expression via both calcium-dependent Molecular Systems Biology 17: e10387 \| 2021 19 of 22 ª 2021 The Authors Molecular Systems Biology Marek Ostaszewski et al and calcium-independent protein kinase C pathways. FASEB J Publ Fed Am Soc Exp Biol 21: 1586 – 1596 Naithani S, Gupta P, Preece J, Garg P, Fraser V, Padgitt-Cobb LK, Martin M, Vining K, Jaiswal P (2019) Involving community in genes and pathway curation. Database 2019: bay146 Nakagawa K, Lokugamage KG, Makino S (2016) Viral and cellular mRNA translation in coronavirus-infected cells. Adv Virus Res 96: 165 – 192 Naldi A, Hernandez C, Abou-Jaoude W, Monteiro PT, Chaouiya C, Thieffry D (2018a) Logical modeling and analysis of cellular regulatory networks with GINsim 3.0. Front Physiol 9: 646 and calcium-independent protein kinase C pathways. GSE152075). [DATASET] Front Microbiol 10: 962 Messina F, Giombini E, Agrati C, Vairo F, Ascoli Bartoli T, Al Moghazi S, Piacentini M, Locatelli F, Kobinger G, Maeurer M et al (2020) COVID-19: viral-host interactome analyzed by network based-approach model to study pathogenesis of SARS-CoV-2 infection. J Transl Med 18: 233 Renz A, Widerspick L, Dr€ager A (2020) FBA reveals guanylate kinase as a potential target for antiviral therapies against SARS-CoV-2. Bioinforma Oxf Engl 36: i813 – i821 Miao G, Zhao H, Li Y, Ji M, Chen Y, Shi Y, Bi Y, Wang P, Zhang H (2020) ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complex-mediated assembly of the SNARE complex required for autolysosome formation. Dev Cell 56: 427 – 442 Rian K, Esteban-Medina M, Hidalgo MR, Çubuk C, Falco MM, Loucera C, Gunyel D, Ostaszewski M, Pe~na-Chilet M, Dopazo J (2021) Mechanistic modeling of the SARS-CoV-2 disease map. BioData Min 14: 5 Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinforma Oxf Engl 26: 139 – 140 Rodchenkov I, Babur O, Luna A, Aksoy BA, Wong JV, Fong D, Franz M, Siper MC, Cheung M, Wrana M et al (2020) Pathway Commons 2019 Update: Rian K, Esteban-Medina M, Hidalgo MR, Çubuk C, Falco MM, Loucera C, Gunyel D, Ostaszewski M, Pe~na-Chilet M, Dopazo J (2021) Mechanistic modeling of the SARS-CoV-2 disease map. BioData Min 14: 5 Rian K, Esteban-Medina M, Hidalgo MR, Çubuk C, Falco MM, Loucera C, Gunyel D, Ostaszewski M, Pe~na-Chilet M, Dopazo J (2021) Mechanistic modeling of the SARS-CoV-2 disease map. BioData Min 14: 5 Mogensen TH (2009) Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev 22: 240 – 273 Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinforma Oxf Engl 26: 139 – 140 Murakami Y, Hoshi M, Imamura Y, Arioka Y, Yamamoto Y, Saito K (2013) Remarkable role of indoleamine 2,3-dioxygenase and tryptophan metabolites in infectious diseases: potential role in macrophage-mediated inflammatory diseases. Molecular Systems Biology Marek Ostaszewski et al integration, analysis and exploration of pathway data. Nucleic Acids Res 48: D489 – D497 T€urei D, Korcsmaros T, Saez-Rodriguez J (2016) OmniPath: guidelines and gateway for literature-curated signaling pathway resources. Nat Methods 13: 966 – 967 Rubio D, Xu R-H, Remakus S, Krouse TE, Truckenmiller ME, Thapa RJ, Balachandran S, Alcamı A, Norbury CC, Sigal LJ (2013) Crosstalk between the type 1 interferon and nuclear factor kappa B pathways confers resistance to a lethal virus infection. Cell Host Microbe 13: 701 – 710 T€urei D, Valdeolivas A, Gul L, Palacio-Escat N, Klein M, Ivanova O, Ölbei M, Gabor A, Theis F, Modos D et al (2021) Integrated intra- and intercellular signaling knowledge for multicellular omics analysis. Mol Syst Biol 17 Sa Ribero M, Jouvenet N, Dreux M, Nisole S (2020) Interplay between SARS- CoV-2 and the type I interferon response. PLoS Pathog 16: e1008737 Urwyler P, Moser S, Charitos P, Heijnen IAFM, Rudin M, Sommer G, Giannetti BM, Bassetti S, Sendi P, Trendelenburg M et al (2020) Treatment of COVID-19 with conestat alfa, a regulator of the complement, contact activation and kallikrein-kinin system. Front Immunol 11: 2072 Salavert F, Hidago MR, Amadoz A, Çubuk C, Medina I, Crespo D, Carbonell- Caballero J, Dopazo J (2016) Actionable pathways: interactive discovery of therapeutic targets using signaling pathway models. Nucleic Acids Res 44: W212 – 216 V’kovski P, Gerber M, Kelly J, Pfaender S, Ebert N, Braga Lagache S, Simillion C, Portmann J, Stalder H, Gaschen V et al (2019) Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling. eLife 8: e42037 Satagopam V, Gu W, Eifes S, Gawron P, Ostaszewski M, Gebel S, Barbosa- Silva A, Balling R, Schneider R (2016) Integration and visualization of translational medicine data for better understanding of human diseases. Big Data 4: 97 – 108 Valencia I, Peiro C, Lorenzo O, Sanchez-Ferrer CF, Eckel J, Romacho T (2020) DPP4 and ACE2 in diabetes and COVID-19: therapeutic targets for cardiovascular complications? Front Pharmacol 11: 1161 Seo GJ, Kim C, Shin W-J, Sklan EH, Eoh H, Jung JU (2018) TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing. Nat Commun 9: 613 van den Berg DF, Te Velde AA (2020) Severe COVID-19: NLRP3 inflammasome dysregulated. Front Immunol 11: 1580 van den Berg DF, Te Velde AA (2020) Severe COVID-19: NLRP3 inflammasome dysregulated. Molecular Systems Biology Front Immunol 11: 1580 Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N, Schwikowski B, Ideker T (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13: 2498 – 2504 Wang Y, An G, Becker A, Cockrell C, Collier N, Craig M, Davis CL, Faeder J, Versypt ANF, Gianlupi JF et al (2020) Iterative community-driven development of a SARS-CoV-2 tissue simulator. BioRxiv https://doi.org/10. 1101/2020.04.02.019075 [PREPRINT] Slayden RA, Jackson M, Zucker J, Ramirez MV, Dawson CC, Crew R, Sampson NS, Thomas ST, Jamshidi N, Sisk P et al (2013) Updating and curating metabolic pathways of TB. Tuberc Edinb Scotl 93: 47 – 59 Wei C-H, Kao H-Y, Lu Z (2015) GNormPlus: an integrative approach for tagging genes, gene families, and protein domains. BioMed Res Int 2015: 1 – 7 Slenter DN, Kutmon M, Hanspers K, Riutta A, Windsor J, Nunes N, Melius J, Cirillo E, Coort SL, Digles D et al (2018) WikiPathways: a multifaceted pathway database bridging metabolomics to other omics research. Nucleic Acids Res 46: D661 – D667 Wilkinson MD, Dumontier M, IjJ A, Appleton G, Axton M, Baak A, Blomberg N, Boiten J-W, da Silva Santos LB, Bourne PE et al (2016) The FAIR Guiding Principles for scientific data management and stewardship. Sci Data 3: 160018 Wimalaratne SM, Juty N, Kunze J, Janee G, McMurry JA, Beard N, Jimenez R, Grethe JS, Hermjakob H, Martone ME et al (2018) Uniform resolution of compact identifiers for biomedical data. Sci Data 5: 180029 Smith JA (2018) Regulation of cytokine production by the unfolded protein response; implications for infection and autoimmunity. Front Immunol 9: 422 Wolstencroft K, Krebs O, Snoep JL, Stanford NJ, Bacall F, Golebiewski M, Kuzyakiv R, Nguyen Q, Owen S, Soiland-Reyes S et al (2017) FAIRDOMHub: a repository and collaboration environment for sharing systems biology research. Nucleic Acids Res 45: D404 – D407 Wolstencroft K, Krebs O, Snoep JL, Stanford NJ, Bacall F, Golebiewski M, Kuzyakiv R, Nguyen Q, Owen S, Soiland-Reyes S et al (2017) FAIRDOMHub: a repository and collaboration environment for sharing systems biology research. Nucleic Acids Res 45: D404 – D407 Stoll G, Caron B, Viara E, Dugourd A, Zinovyev A, Naldi A, Kroemer G, Barillot E, Calzone L (2017) MaBoSS 2.0: an environment for stochastic Boolean modeling. GSE152075). [DATASET] Mediators Inflamm 2013: 391984 Rodchenkov I, Babur O, Luna A, Aksoy BA, Wong JV, Fong D, Franz M, Siper MC, Cheung M, Wrana M et al (2020) Pathway Commons 2019 Update: Rodchenkov I, Babur O, Luna A, Aksoy BA, Wong JV, Fong D, Franz M, Siper MC, Cheung M, Wrana M et al (2020) Pathway Commons 2019 Update: 20 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors Molecular Systems Biology Molecular Systems Biology Bioinforma Oxf Engl 33: 2226 – 2228 Su S, Jiang S (2020) A suspicious role of interferon in the pathogenesis of SARS- CoV-2 by enhancing expression of ACE2. Signal Transduct Target Ther 5: 71 Wong HH, Fung TS, Fang S, Huang M, Le MT, Liu DX (2018) Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin- dependent rapid degradation of interferon regulatory factor 3. Virology 515: 165 – 175 Su Y, Chen D, Lausted C, Yuan D, Choi J, Dai C, Voillet V, Scherler K, Troisch P, Duvvuri VR et al (2020) Multiomic immunophenotyping of COVID-19 patients reveals early infection trajectories immunology. bioRxiv https:// doi.org/10.1101/2020.07.27.224063 [PREPRINT] Xia S, Zhu Y, Liu M, Lan Q, Xu W, Wu Y, Ying T, Liu S, Shi Z, Jiang S et al (2020) Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein. Cell Mol Immunol 17: 765 – 767 Sureda A, Alizadeh J, Nabavi SF, Berindan-Neagoe I, Cismaru CA, Jeandet P, Łos MJ, Clementi E, Nabavi SM, Ghavami S (2020) Endoplasmic reticulum as a potential therapeutic target for covid-19 infection management? Eur J Pharmacol 882: 173288 Xiong R, Zhang L, Li S, Sun Y, Ding M, Wang Y, Zhao Y, Wu Y, Shang W, Jiang X et al (2020) Novel and potent inhibitors targeting DHODH are broad- spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2. Protein Cell 11: 723 – 739 Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805 – 820 Thoms M, Buschauer R, Ameismeier M, Koepke L, Denk T, Hirschenberger M, Kratzat H, Hayn M, Mackens-Kiani T, Cheng J et al (2020) Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369: 1249– 1255 Yang N, Shen H-M (2020) Targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in COVID-19. Int J Biol Sci 16: 1724 – 1731 Zhang H, Tu J, Cao C, Yang T, Gao L (2020) Proteasome activator PA28c-dependent degradation of coronavirus disease (COVID- 19) nucleocapsid protein. Biochem Biophys Res Commun 529: 251 – 256 Triana S, Metz-Zumaran C, Ramirez C, Kee C, Doldan P, Shahraz M, Schraivogel D, Gschwind AR, Sharma AK, Steinmetz LM et al (2021) Single- cell analyses reveal SARS-CoV-2 interference with intrinsic immune response in the human gut. List of affiliations Marek Ostaszewski1,*; Anna Niarakis2,3; Alexander Mazein1; Inna Kuperstein4,5,6; Robert Phair7; Aurelio Orta-Resendiz8,9; Vidisha Singh2; Sara Sadat Aghamiri10; Marcio Luis Acencio1; Enrico Glaab1; Andreas Ruepp11; Gisela Fobo11; Corinna Montrone11; Barbara Brauner11; Goar Frishman11; Luis Cristobal Monraz Gomez4,5,6; Julia Somers12; Matti Hoch13; Shailendra Kumar Gupta13; Julia Scheel13; Hanna Borlinghaus14; Tobias Czauderna15; Falk Schreiber14,15; Arnau Montagud16; Miguel Ponce de Leon16; Akira Funahashi17; Yusuke Hiki17; Noriko Hiroi18; Takahiro G Yamada17; Andreas Dr€ager19,20,21; Alina Renz19,20; Muhammad Naveez13,22; Zsolt Bocskei23; Francesco Messina24,25; Daniela Börnigen26; Liam Fergusson27; Marta Conti28; Marius Rameil28; Vanessa Nakonecnij28; Jakob Vanhoefer28; Leonard Schmiester28,29; Muying Wang30; Emily E Ackerman30; Jason E Shoemaker30,31; Jeremy Zucker32; Kristie Oxford32; Jeremy Teuton32; Ebru Kocakaya33; Gökçe Yagmur Summak33; Kristina Hanspers34; Martina Kutmon35,36; Susan Coort35; Lars Eijssen35,37; Friederike Ehrhart35,37; Devasahayam Arokia Balaya Rex38; Denise Slenter35; Marvin Martens35; Nhung Pham35; Robin Haw39; Bijay Jassal39; Lisa Matthews40; Marija Orlic-Milacic39; Andrea Senff Ribeiro39,41; Karen Rothfels39; Veronica Shamovsky40; Ralf Stephan39; Cristoffer Sevilla42; Thawfeek Varusai42; Jean-Marie Ravel43,44; Rupsha Fraser45; Vera Ortseifen46; Silvia Marchesi47; Piotr Gawron1,48; Ewa Smula1; Laurent Heirendt1; Venkata Satagopam1; Guanming Wu49; Anders Riutta34; Martin Golebiewski50; Stuart Owen51; Carole Goble51; Xiaoming Hu50; Rupert W Overall52,53,54; Dieter Maier55; Angela Bauch55; Benjamin M Gyori56; John A Bachman56; Carlos Vega1; Valentin Groues1; Miguel Vazquez16; Pablo Porras42; Luana Licata57; Marta Iannuccelli57; Francesca Sacco57; Anastasia Nesterova58; Anton Yuryev58; Anita de Waard59; Denes Turei60; Augustin Luna61,62; Ozgun Babur63; Sylvain Soliman3; Alberto Valdeolivas60; Marina Esteban-Medina64,65; Maria Pe~na-Chilet64,65,66; Kinza Rian64,65; Tomas Helikar67; Bhanwar Lal Puniya67; Dezso Modos68,69; Agatha Treveil68,69; Marton Olbei68,69; Bertrand De Meulder70; Stephane Ballereau71; Aurelien Dugourd60,72; Aurelien Naldi3; Vincent No€el4,5,6; Laurence Calzone4,5,6; Chris Sander61,62; Emek Demir12; Tamas Korcsmaros68,69; Tom C Freeman73; Franck Auge23; Jacques S Beckmann74; Jan Hasenauer75,76; Olaf Wolkenhauer13; Egon L Wilighagen35; Alexander R Pico34; Chris T Evelo35,36; Marc E Gillespie39,77; Lincoln D Stein39,78; Henning Hermjakob42; Peter D’Eustachio40; Julio Saez-Rodriguez60; Joaquin Dopazo64,65,66,79; Alfonso Valencia16,80; Hiroaki Kitano81,82; Emmanuel Barillot4,5,6; Charles Auffray71; Rudi Balling1; Reinhard Schneider1; the COVID-19 Disease Map Community,† 1Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg. 2Universite Paris-Saclay, Laboratoire Europeen de Recherche pour la Polyarthrite rhumato€ıde - Genhotel, Univ Evry, Evry, France. 3Lifeware Group, Inria Saclay-Ile de France, Palaiseau, France. 4Institut Curie, PSL Research University, Paris, France. 5INSERM, Paris, France. 6MINES ParisTech, PSL Research University, Paris, France. 7Integrative Bioinformatics, Inc., Mountain View, CA, USA. 8Institut Pasteur, Universite de Paris, Unite HIV, Inflammation et Persistance, Paris, France. 9Bio Sorbonne Paris Cite, Universite de Paris, Paris, France. List of affiliations 10Inserm- Institut national de la sante et de la recherche medicale, Paris, France. 11Institute of Experimental Genetics (IEG), Helmholtz Zentrum M€unchen-German Research Center for Environmental Health (GmbH), Neuherberg, Germany. 12Department of Molecular and Medical Genetics, Oregon Health & Sciences University, Portland, OR, USA. 13Department of Systems Biology and Bioinformatics, University of Rostock, Rostock, Germany. 14Department of Computer and Information Science, University of Konstanz, Konstanz, Germany. 15Faculty of Information Technology, Department of Human-Centred Computing, Monash University, Clayton, Vic., Australia. 16Barcelona Supercomputing Center (BSC), Barcelona, Spain. 17Department of Biosciences and Informatics, Keio University, Yokohama, Japan. 18Graduate School of Media and Governance, Research Institute at SFC, Keio University, Kanagawa, Japan. 19Computational Systems Biology of Infections and Antimicrobial-Resistant Pathogens, Institute for Bioinformatics and Medical Informatics (IBMI), University of T€ubingen, T€ubingen, Germany. 20Department of Computer Science, University of T€ubingen, T€ubingen, Germany. 21German Center for Infection Research (DZIF), partner site, T€ubingen, Germany. 22Institute of Applied Computer Systems, Riga Technical University, Riga, Latvia. 23Sanofi R&D, Translational Sciences, Chilly-Mazarin, France. 24Dipartimento di Epidemiologia Ricerca Pre-Clinica e Diagnostica Avanzata, National Institute for Infectious Diseases ’Lazzaro Spallanzani’ I.R.C.C.S., Rome, Italy. 25COVID-19 INMI Network Medicine for IDs Study Group, National Institute for Infectious Diseases ’Lazzaro Spallanzani’ I.R.C.C.S, Rome, Italy. 26Bioinformatics Core Facility, Universit€atsklinikum Hamburg-Eppendorf, Hamburg, Germany. 27Royal (Dick) School of Veterinary Medicine, The University of Edinburgh, Edinburgh, UK. 28Faculty of Mathematics and Natural Sciences, University of Bonn, Bonn, Germany. 29Center for Mathematics, Chair of Mathematical Modeling of Biological Systems, Technische Universit€at M€unchen, Garching, Germany. 30Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA. 31Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA. 32Pacific Northwest National Laboratory, Richland, WA, USA. 33Stem Cell Institute, Ankara University, Ankara, Turkey. 34Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA. 1Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg. 2Universite Paris-Saclay, Laboratoire Europeen de Recherche pour la Polyarthrite rhumato€ıde - Genhotel, Univ Evry, Evry, France. 3Lifeware Group, Inria Saclay-Ile de France, Palaiseau, France. 4Institut Curie, PSL Research University, Paris, France. 5INSERM, Paris, France. 6MINES ParisTech, PSL Research University, Paris, France. 7Integrative Bioinformatics, Inc., Mountain View, CA, USA. 8Institut Pasteur, Universite de Paris, Unite HIV, Inflammation et Persistance, Paris, France. 9Bio Sorbonne Paris Cite, Universite de Paris, Paris, France. 10Inserm- Institut national de la sante et de la recherche medicale, Paris, France. List of affiliations 11Institute of Experimental Genetics (IEG), Helmholtz Zentrum M€unchen-German Research Center for Environmental Health (GmbH), Neuherberg, Germany. 12Department of Molecular and Medical Genetics, Oregon Health & Sciences University, Portland, OR, USA. 13Department of Systems Biology and Bioinformatics, University of Rostock, Rostock, Germany. 14Department of Computer and Information Science, University of Konstanz, Konstanz, Germany. 15Faculty of Information Technology, Department of Human-Centred Computing, Monash University, Clayton, Vic., Australia. 16Barcelona Supercomputing Center (BSC), Barcelona, Spain. 17Department of Biosciences and Informatics, Keio University, Yokohama, Japan. 18Graduate School of Media and Governance, Research Institute at SFC, Keio University, Kanagawa, Japan. 19Computational Systems Biology of Infections and Antimicrobial-Resistant Pathogens, Institute for Bioinformatics and Medical Informatics (IBMI), University of T€ubingen, T€ubingen, Germany. 20Department of Computer Science, University of T€ubingen, T€ubingen, Germany. 21German Center for Infection Research (DZIF), partner site, T€ubingen, Germany. 22Institute of Applied Computer Systems, Riga Technical University, Riga, Latvia. 23Sanofi R&D, Translational Sciences, Chilly-Mazarin, France. 24Dipartimento di Epidemiologia Ricerca Pre-Clinica e Diagnostica Avanzata, National Institute for Infectious Diseases ’Lazzaro Spallanzani’ I.R.C.C.S., Rome, Italy. 25COVID-19 INMI Network Medicine for IDs Study Group, National Institute for Infectious Diseases ’Lazzaro Spallanzani’ I.R.C.C.S, Rome, Italy. 26Bioinformatics Core Facility, Universit€atsklinikum Hamburg-Eppendorf, Hamburg, Germany. 27Royal (Dick) School of Veterinary Medicine, The University of Edinburgh, Edinburgh, UK. 28Faculty of Mathematics and Natural Sciences, University of Bonn, Bonn, Germany. 29Center for Mathematics, Chair of Mathematical Modeling of Biological Systems, Technische Universit€at M€unchen, Garching, Germany. 30Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA. 31Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA, USA. 32Pacific Northwest National Laboratory, Richland, WA, USA. 33Stem Cell Institute, Ankara University, Ankara, Turkey. 34Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA. 35Department of Bioinformatics - BiGCaT, NUTRIM, Maastricht University, Maastricht, The Netherlands. 36Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, The Netherlands. 37Maastricht University Medical Centre, Maastricht, The Netherlands. 38Center for Systems Biology and Molecular Medicine, Yenepoya (Deemed to be University), Mangalore, India. 39MaRS Centre, Ontario Institute for Cancer Research, Toronto, ON, Canada. 40NYU Grossman School of Medicine, New York, NY, USA. 41Universidade Federal do Parana, Curitiba, Brasil. 42European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Hinxton, Cambridgeshire, UK. 43INSERM UMR_S 1256, Nutrition, Genetics, and Environmental Risk Exposure (NGERE), Faculty of Medicine of Nancy, University of Lorraine, Nancy, France. 44Laboratoire de genetique medicale, CHRU Nancy, Nancy, France. 45Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK. Molecular Systems Biology Molecular Systems Biology Marek Ostaszewski et al Zipeto D, da Palmeira JF, Arga~naraz GA, Arga~naraz ER (2020) ACE2/ADAM17/ TMPRSS2 interplay may be the main risk factor for COVID-19. Front Immunol 11: 576745 Zheng Y, Zhuang M-W, Han L, Zhang J, Nan M-L, Zhan P, Kang D, Liu X, Gao C, Wang P-H (2020) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) membrane (M) protein inhibits type I and III interferon production by targeting RIG-I/MDA-5 signaling. Signal Transduct Target Ther 5: 299 License: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Ziegler CGK, Allon SJ, Nyquist SK, Mbano IM, Miao VN, Tzouanas CN, Cao Y, Yousif AS, Bals J, Hauser BM et al (2020) SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Cell 181: 1016 – 1035 22 of 22 Molecular Systems Biology 17: e10387 \| 2021 Molecular Systems Biology Mol Syst Biol 17: e10232 ana S, Metz-Zumaran C, Ramirez C, Kee C, Doldan P, Shahraz Triana S, Metz-Zumaran C, Ramirez C, Kee C, Doldan P, Shahraz M, Schraivogel D, Gschwind AR, Sharma AK, Steinmetz LM et al (2021) Gene Expression Omnibus GSE156760 (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE156760). [DATASET] Zhang X, Ding M, Zhu P, Huang H, Zhuang Q, Shen J, Cai Y, Zhao M, He Q (2019) New insights into the Nrf-2/HO-1 signaling axis and its application in pediatric respiratory diseases. Oxid Med Cell Longev 2019: 3214196 Molecular Systems Biology 17: e10387 \| 2021 21 of 22 ª 2021 The Authors List of affiliations 46Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Bielefeld, Germany. 47Department of Surgical Science, Uppsala University, Uppsala, Sweden. 48Institute of Computing Science, Poznan University of Technology, Poznan, Poland. 49Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA. 50Heidelberg Institute for Theoretical Studies (HITS), Heidelberg, Germany. 51Department of Computer Science, The University of Manchester, Manchester, UK. 52German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany. 53Center for Regenerative Therapies Dresden (CRTD), Technische Universit€at Dresden, Dresden, Germany. 54Institute for Biology, Humboldt University of Berlin, Berlin, Germany. 55Biomax Informatics AG, Planegg, Germany. 56Harvard Medical School, Laboratory of Systems Pharmacology, Boston, MA, USA. 57Department of Biology, University of Rome Tor Vergata, Rome, Italy. 58Elsevier, Philadelphia, PA, USA. 59Research Collaborations Unit, Elsevier, Jericho, VT, USA. 60Institute for Computational Biomedicine, Heidelberg University, Heidelberg, Germany. 61cBio Center, Divisions of Biostatistics and Computational Biology, Department of Data Sciences, Dana-Farber Cancer Institute, Boston, MA, USA. 62Department of Cell Biology, Harvard Medical School, Boston, MA, USA. 63Computer Science Department, University of Massachusetts Boston, Boston, MA, USA. 64Clinical Bioinformatics Area, Fundacion Progreso y Salud (FPS), Hospital Virgen del Rocio, Sevilla, Spain. 65Computational Systems Medicine Group, Institute of Biomedicine of Seville (IBIS), Hospital Virgen del Rocio, Sevilla, Spain. 66Bioinformatics in Rare Diseases (BiER), Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), FPS, Hospital Virgen del Rocıo, Sevilla, Spain. 67Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. 68Quadram Institute Bioscience, Norwich, UK. 69Earlham Institute, Norwich, UK. 70European Institute for Systems Biology and Medicine (EISBM), Vourles, France. 71Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK. 72Institute of Experimental Medicine and Systems Biology, Faculty of Medicine, RWTH, Aachen University, Aachen, Germany. 73The Roslin Institute, University of Edinburgh, Edinburgh, UK. 74University of Lausanne, Lausanne, Switzerland. 75Helmholtz Zentrum M€unchen – German Research Center for Environmental Health, Institute of Computational Biology, Neuherberg, Germany. 76Interdisciplinary Research Unit Mathematics and Life Sciences, University of Bonn, Bonn, Germany. 77St. John’s University College of Pharmacy and Health Sciences, Queens, NY, USA. 78Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 79FPS/ELIXIR-es, Hospital Virgen del Rocıo, Sevilla, Spain. 80Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. 81Systems Biology Institute, Tokyo, Japan. 82Okinawa Institute of Science and Technology Graduate School, Okinawa, Japan. Richland, WA, USA. Stem Cell Institute, Ankara University, Ankara, Turkey. Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, CA, USA. List of affiliations 35Department of Bioinformatics - BiGCaT, NUTRIM, Maastricht University, Maastricht, The Netherlands. 36Maastricht Centre for Systems Biology (MaCSBio), Maastricht University, Maastricht, The Netherlands. 37Maastricht University Medical Centre, Maastricht, The Netherlands. 38Center for Systems Biology and Molecular Medicine, Yenepoya (Deemed to be University), Mangalore, India. 39MaRS Centre, Ontario Institute for Cancer Research, Toronto, ON, Canada. 40NYU Grossman School of Medicine, New York, NY, USA. 41Universidade Federal do Parana, Curitiba, Brasil. 42European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Hinxton, Cambridgeshire, UK. 43INSERM UMR_S 1256, Nutrition, Genetics, and Environmental Risk Exposure (NGERE), Faculty of Medicine of Nancy, University of Lorraine, Nancy, France. 44Laboratoire de genetique medicale, CHRU Nancy, Nancy, France. 45Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK. 46Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Bielefeld, Germany. 47Department of Surgical Science, Uppsala University, Uppsala, Sweden. 48Institute of Computing Science, Poznan University of Technology, Poznan, Poland. 49Department of Medical Informatics and Clinical Epidemiology, Oregon Health & Science University, Portland, OR, USA. 50Heidelberg Institute for Theoretical Studies (HITS), Heidelberg, Germany. 51Department of Computer Science, The University of Manchester, Manchester, UK. 52German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany. 53Center for Regenerative Therapies Dresden (CRTD), Technische Universit€at Dresden, Dresden, Germany. 54Institute for Biology, Humboldt University of Berlin, Berlin, Germany. 55Biomax Informatics AG, Planegg, Germany. 56Harvard Medical School, Laboratory of Systems Pharmacology, Boston, MA, USA. 57Department of Biology, University of Rome Tor Vergata, Rome, Italy. 58Elsevier, Philadelphia, PA, USA. 59Research Collaborations Unit, Elsevier, Jericho, VT, USA. 60Institute for Computational Biomedicine, Heidelberg University, Heidelberg, Germany. 61cBio Center, Divisions of Biostatistics and Computational Biology, Department of Data Sciences, Dana-Farber Cancer Institute, Boston, MA, USA. 62Department of Cell Biology, Harvard Medical School, Boston, MA, USA. 63Computer Science Department, University of Massachusetts Boston, Boston, MA, USA. 64Clinical Bioinformatics Area, Fundacion Progreso y Salud (FPS), Hospital Virgen del Rocio, Sevilla, Spain. 65Computational Systems Medicine Group, Institute of Biomedicine of Seville (IBIS), Hospital Virgen del Rocio, Sevilla, Spain. 66Bioinformatics in Rare Diseases (BiER), Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), FPS, Hospital Virgen del Rocıo, Sevilla, Spain. 67Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, USA. 68Quadram Institute Bioscience, Norwich, UK. 69Earlham Institute, Norwich, UK. 70European Institute for Systems Biology and Medicine (EISBM), Vourles, France. 71Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK. 72Institute of Experimental Medicine and Systems Biology, Faculty of Medicine, RWTH, Aachen University, Aachen, Germany. List of affiliations 73The Roslin Institute, University of Edinburgh, Edinburgh, UK. 74University of Lausanne, Lausanne, Switzerland. 75Helmholtz Zentrum M€unchen – German Research Center for Environmental Health, Institute of Computational Biology, Neuherberg, Germany. 76Interdisciplinary Research Unit Mathematics and Life Sciences, University of Bonn, Bonn, Germany. 77St. John’s University College of Pharmacy and Health Sciences, Queens, NY, USA. 78Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 79FPS/ELIXIR-es, Hospital Virgen del Rocıo, Sevilla, Spain. 80Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. 81Systems Biology Institute, Tokyo, Japan. 82Okinawa Institute of Science and Technology Graduate School, Okinawa, Japan. 22 of 22 Molecular Systems Biology 17: e10387 \| 2021 ª 2021 The Authors
https://openalex.org/W2979904979	https://www.biorxiv.org/content/biorxiv/early/2019/10/09/799213.full.pdf	English	null	Asymmetric Gait Patterns Alter the Reactive Control of Intersegmental Coordination Patterns during Walking	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	14,917	. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 1 Asymmetric Gait Patterns Alter the Reactive Control of 2 Intersegmental Coordination Patterns during Walking 3 Chang Liu1, and James M. Finley1,2,3 4 5 1Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, 6 90089 7 2Division of Biokinesiology and Physical Therapy, University of Southern California, Los Angeles, 8 CA, 90033 9 3Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, 90089 10 11 * Correspondence: James M. Finley, Ph.D. Locomotor Control Lab, Division of Biokinesiology 12 and Physical Therapy, University of Southern California,1540 E. Alcazar St, CHP 155, Los 13 Angeles, CA, USA 90033 14 jmfinley@usc.edu 15 16 Keywords: intersegmental coordination, asymmetry, locomotion, reactive control, angular 17 momentum 18 19 20 21 22 23 24 1 Asymmetric Gait Patterns Alter the Reactive Control of 2 Intersegmental Coordination Patterns during Walking 3 Chang Liu1, and James M. Finley1,2,3 4 5 1Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, 6 90089 7 2Division of Biokinesiology and Physical Therapy, University of Southern California, Los Angeles, 8 CA, 90033 9 3Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, 90089 10 11 * Correspondence: James M. Finley, Ph.D. Locomotor Control Lab, Division of Biokinesiology 12 and Physical Therapy, University of Southern California,1540 E. Alcazar St, CHP 155, Los 13 Angeles, CA, USA 90033 14 jmfinley@usc.edu 15 16 Keywords: intersegmental coordination, asymmetry, locomotion, reactive control, angular 17 momentum 2 Intersegmental Coordination Patterns during Walking . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 48 provide further insight into how the healthy and impaired nervous system regulates dynamic 49 balance during walking. 25 Abstract 25 Abstract 26 Recovery from perturbations during walking is primarily mediated by reactive control 27 strategies that coordinate multiple body segments to maintain balance. Balance control is often 28 impaired in clinical populations who walk with spatiotemporally asymmetric gait, and, as a 29 result, rehabilitation efforts often seek to reduce asymmetries in these populations. Previous 30 work has demonstrated that the presence of spatiotemporal asymmetries during walking does 31 not impair the control of whole-body dynamics during perturbation recovery. However, it 32 remains to be seen how the neuromotor system adjusts intersegmental coordination patterns to 33 maintain invariant whole-body dynamics. Here, we determined if the neuromotor system 34 generates stereotypical coordination patterns irrespective of the level of asymmetry or if the 35 neuromotor system allows for variance in intersegmental coordination patterns to stabilize 36 whole-body dynamics. Nineteen healthy participants walked on a dual-belt treadmill at a range 37 of step length asymmetries, and they responded to unpredictable, slip-like perturbations. We 38 used principal component analysis of segmental angular momenta to characterize 39 intersegmental coordination patterns before, during, and after imposed perturbations. We found 40 that two principal components were sufficient to explain ~ 95% of the variance in segmental 41 angular momentum during both steading walking and responses to perturbations. Our results 42 also revealed that walking with asymmetric step lengths led to changes in intersegmental 43 coordination patterns during the perturbation and during subsequent recovery steps without 44 affecting whole-body angular momentum. These results suggest that the nervous system allows 45 for variance in segment-level coordination patterns to maintain invariant control of whole-body 46 angular momentum during walking. Future studies exploring how these segmental coordination 47 patterns change in individuals with asymmetries that result from neuromotor impairments can . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . 50 1 Introduction WBAM reflects the net 61 influence of all the body segments’ rotation relative to a specified axis, which is commonly taken 62 to project through the body's center of mass [6–8]. WBAM is highly regulated as its value 63 remains close to zero during normal, unperturbed walking [9,10]. During perturbed walking, 64 angular momentum dramatically deviates from that measured during unperturbed walking [6,7], 65 and this deviation captures the features of body rotation that, if not arrested, would lead to a fall. 66 To regain balance when encountering unexpected perturbations, the central nervous system 67 activates muscles to accelerate body segments and restore angular momentum across multiple 68 recovery steps [11,12]. 51 Bipedal locomotion is inherently unstable due to the small base of support, long single- 52 limb support times, and sensorimotor transmission delays [1]. As a result, we must frequently 53 generate corrective responses to maintain balance in response to both internal and external 54 perturbations [2,3]. For example, to recover from unexpected perturbations such as slips or trips 55 while walking, the nervous system generates reactive control strategies involving simultaneous, 56 coordinated responses of both the upper and lower limbs [4,5]. These reactive, interlimb 57 responses to perturbations can restore stability by generating changes in angular momentum that 58 counteract the body's rotation toward the ground. 59 One conventional method to capture whole-body rotational dynamics during perturbation 60 responses is to compute whole-body angular momentum (WBAM). WBAM reflects the net 61 influence of all the body segments’ rotation relative to a specified axis, which is commonly taken 62 to project through the body's center of mass [6–8]. WBAM is highly regulated as its value 63 remains close to zero during normal, unperturbed walking [9,10]. During perturbed walking, 64 angular momentum dramatically deviates from that measured during unperturbed walking [6,7], 65 and this deviation captures the features of body rotation that, if not arrested, would lead to a fall. 66 To regain balance when encountering unexpected perturbations, the central nervous system 67 activates muscles to accelerate body segments and restore angular momentum across multiple 68 recovery steps [11,12]. 69 Angular momentum can also capture balance impairments in populations with gait 70 asymmetries and sensorimotor deficits such as amputees and stroke survivors. 25 Abstract CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 48 provide further insight into how the healthy and impaired nervous system regulates dynamic 49 balance during walking. 48 49 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 50 1 Introduction 51 Bipedal locomotion is inherently unstable due to the small base of support, long single- 52 limb support times, and sensorimotor transmission delays [1]. As a result, we must frequently 53 generate corrective responses to maintain balance in response to both internal and external 54 perturbations [2,3]. For example, to recover from unexpected perturbations such as slips or trips 55 while walking, the nervous system generates reactive control strategies involving simultaneous, 56 coordinated responses of both the upper and lower limbs [4,5]. These reactive, interlimb 57 responses to perturbations can restore stability by generating changes in angular momentum that 58 counteract the body's rotation toward the ground. 59 One conventional method to capture whole-body rotational dynamics during perturbation 60 responses is to compute whole-body angular momentum (WBAM). WBAM reflects the net 61 influence of all the body segments’ rotation relative to a specified axis, which is commonly taken 62 to project through the body's center of mass [6–8]. WBAM is highly regulated as its value 63 remains close to zero during normal, unperturbed walking [9,10]. During perturbed walking, 64 angular momentum dramatically deviates from that measured during unperturbed walking [6,7], 65 and this deviation captures the features of body rotation that, if not arrested, would lead to a fall. 66 To regain balance when encountering unexpected perturbations, the central nervous system 67 activates muscles to accelerate body segments and restore angular momentum across multiple 68 recovery steps [11,12]. 69 Angular momentum can also capture balance impairments in populations with gait 70 asymmetries and sensorimotor deficits such as amputees and stroke survivors. These individuals 51 Bipedal locomotion is inherently unstable due to the small base of support, long single- 52 limb support times, and sensorimotor transmission delays [1]. As a result, we must frequently 53 generate corrective responses to maintain balance in response to both internal and external 54 perturbations [2,3]. For example, to recover from unexpected perturbations such as slips or trips 55 while walking, the nervous system generates reactive control strategies involving simultaneous, 56 coordinated responses of both the upper and lower limbs [4,5]. These reactive, interlimb 57 responses to perturbations can restore stability by generating changes in angular momentum that 58 counteract the body's rotation toward the ground. 59 One conventional method to capture whole-body rotational dynamics during perturbation 60 responses is to compute whole-body angular momentum (WBAM). 50 1 Introduction PCA reduces the high-dimensional, multi-segmental time series data into a lower- 93 dimensional set of latent variables capable of capturing the variance in the overall behavior. 94 Aprigliano et al. used PCA to show that there is no difference in intersegmental coordination 95 patterns between fall-prone older adults and healthy young adults in response to slip-like 96 perturbations [19]. Other studies used PCA of segmental angular momentum to show that the 97 intersegmental coordination patterns observed during recovery from slip-like perturbations are 98 highly correlated with the patterns observed during unperturbed walking [20,21]. Together, these 99 studies suggest that the central nervous system may adopt a preprogrammed and invariant 100 response to perturbation recovery across different tasks and populations. 101 Here, our objective was to determine how the presence of step length asymmetries 102 influences patterns of intersegmental coordination during slip-like perturbations. Since it has 103 previously been demonstrated that step length asymmetry does not influence the magnitude of 104 whole-body angular momentum, we aimed to determine if this was because the neuromotor 105 system generates stereotypical intersegmental coordination patterns across levels of asymmetry 80 strategy that the central nervous system uses to stabilize whole-body dynamics remains to be 81 determined. There are two distinct hypotheses capable of explaining the negligible influence of 82 asymmetry on whole-body angular momentum. First, the central nervous system may generate 83 stereotypical, invariant intersegmental coordination patterns in response to perturbations, 84 irrespective of the level of asymmetry. Alternatively, the nervous system could use reactive 85 control strategies that covary with asymmetry in a manner that would lead to invariant control of 86 whole-body momentum. This would be consistent with the uncontrolled manifold (UCM) 87 hypothesis, which predicts that the nervous system allows for variability in segmental angular 88 momenta to stabilize a higher-order performance variable such as whole-body angular 89 momentum [18]. 80 strategy that the central nervous system uses to stabilize whole-body dynamics remains to be 81 determined. There are two distinct hypotheses capable of explaining the negligible influence of 82 asymmetry on whole-body angular momentum. First, the central nervous system may generate 83 stereotypical, invariant intersegmental coordination patterns in response to perturbations, 84 irrespective of the level of asymmetry. Alternatively, the nervous system could use reactive 85 control strategies that covary with asymmetry in a manner that would lead to invariant control of 86 whole-body momentum. 50 1 Introduction These individuals 71 often have a higher peak-to-peak range of angular momentum than healthy controls [13–16], and 72 the presence of gait asymmetries may contribute to balance impairments in these populations. 73 For example, the magnitude of step length asymmetry in people-post stroke is negatively 74 correlated with scores on the Berg Balance Scale, indicating that step length asymmetry is 75 associated with increased fall risk [17]. 76 An important question for clinical researchers is whether there is a causal relationship 77 between gait asymmetry and the ability to maintain balance in response to perturbations during 78 walking. Previous work demonstrated that whole-body dynamics, as measured by WBAM, do 79 not change in response to imposed gait asymmetries in healthy individuals [7]. However, the . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 80 strategy that the central nervous system uses to stabilize whole-body dynamics remains to be 81 determined. There are two distinct hypotheses capable of explaining the negligible influence of 82 asymmetry on whole-body angular momentum. First, the central nervous system may generate 83 stereotypical, invariant intersegmental coordination patterns in response to perturbations, 84 irrespective of the level of asymmetry. Alternatively, the nervous system could use reactive 85 control strategies that covary with asymmetry in a manner that would lead to invariant control of 86 whole-body momentum. This would be consistent with the uncontrolled manifold (UCM) 87 hypothesis, which predicts that the nervous system allows for variability in segmental angular 88 momenta to stabilize a higher-order performance variable such as whole-body angular 89 momentum [18]. 90 Dimensionality reduction techniques, such as principal component analysis (PCA), are 91 commonly used to capture how the central nervous system coordinates multiple limb segments 92 [6,19]. 50 1 Introduction This would be consistent with the uncontrolled manifold (UCM) 87 hypothesis, which predicts that the nervous system allows for variability in segmental angular 88 momenta to stabilize a higher-order performance variable such as whole-body angular 89 momentum [18]. 90 Dimensionality reduction techniques, such as principal component analysis (PCA), are 91 commonly used to capture how the central nervous system coordinates multiple limb segments 92 [6,19]. PCA reduces the high-dimensional, multi-segmental time series data into a lower- 93 dimensional set of latent variables capable of capturing the variance in the overall behavior. 94 Aprigliano et al. used PCA to show that there is no difference in intersegmental coordination 95 patterns between fall-prone older adults and healthy young adults in response to slip-like 96 perturbations [19]. Other studies used PCA of segmental angular momentum to show that the 97 intersegmental coordination patterns observed during recovery from slip-like perturbations are 98 highly correlated with the patterns observed during unperturbed walking [20,21]. Together, these 99 studies suggest that the central nervous system may adopt a preprogrammed and invariant 100 response to perturbation recovery across different tasks and populations. 101 Here, our objective was to determine how the presence of step length asymmetries 102 influences patterns of intersegmental coordination during slip-like perturbations. Since it has 103 previously been demonstrated that step length asymmetry does not influence the magnitude of 104 whole-body angular momentum, we aimed to determine if this was because the neuromotor 105 system generates stereotypical intersegmental coordination patterns across levels of asymmetry 106 or because the neuromotor system generates patterns of intersegmental coordination that covary 107 with spatiotemporal asymmetry. Ultimately, our findings extend our understanding of how the 108 healthy central nervous system coordinates intersegmental dynamics to maintain balance during 109 walking. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 2.1 Participant characteristics 112 A total of 19 healthy young individuals (10M, 24 ± 4 yrs old) with no musculoskeletal or 113 gait impairments participated in this study. Lower limb dominance was determined by asking 114 participants which leg they would use to kick a ball. The study was approved by the Institutional 115 Review Board at the University of Southern California, and all participants provided informed 116 consent before participating. All aspects of the study conformed to the principles described in the 117 Declaration of Helsinki. 118 2.2 Experiment protocol It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 139 was remotely triggered by preprogrammed Python code and was characterized by a trapezoidal 140 speed profile in which the treadmill accelerated at foot strike to 1.5 m/s at an acceleration of 1.6 141 m/s2, held this speed for 0.3 s, and then decelerated back to 1.0 m/s during the swing phase of the 142 perturbed leg. Participants were aware that they would experience perturbations during the 143 experiment, but the perturbations were randomly triggered to occur within a range of 20 to 30 144 steps after the previous perturbation to prevent participants from precisely anticipating 145 perturbation timing. This range of steps was also selected to provide participants with sufficient 146 time to reestablish their walking pattern to match with the visual feedback. 147 2.3 Data Acquisition 148 A ten-camera motion capture system (Qualisys AB, Gothenburg, Sweden) recorded 3D 149 marker kinematics at 100 Hz and ground reaction forces at 1000 Hz. We placed a set of 19 mm 150 spherical markers on anatomical landmarks to create a 13-segment, full-body model [26,27]. We 151 placed marker clusters on the upper arms, forearms, thighs, shanks, and the back of heels. 152 Marker positions were calibrated during a five-second standing trial at the beginning of each 153 trial. We removed all joint markers after the calibration. 118 2.2 Experiment protocol 119 Data used here were collected as part of a previous study [7], and we provide a summary 120 of the procedures and setup below. Participants walked on an instrumented, dual-belt treadmill 121 with force plates underneath (Bertec, USA) at 1.0 m/s for six separate trials and reacted to 122 accelerations of the treadmill belts throughout the experiment. Although 1 m/s was slower than 123 the reported average self-selected speed during treadmill walking [22], we chose this speed to be 124 consistent with other investigations of the role of asymmetry during healthy gait [23–25]. For 125 the first trial, participants walked on the treadmill for three minutes (Baseline) to obtain their 126 natural level of step length asymmetry. Then, for subsequent trials, participants were instructed 127 to modify their step lengths according to visual feedback provided via a display attached to the 128 treadmill, and we informed them that random slip-like perturbations would occur during these 129 trials. The visual feedback displayed the target step length for both right and left legs. A 130 “success” message would appear on the screen if the participants were able to step within three 131 standard deviations of the target step length. Participants completed a randomized sequence of 132 five, six-minute trials with target step length asymmetries (SLA, Eq. 1) of 0%, 10%, and ± ± 133 15% where 0% represents each participant’s baseline SLA. 130 “success” message would appear on the screen if the participants were able to step within three 131 standard deviations of the target step length. Participants completed a randomized sequence of 132 five, six-minute trials with target step length asymmetries (SLA, Eq. 1) of 0%, 10%, and ± ± 133 15% where 0% represents each participant’s baseline SLA. 134 (1) 𝑆𝐿𝐴= 100 ∗ SLleft ‒ SLright SLleft + SLright (1) 135 represents left step length and represents the right step length. Each trial SLleft SLright 136 consisted of one-minute of practice walking without any perturbations, and then a total of 20 137 perturbations were applied (10 to each belt) during the remainder of the trial. Foot strike was 138 computed as the point when vertical ground reaction forces reached 150 N. Each perturbation . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 154 2.4 Data processing The model included the following segments: head, thorax, pelvis, upper arms, 176 forearms, thighs, shanks, and feet. The limb segments’ mass was modeled based on 177 anthropometric tables [30], and segment geometry was modeled based on the description in 178 Hanavan [31]. All segments were modeled with six degrees of freedom, and we did not define 179 any constraints between segments. Segmental linear and angular velocity were computed using 180 Eq. 2 [15]. 169 observed. Only minor deviations in WBAM about the roll and yaw axes occurred during the 170 perturbation and recovery steps [7]. 169 observed. Only minor deviations in WBAM about the roll and yaw axes occurred during the 170 perturbation and recovery steps [7]. 172 We created a 13-segment, whole-body model in Visual3D and calculated the angular 173 momentum of each segment about the body’s center of mass. Segmental angular momenta ( ) 𝐿i𝑠 174 captured how the rotational behavior of each body segment changed in response to the treadmill 175 perturbations. The model included the following segments: head, thorax, pelvis, upper arms, 176 forearms, thighs, shanks, and feet. The limb segments’ mass was modeled based on 177 anthropometric tables [30], and segment geometry was modeled based on the description in 178 Hanavan [31]. All segments were modeled with six degrees of freedom, and we did not define 179 any constraints between segments. Segmental linear and angular velocity were computed using 180 Eq. 2 [15]. (2) 𝐿i𝑠 = 𝑚𝑖(𝑟 𝑖 𝐶𝑀‒ 𝑖 × 𝑣 𝑖 𝐶𝑀‒ 𝑖 ) + 𝐼𝑖𝜔𝑖 𝑀𝑉𝐻 (2) 182 Here, mi is segmental mass, rCM-i is a vector from the segment's COM to the body's COM, 183 vCM-i is the velocity of each segment’s COM relative to the body’s COM, Ii is the segmental 184 moment of inertia, is segmental angular velocity, and the index i corresponds to individual ω𝑖 185 limb segments. Lastly, we normalized momentum by the participant’s mass (M), baseline 186 treadmill velocity (V), and the participant’s height (H) (Eq. 2) following previous literature 187 [9,16]. Since our statistical analysis used a within-subject design, the choice of variables used for 188 normalization should not affect the statistical results.The convention for measuring angular 189 momentum was defined such that positive values represented backward rotation. 154 2.4 Data processing 155 We post-processed the kinematic and kinetic data in Visual3D (C-Motion, Rockville, 156 MD, USA) and Matlab 2017a (Mathworks, USA) to compute variables of interest. Marker 157 positions and ground reaction forces were low-pass filtered by 4th order Butterworth filters with 158 cutoff frequencies of 6 Hz and 20 Hz, respectively. We selected the type of filter and cut-off 159 frequency based on previous literature [3,28,29]. We calculated the achieved SLA as follows: 160 first, we calculated the mean SLA of the four strides before each perturbation and then 161 distributed these mean values into five equally spaced bins centered at -15%, -10%, 0, 10%, 15% 162 with bin width equal to 5%. We used this achieved SLA instead of target SLA as the independent 163 variable in our statistical analyses. We categorized Baseline (BSL) steps as the two steps before 164 the perturbation occurred, perturbation (PTB) steps as the step during which the perturbation was 165 applied, and recovery (REC) steps as the steps that followed the perturbation. Since we did not 166 find any differences between left and right perturbations, our current analysis includes only 167 perturbations of the right limb [7]. We also focused our analysis on angular momentum about the 168 pitch axis as this was the direction in which the most prominent changes in WBAM were . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 169 observed. Only minor deviations in WBAM about the roll and yaw axes occurred during the 170 perturbation and recovery steps [7]. 171 2.5 Segmental Angular Momentum 172 We created a 13-segment, whole-body model in Visual3D and calculated the angular 173 momentum of each segment about the body’s center of mass. Segmental angular momenta ( ) 𝐿i𝑠 174 captured how the rotational behavior of each body segment changed in response to the treadmill 175 perturbations. 190 2.6 Principal component analysis (PCA) 191 We used principal component analysis (PCA) to extract intersegmental coordination 192 patterns for each step cycle. Before performing PCA, we first time normalized the time series of 193 segmental angular momenta to 100 points for each step cycle. Then, for each participant, we 194 generated an matrix for each achieved SLA ( step type 𝐿𝑠 ± 15%, ± 10%, ± 5%, %0) and 195 (BSL1, BSL2, PTB, REC1, REC2, REC3, REC4) with n_steps100 rows and 13 columns. On 196 average, we created 6 (achieved SLA) by 7 (step types) matrices per participant as not all 197 participants achieved each desired level of asymmetry. We then standardized each matrix to have . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 198 zero mean and performed PCA to extract subject-specific coordination patterns using the pca 199 function in Matlab’s Statistical and Machine Learning Toolbox. Using PCA, we decomposed the 200 segmental angular momenta data into 1) a weighting coefficient matrix consisting of principal 201 components (PCs) ordered according to their variance accounted for (VAF) and 2) time series 202 scores which represented the activation of each PC throughout the step cycle (Figure 1). We 203 retained the number of PCs necessary to account for at least 90% of variance in . 𝐿𝑠 204 205 Figure 1: (A) Sagittal plane angular momentum (Lx) for 13 segments during one representative 206 baseline stride (black) and one perturbation stride (grey). The segments included the thigh, 207 shank, foot, forearm, and upper arm, bilaterally as well as the head, pelvis, and thorax. The 208 duration of each trace is one full stride from 0 to 100% of the stride cycle. (B) Schematic of 209 principal component analysis (PCA) of segmental angular momentum. The organization of the 210 data used as input to the PCA is illustrated to the left. 190 2.6 Principal component analysis (PCA) PCA extracts weighting coefficient as 211 intersegmental coordination patterns or principal components (PC1 and PC2) and time series 212 scores of each PC (Filled bar plots: PC1; Open bar plots: PC2). 213 214 2.7 Comparison of intersegmental coordination patterns 215 To investigate how intersegmental coordination patterns changed after each perturbation, 216 we compared the PCs extracted from the perturbation and recovery steps to the PCs extracted 217 from baseline steps. We computed the included angle ( , Eq. 3) between each pair of PCs as θstep 218 this is a common method to compare the similarity between vectors in a high-dimensional space. 219 The included angle of the unit vectors was between 0° (parallel and identical) and 90° 220 (orthogonal and most dissimilar) [32]. 198 zero mean and performed PCA to extract subject-specific coordination patterns using the pca 199 function in Matlab’s Statistical and Machine Learning Toolbox. Using PCA, we decomposed the 200 segmental angular momenta data into 1) a weighting coefficient matrix consisting of principal 201 components (PCs) ordered according to their variance accounted for (VAF) and 2) time series 202 scores which represented the activation of each PC throughout the step cycle (Figure 1). We 203 retained the number of PCs necessary to account for at least 90% of variance in . 𝐿𝑠 14 2.7 Comparison of intersegmental coordination patterns ncluded angle of the unit vectors was between 0° (parallel and identical) and 90° (3) θstep = cos ‒ 1 (𝑃𝐶𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒∙𝑃𝐶𝑝𝑜𝑠𝑡) (3) θstep = cos ‒ 1 (𝑃𝐶𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒∙𝑃𝐶𝑝𝑜𝑠𝑡) (3) 222 We then determined if the included angle between perturbation steps and baseline steps 223 was outside the distribution of included angles observed during unperturbed baseline walking. 224 To this end, we performed a permutation test that randomly and repeatedly selected two groups 225 of ten baseline steps for each participant. For each permutation, we first performed PCA for each 226 group of 10 steps and then calculated the included angle between the two PCs. We repeated this 227 shuffling process 10000 times for each participant. We used the median of this distribution as a 228 threshold to determine if the included angle for post-perturbation values was greater than what 229 would be expected from step-to-step variance. 222 We then determined if the included angle between perturbation steps and baseline steps . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 230 Similarly, we computed the included angle between PCs extracted during walking at 231 different levels of asymmetry to those extracted from symmetrical walking to investigate how 232 asymmetry influenced intersegmental coordination patterns. (Eqn. 4). Similarly, we computed the included angle between PCs extracted during walking 230 Similarly, we computed the included angle between PCs extracted during walking at 231 different levels of asymmetry to those extracted from symmetrical walking to investigate how 232 asymmetry influenced intersegmental coordination patterns. (Eqn. 4). 14 2.7 Comparison of intersegmental coordination patterns (4) θasym = cos ‒ 1 (𝑃𝐶𝑠𝑦𝑚∙𝑃𝐶𝑎𝑠𝑦𝑚) (4) 234 We also determined if the differences in coordination observed during walking with 235 different levels of asymmetry were above the level of variance observed during symmetrical 236 walking. As described above, we obtained a reference distribution of included angles from 237 symmetric walking to determine if the included angle for each level of asymmetry was greater 238 than would be expected from natural, step-to-step variance. 259 be expected by chance. We used Welch’s t-test because the included angle was not normally 260 distributed. 261 Lastly, we determined if the included angle between each asymmetric trial and symmetric 262 walking varied with the magnitude or direction of asymmetry. For this analysis, the independent 263 variables were the magnitude of asymmetry, the direction of asymmetry, and the interaction 264 between asymmetry magnitude and direction, and the dependent variable was . We fit θasym 265 separate linear mixed-effect models for each of five steps (Baseline1, Baseline2, Perturbation, 266 Recovery 1 and Recovery 2) and each PC. We performed a log transformation of the dependent 267 variable ( ) to ensure that the residuals were normally distributed. θasym 269 For all steps, two principal components accounted for ~95% of the variance in segmental 270 angular momentum (Table 1). On average, PC1 explained 74 ± 4% of the variance, and PC2 271 explained 22± 1% of the variance, while PC3 accounted for less than 3% of the variance. Thus, 272 the remaining analysis focuses on the first two PCs. 273 Table 1: Variance accounted for (VAF) for PC1, PC2, and PC3 during baseline steps, 274 perturbation steps, and recovery steps. Step Type PC1 PC2 PC3 Sum Baseline steps 74±4% 22±5% 2±1% 98±1% Perturbation steps 75±5% 20±5% 3±1% 98±1% Recovery steps 74±3% 21±4% 3±1% 98±1% All steps 74±4% 22±4% 2±1% 98±1% 275 1: Variance accounted for (VAF) for PC1, PC2, and PC3 during baseline steps, bation steps, and recovery steps. 276 3.1 Patterns of intersegmental coordination when walking with equal step lengths 239 2.8 Statistical analysis We performed a log transformation of the dependent 267 variable ( ) to ensure that the residuals were normally distributed. θasym 239 2.8 Statistical analysis 240 All statistical analyses were performed in R (3.4.3) using linear mixed-effects (LME) 241 models. We used the lme4 package to fit the model, the multcomp comparison for multiple 242 comparisons [33], and lmerTest package to calculate p-values [34]. Residual normality was 243 confirmed using the Shapiro-Wilk test. When computing p-values, we used the Satterthwaite 244 approximation for the degrees of freedom based on differences in variance between conditions. 245 We used the Bonferroni correction for multiple comparisons for all post-hoc analyses. For each 246 model, we determined if random effects were necessary by comparing a model including random 247 intercepts for each participant against a model with only fixed effects. The most parsimonious 248 model was chosen based on the results of a likelihood ratio test. The random effects were 249 included to account for the individual differences between subjects. Significance was set at 250 p<0.05 level. 251 We first determined if the PCs extracted from the recovery steps differed from the PCs 252 extracted from the baseline steps during symmetrical walking. Here, the independent variable 253 was step type, and the dependent variable was . The models were fit for both PC1 and PC2. θstep 254 We performed a log transformation of the dependent variable ( ) to ensure that the residuals θstep 255 were normally distributed. Then, we determined if intersegmental coordination patterns during 256 asymmetrical walking differed from those during symmetrical walking. For this analysis, we 257 used Welch’s t-test to evaluate if the included angle between the PCs extracted from the 258 asymmetrical trials and those extracted from symmetric walking were greater than what would . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 259 be expected by chance. We used Welch’s t-test because the included angle was not normally 260 distributed. 239 2.8 Statistical analysis 261 Lastly, we determined if the included angle between each asymmetric trial and symmetr 262 walking varied with the magnitude or direction of asymmetry. For this analysis, the independen 263 variables were the magnitude of asymmetry, the direction of asymmetry, and the interaction 264 between asymmetry magnitude and direction, and the dependent variable was . We fit θasym 265 separate linear mixed-effect models for each of five steps (Baseline1, Baseline2, Perturbation, 266 Recovery 1 and Recovery 2) and each PC. We performed a log transformation of the dependent 267 variable ( ) to ensure that the residuals were normally distributed. θasym 268 3 Results 269 For all steps, two principal components accounted for ~95% of the variance in segmenta 270 angular momentum (Table 1). On average, PC1 explained 74 ± 4% of the variance, and PC2 271 explained 22± 1% of the variance, while PC3 accounted for less than 3% of the variance. Thus, 272 the remaining analysis focuses on the first two PCs. 273 Table 1: Variance accounted for (VAF) for PC1, PC2, and PC3 during baseline steps, 274 perturbation steps, and recovery steps. Step Type PC1 PC2 PC3 Sum Baseline steps 74±4% 22±5% 2±1% 98±1% Perturbation steps 75±5% 20±5% 3±1% 98±1% Recovery steps 74±3% 21±4% 3±1% 98±1% All steps 74±4% 22±4% 2±1% 98±1% 275 276 3.1 Patterns of intersegmental coordination when walking with equal step lengths 277 Contributions from the lower extremities were typically dominant in the first PC, while 278 contributions from the arms, pelvis, thorax, and head were less prominent (Figure 2). During 279 right steps, the left leg was in the swing phase and generated more positive momentum about th 280 body's COM, while the right leg generated negative momentum. Thus, the weighting coefficien 259 be expected by chance. We used Welch’s t-test because the included angle was not normally 260 distributed. 261 Lastly, we determined if the included angle between each asymmetric trial and symmetric 262 walking varied with the magnitude or direction of asymmetry. For this analysis, the independent 263 variables were the magnitude of asymmetry, the direction of asymmetry, and the interaction 264 between asymmetry magnitude and direction, and the dependent variable was . We fit θasym 265 separate linear mixed-effect models for each of five steps (Baseline1, Baseline2, Perturbation, 266 Recovery 1 and Recovery 2) and each PC. 276 3.1 Patterns of intersegmental coordination when walking with equal step lengths Similarly, during the left step, the right thigh and shank momenta 299 opposed the right foot momentum. Thus, PC2 captured intralimb cancellation of segmental 300 momenta. 285 segments were negative. Overall, the first PC captured the opposing momenta of the two legs 286 resulting from differences in the direction of rotation relative to the body's center of mass. 287 Figure 2: Principal components (PC) extracted from segmental angular momentum during (A) 288 baseline right steps, (B) baseline left steps, (C) perturbation steps, (D) recovery left steps, and 289 (E) recovery right steps when walking symmetrically (N=17). Blue: Right step; Pink: Left step; 290 Filled bars: PC1; Unfilled bars: PC2. The 13 segments include: RTH (right thigh), RSH (right 291 shank), RFT (right foot), LTH (left thigh), LSH (left shank), LFT (left foot), LFA (left forearm), 292 RFA (right forearm), LUA (left upper arm), RUA (right upper arm), H (head), PEL (pelvis), 293 THX (thorax). 294 295 For PC2, weighting coefficients for distal segments were also larger than the weighting 296 coefficients for proximal segments, although the coefficient for the thorax (THX) increased 297 compared to that in PC1. During the right step, the left thigh and left shank’s momenta opposed 298 the momentum of the left foot. Similarly, during the left step, the right thigh and shank momenta 299 opposed the right foot momentum. Thus, PC2 captured intralimb cancellation of segmental 300 momenta. 285 segments were negative. Overall, the first PC captured the opposing momenta of the two legs 286 resulting from differences in the direction of rotation relative to the body's center of mass. 276 3.1 Patterns of intersegmental coordination when walking with equal step lengths 277 Contributions from the lower extremities were typically dominant in the first PC, while 278 contributions from the arms, pelvis, thorax, and head were less prominent (Figure 2). During 279 right steps, the left leg was in the swing phase and generated more positive momentum about the 280 body's COM, while the right leg generated negative momentum. Thus, the weighting coefficients 281 for the left leg segments (left thigh, shank, and foot) were positive while the coefficients for the 282 right leg segments were negative. Similarly, during a left step, the right leg was in the swing 283 phase and generated more positive momentum about COM, while the left leg generated negative 284 momentum. Thus, the weighting coefficients were positive while the coefficients for the left leg . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 285 segments were negative. Overall, the first PC captured the opposing momenta of the tw 286 resulting from differences in the direction of rotation relative to the body's center of ma 287 Figure 2: Principal components (PC) extracted from segmental angular momentum dur 288 baseline right steps, (B) baseline left steps, (C) perturbation steps, (D) recovery left step 289 (E) recovery right steps when walking symmetrically (N=17). Blue: Right step; Pink: L 290 Filled bars: PC1; Unfilled bars: PC2. The 13 segments include: RTH (right thigh), RSH 291 shank), RFT (right foot), LTH (left thigh), LSH (left shank), LFT (left foot), LFA (left f 292 RFA (right forearm), LUA (left upper arm), RUA (right upper arm), H (head), PEL (pe 293 THX (thorax). 294 295 For PC2, weighting coefficients for distal segments were also larger than the we 296 coefficients for proximal segments, although the coefficient for the thorax (THX) increa 297 compared to that in PC1. 276 3.1 Patterns of intersegmental coordination when walking with equal step lengths During the right step, the left thigh and left shank’s momenta 298 the momentum of the left foot. Similarly, during the left step, the right thigh and shank 299 opposed the right foot momentum. Thus, PC2 captured intralimb cancellation of segme 300 momenta. 301 3.2 Effects of perturbations on patterns of intersegmental coordination 302 During the perturbation step, there was a significant increase in the included ang 303 indicated that the intersegmental coordination patterns during perturbation steps differe 304 the coordination patterns during baseline steps (Figure 3). For this analysis, the results o 305 likelihood ratio test revealed that random effects were necessary for the regression mod 306 PC1, we found that the intersegmental coordination patterns were significantly differen 307 patterns during baseline walking for the perturbation steps (t(54)=18.2, p<2e-16), first r 308 steps (t(54)=11.8, p<2e-16), and second recovery steps (t(54)=8.4, p=2.3e-11). Similarl 309 PC2, intersegmental coordination differed during perturbation steps (t(54)=11.8, p<2.0e 310 recovery steps (t(36)=6.7, p<2e-16),and second recovery steps (t(54)=4.9,p=8.9e-6).Th 311 no significant difference between intersegmental coordination patterns during the third 312 steps for either PC1 (p = 0.97) or PC2 (p = 0.14). Thus, participants generally were able 313 restore their coordination patterns to baseline by the third recovery step. 314 285 segments were negative. Overall, the first PC captured the opposing momenta of the two legs 286 resulting from differences in the direction of rotation relative to the body's center of mass. 287 Figure 2: Principal components (PC) extracted from segmental angular momentum during (A) 288 baseline right steps, (B) baseline left steps, (C) perturbation steps, (D) recovery left steps, and 289 (E) recovery right steps when walking symmetrically (N=17). Blue: Right step; Pink: Left step; 290 Filled bars: PC1; Unfilled bars: PC2. The 13 segments include: RTH (right thigh), RSH (right 291 shank), RFT (right foot), LTH (left thigh), LSH (left shank), LFT (left foot), LFA (left forearm), 292 RFA (right forearm), LUA (left upper arm), RUA (right upper arm), H (head), PEL (pelvis), 293 THX (thorax). 294 295 For PC2, weighting coefficients for distal segments were also larger than the weighting 296 coefficients for proximal segments, although the coefficient for the thorax (THX) increased 297 compared to that in PC1. During the right step, the left thigh and left shank’s momenta opposed 298 the momentum of the left foot. 315 Figure 3: Included angle between PCs extracted during each step relative to baseline steps 316 during symmetric walking (* p<0.001). The horizontal bars and corresponding stars indicate 315 Figure 3: Included angle between PCs extracted during each step relative t 316 d i i lki ( 0 001) h h i l b d 301 3.2 Effects of perturbations on patterns of intersegmental coordination 302 During the perturbation step, there was a significant increase in the included angle, which 303 indicated that the intersegmental coordination patterns during perturbation steps differed from 304 the coordination patterns during baseline steps (Figure 3). For this analysis, the results of the log- 305 likelihood ratio test revealed that random effects were necessary for the regression model. For 306 PC1, we found that the intersegmental coordination patterns were significantly different from the 307 patterns during baseline walking for the perturbation steps (t(54)=18.2, p<2e-16), first recovery 308 steps (t(54)=11.8, p<2e-16), and second recovery steps (t(54)=8.4, p=2.3e-11). Similarly, for 309 PC2, intersegmental coordination differed during perturbation steps (t(54)=11.8, p<2.0e-16), first 310 recovery steps (t(36)=6.7, p<2e-16),and second recovery steps (t(54)=4.9,p=8.9e-6).There was 311 no significant difference between intersegmental coordination patterns during the third recovery 312 steps for either PC1 (p = 0.97) or PC2 (p = 0.14). Thus, participants generally were able to 313 restore their coordination patterns to baseline by the third recovery step. 314 315 Figure 3: Included angle between PCs extracted during each step relative to baseline steps 316 during symmetric walking ( p<0.001). The horizontal bars and corresponding stars indicate . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 317 significant differences in the included angle. The data are represented as boxplots such that the 318 lower and upper edges of the box indicate the 25th and 75th percentile of the data, respectively. 319 The horizontal line in each box indicates the median. The whiskers extend to the furthest data 320 point beyond the lower or upper edges of the box that is within a distance of 1.5 times the middle 321 50th percentile of the data. Dots that lie beyond the whiskers indicate outliers. Blue: Right step; 322 Pink: Left step; Filled box plots: PC1; Non-filled box plots: PC2. 301 3.2 Effects of perturbations on patterns of intersegmental coordination Dots that lie beyond the whisk 322 Pink: Left step; Filled box plots: PC1; Non-filled box pl 323 mean of the permutated angle distribution of baseline ste 324 standard deviation. 325 326 3.3 Effects of step length asymmetry on patterns 327 Although the general patterns of intersegmental c 328 of asymmetry, asymmetric walking patterns led to measu 329 distal lower extremity segments (Figure 4). Qualitatively 330 left foot as well as decreased weights of the left shank se 331 during right steps. This likely reflected the need for long 332 positive step length asymmetries. 333 Figure 4: The first intersegmental coordination pattern ( 334 pattern (PC2) during (A) baseline right step, (B) perturba 335 step with -15%, 0% and 15% step length asymmetry. Th 336 across all participants (N=17), and the black lines indica 337 338 As the magnitude of achieved asymmetry increas 339 deviation of intersegmental coordination patterns from s 340 of log-likelihood ratio tests showed that random intercep 341 models. One outlier was removed before fitting the linea 342 for PC2 because it was more than three standard deviatio 343 included angles. Excluding the outlier did not change the 344 differed from the permutated estimate of included angles 345 coordination at each level of asymmetry differed from th 346 symmetrical walking. For all steps, we observed a signif 347 included angle between the PCs from the asymmetric tri 317 significant differences in the included angle. The data are represented as boxplots such that the 318 lower and upper edges of the box indicate the 25th and 75th percentile of the data, respectively. 319 The horizontal line in each box indicates the median. The whiskers extend to the furthest data 320 point beyond the lower or upper edges of the box that is within a distance of 1.5 times the middle 321 50th percentile of the data. Dots that lie beyond the whiskers indicate outliers. Blue: Right step; 322 Pink: Left step; Filled box plots: PC1; Non-filled box plots: PC2. The black line indicates the 323 mean of the permutated angle distribution of baseline steps and the shading indicates the 324 standard deviation. 301 3.2 Effects of perturbations on patterns of intersegmental coordination The black line indicates the 323 mean of the permutated angle distribution of baseline steps and the shading indicates the 324 standard deviation. 325 326 3.3 Effects of step length asymmetry on patterns of intersegmental coordination 327 Although the general patterns of intersegmental coordination were similar across levels 328 of asymmetry, asymmetric walking patterns led to measurable changes in the contributions of the 329 distal lower extremity segments (Figure 4). Qualitatively, we observed increased weights at the 330 left foot as well as decreased weights of the left shank segment for the first principal component 331 during right steps. This likely reflected the need for longer left steps and faster foot swing for 332 positive step length asymmetries. 333 Figure 4: The first intersegmental coordination pattern (PC1) and the second coordination 334 pattern (PC2) during (A) baseline right step, (B) perturbation step, and (C) the second recovery 335 step with -15%, 0% and 15% step length asymmetry. The colored bars indicate the mean value 336 across all participants (N=17), and the black lines indicate the standard deviation. 337 338 As the magnitude of achieved asymmetry increased, we observed an increase in the 339 deviation of intersegmental coordination patterns from symmetrical walking (Figure 5). Results 340 of log-likelihood ratio tests showed that random intercepts were required in the regression 341 models. One outlier was removed before fitting the linear mixed model for the perturbation step 342 for PC2 because it was more than three standard deviations higher than the median of the 343 included angles. Excluding the outlier did not change the statistical outcome. All included angles 344 differed from the permutated estimate of included angles (p<0.05), indicating that intersegmental 345 coordination at each level of asymmetry differed from the coordination pattern during 346 symmetrical walking. For all steps, we observed a significant main effect of asymmetry on the 347 included angle between the PCs from the asymmetric trials and the symmetric trial (Table 2). 317 significant differences in the included angle. The data ar 318 lower and upper edges of the box indicate the 25th and 7 319 The horizontal line in each box indicates the median. Th 320 point beyond the lower or upper edges of the box that is 321 50th percentile of the data. 368 4 Discussion 369 We investigated how step length asymmetry affected intersegmental coordination 370 patterns during responses to treadmill-based slip perturbations during walking. Our central 371 finding was that intersegmental coordination patterns observed during asymmetrical walking 372 differed from symmetrical walking during both unperturbed walking and perturbation recovery. 373 When combined with previous observations that the reactive control of overall WBAM is not 374 influenced by asymmetry [7], these results indicate that healthy people use a flexible 375 combination of intersegmental coordination patterns rather than invariant reactions to maintain 376 WBAM during perturbation responses when walking with asymmetric gait patterns. 377 Variations in coordination patterns during asymmetrical walking likely resulted from 378 changes in the momentum generated by the lower extremities to reach the target asymmetry. 379 Since the distal segments of the lower limbs are relatively far from the body’s center of mass and 380 have a high velocity, they make the largest contribution to changes in intersegmental 381 coordination patterns. For example, to achieve a positive asymmetry, participants placed their 369 We investigated how step length asymmetry affected intersegmental coordination 370 patterns during responses to treadmill-based slip perturbations during walking. Our central 371 finding was that intersegmental coordination patterns observed during asymmetrical walking 372 differed from symmetrical walking during both unperturbed walking and perturbation recovery. 373 When combined with previous observations that the reactive control of overall WBAM is not 374 influenced by asymmetry [7], these results indicate that healthy people use a flexible 375 combination of intersegmental coordination patterns rather than invariant reactions to maintain 376 WBAM during perturbation responses when walking with asymmetric gait patterns. 377 Variations in coordination patterns during asymmetrical walking likely resulted from 378 changes in the momentum generated by the lower extremities to reach the target asymmetry. 379 Since the distal segments of the lower limbs are relatively far from the body’s center of mass and 380 have a high velocity, they make the largest contribution to changes in intersegmental 381 coordination patterns For example to achieve a positive asymmetry participants placed their 369 We investigated how step length asymmetry affected intersegmental coordination 370 patterns during responses to treadmill-based slip perturbations during walking. Our central 371 finding was that intersegmental coordination patterns observed during asymmetrical walking 372 differed from symmetrical walking during both unperturbed walking and perturbation recovery. 326 3.3 Effects of step length asymmetry on patterns of intersegmental coordination Blue: Right 353 step; Pink: Left step; Filled box plots: PC1; Non-filled box plots: PC2. The shaded gray area 354 indicated the standard deviation of permutated included angle for each step, and the black line 355 indicated the mean of the distribution. 351 Figure 5: included angle between PCs extracted during asymmetrical walking (5%, 10%, and 352 15%) and symmetrical walking for each step (* p<0.001, p<0.01, * p<0.05). Blue: Right 353 step; Pink: Left step; Filled box plots: PC1; Non-filled box plots: PC2. The shaded gray area 354 indicated the standard deviation of permutated included angle for each step, and the black line 355 indicated the mean of the distribution. 356 357 The included angle between the PCs extracted during asymmetric walking and symmetric 358 walking increased with the magnitude of achieved asymmetry (Figure 5). Specifically, the 359 difference between intersegmental coordination patterns was greater when walking with 15% 360 asymmetry compared to 5% asymmetry during right baseline steps (Bonferroni corrected 361 p<0.001), perturbation steps (Bonferroni corrected p<0.001), first recovery steps (Bonferroni 362 corrected p=0.03) and second recovery steps (Bonferroni corrected p=0.002) for PC1. The 363 difference in included angles was also significantly different from 5% asymmetry for PC2 when 364 walking with 15% asymmetry during baseline right steps (Bonferroni corrected p=0.01) and 365 perturbation steps (Bonferroni corrected p = 0.003) and second recovery steps (Bonferroni 366 corrected p=0.04). Lastly, there was only an effect of the direction of asymmetry for PC2 (F(1, 367 79), p=0.049) during the baseline right step (Baseline 1). 357 The included angle between the PCs extracted during asymmetric walking and symmetric 358 walking increased with the magnitude of achieved asymmetry (Figure 5). Specifically, the 359 difference between intersegmental coordination patterns was greater when walking with 15% 360 asymmetry compared to 5% asymmetry during right baseline steps (Bonferroni corrected 361 p<0.001), perturbation steps (Bonferroni corrected p<0.001), first recovery steps (Bonferroni 362 corrected p=0.03) and second recovery steps (Bonferroni corrected p=0.002) for PC1. The 363 difference in included angles was also significantly different from 5% asymmetry for PC2 when 364 walking with 15% asymmetry during baseline right steps (Bonferroni corrected p=0.01) and 365 perturbation steps (Bonferroni corrected p = 0.003) and second recovery steps (Bonferroni 366 corrected p=0.04). Lastly, there was only an effect of the direction of asymmetry for PC2 (F(1, 367 79), p=0.049) during the baseline right step (Baseline 1). 326 3.3 Effects of step length asymmetry on patterns of intersegmental coordination . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint Table 2 Statistical results from the ANOVA examining the effects of asymmetry and direction on the included angle for each step type. Step Type PC Factor numDF denDF F-value P value Baseline1 PC1 Asym 2 73 9.7 <0.001 Direction 1 77 2.4 0.13 Asym:Direction 2 74 0.6 0.55 PC2 Asym 2 72 14.4 <0.001 Direction 1 78 4.0 0.049 Asym:Direction 2 74 0.08 0.92 Baseline2 PC1 Asym 2 72 5.7 0.005 Direction 1 75 1.3 0.26 Asym:Direction 2 73 0.1 0.88 PC2 Asym 2 71 11.0 <0.001 Direction 1 74 0.007 0.93 Asym:Direction 2 72 2.2 0.12 Perturbation PC1 Asym 2 73 19.0 <0.001 Direction 1 75 1.9 0.18 Asym:Direction 2 73 0.5 0.59 PC2 Asym 2 72 8.7 <0.001 Direction 1 73 1.3 0.25 Asym:Direction 2 72 0.68 0.51 Recovery1 PC1 Asym 2 74 11.2 <0.001 Direction 1 78 0.1 0.74 Asym:Direction 2 75 1.3 0.29 PC2 Asym 2 72 9.1 <0.001 Direction 1 75 0.1 0.72 Asym:Direction 2 73 0.8 0.45 Recovery2 PC1 Asym 2 72 8.7 <0.001 Direction 1 75 1.8 0.18 Asym:Direction 2 73 0.4 0.67 PC2 Asym 2 73 8.5 <0.001 Direction 1 77 1.9 0.18 Asym:Direction 2 74 0.2 0.84 48 Table 2 Statistical results from the ANOVA examining the effects of asymmetry and direction on 49 the included angle for each step type. 350 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 326 3.3 Effects of step length asymmetry on patterns of intersegmental coordination It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 351 Figure 5: included angle between PCs extracted during asymmetrical walking (5%, 10%, and 352 15%) and symmetrical walking for each step (* p<0.001, p<0.01, * p<0.05). Blue: Right 353 step; Pink: Left step; Filled box plots: PC1; Non-filled box plots: PC2. The shaded gray area 354 indicated the standard deviation of permutated included angle for each step, and the black line 355 indicated the mean of the distribution. 351 Figure 5: included angle between PCs extracted during asymmetrical walking (5%, 10%, and 352 15%) and symmetrical walking for each step (* p<0.001, p<0.01, * p<0.05). Blue: Right 353 step; Pink: Left step; Filled box plots: PC1; Non-filled box plots: PC2. The shaded gray area 354 indicated the standard deviation of permutated included angle for each step, and the black line 355 indicated the mean of the distribution. 356 357 The included angle between the PCs extracted during asymmetric walking and symmetric 358 walking increased with the magnitude of achieved asymmetry (Figure 5). Specifically, the 359 difference between intersegmental coordination patterns was greater when walking with 15% 360 asymmetry compared to 5% asymmetry during right baseline steps (Bonferroni corrected 361 p<0.001), perturbation steps (Bonferroni corrected p<0.001), first recovery steps (Bonferroni 362 corrected p=0.03) and second recovery steps (Bonferroni corrected p=0.002) for PC1. The 363 difference in included angles was also significantly different from 5% asymmetry for PC2 when 364 walking with 15% asymmetry during baseline right steps (Bonferroni corrected p=0.01) and 365 perturbation steps (Bonferroni corrected p = 0.003) and second recovery steps (Bonferroni 366 corrected p=0.04). Lastly, there was only an effect of the direction of asymmetry for PC2 (F(1, 367 79), p=0.049) during the baseline right step (Baseline 1). 351 Figure 5: included angle between PCs extracted during asymmetrical walking (5%, 10%, and 352 15%) and symmetrical walking for each step (* p<0.001, p<0.01, * p<0.05). 368 4 Discussion 373 When combined with previous observations that the reactive control of overall WBAM is not 374 influenced by asymmetry [7], these results indicate that healthy people use a flexible 375 combination of intersegmental coordination patterns rather than invariant reactions to maintain 376 WBAM during perturbation responses when walking with asymmetric gait patterns. . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 382 left foot further in front of the center of mass and increased the extension of their right hip so that 383 the right foot was further behind their COM at heel strike. To achieve this objective, participants 384 had to increase swing velocity. This likely explains why we observed increased weights of the 385 left foot as SLA increased during right steps in the first principal component since positive step 386 length asymmetries required longer left steps and faster foot swing. 382 left foot further in front of the center of mass and increased the extension of their right hip so that 383 the right foot was further behind their COM at heel strike. To achieve this objective, participants 384 had to increase swing velocity. This likely explains why we observed increased weights of the 385 left foot as SLA increased during right steps in the first principal component since positive step 386 length asymmetries required longer left steps and faster foot swing. 387 The observation that reactive control of WBAM is consistent across levels of asymmetry 388 [7] despite the variation in intersegmental coordination observed here may indicate that WBAM 389 is a task-level variable that is stabilized by the nervous system during perturbation recovery. This 390 is consistent with the framework proposed by the uncontrolled manifold hypothesis (UCM), 391 which argues that the central nervous system allows for variability over a manifold of solutions 392 that all successfully stabilize a higher-level performance variable [35]. 368 4 Discussion In contrast, the statistical 415 differences between pre- and post-perturbation coordination patterns may reflect the changes in 416 coordination necessary to maintain balance in response to perturbations. Patterns of 417 intersegmental coordination observed during responses to external perturbations during walking 418 likely capture a combination of passive limb dynamics, stereotypical pattern generation, and 419 reactive balance control responses [37]. 420 We observed that the upper limbs’ contribution to the control of angular momentum in 421 the sagittal plane was negligible compared with lower limb segments during perturbation 422 recovery. Since a stepping response is sufficient to restore balance from the treadmill 423 accelerations used in this study, increases in momentum from the lower extremities may have 424 been sufficient to restore sagittal plane WBAM. Consistent with our findings, Pijnappels et al. 425 also found that arm movements had a small effect on body rotation in the sagittal plane during 426 tripping over obstacles which elicits excessive forward rotation similar to the current study [38]. 427 However, during larger perturbations that trigger backward falls, the arms elevate to shift the 428 body’s center of mass back within the base of support [4]. This difference in the role of the arms 429 across studies of perturbation recovery may result from the use of a larger velocity and 430 displacement of the foot in the Marigold et al. [4] study. However, it remains to be seen how 431 systematic variation of the magnitude and direction of external perturbations influences the role 432 of the upper extremities during balance recovery. 433 Our results may also have implications for understanding the potential effects of 434 interventions designed to reduce gait asymmetries in people post-stroke, as this is a common 435 rehabilitation objective in this population [39]. Based on the current results, we would expect 436 that reducing asymmetry in people post-stroke would also affect their reactive control strategies. 437 However, further investigation is necessary to determine if reductions in asymmetry affect 420 We observed that the upper limbs’ contribution to the control of angular momentum in 421 the sagittal plane was negligible compared with lower limb segments during perturbation 422 recovery. Since a stepping response is sufficient to restore balance from the treadmill 423 accelerations used in this study, increases in momentum from the lower extremities may have 424 been sufficient to restore sagittal plane WBAM. Consistent with our findings, Pijnappels et al. 368 4 Discussion Here, WBAM would 393 serve as a high-level performance variable that is stabilized through covariation of elemental, 394 segmental-level momenta. For example, Papi et al. demonstrated a similar concept when they 395 found no differences between people post-stroke and healthy individuals in COM displacement 396 during the stance phase of walking despite between-group differences in lower extremity joint 397 kinematics [36]. Therefore, it is possible that when dynamic stability is challenged during 398 walking, the central nervous system carefully regulates WBAM while allowing variance in 399 lower-level, intersegmental coordination patterns. 400 In this study, we provided visual information about the desired and actual step lengths at 401 each foot-strike throughout all trials, including the perturbation and recovery steps. Participants 402 were encouraged to achieve the target step lengths for as many steps as possible, and therefore 403 participants may have relied on this feedback during perturbation recovery to return to their pre- 404 perturbation walking patterns faster than they otherwise would without visual feedback. 405 However, participants’ reactive response is unlikely to influence measures of momentum until 406 late into the first recovery step as the step length information was only shown after the foot-strike 407 of the first recovery step. It remains to be seen if patterns of interlimb coordination would differ 408 in the presence of asymmetries that are not guided by online visual feedback. 409 Although the reactive intersegmental coordination patterns were significantly different 410 from those observed during unperturbed locomotion, the overall patterns were qualitatively 411 similar across steps. Taken together, these results may reflect two keys aspects of coordination . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 412 during perturbed walking. First, the qualitative similarity between pre- and post-perturbation 413 patterns observed here and in previous work [21] may reflect the dominant coordination patterns 414 that characterize both unperturbed and perturbed bipedal walking. 368 4 Discussion 425 also found that arm movements had a small effect on body rotation in the sagittal plane during 426 tripping over obstacles which elicits excessive forward rotation similar to the current study [38]. 427 However, during larger perturbations that trigger backward falls, the arms elevate to shift the 428 body’s center of mass back within the base of support [4]. This difference in the role of the arms 429 across studies of perturbation recovery may result from the use of a larger velocity and 430 displacement of the foot in the Marigold et al. [4] study. However, it remains to be seen how 431 systematic variation of the magnitude and direction of external perturbations influences the role 432 of the upper extremities during balance recovery. 433 Our results may also have implications for understanding the potential effects of 434 interventions designed to reduce gait asymmetries in people post-stroke, as this is a common 435 rehabilitation objective in this population [39]. Based on the current results, we would expect 436 that reducing asymmetry in people post-stroke would also affect their reactive control strategies. 437 However, further investigation is necessary to determine if reductions in asymmetry affect 438 interlimb coordination during reactions to perturbations. The data from the current study 439 illustrate how the intact neuromotor system modulates coordination between the upper and lower 440 extremities in response to changes in asymmetry, and these data could serve as useful reference 441 data to understand how sensorimotor impairments such as muscle weakness [40] and . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 442 transmission delays [41] affect the ability to restore WBAM during perturbation recovery in 443 people post-stroke. 444 5 Acknowledgments 445 We thank Natalia Sanchez, Ph.D., for her insights during the design of this experiment 446 and Aram Kim for her assistance with the statistical analysis. 368 4 Discussion 447 6 Author Contributions 448 C.L designed the experiment, collected data, analyzed data, and wrote the manuscript. 449 J.M.F conceived of the experiment, advised in data analyses, and edited the manuscript. 450 7 Conflict of Interest Statement 451 The authors declare that the research was conducted in the absence of any commercial or 452 financial relationships that could be construed as a potential conflict of interest. 448 C.L designed the experiment, collected data, analyzed data, and wrote the manuscript. 449 J.M.F conceived of the experiment, advised in data analyses, and edited the manuscript. 450 7 Conflict of Interest Statement Simoneau GC, Krebs DE. Whole-Body Momentum during Gait: A Preliminary Stud 482 Non-Fallers and Frequent Fallers. J Appl Biomech. 2000;16: 1–13. doi:10.1123/jab. 483 12. Pijnappels M, Bobbert MF, Van Dieën JH. Contribution of the support limb in contr 484 angular momentum after tripping. J Biomech. 2004;37: 1811–1818. 485 doi:10.1016/j.jbiomech.2004.02.038 486 13. Vistamehr A, Kautz SA, Bowden MG, Neptune RR. Correlations between measures 487 dynamic balance in individuals with post-stroke hemiparesis. J Biomech. 2016;49: 3 488 400. doi:10.1016/j.jbiomech.2015.12.047 489 14. Nott CR, Neptune RR, Kautz SA. Relationships between frontal-plane angular mom 490 and clinical balance measures during post-stroke hemiparetic walking. Gait Posture. 491 2014 39 129 34 d i d i 10 1016/j it t 2013 06 008 R l ti hi 453 8 References 454 1. Winter DA. Human balance and posture control during standing and walking. Gait 455 Posture. 1995;3: 193–214. doi:10.1016/0966-6362(96)82849-9 456 2. Tang PF, Woollacott MH, Chong RKY. Control of reactive balance adjustments in 457 perturbed human walking: Roles of proximal and distal postural muscle activity. Exp 458 Brain Res. 1998;119: 141–152. doi:10.1007/s002210050327 459 3. Winter DA. Biomechanics and Motor Control of Human Movement. Motor Control. 2009. 460 doi:10.1002/9780470549148 461 4. Marigold DS, Bethune AJ, Patla AE. Role of the unperturbed limb and arms in the 462 reactive recovery response to an unexpected slip during locomotion. J Neurophysiol. 463 2003;89: 1727–1737. doi:10.1152/jn.00683.2002 464 5. Wang T-Y, Bhatt T, Yang F, Pai Y-C. Adaptive control reduces trip-induced forward gait 465 instability among young adults. J Biomech. 2012/02/28. 2012;45: 1169–1175. 466 doi:10.1016/j.jbiomech.2012.02.001 464 5. Wang T-Y, Bhatt T, Yang F, Pai Y-C. Adaptive control reduces trip-induced forward gait 465 instability among young adults. J Biomech. 2012/02/28. 2012;45: 1169–1175. 466 doi:10.1016/j.jbiomech.2012.02.001 467 6. Martelli D, Luciani LB, Micera S. Angular Momentum During Unexpected 468 Multidirectional Perturbations Delivered WhileWalking. IEEE Trans Biomed Eng. 469 2013;60: 1785–1795. 470 7. Liu C, De Macedo L, Finley MJ. Conservation of Reactive Stabilization Strategies in the 471 Presence of Step Length Asymmetries during Walking. Front Hum Neurosci. 2018;12: 472 251. doi:doi: 10.3389/fnhum.2018.00251 473 8. Pijnappels M, Bobbert MF, Van Dieën JH. Push-off reactions in recovery after tripping 474 discriminate young subjects, older non-fallers and older fallers. Gait Posture. 2005;21: 475 388–394. doi:10.1016/j.gaitpost.2004.04.009 476 9. Herr HM, Popovic M. Angular momentum in human walking. J Exp Biol. 2008;211: 467– 477 81. doi:10.1242/jeb.008573 478 10. Popovic M, Hofmann A, Herr H. 450 7 Conflict of Interest Statement 451 The authors declare that the research was conducted in the absence of any commercial or 452 financial relationships that could be construed as a potential conflict of interest. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 453 8 References 454 1. Winter DA. Human balance and posture control during standing and walking. Gait 455 Posture. 1995;3: 193–214. doi:10.1016/0966-6362(96)82849-9 456 2. Tang PF, Woollacott MH, Chong RKY. Control of reactive balance adjustments in 457 perturbed human walking: Roles of proximal and distal postural muscle activity. Ex 458 Brain Res. 1998;119: 141–152. doi:10.1007/s002210050327 459 3. Winter DA. Biomechanics and Motor Control of Human Movement. Motor Control 460 doi:10.1002/9780470549148 461 4. Marigold DS, Bethune AJ, Patla AE. Role of the unperturbed limb and arms in the 462 reactive recovery response to an unexpected slip during locomotion. J Neurophysiol 463 2003;89: 1727–1737. doi:10.1152/jn.00683.2002 464 5. Wang T-Y, Bhatt T, Yang F, Pai Y-C. Adaptive control reduces trip-induced forwar 465 instability among young adults. J Biomech. 2012/02/28. 2012;45: 1169–1175. 466 doi:10.1016/j.jbiomech.2012.02.001 467 6. Martelli D, Luciani LB, Micera S. Angular Momentum During Unexpected 468 Multidirectional Perturbations Delivered WhileWalking. IEEE Trans Biomed Eng. 469 2013;60: 1785–1795. 470 7. Liu C, De Macedo L, Finley MJ. Conservation of Reactive Stabilization Strategies i 471 Presence of Step Length Asymmetries during Walking. Front Hum Neurosci. 2018; 472 251. doi:doi: 10.3389/fnhum.2018.00251 473 8. Pijnappels M, Bobbert MF, Van Dieën JH. Push-off reactions in recovery after tripp 474 discriminate young subjects, older non-fallers and older fallers. Gait Posture. 2005;2 475 388–394. doi:10.1016/j.gaitpost.2004.04.009 476 9. Herr HM, Popovic M. Angular momentum in human walking. J Exp Biol. 2008;211 477 81. doi:10.1242/jeb.008573 478 10. Popovic M, Hofmann A, Herr H. Angular momentum regulation during human walk 479 biomechanics and control. Robotics and Automation, 2004 Proceedings ICRA’04 20 480 IEEE International Conference on. IEEE; 2004. pp. 2405–2411. 481 11. 450 7 Conflict of Interest Statement Angular momentum regulation during human walking: 479 biomechanics and control. Robotics and Automation, 2004 Proceedings ICRA’04 2004 480 IEEE International Conference on. IEEE; 2004. pp. 2405–2411. 481 11. Simoneau GC, Krebs DE. Whole-Body Momentum during Gait: A Preliminary Study of 482 Non-Fallers and Frequent Fallers. J Appl Biomech. 2000;16: 1–13. doi:10.1123/jab.16.1.1 483 12. Pijnappels M, Bobbert MF, Van Dieën JH. Contribution of the support limb in control of 484 angular momentum after tripping. J Biomech. 2004;37: 1811–1818. 485 doi:10.1016/j.jbiomech.2004.02.038 486 13. Vistamehr A, Kautz SA, Bowden MG, Neptune RR. Correlations between measures of 487 dynamic balance in individuals with post-stroke hemiparesis. J Biomech. 2016;49: 396– 488 400. doi:10.1016/j.jbiomech.2015.12.047 489 14. Nott CR, Neptune RR, Kautz SA. Relationships between frontal-plane angular momentum 490 and clinical balance measures during post-stroke hemiparetic walking. Gait Posture. 491 2014;39: 129–34. doi:doi:10.1016/j.gaitpost.2013.06.008. Relationships 492 15. Silverman AK, Neptune RR. Differences in whole-body angular momentum between . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint 493 below-knee amputees and non-amputees across walking speeds. J Biomech. 2011;44: 494 379–385. doi:10.1016/j.jbiomech.2010.10.027 495 16. Honda K, Sekiguchi Y, Muraki T, Izumi SI. The differences in sagittal plane whole-body 496 angular momentum during gait between patients with hemiparesis and healthy people. J 497 Biomech. 2019;86: 204–209. doi:10.1016/j.jbiomech.2019.02.012 498 17. Lewek MD, Bradley CE, Wutzke CJ, Zinder SM. The Relationship Between 499 Spatiotemporai Gait Asymmetry and Balance in Individuals With Chronic Stroke. J Appl 500 Biomech. 2014;30: 31–36. doi:10.1123/jab.2012-0208 501 18. Latash ML, Ansen GJ, Anson JG. Synergies in Health and Disease: Relations to Adaptive 502 Changes in Motor Coordination. Phys Ther. 2006;86: 1151–1160. 503 19. Aprigliano F, Martelli D, Tropea P, Pasquini G, Micera S, Monaco V. Ageing does not 504 affect the intralimb coordination elicited by slip-like perturbation of different intensities. J 505 Neurophysiol. 2017; jn.00844.2016. doi:10.1152/jn.00844.2016 506 20. Martelli D, Vashista V, Micera S, Agrawal SK. 450 7 Conflict of Interest Statement Direction-Dependent Adaptation of 507 Dynamic Gait Stability Following Waist-Pull Perturbations. IEEE Trans Neural Syst 508 Rehabil Eng. 2016;24: 1304–1313. doi:10.1109/TNSRE.2015.2500100 509 21. Aprigliano F, Martelli D, Micera S, Monaco V. Intersegmental coordination elicited by 510 unexpected multidirectional slipping-like perturbations resembles that adopted during 511 steady locomotion. J Neurophysiol. 2016;115: 728–740. doi:10.1152/jn.00327.2015 512 22. Plotnik M, Azrad T, Bondi M, Bahat Y, Gimmon Y, Zeilig G, et al. Self-selected gait 513 speed - Over ground versus self-paced treadmill walking, a solution for a paradox. J 514 Neuroeng Rehabil. 2015;12. doi:10.1186/s12984-015-0002-z 515 23. Sánchez N, Park S, Finley JM. Evidence of Energetic Optimization during Adaptation 516 Differs for Metabolic, Mechanical, and Perceptual Estimates of Energetic Cost. Sci Rep. 517 2017;7: 7682. doi:10.1038/s41598-017-08147-y 518 24. Finley JM, Bastian AJ, Gottschall JS. Learning to be economical : the energy cost of 519 walking tracks motor adaptation. J Physiol Physiol. 2013;591: 1081–1095. 520 doi:10.1113/jphysiol.2012.245506 521 25. Reisman DS, Block HJ, Bastian AJ. Interlimb coordination during locomotion: What can 522 be adapted and stored? J Neurophysiol. 2005;94: 2403–2415. doi:10.1152/jn.00089.2005 523 26. Song J, Sigward S, Fisher B, Salem GJ. Altered dynamic postural control during step 524 turning in persons with early-stage Parkinson’s disease. Parkinsons Dis. 2012;2012: 525 386962. doi:10.1155/2012/386962 526 27. Havens KL, Mukherjee T, Finley JM. Analysis of biases in dynamic margins of stability 527 introduced by the use of simplified center of mass estimates during walking and turning. 528 Gait Posture. 2018;59: 162–167. doi:10.1016/j.gaitpost.2017.10.002 529 28. Kurz MJ, Arpin DJ, Corr B. Differences in the dynamic gait stability of children with 530 cerebral palsy and typically developing children. Gait Posture. 2012;36: 600–604. 531 doi:10.1016/j.gaitpost.2012.05.029 532 29. Reisman DS, Wityk R, Silver K, Bastian AJ. Split-Belt Treadmill Adaptation Transfers to . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: ioRxiv preprint 533 Overground Walking in Persons Poststroke. Neurorehabil Neural Repair. 2009;23: 735– 534 744. 450 7 Conflict of Interest Statement 533 Overground Walking in Persons Poststroke. Neurorehabil Neural Repair. 2009;23: 735– 534 744. 535 30. Dempster WT. Space requirements of the seated operator: geometrical, kinematic, and 536 mechanical aspects other body with special reference to the limbs. WADC Technical 537 Report. 1955. pp. 1–254. doi:AD 087892 535 30. Dempster WT. Space requirements of the seated operator: geometrical, kinematic, and 536 mechanical aspects other body with special reference to the limbs. WADC Technical 537 Report. 1955. pp. 1–254. doi:AD 087892 538 31. Hanavan EP. A Mathematical Model of the human body. Aerosp Med Rsearch Lab. 1964; 539 1–149. 540 32. Valero-Cuevas FJ, Klamroth-Marganska V, Winstein CJ, Riener R. Robot-assisted and 541 conventional therapies produce distinct rehabilitative trends in stroke survivors. J 542 Neuroeng Rehabil. 2016;13: 1–10. doi:10.1186/s12984-016-0199-5 543 33. Hothorn T, Bretz F, Westfall P. Simultaneous inference in general parametric models. 544 Biom J. 2008;50: 346–363. doi:10.1002/bimj.200810425 545 34. Kuznetsova A, Brockhoff PB, Christensen RHB. lmerTest Package: Tests in Linear 546 Mixed Effects Models. J Stat Softw. 2017;82. doi:10.18637/jss.v082.i13 547 35. Domkin D, Laczko J, Jaric S, Johansson H, Latash ML. Structure of joint variability in 548 bimanual pointing tasks. Exp Brain Res. 2002;143: 11–23. doi:10.1007/s00221-001-0944- 549 1 550 36. Papi E, Rowe PJ, Pomeroy VM. Analysis of gait within the uncontrolled manifold 551 hypothesis: Stabilisation of the centre of mass during gait. J Biomech. 2015;48: 324–331. 552 doi:10.1016/j.jbiomech.2014.11.024 553 37. Cappellini G, Ivanenko YP, Poppele R., Lacquaniti F. Motor Patterns in Human Walking 554 and Running. J Neurophysiol. 2006;95: 3426–3437. doi:10.1152/jn.00081.2006 555 38. Pijnappels M, Kingma I, Wezenberg D, Reurink G, van Dieën JH. Armed against falls : 556 the contribution of arm movements to balance recovery after tripping. Exp Brain Res. 557 2010;201: 689–699. doi:10.1007/s00221-009-2088-7 558 39. Patterson KK, Mansfield A, Biasin L, Brunton K, Inness EL, McIlroy WE. Longitudinal 559 changes in poststroke spatiotemporal gait asymmetry over inpatient rehabilitation. 560 Neurorehabil Neural Repair. 2015;29: 153–162. doi:10.1177/1545968314533614 561 40. Gray V, Rice CL, Garland SJ. Factors that influence muscle weakness following stroke 562 and their clinical implications: a critical review. Physiother Canada. 2012;64: 415–26. 563 doi:10.3138/ptc.2011-03 564 41. Sharafi B, Hoffmann G, Tan AQ, Y. Dhaher Y. Evidence of impaired neuromuscular 565 responses in the support leg to a destabilizing swing phase perturbation in hemiparetic 566 gait. Exp Brain Res. 2016;234: 3497–3508. doi:10.1007/s00221-016-4743-0 . 450 7 Conflict of Interest Statement CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint the preprin The copyrig . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 9, 2019. ; https://doi.org/10.1101/799213 doi: bioRxiv preprint
https://openalex.org/W4322747266	https://www.epj-conferences.org/10.1051/epjconf/202327601009/pdf	English	null	Particle production as a function of underlying-event activity and very forward energy with ALICE	EPJ web of conferences	2,023	cc-by	2,331	∗e-mail: feng.fan@cern.ch © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Particle production as a function of underlying-event activ- ity and very forward energy with ALICE Feng Fan ∗ (for the ALICE Collaboration) Central China Normal University, Wuhan, Hubei, 430079, China Abstract. Measurements of charged-particle production in pp and p–Pb col- lisions at √sNN = 5.02 TeV in the toward, away and transverse regions are discussed. These three regions are defined event by event relative to the track with the largest transverse momentum (ptrig T ). The transverse region is sensi- tive to the underlying event (UE), but it also includes contributions from initial- and final-state radiation (ISR-FSR). Therefore, it is further subdivided in two regions, defined according to their relative multiplicities: trans-max (the sub- transverse region with the larger multiplicity) and trans-min (the sub-transverse region with the smaller multiplicity) regions which have an enhanced sensitivity to ISR-FSR and UE, respectively. KNO-like scaling properties are explored in the three defined regions. Finally, the relationship between ptrig T and the energy detected in a region close to beam rapidity (very forward energy) is reported. EPJ Web of Conferences 276, 01009 (2023) SQM 2022 EPJ Web of Conferences 276, 01009 (2023) SQM 2022 https://doi.org/10.1051/epjconf/202327601009 1 Introduction In models incorporating multi-parton interactions (MPI), particles produced in the hard scat- tering (jet) are accompanied by particles from additional parton-parton interactions, as well as from the proton break-up [1]. This component of the collision makes up the underlying event (UE). The traditional UE analysis focuses on the study of particles in three topological regions depending on their azimuthal angle relative to the leading particle (\|∆φ\| = \|φ −φtrig\|), which is the one with the highest transverse momentum in the event (ptrig T or pleading T ) at mid- pseudorapidity (\|η\| < 0.8). The toward region (\|∆φ\| < π/3 rad) contains the primary jet and UE, while the away region (\|∆φ\| > 2π/3 rad) contains the fragments of the recoil jet and UE. In contrast, the transverse region (π/3 rad < \|∆φ\| < 2π/3 rad) is dominated by the UE dynam- ics, but it also includes contributions from initial- and final-state radiation (ISR-FSR) [2, 3]. A study based on Monte Carlo (MC) simulations shows that at the LHC energies the multi- plicity distributions in the transverse region obey a Koba-Nielsen-Olesen (KNO) scaling [4]. In this contribution, the transverse region is further subdivided into trans-max and trans-min, defined according to their relative multiplicities, in order to increase the sensitivity to ISR- FSR and UE effects, respectively. Measurements of multiplicity distributions in the trans-min region at different centre-of-mass energy allows for the study of the role of MPI to produce such scaling properties. Another way to characterise the event is to look into a region in the very forward pseu- dorapidity (\|η\| > 8.8) using the ALICE zero degree calorimeters (ZDC), which measure the beam remnants. Moreover, the relationship between the very forward energy and ptrig T is EPJ Web of Conferences 276, 01009 (2023) SQM 2022 https://doi.org/10.1051/epjconf/202327601009 complementary to the UE measurements, and provides direct insights into the initial stages and the subsequent evolution of the collision. In this contribution, a set of new measurements aimed at understanding the role of MPI in hadronic interactions at the LHC is presented [5, 6]. ALI-PUB-522465 ALI-PUB-522465 ALI-PUB-522470 Figure 1: Multiplicity density as a function of ptrig T measured in pp (left) and p–Pb (right) collisions at √sNN = 5.02 TeV. Results for the toward, transverse, and away regions are displayed. The shaded boxes and the error bars represent the systematic and statistical uncer- tainties, respectively. 1 Introduction ALI-PUB-522470 ALI-PUB-522465 ALI-PUB-522470 Figure 1: Multiplicity density as a function of ptrig T measured in pp (left) and p–Pb (right) collisions at √sNN = 5.02 TeV. Results for the toward, transverse, and away regions are displayed. The shaded boxes and the error bars represent the systematic and statistical uncer- tainties, respectively. 2 Results and discussion Figure 1 shows the charged-hadron multiplicity density as a function of ptrig T measured in pp and p–Pb collisions at √sNN = 5.02 TeV. Results are presented for the toward, away and transverse regions. For both systems, the distributions behave in the same way. At low ptrig T (< 5 GeV/c), the multiplicity densities rapidly increase with ptrig T for those three topological regions, while at higher ptrig T (> 5 GeV/c), they still increase but less steeply in the toward and away regions, whereas in the transverse region they tend to saturate. This saturation is expected in models including an impact parameter dependence such that the requirement of the presence of a high-pT particle in a pp collision biases the selection of collisions towards those with a smaller impact parameter [7]. Moreover, the increase observed in the toward and away regions at high pT is due to the contributions from jets as well as ISR and FSR. The UE contributions to the toward and away regions are larger in p–Pb than in pp collisions. Figure 2 shows the comparison between the multiplicity density in the transverse re- gion [8] and the self-normalised neutral ZDC energy (ZN) as a function of pleading T (at mid- pseudorapidity) in pp collisions at √s = 13 TeV. The very forward energy reaches its min- imum value at pleading T ≈5 GeV/c and then remains constant for increasing pleading T . The sat- uration occurs at the same pT value observed for UE quantities. The result corroborates the interpretation in terms of a bias towards collisions with small impact parameter, because the correlation between central and forward pseudorapidity can only be attributed to the initial stage of the collisions. In a MC study of UE [4], it was shown that the multiplicity distributions in the transverse region (\|η\| < 2.5, pT > 0 GeV/c) at the plateau obey a KNO scaling at the LHC energies. The KNO scaling was expected in a model which assumes that a single pp collision results from the superposition of a given number of elementary partonic collisions emitting particles independently [9]. MPI could produce such an effect. 2 Results and discussion However, the transverse region not 2 EPJ Web of Conferences 276, 01009 (2023) SQM 2022 https://doi.org/10.1051/epjconf/202327601009 ALI-PUB-495251 Figure 2: Multiplicity density (azure squares) in the transverse region [8] and self-normalised ZN signal (red circles) as a function of pleading T (at mid-pseudorapidity) in pp collisions at √s = 13 TeV. ALI-PUB-495251 ALI-PUB-495251 Figure 2: Multiplicity density (azure squares) in the transverse region [8] and self-normalised ZN signal (red circles) as a function of pleading T (at mid-pseudorapidity) in pp collisions at √s = 13 TeV. 0 1 2 3 4 5 6 7 4 − 10 3 − 10 2 − 10 1 − 10 1 Trans-max region c 40 GeV/ ≤ trig T p ≤ 5 \|<0.8 η , \| c 0.5 GeV/ ≥ T p ALICE Preliminary = 2.76 TeV s pp = 5.02 TeV s pp = 7 TeV s pp = 13 TeV s pp 0 1 2 3 4 5 6 7 1 2 〉 t-max ch N 〈/ t-max ch N ) t-max ch N ( P 〉 t-max ch N 〈 Ratio to 7 TeV ALI-PREL-510847 0 2 4 6 8 10 12 4 − 10 3 − 10 2 − 10 1 − 10 1 Trans-min region c 40 GeV/ ≤ trig T p ≤ 5 \|<0.8 η , \| c 0.5 GeV/ ≥ T p ALICE Preliminary = 2.76 TeV s pp = 5.02 TeV s pp = 7 TeV s pp = 13 TeV s pp 0 2 4 6 8 10 12 1 2 〉 t-min ch N 〈/ t-min ch N ) t-min ch N ( P 〉 t-min ch N 〈 Ratio to 7 TeV ALI-PREL-510857 Figure 3: Top: multiplicity distributions in KNO variables in the trans-max (left) and trans- min (right) regions for pp collisions at √s = 2.76, 5.02, 7 and 13 TeV. Bottom: the KNO multiplicity distributions are normalized to that for pp collisions at √s = 7 TeV. The boxes and the error bars represent the systematic and statistical uncertainties, respectively. 3 Conclusions The multiplicity density as a function of ptrig T for both pp and p–Pb collisions at √sNN = 5.02 TeV shows a remarkably similar behavior, such as in the transverse region the distri- butions increase with ptrig T until about 5 GeV/c where it saturates. On the other hand, the very forward energy as a function of pleading T in pp collisions at √s = 13 TeV decreases with increasing pleading T and then saturates at the same pleading T (≈5 GeV/c). These two saturation effects are commonly interpreted as a bias towards collisions with small impact parameter. In addition, in the trans-max and trans-min regions the charged-particle multiplicity distribu- tions exhibit a KNO-like scaling for 0 < z < 4, which is expected for a single pp collision involving a large number of MPI emitting independently. And for z > 4 the scaling is bro- ken. For the MPI-sensitive region, a higher z reach is achieved, especially for z > 6, a larger violation is observed, which may be attributed to high-multiplicity mini jets. 2 Results and discussion 0 1 2 3 4 5 6 7 4 − 10 3 − 10 2 − 10 1 − 10 1 Trans-max region c 40 GeV/ ≤ trig T p ≤ 5 \|<0.8 η , \| c 0.5 GeV/ ≥ T p ALICE Preliminary = 2.76 TeV s pp = 5.02 TeV s pp = 7 TeV s pp = 13 TeV s pp 0 1 2 3 4 5 6 7 1 2 〉 t-max ch N 〈/ t-max ch N ) t-max ch N ( P 〉 t-max ch N 〈 Ratio to 7 TeV ALI-PREL-510847 0 2 4 6 8 10 12 4 − 10 3 − 10 2 − 10 1 − 10 1 Trans-min region c 40 GeV/ ≤ trig T p ≤ 5 \|<0.8 η , \| c 0.5 GeV/ ≥ T p ALICE Preliminary = 2.76 TeV s pp = 5.02 TeV s pp = 7 TeV s pp = 13 TeV s pp 0 2 4 6 8 10 12 1 2 〉 t-min ch N 〈/ t-min ch N ) t-min ch N ( P 〉 t-min ch N 〈 Ratio to 7 TeV ALI-PREL-510857 Ratio to 7 TeV ALI-PREL-510857 Figure 3: Top: multiplicity distributions in KNO variables in the trans-max (left) and trans- min (right) regions for pp collisions at √s = 2.76, 5.02, 7 and 13 TeV. Bottom: the KNO multiplicity distributions are normalized to that for pp collisions at √s = 7 TeV. The boxes and the error bars represent the systematic and statistical uncertainties, respectively. only includes contributions from UE but also from ISR-FSR. In order to investigate the KNO- like scaling properties, a further treatment of the transverse side is implemented, where the transverse region is further divided in two regions: • transverse-I: π/3 < ∆φ < 2π/3 • transverse-II: −π/2 < ∆φ < −π/3, 4π/3 < ∆φ < 3π/2 These two distinct regions are characterized in terms of their relative charged-particle multi- plicities. Trans-max (trans-min) region refers to the sub-transverse region (I or II) with the larger (smaller) multiplicity which have an enhanced sensitivity to ISR-FSR (UE) [2, 10]. These two distinct regions are characterized in terms of their relative charged-particle multi- plicities. Trans-max (trans-min) region refers to the sub-transverse region (I or II) with the larger (smaller) multiplicity which have an enhanced sensitivity to ISR-FSR (UE) [2, 10]. 2 Results and discussion 3 https://doi.org/10.1051/epjconf/202327601009 EPJ Web of Conferences 276, 01009 (2023) SQM 2022 Figure 3 shows the charged-particle multiplicity distributions in KNO variables for pp collisions at √s = 2.76, 5.02, 7 and 13 TeV. Results are presented for the trans-max and trans-min regions. In the trans-max region, within 20%, the KNO-like scaling is observed in a wider range of multiplicity (0 < z =< 4, where z = NX ch/⟨NX ch⟩, X =‘t-max’ or ‘t-min’ which corresponds to the ‘trans-max’ or ‘trans-min’ region ) relative to the results reported in Ref. [3], while for higher z values (z > 4) the scaling is broken. It is worth noticing that for trans-max both contributions are considered: UE and ISR-FSR. If the effect of ISR-FSR is suppressed, i.e., exploiting the features of trans-min region, the KNO-like scaling also holds for 0 < z < 4, and then for z > 4 the KNO-like scaling is still broken but a higher z reach is achieved, especially for z > 6, a lager violation is observed. Events with high-multiplicity jets can contribute to the large violation of the scaling properties. It was observed that for z > 3, the number of uncorrelated seeds (or MPI) deviate from the linear trend suggesting the presence of high-multiplicity jets [11, 12]. 4 Acknowledgement This work has been supported by CONACyT under the Grants CB No. A1-S-22917 and CF No. 2042. References [1] T. Sjöstrand, M. van Zijl, Phys. Rev. D 36, 2019 (1987) [2] G. Bencedi, A. Ortiz, A. Paz, Phys. Rev. D 104, 016017 (2021), [arXiv]2105.04838 [3] A. Ortiz, Phys. Rev. D 104, 076019 (2021), [arXiv]2108.08360 [3] A. Ortiz, Phys. Rev. D 104, 076019 (2021), [arXiv]2108.08360 [4] A. Ortiz, L. Valencia Palomo, Phys. Rev. D 96, 114019 (2017), [arXiv]1710.04741 [5] S. Acharya et al. (ALICE) (2022), [arXiv]2204.10389 [6] S. Acharya et al. (ALICE) (2022), [arXiv]2204.10157 [7] M. Strikman, Acta Phys. Polon. B 42, 2607 (2011), [arXiv]1112.3834 [8] S. Acharya et al. (ALICE), JHEP 04, 192 (2020), [arXiv]1910.14400 [9] J. Dias de Deus, C. Pajares, C.A. Salgado, Phys. Lett. B 408, 417 (1997), hep-ph/9705425 [10] T.A. Aaltonen et al. (CDF), Phys. Rev. D 92, 092009 (2015), [arXiv]1508.05340 [11] B. Abelev et al. (ALICE), JHEP 09, 049 (2013), [arXiv]1307.1249 [12] A. Ortiz, E.A. Zepeda, J. Phys. G 48, 085014 (2021), [arXiv]2101.10274 4
https://openalex.org/W2291744958	https://digital.csic.es/bitstream/10261/248322/1/compleuy031.pdf	English	null	Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031	Standards in genomic sciences	2,016	cc-by	5,847	© 2016 Guarischi-Sousa et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain’s virulence. genome report, Bacterial wilt, Ralstonia solanacearum, Bacterial plant pathogen, Methylome, Uruguay Keywords: Short genome report, Bacterial wilt, Ralstonia solanacearum, Bacterial plant pathogen, M NCPPB909 [8], and CFIA906 [8]. This is the first gen- ome of this group to be completely sequenced, and the fourth within the R. solanacearum species complex (the other three are strains GMI1000 [9], Po82 [10] , and PSI07 [11]). * Correspondence: setubal@iq.usp.br 1Instituto de Química, Universidade de São Paulo, São Paulo, Brazil 4Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA Full list of author information is available at the end of the article Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031 Rodrigo Guarischi-Sousa1, Marina Puigvert2, Núria S. Coll2, María Inés Siri3, María Julia Pianzzola3, Marc Valls2 and João C. Setubal1,4* Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 DOI 10.1186/s40793-016-0131-4 Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 DOI 10.1186/s40793-016-0131-4 Open Access Introduction Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most devastating plant diseases worldwide [1]. It is a highly diversified bacterial plant pathogen in terms of host range, geographical distribution, pathogenicity, epi- demiological relationships, and physiological properties [2]. Strains are divided in four phylotypes, corresponding roughly to their geographic origin: Asia (phylotype I), the Americas (II), Africa (III), and Indonesia (IV) [3]. Strain UY031 belongs to phylotype IIB, sequevar 1 (IIB1), the group considered mainly responsible for bacterial wilt of potato in cold and temperate regions [4]. Phylotype IIB, sequevar 1 is also traditionally classified as race 3 biovar 2. Classification and features Ralstonia solanacearum UY031 strain is classified within the order Burkholderiales of the class Betaproteobacteria. It is an aerobic, non-sporulating, Gram-negative bacter- ium with rod-shaped cells ranging from 0.5 to 1.5 μm in length (Fig. 1, (a) and (b)). The strain is moderately fast- growing, forming 3–4 mm colonies within 2–3 days at 28 °C. On a general nutrient medium containing tetrazolium chloride and high glucose content, strain UY031 usually produces a diffusible brown pigment and develops pearly cream-white, flat, irregular, and fluidal colonies with char- acteristic pink whorls in the centre (Fig. 1, (c)). Strain UY031 was isolated from a naturally infected potato tuber showing typical brown rot symptoms (creamy exudates from the vascular rings and eyes of the tuber). This strain is highly pathogenic in different solanaceous hosts includ- ing important crops like tomato and potato [5]. Pathogen- icity of this strain was also confirmed in several accessions Strain UY031 was isolated in Uruguay from infected potato tubers in 2003 and displays high aggressiveness both on potato and tomato hosts [5]. This strain is being used as a model in plant-pathogen gene expression stud- ies carried out by our group; having its genome available greatly facilitates the identification of pathogenicity- related genes. Four other IIB1 R. solanacearum strains have been partially sequenced: UW551 [6], IPO1609 [7], * Correspondence: setubal@iq.usp.br 1Instituto de Química, Universidade de São Paulo, São Paulo, Brazil 4Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA Full list of author information is available at the end of the article Page 2 of 8 Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Fig. 1 Images of Ralstonia solanacearum strain UY031 using transmission (a) and scanning (b) electron microscopy, as well as light microscopy to visualize colony morphology on solid media (c) Fig. Classification and features 1 Images of Ralstonia solanacearum strain UY031 using transmission (a) and scanning (b) electron microscopy, as well as light microscopy to visualize colony morphology on solid media (c) Table 1 Classification and general features of Ralstonia solanacearum strain UY031according to the MIGS recommendations [27] MIGS ID Property Term Evidence codea Classification Domain Bacteria TAS [28] Phylum Proteobacteria TAS [29] Class Betaproteobacteria TAS [30, 31] Order Burkholderiales TAS [31, 32] Family Burkholderiaceae TAS [31, 33] Genus Ralstonia TAS [34, 35] Species Ralstonia solanacearum TAS [34, 35] Strain UY031 Gram stain Negative IDA Cell shape Rod IDA Motility Motile IDA Sporulation Non sporulating NAS Temperature range Mesophile IDA Optimum temperature 27 °C IDA pH range; Optimum 5.5 – 8.0; 6.5 NAS Carbon source Dextrose, lactose, maltose, cellobiose IDA MIGS-6 Habitat potato plants, soil TAS [5] MIGS-6.3 Salinity <2.0 % TAS [36] MIGS-22 Oxygen requirement Aerobic IDA MIGS-15 Biotic relationship free-living IDA MIGS-14 Pathogenicity Pathogenic TAS [5] MIGS-4 Geographic location Uruguay, San José TAS [5] MIGS-5 Sample collection 2003 TAS [5] MIGS-4.1 Latitude 34°43′58.17”S NAS MIGS-4.2 Longitude 56°32′2.87”W NAS MIGS-4.4 Altitude 116.7 m NAS aEvidence codes - IDA Inferred from direct assay, TAS Traceable author statement (i.e., a direct report exists in the literature), NAS Non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [37] and general features of Ralstonia solanacearum strain UY031according to the MIGS recommendations [27] aEvidence codes - IDA Inferred from direct assay, TAS Traceable author statement (i.e., a direct report exists in the literature), NAS Non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [37] Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Page 3 of 8 Fig. 2 Phylogenetic tree highlighting the position of the Ralstonia solanacearum UY031 (shown in bold) relative to other strains from the same species. The phylogenetic tree was constructed using four conserved prokaryotic marker genes, namely: recA, rpoA, rpoB and rpoC. Each gene was aligned individually with MUSCLE [25]; the resulting multiple alignments were concatenated. PhyML [26] was used to perform tree reconstruction using the GTR model and 1,000 bootstrap replicas. Strain names are colour-coded according to the correspondent phylotype. Classification and features GenBank accession numbers are displayed within brackets. Strains whose genome was completely sequenced are marked with an asterisk. Ralstonia pickettii 12 J (NCBI accession NC_010682) was used as an outgroup Fig. 2 Phylogenetic tree highlighting the position of the Ralstonia solanacearum UY031 (shown in bold) relative to other strains from the same species. The phylogenetic tree was constructed using four conserved prokaryotic marker genes, namely: recA, rpoA, rpoB and rpoC. Each gene was aligned individually with MUSCLE [25]; the resulting multiple alignments were concatenated. PhyML [26] was used to perform tree reconstruction using the GTR model and 1,000 bootstrap replicas. Strain names are colour-coded according to the correspondent phylotype. GenBank accession numbers are displayed within brackets. Strains whose genome was completely sequenced are marked with an asterisk. Ralstonia pickettii 12 J (NCBI accession NC_010682) was used as an outgroup solanacearum strain UY031 is summarized in Table 1, and a phylogenetic tree is shown in Fig. 2. solanacearum strain UY031 is summarized in Table 1, and a phylogenetic tree is shown in Fig. 2. of Solanum commersonii Dunal, a wild species considered as a valuable source of resistance for potato breeding. Due to its great aggressiveness, strain UY031 is being used for selection of resistant germplasm as part of the potato breeding program developed in Uruguay. This strain has been deposited in the CFBP collection of plant-associated bacteria, and has received code CFBP 8401. Minimum In- formation about the Genome Sequence of R. Growth conditions and genomic DNA preparation R. solanacearum strain UY031 was routinely grown in rich B medium (10 g/l bactopeptone, 1 g/l yeast extract and 1 g/ l casaminoacids). Genomic DNA was extracted from a bac- terial culture grown to stationary phase to avoid over- representation of genomic sequences close to the origin of replication. Twelve ml of a culture grown for 16 h at 30 °C and shaking at 200 rpm (OD600 = 0.87) were used to extract DNA with Blood & Cell Culture DNA Midi kit (Qiagen), following manufacturer’s instructions for gram-negative bacteria. DNA concentration and quality were measured in a Nanodrop (ND-8000 8-sample spectrophotometer). The sequencing plataform used to assemble the genome (PacBio RS II) also gives kinectics information about the se- quenced genome. The presence of a methylated base in the DNA template delays the incorporation of the complemen- tary nucleotide; such modifications in the kinectics may be used to characterize modified bases by methylation includ- ing: 6-mA, 5-mC and 4-mC [18]. The analysis of these modifications in a genome-wide and single-base-resolution scale allowed us to characterize the ‘methylome’ of this strain. These epigenetic marks are commonly used by bac- teria, and its implications vary from a defense mechanism, protecting the cell from invading bacteriophages or other foreign DNA, to the bacterial virulence itself [19–21]. We performed methylome analysis and motif detection using RS_Modification_and_Motif_analysis.1 protocol from SMRT Analysis 2.3. Such epigenetic marks arise from DNA methyl-transferases, sometimes coupled with a restriction endonuclease (a Restriction-Modification System). We Genome sequencing and assembly Whole-genome sequencing was performed on the PacBio RS II platform at the Duke Center for Genomic and Com- putational Biology (USA). P5-C3 chemistry and a single SMRTcell were used, and quality control was performed with DUGSIM. The number of Pre-Filter Polymerase Read Bases was greater than 749 million (>130x genome cover- age). Reads were assembled using RS_HGAP_Assembly.2 protocol from SMRT Analysis 2.3 [12]. This resulted in one circular chromosome (3,412,138 bp) and one circular megaplasmid (1,999,545 bp). These lengths are very simi- lar to those of the corresponding replicons in R. solana- cearum Po82, a IIB sequevar 4 strain, also a potato pathogen and which has also been completely sequenced [10]. The origin of replication was defined for both repli- cons based on the putative origin for reference strain GMI1000 [9]. Table 4 Genome statistics Attribute Value % of total Genome size (bp) 5,411,683 100.00 DNA coding (bp) 4,737,274 87.5 DNA G + C (bp) 3,604,487 66.6 DNA scaffolds 2 100.00 Total genes 4,778 100.00 Protein coding genes 4,683 98.0 RNA genes 95 1.9 Pseudo genes NA NA Genes in internal clusters NA NA Genes with function prediction 3,566 74.6 Genes assigned to COGs 3,586 76.6 Genes with Pfam domains 3,892 83.1 Genes with signal peptides 501 10.6 Genes with transmembrane helices 1132 24.1 CRISPR repeats 0 - An assembly quality assessment was performed before all downstream analyses. All reads were mapped back to the assembled sequences using RS_Resequencing.1 proto- col from SMRT Analysis 2.3. This analysis revealed that chromosome and megaplasmid sequences had 100 % of bases called (percentage of assembled sequence with coverage > = 1) and 99.9999 % and 99.9992 %, respectively, of consensus concordance. Genome sequencing information Genome project history This sequencing project was carried out in 2015; the result is a complete and finished genome. Project data is available from GenBank (Table 2). Accession codes for reads in the Table 2 Project information MIGS ID Property Term MIGS 31 Finishing quality Finished MIGS-28 Libraries used SMRT library (P5-C3 large insert library) MIGS 29 Sequencing platforms PacBio RS II MIGS 31.2 Fold coverage 138× MIGS 30 Assemblers HGAP.2 workflow MIGS 32 Gene calling method Prokka v1.10 (ncRNAs search enabled) Locus tag RSUY Genbank ID CP012687 (chr), CP012688 (pl) GenBank date of release September 28, 2015 GOLD ID NA BIOPROJECT PRJNA278086 MIGS 13 Source material identifier SAMN03402637 Project relevance Plant pathogen Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Page 4 of 8 Page 4 of 8 Sequence Read Archive are SRP064191, SRR2518086, and SRZ132405. Table 3 Summary of genome: one chromosome and one plasmid Label Size (Mb) Topology INSDC identifier RefSeq ID Chromosome 3.41 circular NA NA Megaplasmid 1.99 circular NA NA Table 3 Summary of genome: one chromosome and one plasmid Label Size (Mb) Topology INSDC identifier RefSeq ID Chromosome 3.41 circular NA NA Second, the 15 T3Es annotated as “Type III Effector Pro- tein”, “Probable Type III Effector Protein” or “Putative Type III Effector Protein” by Prokka were manually anno- tated using the first BLAST [15] hits (usually 100 % iden- tity) of their DNA sequences against genome sequences of phylotype IIB strains MOLK2 and Po82. Third, the UY031 genome was uploaded to the “Ralstonia T3E” web interface tool [14] to search for additional T3Es not annotated as such with Prokka. The additional 28 T3E genes identified were manually annotated as above. Homologous Gene Group clustering was performed with get_homologues [16] using the orthoMCL program [17] and requiring a mini- mum sequence identity in BLAST query/subject pairs of 30 %. Second, the 15 T3Es annotated as “Type III Effector Pro- tein”, “Probable Type III Effector Protein” or “Putative Type III Effector Protein” by Prokka were manually anno- tated using the first BLAST [15] hits (usually 100 % iden- tity) of their DNA sequences against genome sequences of phylotype IIB strains MOLK2 and Po82. Third, the UY031 genome was uploaded to the “Ralstonia T3E” web interface tool [14] to search for additional T3Es not annotated as such with Prokka. The additional 28 T3E genes identified were manually annotated as above. Genome sequencing information Genome project history Homologous Gene Group clustering was performed with get_homologues [16] using the orthoMCL program [17] and requiring a mini- mum sequence identity in BLAST query/subject pairs of 30 %. Genome annotation Genome annotation was done using Prokka [13] with the option for ncRNA search. Type III effectors of strain UY031 were identified and annotated in three steps: First, 17 of the T3Es from the R. solanacearum species complex [14] were identified based on the Prokka annotations. Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Page 5 of 8 Table 5 Number of genes associated with general COG functional categories Code Value % Description J 160 3.4 Translation, ribosomal structure and biogenesis A 2 <0.1 RNA processing and modification K 273 5.8 Transcription L 240 5.1 Replication, recombination and repair B 3 <0.1 Chromatin structure and dynamics D 28 0.6 Cell cycle control, Cell division, chromosome partitioning V 45 1.0 Defense mechanisms T 162 3.5 Signal transduction mechanisms M 237 5.1 Cell wall/membrane biogenesis N 119 2.5 Cell motility U 61 1.3 Intracellular trafficking and secretion O 154 3.3 Posttranslational modification, protein turnover, chaperones C 226 4.8 Energy production and conversion G 165 3.5 Carbohydrate transport and metabolism E 342 7.3 Amino acid transport and metabolism F 75 1.6 Nucleotide transport and metabolism H 154 3.3 Coenzyme transport and metabolism I 177 3.8 Lipid transport and metabolism P 176 3.8 Inorganic ion transport and metabolism Q 73 1.6 Secondary metabolites biosynthesis, transport and catabolism R 352 7.5 General function prediction only S 362 7.7 Function unknown - 1097 23.4 Not in COGs The total is based on the total number of protein coding genes in the genome further characterized which genes give rise to the modified motifs using tools available at REBASE [22]. Genome properties The genome of R. solanacearum strain UY031 has one chromosome (3,412,138 bp) and one circular megaplasmid (1,999,545 bp) (Table 3). The average GC content of the chromosome is 66.5 % while that of the megaplasmid is 66.7 %. A total of 4,778 genes (4,683 CDSs and 95 RNAs) were predicted. Of the protein-coding genes, 3,566 (76.1 %) had functions assigned while 1,212 were considered hypo- thetical (Table 4). Of all CDSs, 76.6 % could be assigned to one COG functional category and for 83.1 % one or more conserved PFAM-A domains were identified (Table 5). Insights from the genome sequence We performed a pan-genome analysis of the R. solana- cearum UY031 genome, comparing it to four other ge- nomes: two closely-related R. solanacearum strains (UW551 and IPO1609) and two others with complete genome sequences available (GMI1000 and Po82). The pan-genome consists of 7,594 HGGs while the core gen- ome consists of 2,958 HGGs; the variable genome con- sists of 2,643 HGGs, and the number of strain-specific HGGs ranges from 193 to 774 (Fig. 3). We identified 193 HGGs that are UY031-specific; 75.1 % of them were annotated as hypothetical proteins. Type III effector genes are among the most important for virulence determinants in bacterial plant pathogens such as R. solanacearum [14]. Based on comparisons with effector gene sequences in public databases (see above) we have identified 60 T3Es (Table 6), of which 11 appear to be nonfunctional due to frameshifts or other Fig. 3 Venn diagram of the Ralstonia solanacearum homologous gene groups. The R. solanacearum genomes compared were as follows: strains Po82, GMI1000, IPO1609, UW551, and UY031 Fig. 3 Venn diagram of the Ralstonia solanacearum homologous gene groups. The R. solanacearum genomes compared were as follows: strains Po82, GMI1000, IPO1609, UW551, and UY031 Fig. 3 Venn diagram of the Ralstonia solanacearum homologous gene groups. The R. solanacearum genomes compared were as follows: strains Po82, GMI1000, IPO1609, UW551, and UY031 Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Page 6 of 8 Table 6 List of T3E genes identified in R. Insights from the genome sequence solanacearum UY031 genome and their orthologs Former effector name New effector namea UY031(RSUY_) GM1000(RS) Po82(RSPO_) IPO1609(RSIPO_) UW551(RRSL_) AWR2 RipA2 32720 p0099 m00080 03169 03418 AWR3 RipA3 40320 p0846 m01165 03901 + 05027b - AWR4 RipA4 40330/40b p0847 m01166b 03902/3b - AWR5 RipA5_1 41860 p1024 m01289/90b 04049 01071 AWR5 RipA5_2 19780 - c01821 01281 00546 Rip2 RipB 30390 c0245 c03161 00263 02573 Rip62 RipC1 42590 p1239 m01371 04123 03371 Rip34 RipD 33840 p0304 m01520 04484 00947 Rip26 RipE1 01190 c3369 c00070 03083 00852 - RipE2 35100 - c02513 04353 03923 PopF1 RipF1_1 45370 p1555 m01541 03403 04777 PopF2 RipF2 45510 - m01557 05028/9b 04764 Gala2 RipG2 38790 p0672 m01007 04892 02264 Gala3 RipG3 32420 p0028 m00035 03202 00752 Gala4 RipG4 19910 c1800 c01835 01266/68b 00532 Gala5 RipG5 19920 c1801 c01836 01264 00531 Gala6 RipG6 17940 c1356 c01999 01463 01561 Gala7 RipG7 17950 c1357 c01998 01462 01562 HLK1 RipH1 19380 c1386 c01846 01319 00426 HLK2 RipH2 35470 p0215 m00201/2c 04317 03559 HLK3 RipH3 33320 p0160 m00157 03105 00041b Rip1 RipI 00490 + 32050b c0041 c03319 00098b 02976 + 02040b Rip22 RipJ 24610b c2132 c02749 - - Rip16 RipM 19180 c1475 c01871/2/3 01339 + 05024b 00705 Rip58 RipN 43290 p1130 m00869 04184 04736 Rip35 RipO1 34050 p0323 m01496 04463 00926 Rip63 RipQ 44390b p1277 m00717 04287b 02855b PopS RipR 42640 p1281 m01376 04127 03375 SKWP1 RipS1 00860 c3401 c00036 00017 04182 SKWP2 RipS2 44630 p1374 m00690 04310 - SKWP3 RipS3 41210 p0930 m01229 03993/4b 00237b SKWP5 RipS5 10370 + 10840b p0296 c02546b - - SKWP7 RipS7 35110b - m00383 04352b 03921 Rip59 RipU 43920 p1212 m00805 04243 04660 Rip12 RipV1 17880 c1349 c02006 01470 01554 - RipV2 19160b - c01875/76b 01341 00703 PopW RipW 07010 c2775 c00735 02524 02682 PopA RipX 40640 p0877 m01196 03933 02443 Rip3 RipY 30260 c0257 c03153 00276 01439 Rip57 RipZ 42040 p1031 m01312 04067 00271b AvrA RipAA 26380b c0608 c02748 00659 01581 PopB RipAB 40630 p0876 m01195 03932 02442 PopC RipAC 40620 p0875 m01194 03931 02441 Rip72 ripAD 45790 p1601 m01585 03364 02518 Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Page 7 of 8 Page 7 of 8 Table 6 List of T3E genes identified in R. Insights from the genome sequence We have found sev- eral such cases (Table 6), but in all cases there is at least one other strain that also has the same gene in what ap- pears to be a functional state. annotation is an enterotoxin gene found in Vibrio chol- era [23]; in R. solanacearum the role of this toxin gene is still unclear [24]. Motif GTWWAC is probably associ- ated with the product of gene RSUY_22890 (R. Roberts, personal communication), which is hypothesized to be a solitary DNA methyltransferase (no restriction endonucle- ase linked). This gene does have homologs in other R. solanacearum strains (GMI1000, IPO1609, Po82 and PSI07). To our knowledge this is the first R. solanacearum genome with a methylome profile available. mutations that disrupt the coding sequence. For ex- ample, the effector RipS5 is encoded by a gene that has been clearly interrupted by a 34 kbp prophage. Table 6 also shows the orthologs of these genes in the related strains GMI1000, Po82, IPO1609, and UW551. In the table it can be seen that the genes that code for RipAA and RipAR have frameshifts or truncations in strain UY031 only. The absence of a particular effector may be enough for a pathogen to avoid host defenses, and there- fore cause disease. These two genes are therefore a good starting point for additional investigations of phenotypic differences between these strains. Other effector genes of interest are those that are present and do not have disrupting mutations in UY031 but are absent or appear to be nonfunctional in other strains. We have found sev- eral such cases (Table 6), but in all cases there is at least one other strain that also has the same gene in what ap- pears to be a functional state. Insights from the genome sequence solanacearum UY031 genome and their orthologs (Continued) Rip4 RipAE 29570 c0321 c03085 00343 01625 Rip41 RipAI 40230 p0838 m01156 03894 01021 Rip21 RipAJ 13300 c2101 c01332 04893 01260 Rip38 RipAL 39210b - m01053 - 02221 Brg40 RipAM 02270 c3272 c00191 02968 02810 Rip43 RipAN 40310 p0845 m01164 03900 01013 Rip50 RipAO 40750 p0879 m01206 03944 03105 Rip60 RipAP 43960 p1215b m00800 04247 04655 Rip51 RipAQ 40810 p0885 - 03951 03113 Rip61 RipAR 44220b p1236 m00770 04270 01136 Rip39 RipAV 39280 p0732 m01061 - 02213 Brg13 RipAX1 02040 c3290 m01221 02991 - Rip55 RipAY 41810 p1022 m01283 04046 01066 - RipBH 45880 - m01600 03355 00782 - RipBI 45200b - m00718 03419 00326 - RipTPS 39290 p0731 m01062b - 02212 aAccording to Peeters et al. [14]; b: these genes appear to be nonfunctional due to various reasons (frameshift, truncation, etc.); genes in other columns that appear in the form locus tag x + locus tag y are genes which also appear to be nonfunctional due to frameshifts. c:this gene is duplicated Table 6 List of T3E genes identified in R. solanacearum UY031 genome and their orthologs (Continued) aAccording to Peeters et al. [14]; b: these genes appear to be nonfunctional due to various reasons (frameshift, truncation, etc.); genes in other columns that appear in the form locus tag x + locus tag y are genes which also appear to be nonfunctional due to frameshifts. c:this gene is duplicated mutations that disrupt the coding sequence. For ex- ample, the effector RipS5 is encoded by a gene that has been clearly interrupted by a 34 kbp prophage. Table 6 also shows the orthologs of these genes in the related strains GMI1000, Po82, IPO1609, and UW551. In the table it can be seen that the genes that code for RipAA and RipAR have frameshifts or truncations in strain UY031 only. The absence of a particular effector may be enough for a pathogen to avoid host defenses, and there- fore cause disease. These two genes are therefore a good starting point for additional investigations of phenotypic differences between these strains. Other effector genes of interest are those that are present and do not have disrupting mutations in UY031 but are absent or appear to be nonfunctional in other strains. Abbreviations IIB1: Phylotype IIB, sequevar 1; T3E: Type III effectors; HGG: Homologous gene groups. Conclusions The complete sequence of R. solanacearum UY031 strain presented here should provide a rich platform upon which additional plant-pathogen studies can be carried out. Even though this is the fifth phylotype IIB1 sequenced, we found many differences with respect to the genomes of the other strains. In particular, the repertoire of T3E genes has many variations among these strains, and this may help explain some of the most relevant pathogenicity- related phenotypes described in the literature, opening the way to new control methods for bacterial wilt. Our modification analysis revealed two motifs that are essentially always methylated, namely: CAACRAC and GTWWAC. Both are fairly frequent in the genome, occur- ring respectively 2144 and 716 times. Motif CAACRAC is associated with the product of gene RSUY_11320 (R. Roberts, personal communication), which is hypothesized to be an enzyme of the Restriction-Modification System, with a restriction nuclease and a DNA methyltransferase role. This gene does not have homologs in other R. solanacearum strains and is located close to a region containing phage-related genes. This region contains gene RSUY_11410, which has been annotated as encod- ing a zonular occludens toxin. The provenance of this References Betaproteobacteria class. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. Second ed. New York: Springer; 2005. p. 575. part C. 6. Gabriel DW, Allen C, Schell M, Denny TP, Greenberg JT, Duan YP, et al. Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2. MPMI. 2006;19(1):69–79. 6. Gabriel DW, Allen C, Schell M, Denny TP, Greenberg JT, Duan YP, et al. Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2. MPMI. 2006;19(1):69–79. 31. List Editor. Validation List Number 107. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2006,56:1–6 7. Guidot A, Elbaz M, Carrere S, Siri MI, Pianzzola MJ, Prior P, et al. Specific genes from the potato brown rot strains of Ralstonia solanacearum and their potential use for strain detection. Phytopathology. 2009;99(9):1105–12. 32. Garrity GM, Bell JA, Lilburn T. Order I. Burkholderiales ord. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. Second ed. New York: Springer; 2005. p. 575. part C. 8. Yuan KX, Cullis J, Levesque CA, Tambong J, Chen W, Lewis CT, et al. Draft genome sequences of Ralstonia solanacearum race 3 biovar 2 strains with different temperature adaptations. Genome Announc. 2015;3(4). 33. Garrity GM, Bell JA, Lilburn T. Family I. Burkholderiaceae fam. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. Second ed. New York: Springer; 2005. p. 575. part C. 9. Salanoubat M, Genin S, Artiguenave F, Gouzy J, Mangenot S, Arlat M, et al. Genome sequence of the plant pathogen Ralstonia solanacearum. Nature. 2002;415(6871):497–502. 34. Yabuuchi E, Kosako Y, Yano I, Hotta H, Nishiuchi Y. Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. Nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. and Ralstonia eutropha (Davis 1969) comb. Nov. Microbiol Immunol. 1995;39(11):897–904. 10. Xu J, Zheng HJ, Liu L, Pan ZC, Prior P, Tang B, et al. Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82. J Bacteriol. 2011;193(16):4261–2. 35. List Editor. Validation List No. 57. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. Int J Syst Bacteriol. References 25. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7. 1. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol. 2012;13(6):614–29. 1. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol. 2012;13(6):614–29. 26. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. 2. Genin S, Denny TP. Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol. 2012;50:67–89. 2. Genin S, Denny TP. Pathogenomics of the Ralstonia solanacearum species complex. Annu Rev Phytopathol. 2012;50:67–89. 27. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26(5):541–7. 3. Fegan M, Prior P. How complex is the ‘Ralstonia solanacearum’ species complex? In: Allen CP, editor. Bacterial wilt: The disease and the ralstonia solanacearum species complex. Prior, Hayward AC. St. Paul, MN: American Phytopathological Society; 2005. p. 449–61. 3. Fegan M, Prior P. How complex is the ‘Ralstonia solanacearum’ species complex? In: Allen CP, editor. Bacterial wilt: The disease and the ralstonia solanacearum species complex. Prior, Hayward AC. St. Paul, MN: American Phytopathological Society; 2005. p. 449–61. 28. Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990;87(12):4576–9. 4. Janse JD, van den Beld HE, Elphinstone J, Simpkins S, Tjou-Tam-Sin NNA, van Vaerenbergh J. Introduction to Europe of Ralstonia solanacearum biovar 2, race 3 in Pelargonium zonale cuttings. J Plant Pathol. 2004;86(2):147–55. 29. Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematic bacteriology, vol. 2. Second ed. New York: Springer; 2005. p. Part B:1. 5. Siri MI, Sanabria A, Pianzolla MJ. Genetic diversity and aggressiveness of Ralstonia solanacearum strains causing bacterial wilt of potato in Uruguay. Plant Dis. 2011;95(10):1292–301. 5. Siri MI, Sanabria A, Pianzolla MJ. Genetic diversity and aggressiveness of Ralstonia solanacearum strains causing bacterial wilt of potato in Uruguay. Plant Dis. 2011;95(10):1292–301. 30. Garrity GM, Bell JA, Lilburn T. Class II. Competing interests The authors declare that they have followed all local, national and international guidelines and legislation and obtained the required permissions and/or licenses for this study. The authors declare that they do not have any financial and non-financial competing interests. Page 8 of 8 Page 8 of 8 Page 8 of 8 Guarischi-Sousa et al. Standards in Genomic Sciences (2016) 11:7 Authors’ contributions C d h MV 15. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402. Conceived the project: MV, JCS, RGS. Provided strains and metadata: MIS, MJP. Assembled and annotated the genome: RGS. Performed effector gene Conceived the project: MV, JCS, RGS. Provided strains and metadata: MIS, MJP. Assembled and annotated the genome: RGS. Performed effector gene annotation: MP, NSC. Analyzed and interpreted results: JCS, MV, MP, NSC, RGS, MIS, MJP. Wrote the manuscript: JCS, MV, MP, RGS, MIS, MJP. All authors read and approved the final manuscript. 16. Contreras-Moreira B, Vinuesa P. GET_HOMOLOGUES, a versatile software package for scalable and robust microbial pangenome analysis. Appl Environ Microbiol. 2013;79(24):7696–701. annotation: MP, NSC. Analyzed and interpreted results: JCS, MV, MP, NSC, RGS, MIS, MJP. Wrote the manuscript: JCS, MV, MP, RGS, MIS, MJP. All authors read and approved the final manuscript. 17. Li L, Stoeckert Jr CJ, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 2003;13(9):2178–89. Received: 29 September 2015 Accepted: 10 December 2015 24. Murugaiyan S, Bae JY, Wu J, Lee SD, Um HY, Choi HK, et al. Characterization of filamentous bacteriophage PE226 infecting Ralstonia solanacearum strains. J Appl Microbiol. 2011;110(1):296–303. Acknowledgements 18. Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods. 2010;7(6):461–5. We thank Carlos Balsalobre and Cristina Madrid for their helpful advice and for kindly providing materials and protocols; and Carlos Morais for help with NCBI submission. We also thank COST action Sustain from the European Union for funding and Nemo Peeters and Stéphane Genin for hosting MP for a short stay to carry out UY031 effector annotation. RGS has a Ph.D. fellowship from FAPESP, Brazil. JCS has an investigator fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil. 19. Sanchez-Romero MA, Cota I, Casadesus J. DNA methylation in bacteria: from the methyl group to the methylome. Curr Opin Microbiol. 2015;25:9–16. 20. Garcia-Del Portillo F, Pucciarelli MG, Casadesus J. DNA adenine methylase mutants of Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity. Proc Natl Acad Sci U S A. 1999;96(20):11578–83. fellowship from FAPESP, Brazil. JCS has an investigator fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil. 21. Heithoff DM, Sinsheimer RL, Low DA, Mahan MJ. An essential role for DNA adenine methylation in bacterial virulence. Science. 1999;284(5416):967–70. Author details 1I i d Q 1Instituto de Química, Universidade de São Paulo, São Paulo, Brazil. 2Department of Genetics, University of Barcelona and Centre for Research in Agricultural Genomics (CRAG), Bellaterra, Catalonia, Spain. 3Departamento de Biociencias, Cátedra de Microbiología, Facultad de Química, Universidad de la República, Montevideo, Uruguay. 4Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA. 22. Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE–a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 2015;43(Database issue):D298–9. 23. Di Pierro M, Lu R, Uzzau S, Wang W, Margaretten K, Pazzani C, et al. Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. J Biol Chem. 2001;276(22):19160–5. Received: 29 September 2015 Accepted: 10 December 2015 References 1996,46:625–626 11. Remenant B, Coupat-Goutaland B, Guidot A, Cellier G, Wicker E, Allen C, et al. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence. BMC Genomics. 2010;11:379. 36. Denny TP, Hayward AC, Schaad NW, Jones JB, Chun W. II. Gram negative bacteria. F. Ralstonia. In: Laboratory guide for identification of plant pathogenic bacteria. Thirdth ed. St. Paul, MN, USA: American Phytopathological Society Press; 2001. 12. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods. 2013;10(6):563–9. 37. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The gene ontology consortium. Nat Genet. 2000;25(1):25–9. 13. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9. 14. Peeters N, Carrere S, Anisimova M, Plener L, Cazale AC, Genin S. Repertoire, unified nomenclature and evolution of the type III effector gene set in the Ralstonia solanacearum species complex. BMC Genomics. 2013;14:859.
https://openalex.org/W4392000517	https://jurnalp4i.com/index.php/learning/article/download/2746/2426	Indonesian	null	ANALISIS PEMBELAJARAN BERBASIS BLENDED LEARNING DALAM PERKULIAHAN DI ITBA DIAN CIPTA CENDIKIA	Learning	2,024	cc-by-sa	4,157	ABSTRACT This research aims to determine the teaching model using the blended model after the Covid- 19 pandemic. This research focuses on lecturers as the main data source. The researcher used a qualitative approach with a case study design and used simple analysis which only revealed phenomena that occurred during post-covid-19 pandemic learning at ITBA Dian Cipta Cendikia. The research results show that lecturers try to maximize existing technology to carry out teaching using the blended learning model. Although several obstacles were found from both lecturers and students, including obstacles in teaching the material, difficulty understanding the lecturer's instructions and instructions, limited interaction between lecturers and students, students and students, and problems when sending assignments in the form of videos. because it has a large size. Keywords: Application, Teaching Program, Blended Learning ABSTRAK Penelitian ini bertujuan untuk mengetahui model mengajar dengan menggunakan model blended pasca masa pandemic covid-19. Penelitian ini berfokus pada para dosen sebagai sumber data utamanya. Peneliti menggunakan pendekatan kualitatif dengan desain studi kasus dan menggunakan analisis sederhana yang hanya mengungkapkan fenomena yang terjadi pada pembelajaran masa pasca pandemic covid-19 di ITBA Dian Cipta Cendikia. Hasil penelitian menunjukkan bahwa para dosen berusaha memaksimalkan teknologi yang ada untuk melakukan pengajaran dengan model blended learning. Meskipun beberapa kendala ditemukan baik dari dosen maupun dari mahasiswa, yang diantaranya adalah kendala dalam mengajarkan materi, kesulitan memahami instruksi dan petunjuk dosen, terbatasnya interaksi yang bisa dijangkau baik antara dosen dan mahasiswa, mahasiswa and mahasiswa sampai pada masalah saat melakukan pengiriman tugas yang berbentuk video karena memiliki ukuran yang besar. Kata Kunci: Penerapan, Program Pengajaran, Blended Learning LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 ANALISIS PEMBELAJARAN BERBASIS BLENDED LEARNING DALAM PERKULIAHAN DI ITBA DIAN CIPTA CENDIKIA JUNI HARTIWI ITBA Dian Cipta Cendikia juni_marvel@yahoo.com PENDAHULUAN Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Sebuah aktifitas pembelajaran bisa dilakukan secara tatap muka (offline) dan secara virtual (online). Pembelajaran online dapat menggunakan media teknologi pembelajaran digital untuk mewujudkannya. E-learning di Indonesia telah dikembangkan dibawah naungan Program Telematika Pendidikan atau program e-education. E-education berhubungan dengan pemanfaatan media komunikasi dan teknologi informasi, seperti komputer, internet, telepon, komputer/video, radio, dan alat bantu audiovisual lainnya yang digunakan dalam dunia pendidikan (Rusman 2013: 286). Lebih lanjut Abidin (2012) menjelaskan bahwa dengan tampilan yang dapat mengkombinasikan berbagai unsur penyampaian informasi danpesan, komputer dapat dirancang dan digunakan sebagai media teknologi yang efektif untuk mempelajari dan mengajarkan materiperkuliahan. Sehingga bisa disimpulkan bahwa produk media berbasis computer memerikan manfaat bagi banyak bidang dan sector, termasuk diantaranya dalam dunia pendidikan. Beberapa teknologi pembelajaran yang biasa digunakan adalah Computer-Assisted Instruction (CAI), Intelligent Computer-Assisted Instruction (ICAI), dan Intelligent Tutoring System (ITS). Penggunaan teknologi pembelajaran ini juga sudah 35 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 mulai diterapkan oleh beberapa lembaga pendidikan di Indonesia. Namun penggunaan secara massive lebih banyak pada masa pandemi covid19. Hampir seluruh belahan dunia terdampak covid dalam semua sector, tidak terkecuali sector pendidikan yang mengalami perubahan sistem pengajaran dari pembelajaran di dalam kelas menjadi pembelajaran di dalam jaringan atau online (online). Satu model pembelajaran online yaitu model blended learning, model ini diasumsikan bisa mengatasi kendala pembelajaran yang tidak memungkinkan adanya tatap muka secara langsung antara dosen dan mahasiswa karena ada satu dan lain hal. Keterampilan mengajar dosen ternyata juga berpengaruh terhadap pencapaian mahasiswa. Dosen berperan membimbing mahasiswa dalam mencapai kompetensi belajar yang mandiri, percaya diri, kreatif, aktif, dan kritis. Bagi dosen yang terbiasa menggunakan media elektronik maupun media online mungkin tidak menjadi suatu masalah. Akan tetapi jika dosen tidak terbiasa apalagi kurang menguasai keterampilan dalam menggunakan media pembelajaran yang berbasis online akan berdampak signifikan terhadap pencapaian hasil belajar mahasiswa. Pembelajaran dengan model blended learning dibuktikan mampu melatih kemampuan belajar mandiri pada mahasiswa, hal ini berdasarkan hasil penelitian yang dilakukan sebelum masa pandemic covid-19. Pembelajaran blended learning memfasilitasi dosen dan mahasiswa untuk bisa selalu terhubung kapanpun dan dimanapun. Berdasarkan dua penelitian ini dapat diketahui bahwa pembelajaran dengan model blended learning relevan untuk diterapkan di masa pandemi ini. PENDAHULUAN Peneliti memfokuskan penelitian pada model mengajar dengan blended learning oleh para dosen di perguruan tinggi ITBA Dian Cipta Cendikia, Bandar Lampung. Pengertian Blended Learning Blended learning adalah program pembelajaran efektif yang mencampurkan model pembelajaran tradisional, pembelajaran kolaboratif, pembelajaran mandiri, pembelajaran praktis, dan pembelajaran yang berdasarkan pengalaman. Pencampuran model ini ditujukan agar mahasiswa mendapatkan pengalaman belajar yang efektif dan efisien. Selama era pandemic covid-19 ini, para pakar pendidikan merekomendasikan model blended learning sebagai salah satu program yang menjembatani masalah pendidikan di Indonesia. Blended learning merupakan solusi dari kelemahan-kelemahan dari pembelajaran online karena menggabungkan online, offline dan pembelajaran tatap muka (Abdullah: 2013). Pembelajaran ini sifatnya menggabungkan konsep tatap muka dengan menggunakan platform media online seperti aplikasi WhatsApp, Zoom dan Google Meet. Program blended learning ini juga merupakan langkah bijaksana untuk mengatasi masalah pembelajaran yang terkendala oleh jarak dan tempat. Agar interaksi antara dosen dan mahasiswa tetap dapat terjalin, maka diperlukan sebuah platform yang mampu memfasilitasi pembelajaran online. Selain berinteraksi, dosen juga bisa memberikan materi dengan bentuk visual, audio, maupun audio visual melalui platform tersebut. Seperti pada umumnya saat menggunakan model pembelajaran tertentu, sebelum mengajar seorang dosen harus memperhatikan beberapa hal yang dibutuhkan antara lain memberikan silabus, memberi kesempatan mahasiswa untuk meninjau silabus, menghubungi mahasiswa melalui email atau media komunikasi online lainnya, menyajikan perkembangan pencapaian mahasiswa secara real-time, menjaga interaksi dengan mahasiswa agar hubungan emosional tetap terjalin, dan mengadakan evaluasi. Hal ini berlaku baik pada sekolah tingkat tinggi, maupun tingkat sekolah dasar, hal ini tergantung bagaimana seorang pengajar mampu mengemasnya sesuai tingkat kemampuan pemahaman peserta didik. Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Berikutnya adalah memungkinkan bagi dosen untuk memberikan kesempatan kepada mahasiswa untuk memberikan tinjauan terhadap silabus yang telah diberikan secara online maupun offline. Tahapan ini memberikan waktu kepada dosen untuk menjelaskan tujuan dari pengajaran sehingga bisa memberikan deskripsi kepada mahasiswa tentang tema-tema yang 36 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 akan dipelajari dalam satu semester. Dalam proses pembelajaran dalam satu semester, dosen bisa mengirimkan email atau pesan secara berkala seperti mingguan atau bulanan untuk memberikan motivasi dan sesekali mengingatkan kepada mahasiswa mengenai tugas yang harus diselesaikan dan kemampuan yang harus mereka capai. akan dipelajari dalam satu semester. Dalam proses pembelajaran dalam satu semester, dosen bisa mengirimkan email atau pesan secara berkala seperti mingguan atau bulanan untuk memberikan motivasi dan sesekali mengingatkan kepada mahasiswa mengenai tugas yang harus diselesaikan dan kemampuan yang harus mereka capai. Pengertian Blended Learning p y g p Sejatinya pembelajaran secara online memang membatasi ruang perhatian dosen kepada mahasiswa. Peserta didik yang terlihat aktif saja yang lebih diketahui oleh dosen. Terlebih lagi adanya penggunaan perangkat yang sederhana dan kurang memadai misalnya seperti layar computer atau smartphone yang kecil, maka akan membatasi dosen dalam memantau kegiatan mahasiswa. Lain halnya dalam penilaian, dosen bisa menggunakan penilaian online yang bisa dilihat oleh mahasiswa kapanpun dan dimanapun mereka berada yang merupakan wujud feedback dari seorang dosen terhadap mahasiswanya selama proses pembelajaran berlangsung. Dimana feedback tersebut diharapkan dapat memacu mahasiswa untuk terus memperbaiki kemampuannya, juga bisa menjadi kompetisi antar mahasiswa di dalam kelas. Hal ini dikarenakan beberapa mahasiswa biasanya memiliki tingkat kepercayaan diri yang tinggi untuk menunjukkan keberhasilan dan kompetensi mereka kepada mahasiswa lain. Dengan demikian mahasiswa termotivasi untuk meningkatkan kemampuan mereka agar bisa menyamakan bahkan mengungguli mahasiswa dengan nilai terbaik di kelas tersebut. Hal lainnya adalah ketika terjadi pembelajaran online, seorang dosen sebaiknya mampu menghidupkan suasana belajar dengan terus memberikan stimulus bagi mahasiswa untuk berkomunikasi berkala dengan dosen, terkait pembahasan tugas-tugas yang diberikan. Berikutnya adalah evaluasi yang dilakukan secara online memungkinkan untuk diselenggarakan oleh institusi sendiri atau dari masing-masing dosen yang mengampu mata kuliah. Pada sesi ini diperlukan adanya intervensi pihak institusi saat melakukan pengelolaan dan membantu koordinasi antara dosen dan mahasiswa. Contoh bentuk pengelolaan dan koordinasi ini diantaranya adalah memfasilitasi dosen dalam mengumpulkan jawaban-jawaban ataupun respon dari mahasiswa, menyediakan form penilaian secara online agar lebih mudah dalam menginput nilai mahasiswa, yang pada akhirnya akan melancarkan proses publikasi hasil penilaian melalui pengumuman di website maupun secara pribadi. METODE PENELITIAN Peneliti menggunakan pendekatan kualitatif yang lebih memfokuskan pada suatu fenomena atau kasus. Peneliti menjadikan aktivitas yang dilakukan oleh para dosen dalam mengajar online terhadap mahasiswa-mahasiswanya. Aktivitas yang dianalisa adalah aktivitas yang berkaitan dengan pembelajaran, diantaranya adalah aktivitas persiapan dan pelaksanaan pembelajaran online. Desain penelitian yang digunakan yaitu desain studi kasus yang berpedoman bahwa objek penelitiannya terdapat pada sekumpulan invididu atau kelompok yaitu para dosen di kampus ITBA Dian Cipta Cendikia. Teknik wawancara dan observasi terhadap beberapa dosen dan mahasiswa dengan metode pemilihan sample menggunakan metode snowball sampling merupakan teknik yang digunakan dalam mengumpulkan data. Peneliti lebih mudah dalam menjelajahi subjek penelitian berdasarkan saran dari subjek penelitian sebelumnya. Analisis data penelitian dimulai dari klasifikasi data, menentukan kategori pada data yang telah terklasifikasikan, menghubungkan setiap kategori, analisa berdasarkan landasan teori untuk dijadikan sebagai bahan diskusi, dan menemukan sebuah kesimpulan. Model Mengajar Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Model pembelajaran pada dasarnya memiliki tiga model yaitu model perilaku, model kognitif, dan model interaksi sosial. Model mengajar perilaku berbasis pada prinsip teori belajar behavioristik yang mengutamakan adanya stimulus dan respon antara dosen dan mahasiswa seperti meningkatkan perhatian mahasiswa, memunculkan penguatan, memberikan umpan balik yang korektif, dan memberikan kesempatan kepada mahasiswa untuk memberikan tanggapan. Beberapa instruksi langsung yang bisa digunakan pada model mengajar perilaku adalah mengarahkan mahasiswa ke pembelajaran, meninjau dan melengkapi perangkat pembelajaran, menyajikan informasi baru, memberikan latihan yang terbimbing, dan memberikan latihan atau praktik mandiri. Selanjutnya adalah model mengajar kognitif yang dikembangkan berdasarkan teori belajar kognitif yang lebih mengedepankan proses belajar, yang mana tidak dapat diobservasi secara langsung dan lebih banyak berfokus pada pemahaman, pemikiran, dan penciptaan. Beberapa instruksi langsung yang bisa digunakan pada model belajar kognitif seperti mendapatkan perhatian dari mahasiswa, memberitahu mahasiswa tentang tujuan pembelajaran, merangsang ingatan mahasiswa tentang hal yang diperlukan dalam pembelajaran, mempresentasikan materi, memberikan bimbingan belajar, memperoleh kinerja, memberikan umpan balik tentang latihan yang baik, menilai hasil latihan, dan meningkatkan proses transfer pengetahuan. Yang terakhir adalah model mengajar interaksi sosial bahwa setiap individu berinteraksi dengan sekeliling mereka selama proses pembelajaran, dan interaksi mereka ini menjadi salah satu faktor penting dalam meningkatkan 37 pembelajaran. Beberapa instruksi langsung yang bisa digunakan pada model mengajar interaksi langsung seperti memilih konten dan menentukan subtopic, merencanakan pembelajaran kooperatif, mempelajari pembelajaran kooperatif, analisis; sintesis; rencanakan presentasi, menyajikan proses dan hasil, dan melakukan penilaian. HASIL DAN PEMBAHASAN Meskipun memiliki berbagai keuntungan, akan tetapi penggunaan Whats App Grup sebagai media pembelajaran daring juga memiliki ketebatasan. whatsapp sebagai media pembelajaran adalah tidak dapat bertatap muka (face to face) secara langsung. Meskipun demikian, pada beberapa mahasiswa ada yang beranggapan bahwa jika perkuliahan tidak dilakukan secara tatap muka langsung (synchrous) maka suasana pembelajaran menjadi kurang terasa. (Budiyanti dkk, 2021) p j j g y Berbeda dengan materi yang berbasis teori dan konsep, seperti mata kuliah tata bahasa. Maka dosen hanya memberikan petunjuk kepada mahasiswa untuk mempelajari beberapa halaman, atau untuk lebih jelasnya seorang dosen akan mengambil beberapa foto halaman buku berisikan materi tertentu kemudian diunggah agar mahasiswa terfokus pada materi dan tidak pada halaman sebelum atau sesudahnya. Sedangkan pada materi yang berbasis keterampilan, maka mahasiswa diarahkan untuk membuat dan merancang produk berdasarkan petunjuk- petunjuk yang bisa dilihat pada media online lainnya seperti Youtube, vlog dan sebagainya. Media audio visual atau video tentu memiliki kelebihan dan kekurangan. Kelebihannya adalah mahasiswa mampu memahami dengan lebih baik daripada penyajian materi dengan visual saja. Mahasiswa seringkali perlu mengulang-ulang instruksi dari dosen sebelum mulai mengerjakan tugas yang diberikan. Media audio visual juga disebut Video Tutorial, dalam penggunaannya ada beberapa langkah yang bisa diterapkan saat mempersiapkan bahan ajar untuk pengajaran yaitu tahap persiapan, tahap perekaman, tahap penyelesaian akhir (Batubara&Batubara, 2020). Para dosen di ITBA Dian Cipta Cendikia juga melakukan tahap persiapan materi yang dilakukan dengan memilih dan menentukan materi yang bisa juga di ambil dari sumber belajar lainnya. Lalu menyiapkan alat-alat perekaman untuk menghasilkan kualitas suara dan gambar yang memadai. Ketersediaan alat perekaman biasanya hanya menggunakan smartphone yang dimiliki oleh dosen. Namun demikian mahasiswa dimudahkan dalam mengunduh video tutorial yang diunggah oleh guru, karena ukuran file video tutorial tersebut tidak terlalu besar, meskipun dengan gambar yang tidak terlalu jernih. Dan tahap akhir adalah dengan meninjau ulang dan mengunggah materi tersebut pada WhatApp. Dosen juga bisa menggunakan menu “update status” yang disediakan oleh platform tersebut. Kelemahannya adalah perlu pembagian video dalam durasi setiap 30 detik. Teknik ini jarang digunakan oleh beberapa dosen di ITBA Dian Cipta Cendikia. Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran p Sisi lain pelaksanaan pengajaran berbasis blended learning berbeda dengan yang terjadi pada pembelajaran tatap muka. Dosen cenderung pasif pada sesi pemberian materi. Para dosen tidak memungkinkan untuk menggunakan media online langsung yang interaktif seperti Zoom atau pun Google Meet. HASIL DAN PEMBAHASAN Dalam pengambilan data dilakukan sesuai urutan terjadinya proses mengajar yang dilakukan oleh seorang guru, seperti mempersiapkan komponen-komponen mengajar berupa persiapan materi; persiapan media; persiapan mahasiswa, kemudian pelaksanaan pengajaran yang meliputi pembukaan pengajaran; pemberian materi ajar atau pengetahuan; dan penutup pembelajaran, dan pemberian feedback. Pengambilan data dilakukan dengan teknik wawancara. Sumber datanya adalah beberapa dosen yang mengajar pada beberapa jurusan di ITBA Dian Cipta Cendikia. Hasil pengumpulan data ini disajikan sesuai urutan pengumpulan data yang berdasarkan pada urutan pengajaran yang dilakukan oleh seorang dosen setiap harinya, seperti melakukan persiapan mengajar, pelaksanaan pengajaran, dan feedback setelah proses pembelajaran selesai. Pada tahapan persiapan pengajaran, dosen mempersiapkan materi yang akan dijadikan sebagai bahan ajar. Dosen harus mempertimbangkan cara yang tepat untuk menyampaikan materi tersebut agar bisa diunggah pada platform online. Beberapa dosen menggunakan platform WhatsApp, hal ini didasari asumsi bahwa semua mahasiswa memiliki platform WhatsApp sebagai media komunikasi berbasis internet. Adapun materi pada platform WhatsApp disajikan dalam bentuk visual dan audio, yaitu dengan memberikan foto halaman modul, kemudian menjelaskannya secara verbal melalui voice note. Namun terkadang tidak semua materi bisa di berikan secara visual dan audio, seperti materi keterampilan motorik yang apabila materinya berkaitan dengan keterampilan produktif, maka seorang dosen akan memberikan video pendek yang berisikan rekaman contoh keterampilan sesuai dengan materi pada modul. Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Wawancara juga dilakukan kepada beberapa dosen di ITBA Dian Cipta Cendekia seputar persiapan sebelum memasuki pengajaran. Persiapan pengajaran dimulai dengan menjelaskan gambaran umum materi terlebih dahulu. Beberapa dosen ada yang melakukan rangkuman mandiri untuk mempermudah mahasiswa dalam memahami materi pembelajaran, seperti membuat rangkuman tertulis jika materi terlalu panjang, atau membuat slide singkat agar memudahkan dosen dalam menjelaskan materi. Selain itu dosen harus mampu mengenali karakteristik dari materi yang akan sampaikan. Karakteristik materi tergantung pada jenis mata 38 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 kuliah. Jika mata kuliah eksak seperti logika matematika maka dosen bisa memberikan sebuah rumus atau formula, kemudian menyajikan cara penghitungannya. Setelah mengetahui materi pengoperasian rumus matematika, maka dosen tersebut akan membuat sebuah penghitungan sederhana dalam bentuk tulisan tangan. Hasil tulisan tangan ini kemudian difoto atau direkam dengan suara dosen sebagai cara menjelaskannya. HASIL DAN PEMBAHASAN Mahasiswa terlihat kesulitan untuk fokus ketika dilakukan pembelajaran interaksi langsung secara online. Sehingga para dosen lebih menyukai pemberian materi secara pasif dengan hanya mengupload video tutorial atau memberikan narasi yang disertai foto halaman buku yang akan dipelajari. Model tutorial pada dasarnya sama dengan model bimbingan, yang bertujuan memberikan bantuan pada siswa agar dapat mencapai hasil belajar secara optimal. Tutorial didefinisikan sebagai bentuk pembelajaran khusus dengan pembimbing yang terkualifikasi, penggunaan mikro komputer untuk tutorial pembelajaran dengan memberikan arahan, bantuan, petunjuk, dan motivasi agar siswa belajar secara efisien dan efektif (Rusman, 2013). Hasil observasi menunjukkan bahwa disesi pemberian instruksi tentang materi yang akan dipelajari, kemudian dosen menentukan latihan yang perlu dikerjakan yang ada pada buku ajar yang telah diberikan kepada mahasiswa sebelumnya. Mahasiswa harus mampu untuk belajar secara mandiri di rumah. Metode ini memberikan indikasi yang nyata 39 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 bahwa peran dosen sebagai fasilitator dalam membimbing pemahaman terhadap materi yang disampaikan. bahwa peran dosen sebagai fasilitator dalam membimbing pemahaman terhadap materi yang disampaikan. Selanjutnya fokus sebagian dosen adalah kepada latihan yang diberikan kepada mahasiswa. Strategi perulangan (drills and practice) adalah cara yang tepat untuk memahamkan para mahasiswa. Mahasiswa ditugaskan untuk mengerjakan latihan yang relevan dengan materi hari itu. Proses ini menguntungkan bagi mahasiswa, karena mereka memiliki waktu untuk mengerjakan tugas dengan jenis tugas yang bermacam-macam. Sehingga diharapkan mampu memicu aspek kognitif mahasiswa dalam setiap tugasnya. Lebih lanjut seperti yang dijelaskan oleh Azhar Arsyad dan Rusman dalam Nugroho (2018) bahwa dengan melakukan latihan terus menerus disertai dengan adanya penambahan kecepatan, ketepatan, kesempurnaan dalam mengerjakan, maka akan mengasah siswa untuk menjadi terbiasa sehingga siswa bisa lebih kuat dalam memberikan tanggapan pada pelajaran. Observasi yang dilakukan pada beberapa mahasiswa yang sedang belajar online. Tidak jarang tugas yang mereka dapatkan dikerjakan secara mandiri, namun ada beberapa mahasiwa yang hanya mengcopy tugas yang telah dikerjakan rekan sesama mahasiswa. Hal ini mungkin karena mahasiswa merasa kesulitan dalam memahami materi. Dan hal ini jika dibiarkan terus menerus akan menjadi sebuah kebiasaan buruk. Dan hal ini akan berdampak pada kemampuan kognitif mahasiswa. Egbert dan Hanson Smith didalam Abdullah (2018) berpendapat karakteristik blended learning yaitu siswa dapat bersosialisasi dengan baik dengan sesama, siswa mempuanyai waktu banyak dan dapat melakukan feedback, siswa juga dipandu dengan baik serta siswa belajar dengan atmosfer yang ideal. Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran HASIL DAN PEMBAHASAN Konsep dasar blended learning salah satunya untuk memberi kesempatan kepada mahasiswa agar berlatih belajar secara mandiri yakni menciptakan atau memilih cara belajarnya sendiri. Akan tetapi semua kembali lagi kepada sifat alami dari mahasiswa yang perlu bimbingan dan arahan dalam belajar. Mahasiswa belum sepenuhnya memiliki kesadaran terhadap pentingnya pendidikan. p p g y p Anugrahana menemukan adanya kelebihan dan kekurangan pembelajaran online (Anugrahana, 2020). Kelebihan pertama dalam pembelajaran online adalah lebih praktis dan santai. Praktis karena dapat memberikan tugas setiap saat dan pelaporan tugas setiap saat. Kedua, lebih fleksibel bisa dilakukan kapanpun dan dimanapun. Pembelajaran online memberikan waktu yang lebih fleksibel bagi mahasiswa yang bekerja di luar rumah dan bisa menyesuaikan waktu untuk belajar. Ketiga, menghemat waktu dan dapat dilakukan kapan saja. Mahasiswa dapat mengakses dengan mudah, artinya dapat dilakukan kapan saja dan dimana saja. Informasi dapat diperoleh lebih cepat dan bisa menjangkau banyak mahasiswa lewat WA Group. Keempat, tahapan evaluasi pencapaian mahasiwa lebih praktis dan memudahkan khususnya jika melalui Google Form. Artinya nilai bisa langsung dilihat sehingga mahasiswa lebih tertarik dalam mengerjakan tugas selanjutnya. Selain itu mahasiswa juga dimudahkan dalam mengerjakan evaluasi, dimana mahasiswa tinggal memilih pilihan jawaban yang dianggap benar dengan meng-klik pilihan jawaban yang dimaksud. Kelebihan kelima, dosen dan mahasiswa memperoleh pengalaman baru terkait pembelajaran online. p p g p j Kelemahan pembelajaran online adalah kurangnya keterlibatan mahasiswa dalam mengikuti pembelajaran online secara penuh dari awal pembelajaran sampai akhir pembelajaran. Namun Noer dalam Husamah (2014) bahwa pembelajaran online mempunyai kendala interaksi langsung antara peserta didik dengan pengajar bagaimanapun pengajar perlu feedback dari peserta didik dan peserta didik juga butuh feedback dari pengajar. Dari hasil survey menunjukkan bahwa hanya 80% mahasiswa yang aktif, dan 20% mahasiswa kurang aktif dan kurang berpartisipasi dalam pembelajaran online. Hasil wawancara dan observasi singkat peneliti dengan salah seorang mahasiswa yang menjelaskan bahwa perlu mengulang- 40 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 ulang video tutorial yang dikirimkan ke platform WhatsApp Group. Pengulangan video ini untuk lebih memantapkan pemahaman terhadap materi yang diajarkan. ulang video tutorial yang dikirimkan ke platform WhatsApp Group. Pengulangan video ini untuk lebih memantapkan pemahaman terhadap materi yang diajarkan. Data-data mengarahkan pada satu kesimpulan bahwa terdapat langkah-langkah yang dilakukan oleh mahasiswa dalam mengikuti pembelajaran dan memahami instruksi dari guru. Langkah pertama adalah mengulangi, memahami, dan menerapkan. Kegiatan mengulangi untuk mendapatkan pengalaman dan berusaha memahami instruksi dari gurunya. HASIL DAN PEMBAHASAN Dengan mengulangi maka akan memberikan pengalaman terhadap anggota tubuh dalam mengerjakan hal baru dalam hidupnya, seperti seorang mahasiswa yang sedang menirukan gurunya menyelesaikan penghitungan matematika. Ketika seorang mahasiswa melakukan perulangan, maka aspek kognitif mahasiswa belum sepenuhnya bekerja, karena hanya menirukan secara kinestetik apa yang diterima oleh indera penglihatnya. Kemudian ketika mahasiswa telah mengakhiri kegiatan perulangan, maka timbul keingintahuan tentang apa yang telah ia kerjakan. Stimulus yang diberikan akan memancing perulangan hingga mahasiswa akan terbiasa dalam menyelesaikan tugas yang diberikan. Meskipun aspek kognitif tidak digunakan dengan maksimal, lambat laun akan menjadi sebuah kebiasaan dan menuntun aspek kognitif mahasiswa untuk memahami secara tidak sadar. Dampak negative dari perulangan adalah akan selalu bergantung pada model. Hal ini menjadikan mahasiswa tidak dapat berpikir dan berperilaku mandiri atas apa yang dihadapinya, proses berpikir kreatif dan kritis akan lambat berkembang. Wawancara yang dilakukan pada mahasiswa bahwa mahasiswa akan termotivasi untuk memahami materi atau soal dengan orintasi pada pemecahan masalah atau yang dikenal dengan problem based learning. Jika hanya disuruh membaca saja tanpa adanya tantangan maka mahasiswa kurang termotivasi untuk memahami materi dan soal. Tantangan yang lazim didapatkan adalah kewajiban mengerjakan soal dengan batas waktu yang telah ditentukan. Evaluasi terhadap hasil pembelajaran langsung juga dilakukan oleh dosen beberapa saat setelah pemberian soal. Pemberian nilai secara langsung oleh dosen kepada mahasiswa agar bisa diambil feedback. Terdapat dua temuan tentang feedback yang dilakukan yaitu feedback terhadap ketuntasan dan terhadap ketidaktuntasan. Feedback terhadap ketuntasan bisa dilakukan dengan bentuk apresiasi lewat pujian terhadap hasil karya mahasiswa. Pujian ini diberikan bertujuan untuk meningkatkan kepercayaan diri mahasiswa ketika mendapat pujian secara langsung dari dosen. Disamping itu, terdapat juga feedback terhadap ketidaktuntasan. Hal ini bisa dilakukan dengan cara memberikan kesempatan kepada mahasiswa untuk memperbaiki tugas yang telah mereka selesaikan. Otomatis yang pertama kali menerima informasi ini adalah mahasiswa. Terkadang mahasiswa juga mengeluh karena telah mengerjakan soal, tetapi mendapatkan nilai yang belum mencukupi standar ketuntasan. Biasanya, dosen memberikan informasi tentang kesalahan atau kekeliruan yang telah dilakukan ketika menyelesaikan soal. Sehingga mahasiswa mendapatkan toleransi untuk memperbaiki hanya pada bagian yang salah saja pada tugas yang telah dikerjakan. Informasi mengenai feedback ini disampaikan secara pribadi melalui chat pribadi antara dosen dan mahasiswa agar tercipta privasi, sehingga hal-hal yang berpotensi negative dapat terhindar. KESIMPULAN KESIMPULAN Hasil penelitian menampilkan sebuah urutan pengajaran yang dilakukan oleh dosen dan urutan pembelajaran yang dilakukan oleh mahasiswa. Dosen melakukan pembelajaran dengan urutan yang pertama mempersiapkan bahan ajar, menyampaikan materi, membuat instruksi tutorial, memberikan soal untuk dikerjakan sebagai bahan evaluasi, dan diakhiri dengan pemberian feedback. Sedangkan urutan pembelajaran yang dilakukan oleh mahasiswa adalah dengan perulangan, pemahaman, dan penerapan. opyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran 41 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran Vol. 4 No. 1 Februari 2024 E-ISSN : 2777-0575 P-ISSN : 2777-0583 DAFTAR PUSTAKA Abdullah, Walib. (2018). FIKROTUNA: Jurnal Pendidikan dan Manajemen Islam Volume 7, Nomor 1.; p-ISSN 2442- 2401; e-ISSN 2477-5622 Abdullah, Walib. (2018). Model Blended Learning Dalam Meningkatkan‟, Ejournal.Kopertais4, 7.1. p855-66. Abidin, Zainul. (2016). Pengembangan E-Module dan Tutorial Matakuliah Pengembangan Media Audio Radio Jurusan Teknologi Pendidikan, vol 1, no. 2. Anugrahana, Andri. (2020) Hambatan, Solusi dan Harapan: Pembelajaran Online Selama Masa Pandemi Covid-19 oleh Guru Sekolah Dasar, vol. 10, no. 3. Bakhtiyar, Mokh. Arif. (2017). Promoting Blended Learning In Vocabulary Teaching Trought WhatsApp, Nidhomul Haq, vol. 2, no. 2. Batubara, Delila Sari., Hamdan Husein. (2020). Penggunaan Video Tutorial untuk Mendukung Pembelajaran Online di Masa Batubara Pandemi Virus Corona, Muallimuna: Jurnal Madrasah Ibtidaiyah, vol. 5, no. 2, 2020. yanti et. al. (2021). Penggunaan grup Watsapp sebagai Media Pembelajaran daring selam Pandemi COVID-19. SSEJ, 2021, Vol 1 (No 2) : 42 - 48 Creswell, John W. (2012) Educational Research: Planning, conducting, and evaluating quantitative and qualitative research, (Boston: Pearson Education, Inc., 2012), page. 16 Garrison, d. R. & Vaughan, n., Blended Learning in Higher Education: Framework, Principles, and Guidelines, (San Francisco: Wiley, 2008), page. 9. HARNINGSIH, L. S. (2023). PENINGKATAN KETERAMPILAN MEMBACA NARRATIVE TEXT SISWA KELAS 9H MENGGUNAKAN METODE BLENDED LEARNING DI SMP NEGERI 2 TEMANGGUNG. ACTION : Jurnal Inovasi Penelitian Tindakan Kelas Dan Sekolah, 3(3), 205-211. https://doi.org/10.51878/action.v3i3.2429 Hidayati, Nurul. (2023). Pengaruh Model Pembelajaran Blended Learning Berbantuan Aplikasi Google Classroom terhadap Kemampuan Berpikir Kritis dan Kreatif Matematis Peserta Didik. Universitas Islam Negeri Raden Intan Lampung. Unpublished. ah. (2014). Pembelajaran Bauran, Blended Learning, Jakarta: Prestasi Pustaka Publisher. Nugroho, Oktian Fajar. (2018) MODUL: Pengembangan Media Belajar. Universitas Esa Unggul. Rusman, dkk. (2013). Pembelajaran Berbasis Teknologi Informasi dan Komunikasi. Jakarta: PT. Raja Grafindo Persada. SULYANTARI, H. T. (2023). PENINGKATAN HASIL BELAJAR PESERTA DIDIK KELAS X-IPA2 MAN 1 JEMBRANA PADA MATERI ELEKTROLIT MELALUI MODEL PROJECT BASED LEARNING SECARA BLENDED LEARNING. ACTION : Jurnal Inovasi Penelitian Tindakan Kelas Dan Sekolah, 3(3), 194-204. https://doi.org/10.51878/action.v3i3.2427 p g Susan Ko, Steve Rossen. (2010). Teaching Online: A Practical Guide, (New York: Routledge, 2010), page. 308. Copyright (c) 2024 LEARNING : Jurnal Inovasi Penelitian Pendidikan dan Pembelajaran 42 42
https://openalex.org/W4235730889	https://www.biodiversitylibrary.org/itempdf/111055	German	null	Pteropoda	Das Tierreich	1,913	public-domain	102,518	NEUES ESUEIEEE SEEN TV uuuasvs, ISBUHHGBEITINTUATLILCKEL LET N nn nn ger \|\| WEREIE Ra En ARNTN Fer! wrr „I8g PLA nat wu. j” Bi T v a vr. vr a 0, Sul na, Sw 9, 4 ” Ak, Ah de - . I} 8 Dee Nu, N J TORE BARSERUELUN U SW Tree 3 PE TE] \| ; \| PA \| : \ - \| os Ä IRA UOMELT ;E vreluey BEA ia NR: way: IIY= ed x KIN N Ay 5 A AR your rel 7 77 55 EL} Y T TED TV Frl PFER AL w‘, 5 i u uneran een. v Eng Fi EFT HE ‚ “ & 1 0 nein ann vs I i ia BITTER en pe # L sı GE ır aa LU: PEN au = U 5 Fee. \|) UL ver w Se \| uns surv Tu re a L m .yn ı% =} ’ va L lu X Bu >= EP a : Ran en) ı 22 0 dı, AEG: bi D N Sun, hd AL, TIL MEN - \dtyy N Feuer. er BLITHÄNTN ar et! Nat Le in METEITT TI TI ER EN RR N PRDN \| ? . 3: w ir - x ERST ln ui as Wir: wer 2, vTıy ne” 1 \| Nanenn, N \% ve \| 2 NE 34:2 4 5 =. < > re N LAIEN a IL) DELL ARULN an NSS STE R 1 u u A my F . IN iA ve Re A ne RI uud / Mau % A\| ! v $ STR ey uw. vengglt DE er ee Mer nnt: F; \| \| Sn k K Mm \| A KOOR BAELLN n ARALNNT E M ws LER ; .o vr. > ,„’. L iz Go san nilär ann Werdau er N üme Yav Au = 1087, e ur ySaa6s mine “ Er 4.FI Pit. (hr Eum: un wiare NT IA u \| Yy an “u Rt N vn un FRE Frhr u . Nr } & : PR v PETIS LI Kae \| FI A ERRLERELLIT I aaa 1 Pre En EI HET ANUHTTT TPITETTT Th bu wetten AL, > h NET "N AM. zrtr, f de are ann EN a se BR tur u Etare zu . remere vn yyn® « 8 a aW % yr v v “uj,.V “y9-.% uzery! u ATTTLLA AAN Iuwv BE \ arT, „ MN Pr j JeReprL\| E& , = h . AA Ahd AA hd u wiegt PPPIPEP vr KIERLIG I BITTEN Rare 4 wuy # er \| Gedun ) Ilja a 5 2 5 04 24 MW Ze .% ” AR PLPPT IL ‚ vw r .. . 9 Pre “rVwnur _ 5 c EN b v N %” S b you.’ ’vril® a SL S x N ‚AV B na sr e ı7- ra a v a EU) 1 2 t vu r ve a LIEST TI M we“ ' r % u —i% vun. NEL LRN I es Mir & ’.- u" m n "ug UT rg D WERE: p ‘WW ‘ . “s ei Iı#+ Kid uzery! u A = Ahd AA hd u wieg Un ) Ilja NN‘ & n Zn I A KENN = 3 F ‘ a 5 2 5 04 24 MW Ze I .% ” LIED AR PLPPT IL ‚ vw r .. N“ . : . 9 1 > Pre “rVwnur _ 5 * c EN * Mm b nn v N %” iS al 44 bb "\ you.’ ’vril® vuy a SL I6S N x < Br arg ‚AV Y n £ B na sr ne _ a TITEL ı7- ra ba a. 28 e a EU) 1 2 t = = Si: vu er SR ve a 5 — SE eV LIEST TI ES > Se he ea 2, edb ID = N ll = = M we“ ' r Be % u = MN N —i% vun. NEL LRN I S . [2 es Mir & ’.- u" m Mn f "ug UT rg LED WERE: p ‘WW ET ‘ . “s ei Iı#+ d Kid % yı'".. =. * uzery! u ATTTLLA AAN Iuwv BE \ arT, „ MN Pr j = h . AA Ahd AA hd u wiegt PPPIPEP vr KI AR PLPPT IL ‚ vw r .. . 9 Pre “rVwnur _ 5 c EN b v N %” S b you.’ ’vril® a SL S x ‚AV B na sr e ı7- ra a a EU) 1 2 t vu ve a LIEST TI M we“ ' r % u —i% vun. NEUES ESUEIEEE SEEN TV uuuasvs, ISBUHHGBEITINTUATLILCKEL LET N nn nn ger \|\| WEREIE Ra En ARNTN Fer! wrr „I8g PLA nat wu. j” Bi T v a vr. vr a 0, Sul na, Sw 9, 4 ” Ak, Ah de - . I} 8 Dee Nu, N J TORE BARSERUELUN U SW Tree 3 PE TE] \| ; \| PA \| : \ - \| os Ä IRA UOMELT ;E vreluey BEA ia NR: way: IIY= ed x KIN N Ay 5 A AR your rel 7 77 55 EL} Y T TED TV Frl PFER AL w‘, 5 i u uneran een. v Eng Fi EFT HE ‚ “ & 1 0 nein ann vs I i ia BITTER en pe # L sı GE ır aa LU: PEN au = U 5 Fee. \|) UL ver w Se \| uns surv Tu re a L m .yn ı% =} ’ va L lu X Bu >= EP a : Ran en) ı 22 0 dı, AEG: bi D N Sun, hd AL, TIL MEN - \dtyy N Feuer. er BLITHÄNTN ar et! Nat Le in METEITT TI TI ER EN RR N PRDN \| ? . 3: w ir - x ERST ln ui as Wir: wer 2, vTıy ne” 1 \| Nanenn, N \% ve \| 2 NE 34:2 4 5 =. < > re N LAIEN a IL) DELL ARULN an NSS STE R 1 u u A my F . IN iA ve Re A ne RI uud / Mau % A\| ! v $ STR ey uw. vengglt DE er ee Mer nnt: F; \| \| Sn k K Mm \| A KOOR BAELLN n ARALNNT E M ws LER ; .o vr. > ,„’. L iz Go san nilär ann Werdau er N üme Yav Au = 1087, e ur ySaa6s mine “ Er 4.FI Pit. (hr Eum: un wiare NT IA u \| Yy an “u Rt N vn un FRE Frhr u . Nr } & : PR v PETIS LI Kae \| FI A ERRLERELLIT I aaa 1 Pre En EI HET ANUHTTT TPITETTT Th bu wetten AL, > h NET "N AM. zrtr, f de are ann EN a se BR tur u Etare zu . remere NEUES ESUEIEEE SEEN TV uuuasvs, ISBUHHGBEITINTUATLILCKEL LET N nn nn ger \|\| WEREIE Ra En ARNTN Fer! wrr „I8g PLA nat wu. j” Bi T v a vr. vr a 0, Sul na, Sw 9, 4 ” Ak, Ah de - . I} 8 Dee Nu, N J TORE BARSERUELUN U SW Tree 3 PE TE] \| ; \| PA \| : \ - \| os Ä IRA UOMELT ;E vreluey BEA ia NR: way: IIY= ed x KIN N Ay 5 A AR your rel 7 77 55 EL} Y T TED TV Frl PFER AL w‘, 5 i u uneran een. v Eng Fi EFT HE ‚ “ & 1 0 nein ann vs I i ia BITTER en pe # L sı GE ır aa LU: PEN au = U 5 Fee. \|) UL ver w Se \| uns surv Tu re a L m .yn ı% =} ’ va L lu X Bu >= EP a : Ran en) ı 22 0 dı, AEG: bi D N Sun, hd AL, TIL MEN - \dtyy N Feuer. er BLITHÄNTN ar et! Nat Le in METEITT TI TI ER EN RR N PRDN \| ? . 3: w ir - x ERST ln ui as Wir: wer 2, vTıy ne” 1 \| Nanenn, N \% ve \| 2 NE 34:2 4 5 =. < > re N LAIEN a IL) DELL ARULN an NSS STE R 1 u u A my F . IN iA ve Re A ne RI uud / Mau % A\| ! v $ STR ey uw. vengglt DE er ee Mer nnt: F; \| \| Sn k K Mm \| A KOOR BAELLN n ARALNNT E M ws LER ; .o vr. > ,„’. L iz Go san nilär ann Werdau er N üme Yav Au = 1087, e ur ySaa6s mine “ Er 4.FI Pit. (hr Eum: un wiare NT IA u \| Yy an “u Rt N vn un FRE Frhr u . Nr } & : PR v PETIS LI Kae \| FI A ERRLERELLIT I aaa 1 Pre En EI HET ANUHTTT TPITETTT Th bu wetten AL, > h NET "N AM. zrtr, f de are ann EN a se BR tur u Etare zu . remere ‘ & wg» . . “s ei Iı#+ d Qr - Kid % yı'".. =. * N .. NEUES ESUEIEEE SEEN TV uuuasvs, ISBUHHGBEITINTUATLILCKEL LET N nn nn ger \|\| WEREIE Ra En ARNTN Fer! wrr „I8g PLA nat wu. j” Bi T v a vr. vr a 0, Sul na, Sw 9, 4 ” Ak, Ah de - . I} 8 Dee Nu, N J TORE BARSERUELUN U SW Tree 3 PE TE] \| ; \| PA \| : \ - \| os Ä IRA UOMELT ;E vreluey BEA ia NR: way: IIY= ed x KIN N Ay 5 A AR your rel 7 77 55 EL} Y T TED TV Frl PFER AL w‘, 5 i u uneran een. v Eng Fi EFT HE ‚ “ & 1 0 nein ann vs I i ia BITTER en pe # L sı GE ır aa LU: PEN au = U 5 Fee. \|) UL ver w Se \| uns surv Tu re a L m .yn ı% =} ’ va L lu X Bu >= EP a : Ran en) ı 22 0 dı, AEG: bi D N Sun, hd AL, TIL MEN - \dtyy N Feuer. er BLITHÄNTN ar et! Nat Le in METEITT TI TI ER EN RR N PRDN \| ? . 3: w ir - x ERST ln ui as Wir: wer 2, vTıy ne” 1 \| Nanenn, N \% ve \| 2 NE 34:2 4 5 =. < > re N LAIEN a IL) DELL ARULN an NSS STE R 1 u u A my F . IN iA ve Re A ne RI uud / Mau % A\| ! v $ STR ey uw. vengglt DE er ee Mer nnt: F; \| \| Sn k K Mm \| A KOOR BAELLN n ARALNNT E M ws LER ; .o vr. > ,„’. L iz Go san nilär ann Werdau er N üme Yav Au = 1087, e ur ySaa6s mine “ Er 4.FI Pit. (hr Eum: un wiare NT IA u \| Yy an “u Rt N vn un FRE Frhr u . Nr } & : PR v PETIS LI Kae \| FI A ERRLERELLIT I aaa 1 Pre En EI HET ANUHTTT TPITETTT Th bu wetten AL, > h NET "N AM. zrtr, f de are ann EN a se BR tur u Etare zu . remere NEL LRN I es Mir & ’.- u" m n "ug UT rg D WERE: p ‘WW ‘ . “s ei Iı#+ Kid uzery! u A = Ahd AA hd u wieg n = 3 F ‘ \| 3 LIED 718 we N“ 2 Du : 'yr,. RAR ‚* Tyy 4 i FL we. 1 „> \| u u” AT x (B * “ A x KK) * Mm B rrirmif nt he, 4 nn i « iS al 44 g 4 u» I bb "\ ni 798 Y RE y y‘ N vuy I6S N vr RR, N < Br arg ıg Y n £ Iy - ne _ a TITEL I N ba a. 28 ee „ur werk = = Si: LP Bee y er SR . vrr@rnN I. 5 — SE eV ittn : ES > Se \ uxy he ea 2, edb PET en ID = N ll N v u. wer. T a \ = = Be FE = MN N LEE M S . [2 v ji AG Mi Al Dun Mn f “n u ALT ‘u LED Hi “ & v & N, DESZTNET «& RR & & wg» . d Qr - % yı'".. =. * N .. vn yyn® « 8 a aW % yr v v “uj,.V “y9-.% ATTTLLA AAN Iuwv BE \ arT, „ MN Pr j JeReprL\| E& en, h . AA egt PPPIPEP vr KIERLIG I BITTEN Rare Be z * * nn i b I6 N < Br Y n £ n _ a b a. 28 = = Si: e SR 5 — SE ES > Se he ea 2, ID = N = = Be = S . M LE N Br n £ a 28 Si SR SE Se 2, = N Ds a4 =‘. war Au rl Hr ET HNTr4 aa AT DR ige u. zu" GVUyTyr? H L} -’- “ur gen 4 ru” IE GIG LE ygl uuwıL Bea d ) 7 1° all“ vun AR g RI AN MAL UN Ua y 4 4 ern i AS ; vr 5 DR Pr u Mrd zu ins ih ESSO IRA 1 a T \ S in . v # N = win Se \| LT SE GE BuneRE IIENEREIHEN u III I] Sr. NEUES ESUEIEEE SEEN TV uuuasvs, ISBUHHGBEITINTUATLILCKEL LET N nn nn ger \|\| WEREIE Ra En ARNTN Fer! wrr „I8g PLA nat wu. j” Bi T v a vr. vr a 0, Sul na, Sw 9, 4 ” Ak, Ah de - . I} 8 Dee Nu, N J TORE BARSERUELUN U SW Tree 3 PE TE] \| ; \| PA \| : \ - \| os Ä IRA UOMELT ;E vreluey BEA ia NR: way: IIY= ed x KIN N Ay 5 A AR your rel 7 77 55 EL} Y T TED TV Frl PFER AL w‘, 5 i u uneran een. v Eng Fi EFT HE ‚ “ & 1 0 nein ann vs I i ia BITTER en pe # L sı GE ır aa LU: PEN au = U 5 Fee. \|) UL ver w Se \| uns surv Tu re a L m .yn ı% =} ’ va L lu X Bu >= EP a : Ran en) ı 22 0 dı, AEG: bi D N Sun, hd AL, TIL MEN - \dtyy N Feuer. er BLITHÄNTN ar et! Nat Le in METEITT TI TI ER EN RR N PRDN \| ? . 3: w ir - x ERST ln ui as Wir: wer 2, vTıy ne” 1 \| Nanenn, N \% ve \| 2 NE 34:2 4 5 =. < > re N LAIEN a IL) DELL ARULN an NSS STE R 1 u u A my F . IN iA ve Re A ne RI uud / Mau % A\| ! v $ STR ey uw. vengglt DE er ee Mer nnt: F; \| \| Sn k K Mm \| A KOOR BAELLN n ARALNNT E M ws LER ; .o vr. > ,„’. L iz Go san nilär ann Werdau er N üme Yav Au = 1087, e ur ySaa6s mine “ Er 4.FI Pit. (hr Eum: un wiare NT IA u \| Yy an “u Rt N vn un FRE Frhr u . Nr } & : PR v PETIS LI Kae \| FI A ERRLERELLIT I aaa 1 Pre En EI HET ANUHTTT TPITETTT Th bu wetten AL, > h NET "N AM. zrtr, f de are ann EN a se BR tur u Etare zu . remere N ON : RALN u I \| “ Ba ai PLP PL ı 0 a Dr rar r —mnın. Ne. . Ip IT e‘ , AA RA ANER eng AUT, “» rMULMHTT BR LEN Ara —— PUR l K Finı Teen w 2 hau en ’ DOKN, 2 BAAR, Kr Ft En Keule De wider. r N ‚4 1 PETER Rz [Pi PRrE r A zu= wer.) J x Fr u „an ALDI RER 3,5 SUR, fr r \ %y I5I,8 a ze \| on. aA re 25 MAR aan es: Wdıyn? Sur Wer MlBe: .. Ar era eugen SE De RT NER, PT ET I FHIHEHN a D lt) Lllaal Pe LG ALT 2.7203 ieh AALLIFE IE RIED Pre 4 Neiy" 7 ww € “ 4 -F= eur FF \| ZU \| ung ... TTT re S an A ITLILLEN da TI \| 20 ya ) IH T Sy F Er ar iN ‚> , Ne net? r Da } 4 reger id .-. e“’» p\ % - Am Don ut Bla a je wur Lay Daun .. ”. a ”r a ge RW ZA ‘ Pa a En % 5 I ST ı BL 3) 1 1507 Sogn IR aha FR LK HEN 1) ıy 1 at u 6 TR Ach av Yamı } un. en v an“ UFCHLP RAN \ W “ \ Nm LT Top dan? er Al Nuhuc net S) i . Br Ä Urrrene“ ve - 'rwr. ALER ev "Aal BRD ANON 4 4 ne Wr un v AH, 5 -", ACT “- \erw- han AR N \ N L li MLANBEARAEIEIRE ER Be TER THU EA LI Ar HONG LEGEN U » BEE - - ır : \ Free Wu \| 44 NE rgsse m an un I ana DK, ir el N y Pau eng NG > m. IM 55 - LE >. \| { AM A or I ST vo a er, ge ae We) . DPA Dee (Tr i + 7 IF Mei & ww ea. # “ B’ita vr 1 y JJILLL Ay du U TREE I Vıyn- un = ALLA 444 h n . {1 a) de % R TE brennen N NEN en las ı Du I: .Q 29 sn 05 EMEES: . m... iu: “ BSUZErE R En e ” To ir dr R won s 8 F 2 HAT II zn. 7 TESCH TESCH NEUES ESUEIEEE SEEN TV uuuasvs, ISBUHHGBEITINTUATLILCKEL LET N nn nn ger \|\| WEREIE Ra En ARNTN Fer! wrr „I8g PLA nat wu. j” Bi T v a vr. vr a 0, Sul na, Sw 9, 4 ” Ak, Ah de - . I} 8 Dee Nu, N J TORE BARSERUELUN U SW Tree 3 PE TE] \| ; \| PA \| : \ - \| os Ä IRA UOMELT ;E vreluey BEA ia NR: way: IIY= ed x KIN N Ay 5 A AR your rel 7 77 55 EL} Y T TED TV Frl PFER AL w‘, 5 i u uneran een. v Eng Fi EFT HE ‚ “ & 1 0 nein ann vs I i ia BITTER en pe # L sı GE ır aa LU: PEN au = U 5 Fee. \|) UL ver w Se \| uns surv Tu re a L m .yn ı% =} ’ va L lu X Bu >= EP a : Ran en) ı 22 0 dı, AEG: bi D N Sun, hd AL, TIL MEN - \dtyy N Feuer. er BLITHÄNTN ar et! Nat Le in METEITT TI TI ER EN RR N PRDN \| ? . 3: w ir - x ERST ln ui as Wir: wer 2, vTıy ne” 1 \| Nanenn, N \% ve \| 2 NE 34:2 4 5 =. < > re N LAIEN a IL) DELL ARULN an NSS STE R 1 u u A my F . IN iA ve Re A ne RI uud / Mau % A\| ! v $ STR ey uw. vengglt DE er ee Mer nnt: F; \| \| Sn k K Mm \| A KOOR BAELLN n ARALNNT E M ws LER ; .o vr. > ,„’. L iz Go san nilär ann Werdau er N üme Yav Au = 1087, e ur ySaa6s mine “ Er 4.FI Pit. (hr Eum: un wiare NT IA u \| Yy an “u Rt N vn un FRE Frhr u . Nr } & : PR v PETIS LI Kae \| FI A ERRLERELLIT I aaa 1 Pre En EI HET ANUHTTT TPITETTT Th bu wetten AL, > h NET "N AM. zrtr, f de are ann EN a se BR tur u Etare zu . remere J" Pr + f yrr" D» vw 4% , — ip yı® IT Sim 3 ' ” n) Vor RRLKERHIE LOPEPALEL, gt aaa n a IL LAUT PIEM ae AL PP E iuy. Ah np RN A en di Yan’! un Tel TI v PB hy prrreR wu sit Worms Dre Er \ up. ee 0 Du A Dat MA Division of Molluskg Sectional Library Das Tierreich. Eine Zusammenstellung und Kennzeichnung der rezenten Tierformen. m Begründet von der Deutschen Zoologischen Gesellschaft. a _ Im Auftrage der Königl. Preuß. Akademie der Wissenschaften zu Berlin herausgegeben von Franz Eilhard Schulze. „Havra der.“ „sine systemate chaos.“ 36. Lieferung. Mollusca. Division of Mollusks Pt e OP OÖ d a Sertional Library bearbeitet von Dr. Johan Jacob Tesch in Helder. Mit 108 Abbildungen. rsonian nstr REN y N "Os Erz Hr DD SEP 6 1013.) SEINE X N 1fı; f AıcB\,r Ational F Mu5s— Berlin. Verlag von R. Friedländer und Sohn. Ausgegeben im Juni 1913, Eine Zusammenstellung und Kennzeichnung der rezenten Tierformen. rsonian nstr REN y N "Os Erz Hr SEP 6 1013.) SEINE X N 1fı; f AıcB\,r Ational F Mu5s— R. Friedländer & Sohn, Berlin NW 6, Karlstr. 11. Eine Zusammenstellung und Kennzeichnung der rezenten Tierformen. _ Begründet von der Deutschen Zoologischen Gesellschaft. Im Auftrage der Königl. Preuß. Akademie der Wissenschaften zu Berlin herausgegeben von Franz Eilhard Schulze. —_ Fe Canestrini in Padua (f), Geh.-Rat Prof. C. Chun in Leipzig, Prof. J. Csokor in Wien, P. L. Czerny in Pfarrkirchen, Prof. C, W. v. Dalla Torre in Innsbruck, Prof. F. Doflein in Freiburg B., Dr. G. Enderlein in Stettin, Lehrer H. Fahrenholz in Hannover, Dr. O0. Finsch in Braunschweig, Dr. H, Friese in Schwerin, Prof. A. Giard in Paris, Prof. W. Giesbrecht in Neapel (7), Hofrat Prof. L.v. Graff in Graz, Dr. K. Grünberg in Berlin, Prof. V. Häcker in Halle, Dir. E. Hartert in Tring, Dr. R. Hartmeyer in Berlin, Dr. C. E.Hellmayr in München, Dir. Dr. W.E. Hoyle in Cardiff, Dr. Ihle in Utrecht, Dir. Prof. A. Jacobi in Dresden, Dr. K. Jordan in Tring, Prof. J. J. Kieffer in Bitsch, Dr. W. Kobelt in Schwanheim, Prof. C. A. Kofoid in Berkeley (California), Prof. H.J. Kolbe in Berlin, Dir. Prof. K. Kraepelin in Hamburg, Prof. P. Kramer in Magdeburg (+), Dr. H. Krauss in Tübingen, Prof. W. Kükenthal in Breslau, Dir. Prof. L, Kulezynski in Krakau, Dr. A. Labbe& in Paris, Prof. A. Lameere in Brüssel, Prof. R. Lauterborn in Ludwigshafen a. Rh., Prof. R. v. Lendenfeld in Prag, Prof. H. Lohmann in Hamburg, Geh.-Rat Prof. H. Ludwig in Bonn, Prof. M.Lühe in Königsberg i.Pr., A. D. Michael in London, Prof. W. Michaelsen in Hamburg, Dr. T. Mortensen in Kopenhagen, Geh.-Rat Prof. G. W. Müller in Greifswald, Prof. J. P. Me Murrich in Toronto, Prof. A. Nalepa in Wien, Dr. G. Neumann in Dresden, Prof. L.-G. Neumann in Toulouse, Dr. Nieden in Berlin, Prof. A. Nosek in Caslau, W. R. Ogilvie-Grant in Edinburgh, Dr. Ohaus in Berlin, Prof. A. E. Ortmann in Princeton, Geh. Sanitätsrat Dr. A. Pagenstecher in Wiesbaden, Prof. K. A, Penecke in Graz, Prof. G. Pfeffer in Hamburg, Dir. Dr. R. Piersig in Annaberg (f), Prof. L. Plate in Jena, Prof. A, Reichenow in Berlin, Prof. L. Rhumbler in Hannov.-Münden, H. Riffarth in Berlin, Dir. (Fortsetzung auf Seite 3 des Umschlages.) Bearbeiter: Dr. Graf K. Attems-Petzenstein in Wien, R. $. Bagnall, Oxford, England, Stadtbaurat T. Becker in Liegnitz, Graf H. v. Berlepsch auf Schloß Berlepsch, Prof. Berlese in Florenz, Prof. R. Blanchard in Paris, Prof. F. Blochmann in Tübingen, A. de Bormans in Turin, Dr. K. Börner in Berlin, Prof. E.-L. Bouvier in Paris, Prof. J. Ch. Bradley in Ithaca (N. Y.), Geh.-Rat Prof. K. Brandt in Kiel, Staatsrat Prof. M. Braun in Königsberg i. —_ Fe Seit Linn&s Systema naturae ist die Zahl der bekannten Tierformen so angewachsen, daß eine neue, umfassende Übersicht des Systems, die als Abschluß der bisherigen und als Grundlage künftiger systematischer Forschung dienen kann, ein dringendes Bedürfnis geworden ist. Um diese Aufgabe zu erfüllen, hat die Deutsche Zoologische Ge- sellschaft das vorliegende Werk begründet und dessen wissenschaftliche Leitung Herrn Geh. Reg.-Rat Prof. F. E. Schulze in Berlin anvertraut. Das gewaltige Unternehmen fand die Unterstützung der Königlich Preußischen Akademie der Wissen- schaften, die in Würdigung der Bedeutung des Werkes im Jahre 1902 die Heraus- gabe übernommen hat. Die einheitliche Durchführung des Werkes ist durch eine Reihe wohldurchdachter Die einheitliche Durchführung des Werkes ist durch eine Reihe wohldurchdachter Bestimmungen gesichert. Für die Benennung der Tierformen und ihrer systematischen Kategorien gelten die von dem 5. internationalen Zoologen-Kongreß zu Berlin (1901) angenommenen Regeln. Die Herausgabe findet in Lieferungen statt, die je eine oder mehrere nahe- Die einheitliche Durchführung des Werkes ist durch eine Reihe wohldurchdachter Bestimmungen gesichert. Für die Benennung der Tierformen und ihrer systematischen Kategorien gelten die von dem 5. internationalen Zoologen-Kongreß zu Berlin (1901) angenommenen Regeln. Die Herausgabe findet in Lieferungen statt, die je eine oder mehrere nahe- Die Herausgabe findet in Lieferungen statt, die je eine oder mehrere nahe- stehende Gruppen behandeln, jedoch unabhängig von einer systematischen Folge er- scheinen. Nach Abschluß einer jeden in mehreren Lieferungen behandelten Haupt- abteilung erscheint ein Gesamtregister. Jede Lieferung ist einzeln käuflich. Dem Umfang entsprechend ist der Preis Jede Lieferung ist einzeln käuflich. Dem Umfang entsprechend ist der Preis der Lieferungen verschieden; jedoch wird für die Subskribenten, die sich auf 5 Jahre hinaus für die Abnahme aller in diesem Zeitraum erscheinenden Lieferungen verpflichten, der Berechnung der durchschnittliche Preis von Mark 0,80 für den Druckbogen zugrunde gelegt. Der Einzel-Ladenpreis für jede vollständige Lieferung erhöht sich gegen den Subskriptionspreis um ein Drittel. - Bearbeiter: Dr. Graf K. Attems-Petzenstein in Wien, R. $. Bagnall, Oxford, England, Stadtbaurat T. Becker in Liegnitz, Graf H. v. Berlepsch auf Schloß Berlepsch, Prof. Berlese in Florenz, Prof. R. Blanchard in Paris, Prof. F. Blochmann in Tübingen, A. de Bormans in Turin, Dr. K. Börner in Berlin, Prof. E.-L. Bouvier in Paris, Prof. J. Ch. Bradley in Ithaca (N. Y.), Geh.-Rat Prof. K. Brandt in Kiel, Staatsrat Prof. M. Braun in Königsberg i. Pr., Dr. L. Breitfuss in Katharinenhafen, F. Bryk in lWelylä (Finnland), Prof. O. Bürger in Santiago (Chile), Prof. G. Bearbeiter: Dr. Graf K. Attems-Petzenstein in Wien, R. $. Bagnall, Oxford, England, Stadtbaurat T. Becker in Liegnitz, Graf H. v. Berlepsch auf Schloß Berlepsch, Prof. Berlese in Florenz, Prof. R. Blanchard in Paris, Prof. F. Blochmann in Tübingen, A. de Bormans in Turin, Dr. K. Börner in Berlin, Prof. E.-L. Bouvier in Paris, Prof. J. Ch. Bradley in Ithaca (N. Y.), Geh.-Rat Prof. K. Brandt in Kiel, Staatsrat Prof. M. Braun in Königsberg i. Pr., Dr. L. Breitfuss in Katharinenhafen, F. Bryk in lWelylä (Finnland), Prof. O. Bürger in Santiago (Chile), Prof. G. Canestrini in Padua (f), Geh.-Rat Prof. C. Chun in Leipzig, Prof. J. Csokor in Wien, P. L. Czerny in Pfarrkirchen, Prof. C, W. v. Dalla Torre in Innsbruck, Prof. F. Doflein in Freiburg B., Dr. G. Enderlein in Stettin, Lehrer H. Fahrenholz in Hannover, Dr. O0. Finsch in Braunschweig, Dr. H, Friese in Schwerin, Prof. A. Giard in Paris, Prof. W. Giesbrecht in Neapel (7), Hofrat Prof. L.v. Graff in Graz, Dr. K. Grünberg in Berlin, Prof. V. Häcker in Halle, Dir. E. Hartert in Tring, Dr. R. Hartmeyer in Berlin, Dr. C. E.Hellmayr in München, Dir. Dr. W.E. Hoyle in Cardiff, Dr. Ihle in Utrecht, Dir. Prof. A. Jacobi in Dresden, Dr. K. Jordan in Tring, Prof. J. J. Kieffer in Bitsch, Dr. W. Kobelt in Schwanheim, Prof. C. A. Kofoid in Berkeley (California), Prof. H.J. Kolbe in Berlin, Dir. Prof. K. Kraepelin in Hamburg, Prof. P. Kramer in Magdeburg (+), Dr. H. Krauss in Tübingen, Prof. W. Kükenthal in Breslau, Dir. Prof. L, Kulezynski in Krakau, Dr. A. Labbe& in Paris, Prof. A. Lameere in Brüssel, Prof. R. Lauterborn in Ludwigshafen a. Rh., Prof. R. v. Lendenfeld in Prag, Prof. H. Lohmann in Hamburg, Geh.-Rat Prof. H. Ludwig in Bonn, Prof. M.Lühe in Königsberg i.Pr., A. D. Michael in London, Prof. W. Michaelsen in Hamburg, Dr. T. Mortensen in Kopenhagen, Geh.-Rat Prof. G. W. Müller in Greifswald, Prof. J. P. Me Murrich in Toronto, Prof. A. Nalepa in Wien, Dr. G. Neumann in Dresden, Prof. L.-G. Neumann in Toulouse, Dr. Nieden in Berlin, Prof. A. Nosek in Caslau, W. R. Ogilvie-Grant in Edinburgh, Dr. Ohaus in Berlin, Prof. A. E. Ortmann in Princeton, Geh. Sanitätsrat Dr. A. Pagenstecher in Wiesbaden, Prof. K. A, Penecke in Graz, Prof. G. Pfeffer in Hamburg, Dir. Dr. R. Piersig in Annaberg (f), Prof. L. Plate in Jena, Prof. A, Reichenow in Berlin, Prof. L. Rhumbler in Hannov.-Münden, H. Riffarth in Berlin, Dir. (Fortsetzung auf Seite 3 des Umschlages.) Franz Eilhard Schulze. —_ Fe —_ Fe Pr., Dr. L. Breitfuss in Katharinenhafen, F. Bryk in lWelylä (Finnland), Prof. O. Bürger in Santiago (Chile), Prof. G. Canestrini in Padua (f), Geh.-Rat Prof. C. Chun in Leipzig, Prof. J. Csokor in Wien, P. L. Czerny in Pfarrkirchen, Prof. C, W. v. Dalla Torre in Innsbruck, Prof. F. Doflein in Freiburg B., Dr. G. Enderlein in Stettin, Lehrer H. Fahrenholz in Hannover, Dr. O0. Finsch in Braunschweig, Dr. H, Friese in Schwerin, Prof. A. Giard in Paris, Prof. W. Giesbrecht in Neapel (7), Hofrat Prof. L.v. Graff in Graz, Dr. K. Grünberg in Berlin, Prof. V. Häcker in Halle, Dir. E. Hartert in Tring, Dr. R. Hartmeyer in Berlin, Dr. C. E.Hellmayr in München, Dir. Dr. W.E. Hoyle in Cardiff, Dr. Ihle in Utrecht, Dir. Prof. A. Jacobi in Dresden, Dr. K. Jordan in Tring, Prof. J. J. Kieffer in Bitsch, Dr. W. Kobelt in Schwanheim, Prof. C. A. Kofoid in Berkeley (California), Prof. H.J. Kolbe in Berlin, Dir. Prof. K. Kraepelin in Hamburg, Prof. P. Kramer in Magdeburg (+), Dr. H. Krauss in Tübingen, Prof. W. Kükenthal in Breslau, Dir. Prof. L, Kulezynski in Krakau, Dr. A. Labbe& in Paris, Prof. A. Lameere in Brüssel, Prof. R. Lauterborn in Ludwigshafen a. Rh., Prof. R. v. Lendenfeld in Prag, Prof. H. Lohmann in Hamburg, Geh.-Rat Prof. H. Ludwig in Bonn, Prof. M.Lühe in Königsberg i.Pr., A. D. Michael in London, Prof. W. Michaelsen in Hamburg, Dr. T. Mortensen in Kopenhagen, Geh.-Rat Prof. G. W. Müller in Greifswald, Prof. J. P. Me Murrich in Toronto, Prof. A. Nalepa in Wien, Dr. G. Neumann in Dresden, Prof. L.-G. Neumann in Toulouse, Dr. Nieden in Berlin, Prof. A. Nosek in Caslau, W. R. Ogilvie-Grant in Edinburgh, Dr. Ohaus in Berlin, Prof. A. E. Ortmann in Princeton, Geh. Sanitätsrat Dr. A. Pagenstecher in Wiesbaden, Prof. K. A, Penecke in Graz, Prof. G. Pfeffer in Hamburg, Dir. Dr. R. Piersig in Annaberg (f), Prof. L. Plate in Jena, Prof. A, Reichenow in Berlin, Prof. L. Rhumbler in Hannov.-Münden, H. Riffarth in Berlin, Dir. (Fortsetzung auf Seite 3 des Umschlages.) Das Tierreich. Im Auftrage der Königl. Preuß. Akademie der Wissenschaften zu Berlin herausgegeben von Franz Eilhard Schulze. 36. Lieferung. Mollusca. Pteropoda bearbeitet von Dr. Johan Jacob Tesch in Helder. Mit 108 Abbildungen. — He ——— Das Tierreich. Im Auftrage der Königl. Preuß. Akademie der Wissenschaften zu Berlin herausgegeben von Franz Eilhard Schulze. 36. Lieferung. Mollusca. ” PS 2 ” PS 2 Inhalt. Literatur-Kürzungen . Systematischer Index. . Klasse Pieropoda . Corrigenda . Alphabetisches Hogtiter. Nomencelator generum et nn Literatur-Kürzungen . Systematischer Index. . Klasse Pieropoda . Corrigenda . Alphabetisches Hogtiter. Nomencelator generum et nn Literatur-Kürzungen . Systematischer Index. . Klasse Pieropoda . Corrigenda . Alphabetisches Hogtiter. Nomencelator generum et nn Berücksichtigt ist die Literatur bis zum 1. Juli 1912. Literatur-Kürzungen. Abh. Ver. Hamburg — Abhandlungen aus dem Gebiete der Naturwissenschaften, heraus- gegeben vom naturwissenschaftlichen Verein in Hamburg. Hamburg. 4. Abh. Ver. Hamburg — Abhandlungen aus dem Gebiete der Naturwissenschaften, heraus- gegeben vom naturwissenschaftlichen Verein in Hamburg. Hamburg. 4. Adams, Gen. Moll. — The Genera of recent Mollusea; arranged according to their Organization. By Henry Adams and Arthur Adams. v.l,2; atl. London. 1858 (185358). 8. Agassiz, Nomencl. zoo. — Nomenclator zoologieus, continens Nomina systematica Generum Animalium tam viventium quam fossilium. Auctore L. Agassiz. 1 Vol. & Index universalis. Soloduri. 1842—46, 46. 4. Amer. J. Conch. — American Journal of Conchology. Philadelphia. 8. Amer. J. Sci. — The American Journal of Science and Arts. New Haven (Ne Amer. J. Sci. — The American Journal of Science and Arts. New Haven (New York). 8. Ann. Mus. Paris — Annales du Museum \|national\| d’Histoire naturelle. Paris. 4. Ann. Mus. Paris — Annales du Museum \|national\| d’Histoire naturelle. Paris. 4. Ann. nat. Hist. — The Annals and Magazine of natural History, including Zoology, Ann. nat. Hist. — The Annals and Magazine of natural History, including Zoology, Botany, and Geology. London. 8. Ann. Sei. nat. — Annales des Seiences naturelles. — [Ser. 2—4:] Zoologie ([SEr. 5 & sequ.:] Zoologie et Pal&ontologie). Paris. 8. Annwario Mus. Napoli — Annuario del Museo zoologieo della R. Universitä di Napoli. Annwario Mus. Napoli — Annuario del Museo zoologieo della R. Universitä di Napoli. Napoli. 8. Ann. Wetter. Ges. — Annalen ([Band 4 auch]: Neue Annalen) der Wetterauischen Gesell- schaft für die gesammte Naturkunde. Frankfurt am Main. 4. Band 14: 1809— 1819. Arch. Naturg. — Archiv für Naturgeschichte. Berlin. 8. Arch. Zool. eeper. — Archives de Zoologie experimentale et Arch. Naturg. — Archiv für Naturgeschichte. Berlin. 8. Arch. Zool. eeper. — Archives de Zoologie experimentale et generale. Paris. 8. Arch. Zool. eeper. — Archives de Zoologie experimentale et generale. Paris. 8. Atti Acc. Palermo — Atti della Accademia di Seienze e Lettere di Palermo. Nuova Atti Acc. Palermo — Atti della Accademia di Seienze e Lettere di Palermo. Nuova Serie. (Atti della Reale Accademia di Scienze, Lettere e Belle-Arti di Palermo.) Palermo. 4. Atti Soc. Veneto-Trent. — Atti della Societä Veneto-Trentina di Scienze naturali, Atti Soc. Veneto-Trent. — Atti della Societä Veneto-Trentina di Scienze naturali, residente in Padova. Padova. 8. Bih. Svenska Ak. — Bihang till Kongl. Svenska Vetenskaps-Akademiens Handlingar. —- Afdelning 4, Zoologi. Stockholm. 8. Blainville, Man. Actin. Literatur-Kürzungen. Bull. U. 8. Mus: — Bulletin of the United States national Museum. Washington. 8 Bull. U. 8. Mus: — Bulletin of the United States national Museum. Washington. 8. Busch, Beob. wirbell. Seeth. — Beobachtungen über Anatomie und Entwickelung einiger Bull. U. 8. Mus: — Bulletin of the United States national Museum. Washington. 8. Busch, Beob. wirbell. Seeth. — Beobachtungen über Anatomie und Entwickelung einiger Bull. U. 8. Mus: — Bulletin of the United States national Museum. Washington. 8. Busch, Beob. wirbell. Seeth. — Beobachtungen über Anatomie und Entwickelung einiger wirbellosen Seethiere von Wilhelm Busch. Berlin. 1851. 4. [Calonne] Mus. Calonn. — Museum Calonnianum. Speeification of the various Artieles [Calonne] Mus. Calonn. — Museum Calonnianum. Speeification of the various Artieles which eompose the magnificent Museum of Natural History collected by de Calonne in France, and lately his Property: eonsisting of an Assemblage of the most beautiful and rare Subjeets in Entomology, Conchology, Ornithology, Mineralogy ete. London. 1797. 8. Chenu, Man. Conchyl. — Manuel de Conchyliologie et de Palöontologie eonchylio- logique par J.Ö.Chenu. v.1,2. Paris. 1859 (1859, 60), 62 (1861, 62). 8. Chiaje, Deser. An. Sicilia — Animali senza Vertebre del Regno di Napoli. Deserizione Chiaje, Deser. An. Sicilia — Animali senza Vertebre del Regno di Napoli. Deserizione e Notomia degli Animali invertebrati della Sieilia eiteriore osservati vivi negli Anni 1822—30 da S. delle Chiaje,. Tom. 1—8 (Tom. 6 & 7 = Atl.). Napoli. 1841, 44. 4. Se, Mem. Stor. Not. — Memorie sulla Storia e Notomia degli Animali senza Vertebre del Regno di Napoli di (sceritte da) Stefano delle Chiaje (Chiaie). Vol. 1-4; Atl. Napoli. 1823 [1824], 25, 28, 29; 2. 4&2. Costa, Fauna Reg. Napoli — Fauna del Repno di Napoli ossia Enumerazione di tutti gli Animali che abitano le diverse Regioni di questo Regno e le Acque che le bagnano ... di Oronzio-Gabriele Costa. [Continuato da Achille Costa.] — Animali molli, Pteropodi & Eteropodi. 1873. Napoli. 4. Costa, Microdoride — Microdoride mediterranea o Deserizione de’ poco ben cono- seiuti od affatto ignoti viventi minuti e mieroscopiei del Miditerraneo pel O0. G. Costa. Napoli. 1861. OR. Ac. Sci. — Comptes rendus hebdomadaires des Söances de l’Acad&mie des Sciences. Paris. 4. Cuvier, Regne an. — Le Rögne animal distribu& d’apres son Organisation, pour servir Cuvier, Regne an. — Le Rögne animal distribu& d’apres son Organisation, pour servir de Base & l’Histoire naturelle des Anımaux et d’Introduction & l’Anatomie compar&ee. 1) Diese Ausgabe ist bisher als „ed. 3% aufgeführt gewesen. Es gibt aber eine Ausgabe Bruxelles 1836 die mit „3 Edition“ auf dem Titel bezeichnet ist, so dab die zuerst genannte Ausgabe von jetzt ab als „ed. 4“ zitiert werden wird. Literatur-Kürzungen. — Manuel d’Actinologie ou de Zoophytologie, contenant ... Blainville, Man. Actin. — Manuel d’Actinologie ou de Zoophytologie, contenant ... Avec un Atlas... Par H.M.D. de Blainville. Paris. 1834 [& 1837]. 8. Blainville, Man. Malac. — Manuel de Malacologie et de Conchyliologie; contenant: Blainville, Man. Malac. — Manuel de Malacologie et de Conchyliologie; contenant: ... Par H. M. Ducrotay de Blainville. Avec Planches. Paris. 1825, 27. 8. Bory, Voy. Iles Afr. — Voyage dans les quatre prineipales Iles des Mers d’Afrique, Bory, Voy. Iles Afr. — Voyage dans les quatre prineipales Iles des Mers d’Afrique, fait par Ordre du Gouvernement, pendant les Annees neuf et dix de la Repu- blique (1801 et 1802), avee l’Histoire de la Traversöe du Capitaine Baudin jJasqu’au Port-Louis de I’Ile Maurice. Par J. B. G. M. Bory de St.- Vincent. Tome 1—3. Planches. Paris. 1804. 8, Bose, Hist. Cogu. — Histoire naturelle des Coquilles, eontenant leur Description, les Bose, Hist. Cogu. — Histoire naturelle des Coquilles, eontenant leur Description, les Moeurs des Animaux qui les habitent et leurs Usages. Par L. A. G. Bose. Tome 1—5. Paris. X [1802]. 6. Bowdich, Elem. Conch. — Elements of Conchology, ineluding the fossil genera and the animals. T. Edw. Bowdich. Vol.1,2. Paris. 1820, 1822. 8 Bronn Kl. Ordn. — Die Klassen und Ordnungen des Thier-Reichs wissenschaftlich dargestellt in Wort und Bild. Von H.G.Bronn. Leipzig und Heidelberg 8. Literatur-Kürzungen vII Browne, Hist. Jamaica — The eivil and natural History of Jamaica. In three Parts, Containing, I. An aceurate Description of that Island, ... II. A History of the natural Productions, ... III. An Account of the ns of Climates in general, ... In three Eeeriatihne The Whole illustrated with = Copper- Plates: in Een the most ceurious Productions are represented ... By Patrick Browne. London. 1756. 2. Bull. Ac. Belgique — Bulletins de ’Acadömie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique. Bruxelles. 8. Bull. sei. Nord — Bulletin scientifique, historique et littöraire du De&partement du Nord et des Pays voisins ([Tom. 19:] Bulletin scientifigque du Nord de la France et de la Belgique). Lille (Paris). 8. France et de la Belgique). Lille (Paris). 8. Bull. Soc. philom. — Bulletin de la Soeiete philomathique France et de la Belgique). Lille (Paris). 8. Bull. Soc. philom. — Bulletin de la Soeiete philomathique de Paris. Paris. 4. Bull. U. 8. Mus: — Bulletin of the United States national Museum. Washington. Literatur-Kürzungen. meth., Vers — Eneyelopedie methodique, ou par Ordre de Matieres, par une Soeiete de Gens de Lettres, de Savans et d’Artistes. — Histoire naturelle des Vers. Tom. 1—3. Paris. 1792 [1789, 92], 1830 [1830, 32 IX], 18324. 4. Engelmann, Bibl. hist.-nat. — Bibliotheca historico-naturalis. Verzeichnis der Bücher über Naturgeschichte welche in Deutschland, Scandinavien, Holland, England, Frankreich, Italien und Spanien in den Jahren 1700—1846 erschienen sind, Von Wilhelm Engelmann. Leipzig. 1846. 8. Ergeb. Plankton-Exp. — Ergebnisse der in dem Atlantischen Ozean von Mitte Juli bis Anfang November 1889 ausgeführten Plankton-Expedition der Humboldt- Stiftung. Auf Grund von gemeinschaftlichen Untersuchungen einer Reihe von Fach-Forschern herausgegeben von Vietor Hensen. Kiel und Leipzig. 4. Anfang November 1889 ausgeführten Plankton-Expedition der Humboldt- Stiftung. Auf Grund von gemeinschaftlichen Untersuchungen einer Reihe von Fach-Forschern herausgegeben von Vietor Hensen. Kiel und Leipzig. 4. Ergeb. Schwed. Südp.-Exp. — Wissenschaftliche Ergebnisse der Schwedischen Südpolar- Expedition 1901—1903 unter Leitung von Otto Nordenskjöld. Stockholm. 4. Ergeb. Schwed. Südp.-Exp. — Wissenschaftliche Ergebnisse der Schwedischen Südpolar- Expedition 1901—1903 unter Leitung von Otto Nordenskjöld. Stockholm. 4. Ergeb. Tiefsee-Exp. — Wissenschaftliche Ergebnisse der Deutschen Tiefsee-Expedition auf dem Dampfer „Valdivia‘‘ 1898—1899. Im Auftrage des Reichsamtes des Innern herausgegeben von Carl Chun. Jena. 4, Eschscholtz, Zool. Atlas — Zoologischer Atlas, enthaltend Abbildungen und Be- schreibungen neuer Thierarten, während des Flotteapitäns von Kotzebue zweiter Reise um die Welt, auf der Russisch-Kaiserlichen Kriegsschlupp Predpriaeti& in den Jahren 1823—1826 beobachtet von Friedr. Eschscholtz \|5. Heft, herausgegeben von Martin Heinrich Rathke]. Heft 1—5. Berlin. 1829, 29,.29, 31, 33... 2. Exp. Travailleur & Talisman — Expeditions seientifiques du Travailleur et du Talisman pendant les Annees 1880, 1881, 1832, 1883. Ouvrage publi& sous les Auspices du Ministere de l’Instruction publique sous la Direction de A. Milne- Edwards. Paris. 4. — — — — Moll. test. Mollusques testaces par Arnould Locard. Fabricius, Fauna Groenl. — Fauna Groenlandica, systematice sistens Animalia Groen- landiae occidentalis hactenus indagata, ... seecundum proprias Observationes ÖOthonis Fabrieii. Hafniae et Lipsiae. 1780. 8. Fauna arcetica — Fauna aretica. Eine Zusammenstellung der arktischen Tierformen Fauna arcetica — Fauna aretica. Eine Zusammenstellung der arktischen Tierformen mit besonderer Berücksichtigung des Spitzbergen-Gebietes auf Grund der Ergebnisse der Deutschen Expedition in das Nördliche Eismeer im Jahre 1898. Unter Mitwirkung zahlreicher Fachgenossen herausgegeben von Fritz Römer und Fritz Schaudinn. Nach ihrem Tode fortgesetzt von August Brauer. 4. Fischer, Man. Conchyl. Literatur-Kürzungen. Par [Georges] Cuvier. Tom. 1—4. Paris. 1817. 8. Cwvier, Regne an., ed. 4!) — Le Regne animal distribue d’apres son Organisation, pour Cwvier, Regne an., ed. 4!) — Le Regne animal distribue d’apres son Organisation, pour servir ae Base ä l’Histoire naturelle des Animaux, et d’Introduction & l’Ana- tomie comparee, par Georges Cuvier. [4.] Hdıkıan accompagn&e de Planches gravöes, ... par une Röunion de Diseiples de ÖOuvier. Paris. 4. Danske Selsk. Skr. — Det Kongelige Danske Videnskabernes Selskabs Skrifter. — [Raekke Danske Selsk. Skr. — Det Kongelige Danske Videnskabernes Selskabs Skrifter. — [Raekke 5 & sequ.:] Naturvidenskabelig og mathematisk Afdeling. Kjöbenhavn. 4. Dict. class. Hist. nat. — Dietionnaire elassique d’Histoire naturelle, par Audouin, Danske Selsk. Skr. — Det Kongelige Danske Videnskabernes Selskabs Skrifter. — [Raekke 5 & sequ.:] Naturvidenskabelig og mathematisk Afdeling. Kjöbenhavn. 4. Dict. class. Hist. nat. — Dietionnaire elassique d’Histoire naturelle, par Audouin, et Bory de Saint-Vincent. Tome 1—17 & atl, Paris. 1822—31. 8. 5 & sequ.:] Naturvidenskabelig og mathematisk Afdeling. Kjöbenhavn. 4. Dict. class. Hist. nat. — Dietionnaire elassique d’Histoire naturelle, par Audouin, et Bory de Saint-Vincent. Tome 1—17 & atl, Paris. 1822—31. 8. Dict. class. Hist. nat. — Dietionnaire elassique d’Histoire naturelle, par Audouin, et Bory de Saint-Vincent. Tome 1—17 & atl, Paris. 1822—31. 8. Dict. Sci. nat. — Dietionnaire des Sciences naturelles, .... Par plusieurs Professeurs du Jardin du Roi, et des prineipales Ecoles de Paris. [Red. par F. Cuvier.] Tom. 1—60; Planches. Strasbourg et Paris (Paris). 1816—30. 8. D. Südp.-Exp. — Deutsche Südpolar-Expedition 1901—1903. Im Auftrage des Reichs- D. Südp.-Exp. — Deutsche Südpolar-Expedition 1901—1903. Im Auftrage des Reichs- amtes des Innern herausgegeben von Erich von Drygalski. Zoologie, 4. 1) Diese Ausgabe ist bisher als „ed. 3% aufgeführt gewesen. Es gibt aber eine Ausgabe Bruxelles 1836 die mit „3 Edition“ auf dem Titel bezeichnet ist, so dab die zuerst genannte Ausgabe von jetzt ab als „ed. 4“ zitiert werden wird. vl Literatur-Kürzungen Dunker, Index Moll. Gwine« — Index Molluscorum, quae in itinere ad Guineam in- feriorem collegit Georgius Tams. Auctore Guilielmo Dunker. Accedunt novarum speeierum diagnoses, Cirripedia nonnulla et X tabulae iconum. Cassellis Cattorum. 1853. 4. Ene. Brit., ed. 9 — The Eneyclopaedia Britannica, a Dietionary of Arts, Sciences, and general Literature. 9. Edition. [Edited by T. S. Baynes.] Vol. 1—24. Edinburgh. 1875—88. 4. Enc. !) Cfr.: ©. Davies Sherborn & B.B.Woodward in: P. zool. Soc. London, 1893 p.583 & 1899 p. 595. Literatur-Kürzungen. — Paul Fischer, Manuel de Conchyliologie et de Pal&ontologie conchyliologique ou Histoire naturelle des Mollusques vivants et fossiles suivi d’un Appendice sur les Brachiopodes par D. P. Oehlert. Paris. 1887 (1880—87). 8. Fleming, Phil. Zool. — The Philosophy ot Zoology; or a general View of the Structure, Funetions, and Classifieation of Animals. By John Fleming. Vol.1,2. Edinburgh. 1822. 8. Forbes & Hanley, Brit. Moll. — A History of British Mollusca and their Shells b Edward Forbes and Sylvanus Hanley. Vol.1. London. 1853. 8. Forskäl, Deser. An. — Deseriptiones Animalium Avium, Amphibiorum, Piseium, In Forbes & Hanley, Brit. Moll. — A History of British Mollusca and their Shells by Edward Forbes and Sylvanus Hanley. Vol.1. London. 1853. 8. Forskäl, Deser. An. — Deseriptiones Animalium Avium, Amphibiorum, Piseium, In- sectorum, Vermium; quae in Itinere orientali observavit Petrus Forskäl. Post Mortem Auctoris edidit Carsten Niebuhr. Adjuneta est Materia medica Forbes & Hanley, Brit. Moll. — A History of British Mollusca and their Shells by Edward Forbes and Sylvanus Hanley. Vol.1. London. 1853. 8. Forskäl, Deser. An. — Deseriptiones Animalium Avium, Amphibiorum, Piseium, In- sectorum, Vermium; quae in Itinere orientali observavit Petrus Forskäl. Post Mortem Auctoris edidit Carsten Niebuhr. Adjuneta est Materia medica Kahirina atque Tabula Maris Rubri geographieca. Hauniae. 1775. 4. !) Cfr.: ©. Davies Sherborn & B.B.Woodward in: P. zool. Soc. London, 1893 Forskäl, Deser. An. — Deseriptiones Animalium Avium, Amphibiorum, Piseium, In- sectorum, Vermium; quae in Itinere orientali observavit Petrus Forskäl. Post Mortem Auctoris edidit Carsten Niebuhr. Adjuneta est Materia medica Kahirina atque Tabula Maris Rubri geographieca. Hauniae. 1775. 4. !) Cfr.: ©. Davies Sherborn & B.B.Woodward in: P. zool. Soc. London, 1893 p.583 & 1899 p. 595. Literatur-Kürzungen IX Forskäl, Icon. Ber. nat. — Icones Rerum naturalium, quas in Itinere orientali depingi euravit Petrus Forskäl. Post Mortem Auctoris ad Regis Mandatum Aeri _ ineisas edidit Carsten Niebuhr. Hauniae. 1776. 4. Gegenbaur, Unters. Pter. & Heterop. — Untersuchungen über Pteropoden und Heteropoden. Ein Beitrag zur Anatomie und Entwicklungsgeschichte dieser Thiere von Carl Gegenbaur. Leipzig. 1855. 4. Gioeni, Deser. Test. — Descrizione di una nuova Famiglia, e di un nuovo Genere di Testacei trovati nel Littorale di Catania da Giuseppe Gioeni. Con qualche Össervazione sopra una Spezie di Östriche, per servire alla Conchiologia generale. Napoli. 1783. 8. Gmelin, Syst. Nat. — Caroli a Linn& Systema Naturae per Regna tria Naturae, seeundumı Classes, Ordines, Genera, Species, cum Characteribus, Differentiis, Synonymis, Loeis. Literatur-Kürzungen. Editio XIII, aucta, reformata. Cura Jo. Frid. Gmelin. — Tomus I. Pars 1—7. Lipsiae. 1788 [Pars 6 & 7: 1791]. 8. Gray, Cat. Moll. Brit. Mus. — Catalogue of the Mollusea in the Collection of the British Museum; by J. E. Gray. 12. k Gray, Syn. Brit. Mus. — \|[J. E. Gray] Synopsis of the Contents of the British Museum. Ed. 42, London. 1840. 16. (Ed. 44. London. 1842. 8.) Herrmannsen, Ind. Gen. Malac. — Indieis Generum Malacozoorum Primordia. Nomina Subgenerum, Generum, Familiarum, Tribuum, Ordinum, Ölassium; adjectis Auctoribus, T’emporibus, Loeis systematieis atque literariis, Etymis, Synonymis. Praetermittuntur Cirripedia, Tunicata et Rhizopoda. Conseripsit A. N. Herr- mannsen. Vol. 1. 2. Cassellis. 1846, 47—49. 8. Hübner, Verz. Schmett. — Verzeichniß bekannter Schmettlinge verfaßt von Jacob Hübner. Augsburg. 1816. 8. Isis — Isis \|Eneyklopädische Zeitschrift, vorzüglich für Naturgeschichte, vergleichende Anatomie und Physiologie\| von [Lorenz von] Oken. Leipzig. 4. J. Asiat. Soc. Bengal — Journal of the Asiatie Society of Bengal. — [Vol. 34 & sequ.:] Part II, Natural History ete. Calcutta. 8. J. Asiat. Soc. Bengal — Journal of the Asiatie Society of Bengal. — [Vol. 34 & sequ.:] Part II, Natural History ete. Calcutta. 8. J. Conchyl. — Journal de Conchyliologie. Paris. 8. Part II, Natural History ete. Calcutta. 8. J. Conchyl. — Journal de Conchyliologie. Paris. 8. Jeffreys, Brit. Conch. — John Gwyn Jeffreys, British Conchology; or, an Account of the Mollusea which now inhabit the British Isles and the surrounding Seas. Vol. 15. London. 1862, 64, 65, 67, 69. 8. J. Hist. nat. — Choix de Mömoires sur divers Objets d’Histoire naturelle, par Lamarck, Bruguiere, Olivier, Hauy et Pelletier. Formant les Collections du Journal d’Histoire naturelle. [2. Titel:] Journal d’Histoire naturelle, redige par Lamarck, Bruguiere, Olivier, Hauy et Pelletier.... Paris. 4. J. Phys. Chim. Hist. nat. — Journal de Physique, de Chimie, d’Histoire naturelle let des Arts. Paris. 4. Krauss, Südafr. Moll. — Die Südafrikanischen Mollusken. Ein Beitrag zur Kenntniss Krauss, Südafr. Moll. — Die Südafrikanischen Mollusken. Ein Beitrag zur Kenntniss der Mollusken des Kap- und Natallandes und zur geographischen Verbreitung derselben, mit Beschreibung und Abbildung der neuen Arten von Ferdinand Krauss. Stuttgart. 1848. 4. Krohn, ‚Beitr. Pter. & Heterop. — Beiträge zur Entwicklungsgeschiehte der Pteropoden und Heteropoden von August Krohn. Leipzig. 1860. 4. Lamarck, Hist. An. s. Vert. — Histoire naturelle des Animaux sans Vertebres, presentant les Caracteres göneraux et partieuliers de ces Animaux, ...; pr&cedöe d’une Introduction .... Literatur-Kürzungen. — M&moires de l’Acadömie Imperiale des Sciences de St.-Peters- bourg. — [Ser. 6:] Sciences mathematiques, physiques et naturelles. St.-Pöters- bourg. 4. Mem. Werner. Soc. — Memoirs ofthe Wernerian natural History Soeiety. Edinburgh. 8. Mem. Werner. Soc. — Memoirs ofthe Wernerian natural History Soeiety. Edinburgh. 8. Menke, Syn. Moll. — Synopsis methodiea Molluscorum Generum omnium et Specierum earum, quae in Museo Menkeano adservantur; cum Synonymia critica et novarum Specierum Diagnosibus, Auetore Carolo Theodoro Menke. Pyrmonti. 1828. 8. (Ed.2. Pyrmonti. 1830. 8.) Monber. Ak. Berlin. — Monatsberichte der Königlich Preussischen Akademie der Wissen- schaften zu Berlin. Berlin, 8. Montfort, Conchyl. syst. — Conchyliologie systematique, et Olassification mö&thodique des Coquilles; offrant leurs Figures, .... Coquilles univalves, eloisonn&es (Tome 2: Coquilles univalves, non cloisonnees). Par Denys de Montfort. Tome 1,2. Paris. 1808, 10. 8. Mörch, Cat. Conch. Kierulf — Catalogus Conchyliorum quae reliquit ©. P. Kierulf, nune publica Auctione X Decembris 1850 Hafniae dividendä. Seripsit O. A. L. Mörch. Hafniae. 1850. 8. Müller, Zool. Dan. Prodr. — Zoologiae Danieae Prodromus, seu Animalium Daniae et Norvegiae indigenarum Characteres, Nomina, et Synonyma imprimis popu- larium. Auctore Othone Friderico Müller. Havniae. 1776. 8. N. Acta Ac. Leop. — Nova Acta Academiae Caesarese Leopoldino-Carolinae Germanicae N. Acta Ac. Leop. — Nova Acta Academiae Caesarese Leopoldino-Carolinae Germanicae Naturae Öuriosorum. Verhandlungen der Kaiserlichen Leopoldinisch-Caro- linischen Akademie der Naturforscher. Norimbergae (Erlangen ete.). 4. N. Alpina — Neue Alpina. Eine Schrift der Schweizerischen Naturgeschichte, Alpen- N. Alpina — Neue Alpina. Eine Schrift der Schweizerischen Naturgeschichte, Alpen- und Landwirthschaft gewidmet. Winterthur. 8. Naturh. Tidsskr. — Naturhistorisk Tidsskrift. Kjebenhavn. 8. Naturh. Tidsskr. — Naturhistorisk Tidsskrift. Kjebenhavn. 8. N. Bull. Soc. philom. — Nouveau Bulletin des Seiences, par la Soeiet& philomatique de Paris. Paris. 4. - Tidsskr. — Naturhistorisk Tidsskrift. Kjebenhavn. 8. Soc. philom. — Nouveau Bulletin des Seiences, par la Soeiet& philomatique de Paris. Paris. 4. - eyden Mus. — Notes from the Leyden Museum. Leyden. 8. Fürh.— Öfversigt af Kongl.Vetenskaps- Akademiens Förhandlingar. S Notes Leyden Mus. — Notes from the Leyden Museum. Leyden. 8. Öfv. Ak. Fürh.— Öfversigt af Kongl.Vetenskaps- Akademiens Förhandlingar. Stockholm. 8, OÖken, Lehrb. Naturg. — [Lorenz von] Okens Lehrbuch der Naturgeschichte. — 3. Theil. OÖken, Lehrb. Naturg. — [Lorenz von] Okens Lehrbuch der Naturgeschichte. — 3. Theil. Zoologie. Abtheilung 1, 2. Mit 1 Atlas. Leipzig und Jena (Leipzig). 1815, 16. 8&4. Orbigny, Pal. franc., Terr. Or&t. — Palöontologie francaise. Literatur-Kürzungen. Par [Jean Baptiste] de Lamarck. Tom. 1—5, 61 & 1, 7. Paris. 1815, 16, 16, 17, 18,19, 22,22, 8. Lamarck, Syst. An. s. Vert. -— Systöme des Animaux sans Vertebres, ou Tableau general des Classes, des Ordres et des Genres de ces Animaux. Par J. B. Lamarck. Paris. 1801. 8. Lamouroux, Escpos. Polyp. — Exposition möthodique des Genres de l’Ordre des Poly- piers, avee leur Description ... Par J. Lamouroux. Paris. 1821. 4. atreille, Fam. Rögne an. — Famiiles naturelles du Regne animal, exposdes suceincte- Lamouroux, Escpos. Polyp. — Exposition möthodique des Genres de l’Ordre des Poly- piers, avee leur Description ... Par J. Lamouroux. Paris. 1821. 4. Latreille, Fam. Rögne an. — Famiiles naturelles du Regne animal, exposdes suceincte- ment et dans un Ordre analytique, avec l’Indication de leurs Genres. Par [Pierre Andr&] Latreille. Paris. 1825. 8. Latreille, Fam. Rögne an. — Famiiles naturelles du Regne animal, exposdes suceincte- ment et dans un Ordre analytique, avec l’Indication de leurs Genres. Par [Pierre Andr&] Latreille. Paris. 1825. 8. X Literatur-Kürzungen Linne, Syst. Nat., ed. 10 — Caroli Linnaei Systema Naturae per Regna tria Naturae, seecundum Classes, Ordines, Genera, Species, cum Üharacteribus, Differentüs, Synonymis, Loeis. Editio X, reformata. — Tomus I. Holmiae. 1758. 8. Linne, Syst. Nat., ed. 12 — Caroli a Linn& Systema Naturae per Regna tria Naturae, Linne, Syst. Nat., ed. 12 — Caroli a Linn& Systema Naturae per Regna tria Naturae, secundum Classes, Ordines, Genera, Species, eum Üharacteribus, Differentiis, Synonymis, Loeis. Editio XII, reformata. — Tomus I. Pars 1, 2. Holmiae, 1766, 67. 8. Locard, Prodr. Malac. france. — Prodrome de Malacologie francaise. — Catalogue general des Mollusques vivants de France par Arnould Locard. Lyon. Paris. 1882. 8. London med. Repos. — The London medical Repository, monthly Journal, and Review, Edited by David Uwins, Shirley Palmer and Samuel Frederick Gray. London. 8. Mag. Zool. — Magasin de Zoologie \|[Ser. 2:], d’Anatomie comparöe et de Palöontologie). Paris. 8. Martens, Spitzberg. Reis. — Friederich Martens vom Hamburg Spitzbergische oder Groenlandische Reise Beschreibung, gethan im Jahre 1671. Aus eigner Er- fahrunge beschrieben / die dazu erforderte Figuren nach dem Leben selbst abgerissen / (so hierbey in Kupfer zu sehen) und jetzo durch den Druck mit- getheilet. Hamburg. 1675. 4. Mem. Ac. Belgique — Mömoires de l’Academie Royale des Sciences, des Lettres et des Beaux-Arts de Belgique. Bruxelles. 4. Mö£m. Ac. St.- Petersb. Literatur-Kürzungen. Description zoologique et geologique de tous les Animaux mollusques et rayonneös fossiles de France, par Aleide d’Orbigny. Avee les Figures de toutes les Espöces, lithographi6es d’apres Nature par J. Delarue. Terrains UOretacös. Paris. 8. Literatur-Kürzungen xI Orbigny, Voy. Amer. merid. — Voyage dans l’Amerique möridionale, exöcut& pendant les Annees 1826—33 par Aleide d’Orbigny. — v5nr: Mollusques, par Aleide d’Orbigny. Cum atl. Paris, Strasbourg. 1835—43 [1835 —46]; 46. 4, P. Ac. Philad. — Proceedings of the Academy of natural Seiences of Philadelphia. Philadelphia. 8. Pallas, Spice. z00l. — Spieilegia zoologica quibus novae jimprimis\|) et obseurae Ani- malium Species Iconibus, Deseriptionibus atque Commentariis illustrantur, Cura P.S. Pallas. Faseieulus 1—14. Berolini. 1767—80. 4. P. Boston Soc. — Proceedings of the Boston Society of natural History. Cambridge (Boston), 8. i Peron, Voy. terres Austr. — Voyage de decouvertes aux terres Australes, ex&ceut& pa ordre de S. M. l’empereur Napoleon, roi d’Italie, sur les corvettes le G&o- graphe, le Naturaliste et la go@lette la Casuarina pendant les anndes 1800— 1804; publie par decret de l’empereur sous le ministere de S. E. M. de Champagny et rödig& par M.F. P&ron, naturaliste de l’expedition. 2. vol. in 4° et deux Atlas pet. in fol. ensemble de 37 cartes et pl. Paris. 1807 et 1810. Philippi, Handb. Conch: — Handbuch der Conchyliologie und Malaecozoologie von R, A.Philippi. Halle 1853. 8. Philippi, Moll. Sieil. — Fauna Molluseorum viventium et in Tellure tertiaria- fossilium Regni utriusque Sieiliae, Enumeratio Molluscorum Sieiliae cum viventium tum in Tellure tertiaria fossilium, quae in Itinere suo observavit Auctor Rudolphus Amandus Philippi. ®.1,2. Berolini, Halis Saxonum. 1836, 44. 4. Phipps, Voy. North Pole — A Voyage towards the North Pole undertaken by his Phipps, Voy. North Pole — A Voyage towards the North Pole undertaken by his Majesty’s Command 1773 by Constantine John Phipps. London. 1774. 4. Phipps, Voy. North Pole — A Voyage towards the North Pole undertaken by his Majesty’s Command 1773 by Constantine John Phipps. London. 1774. 4. Poli, Test. Sieil. — Testacea utriusque Sieiliae eorumque Historia et Anatome Tabulis aeneis illustrata a Josepho Xaverio Poli. Tomus 1,2. Parmae. 1791, 95. 2. Poli, Test. Sieil. — Testacea utriusque Sieiliae eorumque Historia et Anatome Tabulis aeneis illustrata a Josepho Xaverio Poli. Tomus 1,2. Parmae. 1791, 95. 2. P. R. Soc. London — Proceedings of the Royal Society of London. London. 8. P. zool. Soc. London — Proceedings of the zoological Society of London. London. Literatur-Kürzungen. — Report on the Collections of natural History made in tbe antaretic Regions during the Voyage of the „Southern Oross“. London. 1902. Result. Voy. Belgica — R£sultats du Voyage du S. Y. Belgica en 1897—1898—1899 sous le Öommandement de A. de Gerlache de Gomery. Rapports scientifiques publies aux Frais du Gouvernement Belge, sous la Direction de la Commission de la Belgiea. Zoologie. Anvers. 4. Retzius, Diss. Test. Gen. — Dissertatio historico-naturalis sistens nova Testaceorum Genera. Quam ... Praeside D. M. Andr. J. Retzio ad publicum Examen defert Laurentius Münter Philipsson ... ad Diem X. Decembris 1788. Lundae. 8. Rev. zool. — Revue zoologique, par la Societ& Cuvierienne. Paris. 8. Ric. Lab. Anat. Roma — Ricerche fatte nel Laboratorio di Anatomia normale della Reale Universitä di Roma ed in altri Laboratori biologie. Roma. 8. Rink, Grönland — Grönland geographisk og statistisk beskrevet af H. Rink. Vol.1,2. Kjöbenhavn. 1852, 55, 57. 8. Sars, Moll. arct. Norvegiae — Bidrag til Kundskaben om Norges Arktiske Fauna. I, Mollusca Regionis areticae Norvegiae. Üversigt over de i Norges arktiske Region Forekommende Bleddyr af G.O.Sars. Christiania. 8. Universitets- programm for förste Halvaar 1878. SB. Ak. Berlin — Sitzungsberichte der Königlich Preussischen Akademie der Wissen- schaften zu Berlin. Berlin. 8. Sci. Inwest. Fish. Ireland — Scientific Investigations. Fisheries Branch. Department of Agrieulture and Technical Instruction for Ireland. Dublin. 8. Siboga-Exp. — R£sultats des Explorations zoologiques, botaniques, oceanographiques et geologiques entreprises aux Indes n&erlandaises orientales en 1899—1900 & Bord du Siboga sous le Commandement de G. F. Tydeman. Publies par Max Weber. 4. Skr. Naturh.-Selsk. — Skrivter af Naturhistorie-Selskabet. Kiobenhavn. 8. Smithson. Collect. — Smithsonian miscellaneous Collections. Washington. 8. Sowerby, Thesaur. Conch. — Thesaurus Conchyliorum, or Monographs of Genera of Shells. Edited by G. B. Sowerby, Jun. Vol. 1-5. London. 1847—87 (1842—87). 8. Tabl. enc. meth. — Tableau encyclopedique et m&thodique des trois Regnes de la Nature. — Vers, Coquilles, Mollusques et Polypiers. Tome 1—3. Paris. 1827 [1791, 97, 98, 1816. 241)]. 4. Tijdschr. Nederl. dierk. Ver. — Tijdschrift der Nederlandsche dierkundige Vereeniging. s’Gravenhage (Rotterdam) (Leiden). 8. Tr. Connect. Ac. — Transactions of the Connectieut Academy of Arts and Sciences. New Haven. 8. Tr. Linn. Soc. London — The Transactions of the Linnean Society of London. — [Ser. 2:] Zoology. London. 4. Tr. N. Zealand Inst. — Transactions and Proceedings of the New Zealand Institute. Wellington (Wellington, London). 8. Troschel, Gebiss Schneck. Literatur-Kürzungen. 8. P. zool. Soc. London — Proceedings of the zoological Society of London. London. 8. Quart. J. Sci. — The quarterly Journal of Science, Literature, and Art. The Royal Institution of Great Britain. London. 8. Quart. J. Sci. — The quarterly Journal of Science, Literature, and Art. The Royal Institution of Great Britain. London. 8. Rafinesque, Anal. Nat. — Analyse de la Nature ou Tableau de l’Univers et des Corps organisös. Par C©.S. Rafinesque. Palerme. 1815. 8. Rafinesque, Specchio Sei. — Rafinesque, Speechio delle Scienze o Giornale Enei- Rafinesque, Specchio Sei. — Rafinesque, Speechio delle Scienze o Giornale Enei- elopedieo di Sieilia, »2 nr. 11. Palermo 1. XI 1814. N (Citiert nach: Wm. G. Binney & George W. Tryon, The complete writings of Constantine Smaltz Rafinesque on recent & fossil Conchology. New York. 1864.) Ramon, Histoire Cuba — Histoire physique, politique et naturelle de l’Ile de Cuba \| par Ramon de la Sagra. — Mollusques. Par Aleide d’Orbigny. v.1,2; atl. Paris. 1853 [1841, 42], 53 [1842]. 8&2. naturelle des Mollusques Pteropodes. Rang & Souleyet, Hist. nat. Pter. — Histoire naturelle des Mollusques Pteropodes. Monographie comprenant la Description de toutes les Especes de ce Groupe de Mollusques par P. ©. A.L. Rang & Souleyet. Paris. 1852. 2. ds of the Australian Museum. Sydney. 8. Rec. Austral. Mus. — Records of the Australian Museum. Sydney. 8. eve, Conch. icon, — Conchologia ieonica: or, [llustrations of the Shells of Mollusco Rec. Austral. Mus. — Records Reeve, Conch. icon, — Conchologia ieonica: or, [llustrations of the Shells of Molluscous Animals. By Lovell AugustusReeve. [v.18—20:] Continued by @.B.Sowerby. v.1—20. London. 1843—78. 4. dell’ Accademia delle Seienze fisiche e matematiche Rend. Acc. Napoli — Rendiconto dell’ Accademia delle Seienze fisiche e matematiche \|Sezione\| della Societä Reale di Napoli. Napoli. 4. ss. — Report ofthe... Meeting of the British Association for the Advance- Rep. Brit. Ass. — Report ofthe... Meeting of the British Association for the Advance- ment of Scienee. London. 8. Report on the seientifie Results of the Voyage of H.M. S. Rep. Voy. Challenger — Report on the seientifie Results of the Voyage of H.M. S. Challenger during the Years 1873—76 under the Command of George 8. Nares and Frank Tourle Thomson. Prepared under the Superintendence of the late! ©. Wyville Thomson and now of John Murray. London, Edinburgh, Dublin. 4. Literatur-Kürzungen XII Rep. Voy. Southern Cross. 1) Cfr.: C. Davies Sherborn & B. B. Woodward in: P. zool. Soc. London, 1893 p. 584. Literatur-Kürzungen. — Das Gebiss der Schnecken zur Begründung einer natür- lichen Classification untersucht. Von F. H. Troschel. Fortgesetzt von J. Thiele. Vol.1,2. Berlin. 1856—63, 66—93. 4. Tr. R. Soc. Edinb. — Transactions of the Royal Society of Edinburgh. Edinburgh. 4. Tr. R. Soc. Edinb. — Transactions of the Royal Society of Edinburgh. Edinburgh. 4. U. 5. expl. Exp. — United States exploring Expedition, during the Years 1838—42, U. 5. expl. Exp. — United States exploring Expedition, during the Years 1838—42, under the Command of Charles Wilkes. — Vol.12. Mollusca & Shells, by Augustus A. Gould. Cum atl. Boston. 1852; 56. 4&2. Verany, Cat. an. invert. — Üatalogo degli animali invertebrati marini del Golfo di Verany, Cat. an. invert. — Üatalogo degli animali invertebrati marini del Golfo di Genova e Nizza. G.B. Verany. (Estratto dalla Guida di Genova.) Genova. 1846. 8. Literatur-Kürzungen XIII Voy. Astrol. — Voyage de Döcouvertes de l’Astrolabe, ex&cut6 par Ordre du Roi, pendant les Anndes 1826—29, sous le Commandement de J. Dumont d’Ur- ville. — Zoologie, par [Jean Rene Constantin] Quoy et [Jean Paul] Gaimard. Tom. 1—4; Atl. Paris. 1830, 32, 34(85), 33; 33. 8&2. &2. Voy. Bonite — Voyage autour du Monde, ex6cute pendant les Anndes 1836 et 1837, sur la Öorvette La Bonite, commandöe par Vaillant. Publi& par Ordre du Roi \|du Gouvernement\|, sous les Auspices du Döpartement de la Marine. — Zoologie, par Eydoux et Souleyet. Tom.1,2;atl. Paris. 1841, 52. 8&2. 52. 8&2. Voy. Coquille — Voyage autour du Monde, ex6&cut& par Ordre du Roi, sur la Corvette de Sa Majeste, La Coquille, pendant les Annees 1822—25. Publie ... par L.1.Duperrey. — Zoologie, par [Ren& Primevere] Lesson (Lesson et [Prosper] Garnot). Tom. lı&u, 21ı&u; Atl. Paris. 1826, 28, 30, 30; 26. 4&2. Voy. Uranie & Physicienne — Voyage autour du Monde, entrepris par Ordre du Roi, . ex&cute sur les Corvettes de S. M. l’Uranie et la Physiecienne, pendant les Annees 1817, 1818, 1819 et 1820; publi& sous les Auspices.... par Louis de Freyeinet. — Zoologie, par [Jean Röen& Constantin] Quoy et [Jean Paul]l Gaimard. Cum atl. Paris. 1824. 4&2. Gaimard. Cum atl. Paris. 1824. 4&2. l. Weiß. Meer. — Die Wirbellosen des Wei Wagner, Wirbell. Weiß. Meer. — Die Wirbellosen des Weißen Meeres. Zoologische Forschungen an der Küste des Solowetzkischen Meerbusens in den Sommer- monaten der Jahre 1877, 1878, 1879 und 1882 von Nicolas Wagner. Vol.1l. Leipzig. 1885. 2. Webb & Berthelot, Hist. Canar. Kosse, Pterop. — De Pteropodum Ordine et novo ipsius Genere. Dissertatio ... publice defendet Joannes Friderieus Julius Kosse. Halae. 1813. 4. Systematischer Index. Systematischer Index. Systematischer Index. [Neue Gattungs- und Artnamen sind durch ein Kreuz (+) bezeichnet] Pteropoda . A. Subord. Thecosomata. a) Trib. Buthecosomata. 1. Fam. Limacinidae . Seite 3 3 15 1. Gen. Limacina (Cuvier) Lamarck 15 1. L. helieina (Phipps) . la.,„ ,„ helicina (Phipps) . lb.,„ ,„ antaretica (Wood- ward) : 2. „ helicoides Jeffr. . 3. „ Inflata (Orb.) . 4. „lesueurii (Orb.) 5. „ rangii (Orb.) 6. „ retroversa (Flem.) 64. ,„ ,„ retroversa (Flem.) 6hb.,„ „ balea (Moll. 6c.„ „ australis (Eydoux & Souleyet) . 7. „ trochiformis (Orb.) 8. „ bulimoides (Orb.) 9. „ cochlostyloides Tesch 2, Gen. Thilea Strebel 1. T.procera Streb. 2. Fam. Cavoliniidae . 1. Gen. Creseis Rang . 1. C. virgula Rang. ; la.,„ ,„ virgula Rang. lb.,„ ,„ conica Esch. 2. „ acicula Rang . . 2&., ,„ acicula Rang . 2b., ,„ celava Rang 3. „ thierchiae (Boas) 4. „ caliciformis Meisenh. 2. Gen. Styliola Lesueur . 16 16 17 18 18 18 19 19 19 20 20 21 21 21 22 22 22 23 24 DD DDR MKD DM DOT Or P Seite 1. 8. subula-(Q. & 6.) 72 27E 3. Gen. Hyalocylis Fl . . . 97 1,. H: »triata. (Rang) sr 4. Gen. Clio Linns . .—. .. 8 1. C. andreae (Boa) . . . 30 2. „ polita (Craven) . . . 30 3. „ ehaptalii (Souleyet) . . 31 4. „ recurva (Children) . . 81 5. „ scheelei (Munthe) . . 82 6. „ euspidata (Bose) . . .„ 82 7. „ antarclica Dall %%. 884 8. „ sulcata (Pfeffer) . . . 85 9. „ pyramidata L. . 277722326 9%, „ pyramidata L.. . 285 9b.,„ ,„ lanceolata (Lesusur) 36 96.5 „ . convexa (Boas) .: 87 5. Gen. Cuvierina Boass . . . 37 1. C. columnella (Rang) . . 37 la.,„ ,„ columnella (Rang) 37 lb., _,„ urceolaris (Mörch) 38 6. Gen; Diacria Gray . . . . 88 1. D.trispinosa (Lesueur) . . 39 ia.„ ,„ trispinosa (Lesueur) 39 1b.„ ' „ major (Boa) SU RET 2. „ quadridentata (Lesueur) 41 24.,„ ,„ quadridentata (Lesueur) . „4 2b. costata (Pfeffer) . 42 ” ” 7. Gen. Cavolinia Abildgaard . 42 1. C. longirostris (Lesueur) . 44 la. 1b. IE 2. 3; 32. ” ”„ „ longirostris (Lesueur) 44 angulata (Souleyet) 46 strangulata Hedley 46 „ globulosa (Rang). . Literatur-Kürzungen. — Histoire naturelle des lles Canaries, par P. Barker Webb et Sabin Berthelot. Tome 1-3; Atl. Paris. 1835—50. 4&2. — Tome 2. Partie 1I. Zoologie. Paris. 1836—44. 4 & 2. Wiegmann & Ruthe, Handb. Zool. — Handbuch der Zoologie. Von Arend Friedrich Wiegmann & Ruthe, Handb. Zool. — Handbuch der Zoologie. Von Arend Friedrich Aug. Wiegmann und Johann Friedrich Ruthe. Berlin. 1832. 8. Woodward, Man. Moll. — A Manual of the Mollusca; or a rudimentary Treatise of Woodward, Man. Moll. — A Manual of the Mollusca; or a rudimentary Treatise of recent and fossil Shells. By S.P.Woodward. London. 1851—56 [1851, 54,56]. 12. Z. Malak. — Zeitschrift für Malakozoologie.e Hannover, Cassel, 8. Zool. Anz. — Zoologischer Anzeiger. Leipzig. 8. Zool. Anz. — Zoologischer Anzeiger. Leipzig. 8. Zool. Anz. — Zoologischer Anzeiger. Leipzig. 8. Kosse, Pterop. — De Pteropodum Ordine et novo ipsius Genere. Dissertatio ... publice defendet Joannes Friderieus Julius Kosse. Halae. 1813. 4. Systematischer Index. Notobranchaea Pelseneer 1. N. macdonaldi Pisnr. 2. „ inopinata Plsnr. 3. „ valdiviae Meisenh. Clio capensis Rang 4. Fam. Clionidae 1. Gen. Clione Pallas . 1. C. limacina (Phipps). la. „ ” limaeina (Phipps) . ll, 110 112 117 3b.C. gihbosa plana Meisenh. . 4. „ tridentata (Forsk.) 42&.,„ ,„ tridentata (Forsk.) 4b.,„ ° ,„ affinis (Orb.) 46.„ ,„ kraussi Tesch + 5. „ uncinata (Rang) 6. „ inflexa (Lesueur) . : 64., ,„ inflex& (Lesueur) . Ener, Jabiata (Orb.) . Cavolinidarum species incertae Hyalaea truncata Lesueur „ rugosa Orb. R Oleodora trifilis Deehel Pleuropus hargeri Verrill b) Trib. Pseudothecoso- mata 5 1. Fam. Peraclididae . 1. Gen. Peracle Forbes P. reticulata (Orb.) . „ brevispira (Plsnr.) „ Lissoides (Tesch) . apicifulva (Meisenh.) . „ bispinosa (Plsnr.) . „. moluccensis (Tesch) . „ depressa (Meisenh.) „ triacantha (Fischer) oonıourkumr 2. Fam. Procymbuliidae l. Gen. Procymbulia Meisen- heimer 1. P. valdiviae en. 3. Fam. Cymbuliidae . 1. Gen. Cymbulia P6ron & Lesueur 1. C. peronii Blainv. 2. „ parvidentata Plsnr. 3. „ Sibogae Tesch . Argivora parva Lesueur . 2. Gen. Corolla W. Dall . 1. C. ovata (9. & G.) 2. „ spectabilis W. Dall . 3. „ ealceola (Verrill) . 4. „ Intermedia (Tesch) 3. Gen. Gleba Forskäl. 1. G. cordata Forsk. e 2. „ chrysosticta (Krohn) . Cymbulia radiata Q. & G. Tiedemannia scyllae Trosch. „ charybdis Trosch. . Seite 49 4. Fam. Desmopteridae . 1. Gen. Desmopterus Chun . 1. D. papilio Chun 2. „ gardineri Tesch Seite 87 88 88 89 B. Subord. Gymnosomata 3 1.Fam. Pneumodermatidae 103 1. Gen. Pneumodermopsis Bronn 104 1. P. ciliata (Gegenb.) . minuta (Plsnr.) „ Simplex (Boas) „ paucidens (Boas) . ‚ polycotyla (Boas). „ macrochira Meisenh. . m . se S ar „ 105 . 106 . 106 . 107 . 108 . 108 2, Gen Spongiobranchia Orbieny 109 1. S. australis (Orb.) 3. Gen. Pneumoderma G. Cuvier mediterraneum (Bened.) all elle „ alılal „ME alla ls eo „JE iR: „ violaceum (Orb.) . :„ Peronil (um. ı „ heterocotylum (Tesch) „ eurycotylum Meisenh. „ boasi (Plsnr.) : „ pacificum (W. Dall.) . „ souleyeti (Plsnr.) . ‚ pygmaeum (Tesch) SW DR co Pneumodermen ruber Q.&G@.. o Aal „ pellueidum Q. & G. 4. Gen. Schizobrachium Meisenheimer 1. 3. polyeotylum Meisenh, 2. Fam. Cliopseidae 1. Gen. Cliopsis Troschel.. 1. ©. krohnii Trosch. 2. „ grandis Boas . 3. Systematischer Index. . 46 55 gibbosa (Bang) :».. .ornarn ” ” gibbosa (Rang) . 47 [Neue Gattungs- und Artnamen sind durch ein Kreuz (+) bezeichnet] Systematischer Index XV 3b.C. gihbosa plana Meisenh. . 4. „ tridentata (Forsk.) 42&.,„ ,„ tridentata (Forsk.) 4b.,„ ° ,„ affinis (Orb.) 46.„ ,„ kraussi Tesch + 5. „ uncinata (Rang) 6. „ inflexa (Lesueur) . : 64., ,„ inflex& (Lesueur) . Ener, Jabiata (Orb.) . Cavolinidarum species incertae Hyalaea truncata Lesueur „ rugosa Orb. R Oleodora trifilis Deehel Pleuropus hargeri Verrill b) Trib. Pseudothecoso- mata 5 1. Fam. Peraclididae . 1. Gen. Peracle Forbes P. reticulata (Orb.) . „ brevispira (Plsnr.) „ Lissoides (Tesch) . apicifulva (Meisenh.) . „ bispinosa (Plsnr.) . „. moluccensis (Tesch) . „ depressa (Meisenh.) „ triacantha (Fischer) oonıourkumr 2. Fam. Procymbuliidae l. Gen. Procymbulia Meisen- heimer 1. P. valdiviae en. 3. Fam. Cymbuliidae . 1. Gen. Cymbulia P6ron & Lesueur 1. C. peronii Blainv. 2. „ parvidentata Plsnr. 3. „ Sibogae Tesch . Argivora parva Lesueur . 2. Gen. Corolla W. Dall . 1. C. ovata (9. & G.) 2. „ spectabilis W. Dall . 3. „ ealceola (Verrill) . 4. „ Intermedia (Tesch) 3. Gen. Gleba Forskäl. 1. G. cordata Forsk. e 2. „ chrysosticta (Krohn) . Cymbulia radiata Q. & G. Tiedemannia scyllae Trosch. „ charybdis Trosch. . Seite 49 4. Fam. Desmopteridae . 1. Gen. Desmopterus Chun . 1. D. papilio Chun 2. „ gardineri Tesch Seite 87 88 88 89 B. Subord. Gymnosomata 3 1.Fam. Pneumodermatidae 103 1. Gen. Pneumodermopsis Bronn 104 1. P. ciliata (Gegenb.) . minuta (Plsnr.) „ Simplex (Boas) „ paucidens (Boas) . ‚ polycotyla (Boas). „ macrochira Meisenh. . m . se S ar „ 105 . 106 . 106 . 107 . 108 . 108 2, Gen Spongiobranchia Orbieny 109 1. S. australis (Orb.) 3. Gen. Pneumoderma G. Cuvier mediterraneum (Bened.) all elle „ alılal „ME alla ls eo „JE iR: „ violaceum (Orb.) . :„ Peronil (um. ı „ heterocotylum (Tesch) „ eurycotylum Meisenh. „ boasi (Plsnr.) : „ pacificum (W. Dall.) . „ souleyeti (Plsnr.) . ‚ pygmaeum (Tesch) SW DR co Pneumodermen ruber Q.&G@.. o Aal „ pellueidum Q. & G. 4. Gen. Schizobrachium Meisenheimer 1. 3. polyeotylum Meisenh, 2. Fam. Cliopseidae 1. Gen. Cliopsis Troschel.. 1. ©. krohnii Trosch. 2. „ grandis Boas . 3. „ modesta (Plsnr.) . 4. „ microcephala (Tesch) 3. Fam. Notobranchaeidae 1. Gen. Systematischer Index. „ modesta (Plsnr.) . 4. „ microcephala (Tesch) 3. Fam. Notobranchaeidae 1. Gen. Notobranchaea Pelseneer 1. N. macdonaldi Pisnr. 2. „ inopinata Plsnr. 3. „ valdiviae Meisenh. Clio capensis Rang 4. Fam. Clionidae 1. Gen. Clione Pallas . 1. C. limacina (Phipps). la. „ ” limaeina (Phipps) . ll, 110 112 117 XVI Seite Seite 1b. C. limaeina antarctica 5. Fam. Thliptodontidae . 134 Edg. Sm.. .128 \| \|], Gen. Thliptodon Bons. . . 185 2. „ gracilis Massy. . . 128 3. „ longicaudata (Souley u 129 4. „ flavescens (Gegenb.). . 129 1. T. gegenbauri Boas. . . 186 A diaphanus (Meisenh.). . 136 Clio pyramidalis Q.&G. . . 130 \| 2. Gen. Thalassopterus Kwiet- 2. Gen. Fowlerina Pelseneer . 130 niewski . 0... . 187 1. F. zetesios Plnr.. . : . 19 1. T. zancleus Kwietn.. . . 138 3. Gen. Paraclione Tech . . 131 \| 6.Fam. Halopsychidae . . 140 1. P. pelseneeri Tesch . . . 132 1. Gen. Halopsyche Bronn . . 141 4. Gen. Paedoclione Danforth . 132 1. H. gaudichaudii (Souleyet) . 141 1. P. doliiformis Danf.. . . 183 Cymodocea diaphana Orb. . . 142 Pteropoda 1804 Pteropoda, G. Cuvier in: Ann. Mus. Paris, v.4 p.223 \| 1814 „Pterobranches, Pteeodibranche“ (laps. typ. pro: Pterodibranche) (part.), Blainville in: Bull. Soc. philom., 1814 p. 177,177 (nota) \| 1821 Stomatopterophora, J. E. Gray in: London med. Repos., v.15 p.235 \| 1824 Aporobranchia (part.), Blainville in: Dict. Sci. nat., v.32 p.271 \| 1825 Class. Pteropoda (part.), Ord. Megapterygia (part.), Fam. Procephala, Latreille, Fam. Regne an., p.168 \| 1832 Cryptobranchia (part.), Deshayes in: Enc. möth., Vers v.2ır p- 552 \| 1862 Coponautae, Dipteronautae, Keferstein in: Bronn’s Kl. Ordn., v.3ı1 p. 584. Rein pelagisch lebende, marine Weichtiere, deren gemein- samer Charakter darin besteht, daß der dorsale Teil des Fußes sich als ein Paar mehr oder weniger mächtiger Flossen entwickelt hat und so in den Dienst der Fortbewegung getreten ist. Innere Organisation asymmetrisch. Zentralnervensystem euthyneur. Ge- schlechtsorgane hermaphroditisch, proterandrisch; Öffnung des eingestülpten Penis mit der des Geschlechtsausführungsganges durch eine Samenrinne verbunden. Eier werden meist abgelegt. Entwicklung mit Metamorphose pelagischer Larven, welche denen der Opisthobranchier sehr nahe stehen, verbunden. Nächste Ver- wandtschaft mit Opisthohranchiern, und zwar mit Tectibranchiern. Das gemeinsame Merkmal der Pteropoden besteht vor allem in den Flossen, bald große und mächtige, bald weniger in die Augen fallende, aber immer vorhandene Gebilde, welche nach Pelseneer mit den Parapodien der tectibranchiaten Opisthobranchiern homologisiert werden müssen. Allen Ptero- poden ist weiter die absolut pelagische Lebensweise im freien Meere gemein- sam; sie erscheinen gewöhnlich periodisch an der Oberfläche, namentlich in den Dämmerungs- und Nachtstunden sind sie hier anzutreffen; einige Arten kommen aber auch am hellen Tage bisweilen massenhaft in den oberen Schichten vor. Es bleibt dabei aber noch vieles zu untersuchen übrig. Die Pteropoden gehören vorzugsweise den wärmeren Gewässern an; in den Polarmeeren kommen sie aber ebenfalls vor, und hier oft in großen Schwärmen, welche aber nur durch wenige Arten gebildet werden. — Es findet Kopu- lation zwischen zwei verschiedenen Individuen statt. Aus den in Schnüren oder einzeln abgelegten Eiern entwickeln sich die mit Velum ausgestatteten Larven, welche denen der ÖOpisthobranchier sehr nahe stehen. Bekanntlich läßt sich von der ganzen Ordnung der Pteropoden keine bis in Einzelheiten gehende morphologische Charakteristik geben, da sie in zwei Unterordnungen zerfällt, deren ganze innere und äußere Organisation so verschieden ist, daß sie unbedingt gesondert behandelt werden müssen. Das Tierreich. 36.Lief.: J. J. Tesch, Pteropoda. !) Wenn ganz ausnahmsweise die Schale normal gänzlich fehlt, läßt doch die innige Verbindung des Kopfes mit den Flossen die Zugehörigkeit zu den Thecosomata erkennen (z. B. Desmopterus). Pteropoda Die beiden Unterordnungen lassen sich sogleich auf folgende Weise unter- scheiden: ' Schale vorhanden, sei es in Form einer kalkigen Hülle oder als knorpelige, durchsichtige, vom Mantel allseitig umhüllte Pseudoconcha. Kopf undeutlich abgesetzt, mit einem Paare Tentakeln und einem Paare Flossen!) . . . --. 2...» ' Schale vorhanden, sei es in Form einer kalkigen Hülle oder als knorpelige, durchsichtige, vom Mantel allseitig umhüllte Pseudoconcha. Kopf undeutlich abgesetzt, mit einem Paare Tentakeln und einem Paare Flossen!) . . . --. 2...» 1 2 Schale fehlend; Körper mehr oder weniger spindel- förmig, wenngleich oft stark zusammengezogen. Kopf fast stets deutlich vom Rumpfe (bei aus- gestreckten Exemplaren) durch eine Einschnürung abgegrenzt, mit zwei Tentakelpaaren. Flossen an der Ventralseite der Halsgegend . . - . - Pteropoda Rein pelagisch lebende, marine Weichtiere, deren gemein- samer Charakter darin besteht, daß der dorsale Teil des Fußes sich als ein Paar mehr oder weniger mächtiger Flossen entwickelt hat und so in den Dienst der Fortbewegung getreten ist. Innere Organisation asymmetrisch. Zentralnervensystem euthyneur. Ge- schlechtsorgane hermaphroditisch, proterandrisch; Öffnung des eingestülpten Penis mit der des Geschlechtsausführungsganges durch eine Samenrinne verbunden. Eier werden meist abgelegt. Entwicklung mit Metamorphose pelagischer Larven, welche denen der Opisthobranchier sehr nahe stehen, verbunden. Nächste Ver- wandtschaft mit Opisthohranchiern, und zwar mit Tectibranchiern. Das gemeinsame Merkmal der Pteropoden besteht vor allem in den Das gemeinsame Merkmal der Pteropoden besteht vor allem in den Flossen, bald große und mächtige, bald weniger in die Augen fallende, aber immer vorhandene Gebilde, welche nach Pelseneer mit den Parapodien der tectibranchiaten Opisthobranchiern homologisiert werden müssen. Allen Ptero- poden ist weiter die absolut pelagische Lebensweise im freien Meere gemein- sam; sie erscheinen gewöhnlich periodisch an der Oberfläche, namentlich in den Dämmerungs- und Nachtstunden sind sie hier anzutreffen; einige Arten kommen aber auch am hellen Tage bisweilen massenhaft in den oberen Schichten vor. Es bleibt dabei aber noch vieles zu untersuchen übrig. Die Pteropoden gehören vorzugsweise den wärmeren Gewässern an; in den Polarmeeren kommen sie aber ebenfalls vor, und hier oft in großen Schwärmen, welche aber nur durch wenige Arten gebildet werden. — Es findet Kopu- lation zwischen zwei verschiedenen Individuen statt. Aus den in Schnüren oder einzeln abgelegten Eiern entwickeln sich die mit Velum ausgestatteten Larven, welche denen der ÖOpisthobranchier sehr nahe stehen. Bekanntlich läßt sich von der ganzen Ordnung der Pteropoden keine Bekanntlich läßt sich von der ganzen Ordnung der Pteropoden keine bis in Einzelheiten gehende morphologische Charakteristik geben, da sie in zwei Unterordnungen zerfällt, deren ganze innere und äußere Organisation so verschieden ist, daß sie unbedingt gesondert behandelt werden müssen. Die Thecosomen stehen den Bulliden unter den Teetibranchiern nahe, die Gymnosomen sind mit den Aplysiiden verwandt, und eine nähere Ver- wandtschaft muß, so lange es innerhalb der formenreichen Abteilung der 1 Das Tierreich. 36.Lief.: J. J. Tesch, Pteropoda. 1 Das Tierreich. 36.Lief.: J. J. Tesch, Pteropoda. 2 Pteropoda: A. Theeosomata Teetibranchier nicht gelingt, eine Form aufzufinden, welche die Merkmale der beiden Gruppen in sich vereinigt, geleugnet werden (Boas, Pelseneer, Meisenheimer). 2 Subordines, 2 Tribus, 12 Familien, 28 Genera, 89 sichere Arten, von denen 13 in 30 Unterarten zerfallen und 13 unsichere Arten. Pteropoda: A. Thecosomata, a) Euthecosomata Pteropoda: A. Thecosomata, a) Euthecosomata Pteropoda: A. Thecosomata, a) Euthecosomata 3 Wie besonders von Pelseneer hervorgehoben wurde, stammen die Thecosomen wohl unzweifelhaft von den Bulliden ab: das Vorkommen eines Operculum bei den Limaciniden, der Bau der Mantelhöhlendrüse, die Zusammensetzung der Radula, das Vorkommen von Kauplatten im Magen, sowie der Aufbau des Zentralnervensystems weisen auf diese Verwandtschaft hin. — Die Thecosomen sind meist Dämmerungs- und Nachttiere, welche sich im wesentlichen von mikroskopischen Pflanzen und Tieren ernähren. Bestimmungstabelle der beiden Gruppen der Thecosomata: Stets mit äußerer Kalkschale versehen, bald spiralig, bald gestreckt, aber nie mit Rostrum. Mund. im Niveau der Flossenfläche, keine 3 ; Rüsselbildung . .. ...... 2... 2 Trib. Euthecosomata Kalkschale, wenn vorhanden, spiralig, mit Rostrum, welches als Fortsetzung der Colu- 1 mella zu betrachten ist; meist eine voll- _ kommen durchsichtige, vom Mantel allseitig umhüllte und leicht vom eigentlichen Körper lösliche Pseudoconcha. Flossen zu einer einheitlichen, mächtigen Scheibe verwachsen, auf welcher sich in der Mitte des Vorder- randes der Mund mit den umgebenden Teilen BuSselsthior BIMEDL. nen ann b. Trib. Pseudothecosomata Stets mit äußerer Kalkschale versehen, bald spiralig, bald gestreckt, aber nie mit Rostrum. Mund. im Niveau der Flossenfläche, keine Rüsselbildung . .. ...... 2... ; 2 Trib. Euthecosomata ... 2... Kalkschale, wenn vorhanden, spiralig, mit Rostrum, welches als Fortsetzung der Colu- 1 mella zu betrachten ist; meist eine voll- _ kommen durchsichtige, vom Mantel allseitig umhüllte und leicht vom eigentlichen Körper lösliche Pseudoconcha. Flossen zu einer einheitlichen, mächtigen Scheibe verwachsen, auf welcher sich in der Mitte des Vorder- randes der Mund mit den umgebenden Teilen BuSselsthior BIMEDL. nen ann a. Trib. Buthecosomata A. Subord.. Thecosomata 1821 Ord. Pterobranchia (part.) + Ord. Dactyliobranchia, J. E. Gray in: London med. Repos., v.15 p.235 \| 1824 Thecosomata, Blainville in: Diet. Sei. nat., 0.32 p.271 \| 1886 Eupteropoda, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 14. Entweder mit äußerer Kalkschale, welche bald spiralig (und dann stets links) aufgerollt, bald gestreckt ist oder aber mit innerer, gänzlich durchsichtiger Knorpelschale ausgestattet. Wo die beiden Flossen in der Mittellinie zusammenstoßen findet sich der Mund, der stets von Lippen (= Seitenlappen des Fußes) umgeben wird. Mantelhöhle stets vorhanden, dorsal oder ventral; am Dache der Mantelhöhle eine wohlentwickelte, komplizierte Manteldrüse Nur ein einziges Paar Tentakeln an der Dorsal- seite des Kopfes, welcher kaum vom Rumpfe gesondert er- scheint: Die Radula (selten fehlend) hat in jeder Querreihe eine Mittelplatte und jederseits eine einzige Seitenplatte; vor der Radula liegt rechts und links ein Kiefer. Ein Paar kleiner Speicheldrüsen. Der Magen ist mit vier starken, symmetrischen Hornplatten versehen; eine fünfte, unpaare, liegt weiter distal und vier oder acht kleinere liegen vor den großen Platten. Die Leber hat nur eine einzige Offnung, höchstens deren zwei, am Hinter- ende des Magens. Der Enddarm beschreibt eine Schlinge; der Anus liegt rechts oder links; mit ihm vergesellschaftet findet sich gewöhnlich eine Analdrüse. Der Kopulationsapparat liegt im vorderen, dorsalen Kopfabschnitt, und zwar rechts, in der Nähe der Tentakeln; eine Samengrube führt von der etwas mehr distal und ebenfalls rechts gelegenen Geschlechtsöffnung hierhin. Im Zentralnervensystem sind die ÜCerebralganglien weit aus- eitander gerückt und durch eine lange supraoesophageale Kom- missur verbunden; die Pleuralganglien scheinen mit den Cerebral- ganglien verschmolzen zu sein. Pteropoda: A. Thecosomata, a) Euthecosomata 4 Hinter der Ansatzstelle von Fuß und Flossen verengt sich der Kopf- abschnitt und geht in den Rumpfabschnitt über, der ein wechselndes Aus- sehen hat, je nach der Form der Schale (Fig. HD Der Rumpfabschnitt wird von der Kalkschale umschlossen, sowie Hinter der Ansatzstelle von Fuß und Flossen verengt sich der Kopf- abschnitt und geht in den Rumpfabschnitt über, der ein wechselndes Aus- sehen hat, je nach der Form der Schale (Fig. HD Der Rumpfabschnitt wird von der Kalkschale umschlossen, sowie von einer Mantelhöhle, deren meist schlitzförmige Öffnung am Vorderrande Mantelhöhle Fa „Schale Flosse ’ “ Fuß” ; A Opereulum gelegen ist. In die Mantelhöhle münden After und Nierenöffnung; sie enthält ferner das Osphradium und die eventuellen Kiemenbildungen (bei Cavolinia). Das Innere des Rumpfabschnittes enthält die Ein- geweide, vor allem Zwitterdrüse, Magen und Leber, sowie Herz und Niere. Das Integument besteht aus der Kalkschale umschlossen, sowie hlitzförmige Öffnung am Vorderrande gelegen ist. In die Mantelhöhle münden After und Nierenöffnung; sie enthält ferner das Osphradium und die eventuellen Kiemenbildungen (bei Cavolinia). Das Innere des Rumpfabschnittes enthält die Ein- geweide, vor allem Zwitterdrüse, Magen und Leber, sowie Herz und Niere. Mantelhöhle Fa „Schale Flosse ’ “ Fuß” ; A Opereulum ‚Mantelhöhle ‚ Schale B Mantelhöhle Schale C Schale „ D Flosse Fuß 4 Schale Mantelhöhle Schale wi ee v Schale 5 m „ ) G Fig. 1. Schematische Darstellung der Formveränderungen der Schale, von links betrachtet. A Limacina. B, © hypothetische Zwischenstadien, D Creseis. EcClio, FClio und junge Cavolinia, G erwachsene Cavolinia. Nach Boas. Mantelhöhle Fuß” ; Opereulum ‚Mantelhöhle ‚ Schale Das Integument besteht aus einem äußerst dünnen, abgeplatteten Epithel, das nur an einzelnen Stellen von Drüsen durchsetzt wird. Der Mantel ist eine Faltenbildung der äußeren Haut; die Höhle reicht asymmetrisch nach hinten, ist also gar nicht an allen Seiten des Rumpfes gleich entwickelt, sondern bei den spiralig gewundenen Formen (Zima- cina, Fig. 1A) als eigentliche Höhle nur dorsal ausgebildet, ventral da- gegen nur als einfache Hautfalte ent- halten, bei den gestreckten Formen (Oreseis, Clio etc. Fig. 1D bis F) verhält es sich gerade umgekehrt. Diese Verhältnisse sind durch die Drehung um 180° von Kopf und Rumpfabschnitt bedingt (Fig. 2) und diese Drehung beherrscht die ganze innere Organisation, wie weiter unten gezeigt werden wird. Während bei den einfacheren gestreckten For- men die Mantelhöhle nicht weit nach hinten reicht, ist sie bei Diaeria und Cavolinia bis ans hintere Körperende vertieft. a. Trib. Buthecosomata 1852 „Famille des Hyales“, Souleyet in: Voy. Bonite, v.2 p. 98 \| 1885 Ptero- cephala, N. Wagner, Wirbell. Weiß. Meer, v.1 p.119 \| 1905 Euthecosomata, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.4. In erster Linie fällt die Schale auf. Dieselbe besteht aus kohlen- saurem Kalk und zeigt sehr verschiedene Gestalt. Bei Limacina (Fig. 1A) ist sie spiralig, und zwar stets links eingerollt; bei Creseis (D) ist sie dagegen mehr gestreckt, im Querschnitt kreisrund; bei Cko (E) ist der Querschnitt mehr dorsoventral abgeplattet, dorsaler und ventraler Vorderrand der Schale treten etwas hervor; dieses Verhalten macht sich in weit höherem Maße bei der erwachsenen Cavolinia (G@) geltend, wo die seitlichen Spalten der Schale bis weit distalwärts reichen und von der Mundöffnung der Schale durch einen zahnartigen Vorsprung in der ventralen Schalenhälfte, welcher mit einer entsprechenden Vertiefung in der dorsalen Hälfte korrespondiert, getrennt wird. Die Schale ist für die Systematik von ganz vorwiegender Bedeutung und wird daher bei den einzelnen Familien, Gattungen und Arten gehörig berücksichtigt werden. Das Tier kann sich ganz in die Schale zurückziehen, gewöhnlich aber ist der Kopf mit den Flossen hervorgestreckt. Der Körper setzt sich im wesentlichen aus zwei Hauptabschnitten Der Körper setzt sich im wesentlichen aus zwei Hauptabschnitten zusammen: einem vorderen, der Mund, Fuß, Flossen und Tentakeln trägt, und einem hinteren, viel umfangreicheren, der von der Schale bedeckt wird und die Eingeweide enthält. Wir finden am Kopf auf der Ventralseite den Mund, begrenzt von zwei Seitenfalten, welche als umgewandelte Fußteile aufzufassen sind; nach hinten schließt sich an diese Lippen der hintere Fußabschnitt an, in Form eines quergestellten Lappens, während nach vorn und seitlich davon gelegene, flächenartig ausgedehnte Verbreiterungen die Flossen darstellen. Ganz vorn liegt weiter an der Dorsalseite ein Tentakelpaar, und unmittelbar rechts neben der Mundöffnung die Mündung des Penis; der Ausführungsgang der Geschlechtsprodukte liegt gleichfalls rechts, aber mehr distal (Fig. 2). Pteropoda: A. Thecosomata, a) Euthecosomata Pteropoda: A. Thecosomata, a) Euthecosomata Der Mantelrand ist ver- diekt; bei ZLimacina links in eine kurze Spitze ausgezogen, rechts findet sich ein rinnenförmig umgekrempelter Schnabel (Fig. 2A), dem zur Seite ein tentakelförmiger Anhang, dessen Oberfläche bewimpert ist. Dieser An- es Gleichgewichtes („balancer“), zumal ung bei den gestreckten Formen vor- . Bei den höchstentwickelten Formen n den Seitenrändern mehr spaltförmig inks schlitzförmige Spalten entstehen Mantelhöhle Schale Fuß 4 Mantelhöhle Fuß 4 Mantelhöhle Schale Fig. 1. Schematische Darstellung der Formveränderungen der Schale, von links betrachtet. A Limacina. B, © hypothetische Zwischenstadien, D Creseis. EcClio, FClio und junge Cavolinia, G erwachsene Cavolinia. Nach Boas. Fig. 1. Schematische Darstellung der Formveränderungen der Schale, von links betrachtet. A Limacina. B, © hypothetische Zwischenstadien, D Creseis. EcClio, FClio und junge Cavolinia, G erwachsene Cavolinia. Nach Boas. hang dient wohl als Organ zur Erhaltung des Gleichgewichtes („balancer“), zumal er nicht oder in weit geringerer Ausbildung bei den gestreckten Formen vor- kommt; zudem liegt er hier links (Eig. 2 3). Bei den höchstentwickelten Formen ist die runde oder ovale Schalenöffnung an den Seitenrändern mehr spaltförmig gestaltet (Fig. 1@), so daß rechts und links schlitzförmige Spalten entstehen 5 Pteropoda: A. Thecosomata, a) Euthecosomata können; die Mantelränder und die Mantelöffnung zeigen aber nichts von diesen Spalten, sondern die Ränder sind in dieser Gegend ganz verwachsen, Bei Cavolinia kommt dazu, daß an den Verwachsungsrändern Längsfalten auftreten, welche stark kontraktil sind, und aus dem seitlichen Schalenschlitz beim ungestörten Tier frei hervorhängen (Gleichgewichtsorgane). Der vordere Mantelrand kann hier über die Schale ausgedehnt werden. Am Dache der Mantelhöhle findet sich die große Drüse, welche sich Am Dache der Mantelhöhle findet sich die große Drüse, welche sich histologisch aus quergestreiften Zellenstreifen zusammenstellt, von denen besonders drei dunkle Querbänder auffallen,. welche aus großen, von einem dunklen, längsgestreiften Protoplasma erfüllten Drüsenzellen bestehen, mit großen, oft gelappten Kernen versehen. Die hellen Streifen zwischen den ul ittellappen —- des Fußes © [7] u © = FR Mittellappen des Fußes Geschlechts- __N J öffnung a8 ee: = &D .--- ze „balancer‘“ f = = " ‚balancer‘‘ Anus -" a e= a ” "Anus R=] >} = B=\| [>] un © {do} .. Zwitterdrüse Zwitter- drüse A Fig. 2. Schematische Darstellung der Organisationsverhältnisse. A bei einer (gerade gestreckten) Limacina, B bei Clio, beide von der Ventralseite betrachtet. Nach Boas. 8 = = a Fig. 2. Schematische Darstellung der Organisationsverhältnisse. A bei einer (gerade gestreckten) Limacina, B bei Clio, beide von der Ventralseite betrachtet. Nach Boas. Pteropoda: A. Thecosomata, a) Euthecosomata Querbändern werden von Reihen großer, kastenförmiger Zellen gebildet, welche 2 oder 3, bisweilen aber mehr, napfförmige Vertiefungen ihrer Wand aufweisen. Diese Differenzierungen finden sich bei den höher entwickelten Formen (Diacria und (avolinia), wo die ganze Drüse symmetrisch ist, weit vollkommener als bei den anderen: bei Zimaeina ist die Gestalt der Drüse asymmetrisch und die napfförmigen Vertiefungen in den hellen Zellen fehlen durchaus. Eine zarte Wimperung scheint dem Drüsenfeld stets zuzukommen. Ihrer Funktion nach muß sie als Schleimdrüse bezeichnet werden. den Rumpf überziehenden Seite des Mantels, und zwar in der Querbändern werden von Reihen großer, kastenförmiger Zellen gebildet, welche 2 oder 3, bisweilen aber mehr, napfförmige Vertiefungen ihrer Wand aufweisen. Diese Differenzierungen finden sich bei den höher entwickelten Formen (Diacria und (avolinia), wo die ganze Drüse symmetrisch ist, weit vollkommener als bei den anderen: bei Zimaeina ist die Gestalt der Drüse asymmetrisch und die napfförmigen Vertiefungen in den hellen Zellen fehlen durchaus. Eine zarte Wimperung scheint dem Drüsenfeld stets zuzukommen. Ihrer Funktion nach muß sie als Schleimdrüse bezeichnet werden. An der den Rumpf überziehenden Seite des Mantels, und zwar in der An der den Rumpf überziehenden Seite des Mantels, und zwar in der Nähe des Afters finden wir die Analdrüse, aus einer Lage zylindrischer Drüsenzellen bestehend. als Differenzierungen des ursprünglichen Fuß und Flossen sind als Differenzierungen des ursprünglichen Gastropodenfußes aufzufassen. Wir können den eigentlichen Fuß zuerst unterscheiden. Dieser (Fig. 3) setzt sich aus einem hinteren Teil oder einem Mittellappen zusammen, der verschiedene Gestalt zeigt (vgl. in Fig.3 A u. B) Pteropoda: A, Thecosomata, a) Euthecosomata 6 und bei den spiralig gewundenen Formen das Operculum an der Außenseite trägt (Fig. 1, Opereulum); hier findet sich zudem an der Innenseite eine lange, gegen den Mund hin gerichtete Rinne (Fig. 3 A). Die anderen Teile des Fußes sind die beiden Seitenlappen, welche konvergierend nach vorn ziehen, und an ihrer Ecke den Mund zwischer sich fassen. Mittel- und Seitenlappen sind durch ein eigentümliches Wimperfeld verbunden, welches sich zuweilen seitlich über die ventrale Fläche der Flossen ausdehnen kann. Während sich auf der dem Munde zugekehrten Innenseite des Mittel- lappens ein Wimperepithel findet, ist die entgegengesetzte Seite von einem einfachen, kubischen Epithel überkleidet, zwischen dessen Zellen zahlreiche, einzellige Drüsen ausmünden. Die Seiten- oder Lippenfalten sind am freien Rande bewimpert und in der Nähe des Mundes am höchsten; hier schließen sie bei Zimaecina (Fig. 3 A) ein kleines, rundliches Läppchen ein. Pteropoda: A. Thecosomata, a) Euthecosomata 3 3); dies kommt aber daher, daß die seitlichen Wimperfelder des Fußes sich am Außenrande der Flosse schärfer gesondert haben. Außerlich sind die Flossen von einem Plattenepithel überzogen, welches eingestreute Drüsenzellen enthält. Die Muskulatur der Flosse läßt sich in zwei Abteilungen zerlegen, eine oberflächliche und eine innere. Die oberflächliche Muskulatur besteht aus zwei senkrecht aufeinander stehenden und der Flossenfläche parallelen Systemen von Faserzügen (Fig. 6D), so daß ein zierliches Netzwerk entsteht; gegen den Rand hin verschwinden allmählich diese Fasern. Bisweilen aber (Creseis, Hyaloeylis) sind diese Faserzüge einander sehr nahe gerückt und verschmolzen, so daß die Trennung in einzelne Bündel verschwunden ist. — Unterhalb dieser oberflächlichen Muskulatur finden wir nun im Innern der Flosse ein zentrales System, das sich nicht wie das vorige direkt von der Haut herleitet, sondern als eine direkte Fortsetzung des Columellarmuskels, der sich in der Höhe des Kopfabschnittes in zwei Aste spaltet, zu betrachten ist. Dies sind die Rückziehmuskeln der Flosse, welche dieselbe ganz ins Innere der Schale zurückzuziehen imstande sind. Zarte Membrane und Fasern sind im Innern der Flosse als Stützsystem angeordnet. Die wichtigste membranöse Scheidewand durchzieht das Blatt der Flosse parallel den oberflächlichen Muskellagen; von hier gehen in regelmäßigen Abständen Querwände ab. Beachtenswert ist weiter noch der tentakelförmige Fortsatz am Vorder- rande der Flosse (Fig. 3 A) bei einzelnen Arten (Limacina helieina, L. retroversa, Creseis). Ein echter Tentakel mit Sinnesfunktion ist dieser Fortsatz nicht: er dient vielmehr als eine Art Stützgewebe zur Erhöhung der Festigkeit des Vorderrandes der Flosse. Mesenchymgewebe erfüllt als ein bald lockeres, bald dichteres Maschennetz das Innere des Körpers. Um den Eingeweidesack herum bildet es eine festere Hülle, welche mit eigentümlicher Pigmentierung (oft metallisch glänzend) ausgestattet ist. Diese glänzende Hülle fällt z. B. bei den lebenden Clio oder Cuvierina sofort auf. In der Halsregion findet sich eine diaphragma- artige Scheidewand; diese zieht (bei Clio pyramıdata) ein wenig oberhalb der Einmündungsstelle des Oesophagus in den Magen quer durch den Körper und hebt sich an den Rändern dorsal etwas empor, wonach sie mit den Körperwandungen verschmilzt. Sie wird vom Oesophagus, von Nerven und Blutgefäßen durchbrochen; übrigens aber sind die Lakunenräume des Kopfes auf diese Weise von denen des Rumpfes geschieden (bei Ureseis scheint das Diaphragma zu fehlen). Pteropoda: A. Thecosomata, a) Euthecosomata Daß tentakelartiger Flossenanhang lappenförmiger Anhang “- Wimperfeld A Mittellappen des Fußes -— _.... Schale Rinne auf dem Mittellappen „x Flosse —— Wimperfeld Mittellappen des Fußes -"/ .... Schale B Fig. 3. Schematische Darstellung der Organisationsverhältnisse von Fuß und Flosse, von der Ventralseite. A bei Limacina, B bei Cavolinia. Nach Meisenheimer. tentakelartiger Flossenanhang lappenförmiger Anhang tentakelartiger Flossenanhang lappenförmiger Anhang “- Wimperfeld A Mittellappen des Fußes -— _.... Schale Rinne auf dem Mittellappen tentakelartiger Flossenanhang nhang lappenförmiger Anhang Wimperfeld Mittellappen des Fußes - Rinne auf dem Mittellappen Flosse Fig. 3. Schematische Darstellung der Organisationsverhältnisse von Fuß und Flosse, von der Ventralseite. A bei Limacina, B bei Cavolinia. Nach Meisenheimer. diese beiden Seitenlappen als differenzierte Teile des Fußes zu betrachten sind, zeigt nicht nur der Umstand, daß sie mit dem Wimperfelde und dem Mittellappen ein durchaus einheitliches System bilden, sondern auch, daß sie vom Pedalganglion innerviert werden. Das Ganze bildet, besonders wenn der Mittellappen nach dem Munde zu eingeschlagen werden kann, einen wirksamen Trichter, zu dem die Cilien Nahrungsteilchen zuführen können. Der dorsale Abschnitt des Fußes hat im Gegensatz zu dem ventralen diese beiden Seitenlappen als differenzierte Teile des Fußes zu betrachten sind, zeigt nicht nur der Umstand, daß sie mit dem Wimperfelde und dem Mittellappen ein durchaus einheitliches System bilden, sondern auch, daß sie vom Pedalganglion innerviert werden. Das Ganze bildet, besonders wenn der Mittellappen nach dem Munde zu eingeschlagen werden kann, einen wirksamen Trichter, zu dem die Cilien Nahrungsteilchen zuführen können. Der dorsale Abschnitt des Fußes hat im Gegensatz zu dem ventralen die Funktion eines Fortbewegungsorganes übernommen: die Flossen stellen dorsal eine einheitliche Fläche dar, schließen also dorsal vom Munde zu- sammen, ventral werden sie vom eigentlichen Fuße unterbrochen. Ein Ein- schnitt, genau median auf den Mund zulaufend, teilt die Flosse in zwei Der dorsale Abschnitt des Fußes hat im Gegensatz zu dem ventralen die Funktion eines Fortbewegungsorganes übernommen: die Flossen stellen dorsal eine einheitliche Fläche dar, schließen also dorsal vom Munde zu- sammen, ventral werden sie vom eigentlichen Fuße unterbrochen. Ein Ein- schnitt, genau median auf den Mund zulaufend, teilt die Flosse in zwei Pteropoda; A. Theeosomata, a) Euthecosomata 7 seitliche Lappen; diese Lappen können sehr verschiedene Gestalt haben; sie sind bald schmal, bald flächenartig verbreitert. Bei den höheren Formen findet sich immer eine Einkerbung des Seitenrandes; bei Cavolinia scheinen zwei solche Einkerbungen vorhanden zu sein (Fig. Pteropoda: A. Thecosomata, a) Euthecosomata Von der Muskulatur ist nur der Spindelmuskel stark entwickelt; vor allem bei den Formen mit gewundenen Gehäusen, wie Limacina; er liegt hier auf der Ventralseite und folgt der Columella der Schale in ihren Windungen; nach vorn hin strahlt er dann in die Flossen aus. Bei den übrigen Euthecosomen liegt er, da hier der Rumpf im Verhältnis zum Kopf um 180° gedreht erscheint, auf der dorsalen Seite; hier verläuft er in der Mittellinie des Rumpfes ganz gerade vom Hinterende der Schale bis in die Kopfregion und teilt sich hier in die beiderseitigen Aste, welche, wie oben besprochen, das zentrale Muskelbündel in den Flossenlappen bilden.. Seine Ursprungsstelle liegt stets am hinteren Ende der Schale, und zwar etwas Pteropoda: A. Thecosomata, a) Euthecosomata 8 asymmetrisch, ein wenig nach rechts verschoben. Hinten und in der Mitte liegt er ganz frei, etwas weiter nach vorn verschwindet er ins Innere des Körpers und gibt dann die beiden seitlichen Aste ab; einzelne Fasern heften sich noch am Fuße, andere an den Seitenwänden des Körpers an; so daß alle diese Teile zurückgezogen werden können. — Die übrige Körpermuskulatur ist sehr schwach ausgebildet. Im allgemeinen findet man überall nur glatte Muskel- fasern, in den Flossen einiger Arten (Creseis acicula, Clio pyramidata, Cavolinia longirostris, C.tridentata) kommen aber deutlich quergestreifte Muskelfasern vor. Das Zentralnervensystem (Fig. 4) ist stark zusammengedrängt und liegt zum größten Teile auf der Ventralseite des Oesophagus. Das am meisten dorsal gelegene Ganglienpaar, das durch eine lange, dorsal den Oesophagus umgreifende Kommissur verbunden ist, stellt die Cerebral- ganglien und die damit verschmolzenen Pleuralganglien dar. Das vordere, ventrale Ganglienpaar wird durch die Pedalganglien gebildet; sie sind an Umfang größer als die anderen Ganglien und, wie die Cerebral- ganglien, links und rechts vollkommen gleich. Sie liegen so dicht neben- einander, daß eine deutliche Kommissur äußerlich kaum nachweisbar ist; in Wirklichkeit ist dieselbe aber nicht nur stets vorhanden, sondern es findet sich sehr häufig etwas mehr proximal noch eine zweite, allerdings viel schmächtigere, vor. Am hinteren Rande der Pedalganglien liegt jederseits eine rundliche Statocyste. Das zweite ventrale Ganglienpaar sind die Visceralganglien, welche, im Gegensatz zu den anderen Paaren, nicht völlig symmetrisch entwickelt sind, indem bei den Limaciniden die rechte, bei den Cavoliniiden die linke Ganglienmasse die auf der andern Seite gelegene bald mehr bald weniger an Größe übertrifft. — Infolge der außer- ordentlich gedrängten Lage des Zentralnervensystemes ist von eigentlichen Konnektiven zwischen den genannten ungleichnamigen Ganglien kaum die Rede. Pteropoda: A. Thecosomata, a) Euthecosomata Im Innern finden sich die aus kohlensaurem Kalk bestehenden Statolithen, meist in großer Zahl; im Jugendstadium des Tieres kommt wahrscheinlich nur ein einziger großer Statolith vor. Das Osphradium ist eine langgestreckte, bewimperte Rinne, welche bei Zimaeina links, bei den Cavoliniiden rechts gelagert ist. Tentakeln liegen auf der Dorsalseite des Kopfes, dicht an der Medianebene; sie sind asymmetrisch ausgebildet, da fast stets der rechte größer ist als der linke (besonders bei Zimacina und Creseis). Der rechte Tentakel ist oft in einer Scheide eingeschlossen. An der Spitze beider Tentakeln finden wir (sehr deutlich bei Cavolinia longirostris) ein rudimentäres Auge, indem das einfache Plattenepithel der Oberfläche hier einen Hohlraum bedeckt, welcher wahrscheinlich von einer durchsichtigen, homogenen Flüssigkeit erfüllt ist und am Boden ein hohes, mehrschichtiges Epithel zeigt (die eigentlichen Sinneszellen); unterhalb dieses Epithels, aber durch eine Membran davon geschieden, liegt ein Komplex großer Ganglienzellen. Pigmentzellen fehlen oder sind vielleicht nur auf jüngerem Stadium vorhanden. Diese Diffe- renzierungen sind am rechten Tentakel deutlicher als am linken. — Die Statocysten liegen als rundliche Bläschen auf dem Hinterrande der Pedal- ganglien; ihre Wandung besteht aus einer äußeren, bindegewebigen Hülle und aus einer inneren Schicht von Sinneszellen, welche wahrscheinlich keine Cilien tragen. Im Innern finden sich die aus kohlensaurem Kalk bestehenden Statolithen, meist in großer Zahl; im Jugendstadium des Tieres kommt wahrscheinlich nur ein einziger großer Statolith vor. Das Osphradium ist eine langgestreckte, bewimperte Rinne, welche bei Zimaeina links, bei den Cavoliniiden rechts gelagert ist. Am Darmkanal (Fig. 4) können wir Vorderdarm, Mitteldarm und Enddarm unterscheiden. Die erste Abteilung fängt mit dem in der Längs- richtung des Körpers verlaufenden, spaltförmigen Munde an, der zwischen den Seitenlappen des Fußes gelegen ist. An den Seitenwänden der Mund- höhle liegt jederseits ein kleines, aus mehreren hintereinander geordneten Lamellen bestehendes Plättchen, der Oberkiefer. Hinter den Oberkiefern liegt ventral die Radulatasche, welche die kurze, aus nur etwa 10 Quer- reihen sich zusammensetzende Radula birgt; jede Querreihe besteht aus drei Zähnchen: aus einem Mittelzahn, der auf einer starken Basalplatte meist einen einzigen kräftigen Fortsatz trägt, und aus zwei hakenartig gebildeten Seitenzähnen. In der Mundhöhle münden jederseits der Radulatasche die Speicheldrüsen, kleine, schlauchförmige Organe, deren Wandung aus großen Drüsenzellen besteht; mit emem kurzen Ausführungsgang münden sie in die Mundhöhle ein. — Der Oesophagus ist sehr verschieden lang und längs- gefaltet; unter ganz allmählicher Erweiterung geht er schließlich in den Magen über; bisweilen aber (Limacina, Creseis) ist die Übergangsstelle scharf markiert. Pteropoda: A. Thecosomata, a) Euthecosomata — Die Buccalganglien liegen unmittelbar vor den Pedalganglien, also auf der Ventralseite des Oesophagus; mit den Cerebralganglien sind sie durch kurze Konnektive verbunden. Meist sind die beiden Ganglien zu einer äußerlich einheitlichen Masse verschmolzen. Die Cerebralnerven innervieren den Schlund, die ventrale und seitliche Kopfpartie und die Tentakeln, wahrscheinlich auch die Statocyste. Die Pedalnerven versorgen die Fußlappen und die Flossen. Die Nerven von der Visceralganglienmasse entspringen asymmetrisch: bei den Cavo- liniiden entspringt vom rechten (kleineren) Ganglion ein einziger Nerv zu der rechten Körperwand und der entsprechenden Mantelseite; auch sendet er einen Ast nach dem rechts gelagerten Osphradium (Fig. 4). Vom linken Visceralganglion gehen verschiedene Nerven ab, einige gehen zu der linken Körperhälfte und der linken Mantelseite, andere senken sich in die Eingeweide hinein. Daß aus dem größeren Visceralganglion mehr Nerven entspringen kommt daher, daß ersteres das ursprünglich getrennte Abdominalganglion in sich aufgenommen hat. Nur bei Ayaloeylis striata kommt jederseits nur ein einziger Visceralnerv vor. Zwischen Pedal- und Visceralganglion, an der Stelle, wo das mit dem Cerebralganglion verschmolzene Pleuralganglion sich den ventralen Ganglienmassen anlegt, geht noch ein zarter Nerv zu den Seitenteilen des Körpers (bei Clio pyramidata und Ayaloeylis striata). Bei den Limaeiniden ist der Ur- sprung der Visceralnerven (3rechts, 1 links) fast genau entgegengesetzt; nur wird das Osphradium ebenfalls vom rechten Ganglion aus innerviert. Die Buccal- ganglien versorgen Schlund, Radulatasche, Oesophagus und Magenwandung. Von Sinnesorganen können wir die paarigen Tentakeln, die gleichfalls paarigen Statocysten und das unpaare Osphradium unterscheiden. Die Pteropoda: A. Thecosomata, a) Euthecosomata g Tentakeln liegen auf der Dorsalseite des Kopfes, dicht an der Medianebene; sie sind asymmetrisch ausgebildet, da fast stets der rechte größer ist als der linke (besonders bei Zimacina und Creseis). Der rechte Tentakel ist oft in einer Scheide eingeschlossen. An der Spitze beider Tentakeln finden wir (sehr deutlich bei Cavolinia longirostris) ein rudimentäres Auge, indem das einfache Plattenepithel der Oberfläche hier einen Hohlraum bedeckt, welcher wahrscheinlich von einer durchsichtigen, homogenen Flüssigkeit erfüllt ist und am Boden ein hohes, mehrschichtiges Epithel zeigt (die eigentlichen Sinneszellen); unterhalb dieses Epithels, aber durch eine Membran davon geschieden, liegt ein Komplex großer Ganglienzellen. Pigmentzellen fehlen oder sind vielleicht nur auf jüngerem Stadium vorhanden. Diese Diffe- renzierungen sind am rechten Tentakel deutlicher als am linken. — Die Statocysten liegen als rundliche Bläschen auf dem Hinterrande der Pedal- ganglien; ihre Wandung besteht aus einer äußeren, bindegewebigen Hülle und aus einer inneren Schicht von Sinneszellen, welche wahrscheinlich keine Cilien tragen. Pteropoda: A. Thecosomata, a) Euthecosomata — Der Magen hat birnförmige Gestalt, liegt gewöhnlich schief zur Längsachse des Körpers und trägt im hinteren Abschnitt die charakteris- tischen Kauplatten. Konstant ist immer ein Kranz von vier großen Kau- platten, von denen zwei ventral und zwei dorsal gelegen sind; sie haben länglich-rechteckige Gestalt und tragen an ihrer Oberfläche eine weit vor- springende Crista. Eine fünfte, kleinere Kauplatte liegt etwas weiter nach hinten, und zwar bei ZLimacina dorsal, bei den anderen Formen ventral. Ziemlich konstant sind weiter 4—8 kleinere Plättchen, welche am Vorder- rande der großen Kauplatten liegen. — Die Leber ist beim lebenden Tier gewöhnlich grün gefärbt; sie liegt unmittelbar hinter dem Magen, und zwar bei Limaecina hauptsächlich links, bei den anderen Formen rechts. Je nach der verschiedenen Gestalt des Eingeweidesackes kann sie langgestreckt, spiralig eingerollt oder rundlich (Fig. 4) erscheinen. Bei Diacria trispinosa zerfällt die Leber in zwei völlig getrennte Lappen, von denen der links gelegene die Darmschlinge enthält. Eine Andeutung einer solchen Zweiteilung finden wir schon bei der Gattung Clio; hier finden wir, ebenso wie bei Diacria, zwei Pteropoda: A. Thecosomata, a) Euthecosomata 10 deutlich getrennte Ausführungsgänge nach dem Hinterende des Magens, sonst kommt nur ein einziger Gang vor. Die Leber selbst ist eine acinöse Drüse, von einer großen Zahl kleinster Läppchen und Gänge gebildet; in den Läppchen liegen auch die eigentlichen sezernierenden Drüsenzellen. Neben der Leber mündet nun in den gleichen Darmabschnitt noch ein besonderes Anhangsorgan, der schmale, zylindrische Darmblindsack, der bei Zimaeina links, bei den anderen Formen aber rechts gelegen ist. — Der übrige Teil des Darmkanales bildet eine eigentümliche Schlinge. Bei Zimacina zieht der Enddarm zuerst nach hinten, wendet sich dann nach rechts und nach oben, kehrt sich dann nach links und nach vorn und kreuzt den Anfangsteil des Enddarmes dorsalwärts, um schließlich, nach vorn gewendet, auf der rechten Körperseite auszumünden (Fig. 2A). Gerade umgekehrt ist es bei den übrigen Euthe- <-- Flosse Dödokkeii ER 3 --- Buccalmasse \ Zentralnervensystem -.. e er En=ek „_ = Wimperlinien Pigmentfleck --------- Mr B nun Osphradium Kiemenhöhle .--- A BI FF RENT NE Kopfaorta After -----I---F.--E-SSP B—--;--3--1----}----- Magen Kammer des Herzens --:--:J- 1: tn 53 Tr Mantelnerven Kieme -----L---J--- Be m we Er leg ""-, äußere Nierenöffnung Doc, Retraktormuskel 5 7 ie Zwitterdrüse Fig. 4. Etwas schematisierte Darstellung der Anatomie einer jungen Cavolinia tridentata /„Hyalaea com= planata‘‘). Dorsale Ansicht. Dorsale Hälfte der Schale entfernt. Nach Gegenbaur, Zentralnervensystem Fig. 4. Pteropoda: A. Thecosomata, a) Euthecosomata Etwas schematisierte Darstellung der Anatomie einer jungen Cavolinia tridentata /„Hyalaea com= planata‘‘). Dorsale Ansicht. Dorsale Hälfte der Schale entfernt. Nach Gegenbaur, cosomen; hier wendet sich der Enddarm bald nach dem Austritt aus dem Magen zuerst nach links, beschreibt dann einen nach dem Hinterende des Körpers zu konvexen Bogen, wendet sich also nach rechts, dann nach vorn, schließlich nach links und kreuzt seinen Anfangsteil ventralwärts; der After (Fig.22, Fig. 4) mündet an der linken Körperseite. Der Anfangsteil der Darm- schlinge ist meist ganz von der Leber umschlossen; wie gesagt liegt er bei Diacria in dem linken Leberlappen. Im Verlaufe der Schlinge tritt im Innern des Darmes ein weit vorspringender Längswulst auf. Uber die Anal- drüse wurde schon oben (S. 5) gesprochen. Das vom Pericard umschlossene Herz besteht aus Kammer und Das vom Pericard umschlossene Herz besteht aus Kammer und Vorhof und liegt auf der einen Körperseite an der Oberfläche des Ein- geweidesackes, in der Mantelhöhle. Bei ZLimaeina (Fig. 5 A) liegt es an der linken Körperseite, der Vorhof ist nach links, die Kammer nach rechts ge- wendet. Bei Creseis (Fig. 5) liegt es rechts und ventral; von hier aus Pteropoda: A. Thecosomata, a) Eutheeosomata 11 läßt sich nun eine allmähliche Verschiebung des Herzens über die Ventralseite hinweg nach links feststellen. Ein Übergangsstadium finden wir bei Hryalocylis (Fig.5 C); ‚hier ist das Herz schon quer gelagert und die bisher (von Creseis an) nach hinten gerichtete Kammer hat sich jetzt nach links gewendet. Völlig vollendet ist der Prozeß bei Clio (Fig. 5D) und bei Cavolinia (Fig. 4); das Herz liegt hier entschieden links, die Kammer direkt nach vorn, der Vorhof - Mantelhöhlendrüse Niere -- Vorhof des Herzens -- N Kammer des Herzens \| Mantelhöhlendrüse ------- Vorhof des Herzens Kammer des Herzens -E Mantelhöhlendrüse " Niere - . Zwitterdrüse ---- C Fig. 5. Schematische Darstellung der Lagerungsverhältnisse von Herz und Niere. A bei Limacina, B bei Creseis, C bei Hyalocylis, D bei Clio. A dorsal, B und C ventral, D von links betrachtet. Nach Boas. nach hinten gerichtet. Während die Wände des Vorhofes äußerst zart und nur von feinen Muskelfasern gestützt erscheinen, besitzt die Wandung der Kammer eine mächtige, innere Muskellage. Zwischen den beiden Abteilungen findet sich eine Atrioventrikularklappe. nach hinten gerichtet. Während die Wände des Vorhofes äußerst zart und nur von feinen Muskelfasern gestützt erscheinen, besitzt die Wandung der Kammer eine mächtige, innere Muskellage. Zwischen den beiden Abteilungen findet sich eine Atrioventrikularklappe. Das periphere Gefäßsystem ist sehr einfach, besondere Venen kommen nur bei Cavolinia an der Basis der Kiemen vor; sonst Öffnet sich der Vorhof unmittelbar in die lakunären Räume des Körpers. Die Arterien sind besser ausgebildet (Fig. 4). Am Anfang der Pteropoda: A. Thecosomata, a) Euthecosomata Das periphere Gefäßsystem ist sehr einfach, besondere Venen kommen nur bei Cavolinia an der Basis der Kiemen Vorhof de N Kammer de Mantelhöhlendrüse ------- Vorhof des Herzens Kammer des Herzens -E Vorhof des Herzens -- Kammer des Herzens \| Vorhof des Herzens Kammer des Herzens - Kammer des Herzens -E Mantelhöhlendrüse " Niere - . Zwitterdrüse ---- C Fig. 5. Schematische Darstellung der Lagerungsverhältnisse von Herz und Niere. A bei Limacina, B bei Creseis, C bei Hyalocylis, D bei Clio. A dorsal, B und C ventral, D von links betrachtet. Nach Boas. Mantelhöhlendrüse " Niere - . Zwitterdrüse ---- C Mantelhöhlendrüse " C arstellung der Lagerungsverhältnisse von Herz und Niere. A bei Limacina, B bei yalocylis, D bei Clio. A dorsal, B und C ventral, D von links betrachtet. Nach Boas. Schematische Darstellung der Lagerungsverhältnisse von Herz und Niere. A bei Limacina, B bei Creseis, C bei Hyalocylis, D bei Clio. A dorsal, B und C ventral, D von links betrachtet. Nach Boas. nach hinten gerichtet. Während die Wände des Vorhofes äußerst zart und nur von feinen Muskelfasern gestützt erscheinen, besitzt die Wandung der Kammer eine mächtige, innere Muskellage. Zwischen den beiden Abteilungen findet sich eine Atrioventrikularklappe. Das periphere Gefäßsystem ist sehr einfach, besondere Venen kommen nur bei Cavolinia an der Basis der Kiemen vor; sonst Öffnet sich der Vorhof unmittelbar in die lakunären Räume des Körpers. Die Arterien sind besser ausgebildet (Fig. 4). Am Anfang der Pteropoda: A. Thecosomata, a) Eutlecosomata 12 Aorta findet sich eine nach der Aorta hin öffnende Klappe; diese Aorta teilt sich, nachdem sie sich gleich rechtwinklig von der Kammer nach dem Eingeweidesack hingewendet hat, in zwei Hauptäste. Der eine derselben versorgt die Eingeweide, ein starker Ast geht zudem zu der Leber; der zweite Ast zieht nach vorn, verteilt sich an den im Kopf gelegenen Organen und gibt namentlich die Gefäße zur Versorgung der Flossen ab. Das Blut gelangt so schließlich in die allgemeinen Lakunenräume des Körpers, aus welchen es endlich zum Vorhof zurückgeführt wird. In dem schon oben erwähnten Diaphragma in der Halsgegend finden wir bei Cavolinia, und zwar auf der linken Seite, besondere Kommunikationsöffnungen zwischen Kopf- und Eingeweidesinus; diese Öffnungen sind durch Klappen verschließbar. Das Blut selbst ist wasserhell und enthält nur wenig Blutkörperchen. Respirationsorgane sind in weitaus den meisten Fällen gar nicht Respirationsorgane sind in weitaus den meisten Fällen gar nicht ausgebildet; die Respiration erfolgt direkt an den Wänden der Mantelhöhle. Pteropoda: A. Thecosomata, a) Euthecosomata Nur bei Cavolinia (Fig. 4) sind wirkliche Kiemen ausgebildet. Die Kieme stellt hier ein langes, schmales, aus zahlreichen Falten sich zusammensetzendes Band dar, welches ganz im Innern der Kiemenhöhle gelegen ist; sie ist hauptsächlich auf der linken Seite (wo das Herz gelegen ist) entwickelt, bildet von hier aus nach unten und rechts einen nach vorn hin konkaven mächtigen Bogen und setzt sich auf der rechten Mantelseite bis in den dorsalen Teil der Mantelhöhle fort; so entsteht ein fast geschlossener Kreis- bogen. Die Kieme besteht aus zahlreichen, büschelförmigen Falten, die durch ein auf dem Kamm der Kieme verlaufendes Blutgefäß (Venenstamm) verbunden sind. Diese Falten, welche bald mehr, bald weniger kompliziert erscheinen, werden durch eine mehrmals gefaltete Ausstülpung des Mantelepithels gebildet. Ganz wie das Herz, weist die mit ihm innig verbundene Niere eine bei Ganz wie das Herz, weist die mit ihm innig verbundene Niere eine bei den einzelnen Gattungen wechselnde Lagerung auf. Ursprünglich liegt sie links- seitig, im vorderen Körperteil, wie bei Zimacina (Fig. 5A). Hier liegt sie proximal vom Herzen; die äußere Nierenöffnung liegt ganz vorn, die Reno- pericardialöffnung am Hinterende. Bei Creseis (Fig. 5 B) ist die Niere rechts gelegen, und zwar vor dem Herzen; von dieser Gattung aus können wir bei der Niere wieder die gleiche Wanderung beobachten, wie sie beim Herzen gefunden wird, nur mit dem Unterschiede, daß sie nie ganz nach der linken Seite hinüberzieht, sondern sich der Mittellinie nähert (Fig. 5 C und D) und schließlich, bei Diaeria und Cavolinia (Fig. 4) ganz quer auf der Ventralseite gelagert ist. Die Öffnung in die Mantelhöhle findet sich am Ende des rechten Zipfels, die Renopericardialöffnung am linken in der Nähe des Vorhofes. Die äußere Gestalt wechselt sehr: bei Limacina (Fig. 5A) stellt sie einen platten, länglich-dreieckigen Schlauch dar, mit der Basis nach vorn gewendet; noch mehr schlauchförmig ist sie bei Oreseis und Ayaloeylis (B u. C) ge- staltet; halbmondförmig gekrümmt, mit der konkaven Seite nach vorn ge- wendet ist sie bei Diacria und Cavolinia (Fig. 4). Bei Clio (Fig. 5.D) besteht sie aus zwei durch eine schmale Brücke verbundenen, nach hinten ziehenden Schenkeln. Beim lebenden Tier führt die Niere regelmäßige Schluekbewegungen aus; das aufgenommene Wasser gelangt aber nicht in den Pericardialsinus, ebenso wenig wie von diesem Sinus aus eine Kom- munikation mit dem Lumen des Herzens besteht. Das Genitalsystem setzt sich aus Zwitterdrüse, Zwittergang. An- hangsdrüsen, Ausführungsgängen und Penis zusammen. Pteropoda: A. Thecosomata, a) Euthecosomata — Die Zwitterdrüse nimmt stets, mit Ausnahme von Creseis acieula, den hinteren Abschnitt des Eingeweidesackes ein (Fig. 4 u. 5). Ihre Gestalt ist, entsprechend der Form des Eingeweidesackes, sehr wechselnd, bald spiralig gewunden, bald gestreckt Pteropoda: A. Thecosomata, a) Euthecosomata 13 und dann rundlich, länglich, sehr lang ausgezogen oder asymmetrisch nach der linken Seite entwickelt. Sie zerfällt in eine Reihe übereinander gelagerter Follikel, die zuweilen äußerlich sehr deutlich hervortreten. Die Anordnung der Geschlechtszellen ist verschieden: bei Zimaeina und bei Creseis liegen die weiblichen Elemente, die Eier, außen an der Peripherie, die männlichen im Zentrum; bei Ayalocylis ist die Anordnung gerade umgekehrt. Dies kommt daher, daß bei den höher entwickelten Formen die anfänglich im Querschnitt rundliche Zwitterdrüse sich seitlich verbreitert, und die seitlichen Flügel, welche nur Samenelemente enthalten, sich um die Eizone herum- schlagen. In der Entwicklung reifen die Samenelemente zuerst. — Der Zwittergang entspringt bei Limacina (Fig. 2A) an der vorderen Ventral- seite der Zwitterdrüse und zieht auf der rechten Seite des Körpers nach vorn, bei allen übrigen Formen nimmt er seinen Ursprung, wenngleich ganz ins Innere der Drüse verlagert, an der Dorsalseite der Zwitterdrüse, zieht an der linken Seite des Körpers nach vorn (Fig. 2), kreuzt den hinteren Abschnitt des Oesophagus ventralwärts und gelangt so gleichfalls auf die rechte Seite hinüber. Bald nach dem Ursprung erweitert sich der Gang zu emer Vesicula seminalis; bei Creseis ist diese als kleiner, sackförmiger Anhang entwickelt, bei Cavolinia finden wir einen langen, aufgerollten Blind- sack. — Weiter hinauf finden wir die Genitalanhangsdrüsen; gewöhnlich sind sie sehr umfangreich und stellen äußerlich einen viel gefalteten Komplex dar. der in sich Schalendrüse, Eiweißdrüse, sowie Receptaculum seminis enthält. Die Schalendrüse ist am weitesten nach links gelegen und bildet die Hauptmasse; rechts liegt die kleinere Eiweißdrüse, welche mit einer Haupt- und mit einer sekundären Öffnung in die Falten der Schalendrüse einmündet; bei Zimacina behält sie noch eine gewisse Selbständigkeit, bei den höheren Formen aber wird sie immer mehr von der Schalendrüse um- schlossen. Rechts mündet in eine Falte der Schalendrüse das verschiedene Gestalt aufweisende Receptaculum seminis, das aber auch fehlen kann. — Meistens findet sich nur ein einziger Geschlechtsausführungsgang, der gewöhnlich ganz nahe der Stelle, wo der Zwittergang in die Anhangsdrüsen einmündet, seinen Ursprung nimmt. Er öffnet sich auf der rechten Seite des Kopfabschnittes (Fig. 2), etwa auf der Höhe der Buccalmasse, nach außen. Pteropoda: A. Thecosomata, a) Euthecosomata Das Velum kann sehr verschieden ausgebildet sein: während bei Creseis jeder der beiden Segellappen sich nochmals durch eine tiefe Einschnürung am freien Rande geteilt hat und so das Ganze eine vierstrahlige Figur bildet, bleiben die Lappen der Cavoliniidae klein und ungeteilt. Die Schale erscheint anfangs als kleines, uhrglasförmiges Gebilde, das sich bald vertieft und becherförmig wird. Der Zuwachs erfolgt dadurch, daß sich an den freien Rand neue Lagen ansetzen. Die ursprüngliche Larvenschale ist bei den gestreckten Formen stets durch eine Einschnürung gegen die definitive Schale abgegrenzt; bei Cavolinia wird die Larvenschale durch eine Quer- wand vom übrigen Teile isoliert, nachdem ac Spitze des Körpers aus dem hinteren Abschnitt zurückgezogen hat, und später häufig abgeworfen. Bei den Lima- einiden ist die Larvenschale gleich spiralig gewunden und geht direkt in die bleibende Schale über. Die Anlage des Fußes läßt seitlich die Flossen hervorsprossen, welche bald beträchtlich an Umfang zunehmen. Am Darmkanal ist bemerkenswert, daß der Mitteldarm links und rechts eine Aussackung liefert, deren große, mit Dotter erfüllten Zellen das Nährmaterial liefern. Die Leber entsteht unabhängig von diesen Säcken als unpaare Ausstülpung des Mitteldarmes. Der After, der bei den erwachsenen Cavoliniiden bekanntlich an der linken Seite liegt, ist hier anfänglich genau in der Mittellinie, hinter dem Fuße gelegen. Mit der zunehmenden Ausbildung der Flossen erfährt das Velum eine allmähliche Rückbildung. Wie schon oben erwähnt wurde, ist es jetzt wohl nicht zweifelhaft mehr, daß Wie schon oben erwähnt wurde, ist es jetzt wohl nicht zweifelhaft mehr, daß wir die gewundenen Limaeiniden als die ursprünglicheren Formen ansehen müssen!) ; sie ermöglichen den Anschluß an verwandte Gastropodenformen (Bulla ete.). Es ist nicht nur die Aufwindung von Schale und Körper, mit der dorsal gelegenen Mantel- höhle und dem Besitz eines Operculum verbunden, welche die Limaciniden allen anderen Euthecosomen gegenüber stellt, sondern auch der Umstand, daß in den beiden Familien bei einer großen Zahl von Organen des Eingeweidekomplexes ein Gegensatz in der Lage nachzuweisen ist, insoweit als das eine Organ hier rechts, dort links, das andere hier dorsal, dort ventral gelagert erscheint. Diese entgegengesetzten Lagerungs- verhältnisse lassen sich nun darauf zurückführen, daß bei den Cavoliniiden im Vergleich mit den Limaciniden eine Drehung um 1800 des ganzen Eingeweidekomplexes um die Längsachse stattgefunden hat (Fig. 2). Pteropoda: A. Thecosomata, a) Euthecosomata Beide Geschlechtsprodukte werden längs dieses Weges entleert; indessen ist die Öffnung wesentlich als weibliche Geschlechtsöffnung zu be- zeichnen, insofern als sie nicht nur die reifen Eier nach außen entleert, sondern auch bei der Begattung den Penis des anderen Individuums auf- nimmt. Die Samenzellen werden zwar ebenfalls durch diese Öffnung nach ‘außen befördert, sie werden aber von hier durch eine auf der Rückenfläche des Kopfabschnittes und der Flossen nach vorn verlaufende Rinne nach der Öffnung des Penis übergeleitet. — Dieser liegt von dem übrigen Genital- apparat völlig getrennt im vorderen Kopfabschnitt und kann im Stadium seiner höchsten Entwicklung einen sehr beträchtlichen Umfang erreichen. In seinem hinteren Abschnitt bildet er verschiedene Blindsäcke, welche im Inneren stilettartige Bildungen einschließen; diese scheinen aber Üreseis vir- gula und der Gattung Cavolinia zu fehlen. Bei Cuvierina besteht ein akzes- sorisches Hilfsorgan für die Begattung, das aber mehr median und ventral verlagert ist, unterhalb des mittleren Fußlappens. Es besteht aus einem Stiel, der sich in zwei blattartige Verbreiterungen fortsetzt, von denen die eine länglich und zugespitzt, die andere fächerförmig und am Rande eingerollt erscheint. — Erwähnenswert ist weiter noch, daß bei Cavolinia longirosiris eine Falte der Schalendrüse mit der ventralen Mantelhöhle 14 Pteropoda: A. Thecosomata, a) Euthecosomata kommuniziert und sich hier auf einer Papille öffnet; diese Öffnung muß als ausschließlich weibliche Geschlechtsöffnung betrachtet werden. Die Nahrung besteht aus Protozoen und planktonischen Algen; nur gelegentlich werden andere Thecosomen oder Copepoden im Magen gefunden. — Die Befruchtung, welche aber nie wahrgenommen wurde, erfolgt sehr wahrscheinlich zwischen zwei verschiedenen Individuen mittels des gut ausgebildeten Penis. Die Eier werden in einfachen, glashellen Schnüren, von ein paar Zentimeter Länge, welche viele Eier enthalten können, abgelegt; diese Schnüre treiben frei herum. Nach der zweiten Furchung, bisweilen schon nach der ersten, treten Differenzen in der Größe der Blastomeren hervor; eine Furchungskugel teilt sich in einer Anzahl kleiner Zellen weiter. Es entsteht eine epibolische Gastrula. Bei der Larve fällt ein Wimperring am Vorderende auf, der sich bald in die beiderseitigen Segellappen sondert; am ent- gegengesetzten Ende des Körpers entsteht durch Ektodermeinstülpung die Schalen- drüse, welche natürlich mit dem gleichnamigen Organe bei dem erwachsenen Tiere (Genitalanhangsdrüse) nichts gemein hat. Der Körper wird allmählich zylindrisch und von einer dünnen Schale umkleidet. In der einen ventralen Einbuchtung des Wimper- segels wächst der Fuß hervor, und gleich vor dieser Stelle, am Rande des Segels, ent- steht der Mund. !) Nur Schiemenz betrachtet Oreseis als die Urform, aus welcher sich einerseits die anderen Cavoliniiden, andererseits die Limaciniden entwickelt haben. Bestimmungstabelle der Familien: Schale spiralig, links gewunden, mit spiraligem Oper- eulum. Mantelhöhle dorsal (Fig. 1A)... .... 1. Fam, Limacinidae Schale etwas gebogen oder gerade gestreckt, ohne 1 Schale spiralig, links gewunden, mit spiraligem Oper- eulum. Mantelhöhle dorsal (Fig. 1A)... .... 1. Fam, Limacinidae Schale etwas gebogen oder gerade gestreckt, ohne Opereulum. Mantelhöhle ventral (Fig. 1D—-G) . . 2. Fam. Cavoliniidae 1 Schale spiralig, links gewunden, mit spiraligem Oper- eulum. Mantelhöhle dorsal (Fig. 1A)... .... 1. Fam, Limacinidae Schale etwas gebogen oder gerade gestreckt, ohne Opereulum. Mantelhöhle ventral (Fig. 1D—-G) . . 2. Fam. Cavoliniidae 1 Schale etwas gebogen oder gerade gestreckt, ohne Opereulum. Mantelhöhle ventral (Fig. 1D—-G) . . 2. Fam. Cavoliniidae 1 1817 „Les Limacines“, G.Cuvier, Rögne an., v.2 p.380 \| 1819 Limacina, Lamarck, !Hist. An. s. Vert., 0.61 p.290 \| 1823 Heterofusus, J. Fleming in: Mem. Werner. Soe., v.4ı1 p.500 \| 1824 Spiratella, Blainville in: Dict. Sei. nat., v.32 p.284 \| 1836 Heliconoides Pteropoda: A. Thecosomata, a) Euthecosomata So kommt es, daß zwar die Organe des vorderen Körperteiles bei den beiden Familien eine entsprechende Lage aufweisen (Fuß ventral, Flossen und Tentakeln dorsal, Mundöffnung und Radulatasche ventral, Geschlechtsöffnung rechts), daß aber das Verhalten im hinteren Körperabschnitt genau entgegengesetzt ist: bei den Limaeiniden liegt die Mantelhöhle dorsal, bei den Cavoli- niiden ventral, und wie schon wiederholt hervorgehoben wurde, ist dieser Gegensatz in der Lage auch bei einer Anzahl anderer Organe (Darmblindsack, Verhalten der 15 Pteropoda: A. Thecosomata, a) Euthecosomata, 1. Limaeinidae, 1. Limacina Darmschlinge, After, Herz, Niere, Ursprung des Zwitterganges) zu beobachten. Die Drehung des Eingeweidesackes muß von rechts nach links erfolgt sein, also über die . Ventralseite hinweg. — Unter den Cavoliniiden ist C'reseis noch den Limaeiniden am nächsten verwandt; diese Gattung zeigt in fast schematischer Weise den eben ge- schilderten Ubergang zu den beiden Familien. Von Creseis geht eine durchgehende Reihe über Styliola nach Clio, um sich dann einerseits nach Diacria, andererseits nach Cavolinia abzuspalten; letztere Gattungen sind am meisten spezialisiert (Fig. 1). Eine andere Entwicklungsreihe führt von Creseis über Hyalocylis nach Cuwvierina, deren abweichend gebaute Schale (Fig. 34, 35), welche im hinteren Teil bauchig auf- getrieben und durch eine Scheidewand in zwei Teile zerlegt wird, einen ganz anderen Typus darstellt; auch die innere Organisation (besonders die gegenseitige Lagerung des Herz-Nierenkomplexes) weist auf eine besondere Entwicklungsreihe hin. Weitverbreitet in allen Ozeanen, besonders aber in den wärmeren Gegenden, an der Oberfläche oder in geringen Tiefen. 2 Familien, 9 Gattungen, 34 sichere Arten, von denen 12 in 28 Unterarten zer- fallen und 4 unsichere Arten. Bestimmungstabelle der Familien: 1. Fam. Limacinidae 1847 Limacinidae, J. E. Gray in: P. zool. Soc. London, p.203 \| 1859 Spirialidae, Chenu, Man. Conchyl., v.1 p. 113. Schale spiralig, links aufgerollt, zerbrechlich, sehr zart, turm- artig oder mehr niedergedrückt, Mundöffnung ohne eigentliches Rostrum, Nabel vorhanden. Operculum (bei erwachsenen Tieren oft fehlend) mit nur wenigen (2 oder 3) Windungen; der Anfangs- teil dieser spiraligen Windungen liegt etwas exzentrisch, und zwar nach der Richtung der Columella der Schale hin verschoben. Tier mitungeteilten Flossen; rechter Tentakelfastimmer bedeutend größer als der linke; Mantelhöhle dorsal, Mantelhöhlendrüse asym- metrisch, rechts weiter nach hinten reichend (Fig. 54). Darm- schlinge dorsal, Anus rechts. Herz und Niere links in der Mantel- höhle. Kiemen fehlend. Im Zentralnervensystem ist die rechte Visceralganglienmasse deutlich umfangreicher als die linke. In allen Meeren. \| 2 Gattungen. Bestimmungstabelle der Gattungen: Schale dünn, durchscheinend; Flossen des Tieres an den Rändern farblos, rechter Tentakel bei weitem größer als deuimkens „ie. ata a a ER Re ..... 1. Gen. Limacina Schale glänzend kastanienbraun; Flossen des Tieres an den Rändern schwarz eingesäumt, beide Tentakeln sehr lang und von gleicher Größe . .. 2: ce... 2. Gen. Thilea 1817 „Les Limacines“, G.Cuvier, Rögne an., v.2 p.380 \| 1819 Limacina, Lamarck, !Hist. An. s. Vert., 0.61 p.290 \| 1823 Heterofusus, J. Fleming in: Mem. Werner. Soe., v.4ı1 p.500 \| 1824 Spiratella, Blainville in: Dict. Sei. nat., v.32 p.284 \| 1836 Heliconoides Pteropoda: A. Thecosomata, a) Euthecosomata, 1. Limaeinidae, 1. Limaeina 16 (part.), Orbigny in: Voy..Amer. merid., v.5ıı p.174 \| 1840 Spirialis (part.), Eydoux & Souleyet in: Rev. zool., v.3 p.235 \| 1842 Helicophora, J. E. Gray, Syn. Brit. Mus., p.59 \| 1844 Scaea, A. Philippi, Moll. Sieil., v.2 p.164 \| 1861 Protomedea (part.), O.G. Costa, Mierodoride, p. 73 \| 1869 Embolus, Jeffreys, Brit. Conch., e.5 p. 114. (part.), Orbigny in: Voy..Amer. merid., v.5ıı p.174 \| 1840 Spirialis (part.), Eydoux & Souleyet in: Rev. zool., v.3 p.235 \| 1842 Helicophora, J. E. Gray, Syn. Brit. Mus., p.59 \| 1844 Scaea, A. Philippi, Moll. Sieil., v.2 p.164 \| 1861 Protomedea (part.), O.G. Costa, Mierodoride, p. 73 \| 1869 Embolus, Jeffreys, Brit. Conch., e.5 p. 114. Schale dünn, durchscheinend; Flossen des Tieres an den Rändern farb- los, rechter Tentakel bei weitem größer als der linke. In allen Meeren, zuweilen in Schwärmen, an der Oberfläche oder in geringen Tiefen. In allen Meeren, zuweilen in Schwärmen, an der Oberfläche oder in geringen Tiefen. 1. Fam. Limacinidae 9 Arten, von welchen eine in zwei, eine andere in drei Unterarten zerfällt. Bestimmungstabelle der Arten: Schale turmartig aufgewunden; die Windungen nehmen allmählich an Umfang zu — 2. Spirale niedergedrückt; die letzte Windung allein nimmt fast den ganzen Schalenumfang ein — 5. Außenrand der Schalenöffnung etwas eckig gebogen, so daß die Öffnung trapezförmig erscheint. Nabel 2 ziemlich weit. . ed en a Ka a Pak rn 6. L. retroversa Außenrand der Schalenöffnung gleichmäßig gerundet. Nabel eng — 3. Schale an der Spitze sanft abgerundet. Nabel zwar eng, aber deutlich vorhanden . . .... .. 2... 9. L. cochlostyloides Schale spitz kegelförmig oder kreiselförmig. Nabel SR sichtbar — 4. Länge der Schale (vom Nabel bis zur Spitze gemessen) 4 nahezu gleich der Breite... . . 2 2.2 2.0. « 7. L. trochiformis Schale deutlich länger als breit...» »..... 8. L. bulimoides Schale mit regelmäßigen Transversallinien, welche FERN wellenförmige Ein- und Ausbiegungen der Wand Schale ohne Transversalstreifung. Flossen ohne zipfel- artigen Fortsatz am Vorderrande. In den wärmeren Meeren — 7. Querstreifung sehr deutlich ausgeprägt. Flossen mit zipfelartigem Fortsatz am Vorderrande. In den Polar- meeren. . .:.. ee er RAT BL AN « 1. L. helicina Querstreifung weniger deutlich, mit Spiralskulptur kom- biniert. Flossen ohne den 'zipfelarligen Fortsatz am Vorderrande. In der Übergangsregion _ zwischen warmen und kalten Strömungen . ». »..... . 5. L. rangii Ein zahnartiger Vorsprung in der Mitte des Außen- randes der Behale : 5, 12 mu 12 u Si an Außenrand der Schale ganzrandig, ohne Vorsprung —_ 8. Letzte Windung sehr stark aufgeblasen, Öffnung viel 7 höher als breit, Innenrand spiralig nach links ge- wunden =... 2 Öffnung der Schale breiter sie ih Inubnand Körkdo 4. E \| entstehen. In den kälteren Meeren — 6. . L. helicoides L. lesueurii 1. L. helicina (Phipps) Diese Art zerfällt in zwei Unterarten: la. L. helieina helicina (Phipps) 1774 Clio h., Phipps, Voy. North Pole, p-. 195 \| 1774 Clione h., Pallas, Spie. zool., fase. 10 p. 38 \| 1780 Argonauta arctica, O. Fabrieius, Fauna Groenl., p.386 \| 1819 Limaecina helicialis, Lamarck, Hist. An. s. Vert., v.61 p.291 \| 1824 Spiratella limacina, Blainville in: Diet. Sei. nat., v.32 p. 284 \| 1832 S. arctica, Deshayes in: Enc. meth., Vers v.3 p.138 \| 1841 Limacina a., H.P. C. 9 Arten, von welchen eine in zwei, eine andere in drei Unterarten zerfällt. Diese Art zerfällt in zwei Unterarten: la. L. 1. Fam. Limacinidae helieina helicina (Phipps) 1774 Clio h., Phipps, Voy. North Pole, p-. 195 \| 1774 Clione h., Pallas, Spie. zool., fase. 10 p. 38 \| 1780 Argonauta arctica, O. Fabrieius, Fauna Groenl., p.386 \| 1819 Limaecina helicialis, Lamarck, Hist. An. s. Vert., v.61 p.291 \| 1824 Spiratella limacina, Blainville in: Diet. Sei. nat., v.32 p. 284 \| 1832 S. arctica, Deshayes in: Enc. meth., Vers v.3 p.138 \| 1841 Limacina a., H.P. C. Pteropoda: A. Thecosomata, a) Euthecosomata, 1. Limaeinidae, 1. Limaeina 17 17 Möller in: Naturh. Tidsskr., v.3 p.488 \| 1852 L. helieina, Souleyet in: Rang & Souleyet, Hist. nat. Pter., p.61 \| 1871 L. pacifica, W. Dall in: Amer. J. Conch., v.7 p.138 \| 1878 L. helicina, @.O.Sars, Moll.aret. Norvegiae, p.328 t.29 {.1 \| 1906 L. h. var. typica, Meisen- heimer in: D. Südp.-Exsp., v.911 p.96 t.5 £.1a,1b,3 \| 1908 L. lesueuri (non Boas 1886), Tesch in: Notes Leyden Mus., ©.29 p.183 t.7 f.1—3. Möller in: Naturh. Tidsskr., v.3 p.488 \| 1852 L. helieina, Souleyet in: Rang & Souleyet, Hist. nat. Pter., p.61 \| 1871 L. pacifica, W. Dall in: Amer. J. Conch., v.7 p.138 \| 1878 L. helicina, @.O.Sars, Moll.aret. Norvegiae, p.328 t.29 {.1 \| 1906 L. h. var. typica, Meisen- heimer in: D. Südp.-Exsp., v.911 p.96 t.5 £.1a,1b,3 \| 1908 L. lesueuri (non Boas 1886), Tesch in: Notes Leyden Mus., ©.29 p.183 t.7 f.1—3. Schale sehr dünn, zerbrechlich, farblos, mit kleiner Spira, welche aber in der Höhe etwas zu variieren scheint; 5 oder 6 Windungen, durch eine ziemlich tiefe Sutur geschieden, letzte Windung sehr groß, im Durchschnitt fast zylindrischh Columella etwas nach rechts gebogen; Außenrand der Schalenöffnung regelmäßig gebogen, Innenrand gerade; Nabel sehr weit und tief, zum Teil vom Innenrande bedeckt und umkreist durch einen Kiel, der Im D ger es 28 © - 25 E8 Be "8 Zwischenplatte 3 Seitenplatte Ri \ \ \ : „balancer“ ---- ER \ \| Flosse i E F Fig. 6. L. helieina helicina. A Schale von oben, B von vorn, C von unten, D Tier in der Schale mit aus- gebreiteten Flossen (Fig. A—D 5/,), E Operculum von der Innenseite (vergr.?), F Querreihe der Radula.. A—E nach Sars, F nach Boas. Zwischenplatte Ri Zwischenplatte latte Ri \ \ \ : \ \| F Z Seitenplatte \ \ E Fig. 6. L. helieina helicina. 1. Fam. Limacinidae Operculum unbekannt, Tier nicht beschrieben. L. u. Br. der Schale bis zu 6 mm. — Fig. 7. Schale sehr dünn, glashell, fast gänzlich von der letzten Windung allein gebildet; 3 oder 4 Windungen, welche durch eine deutliche aber wenig tiefe Sutur voneinander geschieden sind; Innenlippe spiralig in der Richtung der Columella ge- bogen, Mundöffnung der Schale sehr weit, länglich-oval; Nabel sehr eng; keine Skulptur. Operculum unbekannt, Tier nicht beschrieben. L. u. Br. der Schale bis zu 6 mm. — Fig. 7. Fig. 7. L. heliooides. Schale von vorn. Nach Pelseneer (%),)., Fig. 7. heliooides Im östlichen Atlantischen Ozean an einigen Stellen, aber meist als leere Schalen gefunden; nur bei Irland und an der West- küste Afrikas lebend beobachtet. L. heliooides. Schale von vorn. Nach Pelseneer (%),)., 3. L. inflata (Orb.) 1836 Atlanta i. (non Souleyet 1852), Orbigny, Voy. Amer. merid., v. 5ıuı p. 174 t. 12 f. 16—19 \| 1840 Spirialis rostralis, Eydoux & Souleyet in: Rev. zool., v.3 p. 236 \| 1850 Limacina inflata, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.31 \| 1852 Spirialis rostralis, Souleyet in: Voy. Bonite, v.2 p. 216, Moll. t.13 f. 1—10 \| 1852 Lima- cina scaphoidea, A. Gould in: U.S. expl. Exp., v. 12 p. 485 t.51 f.602 \| 1861 Protomedea elata, O. G. Costa, Mierodoride, p. 74 t. 11 f.5 \| 1870 Embolus rostralis, Jeffreys in: Ann. nat. Hist., ser. 4 v.6 p.86 \| 1882 Protomedea r., P. Fischer in: OR. Ac. Sei., v. 94 p. 120. 3. L. inflata (Orb.) 1836 Atlanta i. (non Souleyet 1852), Orbigny, Voy. Amer. merid., v. 5ıuı p. 174 t. 12 f. 16—19 \| 1840 Spirialis rostralis, Eydoux & Souleyet in: Rev. zool., v.3 p. 236 \| 1850 Limacina inflata, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.31 \| 1852 Spirialis rostralis, Souleyet in: Voy. Bonite, v.2 p. 216, Moll. t.13 f. 1—10 \| 1852 Lima- cina scaphoidea, A. Gould in: U.S. expl. Exp., v. 12 p. 485 t.51 f.602 \| 1861 Protomedea elata, O. G. Costa, Mierodoride, p. 74 t. 11 f.5 \| 1870 Embolus rostralis, Jeffreys in: Ann. nat. Hist., ser. 4 v.6 p.86 \| 1882 Protomedea r., P. Fischer in: OR. Ac. Sei., v. 94 p. 120. 1. Fam. Limacinidae A Schale von oben, B von vorn, C von unten, D Tier in der Schale mit aus- gebreiteten Flossen (Fig. A—D 5/,), E Operculum von der Innenseite (vergr.?), F Querreihe der Radula.. A—E nach Sars, F nach Boas. auf dem Boden der letzten Windung vorspringt, auf jüngeren Stadien ist dieser Nabelkiel wenig oder gar nicht entwickelt; Oberfläche der Schale mit zahlreichen transversalen Linien, welche einander in regelmäßigen Abständen folgen. Operculum (Fig. 6 #) länglich-oval; meist bei älteren Tieren, bei welchen die Schale mehr als 3 mm breit ist, fehlend. Tier mit tentakel- artigem Fortsatz am Vorderende der Flossen. L. der Schale 6 mm, Br. 8 mm. — Fig. 6. Arktis, zirkumpolar, an den Küsten Nord-Amerikas bis zu 38° n. Br. vor- dringend. lb. L. helieina antarctica (Woodward) ?1852 L. cucullata, A. Gould in: U.S. expl. Exp., v.12 p.486 t.51 f.601 \| 1854 L. antarctica, S. P. Woodward, Man. Moll., p. 207 t.14 f.4 (deser. nulla) \| 1903 L.a., Pelseneer in: Result. Voy. Belgiea, Moll. p.29 t.6 £.70—74 (f.72 Larve, f.74 Radula) \| 1906 L. helicina var. a., Meisenheimer in: D. Südp.-Exp., v.9ı1 p. 96 1.5 f.2, Unterscheidet sich nur durch eine etwas mehr niedergedrückte Schale, durch das gänzliche Verschwinden des Nabelkieles und durch einen engeren Nabel. L. u. Br. der Schale wie bei der vorigen Unterart. Anm. Die Exemplare aus dem wärmeren Gebiete stimmen fast ganz mit der vorigen Unterart überein, so daß Übergänge tatsächlicb zu bestehen scheinen. Antarktis, an der Westküste Afrikas bis zu 310 s. Br. vordringend, Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 2 Antarktis, an der Westküste Afrikas bis zu 310 s. Br. vordringend, Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 2 18 18 8 Pteropoda: A. Thecosomata, a) Euthecosomata 1. Limaeinidae, 1. Limacina 2. L. helicoides Jeffr. 1877 L. h., Jeffreys in: Ann. nat. Hist., ser.4 v.19 p- 338 \| 1888 L. h., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.23 t.1 f.5. 2. L. helicoides Jeffr. 1877 L. h., Jeffreys in: Ann. nat. Hist., ser.4 v.19 p- 338 \| 1888 L. h., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.23 t.1 f.5. Schale sehr dünn, glashell, fast gänzlich von der letzten Windung allein gebildet; 3 oder 4 Windungen, welche durch eine deutliche aber wenig tiefe Sutur voneinander geschieden sind; Innenlippe spiralig in der Richtung der Columella ge- bogen, Mundöffnung der Schale sehr weit, länglich-oval; Nabel sehr eng; keine Skulptur. 1. Fam. Limacinidae 236 \| 1850 Limacina v., J. E. Gray, Cat. Moll. Brit. Mus., v.2 p. 32 \| 1887 L. rangii, Munthe in: Bih. Svenska Ak., v. 13 nr.2 p.8 £.12, 13. Schale dünn, durchsichtig, farblos, im allgemeinen der von ‚Limacina helieina sehr ähnlich, aber in folgenden Punkten verschieden: die Höhe der Schale ist eine etwas beträchtlichere als sie gewöhnlich bei Limacina helicina vorkommt, wenn auch einige nordische Exemplare dieser Art zuweilen die gleiche Höhe erreichen; weiter tritt die für Zimacina helicina so charakteristische Querstreifung der Schale er- \ heblich zurück, mit ihr findet sich eine Spiral- A B streifung vor, welche durch oberflächlich ein- Fig. 10. gelagerte, dunklere Körnchen hervorgerufen L.rangii. A Schale von vorn, B von wird; drittens ist zu bemerken, daß bei der unten. Nach Meisenheimer (),). hier besprochenen Art ein zipfelartiger Fort- satz am vorderen Flossenrande völlig fehlt. Der Nabel ist sehr weit und tief, ein den Nabel umziehender Kiel, wie er so oft bei ZLimacina helicina gefunden wird, fehlt hier durchaus. Br. der Schale 3 mm, L. etwas weniger. — Fig. 10. \ A B Fig. 10. \ B 10. B Fig. 10. L.rangii. A Schale von vorn, B von unten. Nach Meisenheimer (),). Fig. 10. L.rangii. A Schale von vorn, B von unten. Nach Meisenheimer (),). Fig. 10. L.rangii. A Schale von vorn, B von unten. Nach Meisenheimer (),). Es wurde diese Art vielfach mit L. lesueurii vereinigt; von dieser Art unter- scheidet sie sich aber sogleich durch die etwas mehr zugespitzte Spirale, durch die immer noch deutliche Querstreifung der Schale, welche bei L. lesueuriis so gut wie gar nicht vorkommt, vor allem aber durch den weiten, tiefen Nabel, der bei L. !. eng und spaltförmig erscheint. Eine seltene Art, welche bis jetzt nur aus dem südatlantischen Ozean, und zwar aus den Mischgebieten der warmen und kalten Strömungen nachgewiesen wurde. An der Ostküste Süd-Amerikas dringt sie bis 42°, weiter Östlich bis zu 35° s. Br. vor, an der Westküste Afrikas bis zu 33°s. Br. Die kalten antarktischen Strömungen meidet sie völlig. 6. L. retroversa (Flem.) Zerfällt in drei Unterarten: 6a. L. retroversa retroversa (Flem.) ?1791 Turbo lunaris, J. F. Gmelin in: Syst. Nat., ed. 13 v.6 p.3587 \| 1823 Heterofusus retroversus, J. Fleming in: Mem. Werner Soe., v.4 p.498 t.15 f.2 \| 1844 Peracle flemingii, E. Forbes in: Rep. Brit. Ass., v.13 p.249 \| 1844 Scaea stenogyra, A. Philippi, Moll. 1. Fam. Limacinidae Schale sehr dünn, flach, farblos, niedergedrückt; nur 2 oder 3 Windungen, welche ungefähr in der gleichen Ebene gelegen sind, letzte Windung sehr groß; Öffnung der Schale herzförmig; Innenrand gerade, Außenrand mit einem langen Fortsatz versehen, in dessen Längsachse oft eine rippenartige Verdickung auftritt, die Ausbuchtungen, welche dadurch am Rande der Öffnung entstehen, bisweilen durch eine außerordentlich zarte, glashelle Platte ausgeglichen; Nabel klein, aber deutlich; keine Skulptur, nur zarte Zuwachs- streifen. Tier ohne tentakelartigen Fortsatz am Vorderrande der Flossen. Br. der Schale etwa 1'5 mm, L. etwas weniger. — Fig. 8. Allgemein in tropischen und subtropischen Meeren, auch im Mittelmeer; im warmen Kap-Horn-Strom bis zu 58° s. Br. vordringend. A C A A C Fig. 8. L.inflata. A Schale von oben, B von unten, C von vorn. Nach Souleyet ("J.). Fig. 9. L. lesueurii. Schale von vorn. Nach Boas ('?/,). C C Fig. 8. Fig. 9. L. lesueurii. Schale von vorn. Nach Boas ('?/,). L.inflata. A Schale von oben, B von unten, C von vorn. Nach Souleyet ("J.). L.inflata. A Schale von oben, B von unten, C von vorn. Nach Souleyet ("J.). 4. L. lesueurii (Orb.) 1836 Atlanta I. (non Souleyet 1852), Orbigny in: Voy. Amer. merid., v. 5m p.177 t.20 £.12—15 \| 1886 Limaecina I. (non Tesch 1908), Boas in: Danske Selsk. Skr., ser. 6 v.4 p.46 t.3 f.33,34 \| 1888 L. I., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 24. Schale sehr dünn, glashell, farblos, Spira mehr aus der letzten Windung hervorragend als bei der vorhergehenden Art; 4 Windungen, die letzte große Windung nimmt mehr als ®/, des ganzen Schalenumfanges ein; Öffnung der Schale nicht besonders groß, Innenrand gerade, Außenrand regelmäßig ge- Pteropoda: A. Thecosomata, a) Euthecosomata, 1. Limacinidae, 1. Limacina 19 bogen, ohne Fortsatz; Nabel eng, spaltförmig; keine Skulptur, nur sehr zarte Zuwachsstreifen in der Gegend des Außenrandes, um den Nabel herum ver- laufen einige schwache Spirallinien. Tier wahrscheinlich ohne zipfelartigen Fortsatz am Vorderrande der Flossen. Br. der Schale etwa 2 mm, L. etwas weniger. — Fig. 9. Eine seltene Art, vorwiegend im Atlantischen Ozean vorkommend, aber auch in den wärmeren Gegenden der anderen Weltmeere gefunden. Nicht im Mittelmeere. An der Südspitze Amerikas im warmen Kap-Horn-Strom bis zu 56° s. Br. vordringend. 5. L. rangii (Orb.) 1836 Atlanta r. (non Souleyet 1852), Orbigny in: Voy. Amer. merid., v.5mı p.176 t.12 f.25—28 \| 1840 Spirialis ventricosa, Eydoux & Souleyet in: Rev. zool., v.3 p. 20 Pteropoda: A Schale kege Windungen, durc n m r . Er ro. troversa, Schalce von vom. Nach t rn In der suba 65° n. Br. bis zum Labradorstrom. 6b. L. retro v.3 p.489 \| 1851 Sp fusus balea, Mörch Spirialis b., G.O.Sa Unterscheide 8 oder 9 Windun gemeinen etwas über die ganze tentakelartigen Fo wie bei voriger U i ipe Br. etwas wen er Gelegentlich diesen beiden Unte 5 ? sie streng auseinand In der suba nördlicheren Gegen stromes bis zu 71° 6c. L. retro Souleyet in: Rev. Moll. t.13 £.20—26 Svenska Ak., v.13 n Zool. v.23 pars 65 Unterscheid wesentlichen und etwas weiter, die mehr in die Lä ganzen Schale vo Es ist diese 20 Pteropoda: A. Thecos Schale kegelförmig Windungen, durch eine nimmt der Sch mäßig rand ge tief. D aussehen . Er ro. Außenra troversa, Schalce am Ran von vom. Nach tentakel rn der Sch In der subarktische 20 20 20 Pteropoda: A. Thecosomata, a) Euthecosomata, 1. Limaeinidae, 1. Limacina Schale kegelförmig, turmartig aufgewunden, glashell, farblos; 6 oder 7 Windungen, durch eine deutliche Sutur getrennt, die letzte große Windung nimmt etwa ”/, des ganzen Schalenumfanges ein; Öffnung der Schale nicht auffallend groß; Außenrand nicht regel- mäßig gerundet, sondern deutlich winklig gebogen, Innen- rand gerade, teils den Nabel bedeckend; Nabel eng, aber tief. Die letzte Windung ist mit sehr zarten, wie punktiert aussehenden Spirallinien bedeckt, welche in der Nähe des . Er ro. Außenrandes allmählich verstreichen, bisweilen aber bis ganz troversa, Schalce am Rande der Öffnung deutlich bleiben. Tier mit kleinem, von vom. Nach tentakelartigem Fortsatze am Vorderrande der Flossen. L. rn der Schale etwa 3 mm, Br. etwas weniger. — Fig. 11. . Er ro. troversa, Schalce von vom. Nach rn Er In der subarktischen Übergangsregion des Atlantischen Ozeanes, von etwa 65° n. Br. bis zum Biskayischen Meerbusen; sie meidet aber durchaus den kalten Labradorstrom. 6b. L. retroversa balea (Möll.) 1841 L. b., H.P.C. Möller in: Naturh. Tidsskr., v.3 p.489 \| 1851 Spirialis gouldi, Stimpson in: P. Boston Soe., v.4 p.8 \| 1857 Hetero- fusus balea, Mörch in: Rink, Grönland, v.2 Anh. p. 86 \| 1878 Spirialis b., G.O.Sars, Moll. aret. Norvegiae, p. 329 t.29 £.2. B Unterscheidet sich nur durch etwas höhere Schale; 8 oder 9 Windungen; die Spiralstreifung ist im all- gemeinen etwas stärker ausgeprägt und erstreckt sich über die ganze Schale. Tier mit einem ähnlichen tentakelartigen Fortsatze am Vorderrande der Flossen wie bei voriger Unterart. L. der Schale etwa 7 mm, i iper, — Hie,’29 Br. etwas wen er Fig B Br. etwas wen er Fig Gelegentlich findet man Zwischenformen zwischen diesen beiden Unterarten, und es ist nieht immer möglich 5 ? sie streng auseinander zu halten. 4 Schale von vorn (%,), B OPerculum von der Innen- seite (vergr.?). Nach Sars. In der subarktischen Übergangsregion des Atlantischen Ozeanes, aber meist in nördlicheren Gegenden als die vorhergehende Unterart, mit den Ausläufern des Golf- stromes bis zu 71° n. Br. vordringend. 6c. L. retroversa australis (Eydoux & Souleyet) 1840 Spirialis a., Eydoux & Souleyet in: Rev. zool., v.3 p.237 \| 1852 $S.a., Souleyet in: Voy. Bonite, v.2 p. 222, Moll. 1. Fam. Limacinidae Sieil, ».2 p.164 t.25 f.20 \| 1847 Spirialis s., Loven in: Öfv. Ak. Förh., v.3 p.136 \| 1849 8. flemingii + S. macandrei + S. jeffreysii, E. Forbes & Hanley, Brit. Moll., ..2 p. 3884—386 t.57 f.4—8 \| 1850 Limacina retroversa, J. EB. Gray, Cat. Moll. Brit. Mus., v.2 p.33 \| 1869 Spirialis retro- versus, Jeffreys, Brit. Conch., v.5 p.115 t.4 f.4; t.98 £.4, 5 \| 1872 Heterofusus alexandri, A. E. Verrill in: Amer. J. Sci., ser.3 v.3 p.284 \| 1878 Spirialis retroversus, G. 0, Sars, Moll. aret. Norvegiae, p. 830 t.29 f.3; t.XVI f.19 (Radula). 20 Windun . Er troversa, von vom 20 Pteropoda: Schale k Windungen, d . Er ro. troversa, Schalce von vom. Nach 20 Pteropoda: A Schale kege Windungen, durc n m r . Er ro. troversa, Schalce von vom. Nach t rn In der suba 65° n. Br. bis zum Labradorstrom. 6b. L. retro v.3 p.489 \| 1851 Sp fusus balea, Mörch Spirialis b., G.O.Sa Unterscheide 8 oder 9 Windun gemeinen etwas über die ganze tentakelartigen Fo wie bei voriger U i ipe Br. etwas wen er Gelegentlich diesen beiden Unte 5 ? sie streng auseinand In der suba nördlicheren Gegen stromes bis zu 71° 6c. L. retro Souleyet in: Rev. Moll. t.13 £.20—26 Svenska Ak., v.13 n Zool. v.23 pars 65 Unterscheid wesentlichen und etwas weiter, die mehr in die Lä ganzen Schale vo Es ist diese t.13 £.20—26 \| 1887 Limacina balea (part., non Möller 1841), Munthe in: Bih. Svenska Ak., v.13 nr.2 p.5 £.5—7 \| 1888 L. australis, Felseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.25 t.1 £.6. Unterscheidet sich von der erstgenannten Unterart nur in ganz un- wesentlichen und nicht einmal konstanten Merkmalen: meist ist der Nabel etwas weiter, die Schale verhältnismäßig etwas höher und ihre Öffnung etwas mehr in die Länge gestreckt. Schwache Spiralstreifung kommt auf der ganzen Schale vor. L. der Schale etwa 2:5 mm, Br. etwas weniger. Es ist diese Form eigentlich nur durch ihre Verbreitung auf der entgegen- gesetzten Seite der Erdkugel, kaum aber durch morphologische Merkmale von den beiden anderen Unterarten getrennt. In der subantarktischen Region, und zwar in der Nähe der Südspitze von Süd- amerika, im Mischgebiete warmer und kalter Strömungen, hauptsächlich zwischen 55° und 58° s. Br., vereinzelt bis 42° s. Br. an der Ostküste Südamerikas, weiter in der Nähe der Kerguelen und Crozet-Inseln, etwa zwischen 48° und 63° s. Br. Zwischen den genannten Gebieten ist diese Unterart, welche wir als eine Charakterform der südlichen Übergangsregion betrachten können, bisher nicht nachgewiesen worden. Pteropoda: A. Thecosomata, a) Euthecosomata, 1. Limaeinidae, 1. Limaeina: 21 7. L. trochiformis (Orb.) 1836 Atlanta t., Orbigny in: Voy. Amör. merid., v5 p.177 t.12 £.29—31 \| 1840 Spirialis t., Eydoux & Souleyet in: Rev. zool., v.3 p- 237 \| 1850 Limacina t., J.E. Gray, Oat. Moll. Brit. Mus., v.2 p.33 \| 1852 Spirialis t., Souleyet in: Voy. Bonite, v.2 p.223, Moll. t.13 f.27—34 \| 1852 Limacina naticoides, Rang in: Rang & Souleyet, Hist. nat. Pter., t.10 £.1,2 \| 1887 L. trochiformis, Munthe in: Bih. Svenska Ak., v.13 nr.2 p.7 f.8—10. Schale sehr dünn, farblos, kegelförmig zugespitzt, kreiselförmig, Spitze stumpf; 5 Windungen, durch eine deutliche aber untiefe Sutur abgegrenzt, letzte Windung sehr umfangreich; Schalenöffnung ziemlich klein; Außenrand regelmäßig gebogen, die letzte Windung nicht weit überragend, Innenrand etwas nach links gebogen; Nabel eng aber deutlich; die Skulptur besteht aus sehr zarten Spirallinien auf der letzten Windung, welche durch schwache Zuwachsstreifen gekreuzt werden. Tier mit glattem Vorderrande der Flossen. L. der Schale 1 mm, Br. 075 mm. — Fig. 13. In allen wärmeren Meeren, von etwa 40° n. Br. bis 35° s. Br.; auch im Mittel- meer häufig. häufig. A häufig. A B B Fig. 14. L.bulimoides. Schale von vorn. Nach Munthe (®/,). B A Fig. 14. Bıe1a. L.trochiformis. 20 Pteropoda: A Schale kege Windungen, durc n m r . Er ro. troversa, Schalce von vom. Nach t rn In der suba 65° n. Br. bis zum Labradorstrom. 6b. L. retro v.3 p.489 \| 1851 Sp fusus balea, Mörch Spirialis b., G.O.Sa Unterscheide 8 oder 9 Windun gemeinen etwas über die ganze tentakelartigen Fo wie bei voriger U i ipe Br. etwas wen er Gelegentlich diesen beiden Unte 5 ? sie streng auseinand In der suba nördlicheren Gegen stromes bis zu 71° 6c. L. retro Souleyet in: Rev. Moll. t.13 £.20—26 Svenska Ak., v.13 n Zool. v.23 pars 65 Unterscheid wesentlichen und etwas weiter, die mehr in die Lä ganzen Schale vo Es ist diese A Schale von vorn, B von unten. Nach Munthe (?J,). Bıe1a. L.trochiformis. A Schale von vorn, B von unten. Nach Munthe (?J,). Bıe1a. L.trochiformis. A Schale von vorn, B von unten. Nach Munthe (?J,). Thecos., a) Euthecos., 1. Limaeinidae, 1. Limaeina, 2. Thilea; 2. Cavoliniida 22 endend; Nabel eng, aber deutlich; keine Skulptur, nur einige vom Nabel ausstrahlende quere Zuwachsstreifen. Operculum und Tier unbekannt. L. der Schale 13 mm, Br. 1 mm. — Fig. 15. endend; Nabel eng, aber deutlich; keine Skulptur, nur einige vom Nabel ausstrahlende quere Zuwachsstreifen. Operculum und Tier unbekannt. L. der Schale 13 mm, Br. 1 mm. — Fig. 15. Bisher nur in einigen leeren Schalen im Golfe von Bengalen gefunden. Bisher nur in einigen leeren Schalen im Golfe von Bengalen gefunden. Feuerland-Gebiet, Feuerland-Gebiet, Feuerland-Gebiet, 1 Art. T. procera Streb. 1908 T. p., Strebel in: Ergeb. Schwed. Südp.-Exp., v.6 nr.1 p.85 t.1 f. 14a—e, 14A—C. Schale zart, glatt, glänzend kastanienbraun, nach dem Wirbel zu etwas duukler, Spirale niedrig, Windungen 3%, an der Zahl, schräg aufgerollt und rasch erweitert, Schalenöffnung groß, mit zurück- geschlagenem Innenrande, Nabel und Deckel fehlen. Flossen des Tieres dick, fleischig, ohne tentakelartige Vorsprünge am Vorderrande, die gleichmäßig ausge- bildeten Tentakeln enden in eine schwache Verdickung, „in der ein schwarzer Fleck sichtbar ist“. L. der Schale 10 mm, Br. 11 mm. — Fig. 15a. Fig. 15a. T. procera. Schalevon Anm. Die Schale zeigt, wie auch der Autor hervorhebt, Fig. 15a. T. procera. Schalevon hai ee Strebel 7 Fig. 15a. T. procera. Schalevon hai ee Strebel 7 Anm. Die Schale zeigt, wie auch der Autor hervorhebt, Ähnlichkeit mit Limacina helicoides, von welcher das Tier nicht beschrieben ist. Anm. Die Schale zeigt, wie auch der Autor hervorhebt, Ähnlichkeit mit Limacina helicoides, von welcher das Tier nicht beschrieben ist. Am Nordufer des Beagle-Channel im Feuerland-Gebiet wurde ein einziges Exemplar aufgefunden. Am Nordufer des Beagle-Channel im Feuerland-Gebiet wurde ein einziges Exemplar aufgefunden. Bıe1a. L.trochiformis. A Schale von vorn, B von unten. Nach Munthe (?J,). L.bulimoides. Schale von vorn. Nach Munthe (®/,). 8. L. bulimoides (Orb.) 1836 Atlanta b., Orbigny in: Voy. Amör. meörid., v.5ru p.179 t.12 f.36—88 \| 1840 Spirialis b., Eydoux & Souleyet in: Rev. zool., v.3 p.238 \| 1850 Limacina b., J.E. Gray, Cat. Moll. Brit. Mus,, v.2 p.34 \| 1852 Spirialis b., Souleyetin: Voy. Bonite, v.2 p. 224, Moll. t. 13 f. 35—42 \| 1887 Limaeina b., Munthe in: Bih. Svenska Ak., v. 13 nr.2 p.9 £. 14. Schale sehr dünn, hoch kegelförmig, zugespitzt, zum größten Teil farblos; 6 Windungen, welche regelmäßig an Größe zunehmen; Öffnung ziemlich klein; Außenrand regelmäßig gebogen, nicht die letzte Windung überragend, Innen- rand etwas ausgebuchtet und oft bräunlich angehaucht; Nabel außerordentlich klein; keine Skulptur; Sutur rötlich oder bräunlich gefärbt. Tier mit glattem Vorderrande der Flossen. L. der Schale 2 mm, Br. 1 mm. — Fig. 14. Allgemein in den wärmeren Meeren, etwa zwischen 40° n. Br. und 40° s. Br., auch im Mittelmeer nicht selten; bisweilen bis an die Westküste Irlands. 4 B . ir L. ae Schale von vom, Bvon unten. Nach Tesch @\|,). 9. L. cochlostyloides Tesch 1908 L. c., Tesch in: Notes Leyden Mus., v.29 p.185 t.7 £. 6,7. Schale sehr dünn, breit, turmartig, m Schale sehr dünn, breit, turmartig, m 4 B Umriß annähernd länglich-oval, an der Spitze charakteristisch breit abgerundet; 5 oder 6 . Windungen, durch eine nicht tief einschneidende ir Sunr geschieden; letzte Windung sehr grob, L. ae Schale von etwa °/, der ganzen Schale einnehmend; Öffnung vom, Bvon unten. Nach Tesch @\|,). ziemlich klein, Außenrand nicht die letzte Windung überragend, regelmäßig gebogen, Innenrand gerade, den Nabel teil- weise verdeckend und an der Unterseite in einen kleinen und kurzen Fortsatz Schale sehr dünn, breit, turmartig, m 4 B Umriß annähernd länglich-oval, an der Spitze charakteristisch breit abgerundet; 5 oder 6 . Windungen, durch eine nicht tief einschneidende ir Sunr geschieden; letzte Windung sehr grob, L. ae Schale von etwa °/, der ganzen Schale einnehmend; Öffnung vom, Bvon unten. Nach Tesch @\|,). ziemlich klein, Außenrand nicht die letzte Windung überragend, regelmäßig gebogen, Innenrand gerade, den Nabel teil- weise verdeckend und an der Unterseite in einen kleinen und kurzen Fortsatz B ir L. ae Schale von vom, Bvon unten. Nach Tesch @\|,). ir L. ae Schale von vom, Bvon unten. Nach Tesch @\|,). 2 Pterop.: A. Thecos., a) Euthecos., 1. Limaeinidae, 1. Limaeina, 2. Thilea; 2. Cavoliniidae Pterop.: A. 1908 Thilea, Strebel in: Ergeb. Schwed, Südp.-Exp., v.6 nr. 1 p.84. Schale zart, braun gefärbt; Flossen des Tieres mit schwärzlichem Saume, beide Tentakeln sehr lang und von gleicher Größe, nur der rechte mit Scheide an der Basis. Ubrigens ist die Gattung ganz wenig bekannt. Feuerland-Gebiet, 7 Gattungen, 24 sichere Arten, von denen 8 in 19 Unterarten zerfallen, 5 un- sichere Arten. 2. Fam. Cavoliniidae 1841 Hyalidae (part.), Orbigny in: Ramon, Historia Cuba, Moll. v.1 p.70 \| 1842 Cleodoridae, J. E. Gray, Syn. Brit. Mus., p. 92 \| 1852 „Famille des Hyales“, Souleyet in: Voy. Bonite, v.2 p.98 \| 1854 Hyalaeacea, Troschel in: Arch. Naturg., v.201 p.196 \| 1856 Hyaleidae (part.), S.P. Woodward, Man. Moll., p.348 \| 1869 Cliidae (non Woodward 1856, Fischer 1881), Jeffreys, Brit. Conch., v.5 p.118 \| 1875 „Orthoconques“, Fol in: Arch. Zool. exp£r., v.4 p.177 \| 1886 Hiyalaeidae, Boas in: Danske Selsk. Skr., ser. 6 v.4 p.dl \| 1888 Cavoliniidae, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.4l. Schale gestreckt, bilateral symmetrisch, am Ende oft dorsal gebogen. Kein Operculum. Flossen bei den höheren Formen am freien Rande durch eine kleine Einkerbung geteilt; Mittellappen des Fußes breit, halbmondförmig (Fig. 3B p. 6); Mantelhöhle ventral. Spindelmuskel dorsal. Darmschlinge ventral, Anus links. Im Zentralnervensystem ist der Gegensatz zwischen rechter und linker Visceralganglienmasse meist weniger stark ausgeprägt als bei der vorigen Familie und außerdem entgegengesetzt, da die linke Ganglienmasse hier größer ist als die rechte. In allen Meeren an der Oberfläche oder in geringen Tiefen. 7 Gattungen, 24 sichere Arten, von denen 8 in 19 Unterarten zerfallen, 5 un- sichere Arten. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 1. Creseis. 23 Bestimmungstabelle der Gattungen: Öffnung der Schale im Querschnitt kreisrund — 2. \| Öffnung der Schale im («Juerschnitt nicht kreisrund, sondern mehr oder weniger abgeplattet — 3. Longitudinale Längsgrube mit verdiekten Rändern an der \| Dorsalseite der Schale; letztere immer ganz gerade . 2. Gen. Styliola Keinesalehe Längesprube , =, 2... nam. e. 0... 1. Gen, Creseis Schale durch deutliche Einschnürungen quergeringelt, schwach dorsal gekrümmt, mit ovaler Öffnung, Spitze meist abgebrochen „‚wenn vorhanden abgerundet . . . 3. Gen. Hyalocylis Schale ohne Querringe, Öffnung nieren- oderspaltförmig. — 4. Schale an der Öffnung am weitesten. . 2. 2.2... . 4. Gen. Clio Schale mehr oder weniger weit, hinter der Öffnung am weitesten — 5. Schale ziemlich weit, hinter der Öffnung allseitig bauchig aufgetrieben, so daß die Schale nr aussieht, Öffnung nierenförmig . » ». ... 5. Gen. Cuvierina Schale gleich hinter der Öffnung ventral wie ahfe&hinsen] Öffnung der Schale ein querer Spalt, der sich weit über die Seiten des Körpers ausdehnt, Dorsalrand fast stets ventral gebogen — 6. Dorsalrand der Öffnung der Schale verdickt, wenig lang 6. Gen. 2. Fam. Cavoliniidae Diacria Dorsalrand der Öffnung der Schale nicht verdiekt, ver- De a a hd ta Serremn Ha he ee ha le 7. Gen. Cavolinia 1. Gen. Creseis Rang acicula Schale kurz-kegelförmig oder nadelartig lang, aber ohne Schale im hinteren Drittel der Länge immer dorsal gebogen 1. C, virgula \| dorsale Krümmung » 1. HU RE NEN 2. C. acicula 1. C. virgula Rang Zerfällt in 2 Unterarten: la. C. virgula virgula Rang 1828 C.v., Rang in: Ann. Sci. nat., 2.13 p.316 t.17 £.2 \| 1829 ©. ungwis + (©. cornucopiae + C: caligula, Eschscholtz, Zool. Atlas, v.3 p.17 t.15 £.4,5; p.18 t.15 f.6 \| 1836 Hyalaea corniformis + H. virgula, Orbigny, Voy. Amer. merid., v.5ıu p.120, 121 t.8 f.20—22, 26—28 \| 1850 Styliola c. + 8. v., J. E. Gray, Cat. Moll. Brit..Mus., v.2 p.18, 17 \| 1852 Cleodora v., Souleyet in: Voy. Bonite, v.2 p.196, Moll. t.8 f. 18—25 \| 1852 C. placida + .C. munda + C. falcata (non Pfeffer 1880), A. Gould in: U. S. expl. Exp., v.12 p.489, 490 t.51 f. 606—608 \| 1879 C. fleca, Pfeffer in: Monber. Ak. Berlin, p.241 f.15,16 \| 1886 C. virgula, Boas in: DanskeSelsk. Skr., ser.6 v.4 p.57 t.4 f.40, 41 (Embryonalschale); t.5 £.71 (Fuß); t.6 f. 94a—o (Schale) \| 1888 Clio (Creseis) v., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.48. Schale glasartig, stets. deutlich dorsal. gekrümmt; der vordere Teil, der etwa zwei Drittel der Länge der Schale einnimmt, ist gerade; der hintere Teil zeigt eine mehr oder weniger schroff gegen den anderen Teil der Schale abgesetzte, dorsale Krümmung. In dieser Gegend zeigt die Schale zwei ziemlich deutliche Einschnürungen, welche die Embryonalschäle von dem übrigen Teil sondern; die Spitze ist etwas rötlich gefärbt. Beim Tiere ist der Oesophagus sehr lang, Magen und Leber liegen (nach Pelseneer) ganz hinten in der Schale, noch weit hinter dem distalen Rande der Mantelhöhlen- drüse. L. der Schale 6 mm, Br. an dem Munde 2 mm. — Fig. 16. In allen wärmeren Meeren häufig, vereinzelt im Nordatlantischen Ozean bis 60° n..Br. (Irminger See) beobachtet. Use Use Fig. 17. C virgula conica. Schale von links (etwa°/,). Nach Boas. e Bi 16. Bi 16. €. virgula virgula. A und B Schalen verschiedener Individuen, von links (etwa ®/,), © Embryonalschale (vergr.?), D Flossen (vergr.?). Nach Boas. lb. C. virgula conica Esch. 1829 C.c., Eschscholtz, Zool. Atlas, «3 p.17 t.15 f.3 \| 1830 C. striata (non Rang 1828), Chiaje, Mem. Stor. Not., t.82-f.12 \| 1869 ©. conica, A. Costa in: Rend. Acc. Napoli, v.8 p.58 \| 1872 Styliola vitrea, A.E. Verrill in: Amer. J. 1. Gen. Creseis Rang 1828 Oreseis (part.), Rang in: Ann. Sei. nat., v.13 p.302 \| 1836 Hyalaea (part.), Orbigny, Voy. Amer. merid., v.5ıı p.77 \| 1850 Styliola (part.), J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.17 \| 1852 Cleodora (part.), Souleyet in: Voy. Bonite, v.2 p. 194 \| 1888 Creseis (Subgen.), Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.47. Schale gestreckt, kegel- oder nadelförmig, oft im hinteren Teile dorsal aufgebogen, Öffnung im (Querschnitt kreisförmig. Spitze (Embryonalschale) lang, aber nicht scharf, sondern abgerundet, durch eine oder zwei schwache Einschnürungen von dem übrigen Teile getrennt: Schale fast immer ohne Skulptur. An dem Tiere selbst kommen noch viele Übereinstimmungen mit der Organisation der vorigen Familie vor oder solche, welche direkt davon abzuleiten sind: der linke Tentakel ist verschwindend klein, die Flossen sind ungeteilt; sie tragen am Vorderrande einen kleinen, tentakelartigen Fortsatz, welcher unter den Cavoliniidae charakteristisch für diese Gattung zu sein scheint. Herz und Niere liegen ventral und rechts; der Vorhof des Herzens ist nach vorn, die Kammer nach hinten gewendet. Vorzugsweise in den wärmeren Meeren, an der Oberfläche oder in geringen Tiefen. 4 Arten, von welchen zwei in je zwei Unterarten zerfallen. Bestimmungstabelle der Arten: 1 Schale im Vorderteile mit ganz feinen Querlinien . ... . 3. C. chierchiae Schale ohne derartige Querlinien, ganz glatt — 2. Schale kegelförmig, von der Spitze bis zum Mundrande sich gleichmäßig erweiternd — 3. Schale im vorderen Drittel der Länge plötzlich becher- Taeuyer erweitert. 0 nme neun . . 4. C. caliciformis 2 Vorzugsweise in den wärmeren Meeren, an der Oberfläche oder in geringen Tiefen. Vorzugsweise in den wärmeren Meeren, an der Oberfläche oder in geringen Tiefen. 4 Arten, von welchen zwei in je zwei Unterarten zerfallen. 4 Arten, von welchen zwei in je zwei Unterarten zerfallen. 1 Schale im Vorderteile mit ganz feinen Querlinien . ... . 3. C. chierchiae Schale ohne derartige Querlinien, ganz glatt — 2. Schale kegelförmig, von der Spitze bis zum Mundrande sich gleichmäßig erweiternd — 3. Schale im vorderen Drittel der Länge plötzlich becher- Taeuyer erweitert. 0 nme neun . . 4. C. caliciformis 2 24 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 1. Creseis Schale kurz-kegelförmig oder nadelartig lang, aber ohne Schale im hinteren Drittel der Länge immer dorsal gebogen 1. C, virgula \| dorsale Krümmung » 1. HU RE NEN 2. C. 1. Gen. Creseis Rang Sei., ser.3 v.3 p.284 t.6 f.7 \| 1873 Cleodora conoidea, O. G. Costa, Fauna Reg. Napoli, An. moll. Pter. p.17 t.4 f.6 \| 1888 Clio (Oreseis) conica, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.50 t.2 £.1,2. Schale farblos, sehr schwach dorsal gekrümmt, die er ist nicht scharf abgesetzt, sonderh erfolgt allmählich. Auch die beiden Einschnürungen proximal von der Embryonalschale kommen hier vor, und die Spitze ist gleichfalls rötlich angehaucht. Beim Tiere ist der Oesophagus (nach Pelseneer) kurz; Magen und Leber liegen unmittelbar hinter dem distalen Rande der Mantelhöhlendrüse. L. der Schale 7 mm. — Fig. 17. 25 Pteropoda: A. Thecosomata, a) Eutheeosomata, 2. Cavoliniidae, 1. Creseis Es kommen zahlreiche Übergänge zwischen beiden Unterarten vor, so dab es bisweilen nicht möglich ist, eine genaue Grenze anzugeben. Überall in den tropischen und subtropischen Meeren, meist zusammen mit der vorhergehenden Unterart, aber nicht so weit nach Norden hin gefunden. 2. C. acicula Rang Zerfällt in 2 Unterarten: 2a. C. acicula acicula Rang 1828 CO. a., Rang in: Ann. Sci. nat., v13 p. 318 t.17 £.6 \| 1829 ©. acus, Eschscholtz, Zool. Atlas, v.3 p.17 t.15 f£.2 \| 1836 Hyalaea aciculata, Orbigny, Voy. Amer. merid., v.5ım p.123 t.8 f.29—31 \| 1850 Styliola recta, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.18 \| 1852 Oleodora acicula, Souleyet in: Voy. Bonite, v.2 p.194, Moll. t.8 £.10—17 \| 1886 C.a., Boas in: Danske Selsk. Skr., ser. 6 v.4 p.59 t.4 f.42, 43 (Embryonalschale); t.6 f. 9$p—u (Schale) \| 1888 Clio (C'reseis) «a., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.5l. Schale glashell, immer gerade, langgestreckt, kegelförmig, bisweilen nadelförmig und dann im hinteren Teile sehr schwach. links oder rechts ge- krümmt. Auch hier nahe der Spitze zwei schwache Einschnürungen, von welchen aber die vordere bei sehr langgestreckten Exemplaren vollständig verstreicht (Fig. 18 D, E). Die Spitze ist fast immer. farblos, kann aber auch etwas rötlich angehaucht sein. L. der Schale bis zu 33 mm, Br. am Mundrande 1 mm. — Fig. 18. p Häufig in allen wärmeren Meeren, auch in den Übergangsgebieten, nördlic bis etwa 48° n. Br., allgemein im Mittelmeer. 2b. C.acieula eclava Rang 1828 C.c., Rang in: Ann. Sei.nat., v.13 p.317 t.17 £.5. 2b. C.acieula eclava Rang 1828 C.c., Rang in: Ann. Sei.nat., v.13 p.317 t.17 £.5. Schale gerade gestreckt, aber kurz-kegelförmig. L. der Schale etwa 6 mm. . .3. C. chierchiae (Boas) 1886 Cleodora c., Boas in: Danske Selsk. Skr., ser. 6 v.4 p.62 1.3 f.39 ter (Schale), t.4 £.43 bis—ter (Embryonalschale) \| 1888. Clio (Oreseis) cı, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.53 \| 1905. Creseis c., Meisen- heimer in: Ergeb. Tiefsee-Exp., v.9ı p. 17. 1. Gen. Creseis Rang Auch hier kommen Übergänge zwischen den beiden Unterarten vor, und es ist Schale gerade gestreckt, aber kurz-kegelförmig. L. der Schale etwa 6 mm. Auch hier kommen Übergänge zwischen den beiden Unterarten vor, und es ist Auch hier kommen Übergänge zwischen den beiden Unterarten vor, und es ist schwierig eine genaue Grenze zu ziehen. Verbreitung wie bei der vorigen Unterart. A WIO.DE Br °C Fig. 19. C.chierchiae. ASchale, B Embryonalschale ‘eines kürzeren, C eines längeren Exemplares. Nach Boas (vergr.?). Br °C A WIO.DE Fig. 19. C.chierchiae. ASchale, B Embryonalschale ‘eines kürzeren, C eines längeren Exemplares. Nach Boas (vergr.?). Fig. 18. Fig. 18. C. acicula acieula. A,B,C Schalen verschiedener Exem- plare, D Embryonalschale eines kürzeren, E eines sehr langen Exemplares. Nach Boas(vergr.?). C. acicula acieula. A,B,C Schalen verschiedener Exem- plare, D Embryonalschale eines kürzeren, E eines sehr langen Exemplares. Nach Boas(vergr.?). C. acicula acieula. A,B,C Schalen verschiedener Exem- plare, D Embryonalschale eines kürzeren, E eines sehr langen Exemplares. Nach Boas(vergr.?). . .3. C. chierchiae (Boas) 1886 Cleodora c., Boas in: Danske Selsk. Skr., ser. 6 v.4 p.62 1.3 f.39 ter (Schale), t.4 £.43 bis—ter (Embryonalschale) \| 1888. Clio (Oreseis) cı, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.53 \| 1905. Creseis c., Meisen- heimer in: Ergeb. Tiefsee-Exp., v.9ı p. 17. Schale gerade gestreckt, kegelförmig, farblos; das hervorragendste Merkmal besteht in den feinen Querlinien, welche in den vorderen zwei 26 Pteropoda: A. Thecosomata, a) Eutheeosomata, 2. Cavoliniidae, 1. Creseis, 2. Styliola 26 Dritteln der Schale sichtbar sind und in gleichen Abständen aufeinander folgen; die Reifung kommt durch regelmäßige Fältelung der Schalenoberfläche zustande. Auch hier finden wir vor der Embryonalschale zwei schwache Ein- schnürungen, von welchen wieder die vordere bei etwas gestreckten Exem- plaren fast gänzlich verstreicht (Fig. 18 B, C). Das Tier hat ebenso wie die der anderen Arten dieser Gattung einen kleinen tentakelartigen Fortsatz am Vorderrande der Flossen. L. der Schale 2:5 mm. — Fig. 19. Dritteln der Schale sichtbar sind und in gleichen Abständen aufeinander folgen; die Reifung kommt durch regelmäßige Fältelung der Schalenoberfläche zustande. Auch hier finden wir vor der Embryonalschale zwei schwache Ein- schnürungen, von welchen wieder die vordere bei etwas gestreckten Exem- plaren fast gänzlich verstreicht (Fig. 18 B, C). Das Tier hat ebenso wie die der anderen Arten dieser Gattung einen kleinen tentakelartigen Fortsatz am Vorderrande der Flossen. L. der Schale 2:5 mm. — Fig. 19. 1. Gen. Creseis Rang eine seichte Furche (x) gegen die übrige Schale vande (äußerer Kreis), C Em- abgesetzt, erweitert sich sodann beträchtlich, um on Nach Meisenheimer Sich von neuem einzuschnüren (> x) und mit einer (vergr.?). .. stumpfen, bräunlich gefärbten Endspitze zu enden. Fuß und Flossen des Weichkörpers zeigten bräun- liche Färbung.“ L. der Schale bis zu 10 mm. — Fig. 20. Fig. 20. rmis. AS C. calieiformis. ASchale, 8 nn 13% Haar Kreis) und am Mund. vande (äußerer Kreis), C Em- on Nach Meisenheimer (vergr.?). .. Bisher nur im südlichen Indischen Ozean zwischen Neu-Amsterdam und den Cocos-Inseln gefunden. Bisher nur im südlichen Indischen Ozean zwischen Neu-Amsterdam und den Cocos-Inseln gefunden. 1. Gen. Creseis Rang Die Querfältelung auf der Schale läßt diese Art auf den ersten Blick mit jungen Exemplaren von Hyalocylis striata verwechseln. Sie ist aber sogleich zu unterscheiden, 1. durch die ganz gerade Schale, welche keine dorsale Krümmung aufweist, 2. durch die kreisförmige Schalenöffnung, 3. durch die schmalen, ungeteilten Flossen, und 4. durch den tentakelartigen Fortsatz am Vorderrande derselben. Bisher nur in zahlreichen Exemplaren bei Panama und vereinzelt auf 10° n. Br. 137° w. L, gefunden worden, auch bei Florida. 4. C. caliciformis Meisenheimer 1905 C. c., Meisenheimer in: Ergeb. Tiefsee- Exp., v.9ı p. 308. „Die Schale ist völlig gerade gestreckt, im Querschnitt überall annähernd kreisrund. Ihr Hinter- ende läuft spitz-kegelförmig nach hinten aus und erweitert sich nach vorn hin nur langsam und all- mählich bis etwa zum vorderen Drittel der ganzen Schale. Hier tritt dann eine plötzliche und sehr starke Erweiterung auf (Fig. 20 A), welche sich bis zur Schalenmündung fortsetzt, so daß letztere den doppelten Durchmesser der Stelle, an der die Erweiterung begann, besitzt (Fig.20B, von oben gesehen). Die Schale gewinnt auf diese Weise das zierliche Aussehen eines langgestielten Weinglases. Die Oberfläche der Schale besitzt eine ganz feine Querringelung, ist aber im übrigen glänzend glatt. Die Endspitze der Schale wird gebildet durch die Embryonalschale (Fig. 20 C). Dieselbe ist durch eine seichte Furche (x) gegen die übrige Schale abgesetzt, erweitert sich sodann beträchtlich, um Sich von neuem einzuschnüren (> x) und mit einer stumpfen, bräunlich gefärbten Endspitze zu enden. Fuß und Flossen des Weichkörpers zeigten bräun- Schale bis zu 10 mm. — Fig. 20. B * c Fig. 20. B „Die Schale ist völlig gerade gestreckt, im Querschnitt überall annähernd kreisrund. Ihr Hinter- ende läuft spitz-kegelförmig nach hinten aus und erweitert sich nach vorn hin nur langsam und all- mählich bis etwa zum vorderen Drittel der ganzen Schale. Hier tritt dann eine plötzliche und sehr starke Erweiterung auf (Fig. 20 A), welche sich bis zur Schalenmündung fortsetzt, so daß letztere den doppelten Durchmesser der Stelle, an der die * Erweiterung begann, besitzt (Fig.20B, von oben c gesehen). Die Schale gewinnt auf diese Weise das zierliche Aussehen eines langgestielten Weinglases. Die Oberfläche der Schale besitzt eine ganz feine Fig. 20. Querringelung, ist aber im übrigen glänzend glatt. C. calieiformis. ASchale, 8 Die Endspitze der Schale wird gebildet durch die nn 13% Embryonalschale (Fig. 20 C). Dieselbe ist durch Haar Kreis) und am Mund. Fig. 21. . 4 Sch Fig. 21. . 4 Sch $. subula. 4 Schale von ee Sonleyet «(vergr.?). € Embryonal- De en In den tropischen und subtropischen Meeren, aber in den wärmsten Gegenden, unter dem Aquator, viel seltener. Im Mittelmeer ziemlich häufig. 2. Gen. Styliola Lesueur 1827 „Styliole“, Lesueur MS. in: Blainville, Man. Malac., Planches p. 655 \| 1827 Cleodora (part.), Quoy & Gaimard in: Ann. Sci. nat., «.10 p.233 \| 1828 COreseis (part.), Rang in: Ann. Sei. nat., v.13 p.313 \| 1836 Hyalaea (part.), Orbigny in: Voy. Amer. merid., v.5ı p.119 \| 1888 Styliola (Subgen.), Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 47, 56. Schale kegelförmig, ganz gerade, farblos, ziemlich lang, Oberfläche glatt, Öffnung kreisförmig, auf der Dorsalseite verläuft eine gerade Längsgrube, nicht in der Richtung der Längsachse der Schale, sondern im hinteren Teile Pterop.: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 2. Styliola, 3. Hyaloeylis 27 links von dieser Achse und nur an der Öffnung in der Medianlinie endend. Vor der Embryonalschale zwei schwache Einschnürungen. Beim Tiere sind die beiden Tentakeln im Nacken deutlich sichtbar, die Flossen sind breiter als bei Creseis und tragen keinen tentakelartigen Fortsatz am Vorderrande. Das Herz liegt links in der Mantelhöhle, die Kammer ist etwas nach vorn, der Vorhof nach hinten gewendet; die Niere liegt quer zu der Längsachse ‚des Körpers. In den wärmeren Meeren, an der Oberfläche oder in geringen Tiefen. \| 1. S. subula (9. &G.) ?1827 $. recta (non A. E. Verrill 1880), Lesueur MS. in: Blainville, Man. Malae., Planches p.655 (deser. nulla) \| 1827 Oleodora subula, Quoy & Gaimard in: Ann. Sei. nat., v.10 p.233 t.8D f.1—3 \| 1828 Creseis spinifera + C. subula, Rang in: Ann. Sei. nat., v. 13 p.313 t.17 £.1; t.18 £. 1] \| 1832 Oleodora subulata Quoy & Gaimard in: Voy. Astrol., v.2 p.382, Moll. t.27 f.14—16 \| 1836 Hyalaea subula, Orbigny in: Voy. Amer. mörid., v5 p.119 t.8 £.15—19 \| I 1850 Styliola s., J.E. Gray, Cat. Moll. Brit. Mus., v.2 p.17 \| 1852 Cleodora subulata, Souleyet in: Voy. Bonite, v. 2 p.192, Moll. t.8 £.5—9 \| 1883 Styliola s., P. Fischer, Man. Conchyl., p.437 \| 1888 Clio (S.) subula, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.57 \| 1889 Cleodora ($.) s., W. Dall in: Bull. U. S. Mus., nr. 37 p. 80. ? I A B c Fig. 21. Schale gestreckt, farblos, kegelförmig, der Dorsal- rand der Öffnung ragt etwas hervor, so daß die Schale bei Seitenansicht schräg abgestützt erscheint. Vor der jmbryonalschale zwei schwache Einschnürungen, die vordere nur angedeutet, Embryonalschale spitz. Tier mit ziemlich breiten Flossen. L. der Schale 10 mm, Br am Mundrande 1-5 mm, — ie 91. 3. Gen. Hyalocylis Fol Embryonalschale durch eine deutliche Einschnürung ab- getrennt, an der Spitze stumpf, abgerundet; sie Schale kegelförmig, farblos, leicht an den über der ganzen Oberfläche vorkommenden, regelmäßigen Querfalten kenntlich; im hinteren Drittel ist die Schale leicht dorsalwärts gekrümmt; Öffnung der Schale ohne vorspringende Kanten, im Querschnitt quer- oval; in der hinteren Hälfte ist der Querschnitt der Schale annähernd kreisrund. Embryonalschale durch eine deutliche Einschnürung ab- getrennt, an der Spitze stumpf, abgerundet; sie fehlt aber bei den aller- meisten Exemplaren, da C dieser Teil sehr leicht ab- Fig. 22. bricht und auch wohl ab- H. striata. A Schale mit Tier, ventraler Ansicht, B Schale von geworfen zu werden scheint; links. Nach Souleyet. © Embryonalschale. Nach Pelseneer 5 > - N (vergr.?) der hintere Teil der Schale ist durch ein Septum ver- schlossen. Das Tier ist bemerkenswert dadurch, daß die Flossen sehr breit (etwa ebenso lang wie breit) und am freien Seitenrande durch eine Ein- kerbung geteilt sind; im Winkel zwischen Vorder- und Seitenrand findet sich eine helle, durchsichtige Stelle, da hier keine Muskelfasern vorkommen. L. der Schale 8 mm. — Fig. 22. u IT C Fig. 22. H. striata. A Schale mit Tier, ventraler Ansicht, B Schale von links. Nach Souleyet. © Embryonalschale. Nach Pelseneer N (vergr.?) u IT Fig. 22. H. striata. A Schale mit Tier, ventraler Ansicht, B Schale von links. Nach Souleyet. © Embryonalschale. Nach Pelseneer N (vergr.?) schlossen. Das Tier ist bemerkenswert dadurch, daß die Flossen sehr breit (etwa ebenso lang wie breit) und am freien Seitenrande durch eine Ein- kerbung geteilt sind; im Winkel zwischen Vorder- und Seitenrand findet sich eine helle, durchsichtige Stelle, da hier keine Muskelfasern vorkommen. L. der Schale 8 mm. — Fig. 22. Die Verbindung zwischen Tier und Schale ist so locker, daß häufig Tiere ohne Schalen gefunden werden. Diese Exemplare sind aber stets noch mit Sicherheit zu erkennen, 1. durch die breiten, gekerbten Flossen, mit dem hellen Fleck am Vorder- rande (die Flossen sind nie zusammengezogen, sondern breit entfaltet), 2. durch die Abrundung am Hinterende des zylindrischen Eingeweideknäuels. Gemein in allen tropischen und subtropischen Meeren, auch im Mittelmeer. 3. Gen. Hyalocylis Fol 1828 Creseis (part.), Rang in: Ann. Sci. nat., v. 13 p.315 \| 1836 Hyalaea (part.), Orbigny, Voy. Amer. merid., v.5ıı p. 122 \| 1850 Styliola (part.), J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.18 \| 1852 Cleodora (part.), Souleyet in: Voy. Bonite, v.2 p.191 \| 1875 Hyalocylis, Fol in: Arch. Zool. exper., v.4 2.177 \| 1883 Hyalocylix, P. Fischer, Man. Conchyl., p.436 \| 1888 H. (Subgen.), Pelseneer in: Rep. Voy. Challenger, Zool. v. 23 pars 65 p. 54. Schale kegelförmig, farblos, an der Spitze etwas dorsal gebogen, im Querschnitt im hinteren Teile kreisrund, im Vorderteile quer-oval. Die ganze Oberfläche ist durch die in regelmäßigen Abständen aufeinander folgenden Querfalten in Ringe geteilt, welche sich bis zu der Embryonalschale ver- folgen lassen. Embryonalschale abgerundet. Beim Tiere ist der linke Tentakel im Nacken nicht viel kleiner als der rechte, die Flossen sind breit und mächtig, am freien Rande durch einen Einschnitt gekerbt, der Mittellappen des Fußes ist sehr breit und kurz. Das Herz liegt quer zur Längsachse des Körpers, der Vorhof ist nach rechts, die Kammer nach links gewendet; die Niere ist in der Längsachse des Körpers und etwas nach rechts gelegen. In den wärmeren Meeren, an der Oberfläche oder in geringen Tiefen. 1 Art, 28 1. H. striata (Rang) 1828 Creseis s., Rang in: Ann. Sci. nat., v.13 p.315 t.17 £.3 \| 1829 ©. compressa, Eschscholtz, Zool. Atlas, v.3 p.17 t.15 £.7 \| 1830 ©. zonata (non ©. striata), Chiaje, Mem. Stor. Not., t.82 f.9 \| 1836 Hyalaea striata, Orbigny, Voy. Amir. merid., v.5ım p.122 t.8 f.23—25 \| 1850 Styliola s., J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.18 \| 1852 Cleodora s., Souleyet in: Voy. Bonite, v.2 p.191, Moll. t.8 £.1—4\| 1854 Oreseis phaeostoma + C. monotis, Troschel in: Arch. Naturg., v.201 p. 206 t.8 f.5—7; p.208 t.8 f.8,9 (junges Exemplar ohne Schale) \| 1875 Hyalocylis striata, Fol in: Arch. Zool. exper., v.4 p.177 \| 1888 Clio (Hyalocylix) s., Pelseneer in: Rep. Voy. Chal- lenger, Zool. v.23 pars 65 p.54 t.2f.3 \| 1905 H. s., Meisenheimer in: Ergeb. Tiefsee- Exp., 0.91 p.17 t.1 f.8 (Tier ohne Schale). Schale kegelförmig, farblos, leicht an den über der ganzen Oberfläche vorkommenden, regelmäßigen Querfalten kenntlich; im hinteren Drittel ist die Schale leicht dorsalwärts gekrümmt; Öffnung der Schale ohne vorspringende Kanten, im Querschnitt quer- oval; in der hinteren Hälfte ist der Querschnitt der Schale annähernd kreisrund. 4. Gen. Clio Linne 1767 Clio, Linn& in: Syst. Nat., ed. 12 o.lır p. 1094 (non 1776 C., O. F. Müller, Zool. Dan. Prodr., p. 226) \| 1810 Cleodora, Peron & Lesueur in: Ann. Mus. Paris, v.15 p.66 \| 1823 Balantium (Children, fide Gray) in: Quart. J. Sei., v.15 p.220 (nota) \| 1836 Hyalaea (part.), Orbigny in: Voy. Amer. merid., e.5m p.111 \| 1888 Clio (Subgen.), ' Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 59. Schale farblos, gerade gestreckt oder dorsal gekrümmt, im Querschnitt nicht zylindrisch, sondern stets eckig, mit Seitenkielen, so daß eine dorsale und eine ventrale Seite sich scharf voneinander sondern. Gewöhnlich findet sich wenigstens eine dorsale Längsrippe auf der Schale, welche als eine Hervorwölbung der Schalenfläche durch zwei distal konvergierende Längs- 29 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio gruben begrenzt erscheint; die ventrale Seite der Schale ist gewöhnlich gleichmäßiger gewölbt. Öber- und Unterrand der Öffnung sind nie ein- oder auswärts gebogen und ‚unterscheiden sich nicht von den übrigen Teilen der Schale; der Oberrand ragt mehr oder weniger weit über den Unterrand hervor. Embryonalschale am Ende der Schale spitz oder abgerundet, immer deutlich abgesetzt. Die Seitenränder der Schale können konkave oder fast ganz gerade Linien in ihrem Verlaufe beschreiben, die Seitenkiele sind ent- weder flachgedrückt oder rinnenartig ausgehöhlt und erstrecken sich bis- weilen von der Mundöffnung bis nahe der Embryonalschale. — Die Mantel- ränder beim Tiere sind an den Seiten eine Strecke weit verschmolzen und bilden oft an jeder Seite eine Verlängerung des Mantels, welche sich nur wenig aus der Schale hervorstrecken läßt. Die freien Seitenränder der breiten Flossen sind durch einen einzigen Einschnitt eingekerbt. Die Lippen, welche die Mundöffnung jederseits und dorsal begrenzen (Seitenlappen des Fußes) ragen etwas über den dorsalen Rand der Vereinigungsstelle beider Flossen hervor, so daß es scheint, als ob hier eine dritte unpaare Flosse gelegen wäre. Der Unterschied in der Größe zwischen den beiden Tentakeln ist nicht sehr ausgeprägt. Das Herz liegt im hinteren Teile der Mantelhöhle, ganz auf der linken Seite; der Vorhof ist nach vorn, die Kammer nach hinten gerichtet; die Niere ist quer zur Längsachse des Körpers gestellt. Die meisten Arten sind sehr selten und scheinen gewöhnlich ein ziemlich be- schränktes Gebiet zu bewohnen; sie sind indessen in allen Ozeanen, wenn auch vor- zugsweise in der Übergangsregion zwischen warmen und kalten Gewässern, gefunden worden; ein oder zwei Arten sind in den tropischen Meeren häufig. 4. Gen. Clio Linne Sie leben nahe der Oberfläche oder in geringen Tiefen. 9 Arten, von denen 1 in 3 Unterarten zerfällt. 9 Arten, von denen 1 in 3 Unterarten zerfällt. Bestimmungstabelle der Arten: Sehale mit Seitenkielen über die ganze Länge — 2. l Seitenkiele auf der hinteren Hälfte fehlend — 6. Die dorsalen Längsrippen auf der Schale schwach, wenig ausgeprägt — 8. Die dorsalen Längsrippen auf der Schale scharf abgesetzt, deutlich — 4. 2 Hinterteil der Sehale ziemlich breit, mit dorsaler Krümmung ER ee EN er 1. ©. andreae Hinterteil der Sehale lang und schmal, ganze Schale regel- mäßig dorsal gekrümmt (Fig. 24) . »... 2... 2. C. polita ganz gerade — 5. Schale mit fünf dorsalen Da Seitenkanten konkav Dean ee te 48 Yen ee Aierint 4 3. C. chaptalii Schale an der Spitze schwach dorsal elta, Embryonal- schale zugespitzt (Fig. 265) . -. er... ‚. 4. C. recurva Schale ganz gerade, ohne dorsale Fre Embryonal- schale abgerundet (Fig. 27) . were een au enscheeler Die Seitenränder der Schale enden in Be a freien Stacheln (Fig. 28) . .»....».. . 6, C. cuspidata Die Seitenränder ragen wenig oder gar nicht eh a 2 1. 4 5 6 Seitenränder der Schale fast parallel, sehr wenig diver- PIE (IE. 29) & 0. 400 na een . . . 7. C. antarctica Seitenränder der Schale stark divergierend — 8. J \ \| Schale mit drei dorsalen Längsrippen. Seitenkanten fast i \| 30 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio Querfurchen auf dem Hinterteil der Schale, dorsale Längs- rippen ziemlich zahlreich (etwa 9) (Fig. 30)... .. . 8. C. sulcata \| Keine Querfurchen auf dem Hinterteil der Schale, dorsale 8 rippen ziemlich zahlreich (etwa 9) (Fig. 30)... .. . 8. C. sulcata \| Keine Querfurchen auf dem Hinterteil der Schale, dorsale Längsrippen drei, sehr undeutlich (Fig. 31—33) . . . . 9. C. pyramidata 8 \| Keine Querfurchen auf dem Hinterteil der Schale, dorsale Längsrippen drei, sehr undeutlich (Fig. 31—33) . . . . 9. C. pyramidata 8 \| Keine Querfurchen auf dem Hinterteil der Schale, dorsale Längsrippen drei, sehr undeutlich (Fig. 31—33) . . . . 9. C. pyramidata 8 Längsrippen drei, sehr undeutlich (Fig. 31—33) . . . . 9. C. pyramidata 1. C. andreae (Boas) 1886 Cleodora a., Boas in: Danske Selsk. Skr., ser. 2. C. polita (Craven MS.) 1880 COleodora falcata (non A. Gould 1852), Pfeffer in: Abh. Ver. Hamburg, ©.71 p.96 t.7 £.19 \| 1888 Balantium politum, Craven MS. in: Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.60 \| 1888 Clio polita,, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.60 t.2 £.4—6 (f.6 Embryonal- schale) \| 1905 (©. falcata, Meisenheimer in: Fauna arctica, v.4 p. 422. 4. Gen. Clio Linne Tier durch dunkel schwarzviolette Farbe unterschieden. L. der Schale 12:5 mm, Br. an der Mundöffnung 5 mm. — Fig. 24. markiert. Tier durch dunkel schwarzviolette Farbe unterschieden. L. der Schale 12:5 mm, Br. an der Mundöffnung 5 mm. — Fig. 24. Im Nordatlantischen Ozean, von der Davisstraße bis nahe dem Äquator, auch an der Küste Irlands, aber überall sehr selten. Meist leere Schalen auf dem Boden. A B C Fig. 25. C. chaptalii. A Schale von oben, B von links (3},). Nach Souleyet. C© Embryonalschale. Nach Pel- seneer (vergr.?). C A B A C A C C B C A Fig. 25. Fig. 24. Fig. 25. C. chaptalii. A Schale von oben, B von links (3},). Nach Souleyet. C© Embryonalschale. Nach Pel- seneer (vergr.?). Fig. 24. C. polita. A Schale von rechts, B von oben (2/,), © Embryonalschale. Nach Pelseneer (vergr.?). Fig. 24. C. polita. A Schale von rechts, B von oben (2/,), © Embryonalschale. Nach Pelseneer (vergr.?). Fig. 25. C. chaptalii. A Schale von oben, B von links (3},). Nach Souleyet. C© Embryonalschale. Nach Pel- seneer (vergr.?). 3. C. chaptalii (Souleyet) 1852 Cleodora c., Souleyet in: Voy. Bonite, v.2 p- 183, Moll. t.7 £.1—5 \| 1888 Clio c., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.61 t.2 f.7 (Embryonalschale). Schale sehr breit-dreieckig mit etwas konkaven Seitenrändern, in der hinteren Hälfte dorsal gekrümmt, auf Dorsal- wie auf Ventralseite mit regelmäßigen, wellenförmigen Querfalten. Beide Seiten gleich hervorgewölbt, auf der Dorsallläche fünf Längsrippen, die beiden lateralen sehr schwach angedeutet. Oberrand in der Mitte kaum hervorragend. Seitenkiele flach, nicht rinnenartig ausgehöhlt. Embryonalschale hinter der Einschnürung nicht erweitert, zugespitzt. L. der Schale 19 mm, Br. an der Mundöffnung 16 mm. — Fig. 25. Eine sehr seltene Art, welche bisher nur am Kap der Guten Hoffnung, an der Westafrikanischen Küste und an der Ostküste Australiens gefunden wurde. Ob sie auch am Rande des antarktischen Eises vorkommt (62° 27’ s. Br., 53° 22° 6. L.), wo die „Valdivia“-Expedition ein nicht sicher hierher gehöriges Exemplar von nicht weniger als 23 mm Länge fing, muß dahingestellt bleiben. 4. C. recurva (Children) 1823 Balantium r., (Children, fide Gray) in: Quart. J. Sei., v.15 p.220 (nota) t.7 f£.107 \| 1854 COleodora balantıum, Rang in: Mag. Zool., v.4 1.44 \| 1836 Hyalaea b., Orbigny in: Voy. Amer. merid., »5m p.116 t.8 f.1-—4 \| 1837 Balantium bicarinatum, Benson in: J. Asiat. Soc. 4. Gen. Clio Linne & v4 p.80 t.1 f.1; t.2 £.12; t.4 f.49 (Embryonalschale); t.5 f.92 (Querschnitt der Schale) \| 1888 Clio a., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 59. Schale ziemlich breit, Seitenränder gerade, nur ganz hinten auf einmal stark konvergierend. Die vordere Hälfte ist gerade, die hintere Hälfte dorsal gekrümmt, an der Spitze wieder sehr schwach ventral gebogen; die Seitenkiele sind nicht rinnenförmig ausgehöhlt, sondern flach, breit und A A C Fig. 23. C.andreae. A Schale von oben, B von rechts (!$/,), © Embryonalschale. Nach Boas (vergr.?). Fig. 23. Fig. 23. C.andreae. A Schale von oben, B von rechts (!$/,), © Embryonalschale. Nach Boas (vergr.?). deutlich abgesetzt. Dorsal- und Ventralseite gleich gewölbt, auf der Dorsal- seite finden sich drei ziemlich schwache und dicht beisammen stehende Längsrippen. Querfurchen sind auf dem Vorderteile der Schale nur schwach angedeutet. Embryonalschale abgerundet, hinter der Einschnürung verbreitert. L. der Schale 20 mm, an der Mundöffnung 14 mm breit. — Fig. 23. deutlich abgesetzt. Dorsal- und Ventralseite gleich gewölbt, auf der Dorsal- seite finden sich drei ziemlich schwache und dicht beisammen stehende Längsrippen. Querfurchen sind auf dem Vorderteile der Schale nur schwach angedeutet. Embryonalschale abgerundet, hinter der Einschnürung verbreitert. L. der Schale 20 mm, an der Mundöffnung 14 mm breit. — Fig. 23. Im Südatlantischen Ozean in dem Übergangspebiet zwischen warmen und kühlen: Strömungen, aber bisher nur ein paar Mal beobachtet, nördlich bis 33° s. Br. 2. C. polita (Craven MS.) 1880 COleodora falcata (non A. Gould 1852), Pfeffer in: Abh. Ver. Hamburg, ©.71 p.96 t.7 £.19 \| 1888 Balantium politum, Craven MS. in: Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.60 \| 1888 Clio polita,, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.60 t.2 £.4—6 (f.6 Embryonal- schale) \| 1905 (©. falcata, Meisenheimer in: Fauna arctica, v.4 p. 422. Schale schlank, langgestreckt, fast gänzlich ohne Längsrippen oder Querfurchen, Seitenränder gerade. Auf der Ventralseite, welche etwas mehr hervorgewölbt ist als die dorsale Seite, finden sich vier sehr schwache Längs- furchen, auf der anderen Seite keine. Seitenkiele stark ausgeprägt, nicht rinnenartig ausgehöhlt. Hintere Hälfte der Schale schwach dorsal gekrümmt, Vorderteil gerade. Der Oberrand der Öffnung ragt nicht über den Unterrand hervor. Embryonalschale abgerundet, oval, hinter der Einschnürung nur wenig verbreitert, die Einschnürung ist durch einen hervortretenden Ring Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4, Clio al Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4, Clio al markiert. 6. C. cuspidata (Bose) 1802 Hyalaea c., Bose, Hist. Coqu., v.2 p. 238 (241?) t.9 f.5—7 \| 1820 H. tricuspidata, Bowdich, Elem. Conch., v.1 t.6 f.1 \| 1830 Oleodora lessonti, Rang MS. + C. quadrispinosa, Rang MS. in: Lesson in: Voy. Coquille, v. 21 p.247 4. Gen. Clio Linne Bengal., v.6 p.151 \| 1852 Cleodora inflata, Souleyet in: Voy. Bonite, v.2 p.188, Moll. t.7 £.17—19 (junges Exemplar) \| 1886 C. balantium, Boas in: Danske Selsk. Skr., ser.6 v.4 p.78 t.4 f.48 (Embryonalschale); t.5 f.89 (Querschnitt der Schale am Mundrande) \| 1888 Clio b., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.61 \| 1889 Cleodora recurva, 'W.Dall in: Bull. U. S. Mus., nr. 57 p. 82. Schale ziemlich breit-dreieckig, der größere Teil gerade, nur ganz hinten sehr schwach dorsal gekrümmt. Die Seitenränder verlaufen von der Spitze der Schale aus zunächst ziemlich stark divergierend; in der vorderen Hälfte ihres Verlaufes wird diese Divergenz allmählich geringer. An der Ventralseite findet sich eine sehr breite konvexe Längsrippe, an der Dorsal- seite drei, wenig hervortretende, aber deutliche, nach hinten konvergierende Rippen; die Seitenkanten der Schale sind zusammengedrückt, flach, die Seitenkiele erstrecken sich bis zur Spitze und sind schwach rinnenartig aus- gehöhlt. Die ganze Oberfläche der Schale ist mit wellenförmig gebogenen Querfalten versehen. Embryonalschale durch eine deutliche Einschnürung Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio 32 abgegrenzt, nur wenig erweitert, kurz und breit, und mit einer: kleinen Spitze ausgestattet. — Junge Schalen (bis etwa 6 mm Länge) zeigen von oben wie von unten gesehen einen konkaven Seitenrand, die dorsale Krümmung erstreckt sich auf die ganze hintere Hälfte der Schale, Längsrippen auf der Dorsalseite fehlen durchaus, die Querfalten sind aber deutlich vorhanden. L. der ausgewachsenen Schale 23 mm, Br. am Mundrande 16 mm. — Fig. 26. Sehr konstant scheinen auf der Schale kleine Hydrozoenkolonien vorzukommen. Eine ziemlich seltene Art, hauptsächlich im Atlantischen Ozean, etwa zwischen 30° n. u. s. Br.; auch im Indischen Ozean beobachtet, hier aber bis zum 40.° s. Br. vor- dringend; spärlich sind die Fundorte im Pazifischen Ozean (nördlich von Neuguinea und an den Küsten von Chili). . C EEE TTERNEN A B C Fig. 26. C. recurva. A Schale von oben, B von rechts (@/). Nach Orbigny. C Embryonalschale. Nach Pelseneer (vergr.?). A A C C B Fig. 26. Fig. 97. Fig. 97. j C. scheelei. A Schale von oben, B von rechts (/,), € Querschnitt der Schale etwa in der Mitte. Nach Munthe (vergr.?). Fig. 26. C. recurva. A Schale von oben, B von rechts (@/). Nach Orbigny. C Embryonalschale. Nach Pelseneer (vergr.?). 5. C. scheelei (Munthe) 1887 Cleodora s., Munthe in: Bih. Svenska Ak., v.13 nr.2 p.18 £.15—19 \| 1905 Clio s., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 4. Gen. Clio Linne 9ı p. 21. 5. C. scheelei (Munthe) 1887 Cleodora s., Munthe in: Bih. Svenska Ak., v.13 nr.2 p.18 £.15—19 \| 1905 Clio s., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p. 21. Die Schale von der Seite gesehen ist gerade, vom Rücken gesehen etwas gebogen, so daß die linke Seite leicht konkav, die rechte konvex ist. Die Seitenkanten zeigen einen schmalen, leicht konvexen Rand, ähnlich dem von C. andreae (nicht ausgehöhlt wie bei C. recurva), und erstrecken sich nach hinten bis nahe an die Embryonalschale. Die Bauchseite ist konvex und mit einem breiten und deutlichen Längskiel („Längsköl“) versehen, der ungefähr °/, der Breite einnimmt und, wie gewöhnlich, breiter und flacher nach der ffnung hin wird. Die Rückenseite hat 3 konvexe Längskiele, von denen der mittlere bei weitem stärker entwickelt ist als die beiden seitlichen Kiele, die nahe den Seitenkanten verlaufen. Die Schale ist mit sehr deutlichen Querfurchen versehen, die durch gerundete Querkiele getrennt werden und die gegen die Spitze der Schale niedriger und weniger deutlich werden. Wie gewöhnlich sind auch Zuwachslinien bemerkbar. Die Em- bryonalschale ist durch eine ziemlich schwache Einschnürung vom übrigen Teile der Schale abgetrennt, sie ist kurz, gleichmäßig gerundet und ohne eine kleine Spitze. L. der Schale 16mm, Br. am Schalenrande 7 mm. — Fig. 27, x Eine wie es scheint sehr seltene Art, welche bisher in einem einzigen Exemplare, und zwar an der Westküste Patagoniens, gefunden wurde, 6. C. cuspidata (Bose) 1802 Hyalaea c., Bose, Hist. Coqu., v.2 p. 238 (241?) t.9 f.5—7 \| 1820 H. tricuspidata, Bowdich, Elem. Conch., v.1 t.6 f.1 \| 1830 Oleodora lessonti, Rang MS. + C. quadrispinosa, Rang MS. in: Lesson in: Voy. Coquille, v. 21 p.247 33 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio t.10 £.1; p.248 t.10 £.2 \| 1852 C. cuspidata, Quoy & Gaimard in: Voy. Astrol., v.2 p- 384 t.27 £. 1—5 \| 1886 ©. c., Boas in: Danske Selsk. Skr., ser. 6 ».4 p.81 t.1f.1; t.2 f.13; t.4 £.51 (Embryonalschale); t.5 f. 87,88 (Querschnitt am Mundrande und in der Mitte der Schale) \| 1888 Clio c., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.66, t.10 £.1; p.248 t.10 £.2 \| 1852 C. cuspidata, Quoy & Gaimard in: Voy. Astrol., v.2 p- 384 t.27 £. 1—5 \| 1886 ©. c., Boas in: Danske Selsk. Skr., ser. 6 ».4 p.81 t.1f.1; t.2 f.13; t.4 £.51 (Embryonalschale); t.5 f. Das Tierreich, 36, Lief.: J. J. Tesch, Pteropoda. 4. Gen. Clio Linne Eig \| Seitenkiele sind nicht aus die Umschlagstelle zwischen und Ventralseite wird nach zu weniger deutlich und ve im hinteren Drittel gänzlic Schalenrand erscheint schrä d Ss er D stutzt, weil der Oberrand de Schale gerade gestreekt oder an der Spitze leicht dorsal gebogen (in dieser Hinsicht ist die Figur 29 3 unrichtig), schmal; die Seitenränder divergieren nur in der hinteren Hälfte der Schale deutlich, weiter vorn nur sehr wenig, so daß die Schale sich kaum allmählich erweitert. Bei Seitenansicht fällt deutlich der Gegensatz zwischen Dorsal- und Ventralseite auf. Während erstere stark gewölbt ist, besonders in der vor- deren Hälfte der Schale, und hie drei schwach angedeutete Längs- N > rippen trägt, ist die Ventralseite \| IM bl hinten fast ganz abgeflacht, nach dem \| c Rande zu höhlt sie sich immer mehr aus, hier findet sich eine schwache mediane Längsrippe. Eigentliche \| Seitenkiele sind nicht ausgeprägt; die Umschlagstelle zwischen Dorsal- und Ventralseite wird nach hinten Schale gerade gestreekt oder an der Spitze leicht dorsal gebogen (in dieser Hinsicht ist die Figur 29 3 unrichtig), schmal; die Seitenränder divergieren nur in der hinteren Hälfte der Schale deutlich, weiter vorn nur sehr wenig, so daß die Schale sich kaum allmählich erweitert. Bei Seitenansicht fällt deutlich der Gegensatz zwischen Dorsal- und Ventralseite auf. Während erstere stark gewölbt ist, besonders in der vor- deren Hälfte der Schale, und hier drei schwach angedeutete Längs- N > rippen trägt, ist die Ventralseite \| IM bl hinten fast ganz abgeflacht, nach dem \| c Rande zu höhlt sie sich immer mehr aus, hier findet sich eine schwache mediane Längsrippe. Eigentliche \| Seitenkiele sind nicht ausgeprägt; die Umschlagstelle zwischen Dorsal- und Ventralseite wird nach hinten zu weniger deutlich und verstreicht im hinteren Drittel gänzlich. Der Schalenrand erscheint schräg abge- d Ss er D stutzt, weil der Oberrand den Unter- C. antarctica. FERNE oben, B von rechts (?],). rand überragt. Bei BenauEr Be- Schalenspitze nicht ventral, wie in der Abbildung, trachtung sieht man auf der ganzen sondern leicht dorsal gebogen. Nach Souleyet. Schale, besonders aber in der distalen € Querschnitt der Schale am Mundrande, D Em- = 5 s bryoualenkaie. "Mach Bosatvose in Hälfte, Querstreifen, welche einander in regelmäßigen Abständen folgen; sie werden nicht durch Fältelung, sondern durch Verdickung der Schalen- oberfläche hervorgerufen. Embryonalschale nur undeutlich vom übrigen Teile gesondert, länglich, nicht erweitert, mit einer kleinen Spitze am Ende, welche leicht abbricht. L. der Schale 17 mm. 4. Gen. Clio Linne 87,88 (Querschnitt am Mundrande und in der Mitte der Schale) \| 1888 Clio c., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.66, Schale sofort an den langen quergerichteten Stacheln kenntlich, welche sich an den Ecken der außerordentlich umfangreichen Schalenöffnung finden. Im hinteren Teile ist die Schale zierlich dorsal gebogen, dies ist aber bei einigen Exemplaren wenig ausgeprägt. Die Unterseite, welche fast gänzlich von der Ventralklappe der Schale gebildet wird, ist ziemlich gleichmäßig vorgewölbt, nur in der Mitte findet sich ein nach vorn zu sich allmählich verbreiternder Längs- kiel. Die Dorsalseite wird fast gänzlich von der Dorsalklappe der Schale ein- C Fig, 28. an C. cuspidata. A Schale von oben, B von rechts (/,), © Embryonalschale. Nach Boas (vergr. Du Fig, 28. rechts (/,), C Fig, 28. an C. cuspidata. A Schale von oben, B von rechts (/,), © Embryonalschale. Nach Boas (vergr. Du Fig, 28. an C. cuspidata. A Schale von oben, B von rechts (/,), © Embryonalschale. Nach Boas (vergr. Du genommen, ist dachförmig ausgebildet und trägt in der Mitte einen über die ganze Länge sich erstreckenden und sogar über den freien, dorsalen Vorderrand der Schale eine Strecke weit hervorragenden, sehr schmalen Längskiel, der überall gleich breit ist. Dieser Längskiel bildet die Mitte einer proximal sehr breiten, distal aber sich schnell verjüngenden Längsrippe. Der freie Vorderrand der Schalenoberseite ragt beträchtlich über die Unterseite hervor. Öffnung der Schale sehr breit, klaffend; die beiden Ränder bilden dort wo sie zusammenstoßen jederseits einen langen, fast geraden, quer- gerichteten Stachel, der an der proximalen Fläche rinnenartig ausgehöhlt ist Das Tierreich, 36, Lief.: J. J. Tesch, Pteropoda. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio 34 und die entsprechenden Verlängerungen des Mantels des Tieres in sich aufnimmt. Der Hinterteil der Schale ist kurz, die Seitenränder sind konkav und gehen in die oben erwähnten queren Stacheln über. Die ganze Öber- fläche der Schale hat regelmäßige, wellenförmige Querfurchen, auf der Unter- seite sind sie schwach und undeutlich, auf der Dorsalseite aber stark aus- geprägt. Embryonalschale deutlich durch eine Einschnürung abgetrennt, kugelförmig erweitert und mit einer kleinen Spitze ausgestattet. L. der aus- gewachsenen Schale 16 mm, Br. ohne Stacheln 6 mm. -— Fig. 28. Wie bei der vorigen Art scheinen Kolonien von Hydrozoen bisweilen auf der Schale vorzukommen. In den wärmeren Teilen der Weltmeere nicht selten, besonders im Atlantischen Ozean, und hier sehr vereinzelt bis zu 59° n. Br. 4. Gen. Clio Linne vordringend, auch an der Westküste Irlands, im Biskayischen Meerbusen und im Mittelmeer. In den wärmeren Teilen der Weltmeere nicht selten, besonders im Atlantischen Ozean, und hier sehr vereinzelt bis zu 59° n. Br. vordringend, auch an der Westküste Irlands, im Biskayischen Meerbusen und im Mittelmeer. 7. C. antarctica Dall 1836 Hyalaea australis (non H. A. Peron 1807), Orbigny, Voy. Amer. merid., v. 5m p.117t.8 £.9—11 \| 1850 Balantium australe, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p. 15 \| 1852 Cleodora australis, Souleyet in: Voy. Bonite, v.2 p. 189, Moll. t.7 f£.20—25 \| 1886 C.a., Boas in: Danske Selsk. Skr., ser.6 v.4 p.67 t.4 f.46 (Embryonalschale); t.5 £.80—83 (Querschnitte an verschiedenen Stellen der Schale) \| 1888 Clio a. (non Bruguiere 1792), Pelseneer in: Rep. Voy. Challenger, Zool. v. 23 pars 65 p.62 t.2 f.8 (Embryonalschale) \| 1906 C.a., Meisenheimerin: D. Südp.-Exp., v.9ır p. 106 fig. \| 1908 ©. antarctica, W. Dall in: Smithson. Collect., v.50 p. 501. 7. C. antarctica Dall 1836 Hyalaea australis (non H. A. Peron 1807), y. Amer. merid., v. 5m p.117t.8 £.9—11 \| 1850 Balantium australe, J. E. G l. Brit. Mus., v.2 p. 15 \| 1852 Cleodora australis, Souleyet in: Voy. Bonite, v l. t.7 f£.20—25 \| 1886 C.a., Boas in: Danske Selsk. Skr., ser.6 v.4 p.67 mbryonalschale); t.5 £.80—83 (Querschnitte an verschiedenen Stellen der Schal o a. (non Bruguiere 1792), Pelseneer in: Rep. Voy. Challenger, Zool. v. 23 par f.8 (Embryonalschale) \| 1906 C.a., Meisenheimerin: D. Südp.-Exp., v.9ır p 08 ©. antarctica, W. Dall in: Smithson. Collect., v.50 p. 501. Schale gerade gestreekt oder an der Spitze leicht dorsal gebogen ( nsicht ist die Figur 29 3 unrichtig), schmal; die Seitenränder div r in der hinteren Hälfte der Schale deutlich, weiter vorn nur sehr w ß die Schale sich kaum allmählich erweitert. Bei Seitenansicht fällt r Gegensatz zwischen Dorsal- und Ventralseite auf. Während erste gewölbt ist, besonders in deren Hälfte der Schale, u drei schwach angedeutete N > rippen trägt, ist die Vent IM bl hinten fast ganz abgeflacht, n \| c Rande zu höhlt sie sich imme aus, hier findet sich eine s mediane Längsrippe. Fig. 30. C.sulcata. A Schale von rechts, B von oben (3),), © Embryonalschale.. Nach Pelseneer (vergr.?). C. pyramidata pyramidata. A Schale von oben (?/,), B Querschnitt der Schale am Mund- rande, C Embryonalschale. Nach Boas (vergr.?). C. pyramidata pyramidata. A Schale von oben (?/,), B Querschnitt der Schale am Mund- rande, C Embryonalschale. Nach Boas (vergr.?). 4. Gen. Clio Linne — Fig. 29. N > \| IM bl \| c \| d Ss er D C. antarctica. FERNE oben, B von rechts (?],). Schalenspitze nicht ventral, wie in der Abbildung, sondern leicht dorsal gebogen. Nach Souleyet. € Querschnitt der Schale am Mundrande, D Em- bryoualenkaie. "Mach Bosatvose in Ss d Ss D Ss Ss er RNE ob C. antarctica. FERNE oben, B von rechts (?],). Schalenspitze nicht ventral, wie in der Abbildung, sondern leicht dorsal gebogen. Nach Souleyet. € Querschnitt der Schale am Mundrande, D Em- bryoualenkaie. "Mach Bosatvose in sie werden nicht durch Fältelung, sondern durch Verdickung der Schalen- oberfläche hervorgerufen. Embryonalschale nur undeutlich vom übrigen Teile gesondert, länglich, nicht erweitert, mit einer kleinen Spitze am Ende, welche leicht abbricht. L. der Schale 17 mm. — Fig. 29. Charakteristisch für die Mischungsgebiete der küblen und warmen Strömungen auf der südlichen Halbkugel, ohne jemals in den wärmeren Gewässern vorzukommen, Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio 35 bei Kap Horn im kalten Wasser gefunden. An der Ostküste Südamerikas dringt sie mit der kühlen Strömung bis 30°, vereinzelt sogar bis 20° s. Br. vor. 8. C. sulcata (Pfeffer) 1879 Cleodora s., Pfeffer in: Monber. Ak. Berlin, p.240 f. 11,12 \| 1888 Cko s., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 62 t.2 £.9—11 (f. 11 Embryonalschale). Schale schmal, gestreckt, kaum merkbar im hinteren Teile dorsal gekrümmt; die Seitenränder divergieren in den hinteren zwei Dritteln der Schale nur wenig, im vorderen Drittel auf einmal ziemlich stark, so daß die Schale sich hier beträchtlich erweitert. Ein Seitenkiel ist nur im Bereiche dieser Verbreiterung ausgeprägt. Dorsalfläche stark gewölbt; am Vorderrande finden sich 9 oder 10 schwach angedeutete Längsrippen. Öber- wie Unterrand der Schale sehr zart und zerbrechlich, der Oberrand ist in der Mitte nur wenig vorgebuchtet und ragt nur unbedeutend über den Unterrand hervor. Ventral- fläche weniger gewölbt, mit einer undeutlichen medianen Längsrippe. Die ganze Schalenoberfläche mit undeutlichen Querstreifen. Embryonalschale ganz wie bei der vorigen Art ausgebildet. L. der Schale 20 mm. Kennzeichnend für die antarktischen Meere, wo sie am Rande des Eises bis- weilen in großen Scharen vorkommt. a ‘ C B Fig. 31. C. pyramidata pyramidata. A Schale von oben (?/,), B Querschnitt der Schale am Mund- rande, C Embryonalschale. Nach Boas (vergr.?). a ‘ C B ne m A B C B C Fig. 30. Fig. 30. C.sulcata. 4. Gen. Clio Linne A Schale von rechts, B von oben (3),), © Embryonalschale.. Nach Pelseneer (vergr.?). 9. C. pyramidata 6; TZerfällt in 3 Unterarten: 9a. C. pyramidata pyramidata L. 1756 „Clio I. vagina triquetra p., ore oblique truncato; ?Clio II. vagina compressa caudata; Clio III. vagina triquetra, ore horizontali“, Browne, Hist. Jamaica, p.386 t.48 f.1 \| 1767 €. pyramidata + ?C. cau- data + ?C. retusa (non 1776 ©. r., O. F. Müller, Zool. Dan. Prodr., p.226). Linn@ in: Syst. Nat., ed.12 v.1ır p.1094 \| 1810 Oleodora „pyramidata“, Peron & Lesueur in: Ann. Mus. Paris, v.15 p.69 t.2 f.14 \| 1819 C. caudata, Lamarck, Hist. An. s. Vert.. v.61 p.290 \| 1823 CO. retusa, Deshayes in: Diet. elass. Hist. nat.. v.4 p. 204 \| 1825 & 27 0. brownii, Blainville, Man. Malac., p.481 t.46 f.1 \| 1832 C. lanceolata (non Hyalaea \|., Lesueur 1813), Quoy & Gaimard in: Voy. Astrol., t.27 f.6—8 \| 1836 Hyalaca pyra- midata var. B, Orbigny, Voy. Amör. merid., v.5ıu p.114 t.7 f.25--28 \| 1841 Cleodora lamartinieri (non Orbieny 1843), Orbigny in: Ramon, Historia Cuba. Moll. v.1 p.84 \| 1852 CO. pyramidata, Souleyet in: Voy. Bonite, v.2 p. 180, Moll. t..6 f. 25, 24 \| 1880 ©. martensii, (non C. pyramidata), Pfeffer in: Abh. Ver. Hamburg, v.71 p.95 t.7 £.16 \| 1886 .C. pyramidata var. angusta, Boas in: Danske Selsk. Skr., ser.6 v.4 p.72 t.5 f.84, 85 (Quersehnitte der Schale), t.6 f. 96 a—e. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio 36 Schale gestreckt, schmal, ganz gerade; Seitenränder regelmäßig bis zur Mundöffnung divergierend, nicht gekrümmt. Dorsalseite mit drei Längs- rippen, von welchen nur die mediane stark hervortritt, die beiden seitlichen sind viel schwächer. Die Schale ist an der Öffnung flachgedrückt, so dab die Ecken spaltförmig ausgezogen sind. Der Öberrand ist in der Mitte ziemlich stark vorgebuchtet und ragt beträchtlich über den Unterrand hervor. Die Unterseite ist nur sehr wenig vorgewölbt, am Schalenrande sogar etwas ausgehöhlt und mit einer schwachen, medianen Längsrippe versehen. Keine Skulptur, nur undeutliche Zuwachsstreifen. Embryonalschale wenig gesondert, nicht erweitert, zugespitzt. L. der ausgewachsenen Schale 21 mm. — Fig. 31. Häufig im Nordatlantischen Ozean, zwischen 40° und 60° n. Br., vereinzelt auch bis zur Davisstraße und bei Spitzbergen vorkommend; auch im östlichen Teile des Südpazifischen Ozeanes. 9b. C. pyramidata lanceolata (Lesueur) 1813 Hyalaea l., Lesueur in: Bull. Soe. philom., v.3 p.284 t.5 f.ö \| 1836 H. pyramidata var. A, Orbigny, Voy. Amer. merid., v.5ur p.114 t.7 f.30, 31 \| 1843 Cleodora lamartinieri (non Orbigny 1841), Orbigny, Voy. Amör. In den wärmeren Teilen der Ozeane häufig, von etwa 40° n. Br. bis 300 s. Br., auch im Mittelmeer und im Westpazifischen Ozean. 9. C. pyramidata 6; merid., v.5ın p.688 \| 1852 C. lanceolata (non Quoy & Gaimard 1832), Souleyet in: Voy. Bonite, v.2 p!179, Moll. t.6 £.17—22 \| 1852 C. exacuta, A. (Gould in: U. S. expl. Exp., v.12 p.488 t. 51 £. 605 \| 1877 C. labiata, G. B. Sowerby in: Reeve, Conch. icon.. v.20 Pter. f.26 \| 1880 C. pyramidata (non Peron & Lesueur 1810). Pfeffer in: Abh. Ver. Hamburg, v.71 p.94 t.7 f.15 \| 1886 C. p. var. lata, Boas in: Danske Selsk. Skr., ser.6 v.4 p.72 t.5 f.86 (Querschnitt der Schale am Mundrande); t.6 f.96f, g, 97e. Schale an der Spitze dorsal gekrümmt, aber in dieser Hinsicht indi- viduell variierend; Seitenränder stark konkav und am Schalenrande bis- weilen in mehr oder weniger lange Stacheln ausgezogen. Oberrand weit vorspringend. Oberseite dachförmig, mit einem schmalen, vorn sich etwas erweiternden Längskiel, der sich bis in das hintere Drittel der Schalenfläche fortsetzt. Dieser Kiel bildet die Mitte einer medianen, vorn breiten und PZN G \ B C Fig. 32. C. pyramidata lanceolata. A Schale von oben, B von links (?/,). Nach Souleyet. € Querschnitt der Schale am Mundrande. Nach Boas (vergr.?). C C C Fig. 32. Fig. 32. C. pyramidata lanceolata. A Schale von oben, B von links (?/,). Nach Souleyet. € Querschnitt der Schale am Mundrande. Nach Boas (vergr.?). flachen, nach hinten zu sich allmählich zuspitzenden Längsrippe; zu jeder Seite ein ähnliches, aber weit schwächeres Gebilde. (uerlinien auf der Dorsal- seite in regelmäßigen Abständen, auf der Unterseite weniger deutlich. Unter- seite am Schalenrande etwas ausgehöhlt. Ecken der Öffnung noch mehr spaltförmig als bei er vorhergehenden Unterart. L. der Schale 16—17 mm. — Fig. 32. In den wärmeren Teilen der Ozeane häufig, von etwa 40° n. Br. bis 300 s. Br., auch im Mittelmeer und im Westpazifischen Ozean. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 4. Clio, 5. Cuvierina 37 9e. C. pyramidata convexa (Boas) 1886 Oleodora p. var. c., Boas in: Danske Selsk. Skr., ser.6 v.4 p. 73 t.6 f. 97 a—d. 9e. C. pyramidata convexa (Boas) 1886 Oleodora p. var. c., Boas in: Danske Selsk. Skr., ser.6 v.4 p. 73 t.6 f. 97 a—d. Fig. 33. E C. pyramidata convexa. Schale vorne ln. Nach Boas. 9. C. pyramidata 6; Ist am nächsten mit der erstgenannten Unterart ver- wandt, unterscheidet sich aber habituell dadurch, daß die Seitenränder, welche in ihrem ganzem Verlaufe gerade oder schwach konkav sind, ganz am Mundrande etwas einge- bogen sind und die Öffnung auf diese Weise ein wenig verengern. Die schwache Ausprägung der Längsrippen ist übrigens die gleiche. L. der Schale nur 8 mm, sehr selten bis zu 14 mm. — Fig. 33. Anm. Es können diese Unterarten bisweilen nicht streng . : ; auseinander gehalten werden; auch die Verbreitung hält sich nicht immer an die hier genannten Gebiete. Fig. 33. E Im Südatlantischen und Südindischen Ozean, von etwa 25° bis 40° s. Br. la. C. columnella columnella (Rang) ?1824 Cleodora obtusa, Quoy & Gaimard in: Voy. Uranie & Physicienne, p.415 t.66 £.5 \| 1827 Owvieria columnella (non Souleyet 1852), Rang in: Ann. Sei. nat., v.12 p.323 t.45 f.1—8 \| 1828 ÜOreseis obtusa, Rang in: 5. Gen. Cuvierina Boas Soc. Bengal, v.4 p.698 \| 1839 ©. obtusa, Orbigny in: Webb & Berthelot, Hist. Canar., v.2n Moll. p. 32 \| 1850 Triptera columnella, J. E. Gray, Cat. Moll. Brit. Mus., ©.2 p.23 \| 1852 Cuwvieria ce. var. 1 & 2, Souleyet in: Voy. Bonite, v.2 p.206, Moll. t.12 f.1,2 \| 1879 Triptera columella, Pfeffer in: Monber. Ak. Berlin, p.243 f.39 \| 1886 Cwvierina colum- nella var. typica, Boas in: Danske Selsk. Skr.. ser.6 v.4 p.134 t.3 f.28 (Radula); f. 39; t.4 f.56 (Embryonalschale); t.5 f. 95d—p. a . u ig. 34. Schale gestreckt, Septum gut ent- wickelt. Der angeschwollene Teil ist nicht besonders stark markiert und geht proxi- mal ganz allmählich in den Hals über, der langgestreckt ist; nahe dem Schalen- rande divergieren die Seitenkanten der Schale etwas. L. der unverletzten Schale 16 mm, L. des gewöhnlich allein vor- handenen Vorderteils 9 mm. — Fig. 34. Hauptsächlich in den wärmeren Teilen des Atlantischen Özeanes, auch im Indischen und Östpazifischen Ozean, zwischen etwa 40° n. Br. und 35° s. Br. a . u ig. 34. C. columnella columnella. A unverletzte Schale, von unten (/,), B Vorderteil dr Schale von unten (®/). Nach Boas. a . u ig. 34. C. columnella columnella. A unverletzte Schale, von unten (/,), B Vorderteil dr Schale von unten (®/). Nach Boas. lb. C. columnella urceolaris (Mörch) 1850 Cuvieria urceolaris, Mörch, Cat. Conch. Kierulf, p.32 \| 1852 C. columnella (non Rang 1827), Souleyet in: Voy. Bonite, v.2 p.206, Moll. t.12 f.3 \| 1879 Triptera cancellata, Pfeffer in: Monber. Ak. Berlin, p. 243 f. 19 \| 1886 Cuvierina colum- nella var. urceolaris, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 134 t.6 f. 95a—c. Fig. 35. C. columnella urceo- laris. Vorderteil der 9 eng 9 unten (je). ach Boas. Schale verhältnismäßig etwas kürzer, Septum dünn, schwach. Der angeschwollene Teil der Schale hebt sich etwas stärker hervor, der Hals ist kurz, kaum einge- schnürt, am Rande nicht erweitert. L. des Vorderteiles der Schale 6 mm. — Fig. 35. Fig. 35. Fig. 35. C. columnella urceo- laris. Vorderteil der 9 eng 9 unten (je). ach Boas. Fig. 35. C. columnella urceo- laris. Vorderteil der 9 eng 9 unten (je). ach Boas. C. co ur Im Chinesischen Meere und im Westpazifischen Ozean. laris. Vorderteil der . -, . . . . . . 9 Im Indischen Ozean, im ÖOstindischen Archipel und im Pazi- eng 9 unten (je). 5. Gen. Cuvierina Boas ?1824 Cleodora (part.), Quoy & Gaimard in: Voy. Uranie & Physieienne, p.415 \| 1827 Cuvieria (non Pöron & Lesueur 1807), Rang in: Ann. Sei. nat., v.12 p. 322 \| 1850 Triptera (non Quoy & Gaimard 1825), J. E. Gray, Oat. Moll. Brit. Mus., v.2 p.23 \| 1886 Cuvierina, Boas in. Danske Selsk. Skr., ser. 6 v.4 p.131. ?1824 Cleodora (part.), Quoy & Gaimard in: Voy. Uranie & Physieienne, p.415 \| 1827 Cuvieria (non Pöron & Lesueur 1807), Rang in: Ann. Sei. nat., v.12 p. 322 \| 1850 Triptera (non Quoy & Gaimard 1825), J. E. Gray, Oat. Moll. Brit. Mus., v.2 p.23 \| 1886 Cuvierina, Boas in. Danske Selsk. Skr., ser. 6 v.4 p.131. Schale langgestreckt, gerade, im Querschnitt kreisförmig, nur ganz nahe der Öffnung schwach nierenförmig, da die Unterseite etwas ausgehöhlt ist; hintere Hälfte länglich-kegelförmig, bei erwachsenen Exemplaren fehlt dieser Teil fast immer; vordere Hälfte im hinteren Abschnitte ringsum bauchig erweitert, dann allmählich eingeschnürt bis zur Öffnung, wo wieder eine kleine Erweiterung erscheint. Schalenrand schräg abgestutzt, Oberseite etwas vor- springend, Unterseite schwach ausgehöhlt, der Querschnitt der Schalenöffnung bildet eine bohnen- oder nierenförmige Figur. Etwas hinter der erweiterten Partie der Schale findet sich ein queres, nach vorn konkaves Septum, distal von dieser Scheidewand sieht man die zackigen Reste des abgebrochenen hinteren Schalenteiles. Embryonalschale, an der Spitze der Schale nur ganz wenig gesondert, wahrscheinlich zugespitzt, aber die äußerste Spitze immer fehlend. Schale glashell, durchsichtig, mit schwacher Längsstreifung und mit queren Zuwachsstreifen. — Tier ganz in der vorderen Hälfte der Schale eingeschlossen; die Flossen sind, wie bei Clio, in der Mitte des Seitenrandes durch einen Einschnitt gekerbt, der Mittellappen des Fußes ist in der Mitte eingebuchtet. Niere und Herz liegen beide quer, ganz hinten in der Mantelhöhle; der Vorhof ‘des Herzens ist nach rechts, die Kammer nach links gewendet. Unterhalb des mittleren Fußlappens, also ventral in der Halsgegend, findet sich bei einigen Exemplaren ein eigentümliches, gestieltes, blattartig ausgebreitetes Organ, das mit der rechts gelegenen weiblichen Geschlechtsöffnung durch eine Hautfalte verbunden ist und wohl als akzessorisches Hilfsmittel bei der Begattung betrachtet werden muß (s. auch p. 13). In den wärmeren Teilen der Ozeane. 1 Art, welche in 2 Unterarten zerfällt. 1 Art, welche in 2 Unterarten zerfällt. 1. C. columnella (Rang) Zerfällt in 2 Unterarten: 38 Pterop.: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae. 5. Cuvierina, 6. Diacria 38 Ann. Sei. nat., ©. 13 t.17 f.4 \| 1835 Cuvieria oryza, Benson in: J. Asiat. 5. Gen. Cuvierina Boas 3 - = R L ach Boas. fischen Ozean scheinen Übergänge zwischen den beiden Unter- arten vorzukommen. Im Mittelmeer findet man fast immer nur leere Schalen der erstgenannten Unterart. 6. Gen. Diacria Gray 1821 Hyalaea (part.), Lesueur MS. in: Blainville in: Diet. Sei. nat., v.22 p.82 \| 1850 Diacria (part.), J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.10 \| 1858 Pleuropus (part.), H. & A. Adams, Gen. Moll., v.2 p.611 \| 1886 Hyalaea A., Boas in: Danske Selsk. Skr., ser.6 v.4 p.92 \| 1888 Cavolinia (part.), Pelseneer in: Rep. Voy. Challenger, Zool. v. 23 pars 65 p.76 \| 1904 Diacria (Subgen.), Tesch in: Siboga-Exp., nr. 52 p.35 \| D., Meisen- heimer in: Ergeb. Tiefsee-Exp., v.9ı p. 27. Schale (bei erwachsenen Exemplaren) ziemlich resistent, charakteristisch gefärbt. Öffnung ein langer, gebogener Querspalt, der sich über die ganze vordere Partie der Schale erstreckt; nur der mediane Teil dient dem Tiere zum Herausstrecken der Flossen; die engen, schlitzförmigen Seitenteile sind vorn durch einen eigentümlichen Apparat verschlossen; ein kleiner Vorsprung auf dem Unterrande paßt in eine entsprechende Vertiefung des Öberrandes Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 6. Diacria 39 der Öffnung hinein, so daß eine Art Schlußvorrichtung zustande kommt, die aber noch lange nicht so ausgeprägt ist wie bei der folgenden Gattung. Die ganze Schale wird fast nur von den lippenartigen Teilen der Schale gebildet, die Schalenoberlippe ist sanft gewölbt und trägt fünf mehr oder weniger stark ausgebildete Längsrippen, von welchen die mediane stets die stärkste ist; an der eigentlichen Öffnung ist der Oberrand stets etwas ventral gekrümmt und am freien Rande verdickt; die Schalenunterlippe ist wie auf- geblasen, stärker vorgewölbt als die Oberschale; an der Öffnung ist sie dorsal gekrümmt, so daß der Schalenspalt eingeengt wird, am freien Rande wieder ventral zurückgeschlagen. In den äußeren Ecken der Öffnung sind Ober- und Unterschale entweder wenig oder sehr stark vorgezogen. Der Hinterteil der Schale ist lang, aber sehr schmal, oft abgebrochen, so daß die Schale am Hinterende quer abgestutzt erscheint; eine Querlamelle schließt dann hier den Eingeweideknäuel des Tieres von der Außenwelt ab. Im langen End- stachel findet sich vor der Embryonalschale ein kleines Septum. Embryonal- schale deutlich abgetrennt, gerundet. Bei jungen Tieren findet man eine sehr dünne, langgestreckte, dorsoventral flachgedrückte und im ganzen Clio- ähnliche Schale; die Seitenkanten sind gerade, nur nahe dem Mundrande konkav. Die jungen Schalen können schon gänzlich die Länge der alten erreichen und bilden sich dann nachträglich um. — Beim Tiere ist der freie Seitenrand der Flossen durch einen Einschnitt eingekerbt; der Mittellappen des Fußes ist schmal. Schale flach, in der Mitte etwas aufgeblasen. Enddorn lang, ganz gerade. Zerlegt man die Schale auf der Dorsalseite durch eine Linie, welche 6. Gen. Diacria Gray Niere und Herz liegen ganz links in der Mantelhöhle, der Vorhof des Herzens ist nach hinten, die Kammer nach vorn gerichtet. Die Leber ist nicht einheitlich, sondern in zwei völlig getrennte Lappen zerteilt, von welchen jeder mit besonderem Ausführungsgange in den Darm, unmittelbar hinter dem Magen, einmündet. In den wärmeren Teilen der Ozeane, an der Oberfläche oder in geringen Tiefen. 2 Arten, von welchen beide in 2 Unterarten zerfallen. In den wärmeren Teilen der Ozeane, an der Oberfläche oder in geringen Tiefen. 2 Arten, von welchen beide in 2 Unterarten zerfallen. 2 Arten, von welchen beide in 2 Unterarten zerfallen. Bestimmungstabelle der Arten: Schale flachgedrückt, mit einem quergerichteten Seiten- stachel jederseits und mit langem Enddorne (Fig. 36,37) 1. D. trispinosa Schale wie aufgeblasen, kuglig, ohne Seitenstacheln, hinten quer abgestutzt (Fig. 38) »-..:....- 2. D. quadridentata Schale flachgedrückt, mit einem quergerichteten Seiten- stachel jederseits und mit langem Enddorne (Fig. 36,37) 1. D. trispinosa Schale wie aufgeblasen, kuglig, ohne Seitenstacheln, hinten quer abgestutzt (Fig. 38) »-..:....- 2. D. quadridentata stachel jederseits und mit langem Enddorne (Fig. 36,37) 1. D. trispinosa Schale wie aufgeblasen, kuglig, ohne Seitenstacheln, hinten quer abgestutzt (Fig. 38) »-..:....- 2. D. quadridentata 1. D. trispinosa (Lesueur) Zerfällt in 2 Unterarten: la. D. trispinosa trispinosa (Lesueur) 1821 Hyalaea t. (part.), Lesueur MS. in: Blainville in: Diet. Sei. nat., v.22 p.82 \| 1827 H. mucronata (non Orbigny 1836), Quoy & Gaimard in: Ann. Sei. nat., v.10 p.231 t.8 f.1,2 (figura mala) \| 1832 H. trispinosa, Quoy & Gaimard in: Voy. Astrol., 0.2 p. 378, 1.27 f.17—19 \| 1841 H. cuspidata (non Bose 1802), Chiaje, Deser. An. Sicilia, v.7 t.180 f.1,2 \| 1850 Diacria trispinosa + Clio depressa, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.10; p.14 (junges Exemplar) (descer. nulla) \| 1852 Hyalaea trispinosa + Oleodora compressa, Souleyetin: Voy. Bonite, v.2 p. 161, Moll. t.6 f. 1-6; p. 181, Moll. t. 6 f.26—32 (junges Exemplar) \| 1853 Hyalaea reeviana, Dunker, Index Moll. Guinea, p.2 t.1 £.17—20 \| 1858 Plenropus trispinosus, H. & A. Adams, Gen. Moll., v.2 p. 611 \| 1886 Hyalaea trispinosa var. minor, Boas in: Danske Selsk. Skr., ser.6 v.4 p.95 t.1 f.3; 1.2 £.14; t.4 f.52 (Embryonalschale); t.5 f.93 (Querschnitt der Schale, etwa in der Mitte) \| 1886 Cavo- linia t. (part.), Locard, Prodr. Malac. franc., Moll. mar. p.22 \| 1904 ©. (Diacria) t., Tesch in: Siboga-Exp., nr. 52 p.35 t.2 f. 27—31. 6. Gen. Diacria Gray Schale flach, in der Mitte etwas aufgeblasen. Enddorn lang, ganz gerade. Zerlegt man die Schale auf der Dorsalseite durch eine Linie, welche 40 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 6. Diacria die beiden Seitenstacheln verbindet, dann ist der vordere Teil nur '/, der ganzen Schalenlänge. Die Unterseite ist am meisten hervorgewölbt und hat drei Längsrippen, die mediane ziemlich breit, die beiden seitlichen schmaler; die Oberseite ist viel mehr abgeflacht und zeigt fünf Längsrippen, eine mediane und zwei laterale jederseits, welche sich nur wenig in der Größe unterscheiden. Seitenkanten der Schale scharf, im hinteren Teile ist der Querschnitt fast zylindrisch. Der Rand der Oberschale ist verdickt, deutlich abgesetzt; diese Verdickung erstreckt sich bis nach den Seitenstacheln hin und ist am stärksten in der Mitte; Unterschale in den Seitenteilen ebenso verdickt, der mediane Teil ist hakenförmig ventral umgeschlagen und wird von der ventral gebogenen Oberschale überragt. Die Zahnbildung, welche die seitlichen Teile des Mundspaltes von dem medianen Teile sondert, ist nur schwach ausgebildet. Die Seitenstacheln an den Ecken der Mundöffnung sind kräftig und ziemlich kurz, ganz gerade und nahezu quer gerichtet. Der C Fig. 36. D. trispinosa trispinosa. A Schale von oben, B von rechts (/,), € junge Schale („Cleodora com- pressa“ Souleyet) (/,). Nach Boas. C Fig. 36. D. trispinosa trispinosa. A Schale von oben, B von rechts (/,), € junge Schale („Cleodora com- pressa“ Souleyet) (/,). Nach Boas. lange Enddorn endet in eine deutlich gesonderte, kuglige Embryonalschale. Auf beiden Seiten zeigt die Schale mehr oder weniger deutliche Querfurchen, nebst den üblichen Zuwachsstreifen. Sie ist gänzlich durchsichtig, an den Rändern zeigt sich sehr konstant eine rotbraune Farbe, welche sich auch auf größere Strecken der Ober- und Unterseite ausdehnt. — Die junge Schale (Fig. 36 C) wurde unter dem Namen „Üleodora compressa‘“‘ Souleyet als besondere Art beschrieben. Diese Schale ist sehr langgestreckt, dünn und flach, die Öffnung ist ganz auf die Vorderseite beschränkt und nicht spalt- förmig ausgebildet, eine besondere Lippenbildung fehlt; die Seitenkanten der Schale sind fast über die ganze Länge gerade, nur an der Öffnung allmählich konkav, die Embryonalschale ist gerundet. Drei sehr schwache Längsrippen finden sich auf der Oberseite am Rande. Embryonalschale und die Seiten- kanten der Schale bräunlich, sonst farblos. L. der jungen noch nieht meta- morphosierten Schale („Cleodora compressa“) 5 mm, L. der ausgewachsenen Schale 8 mm. — Fig. 36. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavolinüdae, 6. 6. Gen. Diacria Gray Diacria 4l Oft findet man an der Schalenöffnung Kolonien von kleinen Hydrozoen angesiedelt. In allen wärmeren Meeren, etwa zwischen 40° n. Br. bis 40° s. Br., auch noch an der Westküste Irlands und Schottlands. Im östlichen Mittelmeer und im Ost- indischen Archipel sind nur leere Schalen gefunden worden. lb. D. trispinosa major (Boas) 1821 Hyalaea trispinosa (part.), Lesueur MS. in: Blainville in: Diet. Sci. nat., v.22 p.82 \| 1836 H. mucronata (non Quoy & Gaimard 1827), Orbigny Voy. Amer. merid., v.5ı p.108 t.7 f.6—10 \| 1850 Diacria m., J. E. Gray, Öat. Moll. Brit. Mus., v».2 p.11 \| 1852 Hyalaea trispinosa var., Souleyet in: Voy. Bonite, v.2 p. 163, Moll. t.6 f.7—10 \| 1858 Pleuropus mueronatus, H. &A.Adams, (sen. Moll., v.2 p.611 \| 1886 Hyalaca trispinosa var. major, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 95. u al von oben @],). SR rt; sie kommt Schale etwas größer, Seitenstacheln nach hinten und nicht quer gerichtet. Vorderteil (vor einer Linie, welche die beiden Seitenstacheln verbindet) etwas umfangreicher als bei der vorigen Unterart. Die Unterseite ist etwas weniger stark hervorgewölbt. Querfurchen auf der Oberfläche deutlicher. Die bräunliche Farbe ist nur auf die Mundränder beschränkt, sonst ist die Schale gänzlich farblos. L. der ausgewachsenen Schale 11 mm. — Fig. 37. u Die Verbreitung ist dieselbe wie die der vorhergehenden Unterart; sie kommt mit dieser zusammen vor. 2. D. quadridentata (Lesueur MS.) 38 C) wurde früher unter dem Namen „Gleodora pygmaea‘ als besondere Art beschrieben; sehr wahrscheinlich ge- hört sie aber hierher. Die Schale ist langgestreckt, flachgedrückt, sehr dünn; Fig. 38. D. quadridentata quadridentata. A Schale von oben, B von rechts ("/,), C junges Exemplar (‚„Cleodora pygmaea‘“ Boas) von oben (?/,). Nach Boas. Fig. 38. Fig. 38. D. quadridentata quadridentata. A Schale von oben, B von rechts ("/,), C junges Exemplar (‚„Cleodora pygmaea‘“ Boas) von oben (?/,). Nach Boas. die Öffnung ist ganz auf das Vorderteil beschränkt; auf der Oberseite finden sich drei schwache Längsrippen, die Seitenkanten sind gerade, nur im vorderen Drittel konkav, durch eine bräunliche Linie markiert, die Embryonalschale ist deutlich gesondert, abgerundet. L. der ausgewachsenen Schale 2—4 mm, L. der jungen nicht metamorphosierten Schale 3 mm. — Fig. 38. In den tropischen und subtropischen Teilen des Atlantischen und Pazifischen Ozeanes. 2b. D. quadridentata costata (Pfeffer) 1879 Hyalaea c., Pfeffer in: Monber. Ak. Berlin, p.234 \| 1886 H. quadridentata var. c., Boas in: Danske Selsk. Skr., ser. 6 v.4 p. 100. Unterscheidet sich nur dadurch von der vorigen Unterart, daß die fünf Längsrippen auf der Dorsalseite der Schale viel schärfer ausgeprägt sind; auch ist die Verbreitung in der Hauptsache eine andere. L. der Schale 3—4 mm. Im Indischen Ozean, auch im Östindischen Archipel und im Pazifischen, spärlich im Atlantischen Ozean. 1) Abildgaard 1. e. p. 154 ist im Irrtum, wenn er behauptet, daß Gioeni die Gattung ‚„Cavolina“ genannt hat, er gab ihr den Namen: „celata‘“ (Helm). 2. D. quadridentata (Lesueur MS.) 2. D. quadridentata (Lesueur MS.) Zerfällt in 2 Unterarten: Zerfällt in 2 Unterarten: 2a. D. quadridentata quadridentata (Lesueur MS.) 1821 Hyalaea q.. Lesueur MS. in: Blainville in: Diet. Sei. nat., v.22 p.81 \| 1850 Cavolina q., J. E. Gray, Cat. Moll. Brit. Mus., vo. 2 p.8 \| 1852 Hyalaea inermis, A. Gould in: U. S. expl. Exp., v.12 1.51 f.604 \| 1877 H. minuta + H. intermedia, G. B. Sowerby in: Reeve, Conch. icon., v.20 Pter. f.9, 10 \| 1886 H. quadridentata + Cleodora pygmaea, Boas in: Danske Selsk. Skr., ser.6 v.4 p.99 t.1 £.4; t.2 f.15; p.84 t.4 f.50 (Embryonal- schale); £.57—571u1; t.5 f.90 (Querschnitt am Mundrande) \| 1888 Cavolinia quadri- dentata, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.78 \| 1904 ©. (Di- acria) q., Tesch in: Siboga-Exp., nr.52 p.36 \| 1905 D.q., Meisenheimer in: Ergeb. Tiefsee-Exp., v.91ı p. 29. Schale stark kuglig, aufgeblasen; die Unterseite ist, wie bei den Oavolinia-Arten, sehr stark hervorgewölbt, gleichmäßig gerundet, ohne eine Spur von hervortretenden Längsrippen; die Oberseite ist weniger gewölbt und trägt fünf Längsrippen, welche durch schmale Furchen getrennt sind. Der hintere Teil der Schale ist quer abgestutzt, die Krümmung der Seiten- kanten deutet darauf hin, daß ein ziemlich langer, konisch zugespitzter End- dorn in einem gewissen Lebensstadium vorhanden sein muß; bei den er- wachsenen Tieren wird er aber stets abgeworfen und die dadurch entstandene Öffnung durch eine nach vorn konkave Lamelle verschlossen. Der Rand der Oberschale ist stark verdickt und stark ventral gekrümmt, so daß der quere Spalt fast ganz überdacht wird. Auch der vordere Rand der Ventralschale ist etwas verdickt, mehr aber die seitlichen Teile. Die lateralen Schlitze der Schalenöffnung sind durch eine Zahnbildung an der Unterseite von dem medianen Teile getrennt; diese Zahnbildung ist hier etwas besser ausgebildet als bei Diacria trispinosa. An den Ecken der Öffnung ist die Schale etwas flachgedrückt, aber nicht in Seitenstacheln ausgezogen. Schale, besonders auf der Unterseite, mit feinen, regelmäßigen Querfurchen. Die Farbe ist weißlich 42 2 Pterop.: A. Thecos., a) Euthecosomata, 2. Cavoliniidae, 6. Diacria, 7. Cavolinia oder fehlt gänzlich; der verdickte Rand der Dorsalschale ist stets bräunlich gefärbt. — Die junge Schale (Fig. 38 C) wurde früher unter dem Namen „Gleodora pygmaea‘ als besondere Art beschrieben; sehr wahrscheinlich ge- hört sie aber hierher. Die Schale ist langgestreckt, flachgedrückt, sehr dünn; oder fehlt gänzlich; der verdickte Rand der Dorsalschale ist stets bräunlich gefärbt. — Die junge Schale (Fig. 7. Gen. Cavolinia Abildgaard (corr. Philippi) 1775 Anomia (non Linne 1767), Forskäl, Deser. An., p.124 \| 1791 Cavolina, Abildgaard!) in: Skr. Naturh. Selsk., v.lır p. 171 (non 1792 C., Bruguiere in: Tabl. ene. meth., v1 t.85 f.4,5) \| 1797 Rheda [Calonne], Mus. Calonn., p. 41 (nom. nud.) \| 1801 Hyalaea, Lamarck, Syst. An. s. Vert., p. 139 \| 1810 Archonta, Montfort, Conch. syst., v.2 p.50 \| 1815 Triela, Oken, Lehrb. Naturg., v.1 p.327 \| 1825 Pleuropus, Eschscholtz in: Isis, p. 735 \| 1853 Cavolinia, A. Philippi, Handb. Conch., p. 290 \| 1859 Orbignyia (Subgen.), A. Adams in: Ann. nat. Hist., ser. 3 v. 3 p. 45 \| 1886 Hyalaea (part.), Boas in: Danske Selsk. Skr., ser. 6 v.4 p. 92 \| 1888 Cavolinia (part.), Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. #9. Schale meist stark gewölbt, besonders an der Ventralseite; durch eine Linie, welche die Ecken der Schalenöffnung verbindet, in zwei Teile zerlegbar, von welchen der vordere, weitaus größere Teil von Ober- und Unterschale ge- Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 43 bildet wird, da der Spalt sich über den Vorderrand und über die Seitenteile der Schale bis weit nach hinten erstreckt; der hintere Teil ist sehr kurz, entweder in eine dorsal gebogene Spitze endend oder quer abgestutzt, da der Hinter- teil abgeworfen und durch eine die Öffnung schließende Kalkmasse ersetzt wird. Der Spalt wird durch eine, ähnlich wie bei /Viaeria, aber viel besser ausgebildete Zahnvorrichtung in einen medianen Teil und in zwei seitliche Partien geteilt; ein kleiner Zahn am Rande der Ventralschale paßt in eine entsprechende Aushöhlung am Rande der Dorsalschale hinein. Der mediane ‚Spalt dient als eigentliche Schalenöffnung, durch welche das Tier Flossen und vordere Körperpartie herausstreckt; die beiden lateralen Schlitze sind sehr eng und erstrecken sich gerade nach hinten; sie lassen beim lebenden Tiere eigentümliche schleifenartige und sehr dehnbare Verlängerungen der im Bereich des Schalenschlitzes verwachsenen Mantelränder hervortreten (s. p.5). An den hinteren Enden der Schlitze ist: die Schale etwas flach- gedrückt und zieht sich in einen kurzen, meist halbrinnenförmig gebogenen Fortsatz aus. Die Schalenober- und -unterlippe bilden, da der Schalenspalt so weit nach hinten reicht, fast allein die ganze Schale. Die Oberseite ist weniger stark vorgewölbt und zeigt fünf, vorn breite und abgerundete, nach hinten zu sich verengende und konvergierende Längsrippen, eine größere mediane und zwei etwas schwächere laterale jederseits, von welchen die äußere wieder sekundär durch eine in der Mitte verlaufende Längsfurche geteilt sein kann. Zerfällt in 3 Unterarten: Zerfällt in 3 Unterarten: la. C. longirostris longirostris (Lesueur) 1821 Hyalaea Il. + H. ecaudata, Lesueur MS. in: Blainville in: Diet. Sei. nat., v.22 p.81, 82 \| 1836 A. longirostrd + H. limbata + H. laevigata, Orbigny, Voy. Amer. merid., e.5ım p. 100 t.6 £.6—10; p.101 t.6 £.11—15; p.110 t.7 f.15—19 (junges Exemplar) \| 1850 Cavolina longi- rostra + Diacria laevigata, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.8; p.11 (junges Exemplar) \| 1852 Hyalaeca femorata, A. Gould in: U. S. expl. Exp., «12 t.51 f. 605 \| 1858 Pleuropus laevigatus, H. & A. Adams, Gen. Moll, «.2 p. 611 (junges Exemplar) \| 1861 Hyalaea fissilabris, Benson in: Ann. nat. Hist., ser.8 v.7 p.26 \| 1877 H. obtusa, G. B. Sowerby in: Reeve, Conch. icon.. v.20 Pter. t.2 f.8 \| 1877 Cavolina longirostris + C. limbata, Angas in: P. zool. Soc. London, p.178 \| 1886 Hyalaea longi- rostris, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 102 t.1 £.5; t.2 £.16 \| 1888 Cavolinia l., Pelseneer in: Rep. Voy. Challenger, Zool. v. 23 pars 65 p. 79 \| 1908 Cavolina couthouyi, W.Dall in: Smithson. Collect., v.50 p. 501. Die Schale bildet von oben gesehen annähernd ein gleichseitiges Dreieck. Die Unterseite ist stark hervorgewölbt, in Profilansicht gleichmäßig gerundet. Der Vorderrand der Oberschale ist schräg nach vorn und ventral umgebogen, in der Medianlinie sehr lang vorgezogen in Gestalt einer nach der Schalen- öffnung zu offenen und ganz geraden Halbrinne, der Übergang zwischen dem geraden und dem gebogenen Teile der Oberseite vollzieht sich ganz all- mählich, ohne Wülste oder Querfalten. Die Oberseite hat fünf Längsrippen, die Furchen zu beiden Seiten der medianen sind sehr schwach, dagegen sind die beiden lateralen jederseits, von welchen die äußere sehr undeutlich ausgebildet ist, durch eine tiefe Furche getrennt. Eigentliche Seitenfortsätze, an deren Bildung sich Ober- wie Unterseite beteiligen, sind nieht vorhanden, nur ist am Ende des seitlichen Schalenschlitzes der Rand der Unterschale immer vorgezogen, oft so weit, daß eine dünne, dreieckige Platte vorragt, welche in ihrem Verlaufe gerade oder etwas dorsal gekrümmt sein kann. Die Schale ist hier, im Bereich dieser Platten, beträchtlich breiter als mehr vorn. Die Unterseite hat zahlreiche feine Querrippen, besonders auf der vorderen Hälfte. Der Enddorn fehlt fast ganz, ist an der Basis abgebrochen und durch eine dünne Kalkmasse ersetzt. 7. Gen. Cavolinia Abildgaard (corr. Philippi) Der proximale Rand der Oberseite ist stets ventral gekrümmt, oft in der Mitte halbrinnenförmig vorgezogen. Die Unterseite ist sehr stark gewölbt, aufgeblasen, mit zahlreichen feinen Querfurchen versehen, am proximalen Rande stark dorsal, nach der Schalenöffnung hin, gebogen, so dab der Spalt bedeutend eingeengt wird, dann wieder etwas ventral zurück- geschlagen. Die Ränder der Öffnung sind nie verdickt. Der Hinterteil der Schale ist gewöhnlich kurz, schnell zugespitzt oder quer abgestutzt, ohne scharfe Seitenkanten; die letzteren in ihrem Verlaufe stark konkav. Eine eigentliche Embryonalschale ist nicht scharf abgetrennt, meist fehlend, da häufig die Spitze der Schale abgeworfen wird; wenn vorhanden nicht scharf zugespitzt, sondern gerundet. — Die jungen Schalen können, wie bei Diaeria, schon ganz die Größe der erwachsenen Schale erreichen und bilden sich dann allmählich um; sie sind stark abgeflacht, sehr dünn und zerbrechlich; der Spalt ist weiter, mehr klaffend und nicht so weit nach hinten reichend, eine Zerlegung in drei Teile ist noch nicht ausgebildet; Längsrippen auf den Ober- und Querfurchen auf der Unterseite fehlen fast vollständig; in sehr jugendlichem Stadium ist die Schale noch Clio-ähnlich und erst bei fort- schreitendem Wachstum bildet sie sich allmählich heraus. — Beim Tiere sind die Flossen breit, am freien Rande einmal eingekerbt, das Wimperfeld auf den Flossen nimmt eine besonders scharf umschriebene Stelle ein, der Mittellappen des Fußes ist sehr breit und kurz. Der Mantel kann sich aus der Schale herausstrecken und sich eine Strecke weit über den vorderen Teil der Ventralschale lagern, auch der sehr dehnbare vordere Teil der seit- lichen Mantelverlängerung kann die Schale teilweise bedecken. Die Niere liegt quer und ganz hinten in der Mantelhöhle und hat eine hufeisenförmige, nach vorn konkave Gestalt; das Herz ist links gelagert, mit dem Vorhof nach hinten, die Kammer nach vorn gewendet. In der Mantelhöhle findet sich weiter eine sehr lange, hufeisenförmig gebogene Kieme, deren Falten sich über die hintere Wand der linken, hinteren und rechten Mantelhöhle erstrecken. In den wärmeren Teilen der Ozeane, an der Oberfläche oder in geringen Tiefen. 6 Arten, von welchen drei in je 2, eine in 3 Unterarten zerfallen. 44 Pteropoda: A. Theeosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia Bestimmungstabelle der Arten: Sehale hinten breit abgestutzt; Vorderteil der Oberschale in Form einer schmalen Halbrinne ausgezogen; seitliche Fortsätze der Schale fast nur von dem hinteren Teile der Unterschale gebildet (Fig. 39, 40) . . ». .» ... . 1. ©. longirostris Schale hinten mehr oder weniger spitz endend — 2. 7. Gen. Cavolinia Abildgaard (corr. Philippi) Schale mit schwachen, undeutlichen Seitenfortsätzen — 3. Schale mit deutlichen Seitenfortsätzen — 5. Schale in der Gegend vor den Seitenfortsätzen am breitesten — 4. Schale in der Gegend der Seitenfortsätze ebenso breit wie weiter vorn (Fig. 44, 45) - SE: 4. C. tridentata Unterseite der Schale stark und Aasimäsık gew völbt (Fig. 41) 2. C. globulosa Unterseite der Schale stark hervorgewölbt, der vordere Teil geht mit einem rechten oder spitzen Winkel in den hinteren Teil über (Fig. 42, 43)...» . 2... 3. C©. gibbosa Vorderteil der Oberschale sehr stark ventral Bst Ki 16) 207 21012 BURN 5. C. uncinata Vorderteil der Oberschale eradde, biekk gebogen (Fig. 47 48) 6. C. inflexa 1. C. longirostris (Lesueur) Zerfällt in 3 Unterarten: Die Exemplare aus dem Atlantischen und Ostpazifischen Ozean (bis zu 20° s. Br. sind gewöhnlich größer als in den anderen Meeren, und unterscheiden sich dadurch, daß die seitlichen Fortsätze der Schale ganz gerade oder nur sehr schwach gebogen sind, während sie sonst meist eine deutliche dorsale Krümmung aufweisen. Es kommt mir vor, als ob Dall die Diagnose seiner neuen Art Cavolina couthouyi nach größeren Exempiaren von ©. longirostris longirostris, aufgestellt hat; ich kann wenigstens aus seiner Beschreibung, zu welcher er keine Abbildung gibt, nur die sehr große Übereinstimmung mit derjenigen der betreffenden Unterart herauslesen und finde keine besonderen Artmerkmale. In den wärmeren Teilen der Ozeane, annähernd zwischen 40° n. Br. und 30° s. Br., auch im westlichen Mittelmeer. Zerfällt in 3 Unterarten: Farbe violett, bräunlich oder weißlich; an der Stelle, wo sich die Vertiefung am Rande der Oberschale findet, ist oft ein scharf umschriebener, brauner Fleck vorhanden, eine Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Oavoliniidae, 7. Cavolinia 45 bräunliche, verwaschene Farbe zeigt auch oft die mediane Längsrippe auf der Oberseite. — Die junge Schale (Fig. 39 C—E) ist unter dem Namen „Ayalaea laevigata® Orbigny beschrieben worden; sie ist sehr flach und dünn, der Hinterteil ist ziemlich stark entwickelt und dorsal gekrümmt mit stumpfem Ende, da die Spitze immer abgebrochen ist; der Vorderteil ist nach vorn zu gleichmäßig abgerundet und bildet, von oben gesehen, mehr als einen Halbkreis. Der Vorderrand der Dorsalschale ragt nicht hervor, der der Ventralschale ist in der Medianlinie etwas eingebuchtet. Seitenfortsätze bräunliche, verwaschene Farbe zeigt auch oft die mediane Längsrippe auf der Oberseite. — Die junge Schale (Fig. 39 C—E) ist unter dem Namen „Ayalaea laevigata® Orbigny beschrieben worden; sie ist sehr flach und dünn, der Hinterteil ist ziemlich stark entwickelt und dorsal gekrümmt mit stumpfem Ende, da die Spitze immer abgebrochen ist; der Vorderteil ist nach vorn zu gleichmäßig abgerundet und bildet, von oben gesehen, mehr als einen Halbkreis. Der Vorderrand der Dorsalschale ragt nicht hervor, der der Ventralschale ist in der Medianlinie etwas eingebuchtet. Seitenfortsätze B4 x Fig. 39. C. longirostris longirostris.. A Schale von oben, B von rechts (1%), © junge Schale (,„Hyalaea (laevigata“ von oben, D dieselbe von unten, E dieselbe von rechts (?”/,). Nach Boas. Fig. 39. Fig. 39. C. longirostris longirostris.. A Schale von oben, B von rechts (1%), © junge Schale (,„Hyalaea (laevigata“ von oben, D dieselbe von unten, E dieselbe von rechts (?”/,). Nach Boas. C. longirostris longirostris.. A Schale von oben, B von rechts (1%), © junge Schale (,„Hyalaea (laevigata“ von oben, D dieselbe von unten, E dieselbe von rechts (?”/,). Nach Boas. oder Gebilde, welche bei der erwachsenen Schale an deren Stelle vorkommen, fehlen noch durchaus. Die Oberseite hat drei schwache Längsrippen, die Unterseite ist mit ziemlich deutlichen, dem Umriß parallel verlaufenden und in der Medianlinie eingebuchteten Querlinien ausgestattet. Mundspalt weit nach hinten reichend, bis zu der Stelle, wo der gebogene Umriß des Vorder- teiles der Schale plötzlich in einen rechten Winkel umbiegt und in den Hinter- teil übergeht. — L. der ausgewachsenen Schale sehr wechselnd, von 2—9 mm. — Fig. 39. Die Exemplare aus dem Atlantischen und Ostpazifischen Ozean (bis zu 20° s. Br. In den wärmeren Teilen der Ozeane, annähernd zwischen 40° n. Br. und 30° s. Br., auch im westlichen Mittelmeer. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 465 lb. C.longirostris angulata (Souleyet) 1852 Hyalaea a., Souleyet in: Voy. Bonite, «.2 p.152, Moll. t.5 f. 1—#. lb. C.longirostris angulata (Souleyet) 1852 Hyalaea a., Souleyet in: Voy. Bonite, «.2 p.152, Moll. t.5 f. 1—#. Unterscheidet sich nur dadurch, daß der Vorder- rand der Oberschale sich von dem Rest durch eine tiefe (uerfalte abgrenzt, welche in Profilansicht deutlich sicht- bar ist. Seitenfortsätze der Schale wenig entwickelt. L. der Schale 4 mm. — Fig. 40. — NL , a C. longirostris angu- lata. Schale von rechts (4). Nach Souleyet I). D> r a a C. longirostris angu- lata. Schale von rechts (4). Nach Souleyet I). D> r Zerstreut zwischen Exemplaren der vorhergehenden Unter- art im Indischen und Westpazifischen Ozean. le. C. longirostris strangulata Hedley 1906 ©. Il. var., Hedley in: Tr. N. Zealand Inst.,. » 38 p.76 \| 1907 ©. I. var. strangulata, Hedley in: Rec. Austral. Mus., 0.6 p.299 t.54+ £. 13. Gym Pe. jeta. Schale von unten. Nach Hedley (vergr.?). Unterscheidet sich von der Hauptart dadurch, daß der vorspringende Vorderrand der Dorsalschale an der Basis stark eingeschnürt und vor der Ein- schnürung löffelartig erweitert ist. Wie bei der vor- hergehenden Unterart sind die hinteren Seitenecken der Schale wenig entwickelt. L. unbekannt. — Fig. 40a. Nur in leeren Schalen in der Nähe von Great Barrier Island bei Neu-Seeland und von Sydney bekannt. In der letzten Lokalität scheint sie die Hauptart zu ersetzen und weiter nördlich nicht vorzukommen. Gym Pe. jeta. Schale von unten. Nach Hedley (vergr.?). 2. C. globulosa (Rang) 1850 Cavolina (Hyalaea) g, Gym Pe. Rang MS. in: .J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.8 \| 1850 jeta. Schale von unten. C. pisum, Mörch, Cat. Conch. Kierulf, p.32 t.1 f.7 \| 1852 Nach Hedley (vergr.?). Hyalaea globulosa, Rang MS. in: Souleyet in: Voy. Bonite, v.2 p.142, Moll. t.4 f.20—24 \| 1886 H.g. + H.rotundata, Boas in: Danske Selsk. Skr., ser. 6 «4 p.107 t.1.7; t.2 £.18; p.129 t.4 £.59—61 (junges Exemplar) \| 1888 Cavolinia g., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.81. Die Unterseite der Schale ist hier sehr stark hervorgewölbt und in Profilansicht gleichmäßig gerundet; die Höhe der Schale ist gleich der größten Breite; die Seitenfortsätze sind ziemlich kurz, der Abstand zwischen ihnen ist kleiner als die größte Breite der Schale weiter nach vorn. Zerfällt in 3 Unterarten: sind gewöhnlich größer als in den anderen Meeren, und unterscheiden sich dadurch, daß die seitlichen Fortsätze der Schale ganz gerade oder nur sehr schwach gebogen sind, während sie sonst meist eine deutliche dorsale Krümmung aufweisen. Es kommt mir vor, als ob Dall die Diagnose seiner neuen Art Cavolina couthouyi In den wärmeren Teilen der Ozeane, annähernd zwischen 40° n. Br. und 30° s. Br., auch im westlichen Mittelmeer. In den wärmeren Teilen der Ozeane, annähernd zwischen 40° n. Br. und 30° s. Br., auch im westlichen Mittelmeer. 465 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia Der Enddorn ist sehr kurz und schmal, gleich von der Basis ab fast senkrecht in die Höhe gerichtet; die Endspitze fehlt immer, an deren Stelle findet man eine dünne Querwand. Außer den beiden Seitenkanten der Schale, welche sich bis auf den Enddorn erstrecken, finden sich jederseits noch zwei weitere Kanten, eine oben und eine unten, so daß der Querschnitt an der Basis des Enddornes unregelmäßig sechseckig wird. Der Vorderrand der Oberschale ist sehr stark ventral gebogen und bildet eine senkrechte Überdachung der Öffnung der Schale; der obere Rand der seitlichen Schalenschlitze ist über den Unterrand gebogen, nur der hintere Teil der Schlitze ist von außen sichtbar. Die Oberseite ist ziemlich hervorgewölbt und sieht glatt, wie poliert aus; sie zeigt fünf Längsrippen, von welchen die beiden lateralen jederseits nur durch eine untiefe Furche getrennt sind; die Unterseite hat das gleiche polierte Aussehen, in der vorderen Hälfte trägt sie feine Quer- rippen. Eine zarte Querstreifung kommt auf der ganzen Schale vor. Farbe leicht bräunlich, mit Ausnahme des vorderen Teiles der Unterseite, wo die Farbe meist gänzlich fehlt und die Schale durchsichtig ist. — Die junge Schale (Fig. 41 C°—E) wurde unter dem Namen „Ayalıea votundata“ Boas Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 47 beschrieben. Sie ist sehr dünn, glashell; Ober- und Unterseite sind nahezu gleich stark vorgewölbt; der Vorderteil der Schale ist sehr groß, im Umriß beschreibt er etwa drei Viertel eines breiten Ovales; der Hinterteil ist sehr kurz, der Enddorn gleich nach oben gekrümmt, und man findet hier schon beschrieben. Sie ist sehr dünn, glashell; Ober- und Unterseite sind nahezu gleich stark vorgewölbt; der Vorderteil der Schale ist sehr groß, im Umriß beschreibt er etwa drei Viertel eines breiten Ovales; der Hinterteil ist sehr kurz, der Enddorn gleich nach oben gekrümmt, und man findet hier schon a D Fig. 41. C. globulosa. A Schale von oben, B von rechts (%/,), € junge Schale (,HAyalaea rotundata“) von oben, D von unten, E von rechts (/),). Nach Boas. a D D Fig. 41. C. globulosa. A Schale von oben, B von rechts (%/,), € junge Schale (,HAyalaea rotundata“) von oben, D von unten, E von rechts (/),). Nach Boas. Fig. 41. die im Querschnitt eckige Ausbildung, welche für diese Art charakteristisch ist, die Endspitze fehlt immer. Die beiden Lippen der Schalenöffnung sind von gleicher Länge. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia An der Oberseite sind schon fünf schwache Längsrippen angedeutet; auf beiden Seiten kommen feine, dem Umriß parallel verlaufende Querstreifen vor. — L. der ausgewachsenen Schale 5—6 mm. — Fig. 41. die im Querschnitt eckige Ausbildung, welche für diese Art charakteristisch ist, die Endspitze fehlt immer. Die beiden Lippen der Schalenöffnung sind von gleicher Länge. An der Oberseite sind schon fünf schwache Längsrippen angedeutet; auf beiden Seiten kommen feine, dem Umriß parallel verlaufende Querstreifen vor. — L. der ausgewachsenen Schale 5—6 mm. — Fig. 41. Auf die wärmsten Stromgebiete des Indischen und Pazifischen Ozeanes beschränkt, im Bereich des Kap-Horn-Stromes bis etwa 85°s. Br. vordringend, sonst aber meist innerhalb der Wendekreise.. Im Atlantischen Ozean ist sie äußerst selten; kaum sind hier einige wenige Fundorte innerhalb der tropischen Zone bekannt geworden. 3. C. gibbosa (Rang) Zersällt in 2 Unterarten: 3. C. gibbosa (Rang) 3. C. gibbosa (Rang) Zersällt in 2 Unterarten: Zersällt in 2 Unterarten: 3a. C. gibbosa gibbosa (Rang) 1836 Hyalaea g., Rang MS. in: Orbigny, Voy. Amer. merid.. v.5ur p.95 t.5 f.16—20 \| 1836 H. flava, Orbieny, Voy. Amer. merid., v. 5ıu p.97 t.5 £.21—25 \| 1850 Cavolina gibbosa + ©. f., J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.8 \| 1852 Hyalaca g., Souleyet in: Voy. Bonite, v.2 p.144, Moll. t.4 f. 13—19 \| 1880 H. gegenbauri, Pfeffer in: Abh. Ver. Hamburg, v.71 p.86 t.7 f.7,7a\| 1886 H. gibbosa, Boas in: Danske Selsk. Skr., ser. 6 v.4 p. 109 t.1 f.6; t.2 £.17; t.6 f.99 \| 1888 Cavolinia g., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 82. Diese Art ist am nächsten mit der vorigen verwandt. Sie stimmt mit ihr überein in der ansehnlichen Höhe der Schale, welche nahezu der größten Breite gleichkommt, weiter dadurch, daß die Schale in der Gegend der ziemlich kurzen Seitenfortsätze beträchtlich weniger breit ist als weiter vorn. Der Vorderrand der Oberschale ist, wie bei der vorigen Art, stark Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 48 nach der Öffnung hin gekrümmt; doch ist dieser Vorderrand nicht selbst gebogen, sondern gerade, und schräg nach vorn und ventral gerichtet, länger als bei ©. globulosa. . Zudem weicht die jetzt zu besprechende Art in folgenden Punkten ab. Der vordere Teil der Unterschale geht nicht mit einer sanften Rundung in den hinteren Teil über, sondern bildet, in Profilansicht der Schale, mit diesem Hinterteil einen deutlichen Winkel. Die für ©. Die Exemplare des Indischen und des Südatlantischen Ozeanes unterscheiden sich meist dadurch, daß sie eine kleinere und verhältnismäßig höhere Schale besitzen; der Enddorn ist etwas mehr nach oben gerichtet, und die innere laterale Längsrippe auf der Oberseite ist kaum oder gar nicht durch eine Längsfurche geteilt; auch ist der vordere Teil der Oberschale nicht schräg, sondern fast senkrecht ventral gebogen. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia Br., auch im Mittelmeer, aber merkwürdigerweise in den tropischen Gegenden, etwa zwischen den Wendekreisen, viel seltener, hier meist als leere Schalen gefunden worden. 3b. C. gibbosa plana Meisenheimer 1904 C.g.var., Tesch in: Siboga-Exp., nr.52 p.4l t.2 f.34, 35 \| 1905 C. g. var. plana, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.32 t.I f.2a,b. 3b. C. gibbosa plana Meisenheimer 1904 C.g.var., Tesch in: Siboga-Exp., nr.52 p.4l t.2 f.34, 35 \| 1905 C. g. var. plana, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.32 t.I f.2a,b. Fig. 43. C. gibbosa plana. Schale von rechts (/). Nach Meisenheimer. Unterscheidet sich hauptsächlich dadurch, daß die Unterseite der Schale bei weitem nicht so stark ventral vorgebuchtet ist; die Hervor- wölbung ist namentlich nach der Vorderseite hin entwickelt, so daß hier in Profilansicht ein spitzer Winkel ausgebildet ist. Die Längsrippen Fig. 43. ; : RSS; C. gibbosa plana. Schale von auf der Oberseite sind fast gänzlich ver- rechts (/). Nach Meisenheimer. schwunden. Der Enddorn ist nur schwach dorsal gekrümmt; die Spitze fehlt. L. der Schale etwa 10 mm. — Fig. 43, Im Ostindischen Archipel und im östlichen Teile des Indischen Ozeanes, zwischen Exemplaren der vorhergehenden Unterart vorkommend. ] Unterscheidet sich hauptsächlich dadurch, daß die Unterseite der Schale bei weitem nicht so stark ventral vorgebuchtet ist; die Hervor- wölbung ist namentlich nach der Vorderseite hin entwickelt, so daß hier in Profilansicht ein spitzer Winkel ausgebildet ist. Die Längsrippen ; : RSS; auf der Oberseite sind fast gänzlich ver- schwunden. Der Enddorn ist nur schwach dorsal gekrümmt; die Spitze fehlt. L. der Scha Fig. 43. Fig. 43. C. gibbosa plana. Schale von rechts (/). Nach Meisenheimer. Im Ostindischen Archipel und im östlichen Teile des Indischen Ozeanes, zwischen Exemplaren der vorhergehenden Unterart vorkommend. ] 4. C. tridentata (Forsk.) Zerfällt in 3 Unterarten: 4a. C. tridentata tridentata (Forsk.) ?1767 Monoculus telemus, Liune, Syst. nat., ed. 12 v.lır p.1059 \| 1775 Anomia tridentata, Forskäl, Deser. An., p. 124 \| 1791 Cavolina natans, Abildgaard in: Skr. naturhist. Selsk., v.J p.175 \| 1801 HAyalaea cornea, Lamarck, Syst. An.s. Vert., p. 140 \| 1802 H. tridentata, Bose in: Hist. Coqu., v.2 p.241 t.9 £.4 \| 1804 H. papilionacea, Bory, Voy. Iles Afr., v.1 p.137 t.5 £.1 \| 1807 H. australis (non Orbigny 1836), P&ron, Voy. terres Austr., Atl. t.31 f.5 \| 1810 „Ayale teniobranche“, Peron & Lesueur in: Ann. Mus. Paris, v.15 t.2 f.13 \| 1813 Hyalaca peronii + H. chemnitziana, Lesueur in: N. Buil. Soc. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia globulosa so charakte- ristische Falte an der dorsalen Seite der seitlichen Schalenschlitze, welche den ventralen Rand überragt, ist hier nur angedeutet im vorderen Teile der Schlitze oder fehlt gänzlich; man findet übrigens alle Übergänge von einem vollständigen Mangel bis zu einer deutlichen Entwicklung, wenn auch nie so stark aus- geprägt wie bei der vorigen Art. Der Enddorn ist an der Basis breiter als bei ©. globulosa und nicht so direkt dorsal gebogen, sondern schräg nach hinten und nach oben gerichtet; nur die Spitze ist stärker, oft sehr stark, hakenförmig gekrümmt, sie scheint oft zu fehlen. Die scharfen Seiten- kanten des Enddornes bei €. globulosa fehlen hier, der Querschnitt ist also mehr zylindrisch. An der Stelle der abgebrochenen Spitze findet man Fig. 42. C. gibbosa gibbosa. A Schale von oben, B von rechts (,). Nach Boas. Fig. 42. C. gibbosa gibbosa. A Schale von oben, B von rechts (,). Nach Boas. gewöhnlich eine Querwand, welche sich aber nie bei Exemplaren ausbildet bei welchen diese Spitze noch vorhanden ist. Die Embryonalschale ist, wie bei den übrigen Cavolinia-Arten, durch eine schwache aber deutliche Ein- schnürung von dem übrigen Teile des Enddornes gesondert. Die Oberseite der Schale trägt fünf Längsrippen, die innere laterale ist zuweilen durch eine schwache Längsfurche sekundär geteilt; die Furche zwischen den beiden lateralen Längs- rippen ist deutlicher als bei €. globulosa. An der Unterseite, besonders im vorderen Teile, finden sich transversale Rippen; zudem erstreckt sich hier auf der vorderen, senkrecht umgebogenen Partie eine mediane, flache Grube. Die Schale ist durchsichtig, oft aber auch ganz kreideweiß, von glänzendem, poliertem Aussehen; an den Schalenschlitzen, auch am Vorderrande der Unterseite ist eine schwach rosenrote oder rötlich-braune Farbe vorhanden, welche sich wie ein zarter Hauch auch über die Oberseite, mit Ausnahme des vorderen, gebogenen Teiles, erstrecken kann. — Mit Sicherheit kennt man die junge Schale noch nicht. — L. der ausgewachsenen Schale bis zu 11 mm. — Fig. 42. Die Exemplare des Indischen und des Südatlantischen Ozeanes unterscheiden sich meist dadurch, daß sie eine kleinere und verhältnismäßig höhere Schale besitzen; der Enddorn ist etwas mehr nach oben gerichtet, und die innere laterale Längsrippe auf der Oberseite ist kaum oder gar nicht durch eine Längsfurche geteilt; auch ist der vordere Teil der Oberschale nicht schräg, sondern fast senkrecht ventral gebogen. Pteropoda: A. Thecosomata, a) Euthecosomata, 2, Cavoliniidae, 7. Cavolinia 49 In den wärmeren Teilen aller Ozeane, etwa zwischen 40° n. Br. und 40° s. Das Tierreich, 36. Lief.: J. J. Tesch, Pteropoda. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 44€, D) („Pleuropus longifilis“ Troschel, „Ayalaea complanata“ Gegenbaur) ist im Anfang Clio-ähnlich, länglich-dreieckig, mit einem stumpfen, ziemlich stark dorsal gekrümmten Hinterteil; sehr dünn und ganz durchsichtig, ohne Skulptur; die Schalenöffnung ist noch sehr wenig breit. Beim fortschreitenden Wachs- D E F Fig. 44. C. tridentata tridentata. A Schale von oben, B von rechts (?),). Nach Boas. C junge Schale („Pleuropus longifilis‘‘) mit halb hervorgestrecktem Tiere, von unten, D dieselbe von rechts (%/,), E etwas älteres Stadium mit zurückgezogenem Tiere, van unten, F dasselbe von rechts (j,). Nach Schiemenz. Fig. 44. Fig. 44. C. tridentata tridentata. A Schale von oben, B von rechts (?),). Nach Bo („Pleuropus longifilis‘‘) mit halb hervorgestrecktem Tiere, von unten, D diese E etwas älteres Stadium mit zurückgezogenem Tiere, van unten, F dasse Nach Schiemenz. Fig. 44. C. tridentata tridentata. A Schale von oben, B von rechts (?),). Na („Pleuropus longifilis‘‘) mit halb hervorgestrecktem Tiere, von unten, D E etwas älteres Stadium mit zurückgezogenem Tiere, van unten, F Nach Schiemenz. Nach Boas. C junge Schale D dieselbe von rechts (%/,), F dasselbe von rechts (j,). tum vergrößert sich der Vorderteil immer mehr, wird halbkreisförmig im Umriß, die Öffnung erstreckt sich über den ganzen Vorderrand, welcher sich zugleich schärfer dem Reste der Schale gegenüber abhebt. . Schließlich nähert sich die junge Schale, wie man Schritt für Schritt, besser als bei irgend einer anderen Cavolinia-Art beobachtet hat, immer mehr der erwachsenen Form. Besonders auffallend sind bei dem jungen Tiere die zwei (oder drei?) seitlichen Mantelverlängerungen, welche beim Schwimmen aus den Winkeln der Schalen- öffnung hervorgestreckt werden. — L. der ausgewachsenen Schale sehr wechselnd, 9—20 mm; wir haben es hier mit der größten Cavolinia-Art zu tun. — Fig. 44. tum vergrößert sich der Vorderteil immer mehr, wird halbkreisförmig im Umriß, die Öffnung erstreckt sich über den ganzen Vorderrand, welcher sich zugleich schärfer dem Reste der Schale gegenüber abhebt. . Schließlich nähert sich die junge Schale, wie man Schritt für Schritt, besser als bei irgend einer anderen Cavolinia-Art beobachtet hat, immer mehr der erwachsenen Form. Besonders auffallend sind bei dem jungen Tiere die zwei (oder drei?) seitlichen Mantelverlängerungen, welche beim Schwimmen aus den Winkeln der Schalen- öffnung hervorgestreckt werden. — L. der ausgewachsenen Schale sehr wechselnd, 9—20 mm; wir haben es hier mit der größten Cavolinia-Art zu tun. — Fig. 44. Auf der Schale kommt häufig eine Hydrozoen-Art (Perigonimus repens nach Chun) vor. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia philom., v.3 p.284 \| 1821 AH. forskalii (non Rang & Souleyet 1852), Lesueur MS. in: Blainville in: Diet. Sci. nat., v. 22 p- 79 \| 1821 H. teniobranchia, Blainville in: Diet. Sci. nat., v.22 p.80 \| 1853 Cavolinia tridentata, A. Philippi. Handb. Conch., p.290 \| 1854 Pleuropus longifilis, Troschel in: Arch. Naturg., v.201 p.208 t.8 £.1—3 (junges Exemplar) \| 1855 Hyalaea complanata, Gegenbaur, Unters. Pter. u. Heterop., p.40 t.1 f. 1 (junges Exemplar) \| 1858 Cavolina tridentata, H.& A. Adams, Gen.Moll.,v.1 p.5l t.6 f.1 \| 1859 CO. telemus, A. Adams in: Ann. nat. Hist., ser. 3 v.3 p.44 \| 1877 Hyalaea cumingii + H.affinis (non Orbigny 1836), G. B. Sowerby in: Reeve, Conch. ieou, v.20 Pter. t.1 f.3,5 \| 1886 A. tridentata + H. longifilis, Boas in: Danske Selsk. Skr., ser.6 v.4 p.115 t.1 f.8; t.2 £.19; p.128 t.4 £.64, 65 (junges Exemplar). Die Unterseite der Schale ist in Profilansicht, besonders in der vorderen Hälfte, stark vorgewölbt, nach hinten dagegen flacht sie sich schnell ab. Im Bereich der lateralen Schalenschlitze bleibt die Breite der Schale immer gleich. Der Hinterteil ist sehr kurz, der Enddorn gerade nach hinten gestreckt; nur die Spitze leicht dorsal gekrümmt, meist aber fehlend. Die Oberseite ist nur wenig vorgewölbt und zeigt fünf Längsrippen; die innere laterale ist durch eine longitudinale Furche sekundär geteilt, die mediane ist vorn durch eine halbkreisförmige Furche deutlich abgegrenzt. Gleich vor den proximalen Enden der Längsrippen lagert sich ein deutlicher, in der Mitte des Verlaufes stark vorgebuchteter Querwulst, der den Vorderrand der Dorsalschale von dem Reste trennt; dieser Vorderrand ist schräg nach vorn und ventral umgebogen, stark löffelartig verlängert mit etwas aufgebogenen Rändern und ganz gerade. Die Unterseite ist erheblich kürzer, am Rande des Mundspaltes ventral umgekrempelt. Die lateralen Schalenschlitze werden vorn ein wenig von dem Seitenrande der Oberseite überdacht, hinten aber tritt dieser Rand etwas zurück und wird also, bei dorsaler Ansicht der Schale, Das Tierreich, 36. Lief.: J. J. Tesch, Pteropoda. 4 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 50 der Rand der Unterseite sichtbar. Die Schalenbreite ist in der Gegend der Seitenfortsätze dieselbe wie weiter vorn. Die Unterseite zeigt, besonders in der vorderen Hälfte, zahlreiche feine Querlinien. Die Farbe ist rotbräunlich oder gelblich, an der Unterseite etwas dunkler. — Die junge Schale (Fig. In den wärmeren Teilen der Weltmeere, besonders im Atlantischen Ozean, etwa zwischen 40° n. Br. und 40° s. Br.; auch im Mittelmeer häufig. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia In den wärmeren Teilen der Weltmeere, besonders im Atlantischen Ozean, etwa zwischen 40° n. Br. und 40° s. Br.; auch im Mittelmeer häufig. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 5 51 ‚4b. C. tridentata affinis (Orb.) 1836 Hyalaea a. (non Sowerby 1877), Orbigny, Voy. Amer. mörid., v5 p.91 t.5 f.6—10 \| 1852 H. forskalii (non Lesueur 1821), Rang & Souleyet, Hist. nat. Pter., t.2 £.1—5 \| 1877 H. tridentata (uon Bose 1802), G@.B.Sowerby in: Reeve, Oonch. icon., v.20 Pter. t.1 f.4b \| 1886 H.t. var. affinis, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 116 t.6 f. 100a, b. Fig. 45. ee. m Bo Unterscheidet sich von voriger Unterart dadurch, daß die Schale etwas kleiner und mehr kuglig ist, indem die Unterseite, wie in Profilansicht deutlich zu sehen, auch in der hinteren Hälfte sich stark vorwölbt; der Hinterteil ist verhältnismäßig kürzer, die Spitze ist gewöhnlich vorhanden und dorsal ge- krümmt. L. der ausgewachsenen Schale 9—13 mm. — Fig. 45. Fig. 45. Fig. 45. ee. m Bo Typisch ausgebildet in den wärmeren Teilen des Ostpazifischen Ozeanes, nach Westen zu treten dann allmählich zwischen typischen Exemplaren Übergänge zu der vorhergehenden Unterart hinzu; bis in der östlichen Hälfte des Indischen Ozeanes. 4c. C. tridentata kraussi Tesch. (nom. nov.) 1848 Hyalaea truncata (non Lesueur 1821), F. Krauss, Südafr. Moll., p.84 t.2 f.12 \| 1886 H. tridentata var. truncata, Boas in: Danske Selsk. Skr., ser. 6 v.4 p. 116 t.1f.9 \| 1912 Cavolinia tridentata kraussi, Tesch MS. Unterscheidet sich von der erstgenannten Unterart dadurch, daß die vordere Hälfte der Unterseite der Schale verhältnismäßig noch stärker. ge- wölbt ist, sich nach hinten in Profilansicht aber ebenso schnell abflacht. Längs den Rändern der Schalenschlitze, sowohl oben als unten, findet sich eine dünne, vorragende, zuweilen leicht gekräuselte Leiste. Die Dorsalschale ist vorn in der Medianlinie durch eine Einbuchtung in zwei kurze Hörner zerteilt. Schale leicht bräunlich, besonders an der Unterseite; längs den Schalenschlitzen und den seitlichen Fortsätzen glashell, an der Oberseite ist der vordere Teil der Dorsalschale stärker gefärbt, distal davon folgt eine durchsichtige Stelle, der hintere Teil ist wieder schwach bräunlich. L. der ausgewachsenen Schale etwa 12 mm. Im Südatlantischen Ozean, südlich von etwa 34°s. Br. und in der westlichen Hälfte des Indischen Ozeanes. 5. C. uncinata (Rang) 1836 Hyalaea u. (non Hoeninghaus 1836), Rang MS. in: Orbigny, Voy. Amör. merid., v.5ıuı p.93 t.5 £.11—15 \| 1850 Cavolina u., J. E. Gray, Cat. Moll. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia Brit. Mus., ©.2 p.7 \| 1880 Hyalaea uneinatiformis, Pfeffer in:. Abh. Ver. Hamburg, v.71 p.83 \| 1886 H. uncinata, Boas in: Danske Selsk. Skr., ser.6 v.4 p.119 t.1 f£.10; t.2 £.20 \| 1888 Cavolinia u., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.84. Die Unterseite der Schale ist, auch in der hinteren Hälfte, stärker hervorgewölbt als bei C. iridentata, aber nicht so stark wie bei C. globulosa; auch hier geht in Profilansicht der vordere Teil mit einer gleichmäßigen Rundung in den hinteren Teil über; die Höhe der Schale beträgt verhältnis- mäßig mehr als bei €. tridentata, erreicht aber doch nicht das Maß der größten Breite. Die seitlichen Fortsätze sind lang vorgezogen und zugespitzt, in dieser Gegend ist die Schale am breitesten. Der Enddorn ist kräftig ent- wickelt (namentlich bei kleinen Exemplaren), ziemlich lang, an der Basis breit, schräg nach hinten und dorsal gerichtet (nicht so stark wie bei Ü. globulosa), die Spitze ist nie abgebrochen, leicht hakenförmig gekrümmt, eine Scheidewand im Enddorn kommt nicht vor. Die Embryonalschale ist durch eine schwache Einschnürung von dem Rest abgetrennt. Der vordere Rand der Oberschale ist sehr stark umgebogen und überdacht gänzlich die Öffnung, 4 Pteropoda: A. Theeosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 52 der umgebogene Teil ist nicht, wie bei ©. tridentata, durch einen Querwulst von dem Rest abgetrennt; die Oberseite zeigt fünf Längsrippen, die innere laterale ist durch eine Längsfurche sekundär geteilt, die äußere ist als be- sonderes Gebilde kaum ausgeprägt, die mediane Längsrippe wird nach vorn zu durch eine halbkreisförmige Furche deutlich abgegrenzt. In der Gegend Fig. 46. C. uncinata. A Schale von oben, B von rechts (,). Nach Boas. Fig. 46. C. uncinata. A Schale von oben, B von rechts (,). Nach Boas. der seitlichen Fortsätze ist die Schale stark zusammengedrückt. Auf dem vorderen Teile der Unterseite finden sich ziemlich undeutliche Querfalten, welche distal allmählich verschwinden. Die Schale hat eine gleichmäßige und zarte, bräunliche Farbe. — Die junge Schale ist noch nicht mit Sicher- heit bekannt. — L. der ausgewachsenen Schale bis zu etwa 11 mm, gewöhnlich kleiner. — Fig. 46. Die Art ist am nächsten mit C©. tridentata und zwar mit der Unterart (. t. affinis verwandt. 465 Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia Sie unterscheidet sich aber auf den ersten Blick durch die stärker aus- geprägte Skulptur der Oberseite, durch die kräftigere Entwicklung der seitlichen Fortsätze, in deren Gegend die Schale am breitesten ist, und durch den zurück- geschlagenen Vorderrand der Oberschale, der bei (©. iridentata immer gerade und schräg nach vorn gerichtet ist. In den tropischen und subtropischen Meeren, am häufigsten etwa zwischen den Wendekreisen, jenseits dieser Grenze nur gelegentlich, wenn sie mit den warmen Strömungen bis zu höheren Breiten gelangt. 6. C. inflexa (Lesueur) 6. C. inflexa (Lesueur) Zerfällt in 2 Unterarten: 6a. C. inflexa inflexa (Lesueur) 1813 Hyalaea i., Lesueur in: N. Bull. Soc philom., v.3 p.285 \| 1821 H. elongata, Lesueur MS. in: Blainville in: Dict. Sei. nat., v.22 p.82 \| ?1825 Pleuropus pellucidus, Eschscholtz in: Isis, p.755 t.5 f.2 (junges Exemplar) \| 1835 Hyalea vaginellina, Oantraine in: Bull. Ac. Belgique, v.2 p.380 \| 1836 Hyalaea depressa (non Tesch 1904), Orbigny, Voy. Amer. merid., v. 5ım p. 110 t.7 f. 11—14 (junges Exemplar) \| 1836 H. uncinata (non Rang 1836), Hoeninghaus MS. in: A. Philippi, Moll. Sieil., v.1 p.101 t.6 f.18 \| 1850 Cavolina inflexa + Diacria depressa + Clio pellucida, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.9; p.11 (junges Exemplär) \| 1852 Hyalaea i.—+ Cleodora curvata (non Huxley 1853). Souleyet in: Voy. Bonite, v.2 p- 156, Moll. t.5 f.21—26; p. 185, Moll. t.7 £.6—10 (junges- Exemplar) \| 1852 ©. pleuropus, Rang in: Raug & Souleyet, Hist. nat. Pter., t.10 f.8 (junges Exemplar) \| 1858 Pleuropus depressus, H. & A. Adams, Gen. Moll., v.2 p.61l (junges Exemplar) \| 1877 Hyalaea (Diacria) inflexa, G. B. Sowerby in: Reeve, Conch. icon., v.20 Pter., t.3 f. 17a.b \| 1884 Cleodora i., G. B. Sowerby in: Thesaur. Conch., v.5 p.143 t. 473 f. 21, 22 \| 1886 Cavolinia i., Locard, Prodr. Malac. franc., Moll. mar. p. 22 \| 1886 Hyalaea i. var. longa, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 123 t.6 f. 8a—f. Pteropoda: A. Thecosomata, a) Euthecosomata, 2. Cavoliniidae, 7. Cavolinia 583 In Profilansicht fällt es gleich auf, daß die Schale sic denen anderer Cavolinia-Arten entfernt, indem die Unterse stark hervorgewölbt ist; die Höhe der Schale ist viel gerin Breite. Auffallend ist weiter das beträchtliche Vorherrschen der hier sehr lang‘ und voluminös entwickelt ist. Die se sind ziemlich vorragend, ein wenig nach hinten gerichtet; ist die Schale am breitesten, der Vorderrand der Oberschale liches Halboval und ist gar nicht nach der Öffnung hin erstreckt sich an dieser vorbei ziemlich weit nach vorn un sogar etwas dorsal aufgeschlagen, breit dreieckig und an gezähnelt. Die Längsrippen auf der Oberseite sind immer ausgeprägt, nur die mediane und die innere laterale Längsr deutlicher hervor. Der Schalenschlitz ist in dem vorderen bei C. globulosa, U. gibbosa und C. tridentata) durch eine Falte am Rande der Oberseite bedeckt. Der vordere, wenig umgekrempelte Rand der Unterschale ist dünn und im ganzen breit dreieckig. 6b. C. inflexa labiata (Orb.) 1836 Hyalaea l., Orbigny, Voy. Amer. merid., v.5mı p.104 t.6 f.21—25 \| 1852 H. I, Souleyet in: Voy. Bonite, v.2 p.159, Moll. t.5 f.27—32 \| 1877 H. (Diacria) 1., &. B. Sowerby in: Reeve, Oonch. icon., v.20 Pter. t.3 f. 18a, b \| 1880 H. imitans, Pfeffer in: Abh. Ver. Hamburg, v.71 p.90 t.7 £.9a \| In den wärmeren Teilen des Nordatlantischen Ozeanes, nördlich bis zu der Westküste Schottlands und Irlands, auch im Mittelmeer, südlich bis etwa 25° s. Br.; weiter im Ostpazifischen Ozean. 6. C. inflexa (Lesueur) T'hecos., a) Euthecos., 2. Oavol., 7. Cavol., Cavolinidarum spee. incertae 1884 Diacria labiata, G. B. Sowerby, Thesaur. Conch., v.5 p. 141 t.473 f. 23, 24 \| 1886 Hyalaea inflexa var. lata, Boas in: Danske Selsk. Skr., ser.6 v.4 p.123 t.1 f.11; t.2 f.21; t.6 f. 98g—1l \| 1904 H. depressa (non Orbigny 1836), Tesch in: Siboga-Exp., nr.52 p.47 t.2 f. 74—77 (junge Exemplare). 1884 Diacria labiata, G. B. Sowerby, Thesaur. Conch., v.5 p. 141 t.473 f. 23, 24 \| 1886 Hyalaea inflexa var. lata, Boas in: Danske Selsk. Skr., ser.6 v.4 p.123 t.1 f.11; t.2 f.21; t.6 f. 98g—1l \| 1904 H. depressa (non Orbigny 1836), Tesch in: Siboga-Exp., nr.52 p.47 t.2 f. 74—77 (junge Exemplare). Unterscheidet sich dadurch, daß der vordere Teil der Schale dem End- dorne gegenüber verhältnismäßig viel größer und umfangreicher ist, die seit- lichen Fortsätze sind länger und mehr zugespitzt, in noch stärkerem Maße als bei ©. uneinata; der Enddorn ist viel kleiner und kürzer, schon nahe der Basis schräg nach hinten und dorsal gebogen, am Ende hakenförmig ge- krümmt. Die Oberseite ist mehr gleichmäßig gewölbt, von den Längsrippen ist kaum noch eine Spur zu sehen; dagegen sind die Zuwachsstreifen auf der Schale, auch an der Oberseite deutlicher; der vorspringende Rand der Dorsalschale hat eine mehr oder weniger deutliche schmale Längsrippe, ist breit dreieckig vorgezogen und an den Rändern fein gezähnelt. Die feinen VS C . Fig. 48. C.inflexa labiata. A Schale von oben, B von rechts (%/,). Nach Boas. C junge Schale (,Hyalaea depressa‘‘) von oben, D dieselbe von rechts, E etwas älteres Stadium, von oben, F dasselbe von rechts ('5/,),. Nach Tesch. VS C . VS S Fig. 48. C.inflexa labiata. A Schale von oben, B von rechts (%/,). Nach Boas. C junge Schale (,Hyalaea depressa‘‘) von oben, D dieselbe von rechts, E etwas älteres Stadium, von oben, F dasselbe von rechts ('5/,),. Nach Tesch. Querfurchen auf der Unterseite der Schale sind sehr undeutlich. Farbe weißlich, die Vertiefung am Rande der Oberschale, in welche‘ der kleine Höcker der Unterschale hineinpaßt, ist durch einen leicht bräunlichen Fleck markiert. — Die junge Schale unterscheidet sich im Anfange kaum von der der vorhergehenden Unterart; später stellt sich dadurch der Unterschied deutlich heraus, daß der Vorderteil der Schale, auf Kosten des Hinterteiles, sehr stark an Umfang zunimmt und im Umriß einen Halbkreis beschreibt, welchem gegenüber der kurze, sich schnell zuspitzende Hinterteil sehr zurück- tritt. 6. C. inflexa (Lesueur) Der Übergang zu der erwachsenen Schale ist ein ganz allmählicher. — L. der ausgewachsenen Schale etwa 8 mm. — Fig. 48. Im Indischen und im Pazifischen Ozean, auch in dem südlichen Teile des Atlantischen Ozeanes von etwa 25° bis 37° s. Br. In dem Südatlantischen Ozean kommen zwar Übergänge zwischen den genannten Unterarten vor; im allgemeinen aber sind sie geographisch wie morphologisch genügend scharf gesondert. 6. C. inflexa (Lesueur) Die queren Rippen, welche sich sonst regelmäßig bei anderen Cavolinia-Arten wenigstens auf der vorderen Hälfte der Unter- MEBE B seite finden, fehlen hier gänzlich, ae nur feine Zuwachsstreifen kom- C. inflexa inflexa. A Schale men auf der ganzen Schäle vor. Boas. B junge Schale („Cleodor Der Enddorn ist sehr gut ent- C dieselbe von links (!°/). wickelt und nimmt, in typischer Ansbildung, etwa die Hälfte der ganzen Schalenlänge ei nur wenig, am Ende aber stärker hakenförmig dorsal Embryonalschale ist immer vorhanden, durch eine schwac von dem Reste des Enddornes gesondert; eine Querwand fehlt vollständig. Farbe weißlich, bisweilen am Vorderra schale leicht rosa gefärbt. — Die junge Schale (Fig. 47 dem Namen „Ayalaea depressa Orb., „Cleodora curvata“ worden. Sie gleicht in den jüngeren Stadien („Cleodora cu denen von Ü. tridentata, ist also Clio-ähnlich, von der breit länglich dreieckig, sehr dünn und ohne Skulptur, die Öffnu wenig in die Breite entwickelt; im ganzen hat die junge Gestalt des abgebrochenen Hinterteiles der erwachsenen Sch Stadien kommt allmählich die Form der „yalaea depressa der erwachsenen Schale schon gleichkommt. — L. der er etwa 7 mm. — Fig. 47. ei Die Form „Cleodora curvata“ wurde von Pelseneer als die Jug nata betrachtet; ich stelle sie aber, wie mir scheint mit mehr Wahrsche MEBE B C ae C. inflexa inflexa. A Schale von oben (/,). Nach Boas. B junge Schale („Cleodora curvata“) von oben, C dieselbe von links (!°/). Nach Souleyet. C C C B ae ae C. inflexa inflexa. A Schale von oben (/,). Nach Boas. B junge Schale („Cleodora curvata“) von oben, C dieselbe von links (!°/). Nach Souleyet. ei Die Form „Cleodora curvata“ wurde von Pelseneer als die Jugendform von C. unci- nata betrachtet; ich stelle sie aber, wie mir scheint mit mehr Wahrscheinlichkeit, hierher. In den wärmeren Teilen des Nordatlantischen Ozeanes, nördlich bis zu der Westküste Schottlands und Irlands, auch im Mittelmeer, südlich bis etwa 25° s. Br.; weiter im Ostpazifischen Ozean. 6b. C. inflexa labiata (Orb.) 1836 Hyalaea l., Orbigny, Voy. Amer. merid., v.5mı p.104 t.6 f.21—25 \| 1852 H. I, Souleyet in: Voy. Bonite, v.2 p.159, Moll. t.5 f.27—32 \| 1877 H. (Diacria) 1., &. B. Sowerby in: Reeve, Oonch. icon., v.20 Pter. t.3 f. 18a, b \| 1880 H. imitans, Pfeffer in: Abh. Ver. Hamburg, v.71 p.90 t.7 £.9a \| 54 Pterop.: A. T'hecos., a) Euthecos., 2. Oavol., 7. Cavol., Cavolinidarum spee. incertae 54 54 Pterop.: A. Cavolinidarum species incertae Hyalaea truncata Lesueur 1821 H.t. (non Krauss 1848), Lesueur MS. in: Blainville in: Diet. Sei. nat., v.22 p. 82. Unsicheres Jugendstadium. Vorkommen unbekannt. Hyalaea truncata Lesueur 1821 H.t. (non Krauss 1848), Lesueur MS. in: Blainville in: Diet. Sei. nat., v.22 p. 82. Unsicheres Jugendstadium. Vorkommen unbekannt. Pteropoda: A. Theeosomata, b) Pseudotheecosomata 55 Hyalaea rugosa Orb. 1856 H.r., Orbigny, Voy. Amer. merid., v.5ımı p. 118 t.8 f. 12-14 \| 1850 Balantium rugosum, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p. 15. Nach Pelseneer wahrscheinlich ein Jugendstadium von (. gibbosa. Ostpazifischer Ozean. Cleodora trifilis Troschel 1854 C. t., Troschel in: Arch. Naturg., v. 201 p. 205 t.8 f.4. Cl t.8 f.4. Unsicheres Jugendstadium, vielleicht zu (©. tridentata gehörig. Mittelmeer. Pleuropus hargeri Verrill 1882 P.h., A. E.Verrill in: Tr. Connect. Ac., v.5 p.555. Nach Pelseneer vielleicht ein Jugendstadium von (C. gibbosa. An der Ostküste von Nordamerika. b. Trib. Pseudothecosomata 1905 Pseudothecosomata, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 4. 1905 Pseudothecosomata, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 4. 1905 Pseudothecosomata, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 4. 1905 Pseudothecosomata, Meisenheime Diese Gruppe besteht aus im äußeren Hahitus weit auseinander gehenden Formen und schließt in sich nicht nur Gattungen mit äußerer, gewundener Kalkschale, welche den Limaecinidae nahe stehen, ein, sondern enthält auch die sehr abweichend gebauten Cymbuliiden und Desmopteriden, als deren Vorfahren wir die Gattungen mit spiraliger Kalkschale zu betrachten haben. Allen gemeinsam ist nur, daß die in den Dienst der Nahrungsaufnahme getretenen Teile des Fußes sich auf der Dorsalfläche der Flosse zu einer Art Rüsselbildung erheben und nicht, wie bei den Euthecosomata im Niveau der Flossen gelegen sind; weiter dadurch, daß im Zentralnervensystem die Visceral- ganglienmasse nicht asymmetrisch ist, sondern das unpaare Ab- dominalganglion von den beiden Visceralganglien gesondert bleibt. Eine äußere, spiralig links gewundene Kalkschale kommt bei den Gattungen Peracle und Procymbulia vor. Ein eigentlicher Nabel fehlt; Innen- und Außenrand bilden zusammen in dem unteren Winkel der Schalenöffnung ein mehr oder weniger langes Rostrum, das von einem Kiele durchzogen wird. Die Cymbuliidae haben eine sekundäre, gallertartige innere Schale, die Pseudo- concha, welche in dorso-ventraler Richtung stark ausgedehnt ist und in einer Vertiefung der Oberseite den eigentlichen Körper des Tieres (Fig. 49 D) aufnimmt. Diese Pseudoconcha wird gänzlich von dem zarten Häutchen des äußeren Integumentes des Tieres überzogen, dieses Integument stellt die alleinige Verbindung zwischen Tier und Pseudoconcha dar. Innerlich besteht die Pseudoconcha aus einer konsistenten Gallertmasse, welche zuweilen schichtenweise Struktur zeigt und in der Peripherie zuweilen größere oder kleinere Hohlräume, nie aber Zellenelemente enthält. Ihre äußere Form wechselt sehr nach den Gattungen. Bei Desmopterus fehlt jede Spur einer Schalenbildung. Der Körper des Tieres teilt sich in Kopf- und Rumpf- abschnitt, die Grenze ist aber viel weniger deutlich als bei den Euthecosomata. Der Kopf trägt Fuß und Flossen; der Fuß ist ganz in der Rüsselbildung, welche im Dienste der Nahrungsaufnahme steht, aufgegangen, und erhebt sich als ein mehr oder weniger langes, ventral umgeschlagenes Gebilde von der Dorsalseite der Flossen, welche eine breite, nach den Seiten und ventral- wärts ausgedehnte, einheitliche Fläche darstellen. An der Spitze des Rüssels liegt der Mund, auf seiner hinteren Dorsalseite ein Paar symmetrisch ent- Pteropoda: A. Thecosomata, b) Pseudotheeosomata 56 wickelter Tentakeln.. Der Kopf trägt an der Dorsalseite in der Medianlinie, bei Peracle (Fig. 50) aber rechts, die Mündung des Penis. 1905 Pseudothecosomata, Meisenheime Der Rumpf- abschnitt ist gänzlich in die dorsale Vertiefung der Pseudoconcha ein- gesenkt und größtenteils von einer Mantelhöhle umschlossen, welche ventral- wärts ausmündet und in dem ventralen Abschnitte After und Nierenöffnung, sowie das Osphradium, in dem dorsalen Herz und Niere enthält; der Rumpf selbst wird vom Darmkanal mit seinen Anhängen sowie durch die Gonade gebildet. Sehr abweichend ist: Desmopterus gestaltet, bei welchem mit dem Mangel der Schale auch die Mantelhöhle völlig fehlt. Im folgenden werden nur die übrigen Gattungen berücksichtigt, die Gattung Desmopterus aber in einem Anhange besonders beschrieben werden. Das Integument wird von einem abgeflachten Epithel gebildet, das Das Integument wird von einem abgeflachten Epithel gebildet, das Chromatophoren sowie einzelne Drüsenzellen enthält; bei den Cymbuliiden umschließt es zudem die Pseudoconcha, von welcher oben gesprochen wurde; es bildet weiter eine Mantelhöhle, welche bei Peracle dorsal, bei Procym- Mund Flosse Mund Fa F Ka Flosse \| Mantel- .-“ 63 höhle Be: 7 Einge- \ -- Einge- weidesack Mantel- « 7 weidesack höhle Mund Flosse Mund Fa F Ka Flosse Mund De r Flosse 5 \| . Einge. Mantel- .-“ 63 weidesack höhle Be: 7 .... Pseudo- Einge- \ -- Einge- (35 concha weidesack Mantel- « 7 weidesack SA - höhle BR er Eingeweidesack . ...... Pseudoconcha Fig. 49. Schematische Darstellung einer Ableitung der Organisationsverhältnisse der Cymbuliiden von denjenigen der ursprünglichen Thecosomen. Nach Meisenheimer. A Stadium einer gestreckten Procymbulia, ZB, C hypothetische Zwischenstadien, D Cymbulia. Flosse Mund De r 5 . Ein weide .... Pseud (35 conc SA BR er Flosse Mund De r 5 . Einge. weidesack .... Pseudo- (35 concha SA - BR er Flosse Mund Mund Einge. weidesack Pseudo- concha - - er Eingeweidesack ...... Pseudoconcha Fig. 49. Eingeweidesack Pseudoconcha Fig. 49. Schematische Darstellung einer Ableitung der Organisationsverhältnisse der Cymbuliiden von denjenigen der ursprünglichen Thecosomen. Nach Meisenheimer. A Stadium einer gestreckten Procymbulia, ZB, C hypothetische Zwischenstadien, D Cymbulia. Fig. 49. Fig. 49. Schematische Darstellung einer Ableitung der Organisationsverhältnisse der Cymbuliiden von denjenigen der ursprünglichen Thecosomen. Nach Meisenheimer. A Stadium einer gestreckten Procymbulia, ZB, C hypothetische Zwischenstadien, D Cymbulia. bulia und den Cymbuliiden aber ventral gelagert ist. Wir haben hier mit einem ähnlichen Vorgange zu tun, wie es die phylogenetische Reihe der Euthecosomata zeigt (Fig. 2 p. 5) und wie auch durch Fig. 49 erläutert wird, d. h. 1905 Pseudothecosomata, Meisenheime es findet auch hier eine Drehung des Rumpfabschnittes dem Kopfteile gegenüber um 180° statt, wodurch die ursprünglich dorsale Mantel- höhle sich ventralwärts verlagerte. Nachher bildete sich allmählich die rund- liche Form des Eingeweidesackes bei den Cymbuliiden und die gallertartige Pseudoconcha aus, welche sich ebenso wie die Mantelhöhle stark in dorso- ventraler Richtung ausgedehnt hat. Bei Feracle liegt die Mantelöffnung dorsal und zwar auf der rechten Seite des Tieres, bei Procymbulia und den Cymbuliidae bereits völlig ventral. Die Höhle selbst ist bei Peracle und Procymbulia (Fig. 49 A) einheitlich und ungeteilt, bei den Cymbuliden (D) aber setzt sie sich aus zwei Teilen zusammen, einem äußeren Teile, dessen obere Begrenzung die untere Fläche der weit ventralwärts ausgedehnten Flosse darstellt, und einem inneren Teile, der den aboralen Teil des Ein- geweidesackes umzieht und sich weit dorsalwärts bis in die Höhe von Herz bulia und den Cymbuliiden aber ventral gelagert ist. Wir haben hier mit einem ähnlichen Vorgange zu tun, wie es die phylogenetische Reihe der Euthecosomata zeigt (Fig. 2 p. 5) und wie auch durch Fig. 49 erläutert wird, d. h. es findet auch hier eine Drehung des Rumpfabschnittes dem Kopfteile gegenüber um 180° statt, wodurch die ursprünglich dorsale Mantel- höhle sich ventralwärts verlagerte. Nachher bildete sich allmählich die rund- liche Form des Eingeweidesackes bei den Cymbuliiden und die gallertartige Pseudoconcha aus, welche sich ebenso wie die Mantelhöhle stark in dorso- ventraler Richtung ausgedehnt hat. Bei Feracle liegt die Mantelöffnung dorsal und zwar auf der rechten Seite des Tieres, bei Procymbulia und den Cymbuliidae bereits völlig ventral. Die Höhle selbst ist bei Peracle und Procymbulia (Fig. 49 A) einheitlich und ungeteilt, bei den Cymbuliden (D) aber setzt sie sich aus zwei Teilen zusammen, einem äußeren Teile, dessen obere Begrenzung die untere Fläche der weit ventralwärts ausgedehnten Flosse darstellt, und einem inneren Teile, der den aboralen Teil des Ein- geweidesackes umzieht und sich weit dorsalwärts bis in die Höhe von Herz Pteropoda: A. Thecosomata, b) Pseudothecosomata 57 und Niere umschlägt. Der untere Pol des Eingeweidesackes bildet die Scheidung zwischen diesen Teilen, welche miteinander durch eine besonders nach der rechten Seite des Körpers hin stärker entwickelte, schlitzförmige Öffnung kommunizieren. Auch der hintere Abschnitt der Mantelhöhle dringt auf der rechten Seite weit mehr dorsalwärts vor als links. Die Mantel- höhlendrüse bildet bei Z/eracle die dorsale, bei den übrigen Gattungen (in Fig. 1905 Pseudothecosomata, Meisenheime 49 schwarz angedeutet) die ventrale Begrenzungswand der Mantelhöhle; bei Peracle ist sie stark asymmetrisch nach rechts verschoben und im vorderen Abschnitte in zahlreiche Querfalten gelegt; bei den Cymbuliiden reicht sie bis tief in die innere Mantelhöhle hinein, läßt aber den innersten Teil der- selben vollständig frei, auch hier ist sie stets etwas asymmetrisch, am stärksten bei Cymbulia, sehr wenig bei Corolla. Bei Peracle besteht die Drüse aus gleichartigen, großen Zylinderzellen, bei den Cymbuliiden kommen streifen- artige Bänder vor, wodurch es zur Ausbildung hellerer und dunklerer Quer- streifen kommt. Typisch scheinen drei helle Querstreifen zu sein (bei Corolla und (sleba), von welchen der mittlere breit ist; mehr proximal liegt ein schmalerer Streifen, der in einem großen Bogen von der einen bis zur anderen Seite hinüberzieht, während der distal gelegene kürzer und ge- drungener erscheint, aber nicht völlig von einem bis zum anderen Rande reicht. Bei Cymbulia kommt an der Übergangsstelle zwischen den beiden Teilen der Mantelhöhle eine starke Knickung in der Mantelhöhlendrüse vor (Fig. 49 D bei ), weshalb hier die drei Querstreifen nur mit Mühe zu ent- decken sind, nur der mittlere ist ganz deutlich. Die Zellen in der Mantel- höhlendrüse sind einfache, mittelhohe Zylinderzellen, von denen diejenigen der dunkleren Streifen in ihrer der Mantelhöhle zugekehrten Seite von sehr stark färbbaren Körnchen erfüllt sind, während die Elemente der helleren Streifen ein durchsichtiges, feinkörniges Protoplasma aufweisen. Eine kom- plizierte Struktur, wie sie in den Elementen der Mantelhöhlendrüse bei den Euthecosomata vorkommt, ist hier nicht nachzuweisen. An dem Außenrande der Drüsenzellen, in den helleren Querstreifen, kommen kleine Kerne vor, welche vielleicht besonderen Flimmerzellen angehören. — Es kommt weiter bei den Cymbuliiden noch zu einer Verdickung ihrer Wandung und zwar auf der ventralen Seite des Eingeweidesackes; diese Verdickung bildet ein ziemlich umfangreiches, asymmetrisch nach links hin stärker entwickeltes Feld, das aus hohen, blaß gefärbten Zylinderzellen besteht. Es ist dieses Feld mit dem Analfelde der Euthecosomata homolog, welches übrigens wenigstens bei Cymbudia fehlt, bei Procymbulia aber noch deutlich vor- handen ist. — Äußere Mantelanhänge finden wir nur bei Peracle und Pro- cymbulia; bei ersterer Gattung kommt rechterseits, bei Pr oeymbulia an dem linken Mantelrande oder eigentlich fast gänzlich dorsalwärts ein rinnen- förmiger, braun pigmentierter Lappen mit einem seitlich sich ansetzenden Fortsatze vor (Fig. 62 p. 78); er stimmt mit dem ähnlichen Mantelanhang bei Limacina (Fig. 6D, p. 17) überein. 1905 Pseudothecosomata, Meisenheime — Es kommt nach verschiedenen Autoren, deren Angaben aber von anderen in dieser Hinsicht nicht bestätigt wurden, noch zu einer besonderen Öffnung in dem Hinterteile der Mantel- höhle, welche der Nierenöffnung ähnlich und mit Ring- und Radiarmuskeln versehen sein soll. — Bei Corolla findet sich noch eine ganz eigentümliche Bildung des Integument, nämlich eine dunkelpigmentierte Ringfalte um den aboralen Pol des Eingeweidesackes. Die Eingeweide reichen nicht ganz bis zu dem hinteren Ende des Sackes, die Endspitze wird von Mesenchym- masse erfüllt, deren Fasern sich senkrecht zur Oberfläche stellen; zwischen . dieser Masse und dem äußeren Körperepithel schieben sich dunkelbraun Pteropoda: A. Thecosomata, b) Pseudothecosomata 58 pigmentierte Ringfasern ein, welche die Seitenwände der kegelförmigen End- spitze des Eingeweidesackes sowie die Innenwände der Ringfalte bekleiden. Die Ringfalte selbst wird äußerlich vom Körperepithel umhüllt und ist innerlich von der Mesenchymmasse erfüllt; ihre physiologische Deutung ist noch unbekannt. Der Fuß ist ganz in der Bildung eines für die Pseudothecosomata charakteristischen Mundtrichters aufgegangen und steht im Dienst der Nahrungsaufnahme. Bei Peracie (Fig. 50) und Procymbulia können wir einen - Operculum Mittellappen des Fußes . „N: Flosse 5 -+..-- Wimperfeld Seitenlappen des Fußes 2 N 2 Mund Tentakel Mantelrand Öffnung des Panik Fig. 50. Peracle. Dorsale Ansicht von Kopf, Fuß und Flossen. Nach Meisenheimer (!?],). Operculum Flosse Wimperfeld Seitenlappen des Fußes 2 Mund Öffnung des Panik Tentakel Mantelrand Fig. 50. Peracle. Dorsale Ansicht von Kopf, Fuß und Flossen. Tentakel Mantelrand Öffnung des Panik Fig. 50. Peracle. Dorsale Ansicht von Kopf, Fuß und Flossen. Nach Meisenheimer (!?],). Nach Meisenheimer (!?],). kurzen Trichter erkennen, in dessen Inneren die Mundöffnung des Tieres sich findet; die Ränder des Trichters werden an den Seiten und dorsal von Pseudoconcha .. . Eingeweidesack .. Penis ...- "” Tentakel Mund - Flosse Seitenlappen Milielapren } des Fußes UT ENG) B Fig. 51. Cymbulia peronii. A Tier mit Pseudoconcha, in oraler Ansicht (3/,). Nach Meisenheimer. B Quer- reihe der Radula, Mittelplatte und die beiden Seitenplatten. Nach Boas (vergr.?). Pseudoconcha .. Pseudoconcha . Eingeweidesack Penis ...- "” Tentakel Eingeweidesack Penis Fig. 51. Cymbulia peronii. A Tier mit Pseudoconcha, in oraler Ansicht (3/,). Nach Meisenheimer. B Quer- reihe der Radula, Mittelplatte und die beiden Seitenplatten. Nach Boas (vergr.?). den Seitenlappen des Fußes, an der ventralen Seite von einem unpaaren, medianen Lappen, dem Mittellappen des Fußes, gebildet. 1905 Pseudothecosomata, Meisenheime Am freien Außen- rande vereinigen sich die Seitenfalten mit der medianen Falte, biegen sodann scharf nach hinten um, bilden hier eine gut ausgeprägte, direkt nach hinten ziehende, bewimperte Falte, mit dem Wimperfelde auf der Flosse der Euthecosomata homolog und verschmelzen schließlich mit dem Flossenrande. Der so entstandene Trichter schlägt sich gegen die Ventralseite der Flosse Pteropoda: A. Thecosomata, b) Pseudothecosomata 59 um und diese selbst bildet sich hauptsächlich ventralwärts und seitlich aus, so daß schließlich das Verhalten demjenigen der Euthecosomen direkt ent- gegengesetzt wird (vgl. Fig. 3 p. 6): indem dort die Flosse dorsal, der Fuß ventral gelegen ist, finden wir bei den Pseudothecosomata den Fuß oder den Mundtrichter hauptsächlich dorsal am Vorderrande der Flossen, diese selbst nach der ventralen Seite hin entwickelt. Indem der Mundtrichter oder der Rüssel sich ventralwärts umschlägt, werden einzelne auf der dorsalen Seite des Kopfabschnittes gelegene Teile in diesen Vorgang hineingezogen, namentlich die Tentakeln. Die Innenwände des Trichters flimmern stark. Bei den Cymbuliiden entwickelt sich nun dieser Prozeß der Rüsselbildung all- mählich weiter. So ist bei Cymbulia (Fig. 51) der Rüssel bereits viel stärker ausgeprägt; die Basis ist sehr verbreitert und ventralwärts umgeschlagen. Er wird auch hier durch die Seitenlappen des Fußes und den unpaaren Mittellappen gebildet, welche zusammen den in der Medianlinie gelegenen Mund einschließen. Der Rüssel hat hier eine stark abgeflachte, breit drei- eckige Gestalt; Seitenlappen und Mittellappen verlaufen bogenförmig und schließen in den Seitenteilen eine Wimperrinne ein. Bei (Corolla (Fig. 52) ist die Rüsselbildung noch schärfer ausgeprägt; die den Mund umgebenden Pseudoconcha \| Eingeweidesack ....... Tentakel Mittellappen des Fußes = de = Fig. 52. Corolla caleeola. Orale Ansicht. Nach Meisenheimer (!/,). Eingeweidesack . Tentakel Fig. 52. Fig. 52. Corolla caleeola. Orale Ansicht. Nach Meisenheimer (!/,). Falten machen sich mehr und mehr frei von der Flossenfläche. Der End- punkt wird schließlich durch @leba (Fig. 53) gebildet, wo ein langer, häutiger, völlig frei beweglicher Rüssel vorhanden ist, der an der Spitze aber noch, ebenso wie bei Öymbulia, die beiden Seitenlappen und den Mittellappen des Fußes zeigt, welche zwischen sich in der Medianebene den Mund und an den Seitenteilen eine bewimperte Falte einschließen, die hier aber nur das vordere Drittel des Rüssels einnimmt. 1905 Pseudothecosomata, Meisenheime Die Flosse ist nicht, wie bei den Euthecosomen dorsalwärts stark ausgedehnt und in zwei getrennte Flügel gesondert, sondern hat sich hier zu einer einheitlichen Fläche entwickelt, die sich in gleicher Breite von der einen bis zur anderen Seite hin erstreckt, und ventral und distal von dem eigentlichen Fuße gelegen ist. Besondere Differenzierungen am Rande kommen hier und da vor. Einfach ist die Flosse bei Corolla (Fig. 52) gestaltet, wo sie eine ovale Scheibe darstellt, ähnliches findet sich bei (@leba (Fig. 53), wo aber an den Seitenrändern be- sondere Drüsenfelder vorkommen. Komplizierter ist der Flossenrand bei Cymbulia (Fig. 51), wo die Seitenränder in eine stumpfe Spitze ausgezogen sind, und der ventralwärts gerichtete Rand in der Medianebene einen ge- sonderten, weit vorspringenden Mittellappen aufweist, der wieder einen langen, tentakelförmigen Fortsatz trägt. Eine ähnliche Gliederung der Flosse zeigen Pteropoda: A. Thecosomata, b) Pseudothecosomata 60 auch Peraele (Fig. 50) und Procymbulia (Fig. 62), nur trägt hier der mittlere, unpaare Lappen keinen tentakelförmigen Anhang; bei /’rocymbulia finden sich aber zwei kleine derartige Fortsätze zu beiden Seiten dieses Lappens. Die Flosse wird hauptsächlich durch zwei Muskellamellen, eine obere und eine untere, welche durch eine beträchtliche Stützsubstanz getrennt sind, gebildet. In jeder Muskellamelle sind bei den Cymbuliidae drei verschiedene Systeme von Muskelzügen zu unterscheiden (Fig. 67 p. 84). Die äußerste Schicht ist die schwächste; sie besteht aus parallel der Längsachse der Schale verlaufenden Fasern; die zweite Lage ist viel mächtiger und besteht aus bogenförmig von der einen Seite der Flosse nach der anderen hinüber- ziehenden Fasern, welche im ganzen parallel dem ventralen Rande verlaufer; die dritte Lage endlich strahlt radiär von den medianen Teilen der Flossen- basis nach den Seiten hinaus. Diese Systeme bilden am Rande der Flosse bisweilen Anastomosen und durchflechten sich gegenseitig. Am deutlichsten ist das Verhalten der Muskelsysteme bei Corolla; auch bei @leba sind sie noch deutlich, hier ist die äußere Lage aber etwas stärker entwickelt und in den medianen Teilen haben die verschiedenen Fasern die Neigung zu einer einheitlichen Muskelplatte zu verschmelzen; bei Cymbulia ist diese Ver- -- Pseudoconcha ..Eingeweidesack „I Penis 2 +" > Tentakel EN Flosse ">f= Mantelhöhlendrüse # An Mittellappen des Fußes Rüssel Seitenlappen des Fußes = " Mund Fig. 53. Gleba cordata. Orale Ansicht. Nach Meisenheimer ('/,). = Mantelhöhlendrüse Mittellappen des Fußes Fig. 53. Gleba cordata. Orale Ansicht. Nach Meisenheimer ('/,). 1905 Pseudothecosomata, Meisenheime schmelzung schließlich am stärksten ausgeprägt und sieht man die Fasern des zweiten und dritten Systemes nur noch am Rande der Flosse gesondert; auf Schnitten sind indessen alle drei Muskelsysteme noch vorzufinden. Bei Procymbulia kommt es ebenfalls zu einer Verschmelzung; hier sind außerdem die beiden Flossenhälften durch eine hellere Trennungslinie geschieden; es kommen übrigens hier nur zwei, einander kreuzende Fasersysteme vor. Zwischen den beiden Muskellamellen findet sich Stützgewebe, welches der Hauptsache nach von einer Mittellamelle ausstrahlt. An den Seitenrändern der Flosse von @leba finden sich eine Anzahl (4—6) von Auszackungen (Fig. 53), die von einer dunklen Substanz ausgefüllt sind, und zudem findet man über den ganzen Seitenrand unregelmäßige Flecken. Diese Differen- zierungen werden durch eine dünne Lage bildende Drüsenfelder, welche sich aus einzelligen Drüsenzellen zusammenstellen, auf der unteren, aboralen Fläche der Flosse hervorgerufen. Der ganze Flossenrand ist bewimpert, zerstreut komimen feine Tastborsten vor. Auf der Flossenfläche finden sich mannigfache Pigmentzellen vor. Die Muskelfasern sind quergestreif. Am äußeren Dorsalrande kommt bei Cymbulia (Fig.51 p.58 bei ) ein kleiner, heller Fleck vor, der keine Muskelfasern aufweist. Der fadenförmige Anhang an dem Mittellappen der Flosse bei Cymbulia muß als ein Sinnesorgan betrachtet Pteropoda: A. Thecosomata, b) Pseudotheeosomata 6l werden, da zwei Nerven in diesen Fortsatz eintreten; bei @leba bildet sich dieser Fortsatz, der auf früheren Entwicklungsstadien noch vorhanden ist, später allmählich zurück. Der mittlere Lappen der Flosse der Pseudo- thecosomata ist nicht mit dem mittleren Fußlappen der HEuthecosomata homolog, da alle Fußteile vollständig in der Bildung des Rüssels aufgegangen sind; bei Peracle (Fig. 50) ist erwähnenswert, daß hier der mittlere Lappen der Flosse an der unteren Fläche das Operculum trägt und somit die Funktion des Mittellappens des Fußes übernommen hat. Mesenchymgewebe erfüllt alle Zwischenräume der Organe mit einem Mesenchymgewebe erfüllt alle Zwischenräume der Organe mit einem lockeren Maschenwerk. Es fällt bei den Cymbuliidae besonders die elastische, metallglänzende Membran auf, welche die Eingeweidemasse umhüllt; diese besteht aus einer festen, bindegewebigen Fasersubstanz; in regelmäßigen Abständen wird die Wandung der Hülle von größeren oder kleineren Löchern durchbrochen. Die Muskulatur findet sich hauptsächlich in den Flossen, weiter liegen zwei stärker entwickelte Muskelsysteme zu beiden Seiten des Körpers, in der Gegend des dorsalen Randes der Flosse. 1905 Pseudothecosomata, Meisenheime Jedes Muskelbündel stellt eine flache, seitlich zusammengedrückte Platte dar, welche sich mit ver- breiterter Basis an der unteren Flossenfläche anheftet, von hier nach hinten zieht, sich stark verengt, eine fensterartige Durchbrechung der Faserbündel aufweist und sich dann unter fächerartiger Ausspreizung der Muskelfasern in dorsaler und ventraler Richtung ausdehnt, um schließlich an der Wand der Mantelhöhle zu enden. Lateral von diesen Muskelplatten liegen links und rechts die beiden Schenkel der Niere. Morphologisch betrachtet stellen die Muskelbündel Umbildungen des Spindelmuskels dar. Das Zentralnervensystem aller Pseudothecosomata gleicht dem- Das Zentralnervensystem aller Pseudothecosomata gleicht dem- jenigen der Euthecosomata insoweit, als wir auch hier eine sehr lange Cerebral- kommissur finden und die Ganglien selbst sehr nahe beieinander an den Flanken und an der Ventralseite des Oesophagus gelagert sind — es unter- scheidet sich aber stets darin, daß das Abdominalganglion nicht mit einem der beiden Visceralganglien verschmolzen ist, sondern ein selbständiges, unpaares Ganglion bildet. Die Cerebralganglien sind, wie bei den Euthe- cosomata, langgestreckte, dreieckige Massen zu beiden Seiten des Oesophagus; sie haben die Pleuralganglien in sich aufgenommen und sind (am deut- lichsten bei @leba) durch eine Furche in zwei Hälften zerlegt worden. Die Pedalganglien sind die mächtigsten, von länglich-ovaler Gestalt und an der Ventralseite des Oesophagus gelagert, eine breite hintere und eine längere und zarte vordere Kommissur verbindet die beiden Ganglien. Hinter den Pedalganglien liegen die Visceralganglien und das unpaare Abdominal- ganglion. Die ersteren sind völlig symmetrisch, ein wenig größer als das etwas mehr distal gelegene, mediane Abdominalganglion. Da alle Ganglien dicht aneinander liegen, sind Konnektive äußerlich nicht wahrnehmbar; nur die Buecalkonnektive, welche von der medianen Fläche der Cerebralganglien nach den Bucealganglien hinziehen, sind deutlich zu beobachten. Diese Buccalganglien sind bei Peracle zu einer einheitlichen Masse verschmolzen und liegen hier zwischen Pedal- und Visceralganglien; bei Uymbulia dagegen sind sie getrennt und proximal von den Oerebralganglien auf der Ventral- seite des Oesophagus gelagert; bei (@leba wieder gänzlich miteinander ver- schmolzen. Von den Cerebralganglien gehen zwei (oder drei?) Nerven ab, von welchen einer den Tentakel, der andere den Oesophagus und die den Mund umgebenden Lippen innerviert; vielleicht geht noch ein zarter Nerv von den Cerebralganglien nach der Statocyste. Die Pedalndrven entspringen Pteropoda: A. Thecosomata, b) Pseudothecosomata 62 als drei mächtige Stämme jederseits von dem Vorder- und Seitenrande. 1905 Pseudothecosomata, Meisenheime Der innere Stamm, zugleich der schwächste, innerviert die Seitenteile des Rüssels und den größten Teil der Lippenbildungen des Mundes, die zwei äußeren begeben sich zu der Flosse, wo zuletzt eine feine Verästelung statt- findet. Die zwei Visceralnerven (der rechte der stärkere) versorgen den Mantel, der rechte zudem das rechts gelegene Osphradium. Vom Abdominal- ganglion entspringen zwei Nerven; der stärkere entspringt fast genau ‘in der Mitte des Hinterrandes, der schwächere nahe dem rechten Seitenrande; beide begeben sich zu den Eingeweiden. Buccalmasse, Speicheldrüsen, sowie Oesophagus und Magen werden von den Buccalnerven innerviert; auf dem Magen werden zwei Nervenringe gebildet, welche durch vier zwischen den Kauplatten verlaufende Nervenfäden verbunden sind. Die Tentakeln sind, im Gegensatz zu den Euthecosomata, stets gleich Die Tentakeln sind, im Gegensatz zu den Euthecosomata, stets gleich stark auf beiden Seiten entwickelt und besitzen nie eine scheidenartige Hülle. Ursprünglich liegen sie zu beiden Seiten der Medianebene auf der Dorsal- seite des Tieres, nahe der Mundöffnung (Fig. 50), kommen aber infolge der Ausbildung des Rüssels immer mehr in dessen Bereich zu liegen und sind zuletzt, bei Corolla (Fig. 52) und Gleba (Fig. 53) direkt auf der Dorsalseite des zum Teil ganz freien Rüssels zu finden. Sie haben (am deutlichsten bei Peracle und Procymbulia) an der Spitze ein rudimentäres Auge, das bei den genannten Gattungen noch Reste von Pigmenteinlagerungen enthält, bei den Cymbuliden aber nichts davon erkennen läßt. Der innere Bau kommt im wesentlichen mit demjenigen des Auges bei den Euthecosomata (p. 9) überein. — Die Statocysten liegen an dem Hinterrande der Pedal- ganglien und besitzen eine äußere Bindegewebshülle und einen inneren Sinneszellenbelag, mit feinen Cilien; das Innere enthält die aus kohlensaurem Kalk bestehenden Statolithen. — Das Osphradium liegt rechts vom Schlunde, ziemlich weit vorn an der dorsalen Wandung der Mantelhöhle; es stellt einen länglichen Wulst dar und besteht aus einem Flimmerzellenepithel, dem nach innen sich ein mächtiges Ganglion anlegt. Am Darmkanal unterscheidet man Vorderdarm, Magen, Dünn- und Enddarm. Der Mund liegt an der Spitze des Rüssels und von hier gelangt man in den Schlund, der, ‚entsprechend der verschieden langen Ausbildung des Rüssels, bei Peracle und Procymbulia kurz ist, ebenso bei Cyınbulia, um dann bei Corolla und noch mehr bei @leba in die Länge auszuwachsen. 1905 Pseudothecosomata, Meisenheime Bei dieser letzteren Gattung bildet der äußere Muskelbelag des Schlundes jederseits ein mächtiges Muskelbündel, das nach vorn hin allmählich an Umfang abnimmt; zudem finden wir hier an der Stelle, wo der eigentliche Oesophagus anfängt, einen besonderen Sphinetermuskel entwickelt. In der Mundhöhle, welche an seiner Ventralseite die Buccalmasse trägt, finden wir bei Preracle und Procymbulia einen mächtigen, aus zwei im Querschnitt zahnartigen Platten bestehenden Oberkiefer; die Wandungen sind von einem Chitinbelag ausgekleidet. Die Radula ist wohl entwickelt, sie enthält, wie bei den Euthecosomata, in jeder Querreihe drei Platten. Bei Uymbulia sind alle diese Teile schwächer entwickelt; die Oberkieferplatten sind auf der Ventralseite des Schlundes einander genähert, vor der Radula. Die letztere (Fig. 51 3) besteht aus drei Platten in jeder Querreihe, einer sehr breiten Mittelplatte mit kleinem Mittelzahn und mit Randfranzen und zwei Seitenplatten, die auf breiter Basalplatte einen kurzen, hakenförmigen, nach hinten gerichteten Zahn tragen. Bei Corolla und Gleba fehlt die ganze Bucealmasse vollständig. — Die Speicheldrüsen sind bei /eracle bläschen- förmig, bei Procymbulia keulenförmig, mit einem sezernierenden Abschnitte Pteropoda: A. Thecosomata, b) Pseudotheeosomata 63 und einem mehr oder weniger langen Ausführungsgange. Cymbulia besitzt rundliche Speicheldrüsen, bei Corolla und (Gleba fehlen sie wieder völlig. — Der Oesophagus hat bei Perarle und Procymbulia noch einen gekrümmten Verlauf und ist etwas besser vom eigentlichen Magen abgesetzt; bei den Cymbuliiden ist der Oesophagus gerade und erweitert sich ganz allmählich bis zum Magen. — Der letztere ist in seinem hinteren Abschnitte zu einem besonderen Kaumagen umgebildet. Es kommen überall, wie bei den Euthecosomata, zwei symmetrisch gelegene Plattenpaare, das eine dorsal, das andere ventral, vor, zwischen die sich am hinteren Ende eine unpaare Platte einschiebt; diese unpaare Platte liegt bei den Cymbuliidae dorsal, im Gegensatz zu den gestreckten Formen der Euthecosomata, bei Peracle und Procymbulia ventral. Zwischen den vorderen Enden der großen Kau- platten finden sich bei den Öymbuliiden noch andere kleinere. Der Magen hat in seiner Wandung zahlreiche Längsfalten. — Die Leber nimmt den größten Teil des Eingeweidesackes ein; sie stellt bei /eracle ein zweilappiges, distal zu einer einheitlichen Masse verschmolzenes Gebilde dar, das mit zwei rechts und links gelegenen weiten Offnungen in den Magen mündet; bei den Cymbuliidae ist die Leber ungeteilt, unregelmäßig am Rande gelappt und hat nur einen einzigen, weiten, meist asymmetrisch etwas nach rechts ver- schobenen Ausführungsgang in den hinteren Teil des Magens und zwar an dessen Ventralseite. 1905 Pseudothecosomata, Meisenheime Die einzelnen Läppchen setzen sich aus großen Drüsen- zellen zusammen, die Ausführungsgänge sind von Flimmerepithel ausgekleidet. — Weiter mündet, ebenso wie bei den Euthecosomata, ein Blindsack in den Magen ein, ganz nahe dem Leberausführungsgange. Er ist ganz in die Leber- masse eingebettet und unterscheidet sich dadurch, daß zwei vorspringende Längswülste, wie besonders deutlich bei Peracle und Procymbulia zu sehen ist, das Lumen in einen größeren, rundlichen Abschnitt, der die eigentliche Sekretmasse des Blindsackes enthält, und in eine seitlich gelegene, viel kleinere Rinne verteilen. Beide Abschnitte sind von einem Flimmerepithel ausgekleidet. — Der Dünndarm ist ziemlich scharf von dem Magen ab- gesetzt, und Leber und Blindsack münden hier in Wirklichkeit viel eher in den Magen als in den Anfangsteil des Darmes. Bei /eracle entspringt der Dünndarm, der hier die für alle Thecosomata so charakteristische Längsfalte seiner Wandung nur in schwacher Ausbildung .aufweist, ganz dorsal an dem Magen, biegt dann erst in einer weiten Schlinge nach rechts, kehrt wieder in die Mediane der Dorsalseite zurück und senkt sich, während er bisher fast gänzlich auf der Oberfläche der Leber verlief, ins Innere des Eingeweidesackes ein, um nun zwischen Leber und Zwitterdrüse schräg nach oben und nach links anzusteigen und sehr weit vorn am Magen sich auf die Dorsalseite umzuschlagen; der Verlauf geht dann gleich nach der rechten Seite des Eingeweidesackes, der Enddarm verengt sich beträchtlich und mündet rechterseits durch den After aus. Die dorsale Kreuzung des Dünn- darmes durch den Enddarm, wie sie bei Limacina vorkommt, ist also auch hier, wenn auch nicht so deutlich vorhanden. Bei Cymbulia bildet der Magen an der Ventralseite vor der Abgangsstelle des eigentlichen Darmes eine bruch- sackartige Vorwölbung. Der Dünndarm entspringt von der rechten Seite des Magens und bildet, ebenso wie bei den Cavoliniidae unter den Euthecosomata, die gleiche charakteristische Schlinge; der Enddarm kreuzt also den Anfangsteil auf der Ventralseite. Da bei den Cymbuliidae der Eingeweidesack stark zu- sammengeschoben ist, wird auch die Darmschlinge etwas nach oben geschoben und umfaßt das Hinterende des Magens und die gleichfalls proximal verlagerte Leber. Der After mündet links oder in der Medianebene, er kann selbst Pteropoda: A. Thecosomata, b) Pseudothecosomata 64 rechts von der Mediane des Eingeweidesackes münden, doch ist dies ein ganz sekundäres Verhalten, da der Endabschnitt selbst stets nach links ge- richtet ist. Viel komplizierter ist der Verlauf bei Procymbulia. 1905 Pseudothecosomata, Meisenheime Der Dünn- darm entspringt erst auf der Dorsalseite des Magens, wendet sich dann sogleich nach links und bildet hier eine sehr regelmäßige Spirale; weiter umzieht er dorsalwärts den Kaumagen, schlägt sich an dessen rechter Seite angelangt ventral um, bis er wieder die linke Seite erreicht; der Endabschnitt richtet sich dann wieder direkt nach vorn und nach der Medianebene des Eingeweidenucleus; der After mit dem Endabschnitte des Enddarmes springt frei aus dem Eingeweidesack hervor und bildet so einen ziemlich langen Aftersipho. Der Längswulst in dem vorderen Teile des Dünndarmes ist bei Procymbulia und den Cymbuliiden noch mehr als bei Peracle rückgebildet. — Eine Analdrüse kommt hier nicht vor; zwar sind bei @leba Andeutungen derselben in Form großer Drüsenzellen in der Wand des Enddarmes vor- handen und bei /rocymbulia kommt in den äußersten Rändern des After- siphos etwas ähnliches vor. Das Herz ist bei Peracle, wo es links und dorsal in der Mantelhöhle Das Herz ist bei Peracle, wo es links und dorsal in der Mantelhöhle liegt, noch wohl ausgebildet; auch bei Procymbulia, wo es gleichfalls an der dorsalen Seite, aber nach rechts hin gelagert ist. Bei beiden Gattungen finden wir eine deutliche Sonderung in einen nach vorn gewendeten, dünn- wändigen Vorhof und eine nach hinten gerichtete, muskulöse Kammer; das Pericard ist deutlich vorhanden. Bei den Cymbuliidae ist nun das Herz kaum als solches zu erkennen; auch das allerdings sehr weite Pericard ist sehr schwierig aufzufinden, da es einen äußerst dünnen Zellenbelag besitzt. Das Herz selbst ist eine große, prall gefüllte, birnförmige Blase, welche auf der Dorsalseite des Eingeweidesackes zwischen den beiden Schenkeln der Niere gelegen ist. Die Blase wird durch zwei unvollkommene Scheidewände, welche in longitudinaler Richtung verlaufen, in zwei Abschnitte zerlegt; der kleinere stellt den Vorhof, der größere die Kammer dar. In den Vorhof mündet eine kurze Vene, von der Kammer entspringt eine Aorta, und- der Ursprung beider Gefäße liegt einander sehr genähert an dem einen Ende der birnförmigen Herzblase. Die Aorta ist durch eine Klappe gegen den Ven- trikel hin verschließbar; sie spaltet sich bald in zwei Äste, von welchen einer die Eingeweide versorgt, der andere ist viel länger und spaltet sich, um je einen Ast in die Flosse abzugeben. — Die Blutflüssigkeit ist wasser- klar und enthält sphärische, kernhaltige Blutkörperchen. — Besondere Kiemen kommen nur bei Zeraecle vor. 1905 Pseudothecosomata, Meisenheime Der rechte Schenkel ist stärker als der linke und zeigt etwa in der Mitte seiner Innenwandung die Öffnung in die Mantelhöhle; ein Pericardialnierengang fehlt gänzlich oder ist sehr stark reduziert. Die ur- sprüngliche, asymmetrische Lage der Niere auf der rechten Körperseite, wie sie Procymbulia zeigt, gibt sich bei den Cymbuliidae noch durch die stärkere Entwicklung des rechten Nierenschenkels und durch die etwas nach rechts verschobene Lage des Herzens kund. Ein Drüsengewebe der Niere, wie noch bei den gewundenen Formen gefunden wird, ist bei den Cymbuliidae sehr stark rückgebildet. stark rückgebildet. Das Genitalsystem besteht aus Zwitterdrüse, Zwittergang, Anhangs- drüsen, Ausführungsgängen und Begattungsapparat. — Die Zwitterdrüse umkleidet als schalenförmige, im Leben hellrot gefärbte Kappe den aboralen Pol des Eingeweidesackes, und zwar hauptsächlich auf der linken Seite desselben. Bei Peracle und Procymbulia ist sie spiralig links aufgewunden. Sie besteht aus einer großen Zahl einzelner Follikel, die von einer bindegewebe- artigen Membran umgeben sind; im Innern der Follikel liegen die Geschlechts- produkte, und zwar außen an der gesamten Peripherie die Eizellen, innen die männlichen Elemente. Es kommt auch hier Proterandrie vor. — Nach innen bilden die Follikel in einem bestimmten Reifestadium weite Räume, welche sich schließlich in dem Zwittergange vereinigen. Dieser entspringt von der Innenfläche der Zwitterdrüse, zieht als dünnhäutiger Kanal, bei Peracle zwischen den Leberläppchen, bei Cymbulia zwischen Leber und Zwitterdrüse, nach vorn und erweitert sich dann zu einer Vesicula seminalis. Diese ist bei Peracie sehr umfangreich und dehnt sich sehr weit nach vorn bis zum Vorderrande des Eingeweidenucleus aus; der Ubergang von Zwitter- gang und Samenblase ist ein unvermittelter. Cymbulia zeigt zwar denselben plötzlichen Ubergang, doch umzieht hier die Vesicula bogenförmig von der linken Seite her die an der rechten Körperseite gelegenen Genitalanhangs- drüsen, biegt am proximalen Rande derselben angelangt nach hinten um und erreicht schließlich unter allmählicher Verengung wieder ihre Ursprungsstelle, wo sie jetzt die Mündungen der Anhangsdrüsen aufnimmt und sich in den Geschlechtsausführungsgang fortsetzt. Bei Corolla und Gleba sind die Unter- suchungen noch nicht zu einer Entscheidung der wirklichen Verhältnisse ge- kommen. Bei Peracle kommt es nicht zu einer Umwachsung der Anhangs- drüsen seitens der Vesicula seminalis. — Die Vesicula seminalis geht in den Ausführungsgang über, der scharf umbiegend direkt nach vorn zieht und auf seinem Wege die am rechten Vorderrande des Eingeweidesackes gelegenen Genitalanhangsdrüsen aufnimmt. 1905 Pseudothecosomata, Meisenheime Wir finden hier ein wirkliches Ctenidium vor, das an der Ventralseite der Mantelhöhle, und zwar am linken Rande liegt und sich als ein zipfelartiges, sich allmählich verengendes Band nach der rechten Seite hinüber erstreckt. In ihrem Verlaufe liegt die Kieme meistens frei; ihre Oberfläche ist in 5 oder 6 tiefe Querfalten gelegt. Sie stellt eine einfache Faltung des Epithels der Mantelhöhle dar und besteht aus zwei dünnen Epithelmembranen, die sich unter Bildung der gefalteten Kiemenlamelle eng aneinander legen; zwischen beiden Membranen liegt ein enger Spaltraum, der direkt mit den Bluträumen des Körpers in Ver- bindung steht. Die Niere ist überall aus zwei Schenkeln zusammengesetzt. Sie liegt bei Peracle links und dorsal, bei Procymbulia rechts und dorsal, stets eng mit dem Herzen verbunden. Der laterale Schenkel der Niere trägt an dem proximalen Ende die äußere Nierenöffnung; der mediane Schenkel stellt den wichtigeren, sezernierenden Abschnitt dar und von hier geht, gleichfalls aus dem vorderen Zipfel, der Pericardialnierengang, der sich mit dem bei Pteropoda: A. Thecosomata, b) Pseudothecosomata 65 Peracle ventralwärts, bei Procymbulia dorsalwärts von der Niere gelegenen Pericard vereinigt. Die Niere der Cymbuliiden liegt als ein weiter, häutiger Sack auf der Dorsalseite des Eingeweidenucleus und umfaßt denselben huf- eisenförmig von hinten. Die beiden dorsalwärts in der Medianebene inein- ander übergehenden Schenkel liegen seitlich zwischen Mantelhöhle und äußerer Körperwand und dehnen sich ziemlich weit nach vorn und ventral- wärts aus. Der rechte Schenkel ist stärker als der linke und zeigt etwa in der Mitte seiner Innenwandung die Öffnung in die Mantelhöhle; ein Pericardialnierengang fehlt gänzlich oder ist sehr stark reduziert. Die ur- sprüngliche, asymmetrische Lage der Niere auf der rechten Körperseite, wie sie Procymbulia zeigt, gibt sich bei den Cymbuliidae noch durch die stärkere Entwicklung des rechten Nierenschenkels und durch die etwas nach rechts verschobene Lage des Herzens kund. Ein Drüsengewebe der Niere, wie noch bei den gewundenen Formen gefunden wird, ist bei den Cymbuliidae sehr stark rückgebildet. Peracle ventralwärts, bei Procymbulia dorsalwärts von der Niere gelegenen Pericard vereinigt. Die Niere der Cymbuliiden liegt als ein weiter, häutiger Sack auf der Dorsalseite des Eingeweidenucleus und umfaßt denselben huf- eisenförmig von hinten. Die beiden dorsalwärts in der Medianebene inein- ander übergehenden Schenkel liegen seitlich zwischen Mantelhöhle und äußerer Körperwand und dehnen sich ziemlich weit nach vorn und ventral- wärts aus. Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda, 1905 Pseudothecosomata, Meisenheime Da wo das Ende des Zwitterganges oder der Vesicula seminalis in den Ausführungsgang übergeht, liegt die Mündung des Receptaculum seminis, das sich als rundliches Bläschen an der rechten inneren Wand des Anhangsdrüsenkomplexes findet. — Der Ausführungsgang nimmt dann weiter die Windungen der Anhangsdrüsen (Schalen- und Eiweißdrüse) in sich auf. Die Eiweißdrüse liegt als kleines Gebilde an der rechten inneren Wandung der Schalendrüse;- beide bestehen aus einem kompliziert gebauten, gefalteten Schlauchsysteme. Bei älteren Tieren, die sich schon mehr als weihliche Individuen ausgeprägt haben, wird die Vesicula Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda, 5 Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda, 5 5 ierreich. 36. Lief.: J. J. Tesch, Pteropoda, 5 Pteropoda: A. Thecosomata, b) Pseudothecosomata 66 seminalis stark zurückgebildet, die Anhangsdrüsen selbst sind aber viel um- fangreicher entwickelt, und wir sehen, ganz wie bei den Euthecosomata, die Eiweißdrüse einerseits in den Ausführungsgang, andererseits in die Schalendrüse einmünden. — Von dem auf der rechten Seite gelegenen Ende des Ausführüngs- ganges führt nun eine bewimperte Rinne weiter proximalwärts zu der Mündung des Penis. Dieser liegt bei Peracle (Fig. 50 p.58) auf der rechten Seite, bei den Cymbuliden auf der Dorsalseite des Kopfabschnittes; er mündet in kurzer Entfernung hinter der Mundöffnung. In seiner höchsten Ausbildung stellt der Penis einen gewaltigen, aufgeknäuelten Komplex dar. Der Knäuel besteht aus einem Schlauche, der von seiner Öffnung an erst nach hinten zieht, sich dann nach rechts hin umschlägt, hier sowie auch dorsalwärts zahlreiche Schlingen bildet und endlich auf die linke Seite hinüber gelangt, wo er in einen kurzen, geraden Schlauch endet, der hier mit hohem, fimmerndem Drüsenepithel ausgekleidet ist, zwischen dessen Zellen wir die nämlichen Stilettbildungen antreffen, wie sie bei den Euthecosomata vor- kommen. Mit Eintritt der weiblichen Reife wird der Penis sehr stark reduziert und verschwindet (bei Corolla wenigstens) schließlich ganz. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) Wenn Desmopterus auch ohne Zweifel zu den Pseudotheeosomata zu stellen ist, so bietet doch die ganze Organisation so viel Abweichendes dar, daß diese unbedingt eine gesonderte Besprechung fordert. — Wir finden hier einen zylindrischen Körper, dessen Vorderende in scharfem Winkel ventral- wärts umgeknickt erscheint. Das Hinterende ist abgestutzt und von einem eigentümlichen Drüsenfelde eingenommen, das kegelförmig zugespitzte Vorder- ende trägt ein Paar Tentakeln und an der Spitze die Mundöffnung. Charak- teristisch ist weiter das gänzliche Fehlen einer Schale und Mantel- höhle, auch der Fuß ist außerordentlich reduziert. Dagegen ist die Flosse, welche der Ventralfläche des vorderen Körperteiles ansitzt, mächtig entwickelt und weist an ihrem Hinterrande eine reiche Gliederung auf. Das Integument ist ein stark abgeflachtes Epithel, in welches an Das Integument ist ein stark abgeflachtes Epithel, in welches an verschiedenen Stellen des Körpers einzellige Drüsen eingelagert sind. Diese Drüsenzellen bilden auf der Oberfläche der Flosse unregelmäßig zerstreute, knötchenförmige Verdickungen, welche aus mehreren solcher Drüsenzellen bestehen. Cilien scheinen auf der Körperoberfläche in unregelmäßiger An- ordnung vorzukommen. — Die Schale fehit vollständig, nur findet man am Hinterende des Körpers eine tiefe, von großen und hohen Drüsenzellen ausgekleidete Grube. Die Grube steht quer zur Längsachse des Körpers, nimmt die ganze Breite desselben ein und ist an beiden Seiten, namentlich aber an der rechten, etwas eingerollt. Sie mündet durch einen feinen Quer- spalt, welcher der Ventralseite zugekehrt ist, nach außen. Proximal von dieser Grube findet sich auf der Ventralseite ein zweites Drüsenfeld, das einen Querstreifen bildet. Die Grube muß sehr wahrscheinlich als ein Rest der Mantelhöhlendrüse betrachtet werden; zu dieser Auffassung berechtigt nicht nur die Struktur der Drüsenzellen, sondern auch der Umstand, daß in ihrer unmittelbaren Nähe After und Nierenöffnung sowie das Osphradium gelegen sind. Das vordere Drüsenfeld stellt dann den Rest der Analdrüse dar. — Der Fuß ist nichts mehr als eine verschwindend kleine Hautfalte, welche auf der Ventralseite des Körpers, in der Knickung zwischen Kopf- und Rumpfabschnitt, oberhalb der Flosse gelegen ist. Er bildet hier einen kleinen, asymmetrisch nach rechts gerichteten Zapfen. — Die Flosse ist sehr mächtig Pteropoda: A. T'hecosomata, b) Pseudothecosomata 67 entwickelt und stellt eine einheitliche, in querer Richtung verbreiterte Fläche dar, welche auf der Ventralseite des Körpers, in dem Winkel zwischen Kopf- und Rumpfabschnitt mit dem Tiere verbunden ist; nach hinten reicht sie weit über das Hinterende des Rumpfes hinaus. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) Der Vorderrand der Flosse ist leicht gebogen und geht dann seitlich in die einen stumpfen Bogen beschreibenden Seitenränder über; der Hinterrand ist gegliedert; es lassen sich hier fünf einzelne Lappen unterscheiden; ein unpaarer medianer, der zugleich der mächtigste ist, weiter jederseits zwei Lappen, die etwas kleiner sind; alle werden durch tiefe Einschnitte voneinander getrennt. Zwischen den beiden äußeren Lappen jederseits ist in dem Einschnitte ein langer Tentakel vorhanden, der im Leben, ebenso wie einzelne Flecken auf der Flosse, hochrot gefärbt ist; diese Farbe verschwindet nach dem Konservieren fast immer vollständig. Der lange Flossententakel ist band- förmig zusammengedrückt, die Basis ist etwas verbreitert; die Außenwandung besteht aus hohem Zylinderepithel, das starke Cilien trägt, das innere wird durch muskulöse Fasern sowie durch einen im Zentrum verlaufenden Nerv gebildet, der den Tentakel in der ganzen Länge durchzieht. -- Die Musku- latur der Flosse besteht auch hier auf beiden Flächen aus zwei Lagen sich kreuzender Muskelfasern, welche nur wenig Raum zwischen sich lassen, an den Rändern der Flosse weniger zusammenhängen und in einzelnen Fasern auch in die Lappen des Hinterrandes ausstrahlen. Das Innere der Flosse wird nur durch eine lockere Lage unregelmäßig verzweigter Binde- gewebszellen ausgefüllt. Die Muskeln zeigen deutliche Querstreifung. — Beide Hälften der Flosse sind in der Medianebene durch eine gut entwickelte,. aber zarte, senkrecht auf der Oberfläche stehende Bindegewehsmembran geschieden. Das Zentralnervensystem besteht aus einem den Oesophagus un- mittelbar vor seinem Eintritt in den Magen umgebenden Schlundring. Seitlich liegen die Cerebralganglien, welche durch eine über dem Oesophagus gelagerte Kommissur verbunden sind.” An dem ventralen und hinteren Rande der Cerebralganglien schließen sich die nur durch eine seichte Furche von den letzteren getrennten Visceralganglien an. Am Hinterrande der beiden Visceralganglien geht ein Konnektiv nach dem etwas weiter distal gelegenen, unpaaren Abdominalganglion. Vor den Visceralganglien liegen auf der Ventralseite des Oesophagus die Pedalganglien, die durch eine kurze Kommissur verbunden sind und an ihrem distalen Rande die Statocysten tragen. Die’ Buccalganglien liegen ziemlich weit nach vorn, oberhalb und seitlich von der Radulatasche und sind durch lange Konnektive mit den Cerebralganglien verbunden. Vom Üerebralganglion geht jederseits je ein Nerv ab, der hauptsächlich die Lippenränder des Mundes versorgt; ein anderer geht zum Tentakel. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) Vom Pedalganglion entspringt jederseits ein sehr starker Nervenstamm, der sich sehr bald in drei Aste spaltet, von denen der vordere den Vorderrand der Flosse versorgt, der mittlere mit seinem Hauptaste zu dem Flossententakel geht, im übrigen die Seitenteile der Flosse innerviert, während der hintere Ast sich. den medianen Teilen der Flosse zuwendet. Vom Abdominalganglion geht ein (oder zwei) Nerv ab, der auf der Ventralseite des Körpers nach hinten zieht. Ein Genitalnerv entspringt weiter vom rechten Visceralganglion. Die Sinnesorgane bestehen aus einem Paar von Tentakeln, aus den Die Sinnesorgane bestehen aus einem Paar von Tentakeln, aus den paarigen Statoeysten und aus dem unpaaren Osphradium. — Die Tentakeln sind kleine, ungestielte Knöpfchen auf den Seitenteilen des Kopfes; sie enthalten in ihrem Innern ein rudimentäres Auge, das der Hauptsache nach nr nr Pteropoda: A. Thecosomata, b) Pseudothecosomata 68 einen ähnlichen Bau hat wie bei den Euthecosomata, also finden wir auch hier einen Spaltraum mit einem vorderen, dünnen Epithel und einer hinteren Retinaschicht mit daran gelagerten Ganglienzellen; von Pigment findet man hier keine Spur. — Die Statocysten enthalten zahlreiche Statolithen. — Das Osphradium bildet einen langen, schmalen Wimperstreifen, der auf der rechten Ventralseite nahe dem Seitenrande in der Längsrichtung des Körpers verläuft. Mit dem hinteren Ende reicht es gerade bis an den proximalen Rand der hinteren Drüsengrube. Wir finden am Osphradium die gleichen hohen Wimperzellen, welche innen von einem langgestreckten Ganglion be- gleitet werden. Der Darmkanal setzt sich aus Buccalmasse, Oesophagus, Magen und einen ähnlichen Bau hat wie bei den Euthecosomata, also finden wir auch hier einen Spaltraum mit einem vorderen, dünnen Epithel und einer hinteren Retinaschicht mit daran gelagerten Ganglienzellen; von Pigment findet man hier keine Spur. — Die Statocysten enthalten zahlreiche Statolithen. — Das Osphradium bildet einen langen, schmalen Wimperstreifen, der auf der rechten Ventralseite nahe dem Seitenrande in der Längsrichtung des Körpers verläuft. Mit dem hinteren Ende reicht es gerade bis an den proximalen Rand der hinteren Drüsengrube. Wir finden am Osphradium die gleichen hohen Wimperzellen, welche innen von einem langgestreckten Ganglion be- gleitet werden. Der Darmkanal setzt sich aus Buccalmasse, Oesophagus, Magen und Enddarm zusammen. — Die rundliche Mundöffnung an der Spitze des kegel- förmigen Rüssels ist von einem Kranze einzelliger Drüsen umgeben, während an ihrem Eingange ein aus kleinen Zähnchen sich zusammensetzender Oberkiefer liegt. — Die Buccalmasse wird von einer mächtigen Muskelmasse umkleidet; die Radulatasche ist gut entwickelt; die Reibplatte selbst trägt drei haken- förmige Zähnchen in jeder Querreihe. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) — Von der Dorsalseite her münden in die Mundhöhle die Speicheldrüsen ein, kleine, ovale Gebilde, welche indessen einen deutlichen Ausführungsgang aufweisen. — Der Oesophagus fängt hinter der Einmündung der Speicheldrüsen an und verläuft als dünnes Rohr in einem leichten Bogen nach hinten, um etwa in der Höhe der Ansatz- stelle der Flosse in den Magen einzumünden. — Dieser Magen ist sehr umfangreich, nach vorn zu buchtet er sich weit in den Kopfabschnitt vor, nach hinten zu nimmt er die ganze Ventralseite des Eingeweidenucleus ein. — Hier und da in der Wandung finden sich vorspringende Falten, welche große Drüsenzellen einschließen. Diese Falten stellen die Leber dar, die also vollends mit dem Magen verschmolzen ist. Ein Kaumagen ist nicht vorhanden. — An den Magen schließt sich der dünne Enddarm an, der ganz proximal an der rechten Seite des Magens mit einer trichter- förmigen Erweiterung anfängt und als ein zartes, im Innern stark bewimpertes Rohr erst an der rechten Körperseite nach hinten zieht, sich dann nach der Medianebene hinwendet und schräg hinüber zwischen Magen und Genital- anhangsdrüsen nach der linken Seite verläuft, wo er in der Nähe des Vorderrandes der hinteren Drüsengrube, auf der Ventralseite mit dem After ausmündet. Zwischen den Genitalanhangsdrüsen und der hinteren Drüsengrube Zwischen den Genitalanhangsdrüsen und der hinteren Drüsengrube liegt auf der rechten Körperseite das von einem weiten, dünnwandigen Pericard umschlossene Herz. Der Vorhof ist nach hinten, die Kammer nach vorn gerichtet. — Unmittelbar hinter dem Herzen liegt die quergelagerte Niere, welche einen ziemlich umfangreichen, einheitlichen Sack darstellt und mit einer etwas rechts von der Medianebene des Körpers gelegenen kleinen Offnung auf der Ventralseite ausmündet.. In dem rechten Zipfel mündet die Niere durch einen deutlich ausgeprägten Wimpertrichter in das Pericard ein. Wie bei allen Thecosomen herrscht auch hier in dem Genitalsystem die Proterandrie vor; beide Geschlechtsperioden sind hier aber scharf von- einander gesondert. — Auf dem Stadium der männlichen Reife ist die stets dorsal gelegene Zwitterdrüse vollständig von den Samenelementen erfüllt. In der Mitte zeigt die Zwitterdrüse eine leichte Einschnürung; es dringt von der einen Seite her eine quere Scheidewand vor, welche aber nur wenig in die Masse hineinragt. Die Zwitterdrüse nimmt mit zunehmender Reife mehr und mehr an Umfang zu und preßt dadurch den Magen völlig an die 69 Pteropoda: A. Thecosomata, b) Pseudothecosomata Körperwandung; die hintere Drüsengrube kommt so auf die dorsale Seite zu liegen. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) — Auf der Ventralfläche der Zwitterdrüse entspringt nun der Zwittergang, der als starkes, bewimpertes Rohr zur rechten Seite des Körpers hinüberzieht und etwa in der Mitte des Körpers ausmündet. Die Genitalanhangsdrüsen sind dann noch sehr unvollkommen ausgebildet. — Von der Öffnung aus führt dann eine Wimperrinne proximalwärts bis zur Mündung des Penis, der im vorderen Kopfabschnitte, rechts von der Mund- öffnung, gelegen ist. Der Penis ist auch hier wieder im Ruhezustande ein eingestülpter, gefalteter Schlauch, der in dem Anfangsstadium der männ- lichen Reife noch sehr klein ist, später aber sich, zugleich mit der Zwitter- drüse, ganz außerordentlich vergrößert, so daß schließlich das Tier eigentlich nur noch aus Penis und Zwitterdrüse besteht. — Wenn später die ersten Eizellen sich zu entwickeln anfangen, geschieht dies an der äußeren Peripherie der Zwitterdrüse, das Innere ist noch ganz von Spermatozoen erfüllt; all- mählich werden aber die letzteren völlig verdrängt, und die jüngsten Eier liegen dann in der Umgebung eines sich allmählich ausbildenden, später wieder verschwindenden, zentralen Hohlraumes, aus welchem der Zwittergang entspringt; die älteren, völlig entwickelten Eier findet man an der Peripherie. Der Penis schrumpft zusammen und verschwindet zuletzt gänzlich. — Die Genitalanhangsdrüsen aber, welche bei der männlichen Reife kaum vor- handen sind, beginnen sich nun mächtig auszudehnen und nehmen die rechte Körperseite ein, von der hinteren Drüsengrube bis. zur Mitte des Körpers. — In der Mitte der Ventrallläche der Zwitterdrüse entspringt der Zwittergang, der in der weiblichen Reife an Umfang bedeutend abgenommen hat; dieser zieht nach der rechten Seite hinüber, geht an der inneren Fläche der Anhangs- drüsen vorbei bis zum Vorderende des Komplexes und mündet hier direkt in den Ausführungsgang ein. — Dieser Ausführungsgang nimmt seitlich die Anhangsdrüsen auf, bei welchen wir wieder eine größere Schalendrüse und eine kleinere Eiweißdrüse unterscheiden können. — Die Schalendrüse bildet einen, im hinteren Teile zusammengefalteten Schlauch, der nach vorn hin durch eine spaltförmige Offnung mit dem Ausführungsgang in Verbindung steht. Die Eiweißdrüse kommuniziert auch hier wieder, wie bei allen Theco- somata, an zwei Stellen mit dem Ausführungsgang. Dieser führt schließlich am Vorderrande der Schalendrüse mittels einer stark flimmernden Öffnung auf der rechten Körperseite nach außen. — Erst auf einem späteren Stadium der Reife bildet sich ein Receptaculum seminis aus, das oberhalb der Anhangsdrüsen direkt in den Ausführungsgang einmündet. Die Nahrung der Pseudothecosomata besteht aus animalischem und vegeta- bilischem Plankton, die Oymbuliiden ernähren sich zudem von Copepoden und Sagitten. Die Nahrung der Pseudothecosomata besteht aus animalischem und vegeta- bilischem Plankton, die Oymbuliiden ernähren sich zudem von Copepoden und Sagitten. — Der mächtig entwickelte Penis deutet darauf hin, daß Kopulation zwischen zwei verschiedenen Individuen stattfindet. Bei den Cymbuliiden werden die Eier in ziemlich kurzen, zylindrischen Schnüren abgelegt, in welchen die Eier in einer einzigen spiraligen Reihe liegen; diese Schnüre treiben frei im Meere herum. Die ersten Teilungen des Eies sind denjenigen des Eies der Euthecosomata fast gänzlich ähnlich; nach der zweiten Teilung teilt eine der großen Furchungskugeln sich in eine Anzahl kleiner Zellen weiter, welehe die großen Blastomeren allmählich umwachsen. Es entsteht so eine epibolische Gastrula. Merkwürdig ist bei den Larven der Oymbuliiden das Auftreten einer äußeren Kalkschale, welche spiralig linksgewunden ist und 11/, Um- gänge aufweist; die Schale erweitert sich nach der Mündung zu sehr schnell. Die Öffnung der Schale ist durch ein flaches, konzentrisch gestreiftes Operculum ver- schließbar. Bei Cymbulia schließen die Windungen der Schale fest aneinander, bei Gleba sind sie losgelöst. Ein Wimperring im vorderen Drittel der ovalen Larve läßt alsbald das Velum aus sich hervorgehen und gleich dahinter entsteht ventral die Anlage Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) — Der mächtig entwickelte Penis deutet darauf hin, daß Kopulation zwischen zwei verschiedenen Individuen stattfindet. Bei den Cymbuliiden werden die Eier in ziemlich kurzen, zylindrischen Schnüren abgelegt, in welchen die Eier in einer einzigen spiraligen Reihe liegen; diese Schnüre treiben frei im Meere herum. Die ersten Teilungen des Eies sind denjenigen des Eies der Euthecosomata fast gänzlich ähnlich; nach der zweiten Teilung teilt eine der großen Furchungskugeln sich in eine Anzahl kleiner Zellen weiter, welehe die großen Blastomeren allmählich umwachsen. Es entsteht so eine epibolische Gastrula. Merkwürdig ist bei den Larven der Oymbuliiden das Auftreten einer äußeren Kalkschale, welche spiralig linksgewunden ist und 11/, Um- gänge aufweist; die Schale erweitert sich nach der Mündung zu sehr schnell. Die Öffnung der Schale ist durch ein flaches, konzentrisch gestreiftes Operculum ver- schließbar. Bei Cymbulia schließen die Windungen der Schale fest aneinander, bei Gleba sind sie losgelöst. Ein Wimperring im vorderen Drittel der ovalen Larve läßt alsbald das Velum aus sich hervorgehen und gleich dahinter entsteht ventral die Anlage Pteropoda: A. Thecosomata, b) Pseudothecosomata 70 des Fußes, in der Form eines langen, walzenförmigen Anhanges, der das Opereulum trägt. Das Velum sondert sich in die beiden Segellappen, von welchen jeder durch eine seichte Einbuchtung am Seitenrande zweizipflig wird. Gleich hinter dem Segel entstehen die beiden Flossen; an ihrer dorsalen Vereinigungsstelle bildet sich dorsal die Anlage des Fußes zu dem Rüssel um. Das Velum wird dann bald abgeworfen. Sehr bald fallen von inneren Organen die verhältnismäßig großen Statocysten auf, welche zunächst nur einen einzigen Statolithen enthalten. Der Darmkanal bildet an der ventralen Seite des Magens eine Schlinge; der Enddarm zieht nach vorn und endet in einer noch ganz dorsal gelegenen Mantelhöhle mit dem After. Die Leber liegt anfänglich ganz links vom Magen. Die Flossenflügel bei den Larven von Cym- bulia sind farblos, bei denjenigen von Gleba kommen radiäre Reihen von goldgelben Chromatophoren auf der Oberfläche vor. Der Anhang, der bei C’ymbulia in der Mitte des Hinterrandes der Flosse vorkommt, tritt auch bei der Larve von Gleba auf, ver- schwindet dann aber wieder gänzlich. Die Pseudotheeosomata müssen, ebenso wie die Euthecosomata, von ursprünglich gewundenen Formen abgeleitet werden, und zwar ist es unzweifelhaft die Wattung Peracle, welche den Ausgangspunkt bildet. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) Hier finden wir, wie bei Limacina, eine gewundene Schale, ein Operculum und eine dorsal gelegene Mantelhöhle; andererseits aber zeigt Peracle Beziehungen zu den Pseudothecosomata dadurch, daß die Fußteile sich zu einem Rüssel umbilden, daß die Flossen anfangen sich zu einer einheitlichen Scheibe auszubilden, daß die Tentakein keine Scheidenbildung aufweisen und voll- kommen symmetrisch sind, und daß das Zentralnervensystem zwei gesonderte Visceral- ganglien und ein Abdominalganglion besitzt. Weiter können wir auch jetzt die ge- streckten Formen von den gewundenen herleiten durch eine Drehung des Rumpf- abschnittes um 180° anzunehmen, und die Beziehungen, welche zwischen Limacina und den übrigen Euthecosomata bestehen, gelten ganz in der gleichen Weise auch für Peracle und die übrigen Pseudothecosomata; nur scheint die Drehung um 180° hier keine vollständige zu sein. Hier, bei den Pseudothecosomata, besitzen wir aber ein Mittelglied zwischen gewundenen und gestreckten Formen in der Gattung Procymbulia, da hier zwar eine gewundene Schale mit Operculum vorkommt, die Mantelhöhle aber bereits ventral verlagert ist und die Ausbildung des Rüssels und der Flossenscheibe sich stärker herausgeprägt hat. Daneben weist Procymbulia einige Sonderbildungen (Spiraldarm, Aftersipho) auf. Der Übergang zu den Cymbuliidae (Fig. 49 p.56) geht mit sehr beträchtlichen äußeren Formveränderungen einher, welche dieser Familie ein ganz charakteristisches Aussehen verleihen. In erster Linie schwindet die äußere Schale vollständig und wird durch eine gallertartige, innere Schale (Pseudoconcha) vertreten, weiter dehnt sich die bereits ventrale Mantelhöhle beträchtlich weiter aus. Bei den Cymbuliiden können wir dann, von Oymbulia ausgehend, die phylogenetische Reihenfolge über Corolla nach Gleba sehr klar verfolgen. Bei Cymbulia besteht noch dsr mediane Fortsatz am Hinterrande der Flosse, der auch bei Peracle und Procymbulia vorkommt, weiter ist der Rüssel noch wenig ausgebildet und noch mit der Dorsalfläche der Flosse verwachsen; der Oeso- phagus besitzt noch Öberkiefer, Radulatasche und Speicheldrüsen. Bei Corolla fehlt der mediane Fortsatz am Hinterrande der Flosse; die Pseudoconcha ist viel dünner und einfacher gebaut; der Rüssel ist länger und beginnt sich schon von der Unter- lage abzuheben; im Oesophagus sind Öberkiefer, Radulatasche und Speicheldrüsen gänzlich verschwunden. Bei Gleba ist vor allem der Rüssel noch länger und fast in seiner ganzen Ausdehnung frei, die Pseudoconcha ist noch dünner und einfacher gebaut. — Zu den Pseudothecosomata gehört auch Desmopterus; diese Gattung ist aber sehr beträchtlich differenziert und steht den anderen Gattungen gar nicht nahe. Anhang; Organisation von Desmopterus (Fig. 70, 71 p. 88, 89) Der Besitz eines einzigen, symmetrischen Tentakelpaares, der Bau der Radula mit den drei Längsreihen, die einheitliche Flossenscheibe, die Zusammensetzung der Visceral- und Abdominalganglien, die dorsale Lagerung der Zwitterdrüse weisen auf Ver- wandtschaft mit den Cymbuliidae hin. Doch kommen allerdings beträchtliche Ab- änderungen vor: so das gänzliche Fehlen der Schale, die Reduktion der Mantelhöhle, das fast gänzliche Fehlen des Fußes, das Vorhandensein eines durchaus aus anderen morphologischen Gesichtspunkten zu bewertenden Rüssels, das gänzliche Fehlen des 71 Pteropoda: A. Thecosomata, b) Pseudothecosomata, 1. Peraclididae Kaumagens und die Verschmelzung von Magen und Leber. Diese beiden letzten Merkmale, sowie das Fehlen der Schale, kommen zwar mit Charakteren der Gymno- somata überein, doch kann man Desmopterus unmöglich als ein Mitglied dieser Gruppe bezeichnen; sie ist unzweifelhaft ein Thecosome, der aber stark modifiziert ist, Eine Entwicklungsreihe führt von Peracle nach Cymbulia und dann weiter über Corolla direkt nach Gleba. Procymbulia und Desmopterus stellen mehr oder weniger Seiten- zweige im Anfange der phylogenetischen Reihe dar. Der gemeinsame Ursprung aller Theeosomata ist unzweifelhaft in der Nähe der Gattung Peracle zu suchen, wenn schon wir nicht diese bereits ziemlich spezialisierte Gattung als gemeinsamen Ausgangspunkt betrachten können. Peracle und Limacina haben sich vielmehr aus einer gemeinsamen Urform entwickelt, und während Limacina sich dann weiter zu den gestreckten Eutheeosomata herausgebildet hat, wird durch Peracle eine andere Richtung eingeschlagen, welche schließlich zu den abweichend gebauten Cymbuliidae und zu Desmopterus führte. Im Gegensatz zu den Euthecosomata sind die Pseudothecosomata nur auf die wärmeren, meist sogar auf die tropischen Teile der Ozeane beschränkt. Wenn auch nicht als eigentliche Tiefseebewohner zu bezeichnen, so haben doch die meisten Arten, besonders in den jüngeren Stadien, die Neigung, bis zu beträchtlichen Tiefen (etwa 1000—1200 m) hinabzusteigen. Sie bilden nie Schwärme, wie es die Euthecosomata bisweilen zu tun pflegen und sind meist als ziemlich seltene Tiere zu bezeichnen. 4 Familien, 6 Gattungen, 20 sichere und 4 unsichere Arten. Bestimmungstabelle der Familien: Bestimmungstabelle der Familien: \| Stets eine Schale (äußere Kalkschale oder innere 1 Pseudoconcha) vorhanden — 2. \| Körper nackt, ohne Spur einer Schale. .... . 4. Fam. Desmopteridae 9 j Mit äußerer, spiralig gewundener Kalkschale — 3. \ Mit innerer, gallertartiger Pseudoconcha . . . . . 3. Fam. Cymbuliidae Nbetbelhahlerdorsal, 2 Sa ns. sn. BamnvPeraelididae \ Mantelhöhle ventral . .. . 222.222... 2. Fam. Proeymbuliidae 1. Fam. Peraclididae nov. fam. 1. Fam. Peraclididae nov. fam. Schale spiralig links gewunden, mit meist mäßig hoher bis sehr flacher Spirale Windungen von der Spitze bis zur Schalen- mündung meist schnell an Umfang zunehmend, letzte Windung sehr groß, stark aufgeblasen. Columella (Achse) der Schale leicht spiralig gedreht, nach unten in ein langes, spitz zulaufendes Rostrum ausgezogen, das stets von einem Kiel, einer Verdickung der Wandung, durchzogen wird. Dieser Kiel verläuft entweder sehr nahe am freien Außenrande der Columella oder er wird von einer in der Mitte des Innenrandes der Schalenmündung ansetzenden und sich allmählich nach unten zu verbreiternden Platte überragt, die in der Systematik als Columellarmembran bekannt ist und bei der Unterscheidung der Arten eine wichtige Rolle spielt. Da sich dieselbe in ihrem oberen Anfange stark nach außen, nach links hin, über den Kiel umschlägt, kommt so eine Art Nabel zustande. Oberfläche der Schale oft mit netzartiger Skulptur, welche auf den ersten Windungen am stärksten ausgeprägt ist und auf dem letzten Umgange, gegen die Schalenmündung hin, allmählich ver- streicht; die Skulptur gehört zur äußeren Epidermisschicht der Schale und ist bei den leeren, abgestorbenen Schalen aus Tiefsee- ablagerungen völlig verschwunden. Operculum rundlich, mit einer konzentrischen, regelmäßigen, links gedrehten Spirale — Tier mit einer breiten, einheitlichen Flosse, welche in der Mitte des 2 Pteropoda: A. Thecosomata, b) Pseudothecosomata, 1. Peraclididae, 1. Peracle 72 Hinterrandes einen kurzen vorragenden Lappen zeigt (Fig.50 p. 58); die Fußteile haben sich zu einem kurzen Rüssel vereinigt, der die Mundöffnung trägt; die zwei Tentakeln sind vollkommen symmetrisch und liegen dorsal auf dem Kopfabschnitte. In der dorsal gelegenen Mantelhöhle findet sich ein gefaltetes Ctenidium; die Mantelhöhle selbst öffnet sich auf der rechten Seite und trägt am Rande einen Fortsatz („balancer“),. Herz und Niere liegen ganz vorn und links in der Mantelhöhle; der Vorhof des Herzens ist nach vorn, die Kammer nach hinten gewendet. In den wärmeren Teilen der Ozeane. In den wärmeren Teilen der Ozeane. Enthält nur die einzige Gattung Peracle. l. P. reticulata (Orb.) 1836 Atlanta r., Orbigny, Voy. Amör. merid., v.5ıu p. 178 t. 12 f. 32—34, 39 (Operculum) \| 1840 Spirialis clathrata, Eydoux & Souleyet in: Rev. zool., v.3 p.138 \| 1844 Peracle physoides, E. Forbes in: Rep. Brit. Ass., Meet. 13 l. Gen. Peracle Forbes 1836 Heliconoides (Subgen.) (part.), Orbigny, Voy. Amer. merid., v.5ıı p.174 \| 1840 Spirialis (part.), Eydoux & Souleyet in: Rev. zool., v.3 p.235 \| 1844 Peracle, E. Forbes in: Rep. Brit. Ass., Meet. 13 p.186 \| 1847 Campylonaus (non Benson 1835), J. E. Gray in: P. zool. Soe. London, v.15 p.149 \| 1858 Euromus (Subgen.), H. & A. Adams, Gen. Moll., v.2 p.613 \| 1877 Limacina (part.), Jeffreys in: Ann. nat. Hist., ser.4 v.19 p- 337 \| 1882 Embolus (non Jeffreys 1869). P. Fischer in: J. Conchyl., v.30 p.49 \| 1888 Peraclis, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.32 \| 1897 Protomedea (non Costa 1861), Locard in: Exp. Travailleur & Talisman, Moll. test. v. 1 p. 26. Die Diagnose stimmt mit der der Familie überein. In den wärmeren Teilen aller Ozeane, von etwa 100 m bis etwa 1000 m Tiefe, nur sehr selten an der Oberfläche erbeutet. 8 Arten. Bestimmungstabelle der Arten: Bestimmungstabelle der Arten: Schalenmündung mit nur einem gekielten Zahnfortsatz (Rostrum) — 2. Schalenmündung mit mehr als einem gekielten Zahn- fortsatz — 5. Columellarmembran kaum vorhanden — 38. \| Columellarmembran sehr stark ausgebildet (Fig. 57). . . 4. P. apieifulva Spirale ziemlich hoch; die Windungen nehmen erst all- mählich an Umfang zu (Fig. 56) . . - ... 2.2... 3. P. rissoides Spirale etwas niedriger; die Windungen nehmen sehr schnell an Umfang zu — 4. Netzförmige Skulptur auf der Schalenoberfläche ;Operculum ohne Radiärstreifung (Fig. 54) . . » . » - . 1. P. reticulata Keine netzförmige ai Opereulum radiär gesleikt ig, Boyle a a ER ET 8 TIPS 2. P. brevispira \| Schalenmündung mit zwei gekielten Zahnfortsätzen — 6. Schalenmündung mit drei gekielten Zahnfortsätzen (Fig. 61) 8. P. triacantha Außeurand der Schalenmündung mit einem zipfelförmigen, nach dem Apex aufgeschlagenen, nicht gekielten Fortsatz am oberen Außenwinkel (Fig. 58) . ... 2 22... 5, P. bispinosa Außenrand der Schalenmündung gleichmäßig gerundet — 7. Columellarmembran schmal; Schale glatt, ohne oberfläch- 7 \| liche Siiulpter (Fig.;59) on In RR TE Se 6. P. moluccensis Columellarmembran breit ; Schale mit oberflächlichen Spiral- linien. (ie DON 5 4, 0.8, 250 Gase ee 7. P. depressa l. P. reticulata (Orb.) 1836 Atlanta r., Orbigny, Voy. Amör. merid., v.5ıu p. 178 t. 12 f. 32—34, 39 (Operculum) \| 1840 Spirialis clathrata, Eydoux & Souleyet in: Rev. zool., v.3 p.138 \| 1844 Peracle physoides, E. Forbes in: Rep. Brit. Ass., Meet. 13 Pteropoda: A. Thecosomata, b) Pseudothecosomata, 1. Peraclididae, 1. Peracle 73 73 p- 186 \| 1865 Spirialis recurvirostra, A. Costa in: Rend. Ace. Napoli, v.4 p.125 \| 1870 S. physoides, Jeffreys in: Carpenter & Jeffreys in: P. R. Soc. London, v.19 p.173 \| 1875 S. reticulata, Monterosato in: Att. Acc. Palermo, ser.2 v.5 nr.1 p.50 \| 1877 Limacina r., Jeffreys in: Ann. nat. Hist., ser. 4 v.19 p.338 \| 1888 Peraclis r., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 54 \| 1889 Peracle r., W. Dall in: Bull. U. S. Mus., nr.87 p.80 \| 1904 Peraclis r. var. minor, Tesch in: Siboga-Exp., nr.52 p.15 t.1f.4\| 1906 P. r., Meisenheimer in: D. Südp.-Exp., v. 9ıi p. 120 t.5 £.8. Bestimmungstabelle der Arten: Schale rötlich oder gelblich braun, länglich gestreckt, mit vier Windungen; Sutur dunkler, ziemlich tief; parallel mit dieser Sutur verläuft auf der letzten Windung ein Kiel, der gegen den Rand der Schalenmündung hin am schärfsten ausgeprägt ist, aber sich nicht über diesen Rand hinaus eine Strecke weit fortsetzt; zwischen diesem Kiele und der Sutur der letzten Windung eine schwach angedeutete Querstreifung (Fig. 54). Bei Seitenansicht der Schale sind alle Windungen sichtbar; sie nehmen aber sehr schnell vom Apex nach der Mündung an Umfang zu. Der Außenrand der Schalenmündung ist gleich- mäßig gerundet und geht nach unten zu in das etwas nach links gerichtete Rostrum über, dessen linker Rand durch den Kiel der Columella gebildet wird. Nur im obersten Abschnitt der Columella ist eine Columellarmembran in sehr schwacher Ausbildung vorhanden; sonst fehlt sie völlig. Bei der Ansicht vom Apex aus wird das Rostrum ganz durch die letzte Windung verdeckt. Fast die ganze Oberfläche der Schale wird von einer eigentümlichen Skulptur bedeckt, welche aus einem zierlichen Netzwerke erhabener Linien besteht. Diese Linien bestehen aus winzigen, weißlichen Körnchen und bilden meist regelmäßige Sechsecke, welche aber in der Nähe der Schalenmündung oft in Vier- oder Fünfeeke übergehen und allmählich verschwinden. Operculum mit 5"/, Windungen, ohne Radiärstreifung. L. der Schale 5mm, Br. 3 mm. — Fig. 54. In den wärmeren Teilen der Ozeane, auch im Mittelmeer; nördlich bis zur West- küste Irlands. Columellar- —- membran 4 4 B nn Columellar- membran A B B Fig. 54. P.reticulata. A Schale von vorn, B von oben (?),). Nach Meisenheimer. Fig. 54. P.reticulata. A Schale von vorn, B von oben (?),). Nach Meisenheimer. Fig. 54. P.reticulata. A Schale von vorn, B von oben (?),). Nach Meisenheimer. 2. P. brevispira Plsnr. 1906 P.b., Pelseneer in: Tr. Linn. Soc. London, v. 10 pars 5 p.146 t.12 f.46, 49,51. Schale mehr gedrungen, mit nur wenig hervorragenden Windungen; diese sind 2'/, an der Zahl und nehmen sehr schnell an Umfang zu. Die Sutur trägt, besonders auf der letzten Windung, kleine, senkrecht auf der Sutur gestellte Plättchen. Der Außenrand der Schalenöffnung ist gleichmäßig gerundet und geht nach unten zu in das spitz zulaufende Rostrum über. Eine Columellarmembran scheint nur, wie bei der vorigen Art, im obersten Abschnitt der Columella ausgebildet zu sein, sonst aber völlig zu fehlen. Skulptur wird nicht erwähnt. Operculum mit etwa 5 Windungen, die zentralen mit feinen Radiärstreifen versehen. L. der Schale 1:5 mm, Br. 1 mm — Fig. 55. Bestimmungstabelle der Arten: Nur im Biscayischen Meerbusen beobachtet. 74: 3. P. rissoides Tesch 1903 P. r., Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p.111 \| 1904 P.r., Tesch in: Siboga-Exp., nr. 52 p.15 t.1f.5,6 (Operculum). 3. P. rissoides Tesch 1903 P. r., Tesch in: Tijdschr. Nederl. dierk. Ver. ser.2 v.8 p.111 \| 1904 P.r., Tesch in: Siboga-Exp., nr. 52 p.15 t.1f.5,6 (Operculum). Schale rötlich-gelb, mit abgerundeter Spitze; 4'/,, Windungen, welche von der Spitze bis zur Mündung sich nicht so schnell erweitern, sondern, im Gegensatz zu den andern /eracle-Arten, ziemlich langsam an Größe zunehmen; die Schale hat dadurch eine mehr turmartige Gestalt. Der Außenrand der Öffnung wenig hervorgewölbt, Rostrum bei dem einzigen bekannt gewordenen Exemplare ‘abgebrochen, nach dem „balancer“ am rechten Mantelrande des Tieres zu urteilen, ziemlich gut entwickelt. Columellarmembran wenig aus- gebildet, etwa in derselben Weise wie bei den vorhergehenden Arten. Skulptur genau dieselbe wie bei P. retieulata, also hexagonale Felderung der Oberfläche, welche in der Nähe der Mundöffnung allmählich verstreicht. Opereulum mit regelmäßiger Spirale, welche aus 5 nicht quer gestreiften Windungen besteht. L. der Schale ohne Rostrum 1'75 mm, Br. 1'40 mm. — Fig. 56. Bisher nur ein einziges Exemplar in der Nähe von Banda (Molukken) beobachtet. Columellar- membran Fig. 56. P.rissoides. Schale von vorn (#},). Nach Tesch. 5, "= Columellar- membran A B B B Fig. 57. P. apicifulva. A Schale von vorn, B von oben (®),). Nach Meisenheimer. Fig. 56. P.rissoides. Schale von vorn (#},). Nach Tesch. 4. P. apicifulva Meisenh. 1906 P. a.. Meisenheimer in: D. Südp.-Exp., v.9u p.122 t.5 £.9. Schale an der Spitze dunkel gelbrot, welche Farbe allmählich ins Rot- gelbe und ins Bräunliche übergeht; an der Mündung weißlich. Die allgemeine Form ist die von P. retieulata; mit vier durch eine tiefe Sutur voneinander getrennten Windungen. „Spirale ziemlich hoch, ihre Windungen nehmen schnell an Umfang zu und werden von einem Kiele begleitet, der nicht über den Rand der Schalenmündung zahnartig vorspringt. Zwischen dem Kiel und der Naht der Windungen liegen wohlausgebildete Radiärstrahlen, die, an Größe allmählich abnehmend, vom Mündungsrande bis fast zum Apex verlaufen. Die Radiärstrahlen stellen Erhöhungen der Schalenoberfläche dar, die sich mit ihrem breiteren Ende direkt an der Naht festheften und so dieselbe zierlich gezähnelt erscheinen lassen, mit ihrem anderen Ende dagegen allmählich auf der Schalenoberfläche auslaufen. Bestimmungstabelle der Arten: Die Schalenmündung ist mächtig ausgedehnt; ihr oberer Rand ist etwas gegen den Apex hin empor- geschlagen, der Außenrand ist gleichmäßig gerundet und beschreibt einen weiten Bogen, nach unten läuft die Mündung spitz aus. Der Kiel, welcher das Rostrum durchzieht, ist häufig in dem mittleren Teile seines Verlaufes nur schwach ausgebildet oder kann hier ganz schwinden. Eine Columellar- membran ist sehr mächtig entwickelt. Bei der Ansicht vom Apex aus ist das Rostrum nicht sichtbar. Die Struktur der Schalenoberfläche besteht aus fein gekörnelten Liniensystemen, die in der Regel in ziemlich regelmäßigen, bald enger, bald weiter gestellten Spiralen angeordnet sind und kaum noch Pteropoda: A. Thecosomata, b) Pseudothecos omata, 1. Peraclididae, 1. Peracle 75 eine Andeutung ursprünglicher Felderung aufweisen. Nur zuweilen noch finden sich statt dieser regelmäßigen Spirallinien ganz unregelmäßige, mäander- artig geschlungene Liniensysteme, von denen zwei benachbarte noch deutliche Felder mehr oder weniger vollständig umschließen können und so die Ableitung dieser Spirallinien aus einer Auflösung ursprünglich vorhandener Felderung sehr wahrscheinlich erscheinen lassen. Öperculum mit sechs Windungen, ohne Radiärstreifung“ (Meisenheimer). L. der Schale 4 mm, Br. 33 mm — Fig. 57. eine Andeutung ursprünglicher Felderung aufweisen. Nur zuweilen noch finden sich statt dieser regelmäßigen Spirallinien ganz unregelmäßige, mäander- artig geschlungene Liniensysteme, von denen zwei benachbarte noch deutliche Felder mehr oder weniger vollständig umschließen können und so die Ableitung dieser Spirallinien aus einer Auflösung ursprünglich vorhandener Felderung sehr wahrscheinlich erscheinen lassen. Öperculum mit sechs Windungen, ohne Radiärstreifung“ (Meisenheimer). L. der Schale 4 mm, Br. 33 mm — Fig. 57. Bisher nur im tropischen Teile des östlichen Atlantischen Ozeanes gefunden. 5. P. bispinosa (Plsnr.) ?1875 Spirialis diversa, Monterosato in: Atti Ace. Palermo, ser.2 v.5 nr.1 p.50 \| 1888 Peraclis bispinosa, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.36 t.1 f.9, 10 \| ?1897 Peracle diversa, Locard in: Exp. Travailleur & Talisman, Moll. test. v.l p.29 t.1 f.4—6 \| 1906 Peraclis bispinosa, Meisenheimer in: D. Südp.-Exp., v.9ıı p.123 t.5 f.10 \| ?1909 P. diversa, Massy in: Sci. Invest. Fish. Ireland, 1907 nr.2 p.24, 5. P. bispinosa (Plsnr.) ?1875 Spirialis diversa, Monterosato in: Atti Ace. Palermo, ser.2 v.5 nr.1 p.50 \| 1888 Peraclis bispinosa, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.36 t.1 f.9, 10 \| ?1897 Peracle diversa, Locard in: Exp. Travailleur & Talisman, Moll. test. v.l p.29 t.1 f.4—6 \| 1906 Peraclis bispinosa, Meisenheimer in: D. Südp.-Exp., v.9ıı p.123 t.5 f.10 \| ?1909 P. diversa, Massy in: Sci. Invest. Fish. Bestimmungstabelle der Arten: bispinosa nur bei den größeren Exemplaren auftritt. Einstweilen führe ich hier die Arten noch getrennt auf. scheinlich ein vollständig entwickeltes Stadium von P. reticulata, zumal da der zahn- artige Fortsatz am Oberrande der Schalenöffnung bei P. bispinosa nur bei den größeren Exemplaren auftritt. Einstweilen führe ich hier die Arten noch getrennt auf. scheinlich ein vollständig entwickeltes Stadium von P. reticulata, zumal da der zahn- artige Fortsatz am Oberrande der Schalenöffnung bei P. bispinosa nur bei den größeren Exemplaren auftritt. Einstweilen führe ich hier die Arten noch getrennt auf. Lebend nur im tropischen Teile des östlichen Atlantischen Ozeanes, und zwar nördlich vom Aquator gefunden, Leere Schalen aus diesem Gebiete aber auch von den Bermudas, den Azoren und dem östlichen Mittelmeer bekannt. Lebend nur im tropischen Teile des östlichen Atlantischen Ozeanes, und zwar nördlich vom Aquator gefunden, Leere Schalen aus diesem Gebiete aber auch von den Bermudas, den Azoren und dem östlichen Mittelmeer bekannt. 6. P. moluccensis Tesch 1903 P. m., Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p. 112 \| 1906 P.m., Meisenheimerin: D. Südp.-Exp., v.9ır p. 124 t.5 f.11. 6. P. moluccensis Tesch 1903 P. m., Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p. 112 \| 1906 P.m., Meisenheimerin: D. Südp.-Exp., v.9ır p. 124 t.5 f.11. Schale weißlich, durchsichtig, Spirale niedergedrückt, mit drei Windungen, letzte Windung die beiden vorhergehenden gänzlich umfassend. „Ein Kiel begleitet die Windungen nur im letzten Abschnitt des äußersten Umganges, er springt als starker, der Richtung der Spirale folgender und etwas nach oben gekrümmter Zahnfortsatz weit über den oberen Rand der Schalen- mündung vor. Der Raum zwischen Kiel und Naht ist völlig von dicht- gestellten Radiärstrahlen ausgefüllt. Die Schalenmündung selbst ist im oberen Teile mächtig ausgebuchtet, verengt sich aber im unteren Teile ziemlich stark und unvermittelt, um schließlich spitz auszulaufen. Der Kiel der Columella ist wohlentwickelt, die Columellarmembran ist dünn und schmal, aber deutlich ausgeprägt. Bei der Ansicht vom Apex aus ist das Rostrum völlig von der Spirale verdeckt. Die Oberfläche der Schale ist glänzend glatt, ohne ober- flächliche Skulptur ... Operculum mit fünf Windungen, deren Spirallinien von radiär gestellten Streifen begleitet werden“ (Meisenheimer). L. der Schale 3 mm, Br. 25 mm — Fig. 59. -Im Warmwassergebiet des Atlantischen und des Indischen Özeanes, im Süd- afrikanischen Mischgebiet und bei Banda (Molukken) beobachtet. ">. Golumellar- membran A B h Columellar- A membran B B B B A Fig. 59. P. moluccensis. Bestimmungstabelle der Arten: Ireland, 1907 nr.2 p.24, Schale weißlich, durchsichtig; 4 durch eine tiefe Naht getrennte Win- dungen. „Spirale ziemlich hoch, ihre Windungen nehmen schnell an Umfang zu und werden vom Rande der Schalenmündung bis etwa zum halben Umlauf der äußersten Windung von einem sehr starken Kiel begleitet, der über den Rand der Schalenmündung hinaus in einen die Richtung der Spirale beibe- haltenden, zahnartigen Fortsatz vorspringt. Die leicht gekrümmten Radiärstrahlen sind wohlentwickelt und verhalten sich bei ge- ringerer Anzahl im übrigen ganz so wie bei P. apieifulva. Die Schalenmündung ist sehr weit, der obere äußere Winkel ihres Außenrandes ist in einen dreieckigen, ausgehöhlten Zipfel ausgezogen, welcher nach oben und außen gerichtet ist. Dieser Columellar- zipfelartige Fortsatz ist für die Spezies membran überaus charakteristisch ... Nach unten läuft die Schalenmündung wiederum spitz en B aus. Das Rostrum ist von einem starken Fig. 38. Kiel durchzogen, weiter ist eine Columellar- p, bispinosa. A Schale von vom, B von membran in sehr mächtiger Ausbildung oben (),). Nach Meisenheimer. vorhanden. Bei der Ansicht vom Apex aus ragt das Rostrum weit über die Spirale hinaus, in seinem Verlaufe der Richtung der Spirale folgend... Die Skulptur der Schalenoberfläche besteht aus ziemlich regelmäßigen, langgestreckten Sechsecken, die gleichfalls Neigung zeigen, sich in unregelmäßig gestaltete Fünf- und Vierecke umzuwandeln“ (Meisenheimer). Bei den leeren Schalen des Meeresbodens geht diese Struktur völlig verloren. Operculum mit fünf Windungen, ohne Radiärstreifen. L. der Schale 10 mm, Br. 7 mm. — Fig 58. Columellar- membran en B Fig. 38. Fig. 38. p, bispinosa. A Schale von vom, B von oben (),). Nach Meisenheimer. Nach Massy ist „Spirialis diversa“ Monterosato gänzlich synonym mit der hier besprochenen Art. Diese Ansicht hat, wenn man die Abbildungen Locard’s und Meisen- heimer’s vergleicht, viel Wahrscheinlichkeit für sich; nur scheint nach der Abbildung Locard’s bei „Spirialis diversa“ der Zipfel des Außenrandes der Schalenöffnung sowie der vorspringende Kiel am Oberrande abgebrochen zu sein, auch ist über die Columellar- membran nichts bekannt. Die allgemeine Gestalt der Schale ist aber fast genau die- selbe wie bei P. bispinosa. Sollte es sich bestätigen, daß die beiden Arten identisch sind, dann dehnt sich das Wohngebiet (wenigstens der leeren Schalen) bis zur West- küste Irlands aus. Nach Dall (Smithson Collect., v.50 p. 502) wäre diese Art wahr- Pteropoda: A. Thecosomata, b) Pseudothecosomata, 1. Peraclididae, 1. Peracle 76 scheinlich ein vollständig entwickeltes Stadium von P. reticulata, zumal da der zahn- artige Fortsatz am Oberrande der Schalenöffnung bei P. Bestimmungstabelle der Arten: A Schale von vorn, B von oben (/,). Nach Meisenheimer. Fig. 59. P. moluccensis. A Schale von vorn, B von oben (/,). Nach Meisenheimer. 7. P. depressa Meisenh. 1906 P. d., Meisenheimer in: D. Südp.-Exp., v.9u p. 125 t.5 f.12. Schale weißlich, bei größeren Exemplaren in der Nähe der Spitze leicht gelblich, Kiel horngelb, drei Windungen. „Spirale ziemlich stark nieder- gedrückt, schnell an Umfang zunehmend. Am Innenwinkel des oberen Randes der Schalenmündung springt ein Zahnfortsatz vor, der stark nach oben gegen den Apex hin emporgeschlagen ist, jedoch sehr zerbrechlicher Natur zu sein scheint... Der Fortsatz wird wie stets von einem festen Kiel durchzogen, der hier aber nur eine ganz kurze Strecke weit auf die Oberfläche der Windungen selbst sich fortsetzt. Radiärstrahlen fehlen. Der Außenrand der Mündung ist gleichmäßig gerundet. Die Columella wird von einem starken Kiel durchzogen, der in das Rostrum ausläuft, eine Columellarmembran ist in ziemlich beträchtlicher Breite entwickelt. Der Nabel ist sehr deutlich ausgeprägt. Bei der Betrachtung vom Apex aus ist das Rostrum völlig ver- Pterop.: A. Thecos., b) Pseudothecos., 1. Peraclididae, 1. Peracle, 2. Proeymbuliidae deckt. Die Oberfläche der Schale besitzt eine aus regelmäßigen, gekörnelten Spirallinien sich zusammensetzende Struktur. Kleine Ausbuchtungen der Spirallinien weisen noch auf eine ursprüngliche Felderung hin... Operculum mit fünf Windungen, ohne Radiärstreifen“* (Meisenheimer). L. der Schale 3 mm, Br. 25 mm — Fig. 60. Bisher nur im Warmwassergebiet des Atlantischen Ozeanes gefunden. 8. P. triacantha (Fischer) 1882 Embolus triacanthus, P. Fischer in: J. Gonchyl., v.30 p.49 \| 1888 Limacina triacantha, Pelseneer in: Rep. Voy. Challenger, Zool. v. 23 pars 65 p.20 t.1 f.1,2 \| 1897 Protomedea t., Locard in: Exp. Travailleur & Talisman, Moll. test. v.1 p.27 \| 1904 Peraclis t., Pelseneer in: OR. Ace. Sei., v.139 p. 547 \| 1906 P.t., Meisenheimer in: D. Südp.-Exp., v.9ır p. 126 t.5 f£. 13. Schale weiblich, die drei Kiele sind gelbbraun; drei Windungen, von denen die beiden ersten bei der Seitenansicht der Schale fast vollständig von der letzten Windung verdeckt werden, so daß die Spitze der Schale noch unter dem Niveau der letzten Windung liegt. „Die sehr weite Schalenmündung trägt an ihren Rändern drei spitze, zahnartige Fortsätze, von denen der erste am Innenwinkel des oberen Randes der Mündung gelegen und unter Bei- behaltung der Richtung der Spirale direkt nach oben gewendet ist. Bestimmungstabelle der Arten: Sein Kiel setzt sich nur eine kurze Strecke Schale weiblich, die drei Kiele sind gelbbraun; drei Windungen, von denen die beiden ersten bei der Seitenansicht der Schale fast vollständig von der letzten Windung verdeckt werden, so daß die Spitze der Schale noch unter dem Niveau der letzten Windung liegt. „Die sehr weite Schalenmündung trägt an ihren Rändern drei spitze, zahnartige Fortsätze, von denen der erste am Innenwinkel des oberen Randes der Mündung gelegen und unter Bei- behaltung der Richtung der Spirale direkt nach oben gewendet ist. Sein Kiel setzt sich nur eine kurze Strecke weit auf die Oberfläche der Windungen selbst fort, Radiärstrahlen sind zwischen ihm und der Naht nicht entwickelt. Der zweite Fortsatz sitzt dem unteren Abschnitt des Außenrandes an, er besitzt einen nur kurzen, starken Kiel, der ; \ \ dritte endlich bildet das Rostrum in 4 Columellar- B : . 5 membran der gewöhnlichen Weise und zeichnet en sich durch die sehr mächtig entwickelte p triacantha. A Schale von vorn, B von oben Columellarmembran aus. Der Nabel (2). Nach Meisenheimer. ist sehr deutlich ausgeprägt. Bei der Betrachtung vom Apex aus ragen sowohl Fortsatz des unteren Aubenrandes als auch das der Richtung der Spirale folgende Rostrum weit über den oberen Mündunesrand hinaus ... Die oberflächliche Struktur der Schale setzt sich aus feingekörnelten Spirallinien zusammen, zwischen denen unvoll- ständige Querbrücken wiederum noch auf eine ursprüngliche Felderung hin- weisen... Operculum mit sechs Windungen, deren Spirallinien von radiär gestellten Streifen dicht besetzt sind“ (Meisenheimer). L. der Schale 3 mm, Br. 5 mm. — Fig. 61. ; \ \ 4 Columellar- B membran en p triacantha. A Schale von vorn, B von oben (2). Nach Meisenheimer. \ B n \ B n \ Columellar- membran en B en p triacantha. A Schale von vorn, B von oben (2). Nach Meisenheimer. en p triacantha. A Schale von vorn, B von oben (2). Nach Meisenheimer. Im östlichen Teile des Atlantischen Ozeanes, von Kapstadt bis zur Westküste Irlands. 1. Gen. Procymbulia Meisenheimer 1905 Procymbulia (err. typ. Frocymbulia), Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 13. 1905 Procymbulia (err. typ. Frocymbulia), Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 13. 1905 Procymbulia (err. typ. Frocymbulia), Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 13. Die Diagnose stimmt mit der der Familie überein. In der subantarktischen Übergangsregion? Tiefsee? 1 Art. 1. P. valdiviae Meisenh. 1905 P.v., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p-LEH AT, T: Mit den Merkmalen der Familie: Flossenscheibe breit und hoch, die medianen Teile sowie die Mantelränder tief schokoladenbraun pigmentiert. Mittellappen der Flosse 7 tentakelartiger Fortsatz der Flosse „ Flosse — "* Seitenlappen des Fußes SH ""_Mittellappen des Fußes Wimperfld N" Tentakel Seitenlappen des Fußes’ _ ES \ zipfelartiger Mantelfortsatz Mund Fig. 62. P. valdiviae. Dorsale Ansicht (%/,). Nach Meisenheimer. Mittellappen der Flosse 7 tentakelart Wimperfld Seitenlappen des Fußes’ _ zipfelartiger Mantelfortsatz Mantelfortsatz Mund Fig. 62. P. valdiviae. Dorsale Ansicht (%/,). Nach Meisenheimer. Mantelfortsatz Mund Fig. 62. P. valdiviae. Dorsale Ansicht (%/,). Nach Meisenheimer. Oberkiefer und Radula mächtig ausgebildet. Br. der Flossenscheibe 10 mm. — Fig. 62. Bis jetzt nur ein einziges Exemplar im südlichen Indischen Ozean, aus einer Tiefe von 2000 m bekannt. 2. Fam. Procymbuliidae nov. fam. Schale unbekannt, nach der Gestalt des von ihr bedeckten Eingeweidenueleus des Tieres zu schließen spiralig links gedreht, sehr niedrig, mit wenigen, sehr schnell im Umfange zunehmenden Windungen — Tier mit großer, einheitlicher Flossenscheibe, welche am Ventralrande einen stumpfen, medianen Fortsatz trägt, an dessen Seiten jederseits ein kleiner, tentakelartiger Zipfel inseriert ist (Fig. 62); Rüssel groß, aus Umbildung der Fußteile, ähnlich wie bei Peracle hervorgegangen; Mantelhöhle ventral, am dorsalenRande miteinem gutausgebildetenFortsatz(„balancer“) Pterop: A. Thecos., b) Pseudotheeos., 2. Proeymb., 1. Procymbulia, 3. Cymbuliidae 8 Pterop: A. Thecos., b) Pseudotheeos., 2. Proeymb., 1. Procymbulia, 3. Cymbuliidae 78 versehen. Tentakeln zwei, symmetrisch, gleich groß, ohne Scheide, auf der dorsalen Seite des Rüssels. Keine Kieme in der Mantel- höhle;. Dünndarm mit gut ausgebildeter Spirale auf der linken Seite des Magens, Anus ventral, nahezu median; Herz dorsal und rechts, der Vorhof ist nach vorn, dieKammer nach hinten gerichtet. versehen. Tentakeln zwei, symmetrisch, gleich groß, ohne Scheide, auf der dorsalen Seite des Rüssels. Keine Kieme in der Mantel- höhle;. Dünndarm mit gut ausgebildeter Spirale auf der linken Seite des Magens, Anus ventral, nahezu median; Herz dorsal und rechts, der Vorhof ist nach vorn, dieKammer nach hinten gerichtet. Enthält nur die einzige Gattung Procymbulia. Enthält nur die einzige Gattung Procymbulia. 3. Fam. Cymbuliidae 1841 Cymbulidae, Cantraine in: M&m. Ac. Belgique, v.13 [nr. 7] p.33 \| 1850 Cymbuliadae (part.), J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.24 \| 1852 „Famille des Cymbulies“, Souleyet in: Voy. Bonite, v.2 p.99 \| 1854 C'ymbuliacea, Troschel in: Arch. Naturg., v.20ı p.210 \| 1855 C'ymbuliea, Gegenbaur, Unters. Pter. & Heterop., p. 211 \| 1885 Alata, N. Wagner in: Wirbell. Weiß. Meer., v.1 p.119 \| 1886 Oymbuliidae, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 137. Pteropoda: A. Thecosomata, b) Pseudothecosomata, 3. Cymbuliidae, 1. Cymbulia 79 Im erwachsenen Zustande keine Kalkschale, sondern etne innere, vollkommen durchsichtige, kahnförmige, symmetrische Pseudoconcha von knorpelartiger Konsistenz und allseitig vom Mantel umhüllt; die Hauptachse der Pseudoconcha liegt in der dorso-ventralen Richtung des Tieres, auf der einen Seite findet sich eine nach der dorsalen Richtung geschlossene, ventral mehr oder weniger weit offene Aushöhlung, in welcher das Tier steckt. Verbindung zwischen Tier und Pseudoconcha nur durch die dünne Mantelmembran vermittelt, daher leicht löslich. — Tier mit sehr breiter, flacher Flossenscheibe, welche sich stark in seitlicher und ventraler Richtung erweitert und etwa senkrecht auf der Längs- achse des Eingeweidesackes steht. In der Mitte des dorsalen Randes der Flosse liegt der Rüssel, welcher aus Umbildungen der Fußteile entsteht und bei den verschiedenen Gattungen ver- schiedene Länge aufweist; an der Spitze des Rüssels findet sich die Mundöffnung, auf der dorsalen Seite die beiden symmetrischen, gleich großen Tentakeln ohne Scheide, sowie (bei erwachsenen Tieren in der Medianebene, bei jüngeren etwas nach rechts) die Mündung des Penis. Visceralnucleus klein, kompakt. Darm- schlinge ventral, Anus links-oder in der Medianebene Mantel- höhle geräumig; hauptsächlich ventral, mit großer Mantelhöhlen- drüse; keine Kieme. Herz und Niere dorsal in der Mantelhöhle; der Vorhof des Herzens ist nach links, die Kammer nach rechts gewendet, eine äußere Scheidung dieser Abteilungen besteht nicht, auch die Scheidewand ist sehr unvollkommen ausgeprägt; Niere mit zwei Schenkeln das Herz umfassend, keine Renopericardial- öffnung. In den wärmeren, meist in den tropischen Teilen der Ozeane, an der Ober- fläche, aber offenbar auch bis zu Tiefen von mehr als 1000 m hinabsteigend. 3 Gattungen, 9 sichere und 4 unsichere Arten. 3 Gattungen, 9 sichere und 4 unsichere Arten. Bestimmungstabelle der Gattungen: Bestimmungstabelle der Gattungen: Pseudoconcha dick, pantoffelförmig, an der dorsalen Seite zugespitzt, mit kleiner Höhle. Tier mit kurzem Rüssel 1. Gen. Cymbulia Pseudoconcha dünn, an der dorsalen Seite breit abgerundet, mit geräumiger Höhle — 2. 3. Fam. Cymbuliidae Pseudoconcha noch gut entwickelt, mit zahlreichen Tuberkeln auf der aboralen Seite. Rüssel mittelgroß, nicht bis zu der Mitte der Flossenscheibe reichend . ...... . 2. Gen. Corolla Pseudoconcha sehr flach, wenig ausgebildet, nahezu glatt auf der aboralen Seite. Rüssel lang, bis über die Mitte der Flossenscheibe hinausreichend, frei beweglich . . . 3. Gen. Gleba Pseudoconcha dick, pantoffelförmig, an der dorsalen Seite zugespitzt, mit kleiner Höhle. Tier mit kurzem Rüssel 1. Gen. C Pseudoconcha noch gut entwickelt, mit zahlreichen Tuberkeln auf der aboralen Seite. Rüssel mittelgroß, nicht bis zu der Mitte der Flossenscheibe reichend . ...... . Pseudoconcha sehr flach, wenig ausgebildet, nahezu glatt 1. Gen. Cymbulia Peron & Lesueur 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in: Ann. Mus. Paris, v.15 p. 66. 1. Gen. Cymbulia 1. Gen. Cymbulia 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in Ann. Mus. Paris, v.15 p. 66. 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in: Ann. Mus. Paris, v.15 p. 66. Pseudoconcha mit dieken Wandungen und kleiner, nach der ventralen Seite verstreichender Aushöhlung, pantoffelförmig, am vorderen (dorsalen) Ende zugespitzt, am hinteren (ventralen) Ende abgestutzt oder ausgehöhlt. — Tier mit breiter Flossenscheibe, die in der Mitte des ventralen Randes einen gut abgesetzten, geißeltragenden Lappen zeigt. Rüssel kurz, nicht frei. Oberkiefer und Radula gut ausgebildet. Muskulatur der Flosse nur an den 80 Pteropoda: A. Thecosomata, b) Pseudothecosomata, 3. Cymbuliidae, 1. Cymbulia Rändern in einzelne Bündel gesondert; eine kleine, muskelfreie Stelle jeder- seits am Ende des dorsalen Randes. Die jungen Tiere entbehren oft des Mittellappens der Flosse, weisen dagegen bisweilen Anhänge an der Über- gangsstelle zwischen Seiten- und Ventralrand der Flosse auf; sie sind, da, wie bei den erwachsenen Exemplaren, der Zusammenhang zwischen Tier und Schale sehr leicht zerreißt, als der Gattung Cymbulia zugehörig meist nur an dem kurzen Rüssel und der einheitlichen Flossenmuskulatur zu erkennen. Rändern in einzelne Bündel gesondert; eine kleine, muskelfreie Stelle jeder- seits am Ende des dorsalen Randes. Die jungen Tiere entbehren oft des Mittellappens der Flosse, weisen dagegen bisweilen Anhänge an der Über- gangsstelle zwischen Seiten- und Ventralrand der Flosse auf; sie sind, da, wie bei den erwachsenen Exemplaren, der Zusammenhang zwischen Tier und Schale sehr leicht zerreißt, als der Gattung Cymbulia zugehörig meist nur an dem kurzen Rüssel und der einheitlichen Flossenmuskulatur zu erkennen. In den wärmeren Meeren. In den wärmeren Meeren. 3 sichere Arten, 1 unsichere Art. Bestimmungstabelle der sicheren Arten: Bestimmungstabelle der sicheren Arten: höhlung klein, mit sehr kleinen Zähnchen am Rande 2. C. parvidentata Pseudoconcha nicht in der Mitte der Länge eingeschnürt; Aushöhlung geräumig — 2. „ JS Pseudoconcha mit stumpfer vorderer (dorsaler) Spitze. . 1. C. peronii Pseudoconcha mit schlanker, scharfer vorderer Spitze . . 3. C. sibogae \| Pseudoconcha in der Mitte der Länge eingeschnürt; Aus- höhlung klein, mit sehr kleinen Zähnchen am Rande 2. C. parvidentata Pseudoconcha nicht in der Mitte der Länge eingeschnürt; Aushöhlung geräumig — 2. „ JS Pseudoconcha mit stumpfer vorderer (dorsaler) Spitze. . 1. C. peronii Pseudoconcha mit schlanker, scharfer vorderer Spitze . . 3. C. sibogae \| Pseudoconcha in der Mitte der Länge eingeschnürt; Aus- „ JS Pseudoconcha mit stumpfer vorderer (dorsaler) Spitze. . 1. C. peronii Pseudoconcha mit schlanker, scharfer vorderer Spitze . . 3. C. sibogae 1. C. peronii Blainv. 1818 ©. p.. 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in Ann. Mus. Paris, v.15 p. 66. Blainville in: Diet, Sci. nat., v.12 p.333 t.59 f.a \| 1850 C. proboscidea (non Krohn 1844), J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.25 \| 1855 C. quadripunctata, Gegenbaur, Unters. Pter. & Heterop., p.52 t.3 f.20 (junges Exemplar). \| Pseudoconcha mit stumpfer Vorderspitze, mit zahlreichen Höckerchen besetzt, welche teils unregelmäßig zerstreut, teils in bestimmten Längsreihen vorkommen. Bei Seitenansicht der Pseudoconcha fallen besonders drei \| = A \| Fig. 63. C. peronii. Pseudoconcha. A großes Exemplar, von rechts, B von unten (!/,), ©—-@ kleine Exemplare, alle von rechts (1/,). A—C nach Tesch, D—@ nach Steuer. Fig. 63. C. peronii. Pseudoconcha. A großes Exemplar, von rechts, B von unten (!/,), ©—-@ kleine Exemplare, alle von rechts (1/,). A—C nach Tesch, D—@ nach Steuer. Fig. 63. oconcha. A großes Exemplar, von rechts, B von unten (!/,), ©—-@ kleine Exemplare, alle von rechts (1/,). A—C nach Tesch, D—@ nach Steuer. parallele Reihen auf, die den Rändern parallel verlaufen, von denen aber nur der untere das jederseits in eine Spitze ausgezogene ventrale Ende der Pseudoconcha erreicht, die beiden anderen hören schon in der vorderen Hälfte der Pseudoconcha auf. Diese drei Reihen kommen aber nur bei erwachsenen Exemplaren vor, bei den kleineren existiert nur die untere Reihe. Auf der aboralen Seite verläuft jederseits eine Höckerreihe von der Spitze bis in den hinteren Zipfel der Pseudoconcha; diese beiden Reihen sind in der Mitte ihres Verlaufes etwas median eingebuchtet und werden in der hinteren Hälfte an der medianen Seite je durch eine nicht zusammenhängende Reihe begleitet. Vom Zentrum der Unterseite der Pseudoconcha strahlen weiter drei Reihen aus; die eine verläuft in der Medianlinie nach vorn bis zur Pteropoda: A. Theeosomata, b) Pseudotheeosomata, 3. Oymbuliidae, 1. Cymbulia 81 Spitze, die beiden anderen sind paarig und ziehen divergierend nach hinten bis zu dem hinteren Zipfel. Am Rande der Aushöhlung auf der oberen (oralen) Seite der Pseudoconcha kräftige Zähne, welche bei größeren Exem- plaren rechts bedeutend stärker ausgebildet sind als links. — Tier mit großer, im ganzen ungefähr breit herzförmiger Flosse, mit wohlentwickeltem, in dorsoventraler Richtung stark ausgedehntem, geißeltragendem Mittellappen ; bei den ganz jungen Tieren finden sich zuweilen auf jeder Flossenhälfte zwei rotbraune Punkte und der Mittellappen der Flosse ist zylindrisch, nicht abgeflacht. L. der Pseudoconcha bis zu 62 mm. — Fig. 51 (p. 58) u. Fig. 63. 3. C. sibogae Tesch 1903 C. s., Tesch in: Tijdschr. Nederl. dierk. Ver., ser. 2 v8 p.113 \| 1904 ©. s., Tesch in: Siboga-Exp., nr. 52 p. 54 t.3 f. 88—90 \| 1905 ©. s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 37 t.1 f.3. Bisher nur in der Cook-Straße (Neu-Seeland) gefunden. 3. C. sibogae Tesch 1903 C. s., Tesch in: Tijdschr. Nederl. dierk. Ver., ser. 2 v8 p.113 \| 1904 ©. s., Tesch in: Siboga-Exp., nr. 52 p. 54 t.3 f. 88—90 \| 1905 ©. s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 37 t.1 f.3. Pseudoconcha langgestreckt, schmal, mit langer, dorsaler Endspitze;. nicht in der Mitte eingeschnürt, auch die auf der aboralen Seite verlaufenden Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in Ann. Mus. Paris, v.15 p. 66. Die Gestalt der Pseudoconcha wechselt bei den kleineren Exemplaren (unterhalb etwa 40 mm L.) sehr, wie aus den Figuren ersichtlich; auch scheinen die kleinen Pseudoconchae von den größeren darin zu differieren, daß an den Seiten nur ein oder zwei parallele Höckerreihen vorkommen (gegen drei in den größeren Stücken); zudem ist bei den kleineren Exemplaren konstant eine regelmäßige Bezahnung des Randes der Aushöhlung an der Oberseite zu finden, welehe Bezahnung bei größeren Pseudo- eonchae unregelmäßig, und zwar rechts viel stärker als links ausgeprägt erscheint. Ob hier nur Altersdifferenzen oder Artunterschiede vorliegen, läßt sich noch nicht ent- scheiden. In der westlichen Hälfte des Mittelmeeres, einschließlich der Adria; selten außerhalb dieses Gebietes (im Golfe von Guinea und an der Westküste Irlands). 2. C. parvidentata Plsnr. 1888 C. p., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.99 t.2 f.12, 13. Pseudoconcha gestreckt, verhältnismäßig mehr zusammengedrückt und länger als bei C. peronü, in der Mitte der Länge etwas eingeschnürt; der vordere (dorsale) Teil lang und zugespitzt, mit 5 Höckerreihen auf der oberen (oralen) Seite, einer in der Medianlinie und jeder- seits zwei lateralen, welche nach der Spitze der Pseudoconcha konvergieren. Die äußere von diesen Reihen setzt sich auch auf den hinteren (ventralen) Teil der Pseudoconcha fort und zeigt in der Mitte ihres Verlaufes ebenfalls eine deut- liche Einschnürung, sie endet in die beiden Zipfel des Hinterteiles. Diese Partie ist halbkreisförmig ausgehöhlt und zeigt keine Zähne. Auf der Unterseite vorn drei, hinten ‚vier kurze Höckerreihen, welche nach dem Mittelpunkt der Pseudoconcha konvergieren; alle Höcker, auch die Zähne N erze et - Pe na rec wer rn “enee € EN weine & / E “ b 9 = 02 RE A* Ed 1 u ey ee Segen am Rande der Aushöhlung, sehr klein Ein, 0 ? S C. parvidentata. A Pseudoconcha von unten, und gleichgestaltet. Aushöhlung auf der B von oben (°),). Nach Pelseneer. Oberseite sehr wenig tief, nach hinten zu allmählich verstreichend. — Tier unbekannt. — L. der Pseudoconcha 35 mm. — Fig. 64. \| N erze et - Pe na rec wer rn “enee € EN weine & / E “ b 9 = 02 RE A* Ed 1 u ey ee Segen Ein, 0 C. parvidentata. A Pseudoconcha von unten, B von oben (°),). Nach Pelseneer. Ein, 0 C. parvidentata. A Pseudoconcha von unten, B von oben (°),). Nach Pelseneer. Bisher nur in der Cook-Straße (Neu-Seeland) gefunden. Bisher nur in der Cook-Straße (Neu-Seeland) gefunden. Pseudoconcha langgestreckt, schmal, mit langer, dorsaler Endspitze;. nicht in der Mitte eingeschnürt, auch die auf der aboralen Seite verlaufenden — Fig. 64. \| Bisher nur in der Cook-Straße (Neu-Seeland) gefunden. 3. C. sibogae Tesch 1903 C. s., Tesch in: Tijdschr. Nederl. dierk. Ver., ser. 2 v8 p.113 \| 1904 ©. s., Tesch in: Siboga-Exp., nr. 52 p. 54 t.3 f. 88—90 \| 1905 ©. s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 37 t.1 f.3. Pseudoconcha langgestreckt, schmal, mit langer, dorsaler Endspitze;. nicht in der Mitte eingeschnürt, auch die auf der aboralen Seite verlaufenden Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 6 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in Ann. Mus. Paris, v.15 p. 66. Bisher nur in der Cook-Straße (Neu-Seeland) gefunden. 3. C. sibogae Tesch 1903 C. s., Tesch in: Tijdschr. Nederl. dierk. Ver., ser. 2 v8 p.113 \| 1904 ©. s., Tesch in: Siboga-Exp., nr. 52 p. 54 t.3 f. 88—90 \| 1905 ©. s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 37 t.1 f.3. Pseudoconcha langgestreckt, schmal, mit langer, dorsaler Endspitze;. nicht in der Mitte eingeschnürt, auch die auf der aboralen Seite verlaufenden Pseudoconcha langgestreckt, schmal, mit langer, dorsaler Endspitze;. icht in der Mitte eingeschnürt, auch die auf der aboralen Seite verlaufenden Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 6 82 82 Pterop.: A. Theeosomata, b) Pseudothecosomata, 3. Cymbuliidae, 1. Cymbnlia 2. Corolla Höckerreihen zeigen keine Spur einer Einschnürung. Auf den Seiten jederseits eine Längsreihe, auf der Unterseite zwei seitliche Reihen von der Spitze bis in die beiden ventralen Seitenzipfel der Pseudoconcha reichend; vom Mittel- punkte der Unterseite geht nach vorn eine mediane und gerade, nach hinten zwei divergierende, seitliche Reihen aus, welche ebenfalls bis in die ventralen Zipfel reichen. Hinterrand der Pseudoconcha ausgehöhlt, gezähnelt. Auf B Flosse Mittellappen der Flosse 5 % E Aushöhlung // , Mantelhöhle Mantelhöhle Niere “ Mantelhöhlendrüse Flosse Mittellappen der Flosse 5 % E Aushöhlung // , Mantelhöhle Mantelhöhle Niere “ Mantelhöhlendrüse A B Fig. 65. C.sibogae. A Tier mit Pseudoconcha, von rechts, B Pseudoconcha, von unten (?/,). Nach Tesch, etwas modifiziert. Flosse Mittellappen der Flosse 5 % E Aushöhlung // , Mantelhöhle Mantelhöhle Niere “ Mantelhöhlendrüse Flosse Mittellappen der Flosse B Fig. 65. C.sibogae. A Tier mit Pseudoconcha, von rechts, B Pseudoconcha, von unten (?/,). Nach Tesch, etwas modifiziert. Fig. 65. C.sibogae. A Tier mit Pseudoconcha, von rechts, B Pseudoconcha, von unten (?/,). Nach Tesch, etwas modifiziert. Fig. 65. C.sibogae. A Tier mit Pseudoconcha, von rechts, B Pseudoconcha, von unten (?/,). Nach Tesch, etwas modifiziert. der oberen (oralen) Seite der Pseudoconcha eine tiefe und weite Höhle, deren Ränder mit gleichmäßigen kleinen Zähnen besetzt sind. Nach der ventralen Seite hin enden diese Zahnreihen nicht in die ventralen Endspitzen der Pseudoconcha, sondern wenden sich der Medianlinie zu; eine kleine Furche, welche ventralwärts in ein breites, flaches Feld ausläuft, zwischen sich fassend. — Tier gänzlich dem von €. peroni ähnlich. — L. der Pseudo- concha bis zu 39 mm. — Fig. 65. Im tropischen Indischen Ozean und im Indo-Australischen Archipel, selten im Atlantischen Ozean (Golf von Guinea). ?’Argivora parva Lesueur 1827 A.p., Lesueur MS. in: Blainville, Man. 1810 Cymbulia (non Quoy & Gaimard 1832, Verrill 1880), Peron & Lesueur in Ann. Mus. Paris, v.15 p. 66. Malac., Planches p. 655. Martinique (Antillen). Bestimmungstabelle der Arten: Bestimmungstabelle der Arten: Pseudoconcha am ventralen Ende etwas zugespitzt; Öffnung \| kürzer als die halbe Länge der Pseudoconcha. . . . . 1. C. ovata Pseudoconcha am ventralen Ende abgerundet; Öffnung länger als die halbe Länge der Pseudoconcha — 2. Vorderrand der Flosse weit über den Vorderrand der Pseudo- eoneha hinausreichend . -. . ...... . 2. C. spectabilis Vorderrand der fFlosse kaum über den Vordemand Fe Pseudoconcha hinausreichend — 3. 2 Höcker auf der Pseudoconcha regelmäßig zerstreut; proxi- maler Rand der Mantelhöhlendrüse rechts weiter nach vorn reichend als links... ..... . 5. C. calceola Höcker auf der Pseudoconcha naht ersreul Be sonders auf der Unterseite; proximaler Rand der Mantel- höhlendrüse links weiter nach vorn reichend als rechts 4. C. intermedia \| \| Höcker auf der Pseudoconcha naht ersreul Be sonders auf der Unterseite; proximaler Rand der Mantel- höhlendrüse links weiter nach vorn reichend als rechts 4. C. intermedia \| \| 1. C. ovata (Q. & G.) 1832 C’ymbulia o., Quoy & Gaimard in: Voy. Astrol., v.2 p.373 Moll. t. 27 f. 25—28 \| 1852 C. ovularis, Rang in: Rang & Souleyet, Hist. nat. Pter., t.11 £.1—6 \| 1888 Oymbuliopsis ovata, Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars65 p.100 t.2 £f.15, 16 \| 1905 Corolla o., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 41. =, C.ovata. A Brnssleiteihe, von vorn, B von links (%.). Nach Pelseneer. Pseudoconcha eiförmig, mit einem ab- gerundeten und breiten Hinterteil, nach vorn zu etwas zugespitzt; der Vorderrand ist sehr dünn. Länge der Öffnung kürzer als die halbe Länge der Pseudoconcha, höchstens gleich lang. Höcker regelmäßig zerstreut, ziemlich weit auseinander. Tier mit breiter, ovaler Flossenscheibe (weit über den Vorder- rand der Pseudoconcha hinausreichend?). L. der Pseudoconcha 20—30 mm — Fig. 66. =, C.ovata. A Brnssleiteihe, von vorn, B von links (%.). Nach Pelseneer. Amboina (Molukken). 2. C. spectabilis W. Dall 18%1 C. s, W. Dall in: Amer. J. Conch., v.711 p-137 \| 1888 Gleba s., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.103 \| 1901 Oymbuliopsis vitrea, Heath & Spaulding in: P. Ac. Philad., v. 53 p. 509 tf. 2. C. spectabilis W. Dall 18%1 C. s, W. Dall in: Amer. J. Conch., v.711 p-137 \| 1888 Gleba s., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p.103 \| 1901 Oymbuliopsis vitrea, Heath & Spaulding in: P. Ac. Philad., v. 53 p. 509 tf. 2. Gen. Corolla W. Dall 1832 Cymbulia (part.),, Quoy & Gaimard in: Voy. Astrol., v.2 p.373 \| 1871 Corolla, W. Dall in: Amer. J. Conch., v.7u p.137 \| 1880 Cymbulia (non Peron & Lesueur 1810), A. E. Verrill in: Amer. J. Sei., ser.3 v.20 p.392 \| 1888 Cymbuliopsis + Gleba (part.), Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 100, 103. Pseudoconcha (Fig. 66—69) oval, abgerundet, mit sehr dünnen Wandungen und einer geräumigen Höhle, welche mit einer sehr weiten Öffnung auf der ventralen Hälfte der oralen Seite der Pseudoconcha ausmündet. Höcker nicht in Reihen, sondern unregelmäßig zerstreut, besonders auf der Unter- seite; keine Zähne oder größere Höcker am Rande der Öffnung. — Tier mit breiter, abgerundeter Flossenscheibe, ohne Lappen- oder Fransenbildung ; Muskulatur auf der Flosse deutlich als drei gesonderte Systeme zu unter- scheiden. Rüssel gut ausgebildet, sehr breit, nicht über den Mittelpunkt der Flossenscheibe hinausreichend, mit zwei symmetrischen Tentakeln auf der vorderen Hälfte der Dorsalseite; die vorderen zwei Drittel des Rüssels sind Pteropoda: A. Thecosomata, b) Pseudothecosomata, 3. Oymbuliidae, 2. Corolla 83 frei, sonst ist sie mit der Flosse verwachsen. Oberkiefer und Radula fehlen vollständig. Am aboralen Pole des Eingeweidesackes ein dunkel pigmen- tierter Ring. frei, sonst ist sie mit der Flosse verwachsen. Oberkiefer und Radula fehlen vollständig. Am aboralen Pole des Eingeweidesackes ein dunkel pigmen- tierter Ring. Auf die tropischen und subtropischen Gebiete der Ozeane beschränkt. 4 Arten. Fig. 68. C, calceola, vorn (orale Ansicht) (?],). A Tier mit Pseudoconcha, von hinten (aborale Ansicht) (®/,), B Pseudoconcha, von Nach Peck. Bestimmungstabelle der Arten: Pseudoconcha sehr breit und flach, überall abgerundet, auch am Vorder- rande. Die Länge der Öffnung ist deutlich länger als die halbe Länge der Pseudoconcha. Die Höcker auf der Oberfläche unregelmäßig zerstreut, meist stehen sie ziemlich weit auseinander, auf der hinteren oder Dorsalseite sind sie dichter gedrängt und etwas kleiner als sonst. Tier mit sehr großer, breit-viereckiger Flossenscheibe, mit abgerundeten Ecken und den drei sehr deutlich gesonderten Muskelsystemen; der Vorderrand der Flosse ragt sehr weit über den Vorderrand der Pseudoconcha hinaus. L. der Flosse etwa ?/, der Breite. Rüssel sehr breit und flach, Mantelhöhlendrüse nahezu ganz symmetrisch, Vorderrand sehr nahe an den Vorderrand der Pseudoconcha hinanreichend und mit diesem fast parallel, rechts aber sehr wenig weiter 6* 84 Pteropoda: A. Theeosomata, b) Pseudothecosomata, 3. Cymbuliidae, 2. Corolla 84 nach vorn ausgedehnt als links. L. der Flosse 45 mm, Br. 65 mm — Fig. 67. L. der Pseudoeoncha 40 mm, Br. 25 mm, nach vorn ausgedehnt als links. L. der Flosse 45 mm, Br. 65 mm — Fig. 67. L. der Pseudoeoncha 40 mm, Br. 25 mm, nach vorn ausgedehnt als links. L. der Flosse 45 mm, Br. 65 mm — Fig. 67. L. der Pseudoeoncha 40 mm, Br. 25 mm, Im nordöstlichen Teile des Pazifischen Özeanes, im gefunden, auch im westlichen Atlantischen Ozean. Bereich des Kuro-Shio Im nordöstlichen Teile des Pazifischen Özeanes, im gefunden, auch im westlichen Atlantischen Ozean. Bereich des Kuro-Shio 4‘ Pseudoconcha Fig. 67. " Mantelhöhlendrüse "* Eingeweidesack C. spectabilis.. Tier mit Pseudoconcha, von hinten (aborale Ansicht) (%/,). Nach Heath und Spaulding. Fig. 67. C. spectabilis.. Tier mit Pseudoconcha, von hinten (aborale Ansicht) (%/,). Nach Heath und Spaulding. 3. C. calceola (Verrill) 1880 Cymbulia c., A. E. Verrill in: Amer. J. Sei., ser. 3 v.20 p.394 \| 1882 ©. calceolus, A. E. Verrill in: Tr. Connect. Ac., v.5 p.553 t.53 f.33 \| 1888 COymbuliopsis calceola, Pelseneer in: Rep. Voy. Challenger, Zool. v,23 pars 65 p-101 \| 1905 Corolla e., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 40. Flosse h Mantelhöhlendrüse ” Paanin. concha Mantelhöhle “” i “-. aboraler Ring A Fig. 68. C, calceola, vorn (orale Ansicht) (?],). A Tier mit Pseudoconcha, von hinten (aborale Ansicht) (®/,), B Pseudoconcha, von Nach Peck. Fig. 68. C, calceola, vorn (orale Ansicht) (?],). A Tier mit Pseudoconcha, von hinten (aborale Ansicht) (®/,), B Pseudoconcha, von Nach Peck. Fig. 68. C, calceola, vorn (orale Ansicht) (?],). stehend, hier aber oft durch unregelmäßige Felderung der Oberfläche ver- treten, :wobei die Felder durch seichte Gruben getrennt sind, auf der hinteren (aboralen) Seite dichter gedrängt, besonders im Zentrum, nach dem Vorder- ende zu verschwinden die Höcker ganz; Öffnung länger als die halbe Länge der Pseudoconcha. Tier mit breiter, querovaler Flossenscheibe, der bogen- förmig verlaufende Vorderrand ragt nicht oder kaum über den Vorderrand der Pseudoconcha hinaus. Rüssel kurz und breit, mit zwei symmetrischen Tentakeln auf der vorderen Hälfte; Mantelhöhlendrüse etwas asymmetrisch, auf der linken Seite etwas weiter nach vorn reichend als rechts. L. der Pseudoconcha 40 mm — Fig. 69. 3. Gen. Gleba Forskäl 1776 Gleba, Forskäl, Icon. Rer. nat., t.43 £.D \| 1832 Cymbulia (part.), Quoy & Gaimard in: Voy. Astrol., v.2 p. 375 \| 1839 Tiiedemannia, Chiaje MS. in: P.J. van Beneden in: Mem. Ac. Belgique, v.12 [nr. 13] p. 22. Pseudoconcha sehr flach, fast ohne Aushöhlung, breit, am hinteren (dorsalen) Ende abgerundet und verdickt, vorn (ventral) quer abgestutzt und dünn; Höcker klein, nur in der Mitte der Unterseite. — Tier mit sehr großer, halbkreisförmiger Flossenscheibe, welche auf der distalen Hälfte der Seitenränder einige große Auszackungen von drüsenartiger Natur zeigt; zudem kommen an den Rändern häufig große und kleine Pigmentflecke vor, Muskulatur in drei Systeme, ähnlich wie bei Corolla, zerlegbar. Rüssel schlank und lang, deutlich bis über den Mittelpunkt der Flossenscheibe hinausreichend, schlaff, fast über die ganze Länge frei beweglich, vorn trompetenartig verbreitert, mit zwei kleinen Tentakeln an der Basis. Kiefer und Radula fehlen vollständig. Kein aboraler Ring am unteren Pol des Eingeweidesackes. In den wärmeren Gebieten des Atlantischen und Indischen Ozeanes, bisher noch nicht im Pazifischen Ozean beobachtet. 2 sichere Arten, 3 unsichere Jugendstadien. 2 sichere Arten, 3 unsichere Jugendstadien. Bestimmungstabelle der sicheren Arten: Rüssel sehr lang, etwa 2/; der Länge der Flossenscheibe N einnehmöand: ...x-; Trike 1. G. cordata Rüssel kürzer, goldene Flecke an den Rändern der Flossen- a 7 en EN REN: 2. G. chrysostieta Rüssel sehr lang, etwa 2/; der Länge der Flossenscheibe N einnehmöand: ...x-; Trike 1. G. cordata N einnehmöand: ...x-; Trike 1. G. cordata Rüssel kürzer, goldene Flecke an den Rändern der Flossen- a 7 en EN REN: 2. G. chrysostieta l. G. cordata Forsk. 1776 @. c., Forskäl, Icon. Rer.nat., t.43 f.D \| 1839 Tiedemannia napolitana, Uhiaje MS. in: P. J. van Beueden in: Möm. Ac. Belgique, v.12 (or. 18] p.22 t.2 f.1 \| 1844 T. creniptera + Cymbulia proboscidea (non Gray 1850), Krohn in: Arch. Naturg., v.101 p.324 t.9 f. A; p.325 \| 1847 T. neapolitana, Krohn in: Arch. Naturg., v.13 p.36 t.2 1. A—C. Pseudoconcha sehr flach und breit, am Hinterrande abgerundet, vorn quer abgestutzt und in der Mitte etwas eingekerht; Aushöhlung sehr weit, am deutlichsten noch am verdiekten Hinterrande der Pseudoconcha, vorn sehr seicht und flach. Höcker (nur bei größeren Exemplaren) wenig und klein, nur auf der hinteren (aboralen) Seite der Pseudoconcha und zwar in der Mitte. Bestimmungstabelle der Arten: A Tier mit Pseudoconcha, von hinten (aborale Ansicht) (®/,), B Pseudoconcha, von Nach Peck. Pteropoda: A. Thecosomata, b) Pseudothecosomata, 3. Cymbuliidae, 2. Corolla 85 Pseudoconcha oval, abgerundet, sehr breit, am vorderen Ende nicht zugespitzt; Höcker regelmäßig zerstreut, ziemlich weit auseinander. Öffnung länger als die halbe Länge der Pseudoconcha. Tier mit breiter, quer-ovaler Flossenscheibe, deren Vorderrand nicht oder kaum über den Vorderrand der Pseudoconcha hinausreicht; Rüssel kurz und breit, mit zwei symmetrischen Tentakeln auf der vorderen Hälfte; Mantelhöhlendrüse etwas asymmetrisch, an der rechten Seite etwas weiter nach vorn reichend als links. L. der Pseudoconcha 40 mm, Br. der Flossenscheibe 67 mm — Fig. 68 u. Fig. 52 (p- 59). Im tropischen Gebiete des Atlantischen Ozeanes, in den warmen Anfangsteilen des Golfstromes bis etwa 41° n. Br. vordringend; sonst noch von der ostafrikanischen Küste bekannt. 4. C. intermedia (Tesch) 1903 Cymbuliopsis i., Tesch in: Tijdschr. Neder!. dierk. Ver., ser.2 v.8 p.113 \| 1904 ©. i., Tesch in: Siboga-Exp., nr. 52 p.59 t.4 f. 100—104 \| 1905 Corolla i., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p.41. Pseudoconcha oval, breit, abgerundet an beiden Enden; Höcker unregel- mäßig zerstreut, auf der vorderen (oralen) Seite ziemlich weit auseinander- ”»_ Flosse “ Tentakel SL errwa 7 >». Eingeweide- nucleus .. . Rüssel / ” Pseudoconcha A B Fig. 69. C.intermedia. A Tier mit Pseudoconcha, von vorn (orale Ansicht), B Pseudoconcha, von vorn (?},). Nach Tesch, etwas modifiziert. B Fig. 69. C.intermedia. A Tier mit Pseudoconcha, von vorn (orale Ansicht), B Pseudoconcha, von vorn (?},). Nach Tesch, etwas modifiziert. stehend, hier aber oft durch unregelmäßige Felderung der Oberfläche ver- treten, :wobei die Felder durch seichte Gruben getrennt sind, auf der hinteren (aboralen) Seite dichter gedrängt, besonders im Zentrum, nach dem Vorder- ende zu verschwinden die Höcker ganz; Öffnung länger als die halbe Länge der Pseudoconcha. Tier mit breiter, querovaler Flossenscheibe, der bogen- förmig verlaufende Vorderrand ragt nicht oder kaum über den Vorderrand der Pseudoconcha hinaus. Rüssel kurz und breit, mit zwei symmetrischen Tentakeln auf der vorderen Hälfte; Mantelhöhlendrüse etwas asymmetrisch, auf der linken Seite etwas weiter nach vorn reichend als rechts. L. der Pseudoconcha 40 mm — Fig. 69. 86 Pteropoda: A. Theeosomata, b) Pseudothecosomata, 3. Cymbuliidae, 2. Corolla, 8. Gleba 86 Pteropoda: A. Theeosomata, b) Pseudothecosomata, 3. Cymbuliidae, 2. Corolla, 8. Gleba Es ist diese Art jedenfalls sehr nahe mit der vorhergehenden verwandt. Bestimmungstabelle der Arten: Die einzigen wichtigen Unterschiede scheinen in der Unregelmäßigkeit der Höcker auf der Pseudoeoncha zu liegen, sowie in der Mantelhöhlendrüse, deren Vorderrand bei (. calceola rechts, bei (. intermedia links am weitesten nach vorn reicht. Auch ist vielleicht die Pseudoconcha bei (. calceola verhältnismäßig breiter. Bisher nur im indo-australischen Archipel gefunden. Bisher nur im indo-australischen Archipel gefunden. 3. Gen. Gleba Forskäl Tier mit sehr großer, halbkreisförmiger Flossenscheibe, ohne irgend- welche Anhänge, aber mit 5 oder 6 charakteristischen, groben Auszackungen auf der distalen Hälfte der Seitenränder; in dieser Gegend häufig kleinere und größere Pigmentanhäufungen von weißer und goldgelber Farbe. Rüssel sehr lang und schlaff, etwa ?/, der Längsdurchmesser der Flossenscheibe einnehmend, mit zwei kleinen, symmetrisch gestellten Tentakeln auf der Rückenseite, nahe der Basis des Rüssels. Eingeweidenucleus verhältnismäßig Pterop.: A. Thecos., b) Pseudothecos., 3. Cymbuliidae, 3, Gleba, 4. Desmopteridae 87 sehr klein, spindelförmig. L. der Flossenscheibe 45 mm, Br.55 mm — Fig. 53 (p. 60). Mit Sicherheit nur im westlichen Mittelmeer (nicht in der Adria) und im tropischen Gebiete des östlichen Atlantischen Ozeanes gefunden. Mit Sicherheit nur im westlichen Mittelmeer (nicht in der Adria) und im tropischen Gebiete des östlichen Atlantischen Ozeanes gefunden. 2. G. chrysostieta (Krohn) ?1832 Oymbulia punctata, Quoy & Gaimard in: Voy. Astrol., v.2 p. 377, Moll. t.27 £.35, 36 \| ?1852 Tiedemannia p., Rang in: Rang & Souleyet. Hist. nat. Pter., t.2 f.11, 12 \| 1854 T. chrysosticta, Krohn MS. in: Troschel in: Arch. Naturg., v.201 p.218 \| 1888 Gleba c., Pelseneer in: Rep. Voy. Challenger, Zool. v.23 pars 65 p. 103. Unterscheidet sich von der vorhergehenden Art nur dadurch, daß der hüssel verhältnismäßig kürzer ist und durch große, goldgelbe, radiär an- geordnete Flecke auf den Flossen. Die Maße scheinen nahezu dieselben zu sein. Ubrigens ist diese Art ganz wenig bekannt. Nur im Mittelmeer, und zwar bei Messina und Villefranche, weiter im west- lichen Atlantischen Ozean (40° n. Br., 57° w. I.) beobachtet. Nur im Mittelmeer, und zwar bei Messina und Villefranche, weiter im west- lichen Atlantischen Ozean (40° n. Br., 57° w. I.) beobachtet. Cymbulia radiata Q. &G. 1832 C.r., Quoy & Gaimard in: Voy. Astrol., v.2 p. 375, Moll. t. 27 £.33, 34. - Cymbulia radiata Q. &G. 1832 C.r., Quoy & Gaimard in: Voy. Astrol., v.2 p. 375, Moll. t. 27 £.33, 34. - Unbestimmbare Jugendformen von Gleba. Tiedemannia scyllae Trosch. 1854 T. s., Troschel in: Arch. Naturg., v. 201 p-219 t.9 £.12, 13. Jugendform von @. cordata oder G. chrysosticta? Messina. T. charybdis Trosch. 1854 T.c., Troschel in: Arch. Naturg., v.201 p. 220 t.9 1.145. T. charybdis Trosch. 1854 T.c., Troschel in: Arch. Naturg., v.201 p. 220 t.9 1.145. Jugendform von @. cordata oder G. chrysosticta? Messina. 1. Gen. Desmopterus Chun ?1855 Cymbulia (part.), Gegenbaur, Unters. Pter. & Heterop., p. 53 \| 1889 Desmopterus, Chun in: SB. Ak. Berlin, p. 540. ?1855 Cymbulia (part.), Gegenbaur, Unters. Pter. & Heterop., p. 53 \| 1889 Desmopterus, Chun in: SB. Ak. Berlin, p. 540. Die Diagnose stimmt mit der der Familie überein. In den wärmeren Meeren. _ 2 Arten. Die Diagnose stimmt mit der der Familie überein. In den wärmeren Meeren. _ 2 Arten. Bestimmungstabelle der Arten: Bestimmungstabelle der Arten: Muskulatur in den Flossen ziemlich einheitlich, besonders in der Mitte: u . 0,10, 2 sel. Bee en OA N 1. D. papilio l Muskulatur in den Flossen deutlich in zwei sich kreuzende Systeme von Muskelfasern gesondert . .. 2.2... . . 2. D. gardineri Muskulatur in den Flossen ziemlich einheitlich, besonders in der Mitte: u . 0,10, 2 sel. Bee en OA N 1. D. papilio Muskulatur in den Flossen ziemlich einheitlich, besonders in der Mitte: u . 0,10, 2 sel. Bee en OA N 1. D. papilio l Muskulatur in den Flossen deutlich in zwei sich kreuzende Systeme von Muskelfasern gesondert . .. 2.2... . . 2. D. gardineri l Muskulatur in den Flossen deutlich in zwei sich kreuzende Systeme von Muskelfasern gesondert . .. 2.2... . . 2. D. gardineri 1. D. papilio Chun ?1855 Cymbulia eirroptera, Gegenbaur, Unters. Pter. & Heterop., p.53 t.3 f.21 \| 1889 Desmopterus papilio, Chun in: SB. Ak. Berlin, p. 540 \| 1905 D.p., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.43 t.13 £. 10. Körper zylindrisch, gestreckt, der vordere Teil ventral umgeknickt und kegelförmig zugespitzt; in dem Winkel heftet sich die sehr große Flossen- Tentakel Speicheldrüsen ” _ Tentakel "= Mund \ / * Flosse ‚ Körper Flosse Flossententakel-__ Ant Flossententakel Fig. 70. D. papilio. A Tier, von hinten, B Kopf mit Teil der Flosse, von vorn (etwa °],). Nach Meisenheimer. Tentakel ” Tentakel Flosse Körper Flosse Fig. 70. D. papilio. A Tier, von hinten, B Kopf mit Teil der Flosse, von vorn (etwa °],). Nach Meisenheimer. scheibe an, welehe sonst nirgends mit dem Körper verbunden ist. Der Vorderrand hat in der Mitte eine leichte Einschnürung und verläuft bogen- förmig, die Seitenränder sind ebenfalls schwach bogenförmig gebogen, der Hinterrand ist in fünf Lappen geteilt, die medianen Seitenlappen sind die kleinsten; zwischen den beiden seitlichen findet sich ein langer, meist aber abgerissener, bandförmiger Tentakel. 1889 Desmopteridae, Chun in: SB. Ak. Berlin, p. 546. 1889 Desmopteridae, Chun in: SB. Ak. Berlin, p. 546. Weder Kalkschale, noch innere Schale (Pseudoconcha) vor- handen. Körper (Fig. 70, 71) gestreckt, walzenförmig, der Vorder- teil im scharfen Winkel gegen den Rumpf abgesetzt und über den Vorderrand der Flosse hin gebogen, kegelförmig zugespitzt. Mantelhöhle fehlt. Fuß ganz rudimentär. Flossen zu einer mächtigen Scheibe verwachsen, welche am Hinterrande in fünf Lappen zerteilt ist; einer liegt in der Medianebene, jederseits von diesem unpaaren Lappen stehen zwei seitliche, welche einen langen Tentakel zwischen sich tragen. Am Hinterende des Körpers eine quer gelagerte, umfangreiche Drüsengrube. Tentakeln klein, knopfförmig, ohne Scheide, symmetrisch zu beiden Seiten entwickelt, auf der Dorsalseite des Kopfabschnittess Mund am Ende des zugespitzten Vorderendes, Oberkiefer und Radula (mit Formel 1:1:1) vorhanden; der Magen ist nieht mit Kauplatten ausgerüstet und ist sehr weit und dünnwandig; die Leber ist in seinen Wandungen eingelagert; der Enddarm entspringt rechts, und zwar im proximalen Teile des Magens, Anus distal, auf der linken Ventralseite.e Osphradium auf der rechten Ventralseite, 88 Pterop.: A. Theeosomata, b) Pseudotheeosomata., 4. Desmopteridae, 1. Desmopterus 88 Pterop.: A. Theeosomata, b) Pseudotheeosomata., 4. Desmopteridae, 1. Desmopterus 8 Pterop.: A. Theeosomata, b) Pseudotheeosomata., 4. Desmopteridae, 1. Desmopterus langgestreckt. Herz und Niere rechts, Herzkammer nach vorn, Vorhof nach hinten gerichtet, Zwitterdrüse dorsal, Anhangs- drüsen rechts, Penisöffnung rechts, vor der Mundöffnung. In den tropischen Gebieten der Ozeane, auch im Mittelmeer. Enthält nur die einzige Gattung Desmopterus. In den tropischen Gebieten der Ozeane, auch im Mittelmeer. In den tropischen Gebieten der Ozeane, auch im Mittelmeer. Enthält nur die einzige Gattung Desmopterus. Enthält nur die einzige Gattung Desmopterus. Enthält nur die einzige Gattung Desmopterus. Enthält nur die einzige Gattung Desmopterus. 1. Gen. Desmopterus Chun Muskulatur der Flosse besteht aus zwei Systemen sich rechtwinklig kreuzender, sehr schmaler Muskelfasern ; die beiden Richtungen bilden einen Winkel von etwa 45° mit der Längs- Pterop.: A. Thecos., b) Pseudothecos., 4. Desmopt., 1. Desmopterus, B. Gymnosomata 89 Pterop.: A. Thecos., b) Pseudothecos., 4. Desmopt., 1. Desmopterus, B. Gymnosomata 89 achse des Körpers, nach den Rändern sind die einzelnen Fasern gesondert, in der Mitte aber zu einer einheitlichen Platte verbunden. Körper bisweilen hell orangerot gefärbt, auf der Flossenscheibe vier rosa Flecke, der vordere Flossenrand, die Spitze der inneren Seitenlappen der Flosse, sowie ein Mittelstreifen der Flossententakeln hochrot pigmentiert. L. des Körpers 2 mm, Br. der Flossenscheibe 5 mm — Fig. 70. In den wärmeren Teilen des Atlantischen Ozeanes, etwa zwischen 34°n. Br. und 38°s. Br., selten im westlichen Mittelmeer; im tropischen Indischen Ozean, etwa zwischen 13° n. Br. und 11°s. Br., und im indo-australischen Archipel. 2. D. gardineri Tesch 1910 D. g., Tesch in: Tr. Linn. Soc. London, ser. 2 v.14 pars1l p.168 t.12 £.1,2. Diese Art unterscheidet sich von der vorhergehenden nur dadurch, daß die Muskel- Körper .. Fl fasern in der Flosse zu deutlichen Bündeln nn gesondert sind, diese Bündel sind als ziemlich breite Streifen, welche nicht miteinander ver- Se - Flossententakel schmelzen, auf der ganzen Flosse erkennbar. Der Seitenrand der Flosse scheint etwas kon- Anl. kav, nicht konvex zu sein. Farbe unbekannt. ”- 8®rdineri. Von hinten (}»). Nach Br. der Flossenscheibe 5 mm — Fig. 71. Bisher nur ein einziges Exemplar in der Nähe des Chagos Archipels im Indischen Ozean erbeutet. 2. D. gardineri Tesch 1910 D. g., Tesch in: Tr. Linn. Soc. London, ser. 2 v.14 pars1l p.168 t.12 £.1,2. Körper Körper .. Fl nn Se - Flossententakel Anl. ”- 8®rdineri. Von hinten (}»). Nach Diese Art unterscheidet sich von der vorhergehenden nur dadurch, daß die Muskel- fasern in der Flosse zu deutlichen Bündeln gesondert sind, diese Bündel sind als ziemlich breite Streifen, welche nicht miteinander ver- schmelzen, auf der ganzen Flosse erkennbar. Der Seitenrand der Flosse scheint etwas kon- kav, nicht konvex zu sein. Farbe unbekannt. Br. der Flossenscheibe 5 mm — Fig. 71. Bisher nur ein einziges Exemplar in der Nähe des Chagos Archipels im Indischen Ozean erbeutet. B. Subord. Gymnosomata 1824 Gymnosomata, Blainville in: Diet. Sei. nat., v.32 p.271 \| 1885 Deutocephala, N. Wagner in: Wirbell. Weiss. Meer., v.1 p.119 \| 1886 Pterota, Boas in: Danske Selsk. Skr., ser.6 v.4 p. 14. Schale, Mantel und Mantelhöhle völlig fehlend. Körper spindelförmig oder oval, in Kopf- und Rumpfabschnitt zerlegbar. Am oberen Ende des Kopfabschnittes die Mundöffnung, von hier gelangt man in den Schlund, der mit typischen Organen, ausstülp- baren Saugarmen und Hakensäcken ausgestattet ist, der Schlund kann oft rüsselartig hervorgestreckt werden. Weiter am Kopf- abschnitt die beiden Flossen und der aus Mittellappen und paarigen Seitenlappen zusammengesetzte Fuß, der auf der Ventralseite zwischen den Ansatzstellen der Flossen liegt. Zwei Paare von Tentakeln, das erste Paar zu beiden Seiten der Mundöffnung, das zweite auf der Dorsalseite des Kopfes. Rechts, dorsal von der Ansatzstelle der rechten Flosse liegt die weibliche Geschlechts- öffnung, von hier geht eine Rinne proximalwärts und ventralnach der Mündung des Penis, der am dorsalen Rande des rechten Seiten- lappensdesFußesliegt. Rumpfabschnitt mehr oder weniger walzen- förmig, vom Kopfabschnitt bisweilen scharf abgesetzt; rechts, im Bereich des distalen Randes der rechten Flosse, liegen After, Osphradium und Nierenöffnung, meist dieht zusammen auf dem sogenannten Analfelde. Oft in der Medianebene auf der Dorsal- seite des Rumpfes eine längliche Drüsengrube. Kiemen am unteren Pole des Körpers und auf der rechten Seite, oft nur an einer dieser Stellen oder gänzlich fehlend. Das Innere des Körpers wird bald mehr, bald weniger von dem Eingeweidenucleuserfüllt. Larven spindelförmig mit drei sehr charakteristischen Wimperschnüren. Pteropoda: B. Gymnosomata 90 Das Integument besteht aus Epidermis, Drüsenzellen und Bindegewebs- fasern. oft auch aus einer Muskellage. Die Epidermis ist meist ein Zylinder- epithel, das aus bald hohen, bald niedrigen Zellen besteht. Cilien scheinen an bestimmten Stellen, wenigstens in der Jugend, konstant vorzukommen. Unterhalb der Epidermiszellen liegt oft eine dünne Schicht hyaliner Substanz, mit eingestreuten Kernen und glatten Muskelfasern. Darunter folgt eine mächtige Schieht von Bindegewebsfasern, namentlich im Rumpfe, welche nach außen zu kompakt ist und hier bisweilen verästelte Pigmentzellen enthält, nach innen aber weite Hohlräume umschließt. Das Ganze wird an der Innen- seite durch eine Membran abgeschlossen, der sich nicht selten Ringmuskel- fasern anlegen. — In dem Integument zerstreut kommen weiter zahlreiche einzellige Drüsen vor, welche bald flaschenförmig sind und vorzugsweise in der Nähe von Fuß und Kopf liegen, bald kleiner und birnförmig überall zerstreut zu finden sind. Bisweilen kommt ein Ausführungsgang dadurch zu- stande, daß die Epidermis sich einstülpt. B. Subord. Gymnosomata Weiter finden sich noch im Integument sehr große, prall gefüllte Drüsenzellen, welche meist keinen Ausführungsgang nach außen besitzen, sondern mehr als Stützgewebe zu dienen scheinen und bei Halopsyche z. B. dem Körper eine knorpelhafte Festigung verleihen. Bei Cliopsis dagegen kommt es nie zu der Bildung des blasigen Stützgewebes; hier aber findet sich ein Ersatz durch eine mächtig entwickelte, hyaline Substanz unterhalb der Ringmuskelschicht. Alle Stützelemente finden sich nur im Rumpfabschnitt; Kopf und Fußteile, sowie die Nähe der Kiemen bleiben davon frei. An besonderen Stellen des Körpers häufen sich die Drüsen zu besonderen Feldern an. So finden sich große Drüsenkomplexe auf dem Analfelde, wo Darm und Niere ausmünden und das Osphradium gelegen ist; es findet sich auf der rechten Körperseite. Hier kommen sehr zahlreiche, flaschenförmige Drüsenzellen vor. Bei Clopsis sind auf dem Analfelde stets zwei Zellen zu einer einheitlichen Drüse verschmolzen; ein eigentlicher Ausführungsgang kommt nicht vor. Eine zweite Anhäufung von Drüsenzellen liegt auf der Dorsalseite des Rumpfes; hier wird der Dorsalfleck gebildet, wenigstens bei den Pneumodermatiden und bei Clopsis. Dieser Dorsalfleck besteht aus einer flachen Längsgrube, auf deren Boden zahlreiche einzellige Drüsen ausmünden. — Bei T'hliptodon besteht das ganze Integument aus einem flachen Epithel mit einer strukturlosen Membran als Unterlage; oft kommen hier zudem, ebenso wie bei Thalassopterus, mehrzellige Drüsen in der Haut vor. Der Fuß (Fig. 72 p. 91) liegt stets ventral auf dem Übergange zwischen Kopf- und Rumpfabschnitt; er teilt sich in zwei Hauptteile, in einen vorderen, aus paarigen Lappen sich zusammensetzenden Teil und in einen hinteren unpaaren Abschnitt, den Mittellappen, der meist zipfelförmig ausläuft, bisweilen aber gänzlich fehlt. Meist sind die Seitenlappen durch eine breite Fläche voneinander getrennt und findet nur am Vorderrand eine Berührung statt. am Hinterrande endigen sie frei; so entsteht häufig eine hufeisenförmige Figur. Zwischen den hinteren Abschnitten der Seitenlappen findet sich in der Medianebene ein längsgefalteter Höcker, der aber bisweilen sehr wenig ausgebildet erscheint. Die innere Fläche der Seitenlappen ist von feinen Wimperhaaren bedeckt; auch auf dem Höcker finden sich Cilien, und ebenso auf der ventralen Fläche des Mittellappens des Fußes. An letztgenannter Stelle kommen zudem zahlreiche Drüsen vor, besonders auf der dorsalen Seite (bei Halopsyche liegen sie dagegen an der ventralen Seite). B. Subord. Gymnosomata Die Flossen sind an der seitlichen Körperwand in dorsaler Richtung Die Flossen sind an der seitlichen Körperwand in dorsaler Richtung verschobene Teile des Fußes; sie haben eine blattförmige, außen meist ver- Die Flossen sind an der seitlichen Körperwand in dorsaler Richtung verschobene Teile des Fußes; sie haben eine blattförmige, außen meist ver- Pteropoda: B. Gymnosomata 9 breiterte, an der Basis aber stark eingeschnürte Gestalt. Sie können stark zurückgezogen werden, zuweilen sogar in besondere Hauttaschen. Das ober- flächliche Epithel scheint hier und da Wimperstellen zu haben, Drüsenzellen kommen aber niemals vor. Unter dem Epithel liegt die Flossenmuskulatur, welche auf Ober- und Unterseite aus zwei Lagen sich kreuzender Muskelfasern besteht; nach den Rändern der Flosse zu verschwinden sie allmählich. Das Innere wird häufig von einer in der Mittelebene der Flosse verlaufenden elastischen Membran gebildet, während senkrecht dazu gestellte Quersepten zur Befestigung beitragen. Die Muskulatur der beiden Flossen steht weder miteinander, noch mit der Körpermuskulatur in Verbindung; die Flossen sind nur durch membranöse Häutchen verbunden. Ihre Muskulatur ist quer- gestreift. — Nur Halopsyche weicht in dem Flossenbau stark ab, da hier ein stielartig verlängerter, proximaler Teil schärfer gegen den verbreiterten, distalen Teil abgesetzt erscheint; der freie Außenrand dieses letzten Teiles ist durch ausgestülpter Hakensack --------- ------ Lage des Kiefers ---- ventrale Rüsselpapille Rüssel ----------- —— rechter Saugarm rechter vorderer Tentakel ---------- > Lage des rechten hinteren ------------ Tentakels rechte Flosse -----""""""> m Kopf &-------.... Öffnung des Penis -4----------- rechter Seitenlappen des Fußes weibliche Geschlechtsöffnung --""", Sr Fra NS Satans ruber eV 2 Bselanpen der Tukien Seitenkamm der Seitenkieme ---------H-- Al —f------ Seitenkieme REN Endkieme Fig. 72. Schematische Darstellung von Pneumoderma mit hervorgestrecktem Schlundapparat, von rechts. Nach Pelseneer. ausgestülpter Hakensack Lage des Kiefers ventrale Rüsselpapille rechter Saugarm rechter vorderer Tentakel Lage des rechten hinteren - Tentakels Fig. 72. Schematische Darstellung von Pneumoderma mit hervorgestrecktem Schlundapparat, von rechts. Nach Pelseneer. eine tiefe Furche in zwei nicht gleich große Lappen zerlegt. Auch die innere Struktur ist eine andere; wir finden hier nicht zwei Systeme sich kreuzender Muskelfasern, sondern nur eine Lage von Längsmuskeln, während das Innere von Bindegewebe und von vier mächtigen Muskelbündeln, gleichfalls in longitudinaler Richtung, erfüllt wird. Das Mesenchym nimmt Anteil an dem Aufbau des Integumentes und Das Mesenchym nimmt Anteil an dem Aufbau des Integumentes und durchzieht in Form lockerer Faserbündel und verästelter Zellen die Hohl- räume. Die Leibeshöhle wird durch eine bindegewebige Membran in einen Kopfsinus und einen Eingeweidesinus zerlegt (Fig. B. Subord. Gymnosomata hinten und ventralwärts, einander sehr dicht genähert; auch sie sind asymmetrisch, hier aber ist es das rechte Ganglion, das dem anderen an Größe beträchtlich nachsteht. Das Konnektiv zu den Pleuralganglien entspringt am vorderer Außenrande. Die Buccal- ganglien liegen vor den übrigen Ganglien auf der Ventralseite des Oeso- phagus; sie sind klein und berühren sich mit breiter Fläche in der Median- ebene des Körpers. Am dorsalen Außenrande nehmen sie die Cerebro- bucealkonnektive auf. — Bei HAalopsyche sind alle Ganglien unter starker Verkürzung der Kommissuren und Konnektive einander stark genähert. In der Visceralkommissur treten nur drei Ganglien auf, zwei seitliche und eines in der Medianebene. Das links gelegene ist das kleinste, das rechte viel stärker entwickelt, so groß wie das mediane, das quer-oval ausgezogen ist. Die beiden seitlichen sind die jederseits verschmolzenen Pleural- und Viseeral- ganglien, das hintere ist das unpaare Abdominalganglion. — Das periphere Nervensystem ist sehr kompliziert. Von den Cerebralganglien entspringen jederseits bei Pneumoderma sieben Nerven, welche den Schlund, die vorderen sowie die hinteren Tentakeln, die Saugarme und endlich die Statocyste inner- vieren. Nach dem hinteren Tentakel ziehen je zwei Nerven, welche an der Wurzel ganglionäre Anschwellungen zeigen und nahezu parallel verlaufen. Sie enden beide in ein längliches Ganglion, nachdem sie kurz vorher, wenigstens bei Das Zentralnervensystem besteht "fast stets aus fünf paarigen Ganglien: Cerebral-, Pleural-, Pedal-, Visceral- und Buccalganglien. Pedal- und Pleuralganglien sind stets durch Konnektive mit den Cerebral- ganglien verbunden. Kommissuren bestehen überall, mit Ausnahme zwischen den Pleuralganglien. Die Cerebralbanglien liegen dorsal vom Oesophagus, alle übrigen ventral. — Die Cerebralganglien liegen nahe aneinander, vom Seitenrande entspringen die Konnektive mit den Buccalganglien, un- mittelbar dahinter die Verbindungen mit den Pedalganglien und am weitesten nach hinten die Cerebropleuralkonnektive, Die Konnektive mit den Bucecal- ganglien sind zuweilen sehr lang (Pneumoderma, Cliopsis), was mit der Vorstülpbarkeit des Schlundes zusammenhängt. Die Pedalganglien liegen etwas weiter proximal, auf der Ventralseite des Oesophagus; sie sind meist die umfangreichsten Ganglien und durch zwei Kommissuren miteinander ver- bunden, eine vordere, welche lang und dünn erscheint, und eine hintere, breitere. Die Konnektive entspringen auf dem vorderen Außenrande, die- jenigen zu den Pleuralganglien sind sehr kurz. Die Pleuralganglien liegen am Außenrande des Schlundringes, ganz nahe den Pedalganglien; sie sind die kleinsten Ganglien (mit Ausnahme der Buccalganglien) und deutlich asymmetrisch, indem das linke bedeutend schwächer ausgebildet ist als das rechte. B. Subord. Gymnosomata 73 p. 94). Der Kopfsinus ist undeutlich, er enthält den gesamten Vorderdarm, den Penis und das Zentralnervensystem. Die Scheidewand ist in der dorsalen Hälfte am deutlichsten und bildet hier eine Art Diaphragma. Der Eingeweidesack wird überall von einer festen Hülle umschlossen, die mehr oder weniger dick sein kann, bisweilen Pigmenteinlagerungen enthält und im bestimmten Zwischenräumen von scharf umschriebenen Öffnungen durchbrochen wird. Pteropoda: B. Gymnosomata 92 Bei der Körpermuskulatur können wir stets drei große Längsstämme unterscheiden, von denen zwei zu beiden Seiten der Medianebene, der Dorsal- seite genähert, liegen, während der dritte die mediane Ventralseite einnimmt. Sie entspringen aus losen Faserbündeln am hinteren Körperende und ver- laufen in dem Raume zwischen Integument und Eingeweidesack. Die beiden latero-dorsalen Stämme heften sich an die dorsalen Teile des Schlundes und die umliegenden Körperpartien an und dienen zum Zurückziehen dieser Teile, auch die vorderen Tentakeln werden von ihnen versorgt. Der ventrale Stamm versorgt die entsprechenden ventralen Abschnitte des vorderen Rumpfteiles; auch in den Fuß treten drei Bündel ein. Das Zentralnervensystem besteht "fast stets aus fünf paarigen auch in den Fuß treten drei Bündel ein. Das Zentralnervensystem besteht "fast stets aus fünf paarigen Ganglien: Cerebral-, Pleural-, Pedal-, Visceral- und Buccalganglien. Pedal- und Pleuralganglien sind stets durch Konnektive mit den Cerebral- ganglien verbunden. Kommissuren bestehen überall, mit Ausnahme zwischen den Pleuralganglien. Die Cerebralbanglien liegen dorsal vom Oesophagus, alle übrigen ventral. — Die Cerebralganglien liegen nahe aneinander, vom Seitenrande entspringen die Konnektive mit den Buccalganglien, un- mittelbar dahinter die Verbindungen mit den Pedalganglien und am weitesten nach hinten die Cerebropleuralkonnektive, Die Konnektive mit den Bucecal- ganglien sind zuweilen sehr lang (Pneumoderma, Cliopsis), was mit der Vorstülpbarkeit des Schlundes zusammenhängt. Die Pedalganglien liegen etwas weiter proximal, auf der Ventralseite des Oesophagus; sie sind meist die umfangreichsten Ganglien und durch zwei Kommissuren miteinander ver- bunden, eine vordere, welche lang und dünn erscheint, und eine hintere, breitere. Die Konnektive entspringen auf dem vorderen Außenrande, die- jenigen zu den Pleuralganglien sind sehr kurz. Die Pleuralganglien liegen am Außenrande des Schlundringes, ganz nahe den Pedalganglien; sie sind die kleinsten Ganglien (mit Ausnahme der Buccalganglien) und deutlich asymmetrisch, indem das linke bedeutend schwächer ausgebildet ist als das rechte. Vom Vorderrande gehen Konnektive zu Pedal- und zu Cerebral- ganglien, vom Hinterrande entspringt das mächtige Pleurovisceralkonnektiv, das schräg nach hinten und nach der Medianebene zu den Visceralganglien hinzieht. Diese liegen am weitesten nach. B. Subord. Gymnosomata Vom Vorderrande gehen Konnektive zu Pedal- und zu Cerebral- ganglien, vom Hinterrande entspringt das mächtige Pleurovisceralkonnektiv, das schräg nach hinten und nach der Medianebene zu den Visceralganglien hinzieht. Diese liegen am weitesten nach. hinten und ventralwärts, einander sehr dicht genähert; auch sie sind asymmetrisch, hier aber ist es das rechte Ganglion, das dem anderen an Größe beträchtlich nachsteht. Das Konnektiv zu den Pleuralganglien entspringt am vorderer Außenrande. Die Buccal- ganglien liegen vor den übrigen Ganglien auf der Ventralseite des Oeso- phagus; sie sind klein und berühren sich mit breiter Fläche in der Median- ebene des Körpers. Am dorsalen Außenrande nehmen sie die Cerebro- bucealkonnektive auf. — Bei HAalopsyche sind alle Ganglien unter starker Verkürzung der Kommissuren und Konnektive einander stark genähert. In der Visceralkommissur treten nur drei Ganglien auf, zwei seitliche und eines in der Medianebene. Das links gelegene ist das kleinste, das rechte viel stärker entwickelt, so groß wie das mediane, das quer-oval ausgezogen ist. Die beiden seitlichen sind die jederseits verschmolzenen Pleural- und Viseeral- ganglien, das hintere ist das unpaare Abdominalganglion. — Das periphere Nervensystem ist sehr kompliziert. Von den Cerebralganglien entspringen jederseits bei Pneumoderma sieben Nerven, welche den Schlund, die vorderen sowie die hinteren Tentakeln, die Saugarme und endlich die Statocyste inner- vieren. Nach dem hinteren Tentakel ziehen je zwei Nerven, welche an der Wurzel ganglionäre Anschwellungen zeigen und nahezu parallel verlaufen. Sie enden beide in ein längliches Ganglion, nachdem sie kurz vorher, wenigstens bei Pteropoda: B. Gymnosomata 93 einigen Gattungen, durch einen kurzen Ast miteinander in Verbindung getreten sind. Der proximale Nerv besitzt das größere Ganglion, das wahrscheinlich mit dem Geruchsganglion der Opisthobranchier homolog ist; er tritt nicht in den Tentakel ein. Der distale Nerv innerviert den Tentakel und trägt an der Spitze ein kleines Ganglion opticum. Der Nerv zu der Statocyste verläuft in der Richtung des Öerebropedalkonnektivs nach hinten. Die übrigen Gattungen haben meist eine geringere Zahl Cerebralnerven; besonders ist dies bei Halopsyche der Fall, wo nur drei Nerven von jedem Ganglion ent- springen ; statt des Doppelnerven zum hinteren Tentakel findet sich hier nur ein einziger Stamm. Von den Pedalganglien gehen bei Pneumoderma sechs Nerven jederseits ab, die den Fuß, die Flossen, die seitlichen und ventralen Teile des Integumentes von Kopf und Rumpf, sowie die Ausführungsgänge des (reschlechtsapparates versorgen. Charakteristisch ist eine Plexusbildung zwischen einem starken Pedalnerven, der sich bald in zwei Aste spaltet, einerseits und einem Pleuralnerven und einem kleinen Pedalnerven anderer- seits. B. Subord. Gymnosomata Auch hier finden wir bei den anderen Gattungen meist eine geringere Zahl Pedalnerven; bei Aalopsyche kommen jederseits nur drei Nerven vor. Vom Pleuralganglion entspringt nur ein einziger Nerv, und zwar am Vorder- rande, er spaltet sich bald und versorgt das Integument der hinteren Rumpf- teile. Die Visceralnerven sind asymmetrischh vom linken Ganglion gehen drei, vom rechten zwei Nerven ab (bei T’hliptodon und Paedoclione nur ein einziger). Sie innervieren die Seitenteile des Rumpfes und die Eingeweide- hülle; vom rechten Ganglion geht ein Nerv zu dem Osphradium, vom linken einer zu Herz und Niere, sowie zu der Zwitterdrüse:; ein anderer versorgt die übrigen Teile des Eingeweidesackes. Bei Halopsyche ist wieder die Zahl der Visceralnerven beträchtlich reduziert; von den Pleurovisceralganglien geht jederseits nur ein einziger Nerv ab, der rechte begibt sich zu dem ÖOsphradium. Das Abdominalganglion gibt nur einen Nerven zu den Eingeweiden ab. Die Buccalnerven innervieren Radulatasche, Hakensäcke und Speichel- drüsen; bei P’neumoderma entspringen jederseits drei Nerven, sowie ein unpaarer an der medianen Berührungsfläche der Ganglien. Bemerkenswert ist, daß zwei Nerven, welche die hinteren Zipfel der Hakensäcke versorgen, schließlich miteinander zu verschmelzen scheinen und so eine Schlinge bilden. Bei Halopsyche kommen gar keine Buccalnerven vor. Von Sinnesorganen unterscheiden wir die zwei Paare von Tentakeln, die paarigen Statocysten und das unpaare ÖOsphradium. — Die vorderen Tentakeln (Fig.72) liegen als kleine Zäpfchen zu beiden Seiten der Schlund- öffnung; sie sind bald kurz und breit, bald lang und spitz und sehr kontraktil. An der Spitze tragen sie ein hohes Flimmerepithel. Der an die Tentakeln herantretende Nerv löst sich hier in eine große Zahl von Nerven- fasern auf. Aalopsyche besitzt ganz abweichend gebaute vordere Tentakeln (Fig. 108 p.141). Hier liegen zu beiden Seiten des Mundes zwei mächtige Ge- bilde, die sich je aus zwei Schenkeln zusammensetzen. Der äußere ist der bei weitem umfangreichere und ventralwärts gekrümmt; der andere ist bedeutend kleiner und liegt median, er ist nach vorn gerichtet. Beide Schenkel stellen zusammen einen gekrümmten, walzenförmigen Körper dar, der auf der Oberfläche vier hell erscheinende, etwas gefaltete Längsleisten trägt; auf diesen Leisten finden sich dicht gedrängte, lange Wimperhaare. Die Ober- fläche ist übrigens von einem hohen Zylinderepithel eingenommen. B. Subord. Gymnosomata Unterhalb jeder Längsleiste verläuft auf dem äußeren Schenkel ein Längsmuskel ; zudem kommt ein fünftes, schwächeres Muskelbündel an der medianen Seite des Tentakels vor; übrigens ist das Innere von beiden Schenkeln ganz von Von Sinnesorganen unterscheiden wir die zwei Paare von Tentakeln, die paarigen Statocysten und das unpaare ÖOsphradium. — Die vorderen Tentakeln (Fig.72) liegen als kleine Zäpfchen zu beiden Seiten der Schlund- öffnung; sie sind bald kurz und breit, bald lang und spitz und sehr kontraktil. An der Spitze tragen sie ein hohes Flimmerepithel. Der an die Tentakeln herantretende Nerv löst sich hier in eine große Zahl von Nerven- fasern auf. Aalopsyche besitzt ganz abweichend gebaute vordere Tentakeln (Fig. 108 p.141). Hier liegen zu beiden Seiten des Mundes zwei mächtige Ge- bilde, die sich je aus zwei Schenkeln zusammensetzen. Der äußere ist der bei weitem umfangreichere und ventralwärts gekrümmt; der andere ist bedeutend kleiner und liegt median, er ist nach vorn gerichtet. Beide Schenkel stellen zusammen einen gekrümmten, walzenförmigen Körper dar, der auf der Oberfläche vier hell erscheinende, etwas gefaltete Längsleisten trägt; auf diesen Leisten finden sich dicht gedrängte, lange Wimperhaare. Die Ober- fläche ist übrigens von einem hohen Zylinderepithel eingenommen. Unterhalb jeder Längsleiste verläuft auf dem äußeren Schenkel ein Längsmuskel ; zudem kommt ein fünftes, schwächeres Muskelbündel an der medianen Seite des Tentakels vor; übrigens ist das Innere von beiden Schenkeln ganz von Pteropoda: B. Gymnosomata 94 einer dichten Zellenmasse (gangliöse Elemente?) erfüllt und wird im Zentrum von einem starken Nerven durchzogen. — Die hinteren Tentakeln (Fig. 72), welche bei Paedoeclione fast gänzlich reduziert erscheinen, sind als sehr kleine Höcker in der Nackengegend, der Dorsalseite genähert, zu finden. An der Spitze tragen sie ein rudimentäres Auge. Bei Halopsyche finden wir hier ein kleines Bläschen, das im Inneren mit einer körnigen Masse (Glaskörper oder Linse?) erfüllt ist und an dessen Innenwandung ein Nerv herantritt. Bei Pneumoderma violaceum ist die Augenkapsel linsenartig ver- breitert und zeigt im Inneren vakuolenartige Räume (Glaskörper), an der Hinterwand sehr hohe, senkrecht zur Oberfläche gestellte Zellen (Retina). Pigment ist nicht mit Sicherheit nachgewiesen worden. Unmittelbar hinter dem hinteren Tentakel liegt ein Streifen hoher Zylinderzellen, an welchen eine kolbenartige Anschwellung des mit dem Tentakelnerven parallel ver- laufenden Nerven herantritt. Vielleicht dient dieses Organ, das man als Rhinophor auffaßt, der Vermittlung von Tastempfindungen. — Die Stato- eysten liegen am hinteren Außenrande der Pedalganglien und enthalten kleine „Mund „... B. Subord. Gymnosomata Saugarın Schlund Mittellappen des Fußes-.._\| N Ki Radulatasche ---L..._______ i: - --... Grenze zwischen Schlund und Ne en Se Buccalmasse Oesophagus ---1------------\-- an Oberkiefer BERET r ‘Ber; 7 Grenze zwischen Buccalmasse und Diaphragma ----"\V Fr I BE E Oesophagus -._. . Oesophagus N nn Ge - Hakensack Magen- und Lebersack -—" >» Grenze zwischen Oesophagus und Magen Fig. 73. Sagittalschnitt durch den vorderen Körperabschnitt von Spongiobranchia australis, mit einge- stülptem Schlundapparat. Nach Meisenheimer. x Saugarın Schlund Ki Grenze zwischen Schlund und Buccalmasse Oberkiefer Grenze zwischen Buccalmasse und Oesophagus Oesophagus Magen Fig. 73. Sagittalschnitt durch den vorderen Körperabschnitt von Spongiobranchia australis, mit einge- stülptem Schlundapparat. Nach Meisenheimer. x Statolithen. — Das Osphradium liegt auf der rechten Seite des vorderen Rumpfabschnittes, der Ventralseite genähert. Meist stellt es eine gekrümmte Wimperleiste dar, deren konkave Seite nach außen gerichtet ist; bei Thliptodon ist es eine rundliche Scheibe, bei Cliopsis dagegen ganz gerade gestreckt. An der Oberfläche finden sich Flimmerzellen, darunter ein längliches Ganglion. Am Darmkanal ist der Vorderdarm außerordentlich kompliziert gebaut. Statolithen. — Das Osphradium liegt auf der rechten Seite des vorderen Rumpfabschnittes, der Ventralseite genähert. Meist stellt es eine gekrümmte Wimperleiste dar, deren konkave Seite nach außen gerichtet ist; bei Thliptodon ist es eine rundliche Scheibe, bei Cliopsis dagegen ganz gerade gestreckt. An der Oberfläche finden sich Flimmerzellen, darunter ein längliches Ganglion. Am Darmkanal ist der Vorderdarm außerordentlich kompliziert gebaut. Dieser Vorderdarm (Fig. 73) zerfällt in drei große Hauptabschnitte: Schlund, Buccalmasse und Oesophagus. Der Schlund reicht von der äußeren Mund- öffnung bis zum Eingang zu der eigentlichen Bueealhöhle; als Differenzierungen ihrer Wandung gehören die Saugarme der Pneumodermatiden und. die Buccalkegel der Clioniden hierher. Die Buecalmasse schließt sich unmittelbar an den Schlund an und endet bei der Austrittsstelle des Oesophagus; sie enthält Oberkiefer, Radulatasche, Hakengruben und Hakensäcke und die Mündungen der Speicheldrüsen. Dorsalwärts von der Buccalmasse geht der dünne Öesophagus ab, der in den Magen überführt. — Der Schlund der Pneumodermatiden bildet einen muskulösen, im kontrahierten Zustande stark gefalteten Schlauch, der eine äußere Längsmuskellage und eine stärkere Am Darmkanal ist der Vorderdarm außerordentlich kompliziert gebaut. Dieser Vorderdarm (Fig. 73) zerfällt in drei große Hauptabschnitte: Schlund, Buccalmasse und Oesophagus. Der Schlund reicht von der äußeren Mund- öffnung bis zum Eingang zu der eigentlichen Bueealhöhle; als Differenzierungen ihrer Wandung gehören die Saugarme der Pneumodermatiden und. die Buccalkegel der Clioniden hierher. B. Subord. Gymnosomata Die Buecalmasse schließt sich unmittelbar an den Schlund an und endet bei der Austrittsstelle des Oesophagus; sie enthält Oberkiefer, Radulatasche, Hakengruben und Hakensäcke und die Mündungen der Speicheldrüsen. Dorsalwärts von der Buccalmasse geht der dünne Öesophagus ab, der in den Magen überführt. — Der Schlund der Pneumodermatiden bildet einen muskulösen, im kontrahierten Zustande stark gefalteten Schlauch, der eine äußere Längsmuskellage und eine stärkere Pteropoda: B. Gymnosomata 95 innere Ringmuskelschicht besitzt. Sie kann weit hervorgestülpt werden. Bei den Clioniden und Thliptodontiden, auch bei Notobranchaea und Halopsyche-ist der Schlund stark reduziert und kann nicht hervorgestülpt werden; bei den letztge- nannten Gattungen ist sogar der ganze Schlund fast völlig verschwunden, bei den Clioniden ist nur die Muskulatur stark zurückgebildet. Dagegen ist bei Cliopsis der Schlund außerordentlich lang und in zahlreiche Schlingen gelegt; er nimmt hier fast die Hälfte des ganzen Eingeweidesackes für sich in Anspruch. Der aufgeknäuelte Abschnitt ist an der Oberfläche in regelmäßigen Abständen von hellen Ringen überzogen, welche dadurch entstehen, daß das innere Drüsen- epithel des eingestülpten Schlundes sich in zahlreiche Querfalten legt. — Meist finden sich an der seitlichen Schlundwandung die für die Gymnosomata charakteristischen und systematisch sehr wichtigen Greiforgane entwickelt (Fig. 73). Bei den Clioniden und bei T’halassopterus finden wir die Buccal- kegel, die als konische, zapfenförmige Gebilde zu 1 bis 3 Paaren an der seitlichen inneren Schlundwandung ihren Ursprung nehmen. Diese Kegel sind an der Oberfläche von Sinneszellen, welehe mit Nervenendigungen in Verbindung stehen, besetzt; im Innern sind zahlreiche, in Follikel angeordnete Drüsenzellen vorhanden. Die Buccalkegel können an ihrer Oberfläche eine klebrige Flüssigkeit abscheiden, die das Ergreifen der Beute erleichtert. — In derselben Lagerung wie die Buccalkegel der Clioniden finden wir die Saugarme der Pneumodermatiden. Bei /’neumodermopsis sind deren drei vorhanden, ein unpaarer auf der Ventralseite in der Medianebene und zwei seitliche; bei den übrigen Gattungen dieser Familie finden sich nur die seitlichen Saugarme. Sie zeigen sehr verschiedene Gestalt und eine große Mannigfaltiekeit in der Anordnung der Saugnäpfe, mit welchen die Ränder der Arme besetzt sind. Die Saugarme von Prneumoderma mediterraneum sind breit und plattenförmig und tragen an ihrem Rande eine Reihe von etwa 6 oder 7 Saugnäpfen von verschiedener Größe. Der Saugarm selbst ist dünnwandig und besteht aus einem Plattenepithel und einer darunter gelegenen, hauptsächlich in der Längsrichtung orientierten Muskelfaserschicht; das Innere wird vom Bindegewebe erfüllt; auch verläuft hier ein Blutgefäß, das zu jedem Saugnapfe einen Ast abspaltet. B. Subord. Gymnosomata Im Saugnapfe selbst öffnet sich dieser Ast in die unmittelbar unter der Saugscheibe gelegenen Lakunen- räume. Im Innern des Stieles finden sich große, sternförmig verästelte Bindegewebszellen. Die Grundlage der Saugscheibe „wird durch eine aus radiär zur Oberfläche gestellten Muskelzellen sich zusammensetzende Platte gebildet, die nach außen von dem Epithel überkleidet wird, nach innen durch eine starke Muskelplatte mit dem Stiele in Verbindung steht“ (Meisenheimer). Die innere Radiärmuskelscheibe besteht aus hohen, senkrecht zur Oberfläche gerichteten, prismatischen Elementen, welche im Querschnitt im Zentrum ein helles Protoplasma und einen großen Kern enthalten, an der Peripherie aber von einem Kranze drei- oder vierseitiger, dicht aneinander gepreßter Muskel- fasern umgeben sind. Das Epithel des Stieles schlägt sich am Rande der Saugscheibe um und wandelt sich hier, in Gegensatz zu den abgeplatteten Zellen, die es bisher zusammensetzten, zu einem aus hohen Zylinderzellen gebildeten, mehrschichtigen Epithel um, das an seiner Außenfläche einen hohen, fast ganz durchsichtigen Cuticularsaum trägt, der sich als dünnes Häutchen auf die Saugscheibe selbst fortsetzt. In dem Zylinderepithel des Außenrandes finden sich viele flaschenförmige Drüsenzellen, die mit ihrem langen, dünnen Endstück den hyalinen Saum durchsetzen. Am Rande der inneren Radiärmuskelscheibe finden sich drei Ringmuskelsysteme, zwei an der Oberseite und ein drittes an der Unterseite. Eine aus radialen Fasern zu- Pteropoda: B. Gymnosomata 96 sammengesetzte Muskelplatte liegt unterhalb der Saugscheibe. Die Funktion der Saugnäpfe besteht darin, daß die Ränder fest an eine Unterlage angepreßt werden und dann die innere Saugscheibe durch Kontraktion der Muskeln sich nach innen vorwölbt und einen luftverdünnten Raum entstehen läßt. — Bei den übrigen Formen liegt meist eine große Vereinfachung im Bau des Saugnapfes vor, welche sich namentlich in der Randzone geltend macht, wo meist das zylindrische Drüsenepithel fehlt; dagegen sind die inneren Radiär- muskeln, besonders bei Pneumodermopsis, sehr gut ausgebildet. Bei Spongio- branchia findet sich auf dem Epithel der oberen Saugfläche ein stark vor- springender Ring, der als Verdiekung der Cutieula aufzufassen ist. — Auf der Ventralseite des Schlundes liegt bei Pneumoderma und Spongiobranchia eine bald kürzere, bald längere, zipfelartige Papille (Fig. 72). — Die Buccalmasse schließt sich unmittelbar dem Schlunde an, und zwar unter starker Verengung des inneren Lumen, welche Verengung bei den Pneu- modermatiden durch vorspringende Wülste hervorgerufen wird, die ventralen Wülste sind von einer dicken Cuticula überzogen. Diese Gebilde finden sich wahrscheinlich in Form der sogenannten „falschen Lippen“, welche an der Basis der Buccalkegel als zwei seitliche Wülste an der Schlundwandung liegen, bei den Clioniden wieder. B. Subord. Gymnosomata Bei Aalopsyche fehlen sie völlig. — Im Innern der Bucealhöhle liegt der Oberkiefer (Fig. 73), auf der Ventralseite, unmittelbar vor der Radulatasche, nur bei den meisten Olioniden fehlt er völlig. Er bildet ein bald drei-, bald viereckiges Plättchen, welches auf seiner Oberfläche mit stachelartigen Gebilden überdeckt ist und in der Mittellinie einen Spalt trägt; bisweilen besteht er aus einer Querreihe kleiner Zähnchen. Bei Halopsyche ist der Oberkiefer dorsal gelegen und durch einen sehr tiefen Spalt in zwei Teile zerlegt, welche sich aus hohen Zylinderzellen zusammensetzen; auf der Oberfläche finden sich 1—3 Längsreihen horniger, am Rande gezähnter Platten. Bei Notobranchaea besteht der Oberkiefer nur aus einer einzigen (Querreihe von 15 Zähnchen; bei den Thliptodontidae fehlt er gänzlich. — Die Radulatasche nimmt hinter dem Oberkiefer den ganzen ventralen Abschnitt der Buccal- höhle ein. Die Radula besteht in jeder Querreihe aus einem Mittelzahn (bei den erwachsenen Pneumoderma-Arten fehlend) und einer größeren Zahl von Seitenzähnen. Die Mittelplatte ist bald klein, bald umfangreich und am Vorderrande gezähnelt; die Seitenzähne bestehen aus einer großen Basalplatte und einem zahnartigen Fortsatz. Bei den Thliptodontidae kommt eine ganz andere Radula, wie diejenige der Heteropoden, mit Formel 3:1'1'1°3, vor; hier sind Mittel-, Zwischen- und Seitenplatten zu unterscheiden. Bei Halopsyche finden sich in jeder Querreihe nur drei Zähne. — Die Haken- säcke sind, wie die Saugarme, für die Gymnosomata charakteristisch und ebenfalls von hoher systematischer Bedeutung. Mit Ausnahme von Halopsyehe werden sie bei allen Gattungen beobachtet. Sie bilden an den Seiten- wandungen der Bucealhöhle zwei bald mehr (PPneumoderma, Spongiobranchia, Olione, Thliptodon), bald weniger (Pneumodermopsis, Cliopsis, Notobranchaea, Paedoelione) tiefe Aussackungen von sehr verschiedener Länge und besitzen im Inneren starke, gekrümmte und durch ein mächtiges Muskelpolster gestützte Haken. Besonders bei Pneumoderma mediterraneum erreichen sie eine ansehnliche Länge. Wo sie gut ausgebildet und lang sind, sind die Hinterenden gewöhnlich miteinander oder mit der Buccalmasse mittels Binde- gewebsfasern vereinigt. An dem Hakensack unterscheiden wir eine äußere, nicht ausstülpbare Muskelhülle (welche bei Clione eine äußere mächtige Ringmuskulatur und eine innere Längsmuskulatur besitzt. bei Pneumoderma und Spongiobranchia dagegen in der Lage dieser Muskeln gerade das Pteropoda: B. Gymnosomata 97 entgegengesetzte Verhalten aufweist) und eine innere, ausstülpbare Scheide, welche die Haken enthält. Am Ende dieser inneren Scheide inseriert sich ein Retraktormuskel, der am Grunde der äußeren Muskelscheide entspringt; weiter wird die Scheide durch den sogenannten Scheidenmuskel umkleidet. Der Raum zwischen innerem’ und äußerem Muskelschlauch ist von einer hyalinen Flüssigkeit erfüllt. B. Subord. Gymnosomata Im Innern der inneren Scheide findet sich das Epithel, das die Haken trägt; dieselben sind meist auf der medianen Seite der Scheide eingepflanzt und in regelmäßig übereinander gelegenen Quer- reihen angeordnet. Die innere Hakenscheide kann wie ein Handschuhfinger nach außen vorgestülpt werden, durch den Druck, welchen der sich kontra- hierende äußere Muskelschlauch auf die in seinem Innern enthaltene Flüssig- keit ausübt. Dadurch kommen Scheiden- und Retraktormuskel in die Achse der ausgestülpten, inneren Scheide zu liegen; bei dem Zurückziehen üben diese Muskeln dann ihre Wirkung aus. Bei Schizobrachium ist der Haken- sack äußerlich gut entwickelt, innerlich aber besteht er zum größten Teile aus einem mächtigen Muskelpolster und die innere Scheide ist nur als kleine, flache Grube entwickelt. Bei ZiAlptodon ist ein System von radiär vom Grunde der Hakenscheide an die hinteren Seitenwände der äußeren Muskel- scheide ausstrahlenden Muskelfasern entwickelt. Bei Thalassopterus kommt vom ganzen Hakensack nur noch die äußere Muskelscheide vor. — Weitere Differenzierungen in der Wandung der Bucealhöhle finden sich bei den Thliptodontidae. Hier ist das Vorkommen sehr eigentümlicher, blasiger Gebilde charakteristisch, welche als große, prall gefüllte, nach allen Seiten abgeschlossene Blasen (Schlundblasen) in der äußeren Wandung von Schlund und Buecalmasse eingebettet sind. Vielleicht kommen sie auch bei Notobranchaea inopinata vor. Bei Thalassopierus bildet zudem die dorsale Wand der Buccalhöhle jederseits eine mächtige Querfalte von ausgesprochen drüsiger Natur (Buccaldrüsenlappen, Fig. 107, p. 139); sie scheinen die Funktion der sehr reduzierten Speicheldrüsen übernommen zu haben. — Die Speicheldrüsen, zu jeder Seite der Buccalmasse, stellen einen langen Drüsenschlauch dar, der durch einen besonderen Ausführungsgang (nicht bei Thliptodon) in die Buccalhöhle ausmündet. Die Schläuche sind, mit Ausnahme von Thliptodon und Halopsyche, an ihren Hinterenden auf der Ventralseite des Schlundes miteinander verbunden (wenigstens bei Pneumoderma und Spongiobranchia); hier geht das Drüsengewebe ineinander über. Bei Pneu- moderma violaceum verschmelzen die beiden sehr engen Ausführungsgänge schließlich miteinander und münden gemeinsam unmittelbar hinter dem Ober- kiefer; bei Cliopsis bleiben die Ausführungsgänge in ihrem ganzen Verlaufe getrennt. Die ventrale Wandung der Buccalhöhle ist häufig von einzelligen Drüsen bedeckt. — Die Buccalhöhle führt in den Oesophagus, der weit trichterförmig an der Dorsalseite der Buccalmasse entspringt (Fig. 73), sich bald zu einem dünnen Rohre verengt, dann eine große Schlinge proximal- wärts auf der Ventralseite bildet, um dann wieder zurückzubiegen und unter Durchbrechung des Diaphragmas sich in den Magen zu öffnen. Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. B. Subord. Gymnosomata Bei Uliopsis ist der Oesophagus außerordentlich lang; bei Aalopsyche, infolge des Fehlens eines besonderen Schlundes, sehr kurz, er zieht als leicht gebogenes, dünnes Rohr asymmetrisch auf der linken Seite des Körpers nach hinten. Der Mitteldarm setzt sich aus Magen und Leber zusammen, beide sind, was für die Gymnosomata typisch ist, zu einem einheitlichen, weiten Sacke (Fig. 73) verschmolzen. Außerlich ist der Magen-Lebersack (Fig. 73) vielfach gelappt und gefaltet; dieses Aussehen wird durch die Leberdrüse verursacht. Nur an der Einmündungstelle des Oesophagus in den Magen Der Mitteldarm setzt sich aus Magen und Leber zusammen, beide sind, was für die Gymnosomata typisch ist, zu einem einheitlichen, weiten Sacke (Fig. 73) verschmolzen. Außerlich ist der Magen-Lebersack (Fig. 73) vielfach gelappt und gefaltet; dieses Aussehen wird durch die Leberdrüse verursacht. Nur an der Einmündungstelle des Oesophagus in den Magen 7 Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. Pteropoda: B. Gymnosomata 98 sowie an der Austrittsstelle des Enddarmes wird das Drüsenepithel des Sackes von einem kubischen Flimmerepithel abgelöst. Bei Halopsyche finden sich nur einige wenige Lappen, einer auf der rechten Seite und zwei am Vorder- rande des Sackes. Auch fehlt hier wahrscheinlich die sonst so allgemein vorkommende Flimmerung auf den großen Leberzellen. Zudem kommt hier ein ganz besonderes Gebilde vor, nämlich ein rundliches, dorso-ventral ab- geplattetes Bläschen auf der Dorsalseite des Magen-Lebersackes, das mit einer feinen, spaltförmigen Öffnung genau an der Austrittsstelle des Enddarmes aus dem Magen einmündet; vielleicht ist es mit dem Darmblindsack der Theco- somata vergleichbar. Kauplatten findet man niemals im Magen ausgebildet. Auf der proximalen Hälfte des Magen-Lebersackes, und zwar auf dessen rechter Ventralseite, entspringt der Enddarm, der meist als dünnes Rohr direkt auf der rechten Ventralseite des Körpers, in fast gerader Richtung nach vorn zieht, in seinem Verlaufe eine dicke, aber dünnwandige Auftreibung zeigt und schließlich durch den Anus, ebenfalls auf der rechten Ventralseite des Körpers ausmündet. Meist liegen in der Nähe des Afters äußere Nieren- öffnung und Ösphradium dicht beisammen auf dem sogenannten Analfeld. Eine Analdrüse kommt oft vor. Der Anfangsteil des Enddarmes weist in seinem Inneren zahlreiche Längsfalten der Wandung auf; dieselben treten auch nach der Anschwellung wieder zum Vorschein. Bei Clione ist die Auf- treibung kaum angedeutet. Bei Chopsis fehlt sie gänzlich; ' hier ist zudem der Enddarm enorm in die Länge gezogen und verläuft als einfaches, sehr stark abgeplattetes Rohr nach vorn, um am Ende in scharfem Winkel nach rechts auszubiegen und das Analfeld zu erreichen. B. Subord. Gymnosomata Der After liegt stets auf der proximalen, rechten Ventralseite des Körpers, zuweilen (Schizobrachium, Notobranchaea, Clione) ist er weiter nach hinten verschoben und dann weiter von der äußeren Nierenöffnung und vom Osphradium entfernt. Bei Halopsyche entspringt der Enddarm nicht auf der Ventral-, sondern auf der Dorsalseite des Magens; die Ursprungsstelle liegt fast am Hinterrande des Magens, etwas rechts; von hier beschreibt der Darm eine leichte Krümmung nach rechts, dann wieder ganz nach links hinüber, und biegt schließlich in ziemlich scharfem Winkel wieder nach der rechten Seite um, wo er ziemlich weit vorn durch den After ausmündet. In den äußersten Schenkel des Enddarmes mündet nun ein eigentümliches Anhangsgebilde, das sich als weiter Sack auf der rechten Seite des Eingeweidesackes weit nach hinten erstreckt. Dieser Sack ist im Leben von einer rötlich-gelb durchscheinenden Flüssigkeit erfüllt; die Wandung besteht aus einem sehr stark abeeflachten Epithel, nur in der Umgebung der Einmündungsstelle in den Enddarm erhebt sich ein stark bewimperter Ringwulst. Das Herz liegt in einem weiten, dünnwandigen Pericard, auf der sowie an der Austrittsstelle des Enddarmes wird das Drüsenepithel des Sackes von einem kubischen Flimmerepithel abgelöst. Bei Halopsyche finden sich nur einige wenige Lappen, einer auf der rechten Seite und zwei am Vorder- rande des Sackes. Auch fehlt hier wahrscheinlich die sonst so allgemein vorkommende Flimmerung auf den großen Leberzellen. Zudem kommt hier ein ganz besonderes Gebilde vor, nämlich ein rundliches, dorso-ventral ab- geplattetes Bläschen auf der Dorsalseite des Magen-Lebersackes, das mit einer feinen, spaltförmigen Öffnung genau an der Austrittsstelle des Enddarmes aus dem Magen einmündet; vielleicht ist es mit dem Darmblindsack der Theco- somata vergleichbar. Kauplatten findet man niemals im Magen ausgebildet. Auf der proximalen Hälfte des Magen-Lebersackes, und zwar auf dessen Auf der proximalen Hälfte des Magen-Lebersackes, und zwar auf dessen rechter Ventralseite, entspringt der Enddarm, der meist als dünnes Rohr direkt auf der rechten Ventralseite des Körpers, in fast gerader Richtung nach vorn zieht, in seinem Verlaufe eine dicke, aber dünnwandige Auftreibung zeigt und schließlich durch den Anus, ebenfalls auf der rechten Ventralseite des Körpers ausmündet. Meist liegen in der Nähe des Afters äußere Nieren- öffnung und Ösphradium dicht beisammen auf dem sogenannten Analfeld. Eine Analdrüse kommt oft vor. Der Anfangsteil des Enddarmes weist in seinem Inneren zahlreiche Längsfalten der Wandung auf; dieselben treten auch nach der Anschwellung wieder zum Vorschein. Bei Clione ist die Auf- treibung kaum angedeutet. B. Subord. Gymnosomata Besondere Venengefäße scheinen nicht vorzukommen. Die Respiration erfolgt durch Kiemen und durch die Haut; nur Haut- atmung kommt bei den Clioniden und bei HZalopsyche vor. Kiemen finden sich an der rechten Körperseite und am unteren Pol des Körpers (Fig. 72, p. 91). Die erste, die Seitenkieme, stellt einen dreieckigen Vorsprung dar, etwa auf der halben Länge des Rumpfes, die Basis ist nach vorn, die abgerundete Spitze nach hinten gerichtet. Drüsenzellen und -blasen fehlen in ihrem Bereiche völlig. Bei Pneumoderma (Fig. 72, p. 91) verlaufen vom freien Außenrande drei Längskämme nach vorn, von welchen die beiden seitlichen, ebenso wie der freie Rand selbst, von Fransen bedeckt sind. Die Wandung dieser Kieme ist sehr dünn und besteht aus einem flachen Epithel, während das Innere von weitmaschigem Bindegewebe erfüllt ist; das Lakunensystem wird nach innen durch die vorgelagerte Niere abgeschlossen. Die Endkieme entwickelt sich aus der dritten, larvalen Wimperschnur, sie umgibt bei Pneumoderma das Hinterende des Körpers als viereckige Falte, von deren "Ecken vier radiär nach vorn verlaufende und sich allmählich verjüngende Seitenstrahlen abgehen. Die Seitenränder dieser Radiärfalten, sowie auch das Viereck selbst, sind größtenteils von Fransen bedeckt. Der Bau dieser Endkieme ist ähnlich dem der Seitenkieme. — Bei den anderen Gattungen ist die Seitenkieme einfacher gebaut (Spongiobranchia, Preumodermopsis) oder fehlt gänzlich (Schizobrachium, Cliopsis, Notobranchaea); die Endkieme kann als regelmäßiges Sechseck entwickelt sein (Cliopsis) oder bildet eine dreistrahlige Figur (Notobranchaea); sie kann auch eine kleine, ventral gelegene Hautfalte darstellen (Schizobrachium) oder eine vorspringende Ringfalte bilden (Spongiobranchia). Nebst den Schlundorganen sind die Kiemen von großer systematischer Bedeutung. Die Niere liegt bei Halopsyche als einfacher Schlauch auf der Dorsal- seite des Eingeweidesackes, hinter dem Herzen, das sie mit zwei Schenkeln umgibt; äußere Öffnung und Renopericardialöfinung finden sich stets nahe zusammen auf der rechten Körperseite, und zwar in der Nähe des Afters. — Bei den anderen Gattungen liegt die Niere auf der rechten Körperseite als weiter, dünnwandiger Sack, der in der Nähe des Analfeldes ausmündet und in der Höhe des Pericard, welchem er rechts anliegt, mit diesem kommuni- ziert. Die Niere ist meist nicht gekrümmt, sondern mehr gestreckt, nur bei Paedoclione ist sie ein langer, schleifenartig gebogener Schlauch; die Öffnungen liegen meist weit auseinander, bei Z’hliptodon aber liegen sie nahe zusammen am vorderen Ende. B. Subord. Gymnosomata Bei Chopsis fehlt sie gänzlich; ' hier ist zudem der Enddarm enorm in die Länge gezogen und verläuft als einfaches, sehr stark abgeplattetes Rohr nach vorn, um am Ende in scharfem Winkel nach rechts auszubiegen und das Analfeld zu erreichen. Der After liegt stets auf der proximalen, rechten Ventralseite des Körpers, zuweilen (Schizobrachium, Notobranchaea, Clione) ist er weiter nach hinten verschoben und dann weiter von der äußeren Nierenöffnung und vom Osphradium entfernt. Bei Halopsyche entspringt der Enddarm nicht auf der Ventral-, sondern auf der Dorsalseite des Magens; die Ursprungsstelle liegt fast am Hinterrande des Magens, etwas rechts; von hier beschreibt der Darm eine leichte Krümmung nach rechts, dann wieder ganz nach links hinüber, und biegt schließlich in ziemlich scharfem Winkel wieder nach der rechten Seite um, wo er ziemlich weit vorn durch den After ausmündet. In den äußersten Schenkel des Enddarmes mündet nun ein eigentümliches Anhangsgebilde, das sich als weiter Sack auf der rechten Seite des Eingeweidesackes weit nach hinten erstreckt. Dieser Sack ist im Leben von einer rötlich-gelb durchscheinenden Flüssigkeit erfüllt; die Wandung besteht aus einem sehr stark abeeflachten Epithel, nur in der Umgebung der Einmündungsstelle in den Enddarm erhebt sich ein stark bewimperter Ringwulst. Das Herz liegt in einem weiten, dünnwandigen Pericard, auf der Das Herz liegt in einem weiten, dünnwandigen Pericard, auf der rechten Körperseite, im hinteren Drittel des Rumpfes zwischen Integument und Eingeweidesack. Der proximale Zipfel des Pericard kommuniziert durch einen Pericardialnierengang mit der Niere. Die Längsachse des Herzens liegt etwas schräg zur Körperachse; der Vorhof ist nach hinten, die Kammer nach vorn gerichtet. Auf der Grenze zwischen beiden ist eine Klappen- vorrichtung, bestehend aus zwei trichterförmig ins Innere der Kammer vor- springenden Falten; eine weitere Klappe findet sich an der Abgangsstelle der arteriellen Blutgefäße. Nur bei Zalopsyche liegt das Herz nicht auf der rechten Seite, sondern auf der Dorsalseite, genau in der Medianebene des vorderen Rumpfabschnittes; die Längsachse des Herzens ist quer zur Körper- achse geneigt: der Vorhof ist nach rechts, die Kammer nach links gerichtet. — Nur der Anfangsteil des Gefäßsystemes besteht aus geschlossenen Bahnen. Pteropoda: B. Gymnosomata 99 Die mächtige Aorta, die aus dem nach vorn gerichteten Ende der Herz- kammer entspringt und die Organe des vorderen Kopfabschnittes sowie die Flossen versorgt, gibt gleich an der Abgangsstelle ein kleineres Gefäß in seitlicher Richtung ab, das direkt in den Eingeweidesack eindringt. Die Gefäße gehen schließlich in die lakunären Hohlräume des Körpers über. B. Subord. Gymnosomata Der Pericardialnierengang scheint besonders bei Ulione limacina sehr stark als richtiger Gang ausgebildet zu sein; auch bei /’neumoder- mopsis und Spongiobranchia kommt etwas ähnliches vor, während die Kommu- nikation mit dem Pericard bei Pneumoderma und Cliopsis nur einen kurzen Schlauch darstellt. Die Geschlechtsorgane nehmen die Ventralseite des Eingeweide- nucleus ein; sie bestehen aus Zwitterdrüse, Zwittergang, Genitalanhangsdrüsen und Ausführungsgängen, zudem aus einem besonderen Begattungsapparat. — Die Zwitterdrüse nimmt den hinteren Abschnitt des Eingeweidesackes ein und greift als dünne Überkleidung über die Ventralseite des Magen-Leber- sackes nach vorn hin über. Sie setzt sich aus zahlreichen einzelnen Follikeln 7#* 7#* Pteropoda: B. Gymnosomata 100 zusammen; die weiblichen Geschlechtsprodukte liegen an der Peripherie der Zwitterdrüse, die Spermatozoiden werden im Innern erzeugt. Zuerst reifen, ebenso wie bei den Thecosomata, die männlichen Produkte. Nach dem Zentrum der Zwitterdrüse hin gehen die Follikel allmählich in weite Hohl- räume über, die schließlich sich zu einem in der Mitte des Organes ver- laufenden, bewimperten Gange vereinigen, dem Zwittergang. — Am Vorder- rande der Zwitterdrüse tritt derselbe hervor und wendet sich unter starken Windungen nach vorn. In der Mitte seines Verlaufes kommt zuweilen eine bauchige Auftreibung vor, eine Art Vesicula seminalis, nur bei Halopsyche tritt dieselbe als kleine, seitliche Aussackung an der Austrittsstelle aus der Zwitterdrüse auf. — Der Zwittergang führt nach den Genitalanhangs- drüsen, welche sehr kompliziert sind; sie liegen am ventralen Vorderrande des Eingeweidenucleus, meist der rechten Körperseite etwas genähert. Bei Thliptodon bildet der Zwittergang, bevor er die Anhangsdrüsen aufnimmt, zwei mächtige Anschwellungen, kurz hintereinander; dieser Teil wird durch eine sehr dicke Schicht, aus hohen, zylindrischen Epithelzellen und mächtiger Muskelmasse bestehend, umhüllt und stellt wahrscheinlich eine Art Ejaculations- organ dar. Der Zwittergang erreicht die Anhangsdrüsen an deren rechtem kande und begibt sich nach ihrer Dorsalseite. Hier schnürt er sich plötzlich stark ein und schwillt dann wieder beträchtlich an; dann geht er in den. Ausführungsgang über. Gerade an dieser Stelle öffnen sich die Anhangsdrüsen. * Diese setzen sich aus zwei großen Drüsenkomplexen zusammen, die vielfach ineinander geknäuelt erscheinen; der umfangreichere Komplex bildet die Schalendrüse, welche einen langen, aufgewundenen, platten Schlauch dar- stellt, dessen beide Enden mit dem Ausführungsgang in Verbindung stehen; der kleinere ist die Eiweißdrüse, welche meist zwischen den ventralen Schlingen der Schalendrüse eingeschlossen ist. Ihre Offnung liegt da, wo die Wandung der Schalendrüse sich in diejenige der eigentlichen Ausführungsgänge fortsetzt. B. Subord. Gymnosomata In diesen Vorraum mündet bei Z’hliptodon links ein langer Drüsenschlauch, der im Innern durch eine vorspringende Längsfalte in zwei Teile zerlegt wird, von denen der eine mit Drüsenepithel, der andere mit Flimmerzellen ausgekleidet ist. Ein derartiger Drüsenschlauch, mit mehr einheitlichem Lumen, findet sich auch bei T’halas- sopterus und zwar auf der rechten Seite. Er ist wohl als Prostata des eigent- lichen Penis aufzufassen. Die Wand des Vorraumes ist bei letztgenannter Gattung und bei Clione und Paedoclione von sehr großen, vakuolisierten Zellen bedeckt, welche bisweilen eine besondere Aussackung an der Wand bilden und eine besondere Prostatadrüse darstellen. Der eigentliche Penisschlauch ist gewunden; an seinem blinden Ende mündet bei Paedoclione eine zweite Prostata (die erste mündet in den Vorraum) ein. Alle diese Bildungen erreichen, so- lange das Individuum im männlichen Stadium verkehrt, eine hohe Ausbildung und bilden sich nachher zurück. — Nur bei T’hliptodon kommt weiter auf der Ventralseite des Körpers, etwas rechts von der Medianebene, und zwar auf der Höhe der Flossen ein eigentümliches keulenförmiges, nach innen in einen schlauchförmigen Anhang auslaufendes Gebilde vor, das eine besondere Öffnung nach außen besitzt. Der weite Vorraum dieses Gebildes wird von sehr hohen Zylinderzellen ausgekleidet; der Anhang wird von großen Drüsenzellen ge- bildet; beide Teile sind gegeneinander völlig abgeschlossen. Wahrscheinlich wird bei der Begattung der weite Vorraum wie ein Handschuhfinger um- gestülpt und dient als Saugorgan. Übrigens wird neuerdings von Kwietniewski bei Thalassopterus ein ähnliches Gebilde von überstimmender aber etwas ver- einfachter Struktur beschrieben, das aber nicht gesondert, sondern von der linken Seite her in den Penissack mündet. Die Gymnosomata sind wahre Räuber, die sich mit Hilfe ihrer ausstreckbaren Hakensäcke und Saugarme ihrer Beute bemächtigen und sie mit der gut entwickelten Radula zerkleinern. Sie ernähren sich nieht nur von kleineren Tieren verschiedener Art, sondern greifen auch die oft größeren Thecosomata an; so werden Cavolinia- Arten im Magen von Pneumoderma gefunden. Mehr als die 'Thecosomata sind die Gymnosomata als Tagtiere zu bezeichnen; sie werden, wenngleich sie fast immer, mit Ausnahme von Clione limacina im hohen Norden, seltene Tiere sind und nie in größeren Schwärmen aufzutreten scheinen, in oberflächlichen Schichten ebenso gut am Tage wie in der Dämmerung und nachts gefangen. — Die befruchteten Eier werden stets, mit Aus- nahme von Halopsyche, frei ins Wasser abgelegt. B. Subord. Gymnosomata Ein Receptaculum seminis liegt bei Pneumoderma als stark pigmentiertes, langgestrecktes Gebilde an dem ventralen Vorderrande der Schalendrüse und mündet weiter proximal in den Ausführungsgang. Bei Chopsis bildet es ein umfangreiches, aus zwei getrennten Schläuchen bestehendes Organ, die beiden Teile öffnen sich gesondert. Bei Aualopsyche findet sich gleich vor der Stelle, wo der Zwittergang in den Ausführungsgang übergeht und die An- hangsdrüsen aufgenommen werden, ein kleines Gebilde, das mit zwei Gängen sich in den Zwittergang öffnet; vielleicht haben wir hier ein Receptaculum seminis vor uns. Die Anhangsdrüsen selbst sind bei Halopsyche zwei kleine, getrennte Drüsen; die kleinere und distale stellt vielleicht die Eiweißdrüse, die größere und proximale die Schalendrüse vor, welche sich aber nicht, wie es sonst der Fall ist, in histologischer Hinsicht unterscheiden. — Der Aus- führungsgang bildet meist die direkte Fortsetzung des Zwitterganges und der Schalendrüse und nimmt das Receptaculum seminis auf. Er verengt sich beträchtlich und zeigt im Innern eine charakteristische Längsfalte. Ganz auf der rechten Körperseite verlaufend, mündet der Gang hier auf der Höhe der rechten Flosse in einer Hautfalte nach außen; hier wird eine Art Begattungstasche gebildet, welche besonders bei T’hliptodon sehr gut ent- wickelt ist. Jenseits dieser Öffnung, welche als weibliche Geschlechtsöffnung aufzufassen ist, da hier die Eier direkt nach außen entleert werden, setzt sich nun ein Teil des Ausführungsganges in einer Rinne fort, welche an der rechten Körperseite nach vorn verläuft (Fig. 72, p. 91) und an der Mündung des Penis endet. Die Rinne ist anfangs ziemlich tief, allmählich verflacht sie sich aber und bildet schließlich nur einen Wimperstreifen. Sie dient Pteropoda: B. Gymnosomata 101 zur Leitung des Samens. Sehr eigentümlich ist der Umstand, daß bei Halopsyche, solange das Individuum noch als Männchen funktioniert, der Aus- führungsgang überhaupt keine Öffnung nach außen hat, sondern als geschlossenes Rohr unter der Haut nach dem Penis zieht, und erst nach Eintritt der weib- lichen Reife die weibliche Geschlechtsöffnung sich bildet, welche mittels einer Rinne mit der Öffnung des Penis verbunden ist. — Der Penis ist sehr- umfangreich und oft recht kompliziert gebaut. Er liegt quer auf der Ventral- seite des Schlundes und mündet auf der rechten Körperseite nahe dem Vorder- rande des rechten Seitenlappens des Fußes; das Ganze stellt einen muskulösen Schlauch dar, der sich wie ein Handschuhfinger umzustülpen imstande ist; das Hinterende fungiert als Samenreservoir. In eingestülptem Zustande ist der vordere Teil oft weit und dient als eine Art Vorraum. B. Subord. Gymnosomata Aus den Eiern entwickeln sich die Trochophora-Larven, welche bald eine gestreckte Form annehmen und ein großes, zwei- lappiges Velum am Vorderende zeigen; das Hinterende steckt in einer geraden, anfangs napf-, später eiförmigen Schale, die zu einem Rohre auswächst. Zuwachsstreifen sind auf dieser Schale deutlich zu sehen; das Ende ist kolbenartig angeschwollen. Der Mund liegt zwischen den beiden Segellappen, ventral von ihm entwickelt sich der Fuß als zipfelartiger Anhang. Später wird das Velum abgeworfen und auch die Schale geht ver- loren; dagegen entwickeln sich auf dem spindelförmigen, sehr kontraktilen Körper drei für die Gymnosomata sehr charakteristische Wimperreifen. Der erste liegt Pteropoda: B. Gymnosomata 102 auf dem Kopfe, vor Fuß und Flossen, der zweite auf der Höhe des Afters und der dritte am hinteren Körperende, wo später (bei einigen Gattungen) die Endkieme entsteht. Der erste Reifen sondert sich bald in einzelne Stücke und zerfällt allmählich, der zweite und dritte bleiben sehr lange erhalten (besonders der dritte), bis weit in die Geschlechts- reife hinein, ja bei Thliptodon scheint der dritte Reifen zeitlebens zu bestehen, so auch bei T’halassopterus und Paedoclione. Jedenfalls ist sein Verschwinden eine ganz sekundäre Erscheinung, da sich die Organe des ausgebildeten Tieres dann schon alle gebildet haben. In der Nähe der Ringe ist der Körper, der sonst reich mit Pigment- zellen sowie mit zahlreichen einzelligen Drüsen ausgestattet ist, farblos, so daß die hellen Streifen gleich auffallen. Hinter dem Vorderlappen des Fußes, der aus der zipfelförmigen Anlage hervorgegangen ist, macht sich jederseits die erste Anlage der Flossen bemerkbar. — Die Eier bei Halopsyche entwickeln sich nicht frei, sondern im Muttertiere selbst. Die Art ist also vivipar. Die jungen Embryonen werden in einem großen Behälter, einem umfangreichen Sack des Muttertieres aufbewahrt, auf der ventralen Seite des vorderen Rumpfabschnittes. Dieser Sack scheint sich aus den Genitalanhangsdrüsen zu entwickeln. Die Geschlechtsorgane bilden sich dann all- mählich zurück. Später dehnt sich der Brutsack mit den Embryonen stark zwischen Integument und Eingeweidesack des Muttertieres aus und in diesen Raum kommen nach Platzen des Brutsackes die fast völlig reifen Embryonen zu liegen. Die Organe des Muttertieres, wenigstens im Rumpfe, schrumpfen allmählich zusammen, nicht nur die Geschlechtsorgane. sondern auch die Leber und die Niere verschwinden fast gänzlich; am längsten erhalten sich Vorderdarm und Enddarm. Im Kopfe geht kaum eine Anderung vor sich. Später geht sehr. B. Subord. Gymnosomata wahrscheinlich das Muttertier durch all- mähliche Degeneration der vegetativen Organe zugrunde; die Leibeshülle berstet und läßt die Jungen, welche jetzt aber gänzlich schon die Gestalt des erwachsenen Tieres zeigen, also keine Metamorphose durchmachen, ausschwärmen. Das tote Muttertier, das nur noch als Behälter der Jungen funktionieren könnte, sinkt zu Boden hinab. In ihrer gegenseitigen Verwandtschaft bilden die Gymnosomata keine einheitliche Entwieklungsreihe, sondern es muß hier eher eine Anzahl parallel verlaufender, selbständiger Reihen angenommen werden. — Eine erste Reihe bilden die Pneumoder- matiden. Bei Pneumodermopsis sind die Hakensäcke sowie die Kiemen noch sehr wenig ausgebildet, die Saugnäpfe sitzen größtenteils direkt der Schlundwandung auf und sind histologisch sehr einfach gebaut, der Pericardialnierengang ist ziemlich lang. Bei Spongiobranchia sind die Hakensäcke besser ausgebildet, die Saugnäpfe werden von besonderen Saugarmen getragen, die Seitenkieme ist zwar einfach, dazu ist aber eine Endkieme in Gestalt einer ringförmigen Hautfalte am unteren Körperpol hinzu- getreten. Pneumoderma ist höher entwickelt, sowohl in dem Bau der Hakensäcke und der Saugarme als in der hohen Ausbildung der gefransten Seiten- und Endkieme, der Pericardialnierengang ist sehr kurz geworden. Bei Schizobrachium endlich ist die Ausbildung der Saugarme noch weiter vorgeschritten, die Hakensäcke sind sekundär reduziert, die Kiemen fast völlig verschwunden. — Eine zweite Entwicklungsreihe bildet Cliopsis, mit wenig entwickelten Hakensäcken und einer sechsseitigen End- kieme. Letztere sowie die auch hier, wie bei den Pneumodermatiden, vorkommende dorsale Drüsengrube deuten auf eine Verwandtschaft mit letztgenannter Familie hin. Doch ist bei Ckiopsis der Bau des Integumentes ein ganz anderer, der Mittellappen des Fußes ist ganz oder fast völlig verschwunden, der Schlund hat sich in einen außerordentlich langen, ausstülpbaren Rüssel umgewandelt und enthält keine Saugarme oder ähnliche Organe. — Eine dritte Reihe fängt mit Notobranchaea an. Hier finden wir sehr wenig entwickelte Hakensäcke, wohl ausgebildete Seiten- und Mittellappen des Fußes, der Schlund ist wenig entwickelt und enthält keine besonderen Schlundorgane, eine dorsale Drüsengrube fehlt, die Endkieme ist dreistrahlig.. Auf diese Gattung folgen die Clioniden (Clione, Paraclione, Paedoclione, Fowlerina), wo besondere Schlund- organe (Buccalkegel) aufzutreten anfangen. Der Mittellappen des Fußes ist beträchtlich reduziert oder fehlt gänzlich, ebenso der Oberkiefer und die Kiemen, die Hakensäcke dagegen sind bedeutend stärker ausgebildet. — Mit den Clioniden allerdings verwandt und einen sehr spezialisierten Seitenzweig dieser Familie darstellend sind die Thlipto- dontidae. Der Fuß ist hier noch gut entwickelt. Oberkiefer, Kiemen und dorsale Drüsengrube fehlen, wie bei den Clioniden, vollkommen, auch ist der Schlund nur Pteropoda: B. Gymnosomata, 1. B. Subord. Gymnosomata Pneumodermatidae 103 gering entwickelt, die Hakensäcke aber sind gut ausgebildet. Zuweilen (Thalassopterus) kommen Bucealkegel vor. Doch sind die Thliptodontidae scharf unterschieden durch die hohe Ausbildung des Kopfabschnittes, der nicht gegen den Rumpf abgesetzt ist, durch die Existenz besonderer Hauttaschen für Fuß und Flossen, durch die sehr abweichend gebaute Radula und durch ein besonderes Hilfsorgan bei der Begattung (bei Thliptodon). Eigentümlich sind auch die Schlundblasen. — Halopsyche bildet eine vierte Reihe, die allerdings sehr isoliert dasteht. In vielen Hinsichten ist die Gattung mit typischen Merkmalen der Gymnosomata ausgestattet, so in der Ausbildung des Fußes (Mittel- und Seitenlappen), des Integumentes, der drei Längs- muskelsysteme, in dem Besitz zweier Tentakelpaare und in der Verschmelzung von Magen und Leber. Sie weicht aber gänzlich durch die eigentümliche Ausbildung des vorderen Rumpfabschnittes, in welchen der Kopf zurückgezogen werden kann, ab, weiter durch die eigenartigen Flossen und durch das vordere Tentakelpaar, zudem ist das Zentralnervensystem anders gebaut, besondere Schlundorgane und Hakensäcke fehlen gänzlich, am Enddarm kommen Anhangsdrüsen vor, Herz und Niere liegen auf der Dorsalseite des Körpers, die Genitalanhangsdrüsen sind anders gebaut und endlich ist die Viviparität auffallend. Einige Merkmale sind primitiver Natur, andere nicht. So müssen wir den Ursprung von Halopsyche ganz an die Wurzel der Gymnosomata verlegen. Die Gymnosomata leben frei im offenen Meere, besonders in den oberen Schichten, spezifische Tiefseeformen scheint es bei ihnen nicht zu geben. Sie kommen hauptsächlich in den wärmeren Meeren vor. Die einzelnen Arten besitzen nicht entfernt die weite Verbreitung so vieler Thecosomata; doch ist die Seltenheit von fast allen Arten die Ursache, daß sie so wenig gefangen werden und wir über die Verbreitung noch gar nicht vollständig informiert sind. 6 Familien, 13 Gattungen, 35 sichere und 3 unsichere Arten. Bestimmungstabelle der Familien: 1 \| Kiemen vorhanden — 2. Kiemen ganz fehlend — 4. Seitenkieme und Endkieme fast immer beide vor- \| handen; Schlundorgane in Gestalt von Saug- 2 armen entwickelt . . ».. 2.2.2. .... .. 1. Fam. Pneumodermatidae Nur die Endkieme vorhanden, keine Schlund- organe ausgebildet — 3. Schlund außerordentlich lang; unterer Körperpol abgerundet, mit sechsseitiger Endkieme . . . 2. Fam. Cliopseidae Schlund stark rückgebildet; unterer Körperpol etwas zugespitzt, mit dreistrahliger Endkieme 3. Fam. Notobranchaeidae Integument knorpelartig, hart; vordere Tentakeln außerordentlich stark ausgebildet, zweischenklig 6. Fam. Halopsychidae Integument sehr dünn, durchsichtig, vordere Ten- takeln sehr klein — 5. Kopfabschritt vom Rumpfe durch eine deutliche \| Einsehnürung getrennt, im Schlunde Buceal- \ 3 kegel immer vorhanden . . B. Subord. Gymnosomata . . 4. Fam. Clionidae Kopfabschnitt unverhältnismäßig groB; nieht vom Rumpfe abgesetzt, Buccalkegel im Schlunde vorhanden oder fehlend . . . ........ 5. Fam. Thliptodontidae Bestimmungstabelle der Familien: Bestimmungstabelle der Familien: \ Kopfabschnitt unverhältnismäßig groB; nieht vom Rumpfe abgesetzt, Buccalkegel im Schlunde vorhanden oder fehlend . . . ........ 1, Kam Pneumodermatidae Endkieme immer vorhanden, wenn auch zuweilen sehr schwach entwickelt; zwei seitliche Saug- arme — 2. Endkieme entweder in Form einer Ringfalte oder einer viereckigen Leiste; Seitenkieme vor- handen — 3. \ Endkieme sehr klein, in Form einer kleinen Längs- falte auf der Ventralseite des hinteren Körper- endes; Seitenkieme [ehlt, Saugarme dichotomisch ee Endkieme sehr klein, in Form einer kleinen Längs- falte auf der Ventralseite des hinteren Körper- endes; Seitenkieme [ehlt, Saugarme dichotomisch ee 1, Kam Pneumodermatidae 1840 Pneumodermidae (part., non Orbigny 1842), J. E. Gray, Syn. Brit. Mus., ed. 42 p: 86 \| 1846 Pneumodermoidae, L. Agassiz, Nomenel. zool., Index p.299 \| 1855 Clioidea, Gegenbaur, Unters. Pter. & Heterop., p.212 \| 1858 Pneumodermonidae (part.), H. & A, Adams, Gen. Moll., v.1 p.62 \| 1862 Pneumodermidae. Bronn in: Bronn’s Kl. Ordn., v.311 p. 645 \| 1870 Pneumodermatidae, W. Dall in: Amer. J. Conch., v.611 p.139 \| 1881 Cliidae (part., non Woodward 1856, Jeffreys 1869), P. Fischer, Man. Conchyl, p. 243 \| 1887 Pneumonodermatidae, Pelseneer in: Kep. Voy. Challenger, Zool. v.19 pars 58 p.11. 104 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 1. Pneumodermopsis Körper am Hinterende zugespitzt oder abgerundet, mit deut- lichen Pigmentzellen ausgestattet. Kopf- und Rumpfabschnitt durch eine Einschnürung getrennt. Rumpf mit dorsalem Drüsen- feld. Schlundorgane in Gestalt von zwei oder drei mit Saug- näpfen besetzten Saugarmen entwickelt. Hakensäcke meist tief und gut ausgebildet, selten klein und schwach. Öberkiefer vor- handen. Mittellappen des Fußes deutlich, zipfelförmig. Seiten- kieme fast immer entwickelt. After mit äußerer Nierenöffnung und Osphradium meist dicht beisammen auf dem Analfelde. Herz und Niere auf der rechten Körperseite., Iu allen Ozeanen, meist in der Warmwasserzone. 4 Gattungen, 17 sichere und 1 unsichere Art. Iu allen Ozeanen, meist in der Warmwasserzone. 4 Gattungen, 17 sichere und 1 unsichere Art. Iu allen Ozeanen, meist in der Warmwasserzone. 4 Gattungen, 17 sichere und 1 unsichere Art. 4 Gattungen, 17 sichere und 1 unsichere Art. Bestimmungstabelle der Gattungen: Endkieme fehlend; drei Saugarme, einer median auf der Ventralseite des Schlundes und zwei jederseits; Eingeweidesack sich nicht. bis zum 1 hinteren Körperpol erstreckend. . . . . . . l. Gen. Pneumodermopsis Endkieme immer vorhanden, wenn auch zuweilen sehr schwach entwickelt; zwei seitliche Saug- arme — 2. Endkieme entweder in Form einer Ringfalte oder einer viereckigen Leiste; Seitenkieme vor- handen — 3. \ Endkieme sehr klein, in Form einer kleinen Längs- falte auf der Ventralseite des hinteren Körper- endes; Seitenkieme [ehlt, Saugarme dichotomisch ee 4. Gen. Schizobrachium Endkieme in Form einer vorspringenden Ringfalte 2. Gen. Spongiobranchia E \| Endkieme in Form einer viereckigen Leiste, mit vier gefransten, von den Ecken ausgehenden Strahlen: 4 . u ne ar, lum = Br Den, Endkieme fehlend; drei Saugarme, einer median auf der Ventralseite des Schlundes und zwei jederseits; Eingeweidesack sich nicht. bis zum 1 hinteren Körperpol erstreckend. . . . . . . l. Gen. Pneumodermopsis Vereinzelt in allen Ozeanen, aber nicht in den eigentlichen Polarmeeren gefunden. 6 Arten, 1. Gen. Pneumodermopsis Bronn Alle vier seitlichen Saugnäpfe des medianen Saugarmes von derselben Größe — 3. Die beiden seitlichen Saugarme gut entwickelt, aber kurz, mit je 6—9 Saugnäpfen; die beiden distalen Saugnäpfe des medianen Saugarmes sehr weit die anderen an Größe überragend (Fig. 74) ..... Der el, Prcılhata Die beiden seitlichen Saugarme Eck ebd die ent- sprechenden 16 Saugnäpfe in zwei Reihen angeordnet; der terminale Saugnapf und die beiden distalen auf dem medianen Saugarme etwa gleich groß (Fig.75) . . . . 2. P. minuta ( Medianer Saugarm rückgebildet — 4. \ Medianer Saugarm gut ausgebildet, seitliche Arme rückgebildet —5. Die beiden seitlichen Saugarme rückgebildet, mit wenigen (2) Saugnäpfen (Fig.76) ... . ar. BD simmles Die beiden seitlichen Saugarme gut enwiökelt, mit zahl- reichen (44) Saugnäpfen (Fig.79) . -. ». - » 2... .. 6. P. macrochira Die Saugnäpfe auf der Schlundwandung, welche den seitlichen Saugarmen entsprechen, in einer einzigen Reihe (Fig. 77) 4. P. paucidens Die Saugnäpfe auf der Schlundwandung, welche den seitlichen Saugarmen entsprechen, in einer doppelten Reihe (Fig.78) 5. P. polycotyla _ Bestimmungstabelle der Arten: 1. P. ciliata (Gegenb.) ?1839 Pneumodermon mediterraneum (part.), P.J. Beneden in: M&m. Ac. Belgique, v.12 [nr.13] t.3 f.3 \| 1855 P. ciliatum, Gegenbaur, Unters. Pter. & Heterop., p.74 \| 1862 Pneumodermopsis ciliata, Bronn in: Bronn’s Kl. Ordn., v.311 p.619 \| 1886 Dexiobranchaea c., Boas in: Danske Selsk. Skr., ser.6 v.4 p.159 t.7 £.104; t.8 £.113 (großer Saugnapf) \| 1886 Pneumoderma peroni (non Lamarck 1819), Locard, Prodr. Malac. franc., p. 18. _. vorderer Tentakel Seitenlappen des Fußes After Eu mittlerer h .-" Wimperring Mittellappen des Fußes Seitenkieme ...-""' hayikekar - Wimperring 4 _. vorderer unpaarer Saugnapf des medianen Saugarmes Tentakel große Saugnäpfe des medianen Saugarmes Seitenlappen des Fußes After Eu ... Saugnäpfe der mittlerer £ h .-" Wimperring paarigen Arme Mittellappen des Fußes Seitenkieme ...-""' hayikekar - Wimperring 4 B Fig. 74 P. ciliata. A Ganzes Tier, von der Ventralseite mit halbhervorgestreekten Schlundorganen, der unpaare Saugarm eingezogen (?j,). Nach Boas. B Kopfabschnitt von der Ventralseite mit halb- ausgestreckten Schlundorganen (%/,). Nach Meisenheimer. unpaarer Saugnapf des medianen Saugarmes große Saugnäpfe des medianen Saugarmes ... Saugnäpfe der £ paarigen Arme B Fig. 74 alseite mit halbhervorgestreekten Schlundorganen, der Boas. B Kopfabschnitt von der Ventralseite mit halb- dorganen (%/,). Nach Meisenheimer. unpaarer Saugnapf des medianen Saugarmes große Saugnäpfe des medianen Saugarmes unpaarer Saugnapf des medianen Saugarmes Seitenlappen des Fußes mittlerer Wimperring B 4 Fig. 74 P. ciliata. 1. Gen. Pneumodermopsis Bronn 1855 Pneumodermon (part.), Gegenbaur, Unters. Pter. & Heterop., p.74 \| 1862 Pneumodermopsis, Bronn in: Bronn’s Kl. Ordn., v.3u p.645 \| 1885 Dexwiobranchaea, Boas in: Zool. Anz., v.8 p. 688. Körper gestreckt, am Hinterende zugespiszt. Eingeweidesack nicht bis zum hinteren Körperpol reichend. Vordere Tentakeln ziemlich lang. Drei Saugarme, einer median auf der Ventralseite des Schlundes, immer mit 5 Saugnäpfen ausgestattet und zwei seitliche, mit meist zahlreichen Saug- näpfen. Die fünf Saugnäpfe auf dem medianen Arme sind immer so an- geordnet, daß einer auf der Spitze des Armes in der Medianlinie steht und jederseits von ihm an den Seiten des Armes zwei Saugnäpfe vorkommen. Die Saugarme, sowohl die seitlichen als der mediane, können ganz reduziert sein und die Saugnäpfe sitzen dann an den entsprechenden Stellen direkt der Schlundwandung auf. Hakensäcke wenig ausgebildet, klein. Mittelplatte der Radula immer vorhanden, mit drei Spitzen. Mittellappen des Fußes sehr langgestreckt. Seitenkieme einfach, ohne Kämme oder Fransen, dreieckig, zipfelartig; keine Endkieme. Die beiden hinteren larvalen Wimperreifen bleiben sehr lange erhalten. Vereinzelt in allen Ozeanen, aber nicht in den eigentlichen Polarmeeren gefunden. 6 Arten, Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 1. Pneumodermopsis 105 _ Bestimmungstabelle der Arten: Die beiden distalen Saugnäpfe des medianen Saugarmes größer als die beiden proximalen — 2. Alle vier seitlichen Saugnäpfe des medianen Saugarmes von derselben Größe — 3. Die beiden seitlichen Saugarme gut entwickelt, aber kurz, mit je 6—9 Saugnäpfen; die beiden distalen Saugnäpfe des medianen Saugarmes sehr weit die anderen an Größe überragend (Fig. 74) ..... Der el, Prcılhata Die beiden seitlichen Saugarme Eck ebd die ent- sprechenden 16 Saugnäpfe in zwei Reihen angeordnet; der terminale Saugnapf und die beiden distalen auf dem medianen Saugarme etwa gleich groß (Fig.75) . . . . 2. P. minuta ( Medianer Saugarm rückgebildet — 4. \ Medianer Saugarm gut ausgebildet, seitliche Arme rückgebildet —5. Die beiden seitlichen Saugarme rückgebildet, mit wenigen (2) Saugnäpfen (Fig.76) ... . ar. BD simmles Die beiden seitlichen Saugarme gut enwiökelt, mit zahl- reichen (44) Saugnäpfen (Fig.79) . -. ». - » 2... .. 6. P. macrochira Die Saugnäpfe auf der Schlundwandung, welche den seitlichen Saugarmen entsprechen, in einer einzigen Reihe (Fig. 77) 4. P. paucidens Die Saugnäpfe auf der Schlundwandung, welche den seitlichen Saugarmen entsprechen, in einer doppelten Reihe (Fig.78) 5. P. polycotyla _ Bestimmungstabelle der Arten: Die beiden distalen Saugnäpfe des medianen Saugarmes größer als die beiden proximalen — 2. 1. Gen. Pneumodermopsis Bronn A Ganzes Tier, von der Ventralseite mit halbhervorgestreekten Schlundorganen, der unpaare Saugarm eingezogen (?j,). Nach Boas. B Kopfabschnitt von der Ventralseite mit halb- ausgestreckten Schlundorganen (%/,). Nach Meisenheimer. Körper langgestreckt, am hinteren Pole zipfelartig. Mittellappen des Fußes sehr lang. Seitenkieme ebenfalls sehr lang. Die beiden seitlichen Saugarme außerordentlich kurz, aber noch in der Gestalt zweier Fortsätze zu beiden Seiten der Medianlinie auf.der Ventralseite der Schlundwandung; jeder Fortsatz am freien Rande mit 6—9 mittelgroßen Saugnäpfen. Die fünf Saugnäpfe des medianen Saugarmes sehr ungleich an Größe; der terminale 106 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 1. Pneumodermopsis etwas größer als die Saugnäpfe der seitlichen Arme, das vordere Paar wird von zwei außerordentlich großen, tief-napfförmigen Saugnäpfen gebildet, deren freier Rand nach außen umgekrempelt und an einer Stelle zipfelartig aus- gezogen ist, die Saugnäpfe werden von langen, dünnen Stielen getragen; die beiden hinteren Saugnäpfe des medianen Armes sind etwa gleich groß wie diejenigen der paarigen Arme. Formel der Radula 7. 1. 7. Hakensäcke mit 30—40 Haken. Farbe grau-violett. L.') etwa 10 mm. Fig. 74. etwas größer als die Saugnäpfe der seitlichen Arme, das vordere Paar wird von zwei außerordentlich großen, tief-napfförmigen Saugnäpfen gebildet, deren freier Rand nach außen umgekrempelt und an einer Stelle zipfelartig aus- gezogen ist, die Saugnäpfe werden von langen, dünnen Stielen getragen; die beiden hinteren Saugnäpfe des medianen Armes sind etwa gleich groß wie diejenigen der paarigen Arme. Formel der Radula 7. 1. 7. Hakensäcke mit 30—40 Haken. Farbe grau-violett. L.') etwa 10 mm. Fig. 74. Im Atlantischen Ozean, bis jetzt nur nördlich vom Äquator, von etwa 7° bis 610 n. Br. (südlich von Island), auch in der westlichen Hälfte des Mittelmeeres häufig, und in der Adria. 2. P. minuta (Plsnr.) 1887 Dexiobranchaea m., Pelseneer in: Rep. Voy. Challenger. Zool. v.19 pars58 p.16 t.1 f.2 \| 1905 Pneumodermopsis m., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 91 p. 46. Körper etwas mehr gedrungen als bei der vorhergehenden Art. Mittel- lappen des Fußes sehr lang. Seitenkieme mittellang. Die beiden seitlichen Saugarme rückgebildet, zu beiden Seiten der ventralen Mittellinie sitzen der Schlundwandung etwa 8 Saugnäpfe in zwei Reihen übereinander auf. Der mediane Saugarm ist sehr gut entwickelt; von den fünf Saugnäpfen sind der terminale und die beiden vorderen etwa von der gleichen Größe wie medianer Saugarm -.-..__. IL Saugnäpfe der paarigen Arme --- "Bons Flosse hervorgestreckter Penis ! gs "» mittlerer Wimperring Seitenkieme hinterer Wimperring Fig. 76. P. minuta. Von der Ventralseite, mit hervorgestrecktem Rüssel ('/,). 1) Es wird hier stets nur die Länge des Körpers angegeben, der eventuell aus- gestreckte Rüssel wird dabei also nicht mitgerechnet. Bisher nur im nördlichen Pazifischen Ozean (etwa 370 n.Br., 180° w. L.) gefunden. 3. P. simplex (Boas) 1886 Dexiobranchaea s., Boas in: Danske Selsk. Skr., ser.6 v.4 p. 160 \| 1887 D.s.. Pelseneer in: Rep. Voy. Challenger, Zool. v. 19 pars 58 p-16 t.1f.3 \| 1905 Pneumodermopsis s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.91 p.46. Körper gedrungen. Mittellappen nicht besonders lang. Seitenkieme ziemlich kurz. Saugarme alle völlig rückgebildet; die Saugnäpfe sitzen alle 1) Es wird hier stets nur die Länge des Körpers angegeben, der eventuell aus- gestreckte Rüssel wird dabei also nicht mitgerechnet. 1. Gen. Pneumodermopsis Bronn Nach Pelseneer. Flosse Saugnäpfe der paarigen Arme - hervorgestreckter Penis Saugnäpfe der paarigen Arme - hervorgestreckter Penis Fig. 76. Fig. 76. P. minuta. Von der Ventralseite, mit hervorgestrecktem Rüssel ('/,). Nach Pelseneer. diejenigen der rückgebildeten, seitlichen Saugarme; diese drei Saugnäpfe stehen nahe beisammen auf dem freien Ende des medianen Saugarmes und werden auf kurzen Stielen getragen; die zwei unteren (proximalen) Saugnäpfe stehen nahe der Basis des Saugarmes, sie sind sehr klein und haben sehr dünne Stiele. Radula und Hakensäcke unbekannt. Färbung kaum vorhanden, nur einige wenige braune Pigmentflecke auf der rechten Körperseite. L. etwa 3 mm. — Fig. 75. diejenigen der rückgebildeten, seitlichen Saugarme; diese drei Saugnäpfe stehen nahe beisammen auf dem freien Ende des medianen Saugarmes und werden auf kurzen Stielen getragen; die zwei unteren (proximalen) Saugnäpfe stehen nahe der Basis des Saugarmes, sie sind sehr klein und haben sehr dünne Stiele. Radula und Hakensäcke unbekannt. Färbung kaum vorhanden, nur einige wenige braune Pigmentflecke auf der rechten Körperseite. L. etwa 3 mm. — Fig. 75. Bisher nur im nördlichen Pazifischen Ozean (etwa 370 n.Br., 180° w. L.) gefunden. 3. P. simplex (Boas) 1886 Dexiobranchaea s., Boas in: Danske Selsk. Skr., ser.6 v.4 p. 160 \| 1887 D.s.. Pelseneer in: Rep. Voy. Challenger, Zool. v. 19 pars 58 p-16 t.1f.3 \| 1905 Pneumodermopsis s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.91 p.46. Körper gedrungen. Mittellappen nicht besonders lang. Seitenkieme ziemlich kurz. Saugarme alle völlig rückgebildet; die Saugnäpfe sitzen alle Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 1. Pneumodermopsis 107 direkt der Schlundwandung auf und bilden drei Gruppen. Die beiden jeder- seits der ventralen Mittellinie gelegenen setzen sich je nur aus zwei kleinen, kaum gestielten Saugnäpfen zusammen. Von den fünf Saugnäpfen, welche dem medianen Saugarm entsprechen, ist der terminale (bei hervorgestrecktem Rüssel ganz vorn gelegen) weitaus der größte, er zeigt einen umgekrempelten großer Saugnapf des medianen Saugarmes .„.» Saugnäpfe der seitlichen Saugarme Mündungsstelle des Penis Me ar ea After . mittlerer Wimperring Seitenkieme -", _ .. binterer Wimperring Fig. 76. P.simplex. Von der Ventralseite, mit hervorgestrecktem Rüssel (1).). Nach Pelseneer. großer Saugnapf des medianen Saugarmes Mündungsstelle des Penis M Fig. 76. Fig. 76. P.simplex. Von der Ventralseite, mit hervorgestrecktem Rüssel (1).). Nach Pelseneer. Fig. 76. P.simplex. Von der Ventralseite, mit hervorgestrecktem Rüssel (1).). Nach Pelseneer. 1. Gen. Pneumodermopsis Bronn Rand und steht auf einem kurzen Stiele; die vier seitlichen Saugnäpfe sind alle gleich groß wie die Saugnäpfe der rückgebhildeten, lateralen Saugarme, die beiden vorderen liegen bei eingezogenem Rüssel auf der gleichen Höhe wie der große mediane. Formel der Radula 6. 1.6. Hakensäcke mit 40 bis 50 Haken. Farbe bräunlich. L. 45 mm. — Fig. 76. Bisher nur an der Westküste Südamerikas, bei Caldera (Chili) gefunden. 4. P. paucidens (Boas) 1886 Dexiobranchaea p., Boas in: Danske Selsk. Skr., ser.6 v.4 p.160 t.7 £.105, 106 \| 1905 Pneumodermopsis p., Meisenheimer in: are Tiefsee-Exp., v. 91 p. 46. medianer Saugarm en Saugnäpfe der paarigen Saugarme ---z9® vorderer Tentakel Flosse Mittellappen des Fußes a _.. mittlerer Wimperring Seitenkieme _. hinterer Wimperring Fig. 77. P. paucidens. Von der Ventralseite, Rüssel hervorgestreckt ('J,). Nach Boas. medianer Saugarm e Saugnäpfe der paarigen Saugarme - Fig. 77. Fig. 77. P. paucidens. Von der Ventralseite, Rüssel hervorgestreckt ('J,). Nach Boas. Körper gestreckt, schlank. Mittellappen des Fußes ziemlich kurz. Seitenkieme sehr klein, offenbar weit nach dem unteren Körperpol hin ver- schoben. Die Saugnäpfe, welche den gänzlich rückgebildeten, seitlichen Saug- armen entsprechen, sitzen der ventralen Schlundwandung in einer einzigen, Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 1. Pneumodermopsis 108 halbkreisförmigen Reihe auf; es sind deren im ganzen 11—13 vorhanden, welche auf kurzen Stielen stehen. Der mediane Saugarm ist gut ausgebildet und trägt auch hier wieder fünf Saugnäpfe, welche sämtlich größer sind als die den paarigen Saugarmen entsprechenden. Die vier seitlichen Saugnäpfe sind gleich groß und zeigen sehr lange und dicke Stiele, der terminale oder mediane Saugnapf ist wieder größer als die vier anderen. Formel der Radula 2.1.2, die Mittelplatte hat hier nicht, wie sonst, drei, sondern nur zwei Zähne, da der mittlere (kleinere) gänzlich reduziert ist. Hakensäcke mit nur 4—6 Haken. Farbe unbekannt. L. 5 mm. — Fig. 77. Atlantischer Ozean, zwischen Brasilien und Europa gefangen, außerdem im Mittel meer bei Messina. Atlantischer Ozean, zwischen Brasilien und Europa gefangen, außerdem im Mittel- meer bei Messina. 5. P. polycotyla (Boas) 1886 Dexiobranchaea p., Boas in: Danske Selsk. Skr., ser.6 v.4 p.161 \| 1887 D.p., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars58 p.17 t.1 f.4,5 \| 1905 Pneumodermopsis p., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.46. Körper gedrungen. Mittellappen des Fußes lang. Seitenkieme ebenfalls lang. Die Saugnäpfe, welche den gänzlich rückgebildeten, seitlichen Saugarmen entsprechen, sitzen der ventralen Schlundwandung auf. 1. Gen. Pneumodermopsis Bronn Sie bilden hier jederseits eine besondere Gruppe, in denen die 10—12 Saugnäpfe auf kurzen Stielen in -- medianer Saugarm Saugnäpfe der seitlichen Saugarme vorderer Tentakel After Seitenkieme “\| Fig. 78. P. polycotyla. Von der Ventralseite, mit hervorgestrecktem Rüssel (°),). Nach Pelseneer. Fig. 78. P. polycotyla. Von der Ventralseite, mit hervorgestrecktem Rüssel (°),). Nach Pelseneer. zwei alternierenden Reihen übereinander stehen. Der mediane Saugarm ist gut ausgebildet; seine fünf Saugnäpfe sind auch hier verschieden groß. Der mediane oder terminale Saugnapf ist der größte, die vier seitlichen haben ungefähr die Größe der den paarigen Saugarmen entsprechenden. Formel der Radula 3°1°3. Hakensäcke mit etwa 10 Haken. Farbe bräunlich. L. 5 mm. — Fig. 78. Bisher nur im östlichen Pazifischen Ozean, an der Küste von Chili bei Caldera, und weiter bei Panama gefangen, auch im Mittelmeer bei Messina beobachtet. 6. P. macrochira Meisenh. 1905 P. m., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.47. Infolge starker Kontraktion der einzigen bis jetzt bekannten Exemplare ist der allgemeine Körperbau, sowie die relative Länge von Fußzipfel und Seitenkieme, schwierig zu bestimmen. Die paarigen Saugarme sind im Gegensatz zu fast allen übrigen Arten dieser \ Gattung sehr stark entwickelt; jeder trägt 44 großer Saugnapf Saugnäpfe, von denen die distalen auf kurzen, Fig. 79. die proximalen auf langen Stielen sitzen. „Unter . macrochira. Seitlicher Saug- «en distalen Saugnäpfen zeichnet sich einer rm mit den Saugnäpfen. Nach ä & Pr Meisenheimer (vergt.). + durch seine besondere Größe aus, während \ großer Saugnapf Fig. 79. Fig. 79. a. Seitl P. macrochira. Seitlicher Saug- arm mit den Saugnäpfen. Nach Meisenheimer (vergt.). P. macrochira. Seitlicher Saug- arm mit den Saugnäpfen. Nach Meisenheimer (vergt.). Pterop.: B. Gymnos., 1. Pneumodermatidae, 1. Pneumodermopsis, 2. Spongiobranchia 109 Pterop.: B. Gymnos., 1. Pneumodermatidae, 1. Pneumodermopsis, 2. Spongiobranchia 109 die übrigen in ihrem Umfang etwas variieren. Der Mittelarm ..... ist kaum besonders hervorgehoben, er trägt die typischen, hier lauggestielten 5 Saugnäpfe, von denen der mittlere der größte ist“ (Meisenheimer). Formel der Radula 6'1°6. Hakensäcke mit 16—20 mäßig langen Haken. Farbe unbekannt. L. 7 mm. — Fig. 79. Nur im südöstlichen Atlantischen Ozean, westlich von Angra Pequena und im südlichen Indischen Ozean, zwischen Neu-Amsterdam und den Üocos-Inseln angetroffen. 2. Gen. Spongiobranchia Orbigny 1824 Clhiodita (part.), Quoy & Gaimard in: Voy. Uranie & Physicienne, p.413 \| ?1825 Trichocyclus (non A. Costa 1865), Eschscholtz in: Isis, p.735 \| 1840 Spongiobranchea (part.), Orbigny, Voy. Amer. mörid., v.5ın p. 130 \| 1842 Spongiobranchia, Orbigny, Pal. franc., Terr. Oret. v.2 p.4 \| 1854 Spongiobranchaea, S. P.Woodward, Man. Moll., p. 209 \| 1862 Spongobranchia, Bronn in: Bronn’s Kl. Ordn., v. 3ı p. 645 \| 1885 Spongobranchus, N. Wagner in: Wirbell. Weiss. Meer., v.1 p. 120. Körper oval, am unteren Pol nicht zugespitzt, sondern abgerundet. Eingeweidesack bis zum Hinterende des Körpers reichend. Vordere Tentakeln erste Seitenplatte Mittelplatte Ä er N erste Seitenplatte Mittelplatte Ä er ) „_ Saugarın vorderer Tentakel N L_>---" After ventrale Rüssel- ____. ---. - Hakensack papille Rüssel -------? BE En I EN Ne sfr Basis des Saug- Seitenkieme -“ armes vorderer__.. Ber Endkieme Tentakel --+-------- Kopfabschnitt A C Fig. 80. S. australis. A Tier von der Ventralseite, mit, hervorgestreckten Saugarmen (®),). Nach Pelseneer. B Teil der Radula. Nach Boas (vergr.?). C ausgestülpter Rüssel und vorderer Teil des Kopf- abschnittes (%/,). Nach Pelseneer. ) „_ Saugarın vorderer T L_>---" After ve BE En Seitenkieme -“ Ber Endkieme A Mittelplatte vorderer Tentakel ventrale Rüssel- ____. ---. - Hakensack papille Rüssel -------? I EN Ne sfr Basis des Saug- armes vorderer__.. Tentakel --+-------- Kopfabschnitt C After A Fig. 80. S. australis. A Tier von der Ventralseite, mit, hervorgestreckten Saugarmen (®),). Nach Pelseneer. B Teil der Radula. Nach Boas (vergr.?). C ausgestülpter Rüssel und vorderer Teil des Kopf- abschnittes (%/,). Nach Pelseneer. lang. Zwei Saugarme an der vorderen, ventralen Schlundwandung, jeder trägt an der während der Ausstülpung medianen Seite die Saugnäpfe in einer einzigen Reihe, die Größe nimmt allmählich zu von der Basis bis zur Spitze des Saugarmes. Der hervorstreckbare Rüssel ist ziemlich lang und zeigt ganz vorn (wenn ausgestreckt) eine ventrale Papille. Hakensäcke kurz, mit ungleich großen Haken; Radula mit dreizähniger Mittelplatte. Mittel- lappen des Fußes gut ausgebildet, lang, zipfelartig. Seitenkieme sehr klein, nichts als eine kleine Hervorwölbung auf der rechten Rumpfseite; End- lang. Zwei Saugarme an der vorderen, ventralen Schlundwandung, jeder trägt an der während der Ausstülpung medianen Seite die Saugnäpfe in einer einzigen Reihe, die Größe nimmt allmählich zu von der Basis bis zur Spitze des Saugarmes. Der hervorstreckbare Rüssel ist ziemlich lang und zeigt ganz vorn (wenn ausgestreckt) eine ventrale Papille. Hakensäcke kurz, mit ungleich großen Haken; Radula mit dreizähniger Mittelplatte. 2. Gen. Spongiobranchia Orbigny Mittel- lappen des Fußes gut ausgebildet, lang, zipfelartig. Seitenkieme sehr klein, nichts als eine kleine Hervorwölbung auf der rechten Rumpfseite; End- 110 10 Pterop.: B. Gymnos., 1. Pneumodermatidae, 2. Spongiobranchia, 3. Pneumoderma kieme eine einfache, ringförmige Hautfalte am unteren Körperpol, ohne Strahlen oder Fransen. kieme eine einfache, ringförmige Hautfalte am unteren Körperpol, ohne Strahlen oder Fransen. kieme eine einfache, ringförmige Hautfalte am unteren Körperpol, ohne Strahlen oder Fransen. 1. S. australis (Orb.) ?1824 Oliodita caduceus, Quoy & Gaimard in: Voy. Uranie & Physieienne, p. 413 \| ?1825 Trichocyelus dumerilii, Eschscholtz in: Isis, p. 735 1.5 f.4 (Larve) \| ?1825 Cliodita caduceus, Quoy & Gaimard in: Ann. Sci. nat., ser. 1 o.6 p. 74 t.2 f.4 \| 1840 Spongiobranchea australis (non CUhenu 1859), Orbigny, Voy. Amer. mörid., v.5ıı p.131 t.19 f.1—7 \| 1854 Spongiobranchaea a., S. P. Woodward, Man. Moll., p.209 \| 1887 8. a., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars58 p.19 t.1 £.6,7 \| 1905 S.a., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.47 t.16 f.4 \| 1906 S.a., Meisenheimer in: D. Südp.-Exp., v.9ır p. 99. Körper auf der Höhe der kleinen Seitenkieme mit einer charakteristischen, ringförmigen Einschnürung. Saugarme mit 8—10 kurzgestielten Saugnäpfen auf der medianen und ventralen Seite (wenn hervorgestreckt). Die drei oder vier proximalen auf der medianen Seite des Saugarmes sind sehr klein, die distalen sitzen der ventralen Seite des Armes auf und nehmen nach der Spitze an Größe zu; jeder Saugnapf trägt einen deutlichen Hornring. Formel der Radula 8. 1. 8. Hakensäcke mit etwa 20 langen Haken auf dem Boden des Sackes und einige kürzere auf den Seitenwandungen. „Der Rumpf be- sitzt größtenteils eine dunkelbraune, zuweilen etwas ins Violette spielende Farbe, die nur an zwei Stellen durch ringförmige, fast farblose, ein wenig ins Bläuliche übergehende Streifen unterbrochen wird. Die vordere und umfang- reichere dieser hellen Zonen liegt in der Höhe der Seitenkieme, die zweite wird direkt durch die Endkieme dargestellt, wogegen das eigentliche Körper- ende wieder dunkelbraın erscheint. Die Flossen sind bläulich-grau, der Fuß zeigt leicht rötliche Färbung, die vorderen Kopfabschnitte mit den Buceal- organen sind farblos. Der dunkle Eingeweidesack schimmert innerhalb der vorderen hellen Zone mit blaugrauen Tönen durch das Integument hindurch“ (Meisenheimer). L. bis 22 mm. — Fig. 80 und 73 p. 109, 94. Im antarktischen Eismeer nicht selten, zirkumpolar; von der Eisgrenze bis 35°s. Br. (im südlichen Atlantischen Ozean, an der afrikanischen Küste) in das Warm- wassergebiet vordringend. 3. Gen. Pneumoderma G. Cuvier Endkieme eine viereckige, vorspringende Leiste am unteren Körperpol, dünn und durchsichtig; von den vier Ecken, zwei rechts und zwei links gelegen, strahlen vier gerade Strahlen auf der Körperoberfläche proximalwärts aus, die beiden Strahlen auf der rechten Körperseite immer kürzer als die anderen. Alle Strahlen sowie die Vorder- und die Hinterseite der viereckigen Leiste zeigen mehr oder weniger zahlreiche, oft sekundär wieder zerteilte Fransen. In den wärmeren Meeren. In den wärmeren Meeren. 9 sichere, 2 unsichere Arten. Bestimmungstabelle der sicheren Arten: Die vier Strahlen der Endkieme lang, mit zahlreichen, sekundär zerteilten Fransen — 2. Die vier Strahlen der Endkieme kurz, mit wenigen Fransen — 6. Jeder Saugarm mit etwa 7 großen Saugnäpfen (Fig. 81) 1. P. mediterraneum \| Jeder Saugarm mit zahlreichen (16—180) Saugnäpfen — 8. : Saugnäpfe auf sehr verschieden langen Stielen und un- gleich gebaut: jederseits auf dem Saugarme eine Gruppe sehr kleiner, kurzgestielter Saugnäpfe, am Ende ein Bündel lang-becherförmiger Saugnäpfe auf semlanpen Stielen (His: 84). un. 000. 0. 4. P. heterocotylum Saugnäpfe auf ungefähr iikasen Selen und gleich gebaut — 4. Jeder Saugarm mit 16—30 Saugnäpfen, welche zwei- zeilig angeordnet sind und von der Basis bis zur Spitze des Armes an Größe abnehmen (Fig. 85) . EL 5. P. eurycotylum \| Jeder en er: Mchr a 30 Sluenäpfen, le ar "von gleicher Größe — 5. Jeder Saugarm mit etwa 40 Saugnäpfen (Fig. 82) .. . 2. P. violaceum { Jeder Saugarm mit etwa 80—100 Saugnäpfen (Fig. 83) 3. P. peronii Fransen an den Strahlen der Endkieme wohl markiert, scharf geschieden und an der Basis eingeschnürt(Fig.86) 6. P. boasi Fransen an den Strahlen der Endkieme nicht scharf markiert, nur kleine Erhebungen der Ränder dar- stellend — 7. Saugarme sehr kurz, jederseits mit etwa 8 Saugnäpfen (Fig. 89) . ar » » 9. P. pygmaeum Saugarme er mit zuhlr Ahern (50—7 5) en —8. 4 5 6 7 9 sichere, 2 unsichere Arten. 9 sichere, 2 unsichere Arten. Bestimmungstabelle der sicheren Arten: Bestimmungstabelle der sicheren Arten: Bestimmungstabelle der sicheren Arten: Die vier Strahlen der Endkieme lang, mit zahlreichen, sekundär zerteilten Fransen — 2. Die vier Strahlen der Endkieme kurz, mit wenigen Fransen — 6. Jeder Saugarm mit etwa 7 großen Saugnäpfen (Fig. 81) 1. P. mediterraneum \| Jeder Saugarm mit zahlreichen (16—180) Saugnäpfen — 8. 3. Gen. Pneumoderma G. Cuvier 1804 „Pneumoderme“, G.Cuvier in: Ann. Mus. Paris, v.4 p.232 \| 1810 Pneumoderma, Peron & Lesueur in: Ann. Mus. Paris, v. 15 p.65 \| 1815 Pneumodermis + Aegle, Oken, Lehrb. Naturg., v.1 p.326 \| 1817 Pneumodermon, G. Cuvier, Rögne an., v.2 p. 380 \| 1846 Pneumonoderma, L. Agassiz, Nomenel. zool., Index p.299 \| 1847 Pneumonodermum, Herrmannsen, Ind. Gen. Malac., v.2 p.309 \| 1879 Cirrifer, Pfeffer in: Monber. Ak. Berlin, p. 249. 1804 „Pneumoderme“, G.Cuvier in: Ann. Mus. Paris, v.4 p.232 \| 1810 Pneumoderma, Peron & Lesueur in: Ann. Mus. Paris, v. 15 p.65 \| 1815 Pneumodermis + Aegle, Oken, Lehrb. Naturg., v.1 p.326 \| 1817 Pneumodermon, G. Cuvier, Rögne an., v.2 p. 380 \| 1846 Pneumonoderma, L. Agassiz, Nomenel. zool., Index p.299 \| 1847 Pneumonodermum, Herrmannsen, Ind. Gen. Malac., v.2 p.309 \| 1879 Cirrifer, Pfeffer in: Monber. Ak. Berlin, p. 249. Körper gestreckt, aber infolge starker Kontraktilität sehr veränderlich; unterer (distaler) Körperpol abgerundet, bis hierher erstreckt sich der Ein- geweidesack. Haut pigmentiert, besonders auf der rechten Seite, namentlich am Kopfabschnitt. Kopf vom Rumpfe durch einen mehr oder weniger entwickelten Nacken getrennt. Vordere Tentakeln kurz, meist breit-dreieckig. Zwei Saug- arme, in Gestalt abgeplatteter Organe, an der latero-ventralen Schlundwandung inseriert, mit gestielten Saugnäpfen auf derjenigen Seite, welche bei der Hervorstreekung des Armes median liegt. Die Zahl der Saugnäpfe ist bei jungen Exemplaren einer Art meist geringer als bei den erwachsenen Tieren. Rüssel kürzer als bei Spongiobranchia, mit einer ventralen Rüsselpapille am Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma 111 Vorderrande (bei ausgestülptem Rüssel). Die Mittelplatte der Radula fehlt immer bei den erwachsenen Tieren; die Zahl der Seitenzähne variiert von 4—6. Hakensäcke zylindrisch, sehr lang, mit zahlreichen Haken von ungleicher Größe; die längeren liegen an der Spitze, die kürzeren an den Seitenwandungen und an der Basis des (hervorgestreckten) Sackes. Mittel- lappen des Fußes lang, aber nie zipfelartig ausgezogen, sondern an der Spitze abgerundet und fleischig; Seitenlappen in ganzer Länge dem Kopfe angeheftet, zwischen ihnen ein gefalteter Höcker. Seitenkieme aus drei Längs- kämmen (Mittelkamm und zwei Seitenkämme) bestehend, sowie aus einer dreieckigen Partie dicht hinter diesen Kämmen am freien Rande der Kieme und mit der Spitze nach hinten gerichtet; der letztere Teil sowie die beiden seitlichen Längskämme, die von seinen Enden ausstrahlen, sind häufig ge- franst; der Mittelkamm trägt nie Fransen und stellt eine gerade verlaufende, erhabene Leiste auf der Mitte der deutlich abgesetzten und hervorgewölbten Seitenkieme dar. 3. Gen. Pneumoderma G. Cuvier Die vier Strahlen der Endkieme lang, mit jederseits etwa sieben, selbst sekundär wieder zerteilten Fransen, derartige Fransen auch auf der Dorsal- und Ventralseite der viereckigen Leiste der Endkieme. Saugarme ziemlich lang und schlank, jeder mit meist 7, selten 6 Saugnäpfen, von welchen die vier proximalen, nahe der Basis, die größeren sind und sich auf der ventralen Seite des (ausgestreckten) Saugarmes inserieren, während die drei distalen sehr klein und auf der Dorsalseite und an der Spitze des Armes angeheftet sind. Formel der { : s p Radula 6. 0. 6. Hakensäcke sehr lang. Farbe braunviolett oder rot- braun. L. bis 17 mm — Fig. 81. Körper gestreckt. Mittellappen des Fußes lang, mehr gestreckt als es bei den anderen Arten dieser Gattung der Fall ist. Seitenkieme kurz, mit gefransten Seitenkämmen. Die vier Strahlen der Endkieme lang, mit jederseits etwa sieben, selbst sekundär wieder zerteilten Fransen, derartige Fransen auch auf der Dorsal- und Ventralseite der viereckigen Leiste der Endkieme. Saugarme ziemlich lang und schlank, jeder mit meist After 7, selten 6 Saugnäpfen, von welchen die vier proximalen, nahe der Basis, die größeren sind und sich auf der ventralen Seite des (ausgestreckten) u. Ben. Saugarmes inserieren, während die Seitenkieme drei distalen sehr klein und auf der Dorsalseite und an der Spitze des Auer Armes angeheftet sind. Formel der ig. 81. { : s p P. mediterraneum. Von der Ventralseite, mit Radula 6. 0. 6. Hakensäcke sehr hervorgestreckten Schlundorganen (%,). Nach lang. Farbe braunviolett oder rot- Pelseneer. braun. L. bis 17 mm — Fig. 81. - Endkieme Körper gestreckt. Mittellappen des Fußes lang, mehr gestreckt als es bei den anderen Arten dieser Gattung der Fall ist. Seitenkieme kurz, mit gefransten Seitenkämmen. Die vier Strahlen der Endkieme lang, mit jederseits etwa sieben, selbst sekundär wieder zerteilten Fransen, derartige Fransen auch auf der Dorsal- und Körper gestreckt. Mittellappen des Fußes lang, mehr gestreckt als es bei den anderen Arten dieser Gattung der Fall ist. Seitenkieme kurz, mit gefransten Seitenkämmen. Die vier Strahlen der Endkieme lang, mit jederseits etwa sieben, selbst sekundär wieder zerteilten Fransen, derartige Fransen auch auf der Dorsal- und Ventralseite der viereckigen Leiste der Endkieme. Saugarme ziemlich lang und schlank, jeder mit meist After 7, selten 6 Saugnäpfen, von welchen After u. Ben. Seitenkieme Auer ig. 81. P. mediterraneum. Von der Ventralseite, mit hervorgestreckten Schlundorganen (%,). Nach Pelseneer. - Endkieme After Auer ig. 81. P. mediterraneum. Von der Ventralseite, mit hervorgestreckten Schlundorganen (%,). Nach Pelseneer. 3. Gen. Pneumoderma G. Cuvier : Saugnäpfe auf sehr verschieden langen Stielen und un- gleich gebaut: jederseits auf dem Saugarme eine Gruppe sehr kleiner, kurzgestielter Saugnäpfe, am Ende ein Bündel lang-becherförmiger Saugnäpfe auf semlanpen Stielen (His: 84). un. 000. 0. 4. P. heterocotylum Saugnäpfe auf ungefähr iikasen Selen und gleich gebaut — 4. Jeder Saugarm mit 16—30 Saugnäpfen, welche zwei- zeilig angeordnet sind und von der Basis bis zur Spitze des Armes an Größe abnehmen (Fig. 85) . EL 5. P. eurycotylum \| Jeder en er: Mchr a 30 Sluenäpfen, le ar "von gleicher Größe — 5. Jeder Saugarm mit etwa 40 Saugnäpfen (Fig. 82) .. . 2. P. violaceum { Jeder Saugarm mit etwa 80—100 Saugnäpfen (Fig. 83) 3. P. peronii Fransen an den Strahlen der Endkieme wohl markiert, scharf geschieden und an der Basis eingeschnürt(Fig.86) 6. P. boasi Fransen an den Strahlen der Endkieme nicht scharf markiert, nur kleine Erhebungen der Ränder dar- stellend — 7. Saugarme sehr kurz, jederseits mit etwa 8 Saugnäpfen (Fig. 89) . ar » » 9. P. pygmaeum Saugarme er mit zuhlr Ahern (50—7 5) en —8. 4 5 6 7 Die vier Strahlen der Endkieme lang, mit zahlreichen, sekundär zerteilten Fransen — 2. 112 112 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma Saugarme sehr lang, mit aba Dragon Rändern, jederseits mit etwa 75 Saugnäpfen (Fig. 88) . . . 8. P. souleyeti 1. P. mediterraneum (Bened.) 1838 Pneumodermon m., P. J. Beneden in: Möm. Ac. Belgique, v.11 \|nr.20] p.13 t.3 f.1,2 \| 1852 P. peronii (part., non Lamarck 1819), Souleyet in: Voy. Bonite, v.2 p.274, Moll. t. 14 f. 13, 14,16 \| ?1852 P.p. (part., non Lamarck 1819), Rang in: Rang & Souleyet, Hist. nat. Pter., t.11 f.14—19 \| 1859 Spongiobranchia australis (part., non Orbigny 1840), Chenu, Man. Conchyl., «.1 p. 116 f.509 \| 1873 Pneumodermon peronii (non Lamarck 1819), O. G. Costa, Fauna Reg. Napoli, An. moll. Pter. p. 22 t.5 f.1—3 \| 1885 Pneumoderma mediterraneum, P. Fischer, Man, Conchyl., p.423 \| 1886 Pneumodermon macrocotylum, Boas in: Danske Selsk. Skr., ser.6 v.4 p.152 \| 1886 P. audebardi (non Rang 1852), Locard, Prodr. Malae. franc., Moll. mar. p. 19 \| 1887 Pneumonoderma mediterraneum, Pelseneer in: Rep. Voy. Challenger, Zool. v. 19 pars58 p.26 t.1f 8; t.2 f.1. s Fußes lang, mehr gestreckt als es der Fall ist. Seitenkieme kurz, mit gefransten Seitenkämmen. 3. Gen. Pneumoderma G. Cuvier In der westlichen Hälfte des Mittelmeeres und in der Adria, weiter in der Warmwasserzone des Atlantischen und Indischen Ozeanes, auch im Indo-Australischen Archipel und in der China-See. 2. P. violaceum (Orb.) ?1815 Pneumodermis atlantica, Oken, Lehrb. Naturg., v.1 p.327 \| 1832 Pneumodermon peronii (non Lamarck 1819), Quoy & Gaimard in: Voy. Astrol., v.2 p.888t.28 f. 1—6 \| 1840 P. violaceum, Orbigny, Voy. Amer. merid., v.5ıu p. 129 t.9 f. 10—15 \| 1850 P. cucullatum, J. E. Gray, Cat. Moll. Brit. Mus., p-39 \| 1852 P. peroni (part., non Lamarck 1819), Souleyet in: Voy. Bonite, v.2 p.274, Moll. t.14 f. 12,15 \| 1352 P. audebardii (non Locard 1886), Rang in: Rang & Souleyet, Hist. nat. Pter., t.10 f.13 \| 1859 Spongiobranchia australis (part., non Orbigny 1840), Chenu, Man. Conchyl., v.1 p.116 f.508 \| 1879 Cirrifer paradoxus, Pfeffer in: Monber. Ak. Berlin, p. 249 f.20 \| 1887 Pneumonoderma violaceum, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars58 p.28 t.1 f.9 \| 1905 Pneumoderma v., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 49. Körper gestreckt. Mittellappen des Fußes lang, aber nicht so ausgezogen wie bei der vorhergehenden Art. Seitenkieme kurz, die Seitenkämme ohne Körper gestreckt. Mittellappen des Fußes lang, aber nicht so ausgezogen wie bei der vorhergehenden Art. Seitenkieme kurz, die Seitenkämme ohne 113 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma Fransen, Strahlen der Endkieme lang, jeder mit etwa sieben sekundär wieder zerteilten Fransen jederseits, die viereckige Leiste der Endkieme ohne Fransen. Saugarme dreieckig, ziemlich lang, jeder mit etwa 40 kleinen, wenig an Größe Flosse .-. After "7 Seitenkieme Endkieme A Flosse .-. After "7 Saugarm Di Flosse Seitenkieme Endkieme A B Fig. 82. P. violaceum. A von der rechten Seite (#/,). Nach Pelseneer. B vorderer Körperteil, von der Ventralseite, mit hervorgestrecktem Schlundapparat (2/,).,. Nach Souleyet. B A Fig. 82. P. violaceum. A von der rechten Seite (#/,). Nach Pelseneer. B vorderer Körperteil, von de Ventralseite, mit hervorgestrecktem Schlundapparat (2/,).,. Nach Souleyet. . violaceum. A von der rechten Seite (#/,). Nach Pelseneer. B vorderer Körperteil, von der Ventralseite, mit hervorgestrecktem Schlundapparat (2/,).,. Nach Souleyet. abweichenden Saugnäpfen auf der medianen Seite. Die vollständige Zahl der Saugnäpfe wird erst bei ganz erwachsenen Tieren erreicht; Exemplare von 4—6 mm Länge haben jederseits nur 15—20 Saugnäpfe. Farbe bräunlich, Pigment namentlich auf der rechten Seite des Körpers, bisweilen bis auf geringe Reste fehlend. L. bis 13 mm. — Fig. 82. abweichenden Saugnäpfen auf der medianen Seite. 3. Gen. Pneumoderma G. Cuvier Die vollständige Zahl der Saugnäpfe wird erst bei ganz erwachsenen Tieren erreicht; Exemplare von 4—6 mm Länge haben jederseits nur 15—20 Saugnäpfe. Farbe bräunlich, Pigment namentlich auf der rechten Seite des Körpers, bisweilen bis auf geringe Reste fehlend. L. bis 13 mm. — Fig. 82. In der Warmwasserzone des Atlantischen Ozeanes, von etwa 15°s. Br. bis 45° n. Br., auch in der westlichen Hälfte des Mittelmeeres. 3. P. peronii (Lm.) ?1815 Aegle cucullata, Oken, Lehrb. Naturg., v.1 p. 327 \| 1819 Pneumodermon peronii (non Quoy & Gaimard 1832, Souleyet 1852, Rang 1852, Costa 1873, Verrill 1885), Lamarck, Hist. An. s. Vert., v.61 p.294 \| 1883 Pneumoderma D., P. Fischer, Man. Conchyl., p.423 \| 1887 Pneumonoderma peroni, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars58 p.29 t.2 f.2 \| 1905 Pneumoderma p., Meisen- heimer in: Ergeb. Tiefsee-Exp., v.91 p. 50. Körper gedrungen. Mittellappen des Fußes ziemlich kurz. Seitenkieme kurz, mit gefransten Seitenkämmen, Endkieme mit langen Strah- len, welche jederseits etwa 12 dichtgedrängte und sekun- där wieder verteilte Fransen tragen, keine oder undeut- liche Fransen auf der vier- eckigen Leiste der Endkieme. Saugarme dreieckig und breit, aber in der allgemeinen Ge- stalt ziemlich veränderlich, die mediane Seite des (her- vorgestreckten) Armes dicht \ mit etwa 80—100 kleinen seitenkieme Saugnäpfen besetzt, welche noch kleiner sind als bei ‚Seitenlappen des Fußes „ Flosse 2 Se N Mittellappen After - des Fußes S -------Endkieme Fig. 83. der vorhergehenden Art und P. peronii. Von der Ventralseite (4/,). Nach Pelseneer. Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 8 Körper gedrungen. Mittellappen des Fußes ziemlich kurz. Seitenkieme kurz, mit gefransten Seitenkämmen, Endkieme mit langen Strah- len, welche jederseits etwa 12 dichtgedrängte und sekun- där wieder verteilte Fransen tragen, keine oder undeut- liche Fransen auf der vier- eckigen Leiste der Endkieme. Saugarme dreieckig und breit, aber in der allgemeinen Ge- stalt ziemlich veränderlich, die mediane Seite des (her- vorgestreckten) Armes dicht \ mit etwa 80—100 kleinen seitenkieme Saugnäpfen besetzt, welche noch kleiner sind als bei ‚Seitenlappen des Fußes „ Flosse 2 Se N Mittellappen After - des Fußes S -------Endkieme Fig. 83. der vorhergehenden Art und P. peronii. Von der Ventralseite (4/,). Nach Pelseneer. Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 8 \ seitenkieme ‚Seitenlappen des Fußes „ Flosse 2 Se N Mittellappen After - des Fußes S -------Endkieme Fig. 83. P. peronii. Von der Ventralseite (4/,). Nach Pelseneer. esch, Pteropoda. 3. Gen. Pneumoderma G. Cuvier 8 ‚Seitenlappen des Fußes „ Flosse ‚Seitenlappen des Fußes „ Flosse Mittellappen des Fußes noch kleiner sind als bei Fig. 83. der vorhergehenden Art und P. peronii. Von der Ventralseite (4/,). Nach Pelseneer. Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 8 Das Tierreich. 36. Lief.: J. J. Tesch, Pterop Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 114 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma auf sehr kontraktilen Stielen sitzen. Formel der Radula 4. 0.4. Farbe blaugrau, Pigment auf zahlreiche, kleine Flecke verteilt. L. bis etwa 15 mm. — Fig. 83. auf sehr kontraktilen Stielen sitzen. Formel der Radula 4. 0.4. Farbe blaugrau, Pigment auf zahlreiche, kleine Flecke verteilt. L. bis etwa 15 mm. — Fig. 83. auf sehr kontraktilen Stielen sitzen. Formel der Radula 4. 0.4. Farbe blaugrau, Pigment auf zahlreiche, kleine Flecke verteilt. L. bis etwa 15 mm. — Fig. 83. Scheint im südlichen Atlantischen Ozean P. violaceum zu vertreten, da sie hier nur südlich vom Aquator und zwar von 26° bis 38° s. Br. vorkommt. Weiter im ganzen Indischen Ozean bis 40° s. Br., im Indo-australischen Archipel und nördlich von Neuguinea. 4. P. heterocotylum (Tesch) 1903 Pneumonoderma h., Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p.114 \| 1904 P. h., Tesch in: Siboga-Exp., nr. 52 p-68 t.5 £.118, 119 \| 1905 Pneumoderma h., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p.50. Körper gedrungen (bei dem einzigen bekannten Exemplar stark zusammen- gezogen). Mittellappen des Fußes mäßig lang. Seitenkieme kurz, mit ge- fransten Seitenkämmen, die vier Strahlen der Endkieme lang, jeder mit 5 oder 6 sekundär wieder fein zerteilten Fransen, Dorsal- und Ventralseite der vier- eckigen Leiste der Endkieme mit sehr kleinen und zahlreichen Fransen. Saug- arme sehr kurz und breit, Saugnäpfe verschieden gebaut. Jederseits an der Körper gedrungen (bei dem einzigen bekannten Exemplar stark zusammen- gezogen). Mittellappen des Fußes mäßig lang. Seitenkieme kurz, mit ge- fransten Seitenkämmen, die vier Strahlen der Endkieme lang, jeder mit 5 oder 6 sekundär wieder fein zerteilten Fransen, Dorsal- und Ventralseite der vier- eckigen Leiste der Endkieme mit sehr kleinen und zahlreichen Fransen. Saug- arme sehr kurz und breit, Saugnäpfe verschieden gebaut. Jederseits an der A C D Fig. 84. P. heterocotylum. A Tier von der Ventralseite (%/,), 3 Seitenkieme, C Endkieme von der Dorsal- seite, D Saugarm, hervorgestreckt, von der Medianseite. Nach Tesch (vergr.?). A C D Fig. 84. P. heterocotylum. 3. Gen. Pneumoderma G. Cuvier A Tier von der Ventralseite (%/,), 3 Seitenkieme, C Endkieme von der Dorsal- seite, D Saugarm, hervorgestreckt, von der Medianseite. Nach Tesch (vergr.?). C A D D A Fig. 84. P. heterocotylum. A Tier von der Ventralseite (%/,), 3 Seitenkieme, C Endkieme von der Dorsal- seite, D Saugarm, hervorgestreckt, von der Medianseite. Nach Tesch (vergr.?). Basis des Saugarmes eine Gruppe von sehr kleinen, kurzgestielten, napfförmigen Saugnäpfen, etwa 75 an der Zahl. An der Spitze des Armes etwa 30 außer- ordentlich langgestielte, becherförmige, dünnwandige Saugnäpfe. Formel der kadula unbekannt. Hakensäcke lang, innere Hakenscheide halb so lang wie die äußere, mit etwa 20 stark gekrümmten Haken. Farbe gelblich, Kopf etwas mehr bräunlich. L. 5 mm. — Fig. 84. Bisher nur ein einziges Exemplar im östlichen Teile des Indo-australischen Archipels beobachtet. 5. P.eurycotylum Meisenh. 1905 P.e., Meisenheimer in: Ergeb. Tiefsee- Exp., v.9ı p.5l. 5. P.eurycotylum Meisenh. 1905 P.e., Meisenheimer in: Ergeb. Tiefsee- Exp., v.9ı p.5l. „Seitenlappen (des Fußes) stark entwickelt, langgestreckt, Mittellappen ziemlich lang, abgestumpft. Seitenkieme mit langer Seiten- wie Mittelfalte, aber ohne Fransen, Endkieme mit vier ziemlich langen Strahlen, die deutliche Fransenbildung aufweisen. Jeder Saugarm besitzt 16—20, bei einem größeren Exemplar 25—30 Saugnäpfe, die mit langen Stielen zweizeilig dem Arme aufsitzen und an der Basis sehr breit und mächtig sind, nach der Spitze hin „Seitenlappen (des Fußes) stark entwickelt, langgestreckt, Mittellappen ziemlich lang, abgestumpft. Seitenkieme mit langer Seiten- wie Mittelfalte, aber ohne Fransen, Endkieme mit vier ziemlich langen Strahlen, die deutliche Fransenbildung aufweisen. Jeder Saugarm besitzt 16—20, bei einem größeren Exemplar 25—30 Saugnäpfe, die mit langen Stielen zweizeilig dem Arme aufsitzen und an der Basis sehr breit und mächtig sind, nach der Spitze hin 115 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma dagegen stetig und bedeutend an Größe abnehmen. Radula und Hakensäcke unbekannt. L. 3—6 mm“ (Meisenheimer). — Fig. 85. dagegen stetig und bedeutend an Größe abnehmen. Radula und Hakensäcke unbekannt. L. 3—6 mm“ (Meisenheimer). — Fig. 85. Nur in zwei Exemplaren im inneren Teile des Golfes von Guinea erbeutet, ein weiteres Exemplar auf 11° s. Br. 19° w. L. = = \ Fig. 85. P. euryeotylum. Saugarm, von der Seite. Nach Meisenheimer (vergr.?). Seitenkieme ” Endkieme Fig. 86. P.boasi. Von der Ventralseite (5/,). Nach Pelseneer. Fig. 85. Fig. 85. Fig. 86. P.boasi. Von der Ventralseite (5/,). Nach Pelseneer. Fig. 85. P. euryeotylum. Saugarm, von der Seite. Nach Meisenheimer (vergr.?). 6. P. boasi (Plisnr.) 1886 Pneumodermon violaceum (part.), Boas in: Danske Selsk. Skr., ser.6 v.4 p.154 \| 1887 Pneumonoderma boasi, Pelseneer in: Rep. Voy. Challenger, Zool: v.19 pars58 p.30 t.2 f.3 \| 1905 Pneumoderma b., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p. 50. Formel der Radula 4. 0. 4. Hakensäcke lang. Farbe purpurbraun, namentlich auf der rechten Körper- seite, Pigment als kleine Flecke zerstreut. L. bis etwa 25 mm. — Fig. 87. Im nördlichen Teile des Pazifischen Ozeanes, zwischen 33° und 58° n. Br., an der Westamerikanischen Küste am weitesten, bis zur Küste von Alaska, vordringend. 8. P. souleyeti (Plsur.) 1887 Pneumonoderma s., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.31 t.2 f.6 \| 1905 Pneumoderma s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 50. 8. P. souleyeti (Plsur.) 1887 Pneumonoderma s., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.31 t.2 f.6 \| 1905 Pneumoderma s., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 50. h im vorderen Abschnitt des Rumpfes ziemlich breit. Mittellappen des Fußes mäßig lang. Seitenkieme langgestreckt, mit kaum gefransten Seitenkämmen, Endkieme mit sehr großer, viereckiger, vorspringender Leiste und außerordent- lich kurzen Strahlen, welche nur wellen- förmige Erhebungen ihres Randes, keine eigentlichen Fransen tragen. Saug- arme sehr lang und schlank, ebenso lang wie der Körper, mit nahezu parallel verlaufenden Rändern, jeder trägt etwa 75 kleine, kurzgestielte Saugnäpfe, welche von der Basis bis zur Spitze allmählich an Größe ab- nehmen. Radula und Hakensäcke un- pekannt. Farbe (bei dem einzigen be- kannten Exemplar) bräunlich, namentlich “ . auf der rechten Körperseite. L. 4 mm. — Fig. 88. Körper gedrungen, namentli „„. Endkieme After Seitenkieme Fig. 88. Körper gedrungen, namentlich im vorderen Abschnitt des Rumpfes ziemlich breit. Mittellappen des Fußes mäßig lang. Seitenkieme langgestreckt, mit kaum gefransten Seitenkämmen, Endkieme mit sehr großer, viereckiger, vorspringender Leiste und außerordent- lich kurzen Strahlen, welche nur wellen- förmige Erhebungen ihres Randes, keine eigentlichen Fransen tragen. Saug- arme sehr lang und schlank, ebenso lang wie der Körper, mit nahezu parallel verlaufenden Rändern, jeder trägt etwa 75 kleine, kurzgestielte Saugnäpfe, welche von der Basis bis zur Spitze allmählich an Größe ab- nehmen. Radula und Hakensäcke un- „„. Endkieme pekannt. Farbe (bei dem einzigen be- kannten Exemplar) bräunlich, namentlich After Seitenkieme Fig. 88. “ . P.souleyeti. Vonder Ventralseite, mit hervor- auf der rechten Körperseite. L. 4 mm. gestreckten Saugarmen (?),). Nach Pelsener. — Fig. 88. Fig. 88. P.souleyeti. Vonder Ventralseite, mit hervor- gestreckten Saugarmen (?),). Nach Pelsener. P.souleyeti. Vonder Ventralseite, mit hervor- gestreckten Saugarmen (?),). Nach Pelsener. Nur aus dem nordwestlichen Teile des Pazifischen ÖOzeanes (35° 13° n. Br., 154° 43‘ w. L.) bekannt. 9. P. pygmaeum (Tesch) 1903 Pneumonoderma p., Tesch in: Tijdschr. Neder!l. dierk. 6. P. boasi (Plisnr.) 1886 Pneumodermon violaceum (part.), Boas in: Danske Selsk. Skr., ser.6 v.4 p.154 \| 1887 Pneumonoderma boasi, Pelseneer in: Rep. Voy. Challenger, Zool: v.19 pars58 p.30 t.2 f.3 \| 1905 Pneumoderma b., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p. 50. 6. P. boasi (Plisnr.) 1886 Pneumodermon violaceum (part.), Boas in: Danske Selsk. Skr., ser.6 v.4 p.154 \| 1887 Pneumonoderma boasi, Pelseneer in: Rep. Voy. Challenger, Zool: v.19 pars58 p.30 t.2 f.3 \| 1905 Pneumoderma b., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p. 50. Körper gedrungen. Mittellappen des Fußes ziemlich lang. Seitenkieme kurz, mit kurzen Fransen am freien Rande und gefransten Seitenkämmen. Endkieme mit kurzen Strahlen, welche jederseits drei ziemlich weit auseinander gestellte, an der Basis eingeschnürte und nicht sekundär zerteilte Fransen tragen. Saugarme mit etwa 40 Saugnäpfen in derselben Anordnung und von der gleichen Größe wie diejenigen von /. violaceum. Hakensäcke ziemlich kurz. Radula unbekannt. Farbe bräunlich, Pigmentflecke zerstreut, mit dem bloßen Auge sichtbar. L. etwa 8 mm. — Fig. 86. Nur an der Westküste Südamerikas, bei Caldera (Chili) und im Golfe von Guinea gefunden. 7.P.pacificum(W.Dall) 1871 Pneumodermon p., W.Dallin: Amer. J. Conch.,v.7 11 p. 189 \| 1887 Pneumonoderma p., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p-30 t.2£.4, 5 \| 1905 Pneumoderma p., Meisenheimer in: Ergeb. Tiefsee-Exp., v.91ı p.50. ventrale Rüsselpapille Rüssel ------\-.. PR % 4 Saugarım vorderer Tentakel --"""") IA Öffnung des Penis Flosse -.------ "=. Seitenlappen des Fußes Re Mittellappen des Fußes Seitenkamm der Seitenkieme ..._..\ E W 7 ee Seitenkieme RE Endkieme Fig. 87. P. pacificum,. Von der rechten Seite mit hervorgestrecktem Schlundapparat (2). Nach Pelseneer. Mittelkamm der Seitenkieme eme Fig. 87. P. pacificum,. Von der rechten Seite mit hervorgestrecktem Schlundapparat (2). Nach Pelseneer. Körper ziemlich gedrungen. Mittellappen des Fußes mäßig lang. Seiten- kieme langgestreckt-dreieckig, mit gefransten Seitenkämmen, Endkieme mit 8* 116 Pteropoda: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma vier kurzen Strahlen, mit wenigen (2 oder 3 jederseits), nicht sekundär wieder zerteilten Fransen, von welchen diejenigen auf der einen Seite des Strahles mit denen auf der anderen Seite alternieren. Saugarme dick und breit an der Basis, dreieckig, mit etwa 50 kleinen, sehr kurzgestielten Saugnäpfen auf der medianen Seite (bei hervorgestrecktem Arme). Formel der Radula 4. 0. 4. Hakensäcke lang. Farbe purpurbraun, namentlich auf der rechten Körper- seite, Pigment als kleine Flecke zerstreut. L. bis etwa 25 mm. — Fig. 87. vier kurzen Strahlen, mit wenigen (2 oder 3 jederseits), nicht sekundär wieder zerteilten Fransen, von welchen diejenigen auf der einen Seite des Strahles mit denen auf der anderen Seite alternieren. Saugarme dick und breit an der Basis, dreieckig, mit etwa 50 kleinen, sehr kurzgestielten Saugnäpfen auf der medianen Seite (bei hervorgestrecktem Arme). 6. P. boasi (Plisnr.) 1886 Pneumodermon violaceum (part.), Boas in: Danske Selsk. Skr., ser.6 v.4 p.154 \| 1887 Pneumonoderma boasi, Pelseneer in: Rep. Voy. Challenger, Zool: v.19 pars58 p.30 t.2 f.3 \| 1905 Pneumoderma b., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p. 50. Nicht sicher ob hierher oder zu Oliopsis gehörig. 6. P. boasi (Plisnr.) 1886 Pneumodermon violaceum (part.), Boas in: Danske Selsk. Skr., ser.6 v.4 p.154 \| 1887 Pneumonoderma boasi, Pelseneer in: Rep. Voy. Challenger, Zool: v.19 pars58 p.30 t.2 f.3 \| 1905 Pneumoderma b., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p. 50. Ver., ser.2 v.8 p.115 \| 1904 P. p., Tesch in: Siboga-Exp., nr. 52 p-68 t.5 f. 120—122 \| 1905 Pneumoderma p., Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 50. SR: SR R: S Fig. 89. ygmaeum. A von der Ventralseite (%/,), B Endkieme, von unten, € linker Saugarm, von der Ventralseite. Nach Tesch (vergr.?). Fig. 89. P.pygmaeum. A von der Ventralseite (%/,), B Endkieme, von unten, € linker Saugarm, von der Ventralseite. Nach Tesch (vergr.?). Körper gedrungen. Mittellappen des Fußes ziemlich lang, an der Basis breit. Seitenkieme sehr kurz, mit gefransten Seitenkämmen, viereckige Leiste der Pterop.: B. Gymnosomata, 1. Pneumodermatidae, 3. Pneumoderma, 4.Schizobrachium 117 Endkieme an der Dorsal- und an der Ventralseite gefranst, auch die vier kurzen Strahlen je mit zwei sehr kurzen, nicht sekundär wieder zerteilten Fransen, aber nur auf der medianen Seite. Saugarme kurz, jeder mit etwa 8 Saug- näpfen, welche auf langen Stielen sitzen (Fig. 89 C) und meist ziemlich groß sind, ohne daß aber eine bestimmte Regelmäßigkeit in der Anordnung nach der Größe vorkommt. Radula unbekannt. Hakensäcke außerordentlich lang, fast bis zum Körperende reichend, mit etwa 40 großen Haken. Farbe grau- selb. L. 3 mm. — Fig. 89. Bisher nur in einigen Exemplaren im östlichen Teile des Indo-australischen Archipels erbeutet. Endkieme an der Dorsal- und an der Ventralseite gefranst, auch die vier kurzen Strahlen je mit zwei sehr kurzen, nicht sekundär wieder zerteilten Fransen, aber nur auf der medianen Seite. Saugarme kurz, jeder mit etwa 8 Saug- näpfen, welche auf langen Stielen sitzen (Fig. 89 C) und meist ziemlich groß sind, ohne daß aber eine bestimmte Regelmäßigkeit in der Anordnung nach der Größe vorkommt. Radula unbekannt. Hakensäcke außerordentlich lang, fast bis zum Körperende reichend, mit etwa 40 großen Haken. Farbe grau- selb. L. 3 mm. — Fig. 89. Bisher nur in einigen Exemplaren im östlichen Teile des Indo-australischen Archipels erbeutet. Pneumodermon ruber Q.&G. 1832 P.r., Quoy & Gaimard in: Voy. Astrol., v.2 p.889 1.27 f. 19—24. Pneumodermon ruber Q.&G. 1832 P.r., Quoy & Gaimard in: Voy. Astrol., v.2 p.889 1.27 f. 19—24. Pneumodermon ruber Q.&G. 1832 P.r., Quoy & Gaimard in: Voy. Astrol., v.2 p.889 1.27 f. 19—24. Nicht sicher ob zu Pneumoderma oder zu Cliopsis gehörig. Nicht sicher ob zu Pneumoderma oder zu Cliopsis gehörig. Amboina. P, pellueidum Q.&G. 1832 P. p,, Quoy & Gaimard in: Voy. Astrol, v2 p. 390 t. 27 8.25. Nicht sicher ob hierher oder zu Oliopsis gehörig. 1903 Schizobrachium, Meisenheimer in: Zool. Anz., v.26 p. 410. Nach Meisenheimer (vergr.?). Fig. 90. S. polycotylum. A Tier von der Ventralseite, mit ausgestreckten Saugarmen (?,), B Schema der Verzweigung der Saugarme. Nach Meisenheimer (vergr.?). Nur ein einziges Exemplar im südlichen Indischen Ozean (34° 14‘ s. Br., 80% 31’ ö. L.), nördlich von der Insel Neu-Amsterdam erbeutet. 1903 Schizobrachium, Meisenheimer in: Zool. Anz., v.26 p. 410. 1903 Schizobrachium, Meisenheimer in: Zool. Anz., v.26 p. 410. Körper lang gestreckt, in der Mitte des Rumpfes bauchig erweitert, am Hinterende zugespitzt; mit wohlentwickeltem Drüsenfeld auf dem Rücken. Eingeweidesack sehr wahrscheinlich nicht bis zum aboralen Körperpol reichend. Vordere Tentakeln mäßig lang. Zwei Saugarme, welche sich sehr stark ver- zweigen und mit zahlreichen Saugnäpfen ausgestattet sind. Hakensäcke äußerlich gut ausgebildet, innere Hakenscheide aber sehr klein und schwach. Radula mit Mittelplatte und zahlreichen Seitenplatten in jeder Querreihe. Mittellappen des Fußes wohl entwickelt, an der Spitze abgerundet. Seitenkieme fehlt vollständig, Endkieme in Gestalt einer sehr kleinen Längsfalte auf der Ventral- seite des hinteren Körperendes. Anus weit vom Osphradium entfernt, auf der rechten Körperseite nach hinten verschoben. Im südliehen Indischen Ozean. 1 Art. Im südliehen Indischen Ozean. 1 Art. 1. S. polycotylum Meisenh. 1903 $. p., Meisenheimer in: Zool. Anz., ©.26 p-410 \| 1905 S. p., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p.51 t.16 £.5, 6; t.27 f.8, 11 (Verzweigung der Saugarme) 14 (Radula). 1. S. polycotylum Meisenh. 1903 $. p., Meisenheimer in: Zool. Anz., ©.26 p-410 \| 1905 S. p., Meisenheimer in: Ergeb. Tiefsee-Exp., v. 9ı p.51 t.16 £.5, 6; t.27 f.8, 11 (Verzweigung der Saugarme) 14 (Radula). Mit den Merkmalen des Genus. Seitenlappen des Fußes ohne gefalteten Mittelhöcker. Saugarme zwei umfangreiche, abgeplattete, sich dichotomisch verästelnde Organe, überall von Saugnäpfen besetzt, welche von der Basis bis zur Spitze des Armes stetig an Umfang abnehmen. Formel der Radula 7. 1. 7., Mittelplatte sehr klein, mit zwei nach hinten gerichteten Zähnchen, Seitenplatten mit einem sehr langen, kräftigen Zahne; äußere Hakenscheide aus einer umfangreichen Muskelmasse bestehend, die allein fast den ganzen 18 Pterop.: B. Gymnosomata, 1. Pneumodermatidae, 4. Schizobrachium, 2. Cliopseidae 118 Hakensack ausmacht, während die innere Scheide eine kleine, sehr wenig tiefe Grube darstellt. Farbe unbekannt. L. bis 32 mm Br. bis 11 mm. — Fig. 90. Saugarme . vorderer Tentakel - Flosse Seitenlappen des Fußes ..-. Osphradium ----- --- Mittellappen des Fußes After { Endkieme A B Fig. 90. Hakensack ausmacht, während die innere Scheide eine kleine, sehr wenig tiefe Grube darstellt. Farbe unbekannt. L. bis 32 mm Br. bis 11 mm. — Fig. 90. Saugarme . vorderer Tentakel Flosse Mittellappen des Fußes Fig. 90. S. polycotylum. A Tier von der Ventralseite, mit ausgestreckten Saugarmen (?,), B Schema der Verzweigung der Saugarme. Nach Meisenheimer (vergr.?). Fig. 90. S. polycotylum. A Tier von der Ventralseite, mit ausgestreckten Saugarmen (?,), B Schema der Verzweigung der Saugarme. 2. Fam. Cliopseidae 1855 Clioidea (part.), Gegenbaur, Unters. Pter. & Heterop., p.212 \| 1856 Clio- nacea (part.), Troschel, Gebiss Schneck., v.1 p.54 \| 1858 Pneumodermidae (part.), H. & A. Adams, Gen. Moll., v.2 p.613 \| 1862 Clionidae (part.), Bronn in: Bronn’s Kl. Ordn., v.31ı1 p. 645 \| 1873 Clionopsidei, O. G. Costa, Fauna Reg. Napoli, An. moll. Pter. p. 24 \| 1887 Olionopsidae, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.33 \| 1889 Cliopsidae, W.Dall in: Bull. U. S. Mus., nr. 37 p. 82. Körper zylindrisch, am Hinterende breit abgerundet. Kopf- abschnitt verhältnismäßig klein, durch eine Einschnürung vom Rumpfabschnitt getrennt. Haut durchsichtig, mit zahlreichen Drüsen, nieht pigmentiert. Dorsales Drüsenfeld fast immer vor- handen. Der Eingeweidesack reicht als ein dicker Stab bis zum aboralen Körperpol. Vordere Tentakeln kurz und dick. Saugarme oder sonstige Schlundorgane fehlen vollständig, dagegen ist der Schlund selbst außerordentlich lang und kann ganz hervorge- streckt werden, wobei dann Radula, Hakensäcke und Speichel- drüsen ganz an die Spitze zu liegen kommen. Radula immer mit dreispitziger Mittelplattee Hakensäcke sehr kurz, schwach ent- wickelt. Oberkiefer vorhanden. Seitenlappen des Fußes wohl entwickelt, auf ihrer ganzen Länge mit dem Körper verwachsen, in der distalen Hälfte einen gefältelten Höcker zwischen sich fassend, Mittellappen des Fußes vollständig fehlend. Flossen verhältnismäßig klein. Seitenkieme fehlt, Endkieme eine ungefähr sechseckige, in der dorso-ventralen Richtung ausgezogene, vor- springende Leiste darstellend, von deren dorsalen und ventralen Ecken je ein kurzer, nach vorn gerichteter Strahl entspringt. Pteropoda: B. Gymnosomata, 2. Cliopseidae, 1. Cliopsis 119 After mit Osphradium und äußerer Nierenöffnung zusammen auf dem Analfelde Herz und Niere auf der rechten Körperseite. After mit Osphradium und äußerer Nierenöffnung zusammen auf dem Analfelde Herz und Niere auf der rechten Körperseite. In den warmen Stromgebieten der Ozeane. Enthält nur die einzige Gattung Cliopsis. 1. Gen. Cliopsis Troschel Seiten- lappen des Fußes in ganzer Länge mit dem Körper verwachsen, am distalen Rande nicht hervorragend; der gefältelte Höcker zwischen den Seitenlappen nicht durch eine mediane Falte in zwei Teile zerlegt. Flossen klein, am freien Rande abgerundet. Endkieme mit vier nach vorn gerichteten Strahlen, welche ebenso wie die sechseckige Leiste der Endkieme ganz ohne Fransen sind. Schlund sehr lang, wenn hervorgestreckt etwa dreimal so lang wie der Körper selbst. Formel der Radula 6. 1. 6, die Zahl der Seitenplatten nimmt zu, bis das Tier erwachsen ist. Hakensäcke klein, jeder mit etwa 60, in einem Bündel zusammengestellten Haken. Farbe glashell, mit zahlreichen hellen und braunen Stellen, wo einzellige Drüsen, meist in Gruppen von 3 oder 4 beieinander, gelegen sind. Der stabförmige Eingeweidesack schimmert bräunlich durch. L. bis etwa 24 mm. — Fig. 91. 4 beieinander, gelegen sind. Der stabförmige Eingeweidesack s h durch. L. bis etwa 24 mm. — Fig. 91. n der westlichen Hälfte des Mittelmeeres. C. grandis Boas 1885 Pneumodermon peronii (non Lamarck .1 in: Tr. Connect. Ac., v.6 p.431 \| 1886 Cliopsis grandis, Boas in: Da r.6 v.4 p.170 \| 1887 Clionopsis g., Pelseneer in: Rep. Voy. Challe s 58 p.36 t.2 f.7,8. örper sehr breit, am Hinterende noch breiter abgerundet al ehenden Art. Kopfabschnitt klein. Hintere Tentakeln kleine nü. Seitenlappen des Fußes verhältnismäßig länger als ehenden Art und am distalen Rande ein Stück über die Verwa t dem Körper vorspringend, so daß hier der Rand einen scharf Der Höcker zwischen den Seitenlappen ist durch eine median grube in zwei Teile zerlegt kurz und breit, am fre abgerundet. Endkieme e eckige, vorspringende L etwas längeren Strahle “ Höcker der vorhergehenden Ar und Strahlen auf beid überall mit kurzen, nicht zerteilten Fransen bede des Penis .. „- Flosse After ur B. welchen diejenigen d ""—. Endkieme DNeite mit denen der Fig. 9. alternieren. Schlund e s. Von der Ventralseite (°/,). Nach Pelseneer. mal so lang wie der Formel der Radula 5 In der westlichen Hälfte des Mittelmeeres. In der westlichen Hälfte des Mittelmeeres. 2. C. grandis Boas 1885 Pneumodermon peronii (non Lamarck .1819), A. E. Verrill in: Tr. Connect. Ac., v.6 p.431 \| 1886 Cliopsis grandis, Boas in: Danske Selsk. Skr., ser.6 v.4 p.170 \| 1887 Clionopsis g., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.36 t.2 f.7,8. Körper sehr breit, am Hinterende noch breiter abgerundet als bei der vorhergehenden Art. Kopfabschnitt klein. 1. Gen. Cliopsis Troschel 1854 Clopsis, Troschel in: Arch. Naturg., v.201 p.222 \| 1855 Clio (part.), Gegenbaur, Unters. Pter. & Heterop., p.212 \| 1862 Clionopsis, Bronn in: Bronn’s Kl. ÖOrdn., v.3ı1 p.645 \| 1869 Trichocyclus (part., non Eschscholtz 1825), A. Costa in: Annuario Mus. Napoli, v.5 p.46 \| 1885 Pneumodermon (part., non Lamarck 1819), A.E, Verrill in: Tr. Connect. Ac., v.6 p. 431. Die Diagnose stimmt mit der der Familie überein. In den warmen Stromgebieten der Ozeane. 4 Arten. Bestimmungstabelle der Arten: 1 f Endkieme fehlend (Fig.99) . ... 2... 0000. 4. C. microcephala \ Endkieme vorhanden — 2. Tier ziemlich groß (bis mehr als 20 mm); Endkieme mit vier deutlichen Strahlen — 3. Tier sehr klein (etwa 3 mm); Endkieme mit kaum sicht- baren, sekr kurzen Strahlen (Fig.98) . . . ».... . 3. ©. modesta Strahlen der Endkieme nicht gefranst (Fig.91). .. . . 1. C. krohnii ' Strahlen der Endkieme gefranst (Fig. 92). . . .»..... 2. C. grandis 1. C.krohnii Troschel 1854 C.k., Troschel in: Arch. Naturg., v.201ı p.222 t.10 £.2—4 \| 1855 Clio mediterranea, Gegenbaur, Unters. Pter. & Heterop., p.212 t.4 f£.14 \| 1869 Trichocyclus mediterraneus, A. Costa in: Annuario Mus. Napoli, v.5 p.46 t.1 f.3 (Larve) \| 1873 Clionopsis krohniüi, O. G. Costa, Fauna Reg. Napoli, An. moll. Pter. p.25 t.5 £.7—9. . Rüssel Mittelplatte «ei 2 „Seitenplatte Seitenlappen des Fußes ; Nr eZ nr Elosse After * Endkieme A B Fig. 91. C. krohnii. A Tier von der Ventralseite, mit hervorgestrecktem Rüssel (3/,). Nach Pelseneer. B Querreihe der Radula. Nach Boas (vergr.?). Körper dick, gerundet. Haut dünn, sehr durchsichtig, mit zahlreichen . Rüssel «ei Seitenlappen des Fußes nr Elosse After * Endkieme A F . Rüssel Mittelplatte «ei 2 „Seitenplatte Seitenlappen des Fußes ; Nr eZ nr Elosse After * Mittelplatte 2 „Seitenplatte Nr eZ Rüssel . Rüssel Seitenlappen des Fußes nr Elosse Seitenlappen des Fußes nr Elosse Seitenlappen des Fußes nr Elosse After A B Fig. 91. C. krohnii. A Tier von der Ventralseite, mit hervorgestrecktem Rüssel (3/,). Nach Pelseneer. B Querreihe der Radula. Nach Boas (vergr.?). Körper dick, gerundet. Haut dünn, sehr durchsichtig, mit zahlreichen einzelligen Drüsen. Kopfabschnitt klein. Hintere Tentakeln hervorragend, Pteropoda: B. Gymnosomata, 2. Cliopseidae, 1. Oliopsis 120 mehr als bei irgendeiner anderen Art der Gymnosomata entwickelt. Körper oval, weniger breit am Hinterende abgerundet als bei den vorhergehenden Arten. Kopfabschnitt klein. Vordere Tentakeln kurz. Seiten- 3. C. modesta (Plsnr.) 1887 Clionopsis m., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars58 p.37 t.3 £.2. 1. Gen. Cliopsis Troschel Hintere Tentakeln kleiner als bei C. krohnü. Seitenlappen des Fußes verhältnismäßig länger als bei der vorhergehenden Art und am distalen Rande ein Stück über die Verwachsungs- linie mit dem Körper vorspringend, so daß hier der Rand einen scharfen Winkel bildet. Der Höcker zwischen den Seitenlappen ist durch eine mediane Längs- . grube in zwei Teile zerlegt. Flossen kurz und breit, am freien Rande abgerundet. Endkieme eine sechs- eckige, vorspringende Leiste, mit etwas längeren Strahlen als bei “ Höcker der vorhergehenden Art; Leiste und Strahlen auf beiden Seiten überall mit kurzen, nicht sekundär zerteilten Fransen bedeckt, von Öffnung des Penis .. „- Flosse After ur B. welchen diejenigen der einen ""—. Endkieme DNeite mit denen der anderen Fig. 9. alternieren. Schlund etwa drei- C. grandis. Von der Ventralseite (°/,). Nach Pelseneer. mal so lang wie der Körper. Formel der Radula 5.1.5 bei erwachsenen Exemplaren. Hakensäcke kurz, mit etwa 60 in einem Bündel zusammengestellten Haken. Farbe leicht bräunlich, mit zahlreichen hellen und ‘braunen Stellen, von einzelligen Drüsen herrührend, welche meist auf der hinteren Hälfte des Körpers und innerhalb der Leiste der Endkieme zerstreut sind. Übrigens zeigen Kopf, Fuß und Flossen eine etwas hläuliche, - opalisierende Färbung, der Schlund ist stärker braun, der Eingeweidesack braunviolett gefärbt. L. bis 35 mm. — Fig. 92. Körper sehr breit, am Hinterende noch breiter abgerundet als bei der vorhergehenden Art. Kopfabschnitt klein. Hintere Tentakeln kleiner als bei C. krohnü. Seitenlappen des Fußes verhältnismäßig länger als bei der vorhergehenden Art und am distalen Rande ein Stück über die Verwachsungs- linie mit dem Körper vorspringend, so daß hier der Rand einen scharfen Winkel bildet. Der Höcker zwischen den Seitenlappen ist durch eine mediane Längs- . grube in zwei Teile zerlegt. Flossen kurz und breit, am freien Rande abgerundet. Endkieme eine sechs- eckige, vorspringende Leiste, mit etwas längeren Strahlen als bei “ Höcker der vorhergehenden Art; Leiste und Strahlen auf beiden Seiten überall mit kurzen, nicht sekundär zerteilten Fransen bedeckt, von Öffnung des Penis .. „- Flosse After ur B. welchen diejenigen der einen ""—. Endkieme DNeite mit denen der anderen Fig. 9. alternieren. Schlund etwa drei- “ Höcker Öffnung des Penis .. „- Flosse After ur B. ""—. Endkieme Fig. 9. C. grandis. Von der Ventralseite (°/,). Nach Pelseneer. Öffnung des Penis . Fig. 9. C. grandis. Von der Ventralseite (°/,). Fig. 9. C. grandis. Von der Ventralseite (°/,). Nach Pelseneer. erwachsenen Exemplaren. Chinasee, tropischer Indischer Ozean, im Golfe von Guinea und an der atlantischen Küste der Vereinigten Staaten von Nordamerika bis 40° n. Br. 1. Gen. Cliopsis Troschel Hakensäcke kurz, mit etwa 60 in einem Bündel zusammengestellten Haken. Farbe leicht bräunlich, mit zahlreichen hellen und ‘braunen Stellen, von einzelligen Drüsen herrührend, welche meist auf der hinteren Hälfte des Körpers und innerhalb der Leiste der Endkieme zerstreut sind. Übrigens zeigen Kopf, Fuß und Flossen eine etwas hläuliche, opalisierende Färbung, der Schlund ist stärker braun, der Eingeweidesack braunviolett gefärbt. L. bis 35 mm. — Fig. 92. Chinasee, tropischer Indischer Ozean, im Golfe von Guinea und an der atlantischen Küste der Vereinigten Staaten von Nordamerika bis 40° n. Br. Pteropoda: B. Gymnosomata, 2. Cliopseidae, 1. Cliopsis, 3. Notobranchaeidae 121 lappen des Fußes ziemlich breit, der Hinterrand springt in einem scharfen Winkel vor. Der Höcker zwischen den Seitenlappen einheitlich, länglich-oval. Flossen an der Spitze etwas zusammengezogen. Endkieme nur als eine kaum angedeutete, hexagonale Leiste und äußerst kurzen Strahlen vorhanden, ohne Fransen. Länge des Schlundes unbekannt. Radula und Hakensäcke unbekannt. Farbe sehr leicht rosa, der Eingeweidesack bräunlich; die zahl- reichen kleinen, braunen Flecke, welche bei den vorhergehenden Arten vor- kommen, fehlen. L. 3 mm. — Fig. 93. Nur ein einziges Exemplar im nördlichen Pazifischen Ozean (35° 13‘ n. Br., 154° 43' w. L.). it d ß Beuenlappen des Zinübs m . Flosse LRY EEE ANNIE N 9 Een N After Pl, N 2 f \| \ \ x E = Endkieme it d ß Beuenlappen des Zinübs it d ß Beuenlappen des Zinübs zusammengefaltete Flosse m . Flosse Seitenlappen - LRY des Fußes “x. a 4 EEE ANNIE ZN ‘ N 9 1er) Een N SL After Pl, N 2 e: 2 f \ Höcker \| ) \ \| \ \| x ) E 24 = Endkieme Fig. 93. Fig. 94. C. modesta. Von der Ventralseite (?/,). . C. microcephala. Von der Ventralseite, - Nach Pelseneer. etwas modifiziert (?/,). Nach Tesch. zusammengefaltete Flosse - zusammengefaltete Flosse Seitenlappen - des Fußes “x. a 4 ZN ‘ 1er) SL 2 e: \ Höcker ) \| \| ) 24 Fig. 94. C. microcephala. Von der Ventralseite, etwas modifiziert (?/,). Nach Tesch. After 2 Fig. 93. C. modesta. Von der Ventralseite (?/,). - Nach Pelseneer. Fig. 93. C. modesta. Von der Ventralseite (?/,). - Nach Pelseneer. Fig. 94. C. microcephala. Von der Ventralseite, etwas modifiziert (?/,). Nach Tesch. 4. C. mierocephala (Tesch) 1903 Olionopsis microcephalus, Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p.115 \| 1904 ©. m., Tesch in: Siboga-Exp., nr.52 p.73 t.5 f.126—128 (128: Radula). 1. Gen. Cliopsis Troschel Körper gestreckt, zylindrisch, von rechts und links etwas zusammen- gedrückt, hinter den Flossen etwas eingeschnürt. Dorsales Drüsenfeld nicht vorhanden? Kopfabschnitt klein. Seitenlappen des Fußes dick, fleischig, am Hinterrande in einen scharfen Winkel endend. Der Höcker zwischen den Seitenlappen einheitlich, abgerundet, mit fünf oder sechs Längsfalten. Flossen klein, abgerundet. Endkieme gänzlich fehlend [am Hinterende des Körpers beschrieb ich früher ein traubenförmiges, gestieltes Organ, das für die Zwitterdrüse gehalten wurde, das aber eher, wie Meisenheimer annimmt, ein Parasit oder ein sonstiger Fremdkörper ist]. Schlund etwa so lang wie der Körper selbst. Formel der Radula 4. 1.4. Hakensäcke klein, mit etwa 20 in einem Bündel zusammengestellten Haken. Farbe gelblich, der Nucleus schimmert als ein dieker Stab. bräunlich hindurch. L. 15 mm. — Fig. 94. Ein einziges Exemplar im östlichen Teile des Indo-australischen Archipels. Ein einziges Exemplar im östlichen Teile des Indo-australischen Archipels. 1886 Notobranchaeidae, Pelseneer in: Bull. sei. Nord, ser.2 v.9 p. 224. 1886 Notobranchaeidae, Pelseneer in: Bull. sei. Nord, ser.2 v.9 p. 224. Körper oval, am Hinterende zugespitzt. Kopfabschnitt an- geschwollen, gerundet, durch einen deutlichen, kurzen Halsteil vom Rumpfabschnitt gesondert. Haut stark pigmentiert, nament- lich auf der rechten Körperseite Der eigentliche Körper am Hinterende sanft abgerundet; die Spitze wird durch die ver- längerte Endkieme dargestellt; bis hierher reicht der Eingeweide- sack. Ein dorsales Drüsenfeld fehlt. Schlund sehr kurz. Schlund- organe fehlen vollständig [oder sind in Gestalt besonderer Pteropoda: B. Gymnosomata, 3. Notobranchaeidae, 1. Notobranchaea 122 konischer Zapfen (Buccalkegel) entwickelt?]\| Oberkiefer, sowie Mittelplatte der Radula, vorhanden [oder fehlend?]. Hakensäcke klein, die eigentliche Hakenscheide stellt nur eine schwache Vertiefung mit wenigen, aber großen Haken dar. Seitenlappen des Fußes wohl ausgebildet, nur im proximalen Teile mit dem Körper verwachsen, größtenteils frei zipfelartig herabhängend, ein Höcker ist zwischen ihnen ausgebildet. Mittellappen ziemlich kurz, breit an der Basis, von hier sehr schnell zugespitzt. Flossen breit und groß, am freien Rande gerundet. Keine Seitenkieme, Endkieme aus drei häutigen Falten bestehend, von denen eine unpaare auf der Dorsalseite des Körpers in der Mittellinie ge- legen ist, die beiden paarigen rechts und links zu beiden Seiten des Körpers, alle drei Falten vereinigen sich am Hinterende zu einer kurzen Spitze. After nicht in der Nähe von Osphradium und äußerer Nierenöffnung, im Bereich des hinteren Randes der rechten Flosse, sondern weiter nach hinten auf der rechten Körperseite verschoben. Herz und Niere auf der rechten Körperseite. In der Warmwasserzone der ÜOzeane. Enthält nur die einzige Gattung Notobranchaea. Enthält nur die einzige Gattung Notobranchaea. 1. Gen. Notobranchaea Pelseneer 1. Gen. Notobranchaea Pelseneer 1825 Clio (part., non Linn& 1767, O.F. Müller 1776), Rang in: Ann. Sci. nat., v.5 p.286 \| 1886 Notobranchaea, Pelseneer in: Bull. sci. Nord, ser.2 v.9 p. 224. 1825 Clio (part., non Linn& 1767, O.F. Müller 1776), Rang in: Ann. Sci. nat., v.5 p.286 \| 1886 Notobranchaea, Pelseneer in: Bull. sci. Nord, ser.2 v.9 p. 224. 1825 Clio (part., non Linn& 1767, O.F. Müller 1776), Rang in: Ann. Sci. nat., v.5 p.286 \| 1886 Notobranchaea, Pelseneer in: Bull. sci. Nord, ser.2 v.9 p. 224. Die Diagnose stimmt mit der der Familie überein. In der Warmwasserzone der Ozeane. 3 sichere Arten, 1 unsichere Art. Bestimmungstabelle der Arten (nach Meisenheimer): falten — 2. Hintere Falte der Kieme bedeutend kürzer als die Seiten- Taten Fi DT) REIHE EI TEN ER 3. N. valdiviae Hintere Falte mit langen und breiten Fransen (Fig. 95). . i. N. macdonaldi \| Hintere Falte mit kurzen und schmalen Fransen (Fig. 96). 2. N. inopinata Hintere Falte der Kieme stärker entwickelt als die Seiten- Bestimmungstabelle der Arten (nach Meisenheimer): Bestimmungstabelle der Arten (nach Meisenheimer): falten — 2. Hintere Falte der Kieme bedeutend kürzer als die Seiten- Taten Fi DT) REIHE EI TEN ER 3. N. valdiviae Hintere Falte mit langen und breiten Fransen (Fig. 95). . i. N. macdonaldi \| Hintere Falte mit kurzen und schmalen Fransen (Fig. 96). 2. N. inopinata Hintere Falte der Kieme stärker entwickelt als die Seiten- Hintere Falte der Kieme bedeutend kürzer als die Seiten- Taten Fi DT) REIHE EI TEN ER 3. N. valdiviae Hintere Falte mit langen und breiten Fransen (Fig. 95). . i. N. macdonaldi \| Hintere Falte mit kurzen und schmalen Fransen (Fig. 96). 2. N. inopinata Hintere Falte mit langen und breiten Fransen (Fig. 95). . i. N. macdonaldi \| Hintere Falte mit kurzen und schmalen Fransen (Fig. 96). 2. N. inopinata 1. N. macdonaldi Plsnr. 1884 Clione longicaudatus (non Souleyet 1852), A E. Verrill in: Tr. Connect. Ac., v.6 p.215 \| 1886 Notobranchaea macdonaldi, Pelseneer in: Bull. sei. Nord, ser.2 v.9 p- 225 \| 1887 N. m., Pelseneer in: Rep. Voy. a... Zool. v.19 pars 58 p.39 t.3 f.3,4. dorsale Falte.-- der Endkieme B Seitenlappen des Fußes Flosse Mittellappen des Fußes A Fig. 9. Seitenlappen des Fußes Flosse Mittellappen des Fußes dorsale Falte.-- der Endkieme A B Fig. 9. N. macdonaldi. A von der Ventralseite, B von der Dorsalseite (%),). Nach Pelseneer. 1. Gen. Notobranchaea Pelseneer dorsale Falte.-- der Endkieme B A A B Fig. 9. N. macdonaldi. A von der Ventralseite, B von der Dorsalseite (%),). Nach Pelseneer. Pteropoda: B. Gymnosomata, 3. Notobranchaeidae, 1. Notobranchaea 123 Körper oval. Halsteil kurz, deutlich, Seitenlappen des Fußes lang, zwei Drittel derselben frei, mit langen Zipfeln; zwischen den festgehefteten Teilen der Seitenlappen ein kleiner Höcker; Mittellappen des Fußes ziemlich gestreckt, zipfelartig auslaufend. Dorsaler Strahl der Endkieme jederseits mit 8—10 Fransen, diejenigen der einen Seite mit denen der anderen alternierend. Im Schlunde zwei Buccalkegel? Radula und Hakensäcke un- bekannt. Farbe graubraun. L. bis 1U mm. — Fig. 95. Körper oval. Halsteil kurz, deutlich, Seitenlappen des Fußes lang, zwei Drittel derselben frei, mit langen Zipfeln; zwischen den festgehefteten Teilen der Seitenlappen ein kleiner Höcker; Mittellappen des Fußes ziemlich gestreckt, zipfelartig auslaufend. Dorsaler Strahl der Endkieme jederseits mit 8—10 Fransen, diejenigen der einen Seite mit denen der anderen alternierend. Im Schlunde zwei Buccalkegel? Radula und Hakensäcke un- bekannt. Farbe graubraun. L. bis 1U mm. — Fig. 95. An der Ostküste Nordamerikas, nördlich bis etwa 40° n. Br. An der Ostküste Nordamerikas, nördlich bis etwa 40° n. Br. 2. N. inopinata Plsnr. 1887 N.i., Pelseneer in: Rep. Voy. Challenger, Zcol. v.19 pars 58 p.40 t.3 8.5, 6. \ 2. N. inopinata Plsnr. 1887 N.i., Pelseneer in: Rep. Voy. Challenger, Zcol. v.19 pars 58 p.40 t.3 8.5, 6. \ Körper oval. Halsteil kurz, deutlich. Seitenlappen des Fußes kürzer und breiter als bei der vorhergehenden Art, nur über die Hälfte ihrer Länge frei und zipfelartig ausgezogen; zwischen den proximalen Teilen der Seiten- lappen ein kleiner Höcker; Mittellappen des Fußes verhältnismäßig kurz, mit einer kleinen Längsgrube, zugespitzt. Flossen breit, abgerundet. Dorsaler ICE ARD dorsaler Strahl __ der Endkieme A B Fig. 96. N. inopinata. A Tier von der Dorsalseite (°),). Nach Pelseneer. B Querreihe der Radula. Nach Tesch (vergr.?). B A B Fig. 96. N. inopinata. A Tier von der Dorsalseite (°),). Nach Pelseneer. B Querreihe der Radula. Nach Tesch (vergr.?). Fig. 96. N. inopinata. A Tier von der Dorsalseite (°),). Nach Pelseneer. B Querreihe der Radula. Nach Tesch (vergr.?). Strahl der Endkieme mit kleinen Fransen jederseits. Im Schlunde jederseits zwei Buccalkegel? Formel der Radula 8.0.8, Seitenplatten mit kleinen Basalplatten, nach außen zu an Größe abnehmend, Mittelplatte fehlend ? Hakensäcke klein, wenig tief, mit einer geringen Zahl großer Haken. „Schlund- blasen“ wie bei 7’Aliptodon, in der Außenwandung der Buccalmasse. Farbe? L. 4 mm. — Fig. 96. 1. Gen. Notobranchaea Pelseneer Muskulatur der Hakensäcke vorderer -" Tentakel --Mund Seitenlappen - des Fußes Flosse --- Flosse "». Höcker des 7 ußes “ Mittellappen des Fußes After NE Endkieme Muskulatur der Hakensäcke vorderer Tentakel Ba > 3 “ linke Falte der Endkieme B Mittelplatte 3 “ linke Falte der Endkieme B Endki B Mittelplatte Mittelplatte Seitenplatte C Fig. 97. Fig. 97. Fig. 97. N. valdiviae. A Tier von der Ventralseite (!%/,), B hinteres Körperende von der linken Seite ('/,), C Querreihe der. Radula ('%/,). Nach Meisenheimer. Fig. 97. N. valdiviae. A Tier von der Ventralseite (!%/,), B hinteres Körperende von der linken Seite ('/,) C Querreihe der. Radula ('%/,). Nach Meisenheimer. Zahnfortsätzen, nach außen schnell an Größe abnehmend. Farbe am Rumpfe hell-gräulich, an Hals- und Kopfabschnitt weißlich. L. 55 mm, Br. (in der Nähe der Flossen) 2:5 mm“ (Meisenheimer). — Fig. 97. Ein einziges Exemplar, nördlich vom Chagos-Archipel, im Indischen Ozean. Clio capensis Rang 1825 ©. c., Rang in: Ann. Sci. nat., v.5 p.286 t.7 f.3,4. Kap der Guten Hoffnung. Clio capensis Rang 1825 ©. c., Rang in: Ann. Sci. nat., v.5 p.286 t.7 f.3,4. Kap der Guten Hoffnung. 1. Gen. Notobranchaea Pelseneer Im Pazifischen Ozean (östlich von Japan, im Indo-australischen Archipel und bei Sydney). Im Pazifischen Ozean (östlich von Japan, im Indo-australischen Archipel und bei Sydney). 3. N. valdiviae Meisenh. 1905 N.v., Meisenheimer in: Ergeb. Tiefsee-Exp.. v.9ı p.55 t.27 f.1,2,7 (Radula). 3. N. valdiviae Meisenh. 1905 N.v., Meisenheimer in: Ergeb. Tiefsee-Exp.. v.9ı p.55 t.27 f.1,2,7 (Radula). „Kopfabschnitt gerundet, vorn etwas zipfelförmig ausgezogen. Hinter- lappen des Fußes kurz, Seitenlappen mäßig lang, nur in ihrem vordersten Teil mit dem Körper verwachsen, im übrigen frei herabhängend. Seiten- falten der Endkieme sehr lang, hintere Falte kurz, alle am Hinterende in eine scharf ausgeprägte, dorsalwärts aufgebogene, gemeinsame Endspitze zu- sammenlaufend. Alle Falten ohne besonders auffällige Fransen. Keine Buccal- kegel. Oberkiefer aus einem unpaaren, medianen Zahn und je 7 Zähnchen zu beiden Seiten bestehend. Hakensäcke mit je 9 an der Basis verbreiterten, an der Spitze gekrümmten Haken. Formel der Radula 6. 1. 6, Mittelzahn mit einigen sehr kleinen Zähnchen in der Medianebene des Vorderrandes und „Kopfabschnitt gerundet, vorn etwas zipfelförmig ausgezogen. Hinter- lappen des Fußes kurz, Seitenlappen mäßig lang, nur in ihrem vordersten Teil mit dem Körper verwachsen, im übrigen frei herabhängend. Seiten- falten der Endkieme sehr lang, hintere Falte kurz, alle am Hinterende in eine scharf ausgeprägte, dorsalwärts aufgebogene, gemeinsame Endspitze zu- sammenlaufend. Alle Falten ohne besonders auffällige Fransen. Keine Buccal- kegel. Oberkiefer aus einem unpaaren, medianen Zahn und je 7 Zähnchen zu beiden Seiten bestehend. Hakensäcke mit je 9 an der Basis verbreiterten, an der Spitze gekrümmten Haken. Formel der Radula 6. 1. 6, Mittelzahn mit einigen sehr kleinen Zähnchen in der Medianebene des Vorderrandes und 124 124 Pteropoda: B. Gymnosomata, 3. Notobranchaeidae, 1. Notobranchaea, 4. Clionidae ebensolchen. etwas größeren, zu beiden Seiten desselben, Seitenzähne mit mächtiger Basalplatte und langen, an der Spitze leicht auswärts gebogenen ebensolchen. etwas größeren, zu beiden Seiten desselben, Seitenzähne mit mächtiger Basalplatte und langen, an der Spitze leicht auswärts gebogenen dorsale Falte --" der Endkieme Ba > 3 “ linke Falte der Endkieme B Mittelplatte Muskulatur der Hakensäcke vorderer -" Tentakel --Mund Seitenlappen - des Fußes dorsale Falte --" der Endkieme Flosse --- Flosse "». Höcker des Ba > 7 ußes 3 “ “ Mittellappen linke Falte der Endkieme des Fußes B After Mittelplatte NE Endkieme Seitenplatte A C Fig. 97. N. valdiviae. A Tier von der Ventralseite (!%/,), B hinteres Körperende von der linken Seite ('/,), C Querreihe der. Radula ('%/,). Nach Meisenheimer. 4. Fam. Clionidae 1840 Clionidae, J. E. Gray, Syn. Brit. Mus., ed. 42 p.86 \| 1842 Pneumodermidae (part., non J.E. Gray 1840), Orbigny in: Pal. franc., Terr. Cret. v.2 p.4 \| 1846 Clionoidae (part.), L. Agassiz, Nomenel. zool., Index p. 90 \| 1850 C’ioneidae, J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.35 \| 1854 Cliidae (part., mon Jeffreys 1869, non Fischer 1881), S. P. Woodward, Man. Moll., p. 208 \| 1855 Clioidea (part.), Gegenbaur, Unters. Pter. & Heterop., p- 212 \| 1856 Clionacea (part.), Troschel, Gebiss Schneck., v.] p.54 \| 1887 Clionidae, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.4l. Körper gestreckt, nach hinten immer mehr oder weniger zugespitzt, aber stark kontraktil. Eingeweidesack sich nur sehr selten bis zum aboralen Körperpol ausdehnend, beim erwachsenen Tiere meist nur die vordere Hälfte des Rumpfabschnittes ein- nehmend. Haut gewöhnlich nicht pigmentiert, durchsichtig, mit zahlreichen einzelligen Drüsen. Kein dorsales Drüsenfeld. Kopf- abschnitt abgerundet, kurz, Halsteil gedrungen. Vordere Tentakeln lang. Schlund sehr kurz, nicht als Rüssel ausgebildet, sondern Pteropoda: B. Gymnosomata, 4. Clionidae, 1. Clione 125 als ein weiter Vorraum hinter der Mundöffnung; auf der Grenze zwischen Schlund und Buccalhöhle rechts und links ein vor- springender Wulst („falsche Lippen“); diese Wülste, welche den schmalen Zugang zu der Bucealhöhle begrenzen, täuschen bei ausgestrecktem Schlunde die Umrahmung der eigentlichen Mund- öffnung vor. An den Seitenwandungen des Schlundes jederseits 1—3 konische, kurze, mit zahlreichen Sinneszellen und einzelligen Drüsen besetzte Buccalkegel, fast immer symmetrisch, selten asymmetrisch angeordnet. Oberkiefer meistens fehlend. Radula mit großer, halbmondförmiger Mittelplatte und einer mit dem Alter wechselnden Zahl von Seitenplatten. Hakensäcke meist ziemlich gut entwickelt, selten reduziert. Seitenlappen des Fußes gut entwickelt, fast in ihrer ganzen Länge mit dem Körper ver- wachsen; zwischen den Seitenlappen fehlt der Höcker. Mittel- lappen des Fußes fast immer klein, zugespitzt, zuweilen fehlt er gänzlich. Kiemen fehlen vollständig. Anus meist mit Ösphradium und äußerer Nierenöffnung auf dem Analfelde Herz und Niere auf der rechten Körperseite. Penis sehr kompliziert, aus einem Vorderraum und einem schlauchförmigen Hinterteile bestehend; im Vorraume finden sich an einer bestimmten Stelle in der Wandung große, vakuolisierte Zellen, die zuweilen eine Aus- sackung hervorrufen, zudem mündet in den Vorderraum oder in den Penis eine schlauchförmige Prostata. . In allen Meeren. In allen Meeren. 4 Gattungen, 7 sichere Arten, von denen eine in 2 Unterarten zerfällt und l unsichere Art. Bestimmungstabelle der Gattungen: als kleine Querfalte ausgebildet — 2. Mittellappen des Fußes vollkommen fehlend .... . 3. Gen. 4. Fam. Clionidae Paraclione f Mittellappen des Fußes gut ausgebildet; Buccalkegel im 2 ganzen nur & (rechts, links) =. 2...» ...» 4. Gen. Paedoclione \ Mittellappen des Fußes sehr klein — 3. Körper zipfelartig ausgezogen; 2 oder 3 Paare von Buccal- kegeln; Oberkiefer fehlend; Mittelplatte der Radula mit einem großen, vorspringenden Zahn in der Mitte, ZIWellenMahnedAuhri.H sh ee ee l. Gen. Clione Körper am Hinterende etwas mehr abgerundet; 1 Paar von Buccalkegeln; Oberkiefer vorhanden, Mittelplatte der Radula mit etwa 12 gleich großen Zähnchen TImLCnTIdeN S2 3: Gh nenn el, . . 2. Gen. Fowlerina \| Mittellappen des Fußes immer vorbanden, bisweilen nur 1 3 1. Gen. Clione Pallas 1774 Clio (part., non Linne 1767), Phipps, Voy. North Pole, p.195 \| 1774 Clione (part.), Pallas, Spie. zool.. fasc. 10 p.28 \| 1776 Clio, O. F. Müller, Zool. Dan. Prodr., p- 226 \| 1824 Chiodita (part.), Quoy & Gaimard in: Voy. Uranie & Physicienne, p.413 \| 1825 C. (part.), Quoy & Gaimard in: Ann. Sei. nat., v.5 p.74 \| 1840 Spongiobranchea (part.), Orbigny, Voy. Amer. merid., v.5ıı p.132. Körper gestreckt, mit zugespitztem, aboralem Körperpole. Haut durch- sichtig, mit zahlreichen einzelligen Drüsen, nicht pigmentiert, nur das Körper- ende kann eine lebhafte Farbe aufweisen. Der Eingeweidesack nimmt bei Pteropoda: 8. Gymnosomata, 4. Olionidae, 1. Clione 126 erwachsenen Tieren nur die proximale Hälfte des Rumpfabschnittes ein. Kopfabschnitt breit, Halsteil kurz. Im Schlunde 2 oder 3 Paare von Buccalkegeln. Öberkiefer fehlt. Mittelplatte der Radula mit großem Mittelzahne, bisweilen ohne Zähne. Hakensäcke ziemlich lang, gut entwickelt, mit mehr oder weniger zahlreichen Haken. Seitenlappen des Fußes groß, am distalen Ende meist dreieckig vorspringend, wenn zusammengelegt fast gänzlich den kleinen, am Ende zugespitzten Mittellappen bedeckend. Anus mit dem Osphradium nach hinten verschoben, von der äußeren Nierenöffnung am Hinterrande der rechten Flosse getrennt. Penis zuweilen zweiästig. In allen Meeren. 4 Arten, von welchen eine in 2 Unterarten zerfällt. Bestimmungstabelle der Arten: Mit drei Paaren von Buccalkegeln — 2. i Mit zwei Paaren von Buccalkegeln — 3. Kopf breiter als lang, Körper schlank, nicht pigmentiert, \| Seitenplatten der Radula sehr lang und schlank (Fig.99) 2. C. graeilis 2 7 Kopf etwa ebenso breit wie lang, Körper gedrungener, an der Spitze gefärbt, Seitenplatten der Radula kürzer und dicker (Fig.98). ..... BT Te « . 1. C. limacina Tier klein (bis zu etwa 10 mm), Mittellappen des Fußes sehr kurz, Mittelplatte der Radula ohne Zähne (Fig. 100) 3. C. longieaudata 3 ? Tier größer (bis zu etwa 22 mm), Mittellappen des Fußes etwas länger, Mittelplatte der Radula mit einem vor- springender Zahne in der Medianlinie (Fig. 101). . . 4. C. flavescens In allen Meeren. l. C. limaeina (Phipps) Zerfällt in 2 Unterarten: la. C. limacina limaecina (Phipps) 1774 Clio l., Phipps, Voy. North Pole, p-195 \| 1774 Olione borealis, Pallas, Spie. zool., fase. 10 p.28 t.1 f.18.19 \| 1776 Clio retusa (non Linn& 1767), O. F. Müller, Zool. Dan. Prodr., p.226 \| 1789 CO. borealis, Bruguiere in: Ene. möth., Vers v.1 p.506 \| 1791 C. b., Bruguiere in: Tabl. enc. meth., Vers v.1 t.75 f.3,4 \| 1825 C. 1. Gen. Clione Pallas miquelonensis, Rang in: Ann. Sei. nat., 0.5 p.285 1.7 f.2\| 1857 Clione limacina, Mörch in: Rink, Grönland, v.2 Anh. p.79 \| 1869 ©. papilionacea, ‚Jeffreys, Brit. Conch., v.5 p.121 \| 1870 C. elegantissima, W.Dall in: Amer. J. Conch., 0.611 p-139 \| 1878 ©. limacina, G. O. Sars, Moll. arct. Norvegiae, p. 352 t.29 f.4a—c \| 1885 ©. dalli, Arth. Krause in: Arch. Naturg., v.ölı p.299 t.18 f.19a \| 1886 C. limacina, Boas in: Danske Selsk. Skr., ser.6 v.4 p.162 t.7 f. 101—103 \| 1905 C©. !., Meisen- heimer in: Fauna arctica, v.4 p.413 f.2 \| 1906 ©. I. var. typica, Meisenheimer in: D. Südp.-Exp., v.9ıı p. 101 t.6 f.4. Körper langgestreckt, hinten zugespitzt. Kopf rundlich, Halsteil kurz. Rumpf im proximalen Abschnitt bedeutend angeschwollen, diese Auftreibung wird durch den rundlichen Eingeweidesack bedingt, der die hintere Hälfte des Rumpfes, bei erwachsenen Exemplaren wenigstens, völlig frei läßt. Seiten- lappen des Fußes groß, fast ihrer ganzen Länge nach festgewachsen; nach hinten laufen sie in eine Spitze aus; Mittellappen sehr kurz, zipfelartig, zugespitzt, bisweilen bis auf eine kleine Querfalte (bei Exemplaren aus der Behring-Straße) reduziert. Flossen zugespitzt, etwa dreieckig. Im Schlunde jederseits drei Buccalkegel, kurz, zapfenförmig, die Länge nimmt von der Dorsal- zu der Ventralseite ab, sie berühren sich an der Basis miteinander, an ihrer medianen Seite findet sich der Eingang in die Buccalhöhle, von „falschen Lippen“ umgrenzt. Mittelplatte der Radula sehr groß, halbmond- förmig, mit einem großen, vorspringenden Zahn in der Mitte, zu beiden Seiten Pteropoda: B. Gymnosomata, 4. Olionidae, 1. Olione 127 desselben fein gezähnelt; Seitenplatten kurz, hakenförmig gekrümmt, nach den Seiten an Größe abnehmend; Formel der Radula bis 14. 1. 14, die Zahl der Seitenplatten ist bei den erwachsenen Exemplaren größer als bei den jüngeren. Hakensäcke mäßig lang, Haken zahlreich, leicht gebogen. Wimper- reifen der Larve (Fig. 98B) können noch lange erhalten bleiben; bei Exemplaren von 12, sogar bei solchen von 16 mm sind zuweilen noch alle drei Reifen falsche Lippen : Br Buccalkegel 2 .. vorderer Tentakel “ Seitenlappen des Fußes . “ _ Flosse Penis .. vorderer Wimper- N ring Mittelplatte R e = E a N mittlerer = " Wimperring = . Een a e ED hinterer N © Wimperring Seitenplatten = C A B Fig. 98. C.limacina limacina. A Tier von der Ventralseite, mit ausgestülpten Buccalkegeln und hervor- gestrecktem Penis (?/,), B Larve mit drei Wimperringen (etwa °/,). Nach Boas. C Teile der Radula 4 (#9/,). Nach Sars. 1. Gen. Clione Pallas falsche Lippen : falsche Lippen : Buccalkegel . vorderer Tentakel Seitenlappen des Fußes _ Flosse Buccalkegel vorderer Tentakel Seitenlappen des Fußes _ Flosse .. vorderer Wimper- ring N mittlerer " Wimperring . hinterer Wimperring B vorderer Wimper- ring R = E a = = a ED © = Mittelplatte e B C Fig. 98. C.limacina limacina. A Tier von der Ventralseite, mit ausgestülpten Buccalkegeln und hervor- gestrecktem Penis (?/,), B Larve mit drei Wimperringen (etwa °/,). Nach Boas. C Teile der Radula 4 (#9/,). Nach Sars. vorhanden, bei anderen von 26—27 mm kann noch ein Rest des hinteren Reifens nachweisbar sein. Farbe der Haut fehlend, nur das Hinterende des Körpers „intensiv orange- oder himbeerrot gefärbt“ (Meisenheimer); Buccal- kegel tief rot, Flossen schwach gelblich, vorderer Darmabschnitt braun oder orange, Leber stark dunkelbraun, Zwitterdrüse lebhaft rot. L. bis etwa 35 — 4) mm. — Fig. 98. vorhanden, bei anderen von 26—27 mm kann noch ein Rest des hinteren Reifens nachweisbar sein. Farbe der Haut fehlend, nur das Hinterende des Körpers „intensiv orange- oder himbeerrot gefärbt“ (Meisenheimer); Buccal- kegel tief rot, Flossen schwach gelblich, vorderer Darmabschnitt braun oder orange, Leber stark dunkelbraun, Zwitterdrüse lebhaft rot. L. bis etwa 35 — 4) mm. — Fig. 98. Die Nahrung scheint ausschließlich aus Limacina helicina zu bestehen; Clione selbst bildet wieder die Hauptnahrung der Bartenwale. Vorzugsweise erscheint sie an hellen, sonnigen Tagen an der Oberfläche. — Sie ist die einzige Art unter den Gymnosomata, bei welcher die Begattung direkt wahrgenommen wurde: zwei Individuen stellen sich vertikal und berühren sich mit ihren Ventralseiten; der kurze Arm des zweiästigen Penis dringt in die weibliche Geschlechtsöffnung des anderen Tieres, der längere Arm ist nach hinten gewendet und dient wohl nur als Reizorgan. Die Begattung ist eine wechselseitige und dauert etwa 4 Stunden, nach 20—24 Stunden erfolgt die Eiablage. Der Laich besteht aus ziemlich großen, gallertartigen Klumpen oder Schnüren, welche sehr zahlreiche Eier enthalten. Im arktischen Eismeere, zirkumpolar, hier oft in ungeheuren Schwärmen an der Oberfläche des Meeres; vereinzelt aber auch in südlicheren Gebieten, wenn schon stets in kleineren Exemplaren, so bei Kap Hatteras (35° s. Br.) an der Ostküste von Nordamerika, im Norwegischen Nordmeere, in der nördlichen Nordsee und sogar bei Falmouth an der Südwestspitze Englands. Pteropoda: B. Gymnosomata, 4. Clionidae, 1. Clione 128 1b. C. limacina antarctica E. A. Smith ?1789 Clio australis (non Pelseneer 1888), Bruguiere in: Enc. 1. Gen. Clione Pallas meth., Vers v.1 p.507 \| 1791 C.a., Bruguiere in: Tabl. ene. meth., Vers v.1t.75 f.1,2 \| 1902 Clione antarctica, E.A.Smith in: Rep.Voy. Southern Cross, p.210 t.25 f.7,8 \| 1905 Clione limacina, Meisenheimer in: Ergeb. Tiefsee-Exp., v.9ı p. 55 t.16 £.1,2 \| 1906 ©. 1. var. antarctica, Meisenheimer in: D. Südp.-Exp., v.911 p. 101.6 £.5. Unterscheidet sich von voriger Unterart nur dadurch, daß hier zwischen den dorsalen und den mittleren Buccalkegeln konstant ein Zwischenraum an der Basis besteht, während bei der vorhergehenden Unterart die Buccalkegel sich immer an der Basis berühren. In allen anderen Hinsichten sind die beiden Unterarten einander völlig gleich, vielleicht sind bei ©. L. antaretica die Hautdrüsen meist etwas stärker entwickelt. Farben und Maße sind ebenfalls gleich. Im antarktischen Eismeer, meist nahe am. Eisrande, zuweilen auch nördlicher, im südafrikanischen Mischgebiete bis zu 35° s. Br. vordringend. Wahrscheinlich zirkumpolar, bis jetzt aber nur zwischen etwa 15° und 91° ö.L. beobachtet. 2. C. gracilis Massy 1909 C. g., Massy in: Sei. Invest. Fish. Ireland, 1907 nr.2 (1909) p.48 t.1 f. 1—4. Körper gestreckt, sehr schlank, hinten zugespitzt. Kopf breiter als lang, ebenso breit wie die vordere Hälfte des Rumpfes, welche allein von ...... Buccalkegel - Eingang in die Buccalhöhle Seitenlappen des Fußes ; Floss Mittellappen des Fußes Er . Seitenplatten Mittelplatte -. . Fig. 99. C. gracilis. A Tier von der Ventralseite, mit hervorgestrecktem Schlundapparat (/,), B Teil einer Querreihe der Radula (%/,). Nach Massy. Buccalkegel Eingang in die Buccalhöhle Seitenlappen des Fußes Eingang in die Buccalhöhle Seitenlappen des Fußes Fig. 99. C. gracilis. A Tier von der Ventralseite, mit hervorgestrecktem Schlundapparat (/,), B Teil einer Querreihe der Radula (%/,). Nach Massy. dem Eingeweidesack eingenommen wird, während die hintere Hälfte frei bleibt. Seitenlappen des Fußes breit, am unteren Ende in einen kurzen, freien Zipfel ausgezogen; Mittellappen an der Basis sehr breit, aber schnell an Umfang abnehmend und zugespitzt. Flossen am freien Rande abgerundet. dem Eingeweidesack eingenommen wird, während die hintere Hälfte frei bleibt. Seitenlappen des Fußes breit, am unteren Ende in einen kurzen, freien Zipfel ausgezogen; Mittellappen an der Basis sehr breit, aber schnell an Umfang abnehmend und zugespitzt. Flossen am freien Rande abgerundet. Pteropoda: B. Gymnosomata, 4. Clionidae, 1. Clione 129 Im Schlunde jederseits drei zapfenförmige Buccalkegel, welche, wie es scheint, keine Differenz in der Größe aufweisen. Mittelplatte der Radula groß, halb- mondförmig [ohne Mittelzahn?], Seitenplatten schlank, gestreckt, kaum ge- bogen. Hakensäcke mit etwa 30 Haken. 4. C. flavescens (Gegenbaur) 1855 Clio f., Gegenbaur, Unters. Pter. & Heterop., p- 73 t.4 f.15 \| 1875 ©. aurantiaca, Fol in: Arch. Zool. exper., v.4 p.178 t.10 £.10 Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 9 Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 9 Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. 1. Gen. Clione Pallas Fowlerina (Larve) \| 1887 Clione flavescens, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.49 \| 1903 C. f., Kwietniewski in: Ric. Lab. Anat. Roma, v.9 p. 73 t. 14 f. 14 (Larve). (Larve) \| 1887 Clione flavescens, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.49 \| 1903 C. f., Kwietniewski in: Ric. Lab. Anat. Roma, v.9 p. 73 t. 14 f. 14 (Larve). Körper gestreckt, schlank, nach hinten zugespitzt, aber nicht stark zipfelartig verlängert, mit zahlreichen einzelligen Hautdrüsen. Kopf wenig vom Rumpfe abgesetzt. Seitenlappen des Fußes nach hinten zu in einem kurzen Zipfel frei hervorragend; Mittellappen sehr klein und kurz, aber doch deutlich nachweisbar. Flossen sehr breit an der Basis, am freien Rande schräg abgestutzt, so daß der Vorderrand länger ist als der Hinterrand [in der Abbildung sind die Flossen gänzlich zurückgezogen]. Jederseits zwei Bucealkegel im Schlunde [nach der Abbildung von Kwietniewski zu schließen sind diese, wenn hervorgestreckt, verhältnismäßig sehr groß, zylindrisch und nach dem Gipfel zu mit zahlreichen Höckern, denen lange Wimpern aufsitzen, besetzt. Formel der Radula (bei der Larve) 3.1.3; Mittel- platte groß, mit deutlichem Mittelzahne. Hakensäcke schwach entwickelt, kurz, mit etwa 10 kurzen Haken. Farbe fehlend, Haut durchsichtig, Magen- Lebersack hell-rötlich, bräunlich und orange, Zwitterdrüse weiß. L. bis etwa 22 mm, die erwachsenen Exemplare scheinen aber äußerst selten. — Fig. 101. Nur vom Mittelmeer und zwar ausschließlich von Messina bekannt. Clio pyramidalis Q. &G. 1832 C.p., Quoy & Gaimard in: Voy. Astrol., v.2 p.371 t. 27 £.37. 1. Gen. Clione Pallas Farbe gänzlich fehlend, der ganze Körper im Leben wahrscheinlich völlig durchsichtig. L. etwa 10 mm. — Fig. 99. Bisher nur in einigen Exemplaren an der Südwestküste Irlands (55°1'n. Br. 10° 45‘ w. L.) beobachtet. Bisher nur in einigen Exemplaren an der Südwestküste Irlands (55°1'n. Br., 10° 45‘ w. L.) beobachtet. 3. C.longieaudata (Souleyet) ?1824 Oliodita fusiformis, Quoy & Gaimard in: Voy. Uranie & Physieienne, p. 413 \| ?1825 C. f., Quoy & Gaimard in: Ann. Sei. nat., v.5 p.74 t.2 f.3, 4 \| ?1840 Spongiobranchea elongata, Orbigny, Voy. Amer. merid., v.5ın p.132 t.9 f.8,9 \| ?1850 Olione caudata, J.E.Gray, Cat. Moll, Brit. Mus., v.2 p.37 \| ?1852 Clio limacella, Rang in: Rang & Souleyet, Hist. nat. Pter., t.10 f. 10—12 \| 1852 C. longicaudatus, Souleyet in: Voy.Bonite, v.2 p.286, Moll. t. 14 £.17—21 \| 1864 ©. caudata, Macdonald in: Tr. R.Soe. Edinb., v.23 p.185 \| 1887 Clione longicaudata, Pelseneer in: Rep.Voy. Challenger, Z001.v.19 pars 58 p.48 \| 1903 C.1., Kwietniewski in: Rie. Lab. Anat. Roma, v.9 p.76 t.14 £.15, 16. 3 7" h Buecalkegel "erster Wimperring Rs Hautdrüsen > zweiter Wimperring .. dritter Wimperring Fig. 101. C. flavescens. Von der Ventralseite, mit ausgestreckten Bucealkegeln und eingezogenen Flossen (°/,). Nach Kwietniewski. Körper schlank, gestreckt, wenigstens bei erwachsenen Exem- plaren nach hinten zipfelartig aus- laufend ; Eingeweidemasse nur in der vorderen Hälfte des Rumpfes. Kopf breit, wenig vom Rumpfe abgesetzt. Seitenlappen des Fußes breit, am distalen Ende frei vorspringend, Buecalkegel rster Wimperring » > Fig. 100. C.longicaudata. Von der Ventralseite (5/,). Nach Kwietniewski. Fig. 101. C. flavescens. Von der Ventralseite, mit ausgestreckten Bucealkegeln und eingezogenen Flossen (°/,). Nach Kwietniewski. Fig. 100. C.longicaudata. Von der Ventralseite (5/,). Nach Kwietniewski. wenn zusammengelegt fast gänzlich den äußerst kleinen Mittellappen bedeckend (in der Abbildung nicht angegeben). Flossen sehr breit an der Basis, fast viereckig. Zwei Paare von Buccalkegeln. Formel der Radula 3. 1. 3, Mittel- platte ohne Zahn. Hakensäcke mit etwa 12 fast geraden Haken. Die drei Wimperreifen existieren noch bei Larven von etwa 1 mm; bei 15 mm Länge sind der vordere und der mittlere Ring verschwunden, und erst dann bildet sich der anfangs tönnchenförmige Körper zu der langgestreckten Form des erwachsenen Tieres um. Farbe graublau L. etwa 10 mm. — Fig. 100. In den tropischen Teilen aller Ozeane, namentlich im Atlantischen Ozean auch im Mittelmeer bei Messina und Neapel, aber immer eine seltene Erscheinung. 9 130 Pteropoda: B. Gymnosomata, 4. Clionidae, 1. Clione, 2. 1. F. zetesios Plsnr. ?1903 Clione punctata, Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 «8 p.116 \| ?1904 ©. p., Tesch in: Siboga-Exp., nr. 52 p.79 t.5 f.149 \| 1906 Fowlerina zetesios, Pelseneer in: Tr. Linn. Soe. London, v.10 pars5 p.149 t.12 f.53—55, 57 (Radula), 58 (Haken), 60 (Oberkiefer). 2, Gen. Fowlerina Pelseneer ?1903 Clione (part.), Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p.116 \| 1906 Fowlerina, Pelseneer in: Tr. Linn. Soc. London, v. 10 pars 5 p. 149. ?1903 Clione (part.), Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v.8 p.116 \| 1906 Fowlerina, Pelseneer in: Tr. Linn. Soc. London, v. 10 pars 5 p. 149. Körper ganz demjenigen von Clone ähnlich, gestreckt, am Hinterende zugespitzt. Eingeweidesack, wenigstens beim erwachsenen Tiere, nur in der vorderen Hälfte des Rumpfes, Kopf breit und flach. Haut durchsichtig, mit Chromatophoren ausgestattet. Nur ein einziges Paar Buccalkegel im Schlunde. Öberkiefer vorhanden. Mittelplatte der Radula ohne vorspringenden Mittel- zahn, nur am distalen Rande fein gezähnelt. Hakensäcke kurz, mit wenigen Haken. Seitenlappen des Fußes gut entwickelt, breit; Mittellappen sehr kurz, zugespitzt. Flossen sehr breit. Die innere Anatomie, soweit bekannt, gleicht der von Clione. Selten, in der Warmwasserzone des Atlantischen Ozeanes [und im Indo-australischen Archipel?]. l Art. 1. F. zetesios Plsnr. ?1903 Clione punctata, Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 «8 p.116 \| ?1904 ©. p., Tesch in: Siboga-Exp., nr. 52 p.79 t.5 f.149 \| 1906 Fowlerina zetesios, Pelseneer in: Tr. Linn. Soe. London, v.10 pars5 p.149 t.12 f.53—55, 57 (Radula), 58 (Haken), 60 (Oberkiefer). 1. F. zetesios Plsnr. ?1903 Clione punctata, Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 «8 p.116 \| ?1904 ©. p., Tesch in: Siboga-Exp., nr. 52 p.79 t.5 f.149 \| 1906 Fowlerina zetesios, Pelseneer in: Tr. Linn. Soe. London, v.10 pars5 p.149 t.12 f.53—55, 57 (Radula), 58 (Haken), 60 (Oberkiefer). Körper ziemlich gedrungen, zugespitzt, aber stark kontraktil. Haut pigmentiert. Kopf wenig vom Rumpfe gesondert, umfangreich, dorso-ventral zusammengedrückt. Seitenlappen des Fußes breit, dick, hinten frei von der Unterlage vorspringend. Mittellappen deutlich, aber kurz, zugespitzt. Flossen sehr breit, flächenartig ausgedehnt, am freien Rande sanft abgerundet, aber Pteropoda: B. Gymnosomata, 4. Clionidae, 2. Fowlerina, 3. Paraclione 131 durch starke Kontraktilität veränderlich in der Gestalt. Im Schlunde jeder- seits ein einziger Buccalkegel, welcher zylindrische, nicht zapfenförmige Gestalt zeigt, und mit zahlreichen Papillen besetzt ist. Schlund sehr geräumig. Öberkiefer halbmondförmig, mit der Konkavität nach vorn gerichtet, aus etwa 18 gleich großen, mit der Spitze nach vorn gerichteten Zähnen zu- sammengesetzt. Radula mit einer großen, halbmondförmigen Mittelplatte, an deren Hinterrande etwa 12 wenig in Größe abweichende Zähnchen nach- weisbar sind, ohne Mittelzahn; Seitenplatten je nach dem Alter des Tieres 4—6 an der Zahl, ziemlich gedrungen, hakenförmig gebogen. Hakensäcke kurz, mit etwa 15 kurzen, dreieckigen, an der Spitze meist stark gebogenen. Haken. 2, Gen. Fowlerina Pelseneer Farbe? L. bis etwa 85 mm. — Fig. 102. Buccealkegel Seitenlappen & Bucealkegel “ des Fußes N Mittellappen - des Fußes vorderer ‚ Tentakel = S - 3 SEE: E Flosse A F: GER Seitenlappen des Fußes Mittellappen des Fußes Sa Be B > Ze Mittelplatte Seitenplatte F= rd ee < unterer Pol "" des Einge- weidesackes Fig. 102. F. zetesios. A von der Ventralseite, stark zusammengezogen, Flossen ausgebreitet (?/), B orale Ansicht eines Exemplares mit eingezogenen Flossen (/,), € Querreihe der Radula (1%). Nach Pelseneer. Buccealkegel & Buccealkegel & Buccealkegel & Seitenlappen des Fußes Mittellappen des Fußes Seitenlappen “ des Fußes Mittellappen - des Fußes Flosse A Seitenlappen des Fußes B Mittelplatte Seitenplatte rd ee Fig. 102. F. zetesios. A von der Ventralseite, stark zusammengezogen, Flossen ausgebreitet (?/), B orale Ansicht eines Exemplares mit eingezogenen Flossen (/,), € Querreihe der Radula (1%). Nach Pelseneer. Es ist nicht unwahrscheinlich, daß die früher von mir beschriebene Clione punctata mit dieser Form identisch ist. Clione punctata, welehe unterhalb der Haut auf dem Eingeweidenucleus und auf Kopf und Buccalkegel zahlreiche sternförmige, schwarze Chromatophoren zeigt und eine blaßgelbliche Farbe auf dem Eingeweidesack, eine dunklere Farbe auf dem Kopfe aufweist, wurde einmal im Indo-australischem Archipel, später von der deutschen Südpolar-Expedition auch an der Westküste Afrikas beob- achtet. Da aber die Radula und die Hakensäcke nicht untersucht und auch das Vor- handensein eines Oberkiefers nicht festgestellt wurde, läßt sich die Identität der beiden Arten nicht beweisen; jedenfalls hat C. punctata auch nur ein einziges Paar Buccalkegel. Im Golfe von Biscaya. Im Golfe von Biscaya. 3. Gen. Paraclione Tesch 1903 Paraclione, Tesch in: Tijdschr. Nederl. dierk. Ver., ser.2 v8 p. 117. Körper ziemlich gedrungen, aboraler Pol stumpf-kegelförmig, durch eine unvermittelte Einschnürung vom Rumpfe getrennt. Kopf breit, durch eine mediane, dorso-ventrale Grube in zwei Teile zerlegt. Haut pigmentiert. Im Schlunde jederseits zwei zapfenförmige Buccalkegel. Radula mit ge- g g* 132 Pteropoda: B. Gymnosomata, 4. Clionidae, 3. Paraclione, 4. Paedoclione zähnter Mittelplatte. Mittellappen des Fußes gänzlich verschwunden. Flossen breit. Anus mit Osphradium und äußerer Nierenöffnung zusammen auf dem Analfelde. zähnter Mittelplatte. Mittellappen des Fußes gänzlich verschwunden. Flossen breit. Anus mit Osphradium und äußerer Nierenöffnung zusammen auf dem Analfelde. Im Indo-australischen Archipel. 1. P. pelseneeri Tesch 1903 P. p., Tesch in: Tijdschr. Nederl. dierk. Ver., ser. 2 c.8 p.117 \| 1904 P. p., Tesch in: Siboga-Exp., nr. 52 p.82 t.5 £. 147—150, 151 (Radula und Haken). Körper gedrungen, zylindrisch oder spindelförmig, am unteren Pol mit einem stumpfen, kegelförmigen Fortsatz. Haut mit kleinen Chromatophoren ausgestattet, auch mit zahlreichen einzelligen Drüsen. Kopf abgerundet, breit; eine sehr flache mediane, dorso-ventrale Grube teilt ihn in zwei Teile. Seiten- lappen des Fußes für etwa die Hälfte der Länge frei, distalwärts mit kurzem äußere Lippen NE Schlundhöhle y.\..... Buccalkegel Buccalhöhle +-8> --- vorderer Tentakel innere Lippen Radulatasche B Mittelplatte Seitenplatte RV2SST C Fig. 103. äußere Lippen A RV2SST C Fig. 103. P. pelseneeri. A von der Ventralseite (!/,), B Oralansicht des Kopfes (etwa /,), © Querreihe der Radula (vergr.?). Nach Tesch. Fig. 103. P. pelseneeri. A von der Ventralseite (!/,), B Oralansicht des Kopfes (etwa /,), © Querreihe der Radula (vergr.?). Nach Tesch. Zipfel. Flossen sehr breit, fast rundlich. Im Schlunde jederseits zwei zapfen- förmige Buccalkegel, von denen der dorsale_der kleinere ist. Formel der Radula 3.1. 3, Mittelplatte halbmondförmig mit einem langen Zahn in der Mitte. Hakensäcke kurz, mit wenigen, an der Basis sehr breiten und an der Spitze scharf gebogenen Haken. Farbe gelblich oder grau, die Chroma- tophoren sind schwarz. L. bis 3 mm. — Fig. 103. Im Indo-australischen Archipel. Im Indo-australischen Archipel. 1907 Paedoclione, Danforth in: P. Boston Soe., v.34 p.2. 1907 Paedoclione, Danforth in: P. Boston Soe., v.34 p.2. Körper länglich-oval, mit larvalen Wimperringen; in der Nähe des hinteren Wimperringes ist der Körper etwas aufgeblasen und endet dann schnell in eine stumpfe Spitze. Nur die vorderen Tentakeln sind deutlich vorhanden, die hinteren sehr reduziert. Buccalkegel asymmetrisch, zwei rechts, einer links. Hakensäcke sehr wenig entwickelt („ankistrophores“ Danforth), nur als zwei muskulöse, verdickte Stellen der Buccalwandung entwickelt, mit Pteropoda: B. Gymnosomata, 4. Olionidae, 4. Paedoclione 133 wenigen Haken. Mittelplatte der Radula ohne Zähne. Seitenlappen des Fußes im Bereiche des vorderen Wimperringes, Mittellappen weiter distal, gut entwickelt, etwas zugespitzt. Herz und Niere rechts, Niere ein langer, ge- faltelter Schlauch, der sich von der Pericardialöffnung erst nach hinten bis zum Ende des Magen-Lebersackes erstreckt, sich dann nach vorn wendet und ganz vorn am Hinterrande der rechten Flosse ausmündet, wo sich auch der After und das Osphradium finden. Die großen Zellen an der Wandung des Vorraumes des Penis haben sich zu einer besonderen großen Drüse („first penial gland“ Danforth) entwickelt, in das Hinterende des Penis mündet eine schlauchförmige, prostatische Drüse („second penial gland“ Danforth). Nördlicher Atlantischer Ozean. 1 Art. Nördlicher Atlantischer Ozean. Nördlicher Atlantischer Ozean. 1 Art. 1. P. doliiformis Danf. 1907 P. d., Danforth in: P. Boston Soe., v.34 p.2 t.1 £.1,2,5 (Radula). Körper spindelförmig, gestreckt, mit drei larvalen Wimperringen, von denen der vordere in gesonderte Stücke zerlegt, die anderen, wie bei allen --- vorderer Tentakel ... vorderer Wimperring Seitenlappen des Fußes AAF/ Öffnung des Penis ----< Sn ---- Flosse Osphradium -- 5 ---- Mittellappen des Fußes After oe a ---- --- hinterer Wimperring Fig. 104. P. doliiformis. Von der Ventralseite, mit eingezogenen Flossen (etwa 5%,). Nach Danforth. a Fig. 104. Fig. 104. P. doliiformis. Von der Ventralseite, mit eingezogenen Flossen (etwa 5%,). Nach Danforth. Gymnosomata-Larven, kontinuierlich sind; im Bereiche des hinteren Ringes ist der Körper etwas aufgeblasen und endet dann in eine sehr kurze, kegel- förmige Spitze. An beiden Polen kann der Körper stark verkürzt werden durch Kontraktion von sechs Längsmuskelbändern (2 ventral, 2 dorsal und 2 lateral), die Bänder sind im Bereiche des mittleren und hinteren Wimper- ringes durch zirkuläre Muskelfasern verbunden. Eingeweidesack reicht bis zum unteren Körperpol. Kopf breit, abgerundet, die vorderen Tentakeln sind Pteropoda: B. Gymnosomata, 4. Clionidae, 4. Paedoclione, 5. Thliptodontidae 134 deutlich, die hinteren, im Nacken, fast gänzlich rückgebildet. 1907 Paedoclione, Danforth in: P. Boston Soe., v.34 p.2. Seitenlappen des Fußes im Bereiche des vorderen Wimperringes, klein, kontraktil, an der ventralen Seite bewimpert, Mittellappen groß, gut entwickelt, deutlich von den Seitenlappen distalwärts verschoben, zipfelartig verlängert und nicht am Ende scharf zugespitzt, sondern abgerundet. Flossen klein, ebenfalls kontraktil, oval, am freien Rande bewimpert. Buccalkegel asymmetrisch an- geordnet, zwei auf der rechten Seite der Schlundwandung, ein einziger an der linken Seite; Unterschiede in der Größe nicht vorhanden. Formel der Radula 5.1.5, Mittelplatte schmal, stark in die Quere ausgedehnt, ohne Zahn, Seitenplatten schmal, an der Spitze nur wenig gebogen. Hakensäcke nur als verdickte Stellen in der Buccalwandung bemerkbar, mit einer sehr seichten Vertiefung, aus welcher 5—8 große, wenig gekrümmte Haken in die Buccalhöhle vorragen. Farbe unbekannt. L. ungefähr 15 mm. — Fig. 104. Es wurde diese Art nur in Casco Bay, an der nordöstlichen Küste der Ver- einigten Staaten gefunden, und zwar nur als Larven mit den drei charakteristischen Wimperreifen. Obgleich alle geschlechtlichen Entwicklungsstadien bis zum reifen Weibchen beobachtet wurden, blieben die Wimperringe stets erhalten, so daß diese wohl ein permanentes Charakteristikum der Art bilden dürften. 2 Gattungen, 3 Arten. In den wärmeren Meeren. 5. Fam. Thliptodontidae 1902 Thliptodonidae, Kwietniewski in: Atti Soc. Veneto-Trent., ser,2 v.4 fasc. 2 p.54 \| 1902 Pteroceanidae, Meisenheimer in: Zool. Anz., v.26 p.'93 \| 1905 Thliptodon- tidae, Meisenheimer in: Ergeb. Tiefsee-Exp., v. 91 p. 57. . Körper vorn stark verbreitert, nach hinten zugespitzt oder breit abgerundet. Kopfabschnitt außerordentlich umfangreich, er nimmt fast die Hälfte des ganzen Körpers ein und ist nicht durch eine Einschnürung vom Rumpfe abgesetzt. Eingeweidesack läßt meist den hinteren Teil des Körpers frei. Haut nicht pigmentiert, gänzlich durchsichtig. Kein dorsales Drüsenfeld. Schlund weit. und dünnwandig. Keine Rüsselbildung. Bueccalkegel vorhanden oder fehlend, das gleiche gilt von den Hakensäcken. Oberkiefer fehlt. Radula kräftig ausgebildet und von derjenigen aller anderen Pteropoden dadurch abweichend, daß in einer Querreihe drei Arten von Platten (Mittelplatte, Zwischenplatten und Seitenplatten) vor- kommen, statt nur zwei. An der Buccalwandung mächtige, in sich geschlossene Schlundblasen. Flossen auf der halben Körper- länge inseriert, und ebenso wie der zwischen ihnen befindliche Fuß, sowie dieam Kopfabschnitt gelegenen Tentakeln, in besondere Hauttaschen zurückziehbar. Fuß klein, aus Seitenlappen und Mittellappen zusammengesetzt. Kiemen fehlen vollständig. After, äußere Nierenöffnung und ÖOsphradium dicht beisammen im Bereich des distalen Randes der rechten Flosse Herz und Niere links, letztere zuweilen größtenteils ventral des Magen- Lebersackes verlagert: Penis mit weitem Vorraum und einer einzigen Anhangsdrüse (Prostata), zu der zuweilen noch eine zweite hinzutritt. In den wärmeren Meeren. 2 Gattungen, 3 Arten. Pteropoda: B. Gymnosomata, 5. Thliptodontidae, 1. Thliptodon 135 Bestimmungstabelle der Gattungen: Körper spindelartig verlängert, Buccalkegel nicht vorhanden, Hakensäcke mächtig ausgebildet. . Körper spindelartig verlängert, Buccalkegel nicht vorhanden, Hakensäcke mächtig ausgebildet. . 1. Gen. Thliptodon Körper tonnenförmig, gedrungen, Buccalkegel vor- Körper tonnenförmig, gedrungen, Buccalkegel vor- handen, Hakensäcke fast gänzlich rückgebildet . 2. Gen. Thalassopterus 1. Gen. Thliptodon Boas ?1832 Pelagia, Quoy & Gaimard in: Voy. Astrol., v.2 p.392 \| ?1862 Ptero- pelagia, Bronn in: Bronn’s Kl. Ordn., v. 311 p.645 \| 1886 Thliptodon, Boas in: Danske Selsk. Skr., ser.6 v.4 p.174 \| 1902 Pteroceanis, Meisenheimer in: Zool. Anz., v.26 p. 93. Körper in der proximalen Hälfte sehr umfangreich, nach hinten spitz zulaufend. Kopfabschnitt nicht vom Rumpfe abgesetzt, sehr mächtig ent- wickelt, so daß er mehr als die Hälfte des ganzen Körpers einnehmen kann. Eingeweidesack reicht nieht bis zum hinteren Körperpol und bildet eine kompakte, spindelförmige Masse. Haut durchsichtig, nicht pigmentiert, sehr dünnwandig, mit eingestreuten Drüsen, welche sich oft aus mehreren Zellen zusammensetzen; solche Gruppen, welche sich bald als geschlossene Bläschen, bald als mit körniger Sekretmasse gefüllte und sich nach außen öffnende Säckehen darstellen, weisen zwar mehrere Kerne in der Wandung, aber keine Zellgrenzen zwischen diesen Kernen auf; andere Drüsen (auf dem Analfelde) bestehen aus mehreren, deutlich voneinander gesonderten Zellen, welche sich zu einem rundlichen Bläschen vereinigen. Schlund weit, dünnwandig. Buccal- kegel fehlen völlige. Hakensäcke mächtig ausgebildet, innere und äußere Hakenscheide sehr dünnwandig, zwischen ihnen ein System radiär ausstrahlender Muskelfasern, welche vom Grunde der inneren Scheide nach den hinteren Seitenwandungen der äußeren Scheide verlaufen. Zwischen den Falten der Wandung der Buceal- und Schlundhöhle umfangreiche, lang-schlauchförmige, von einer lymphatischen Flüssigkeit erfüllte Schlundblasen, welche dem Schlundapparat bei seiner Tätigkeit als eine Art Polster einen guten Rückhalt bieten. Derartige, in der hinteren Hälfte durch unvollständige Quersepten in mehrere Abschnitte zerlegte Blasen finden sich auch in der Radulatasche. Speicheldrüsen als keulenförmige Gebilde vorhanden. Mittel- und Seitenlappen des Fußes sehr klein, zuweilen weit auseinander gelagert. Flossen breit, an der Basis etwas eingeschnürt und gestielt, bei ganz jungen Larven noch fehlend. Herz links, Niere fast ganz ventral des Magen-Lebersackes, nur die Öffnung nach außen auf der rechten Körperseite in der Nähe des Afters. Penis mit einem sehr weiten Vorraum, in welchen von der linken Seite her eine lange, gewundene, schlauchförmige Prostatadrüse einmündet; außerdem liegt auf der ventralen Körperseite, zwischen den Ansatzstellen der Flossen und etwas rechts von der Mediane, ein besonderer Apparat, der wohl als Hilfsorgan bei der Begattung funktioniert und sich als ein gewundener, nahe der Öffnung mächtig angeschwollener Schlauch darstellt (p. 101). In den wärmeren Meeren. 2 Arten. Bestimmungstabelle der Arten: N und Mittellappen des Fußes zu einem einzigen PDIE Vereinge ee ee A 1. T. gegenbauri \| Seiten- und Mittellappen des Fußes weit auseinander gerückt 2. T. 1. Gen. Thliptodon Boas diaphanus Bestimmungstabelle der Arten: N und Mittellappen des Fußes zu einem einzigen PDIE Vereinge ee ee A 1. T. gegenbauri \| Seiten- und Mittellappen des Fußes weit auseinander gerückt 2. T. diaphanus 13 Pteropoda: B. Gymnosomata, 5. Thliptodontidae, 1. Thliptodon 136 1. T. gegenbauri Boas ?1832 Pelagia alba, Quoy & Gaimard in: Voy. Astrol., v.2 p.392 Moll. t.28 f.7 \| ?1862 Pteropelagia a., Bronn in: Bronn’s Kl. Ordn., v.3ır p-645 \| 1886 Tihliptodon gegenbauri, Boas in: Danske Selsk. Skr., ser.6 v.4 p.174 \| 1903 T. g., Kwietniewski in: Rice. Lab. Anat. Roma, v.9 p. 325 t. 17—19. Körper vorn stark. verbreitert, nach hinten zugespitzt, von der Seite gesehen spindelförmig, in dorsaler und ventraler Ansicht breit-oval, Kopf- abschnitt, besonders im proximalen Teile, dorso-ventral zusammengedrückt. Vordere Tentakeln besonders bei den ganz jungen Larven deutlich. Fuß klein, etwa an der Übergangs- „ vorderer Tentakel stelle zwischen Kopfabsehnitt und Rumpf eingepflanzt, Seitenlappen und kurzer Mittellappen nahe zu- sammen und zu einem einzigen (Gebilde vereinigt, das sich ganz in eine Vertiefung des Integumentes zurückziehen kann. Flossenschmal, nach den Enden zu etwas ver- breitert, distal von der Ansatz- stelle des Fußes inseriert. Formel der Radula 3.1.1.1. 3, Mittel- platte klein, ohne Zähne, Zwischen- platte (zu beiden Seiten der Mittel- platte) langgestreckt, etwas ge- es Seitenlappen des Fußes ..}„.." Mittellappen des Fußes 2. Flosse ‘. -—“ hinterer Wimperring Fig. 108. bogen, am Innenrande mit einem T,gegenbauri. Von der Ventralseite (etwa '/,). Nach Starken, gekrümmten Fortsatz, Kwietniowski. der an der medianen Seite fünf kleine Zähnchen zeigt, Seitenplatten ebenso lang wie die Zwischenplatte, sichelförmig gebogen. Hakensäcke mit zahlreichen (etwa 30) Haken. Larven von weniger als l mm L. noch mit den drei Wimperreifen, der hintere Reifen bleibt noch bei Tieren von etwa 5 mm L. bestehen. Farbe fehlt vollständig, der Magen-Lebersack schimmert mit bräunlicher Farbe hindurch. — L. 5 mm. — Fig. 105. „ vorderer Tentakel es Seitenlappen des Fußes ..}„.." Mittellappen des Fußes 2. Flosse ‘. -—“ hinterer Wimperring Fig. 108. T,gegenbauri. Von der Ventralseite (etwa '/,). Nach Kwietniowski. Fig. 108. T,gegenbauri. Von der Ventralseite (etwa '/,). Nach Kwietniowski. Fig. 108. T,gegenbauri. Von der Ventralseite (etwa '/,). Nach Kwietniowski. fünf kleine Zähnchen zeigt, Seitenplatten ebenso lang wie die Zwischenplatte, sichelförmig gebogen. Hakensäcke mit zahlreichen (etwa 30) Haken. Larven von weniger als l mm L. noch mit den drei Wimperreifen, der hintere Reifen bleibt noch bei Tieren von etwa 5 mm L. bestehen. 1. Gen. Thliptodon Boas Farbe fehlt vollständig, der Magen-Lebersack schimmert mit bräunlicher Farbe hindurch. — L. 5 mm. — Fig. 105. fünf kleine Zähnchen zeigt, Seitenplatten ebenso lang wie die Zwischenplatte, sichelförmig gebogen. Hakensäcke mit zahlreichen (etwa 30) Haken. Larven von weniger als l mm L. noch mit den drei Wimperreifen, der hintere Reifen bleibt noch bei Tieren von etwa 5 mm L. bestehen. Farbe fehlt vollständig, der Magen-Lebersack schimmert mit bräunlicher Farbe hindurch. — L. 5 mm. — Fig. 105. Bisher im Mittelmeer und zwar nur bei Messina beobachtet, außerdem an der Westküste Irlands. 2. T. diaphanus (Meisenh.) 1902 Pteroceanis diaphana, Meisenheimer in: Zool. Anz.. v.26 p.93 \| 1905 Thliptodon diaphanus, Meisenheimer in: Ergeb. Tiefsee- Exp., 0.91 p.59 t.16 £.10. 2. T. diaphanus (Meisenh.) 1902 Pteroceanis diaphana, Meisenheimer in: Zool. Anz.. v.26 p.93 \| 1905 Thliptodon diaphanus, Meisenheimer in: Ergeb. Tiefsee- Exp., 0.91 p.59 t.16 £.10. Körper vorn stark verbreitert, nach hinten zugespitzt. Vordere und hintere Tentakeln in besondere Hauttaschen zurückziehbar. Seitenlappen des Fußes nahe zusammen, an der Übergangsstelle zwischen Kopf- und Rumpf- abschnitt eingepflanzt und wie bei der vorigen Art, in eine besondere Hauttasche zurückziehbar, im gleichen Niveau mit dem Vorderrande der Flossen, Mittellappen ziemlich weit von den Seitenlappen und von diesen völlig getrennt, nach hinten zwischen die Ansatzstellen der Flossen verschoben und gleichfalls retraktil. Flossen an der Basis verschmälert, distal beträchtlich erweitert. Formel der Radula wie bei der vorigen Art, Mittelplatte mit verdicktem Vorderrande und mit zwei stumpfen Höckern an den Seiten, Zwischenplatte mit einem nicht gezähnten, gekrümmten Fortsatz am Innenrande, Seitenplatten wie bei der vorigen Art. Hakensäcke mit zahlreichen Haken. Bei Exemplaren von 7—8 mm L. existiert noch der Pteropoda: B. Gymnosomata, 5. Thliptodontidae, 1. Thliptodon, 2. Thalassopterus 137 Pteropoda: B. Gymnosomata, 5. Thliptodontidae, 1. Thliptodon, 2. Thalassopterus 137 hintere Wimperring. Farbe fehlt, der Eingeweidesack schimmert mit dunkler Farbe hindurch. L. bis 12 mm. — Fig. 106. nnnanen? Mundöffnung IN_----- vorderer Tentakel Hakensack "f- Schlund Schlundblasen .::---.. ber Radulatasche Speicheldrüse -- ; Bucealganglien --.-}.-....\ „0 Hakensack Anlage des Penis ... Oesophagus .__K._____. ae = >-.. ninterer Tentakel ...... Seitenlappen des Fubes Pedalganglien Si Statolithen '"""""”"- q Anlage des Hilfsorzanes BErerIBERe tn I TEA A A = Flosse Anlage der Zwitterdrüse """-- "7 ... Mittellappen Osphradium ---------- D: des Fußes ">... Maeen-, Lebersack Be Ne Aber äußere Nierenöffnung “ ? "» Enddarm Renopericardialgang Kammer des Herzens“ Pericardialhöhle”” _ Vorhof des Herzens hinterer Wimperring Fig. 106. T. diaphanus. 1. Gen. Thliptodon Boas Von der Ventralseite, innere Organisation in den Hauptzügen dargestellt (etwa 3/,). Nach Meisenheimer. Hakensack Bu Anlage des Penis Be Aber äußere Nierenöffnung “ ? Renopericardialgang Kammer des Herzens“ Pericardialhöhle”” Vorhof des Herzens Be Aber äußere Nierenöffnung “ Renopericardialgang Kammer des Herzens“ Fig. 106. T. diaphanus. Von der Ventralseite, innere Organisation in den Hauptzügen dargestellt (etwa 3/,). Nach Meisenheimer. In den warmen Stromgebieten des Indischen und Atlantischen Özeanes, auch 'an der Westküste Irlands, nicht im Mittelmeer, ein einziger Fund aus dem antarktischen Meer, in der Nähe des Eisrandes (61° 58's. Br., 95°8'6.L.). Der letztere Fund, von der deutschen Südpolar-Expedition gemacht, ist sehr bemerkenswert. Das Exemplar zeigte die drei Teile des Fußes näher aneinander gerückt als es sonst bei dieser Art der Fall ist und erreichte zudem die beträchtliche Länge von 12 mm. Meisenheimer hält es für nicht unwahrscheinlich, daß hier eine besondere Abart (antarcticus) von T. diaphanus vorliegt. 2. Gen. Thalassopterus Kwietniewski 1910 Thalassopterus, Kwietniewski in: Zool. Anz., v.36 p. 271. Körper sehr breit, tonnenförmig, am Hinterende sehr breit abgerundet, nicht zugespitzt, mit drei larvalen Wimperreifen. Kopfabschnitt sehr umfang- Pteropoda: B. Gymnosomata, 5. Thliptodontidae, 2. Thalassopterus - 138 reich, vom Rumpfe nicht abgesetzt. Vordere Tentakeln lang, konisch, hintere Tentakeln sehr kurz. Haut völlig durchsichtig, nieht pigmentiert, mit zahl- reichen Drüsen, welche häufig aus wenigstens zwei Abteilungen, durch eine Scheidewand getrennt, bestehen; jede Abteilung enthält mehrere Kerne und während die eine die Schleimsekretion unterhält, sondert die andere eine körnige Masse ab; beide Abteilungen münden durch einen besonderen Porus nach außen. Einfachere einzellige Drüsen kommen ebenfalls vor, besonders auf den Seitenlappen des Fußes. Unterhalb des Körperepithels findet sich eine mächtige Lage von großen, blasigen Bindegewebszellen. Der Eingeweide- sack reicht ganz bis zum aboralen Körperpol. Schlund weit. Keine Rüssel- bildung. An der Schlundwandung jederseits zwei konische Buccalkegel, welche eine breite Basis haben und am Ende zugespitzt sind, hier sind sie mit einem Härchenschopf versehen; die Oberfläche der Buccalkegel ist glatt; das dorsale Paar ist größer als das ventrale. Dorsale Wandung des Schlundes und der Buccalmasse bildet jederseits eine mächtige, drüsige Falte (Buecaldrüsenlappen, Fig. 107), welche dorsalwärts und nach den Seiten hervorragen und funktionell die Speicheldrüsen vertreten; eigentliche Speicheldrüsen nicht ausgebildet. Blasen in der Radulatasche fehlen. Hakensäcke sehr rückgebildet, nur als zwei muskulöse Schläuche, ohne innere Scheide und ohne Haken, zu beiden Seiten der Buccalmasse. Mit den vorderen Enden dieser Muskelscheiden ist jeder- seits ein Schenkel der beiden „Schlundschläuche“ verbunden, welche allseitig geschlossen sind, sich von der Buccalmasse, zwischen Hakensackrudiment und Buecaldrüsenlappen distalwärts erstrecken, dann wieder dorsalwärts nach vorn umbiegen und so einen dorsalen Schenkel bilden. Etwa in der halben Länge sind die Flossen inseriert, welche in besondere Hauttaschen zurückgezogen werden können und sehr schmal sind, bei jungen Larven sind sie noch gar nicht entwickelt. Der Fuß liegt proximalwärts von der Ansatzstelle der Flossen, ist sehr klein und besteht aus eng verbundenen Seitenlappen und Mittellappen. After, Osphradium und äußere Nierenöffnung nahe zusammen am Eingang der Hauttasche für die rechte Flosse. Herz und Niere rechts. Penis mit sehr weitem Vorraum, welcher von vakuolisierten, blasigen Zellen umgeben ist, und mit zwei in diesen Vorraum einmündenden Prostatadrüsen, von denen die linke in histologischer Hinsicht mit dem Saugapparat von Thlipiodon (p. 135) viel Übereinstimmung zeigt; ein gesondertes Saug- und Hilfsorgan bei der Begattung fehlt hier übrigens ganz. Mittelmeer. 1 Art. Mittelmeer. 1 Art. 1. T. zancleus Kwietn. 2. Gen. Thalassopterus Kwietniewski 1910 T.z., Kwietniewski in: Zool. Anz,, v.386 p. 271. Körper sehr breit, tonnenförmig. Kopfabschnitt in dorso-ventraler Richtung abgeplattet. Vordere Tentakeln lang, auf ihrer Oberfläche verläuft eine Leiste gegen die Mundöffnung hin. Seitenlappen des Fußes unmittelbar hinter dem vorderen Wimperreifen, breit, Mittellappen eng mit den Seiten- lappen verbunden, kurz und zugespitzt. An der Basis ist der Fuß wie ge- stielt; er kann ganz zurückgezogen werden. Flossen klein, bei ganz kleinen Larven noch gar nicht entwickelt, in besondere Taschen zurückziehbar, hinter der Ansatzstelle des Fußes. Buccaldrüsenlappen auf der Dorsalwandung der Schlund- und Buccalhöhle mächtig entwickelt. Hakensäcke stark rückgebildet, nur zwei muskulöse Schläuche zu beiden Seiten der Buccalmasse, ohne Haken. Formel der Radula 3. 1. 1. 1. 3., ähnlich wie der von T’Aliptodon gebaut, Mittelplatte halbmondförmig, ohne Zähne, Zwischenplatte langgestreckt, mit 139 Pteropoda: B. Gymnosomata, 5. Thliptodontidae, 2. Thalassopterus einem starken Fortsatz am Innenrande, welcher keine sekundären Zähnchen aufweist, Seitenplatten lang, sichelförmig. Farbe fehlt. L. bis etwa 2 mm. —. ‚Fig. 107. einem starken Fortsatz am Innenrande, welcher keine sekundären Zähnchen aufweist, Seitenplatten lang, sichelförmig. Farbe fehlt. L. bis etwa 2 mm. —. ‚Fig. 107. ‚„. vorderer Tentakel E 2 X---- Buccaldrüsenlappen Bucealkegel ...::-7 7 8 .. Muskelscheide der Hakensäcke ß S' vorderer Wimperring jo Z, STÄ\n - Radulatasche WW Ag ---- Seitenlappen des Fußes akzessorische Penisdrüse -----H=-1- I) EIS) os... akzessorische Penisdrüse Mittellappen des Fußes ----- - n==Rl] Osphradium ....__ = Flosse Hautdrüsen ----7.. a Niere ------- i ‘Gegend der Anhangsdrüsen der -------- Ne, A Zwitterdrüse Blasen der Körperhülle <7--------X ELTT S “"----. hinterer Wimperring dorsaler Buccalkegel --.._ N Muskelscheide des Haken- "., £ sackes y ventraler Schenkel der} Schlundschläuche an na ML ee Haken- ___d_ ventraler Schenkel der Buccaldrüsenlappen ---4 Schlundschläuche - Bucealdrüsenlappen dorsale Schenkel der Schlundschläuche B Fig. 107. T. zancleus. A Larve, von der Ventralseite (#/,), B Querschnitt auf der Höhe des Schlundes (#/,). Nach Kwietniewski. Es wurden bis jetzt nur Larven mit larvalen Wimperringen gefangen, welche aber schon geschlechtlich entwickelt waren und Eier ablegten. Mit Sicherheit nur im Mittelmeer und zwar bei Messina beobachtet, vielleicht . vorderer Tentakel Fig. 107. T. zancleus. A Larve, von der Ventralseite (#/,), B Querschnitt auf der Höhe des Schlundes (#/,). Nach Kwietniewski. Fig. 107. T. zancleus. A Larve, von der Ventralseite (#/,), B Querschnitt auf der Höhe des Schlundes (#/,). Nach Kwietniewski. Es wurden bis jetzt nur Larven mit larvalen Wimperringen gefangen, welche aber schon geschlechtlich entwickelt waren und Eier ablegten. 6. Fam. Halopsychidae 1850 Cymbuliadae (part.), J. E. Gray, Cat. Moll. Brit. Mus., v.2 p.24 \| 1854 Hyaleidae (part.), S. P. Woodward, Man. Moll., p.204 \| 1856 Euribiacea, Troschel in: Gebiss Schneck., v.1 p.54 \| 1859 Eurybidae, Chenu, Man. Conchyl., ®.1 p. 115 \| 1884 Eurybiidae, P. Fischer, Man. Conchyl., p. 426 \| 1887 Halopsychidae, Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p. 52. Körper gedrungen, eiförmig oder oval, am Hinterende breit abgerundet. Kopf- und Rumpfabschnitt durch eine dorsale und eine ventrale Falte des Integumentes scharf geschieden. Durch diese Falten wird eine Vertiefung gebildet, in welche sich der Kopf samt Fuß nnd Flossen ganz zurückziehen kann, es bleibt dann am Vorderende des Körpers nur ein enger, querer Spalt sichtbar. Der Eingeweidesack reicht bis zum hinteren Körperpol. Kopfabschnitt sehr klein. Rechts und links von der Mundöffnung ein Paar sehr mächtiger vorderer Tentakeln; jeder Tentakel be- steht aus einem Stamm, welcher sich in zwei Schenkel teilt, von denen der mediane klein bleibt, der laterale aber sehr lang und zylindrisch ist. Hintere Tentakeln auf den Seiten des Kopfes etwas dorsal verlagert. Haut auf dem Rumpfe sehr dick, nicht pigmentiert, mit zahlreichen, flaschenförmigen, einzelligen Drüsen und vielen großen, prallgefüllten und allseitig geschlossenen Blasenzellen, wie sie in dieser Ausbildung sonst nicht bei Gym- nosomata vorkommen und in dieser Familie der Haut eine knorpelhafte Konsistenz verleihen!). Kein dorsales Drüsenfeld. Schlund eng und dünn, die Wandung in zahlreiche Falten gelegt. Keine Rüsselbildung. Buccalkegel fehlen vollständig, ebenso die Hakensäcke. Radula in jeder Querreihe nur aus drei Platten zusammengesetzt: Fuß unmittelbar ventral der Mundöffnung; er besteht aus eng miteinander verbundenen Seiten- und Mittel- lappen. Flossen auf der Höhe des Fußes am Kopfabschnitt inseriert, lang, an der Basis schmal, nach der Spitze zu allmählich verbreitert und in der Mitte des freien Randes mit einer Ein- kerbung ausgestattete Kiemen fehlen vollständig. After und äußere Nierenöffnung dicht beisammen, etwas distal von der An- satzstelle der rechten Flosse. Osphradium mehr dorsal und proximalwärts verlagert: Niere auf der Dorsalseite des Ein- geweidesackes; die Öffnungen nach dem Pericard und nach außen nahe beieinander an den beiden Enden des bogenförmig ver- laufenden Nierenschlauches, auf der rechten Seite des Kopf- abschnittes. Herz ebenfalls dorsal, ventral von der Niere, quer- gelagert, Vorhof nach rechts, Kammer nach links gewendet. !) Die harte Umhüllung des Rumpfes, in welche sich der Kopfabschnitt ganz zurückziehen kann, hat schon früher zu der irrtümlichen Auffassung Veranlassung gegeben, daß hier eine wirkliche Schale vorhanden war, weshalb man dann den einzigen Repräsentanten der Familie bei den Theeosomata und zwar in die Gattung C'ymbulia einreihte. 2. Gen. Thalassopterus Kwietniewski Es wurden bis jetzt nur Larven mit larvalen Wimperringen gefangen, welche aber schon geschlechtlich entwickelt waren und Eier ablegten. Mit Sicherheit nur im Mittelmeer und zwar bei Messina beobachtet, vielleicht auch bei Madeira, 140 Pteropoda: B. Gymnosomata, &. Halopsychidae 6. Fam. Halopsychidae Der Enddarm entspringt auf der dorsalen Seite des Magens und weist nahe dem After einen sich weit auf der rechten Seite des Einge- weidesackes distalwärts erstreckenden Sack auf, welcher von einer rötlich-gelben Flüssigkeit erfüllt ist. Genitalanhangsdrüsen räumlich gesondert, klein, histologisch nicht abweichend gebaut !) Die harte Umhüllung des Rumpfes, in welche sich der Kopfabschnitt ganz Pteropoda: B. Gymnosomata, 6. Halopsychidae, 1. Halopsyche 141 Ovo-vivipar, die Embryonen, welche eine mehr zugespitzte Gestalt besitzen als dieerwachsenen Individuen, entwickeln sich anfänglich in einem Brutsack, der aus einer umgewandelten Genitalanhangs- drüse hervorgeht und werden später durch Platzen der Körper- hülle des Muttertieres frei (p.102). Ovo-vivipar, die Embryonen, welche eine mehr zugespitzte Gestalt besitzen als dieerwachsenen Individuen, entwickeln sich anfänglich in einem Brutsack, der aus einer umgewandelten Genitalanhangs- drüse hervorgeht und werden später durch Platzen der Körper- hülle des Muttertieres frei (p.102). In der Warmwasserzone des Indischen und Pazifischen Özeanes. 1 Gattung, 1 Art. 1 Gattung, 1 Art. 1. Gen. Halopsyche Bronn ?1825 Psyche, Rang in: Ann. Sei. nat., v5 p.284 \| ?1827 Euribia, Rang in: Ann. Sei. nat., v. 12 p.328 \| ?1852 Oymbulia (part.), Quoy & Gaimard in: Voy. Astrol., v.2 p.376 \| 1853 Eurybia (non Hübner 1816), A. Philippi, Handb. Conchyl., p.293 \| 1854 E., S. P. Woodward, Man. Moll., p.206 \| 1862 Zalopsyche + Theceurybia, Bronn in: Bronn’s Kl. Ordn., v.311 p. 645. ?1825 Psyche, Rang in: Ann. Sei. nat., v5 p.284 \| ?1827 Euribia, Rang in: Ann. Sei. nat., v. 12 p.328 \| ?1852 Oymbulia (part.), Quoy & Gaimard in: Voy. Astrol., v.2 p.376 \| 1853 Eurybia (non Hübner 1816), A. Philippi, Handb. Conchyl., p.293 \| 1854 E., S. P. Woodward, Man. Moll., p.206 \| 1862 Zalopsyche + Theceurybia, Bronn in: Bronn’s Kl. Ordn., v.311 p. 645. Die Diagnose stimmt mit der der Familie überein. In der Warmwasserzone des Indischen und Pazifischen ÖOzeanes. 1 Art. 1. H. gaudichaudii (Souleyet) ?1825 Psyche globulosa, Rang in: Ann. Sei. nat., v.5 p.284 1.7 f.1 \| ?1827 Ewuribia hemispherica, Rang in: Ann. Sci. nat., v.12 p.829 t.14B £.9—11 \| ?1832 Oymbulia norfolkensis, Quoy & Gaimard in: Voy. Astrol., v.2 p.376 Moll. t.27 f.31,32 \| 1852 Buribia gaudichaudii, Souleyet in: Voy. Bonite, v.2 p.253, Moll. t.14 f.1—6 \| 1858 Eurybia g., Macdonald in: Tr. Linn. Soc. London, lateraler Schenkeldes vorderen Tentakels hinterer Tentakel . dorsale Falte des In- »---- . tegumentes Osphradium --- lateraler Schenkeldes vorderen Tentakels ""——.. hinterer Tentakel . dorsale Falte des In- »---- . tegumentes Osphradium --- = Flosse menanen Mittellappen des Fußes mn. After dorsale Falte des Integumentes Mundöffnung .- ventrale Falte des ; £ Integumentes lateraler Schenkel des “ vorderen Tentakels Seitenlappen des Fußes _.__\ Mittellappen des Fußes 2... Flosse ventrale Falte des Integumentes A B Mundöffnung .. vorderer Tentakel hinterer Tentakel Fig. 108, H. gaudichaudii. A junges Exemplar, von links, mit hervorgestrecktem Kopfabschnitt (vergr. 9), B orale Ansicht eines Exemplares (vergr.?), A und B nach Meisenheimer. C von der Ventralseite ($/,). Nach Pelseneer. er Schenkeldes ren Tentakels ""——.. nterer Tentakel . le Falte des In- »---- . tegumentes sphradium --- = Flosse menanen Mittellappen des Fußes mn. After dorsale Falte des Integumentes Mundöffnung .- ventrale Falte des ; £ Integumentes lateraler Schenkel des “ vorderen Tentakels Seitenlappen des Fußes _.__\ Mittellappen des Fußes 2... lateraler Schenkeldes vorderen Tentakels hinterer Tentakel . dorsale Falte des In- »---- . tegumentes Osphradium --- 1. Gen. Halopsyche Bronn Flosse ventrale Falte des Integumentes A B Mundöffnung £ lateraler Schenkel des vorderen Tentakels lateraler Schenkel des “ vorderen Tentakels Flosse A Mundöffnung Mundöffnung Fig. 108, Fig. 108, H. gaudichaudii. A junges Exemplar, von links, mit hervorgestrecktem Kopfabschnitt (vergr. 9), B orale Ansicht eines Exemplares (vergr.?), A und B nach Meisenheimer. C von der Ventralseite ($/,). Nach Pelseneer. 142 Pteropoda: B. Gymnosomata, 6. Halopsychidae, 1. Halopsyche v.22ıı p.245 t.43 \| 1862 Theceurybia g., Bronn in: Bronn’s Kl. Ordn., v.311 1.48D f.1—4 \| 1883 T. norfolkensis, Lankester in: Ene. Brit., ed.9 v.16 p. 666 f.83 \| 1886 Halopsyche gaudichaudi, Boas in: Danske Selsk. Skr., ser.6 v.4 p.173 t.8 f.119 \| 1887 H.g., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.55 t.3 f.7—9. v.22ıı p.245 t.43 \| 1862 Theceurybia g., Bronn in: Bronn’s Kl. Ordn., v.311 1.48D f.1—4 \| 1883 T. norfolkensis, Lankester in: Ene. Brit., ed.9 v.16 p. 666 f.83 \| 1886 Halopsyche gaudichaudi, Boas in: Danske Selsk. Skr., ser.6 v.4 p.173 t.8 f.119 \| 1887 H.g., Pelseneer in: Rep. Voy. Challenger, Zool. v.19 pars 58 p.55 t.3 f.7—9. Körper eiförmig oder oval. Kopfabschnitt ganz in die vordere Hälfte des Rumpfes zurückziehbar. Vordere Tentakeln sehr lang, zweischenklig. Flossen an der Spitze membranös, an der Basis muskulös, die Einkerbung am freien, distalen Rande mehr oder weniger tief. Mittellappen des Fußes ziemlich kurz, zugespitzt. Mittelplatte der Radula dreieckig, ohne Zahn; die beiden Seitenplatten mit breiter Basis und nach oben gekrümmter Spitze. Farbe der Haut fehlt oder ist leicht gelblich, die Blasenzellen in der Haut als zahlreiche weiße Pünktchen sichtbar. Magen-Lebersack braunschwarz. Blindsack des Enddarmes rötlich-gelb, Zwitterdrüse hellrot. L. bis etwa 8 mm. — Fig. 108. Auf die wärmsten Gebiete des Indischen und Pazifischen Ozeanes beschränkt, auch im Indo-australischen Archipel, mit Sicherheit noch nicht aus dem Atlantischen Ozean bekannt. Gänzlich unbekannter Herkunft: Cymodocea diaphana Orb. 1840 (©. d., Orbigny in: Voy. Amör. mörid., v.5ıu p.133 t. 9 £. 16, 17. Cymodocea diaphana Orb. 1840 (©. d., Orbigny in: Voy. Amör. mörid., v.5ıu p.133 t. 9 £. 16, 17. Cymodocea diaphana Orb. 1840 (©. d., Orbigny in: Voy. Amör. mörid., v.5ıu p.133 t. 9 £. 16, 17. Pazifischer Ozean. Alphabetisches Register Seite Alben eV are To vorne oe 150 " acieula acieula (Creseis). . . . . 25 acicula clava (Creseis) . . .. . 25 acieula (Cleodora) -. »- - 2... 25 acicula (Clio (Creseis)) . ». »... 25 seieula (Creseis) -. . -. »'o... 25 acicula (Creseis acicula) . . .».. 325 acieulata (Hyalaea) . .... . .25 ana RER ee EB 25 haar ale hard”. rk 110 affinis (Cavolinia tridentata) . . 51 Bing (Eiyalaea) Orb... .... 2... . 51 affınis (Hyalaea) Sow. Il ..... 49 affınis (Hyalaea tridentata var) .. 51 EN a ee . 150 Alate » . ee ee a he 78 3 BEL GE EI) Er 136 alba (Bieropelagia) .\. ... ..'. >. 136 alexandri (Heterofusus) . . .» ... - 19 Bespliten te mania alas 8 ine 150 andreae (Uleodora) . . . 2... 30 andreae (CO) +... : an. 30 angulata (Cavolinia longirostris) 46 Beewlata, (Eyalaea)'. „u. 2 nl 46 angusta (Cleodora pyramidata var.). 35 Anomia . . ee antarchiea, (Clio), ..: a. 2. ..0.0.84 imtaretiea (Lone) » « o... 0... antarctica (Clione limacina).. . . 128 antaretica (Clione limaeina var.) . . 128 antarctica (Limaecina) antarctica (Limaecina helieina). . 17 antarctica (Limaeina helieina var.) . 17 apicifulva (Peracle) . ... . A Boirıtulvar(beracls). ». . „00.0 74 erorabranehia - . 0... L Ben an re 42 Breticn (Arsonauta). 2... ... 16 Bretiea (Limacina) - .»..... 16 arctica er HR 16 are ran 82 Argonauts r .. .; ER VEN as 16 SEEN NS Die EEE FRE 150 Seite Ablanlare en te einen She 18 atlantica (Pneumodermis) . . .. . 112 Allantise sur. Sr var lee a ma 150 audebardii (Pneumodermon) Rang 112 audebardi (Pneumodermon) Loe. . „ 112 Alina ee a ehe. 150 Auranilarar (Uliop cz. 2 a En kun 129 australe (Balantium) . ...... 34 australis (Olendora) sn. nn 2.0 australis, (Cho) Bru@- „= 2.2... 128 australis (Clioy Plsun 2 2,2...2.3. 34 australis (Hyalaea) Orb... .... 34 australis (Hyalaea) Per... ..... 49 australis (Limaeina). . . ... .. el) australis (Limacina retroversa) . 20 austzalis (Spimalis) = Calw. .,.00 australis (Spongiobranchaea) . . . . 110 australis (Spongiobranchea) 110 australis (Spongiobranchia) Orb. 110 australis (Spongiobranchia) Chenu 118, 112 Balanfiume 9. nalen nel ee balantumz(Gleodora) =. . ou. al balantiume (Choy n » az ae öl balantium (Hyalaea) . ...... öl balea (Heterofusus) . ... Corrigenda Thilea (p.15, 22) muß in Thielea verbessert werden, da Strebel, Autor der Gattung, angibt, daß er sie nach J. Thiele in Berlin benannt hat. Alphabetisches Register 2 ...2.2.20 balear (lumacına) Moll, .. » 2 .% 20 balea (Limaeina) Munihe ..... 20 balea (Limacina retroversa). . . 20 balea(Smsalis)‘ . „ aan rs 20 bicarinatum (Balantium). ..... al bispinosa (Peracle) ....:..:...7 Dispingsagi(beraclis) „u... 00a 000 75 boasi (Pneumoderma). . .... 115 boasi (Pneumonoderma) . . ». . » 115 barealis (Clio) au... 0. Br te 126 Borealis (Olone) 2 ya... 126 brevispira (Peracle) . ..... 73 brevispmatPeraclis)s . 0. ala, 75 brawamı (Wleodoraı m 7 ne... 35 bulimoides (Atlanta) . ı..... 21 bulimoides (Limacina) . .... al bualmgides (Spirialis) : » . „= % » 21 eaduceus (Cliodita) . . 2. 22... 110 Alphabetisches Register 144 Seite galeceola (Corolla) Fri are 84 calceola (Oymbulia) . . . .. ee ealceola (Cymbuliopsis) . . » 0... 84 calceolus (Cymbulia) . » 2... 84 caliciformis (Creseis) . » » ....2%6 calignla (Lreseis) » =... ee! \| VRIDYIOBRURM N aa ee Le 72 cancellata (Triptera) . . .....» 38 CEDENSISFLGLO), u er ie 124 Geroinat aa Dry N are: 150 eaudats (Oleodora) . tm. 4 2 88 Caudata. (io) Er seirun ea ne 35 caudata (Clio) M’Donald . .... 129 eandata (Olione); . ;» vs"... 129 Ra ee Kae an 150 Gavalna. Us AT er rear 42 Cavolinia ENT RIITUENEN, 38 SESHHITINER EL un ut ne Eee es Cavöliniidae..... ra we NEREEE ee e Reae rter alhe 150 chaptalii (Cleodora) . . . . ». . FRE: \| CHaptaliis(@Ho)r. use ee 31 charybdis (Tiedemannia) . . .. 87 chemnitziana (Hyalaea) ...... 49 chierchiae (Cleodora) . . ..... 25 chierchiae (Clio(Creseis)) . . ... 3. chierchiae (Creseis) . chrysosticta (Gleba) . . . ... 87 chrysostieta (Tiedemannia). . . . . 87 eiliata (Dexiobranchaea). . ... . 105 ciliata (Pneumodermopsis) . . . 105 eiliatum (Pneumodermon) . . . . . 105 Crrikhar are ee a 110 eirroptera (Cymbulia) . ...... 88 Gathrata (Spirialisy u „eu ee Ti clava (Ureseis) ,. .. » RS gi: clava (Creseis aelchıläy nn alenke 25 Oleodors—+ 27.7.» . 23, 26, 27, 28, 37 Glevdaoridaen un Ina u 22 VlErgdoRE. aa ee 150 Ida Sn En IE WENDE 22,103, 124 BON a er ne 28 KUBR e Ee 119, 122, 125 VRR er an as -. 109,135 Chodora ua ee en ORT 150 ER ER EEE - 103, 118, 124 Olotapen I er 118,124 HOMO. Alphabetisches Register le 2 So al Ne 0a ED IDEE ara em karge, vn e 130 Gironeidsen.. us sin a IE GHDIIdeB u en a Ei CHORTdBE. SE. neue 124 Cihotozdaey. 7, a 124 OBondpsidae AN. NS 4, Olionopnidel. a X. u rt 0 2 ER GlpnGpkis u oc . 119 Chopssidad..... nad Fl N) , GymBulisa',. „1.2 u we Cliopsidae . 00: ui Gliopsiß...... 0. 0a 2 6 cochlostyloides (Limacina) columella (Triptera). . ... . columnella columnella (Ouvieriunh columnella (Öuvieria) Rang eolumnella (Cuvieria) Soul. . .. . columnella (Cuvierina) . . ».. columnella (Cuvierina columnella) columnella (Triptera) .. .. 2... columnella urceolaris (Cuvierina) columnella var. typica (Öuvierina) columnella var. urceolaris (Öuvierina) complanata (Hyalaea). ...... compressa (Oleodora) . . . ... compressa (Oreneis) - . ..+ 7 msun eoniea (Olio (Creseis)). . - .... conica (ÜUreseis). . = N conica (Creseis virgula). . . . . conoidea (ÜUleodora). . convexa (Cleodora pyramidata var.). convexa (Clio pyramidata) Coponautae cordata (Gleba). . . .. eornea (Hyalaea) , . . TeTisee corniformis (Hyalaea) . . ..... corniformis (Styliola) cornucopiae (Creseis) - » 2.0... Corolla . rn. „NE Terre costata (Diacria quadridentata) . costata (Hyalada) ---. 7. more costata (Hyalaea quadridentata var.) couthouyi (Cavolina) creniptera (liedemannia) Greseis +1... "re ee Creseis OrBia 2. A TE Cryptobranchia .. Oteniurus cucul'ata (Aegle) . cucullata (Limacina) cueullatum (Pneumodermon) . . . . cumingii (Hyalaea) curvata (Cleodora) cuspidata (ÜUleodora) cuspidats (Co. 7 rn Merk ren cuspidata (Hyalaea) Bose cuspidata (Hyalaea) Chiaje. .. . . Ouyieria. u. 2229. SE DR » mr, naar s ..0, Ar er nu Te dB Se Do ee Cymboulia 2227... 222 So Cymbulia Eymbulisacen.'. » 1. = Cymbulisdae...".., va . 78, 140 Cymbulidae Gymbnlisidger. er. er Alphabetisches Register 145 e Seite Cymbuliopsis.. . - -» 82 Cymodocea wur 142 Dactyliobranchia a N: 2 dalli (Clione) . 126 depressa (UOlio) . 39 depressa Da 52 depressa (Hyalaea) Or 52 depressa (Hyalaea) Tesch 54 depressa (Peracle) 76 depressa (Peraclis) 76 depressus (Pleuropus) . . 52 Desmopteridae 87 Desmopterus . . . 83 Deutocephala 89 Dexiobranchaea 104 Diacria . . 38 diaphana Een odrea), 142 diaphana (Pteroceanis) 156 diaphanus a 136 Dieroptera . 151 Win heromautse. cyan nn 1 diversa (Peracle) . . 75 diversa (Peraclis) . 75 diversa (Spirialis) . 75 doliiformis (Paodoclione) 133 dumerilii (Trichocyelus) . 110 ecaudata (Hyalaea) . 44 Eione . 151 elata ea : 18 elegantissima Ge AR: 126 elongata (Hyalaea) . Alphabetisches Register 52 elongata aa 129 Embolus Jeffr. . . 16 Embolus P. Fisch. re a Rupseronada sn ts arena 2 Euribia TEE 141 Mearibineea eo. nat 140 in ee RER 72 Eurybia . 141 Eurybidae . 140 Eurybiidae a 140 eurycotylum eodern) 114 IntBocosomafta;.:'., er. ua 8 exacuta (Cleodora) . - 36 faleata (Cleodora) A. Ga. h 24 falcata (Uleodora) Pfeff. . 30 falcata (Clio). . : 30 femorata (Hyalaea) . . . 44 fissilabris (Hyalaea). . . . . 44 Bava(Cavolins) .. „u. .t-,= 47 keiva(Hvalaga)ı. -, erenalıe a erläet, 47 flavescens (Clio) . . ..» . 129 Havescens (Clione) . . . .... 12% flemingii (Peracle) 19 flemingii (Spirialis) . 19 BEE KÜleodora) 0 ee 24 forskalii (Hyalaea) Lsr. . 49 Das Tierreich. 36. Lief.: J. J. Tesch, Pteropoda. Seite forskalii (Hyalaea) Pa & Soul. . 5l Fowlerina . 150 Frocymbulia . ren 78 fusiformis (Oliodita) . 129 gardineri en ; 89 Gastropteron . . . el gaudichaudi elosehe) 142 eaudichaudii (Kuribia) . 141 saudichaudii (Eurybia). . 141 gaudichaudii (Halopsyche) 141 gaudichaudii (Theceurybia). . . 142 gegenbauri (Hyalaea) . fe 47 gegenbauri (Thliptodon). . 136 gibbosa (Cavolina) 47 geibbosa (Cavolinia) . 47 gibbosa (Cavolinia) . : 47 gibbosa (Cavolinia ibBda). 47 eibbosa gibbosa (Cavolinia) . 47 gibbosa (Hiyalaea)ı ut u 47 gibbosa plana (Cavolinia) . 49 gibbosa var. plana (Cavolinia) . . 49 Gleba . a 82 Gleba. i 5 globulosa Keane KEyalaea)) Mr 46 globulosa (Cavolinia) . i 46 elobulosa (Hyalaea). . 46 globulosa (Psyche) 141 gouldii (Spirialis).. . - 20 gracilis (Clione) . . . 128 grandis (Clionopsis) . 120 grandis (Cliopsis) . 120 Gymnosomata 89 Halopsyche. . N: 141 Halopsychidae.. 140 hargeri (Pleuropus) . 55 helicialis (Limaeina) . 16 helieina antarctica iinaeina). 17 helieina (Olio) - 16 helieina (Clione) . .. . Sue 16 helieina helicina imasına) 16 helieina (Limacina) . 16 helicina (Limacina) . > 17 heliecina (Limaecina Beleinaye: 16 helieina var. antarctica (Limacina) . 17 helieina var. typica (Limacina) . li helieoides (Limacina) . 18 Iklelieonoidese. su cn nee 15,72 Helicophora . 16 hemispherica (Baribia) - 141 heterocotylum ne) 114 heterocotylum (Pneumonoderma) . 114 Heterofusus a errdd Hleteroptera ale, va were ae Hialaeus. . - 151 Honioderme, 2 u. a0 a 151 Hyalaea . 23, 26, 27, 28, 38, 42 Hiyalseacsasds.n ara ae ı 22 Alphabetisches Register 146 Seite Hyslasidse... Sich nal 22 Fyala u vr ee ON 49 Byalon -- u EM . Alphabetisches Register 152 Byaleidae- u star . 22, 140 Byakdae.. WERD RE IR 22 Eiyalsoyla!.. 10, Bean Er HAIR 27 Hysloeyliz . .: 2... 2.04% 27 year a er 152 Biyaitarda > 2 2 SEN NER 152 imitans (Hyalaea). . .... . 53 inermis (Hyalaea). .. ... . RT - inflata (Atlanta) . -... . . 18 inflata (Cleodorae) . „ ......%. 31 inflata (Limaeina). . . .- - SEHE inflexa (Cavolina). .... . CERIE uflexa'(Cavolinia) . sa, 2. Er AUB2 Elera (Osvolii! ar Er arte inflexa (Cavolinia PER u SEHR alexa (Oleodsra)’- -;.. u wink 52 inflexa (Hyalaea) . ..... 52 inflexa (Hyalaea (Diacria)). . -. . . 52 inflexa inflexa (Cavolinia). . .. 52 inflexa labiata (Cavolinia). ... 53 inflexa var. lata (Hyalaea). ... . 54 inflexa var. longa (Hyalaea) .... 52 inopinata (Notobranchaea) . . . 123 intermedia (Corolla) - .. .... 85 intermedia (Cymbuliopsis) . . . . - 85 intermedia (Hyalaea) .. . .... 41 jeffreysii (Spirialis) . . - - - TER a Er Fr 152 kraussi (Cavolinia tridentata). . 51 krohnii (Clionopsis) . . -» .». : ... 119 #r0hnii (CHoPBis) . SE NZ RT 119 labiata (Cavolinia inflexa) ... 53 labiata (Cleodora) . » .. 2... 36 biste (Dineriay" ud ae 54 Iabista (Eiyalaea) : 5) „2% 53 labiata (Hyalaea Rrpri BE laevigata (Diacria) - .:...2..4 Iuevigata (Hyalses) : 3... wa laevigatus (Pleuropus). . . . ... 44 lamartinieri (Cleodora) . . . . - 35, 36 lanceolata (Cleodora) . . - - » 35, 36 lanceolata (Clio pyramidata) . . 36 lanceolata (Hyalaea) . ...... 36 lata (Cleodora pyramidata var.) . . 36 lata (Hyalaea inflexa var.). . ... 54 Les Limaeines: -. ..- .: en 5; lessonii (Cleodora) - lesueurü (Atlanta) .........18 lesueurii (Limaeina) Boas . ... 18 lesueuri (Limaeina) Tesch . . . . - 17 limneells (CHo) .. . -: +7... 129 he Ba ME ER EB Fire in ee 72 limacina teen (Clione). . . 128 Seite lmacına (Olio)'.». u... = SR 126 limaeina (Clione) . ..... Pen.) limaeina (Clione) . . ..... 126, 128 limaeina (Clione limaeina) . . . 1% limacina limaeina (Clione) . . . 126 limacina (Spiratella). -. ». »..... 16 limaeina var. antaretica (Clione) . . 128 limaecina var. typica (Clione). . . . 126 Limacinidae . :'. . v7 15 limbata (Cavolina) . . 2». ..°. 44 limbata (Hyalaea) . ... . Alphabetisches Register RE longa (Hyalaea inflexa var.) . . . . 52 longicaudata (Clione) . . . .. . 129 longieaudatus (Clio) - - 2» 2... 129 longieaudatus (Clione). . . ».. . 122 longifilis (Hyalaea) . .. ..... 49 longifilis (Pleuropus) . » 2»... 49 longirostra (Cavolina) . . .». ....» 44 longirostra (Hyalaea) . ...... 44 longirostris angulata (Cavolinia). 46 longirostris (Cavolina). ». . » .. » 44 longirostris (Cavolinia) . . .... 44 longirostris (Cavolinia) - » .... 44 longirostris(Cavolinialongirostris) 44 longirostris (Hyalaea) . . ..... 44 longirostrislongirostris(Cavolinia) 44 longirostris strangulata (Cavolinia) 46 longirostris var. strangulata (Cavolinia) 46 lunaris (Turbo) . macandrei (Spirialis) macdonaldi (Notobranchaea) . . 122 macrochira (Pneumodermopsis) . 108 macrocotylum (Pneumodermon) . . 112 major (Diacria trispinosa). . . . 4l major (Hyalaea trispinosa var.) . . 41 martensii (Cleodora) mediterranea (Clio) -. ... 2»... 119 mediterraneum (Pneumoderma) . 112 mediterraneum (Pneumodermon) 105, 112 mediterraneum (Pneumonoderma) . 112 mediterraneus (Triehoeyelus). . . . 119 Megapterygia microcephala (Cliopsis) . . - . . 121 mierocephalus (Ölionopsis). . « . . 121 minor (Hyalaea trispinosa var.). . . 89 minor (Peraelis retieulata var.). . . 73 minuta (Dexiobranchaea) . . . + . 106 minut4 (Hyalaea). - :. 2.2... 41 minuta (Pneumodermopsis) . . . 106 miquelonensis (Olio) ... ..... 126 modesta (Clionopsis) . » » 2». . 120 modesta (Cliopsis) - - » »... 120 moluccensis (Peracle). . . .. . 76 molucecensis (Peraclis). . » »..» » 76 Monoculus .: 1... 1 a a 49 monotis- (Oreseis) » . -. + 28 mucronata (Diacria). » » 2 .... 4 Alphabetisches Register 147 Seite mucronata (Hyalaea) Orb. . . 41 mucronata (Hyalaea) Q.&&.. 39 mucronatus (Pleuropus) . . 41 munda (Cleodora) 24 napolitana (Tiedemannia) 86 natans (Cavolina) . 49 naticoides (Limacina) . - 1 neapolitana (Tiedemannia) . 86 norfolkensis (Cymbulia) . 141 norfolkensis (Theceurybia) . 142 Notobranchaea . . 122 Notobranchaeidae. 121 obtusa (Cleodora) . 37 obtusa (Creseis) . . 37 obtusa (Cuvieria) . 38 obtusa (Hyalaea) . 44 Orbignyia 42 oryza (ÜCuvieria) 38 ovata (Corolla) 83 ovata (Cymbulia) . 83 ovata (Cymbuliopsis) 83 ovularis (Öymbulia) . 83 pacifiea (Limacina) . : 17 pacifieum ee nadlasına) ; 115 pacifiecum (Pneumodermon) 115 pacificum a : 115 Paedoclione 132 papilio Derminpterus).. 88 papilionacea (Clione) 126 papilionacea en Vale zane 49 Paraclione : 131 paradoxus (Cirrifer) . Alphabetisches Register wesE 27 Spiratella . :. .. . A 15 Spirialidas "1.7: 2 ok 15 Spirale, 2 RT u nt 2 DR Spongiobranchaes. . . ... 1... 225 109 Spongiobranchea . ...... 109, 125 Spongiobranchia, . . .‚Vemerze 109 Spongobranchia . ..... . 109 Spongobranchus ..'. . 1. Sn sez 109 stenogyra (Scaea). . ©. oe .... 19 stenogyra (Spirialis). -»- » » »... 19 Stomatopterophora ...... 1 strangulata(Cavolinialongirostris) 46 strangulata (Öavolinialongirostrisvar.) 46 striata (Oleodora) . . .». » : .ıo. 28 striata (Clio (Hyalocylix)) . . . - - 28 striata (Creseis) Chiaje . . . .'.. 24 striata (Oreseis) Rang. . » » 2.28 striata (Hyalaea) .... . + ee striata (Hyalocylis) - 2% zz 28 striata (Hyalocylix) . . ».. 2... 28 striate (Styliola) - 2... 725 28 Styliole 0. 23, 27 Btyliola : . «U. se na 2 26 Btylivle 0 275% Korg Vo subuls (@leodore). .. 1.0. Foren 27 subula (Cleodora (Styliola)) -. - » » 27 subula (Olio (Styliola)) ». » » ».. 27 subula (Oreseis). +’. x. 2 3 u Sum 27 subule. .(Hyalaca) . = . 7 2m Ride 27 subula (Styliola): = unse 27 subulata (Cleodora) . . . 2... 27 subulata (Styliola). . .».. 2.2. 27 suleata (Oleodora) . ... .» 35 sulcata «(O110) (12. AU R Mae 35 telemus (Cavolina) . ... - 49 telemus (Monoeulus) . ». 2... 49 teniobranche (Hyale) . ...... 49 teniobranchia (Hyalaea) . .... . 49 Tihnalassopterus... 1: “ikue BiniEH 137 Thoosurybis 1% wi toRegVE 141 Thecosomata.. .:.c..... 2 Thieloa +. 0 7:0 I A . 153 TDhilear's uw. 0 0“ 1 RUNTER Bu 22 Seite pyramidata lanceolata (Clio) . . 36 pyramidata pyramidata (Clio). . 35 pyramidata var. angusta (Cleodora) . 35 pyramidata var. convexa (Cleodora) . 37 pyramidata var. lata (Cleodora) . . 86 quadridentata (Cavolina). . . . . - 41 quadridentata (Cavolinia) . . . 41 quadridentata (Cavolinia ia 4l quadridentata costata (Diacria) . 42 quadridentata (Diacria) . . . . . 41 quadridentata (Diacria) . . .» .. .» 41 quadridentata (Diacria quadri- dentata)ben.. aa ars nee 41 quadridentata (Hyalaea). .. ... 4 quadridentata quadridentata Diaeriayr un -20. der r 41 quadridentata var. costata (iyalaca) 42 quadripunctata (Cymbulia) . ARUHBGE (010) quadrispinosa (Cleodora) . . .. .» 32 Tadıata(Cymbulia) „uni ie ser 87 rangü (Atlanta) . .... a ! ran) (Limacına) =. was, RN 13) recta (Styliola) Gray . . Alphabetisches Register .»..... 25 recta (Styliola) Lsr.. .. .... 927 recurva (Balantium) . . .. 2... al recurva (Olendora) "EN N). 5 3l FOLULVB (@HO).MLE a al recurvirostra (Spirialis) - » » » » . 73 reeviana (Hyalaea) SITE RER 39 retieulata (Atlanta) . „. .» . 2... 72 reticulata (Limaeina) . ..... 73 reticulata (Peracle) ». ». ....72 reticulata (Peraclis). .. .. . au; retieulata (Spirialis). -. . ..... 7 reticulata var. minor (Peraclis). .. 73 retroversa australis (Limacina) . 20 retroversa balea (Limaecina).. . . 20 retroversa (Limaeina). . .... 19 retroversa (Limaeina) . . ..... 19 retroversa (Limacina retroversa) 19 retroversa retroversa (Limacina) 19 retroversus (Heterofusus) . » . . » 19 retroversus (Spirialis) . . ». 2... .» 19 rotnsa (Bleodora) >... „Wish 35 retune TOLo) Aa: ea EHEN, WS Be rebusa (Ole) ML UNIERT 126 Bed nur ; 42 rissoides (Peracle) . .» :. .»... 74 rissoides (Peraclis) . . ». 0... 74 rostralis (Embolus) . . .. . . 18 rostralis (Protomedea) . »..... 18 rostralig (Spirialis) KU "ra un 18 rotundata (Hyalaea). ....... 46 ruber (Pneumodermon) . . . . . 117 rugosa (Hyalaea) . . .».....x. 55 rugosum (Balantium) . ». . 2... 55 Sareopterus 4... IE We ni 153 Seite Seaea . cr 2 Mi 16 scaphoidea (Limaeina). . . .».... 18 schöelei (Cleodora) . . . ... uns 32 schöelei (Clio)... „AT FE 32 Schizobrachium% . HH 7. RE 117 scyllae (Tiedemannia). . ... . 87 sibogae (Cymbulia) . . .... Sul simplex (Dexiobranchaea) . . .. . 106 simplex (Pneumodermopsis) . . . 106 souleyeti (Pneumoderma) . . . . 116 souleyeti (Pneumonoderma) . . . . 116 spectabilis (Corolla). . . ... .« 83 spectabilis (Gleba) ... , WIE 83 spinifera (Creseis). . . ... wesE 27 Spiratella . :. .. . A 15 Spirialidas "1.7: 2 ok 15 Spirale, 2 RT u nt 2 DR Spongiobranchaes. . . ... 1... 225 109 Spongiobranchea . ...... 109, 125 Spongiobranchia, . . .‚Vemerze 109 Spongobranchia . ..... . 109 Spongobranchus ..'. . 1. Sn sez 109 stenogyra (Scaea). . ©. oe .... 19 stenogyra (Spirialis). -»- » » »... 19 Stomatopterophora ...... 1 strangulata(Cavolinialongirostris) 46 strangulata (Öavolinialongirostrisvar.) 46 striata (Oleodora) . . .». » : .ıo. 28 striata (Clio (Hyalocylix)) . . . - - 28 striata (Creseis) Chiaje . . . .'.. 24 striata (Oreseis) Rang. . Alphabetisches Register 112 parva (Argivora) 82 parvidentata ymbulis), 81 paucidens (Dexiobranchaea) rl paueidens San 10 MBelasia ..... 135 pellucida er, ee u 52 pellueidum naeh 117 pellueidus (Pleuropus) . 52 pelseneeri (Paraclione) 132 Peracle . 72 Peraclididae . 71 nee Rn er FE 72 peronii (Ermbulia) N 80 peronii (Hyalaea). 49 peronii oe) rs aj.cphe 113 peronii (Pneumodermon) Lm. 115 peronii (Pneumodermon) Q. & ©.. 112 peronii (Pneumodermon) Rang . 112 peronii (Pneumodermon) Soul. . 112 peronii (Pneumodermon) Verrill 120 peroni (Pneumoderma) 105,113 peroni (Pneumonoderma) 113 phaeostoma (Ureseis) . » » co... 828 physoides (Peracle) . 72 physoides (Spirialis) . 73 Seite pisum (Cavelina) 0a). wir 46 placida (Cleodora) 24 plana (Cavolinia N 49 plana (Oavolinia gibbosa var.) . 49 Pleuropus . . Sk 38,42 pleuropus (Cldodors) 52 Pneumoderma 110 N hl 105 Pneumodermei KR er nen 110 Pneumodermidae 103, 118, 124 Pneumodermis 110 Pneumodermo 152 Pneumodermoidae. 103 Pneumodermon . 104, 110,119 Pneumodermonidae . 105 Pneumodermopsis. . . . 104 Pneumodermum 152 Pneumonoderma 110 ER 3 103 Pneumonodermum. 110 polita-(Clio) . 30 politum (Balantium) U Rdn We 30 polyeotyla (Dexiobr RR 108 polycotyla (Pneiihodermepsik) 108 polyeotylum (Schizobrachium) . 117 probosceidea (Cymbulia) Gray 80 proboseidea a Krohn . 86 Procephala . 1 procera (Thilea) . 22 Procymbulia 78 Proeymbuliidae rl Protomedea O. Costa . 16 Protomedea lioey. re Pseudothecosomata . 55 Psyche ak 141 Piero branichiar se es ee 2 Pteroceanidae. . 134 Pteroceanis uk 135 Pterocephala .. 3 Pteroeymodocea 153 Pteropelagia . 135 Pteropoda il Pterota 89 Pueumonoderma . . 153 punctata (Clione) . 130 punetata (Cymbulia) . . 87 punctata (Tiedemannia) . 87 pygmaea (Cleodora) . 41 pygmaeum oe 116 pygmaeum (Pneumonoderma) . 116 pyramidalis (Clio) Zr. ER 130 pyramidata (Cleodora) Per. & Lsr. 35 pyramidata (Cleodora) Pfeff. . 36 pyramidata (Clio). . 35 pyramidata (UOlio) . Ei) pyramidata (Clio Arniadal) mr, aD pyramidata convexa (Clio) 37 10* Alphabetisches Register 148 Seite Seaea . cr 2 Mi 16 scaphoidea (Limaeina). . . .».... 18 schöelei (Cleodora) . . . ... uns 32 schöelei (Clio)... „AT FE 32 Schizobrachium% . HH 7. RE 117 scyllae (Tiedemannia). . ... . 87 sibogae (Cymbulia) . . .... Sul simplex (Dexiobranchaea) . . .. . 106 simplex (Pneumodermopsis) . . . 106 souleyeti (Pneumoderma) . . . . 116 souleyeti (Pneumonoderma) . . . . 116 spectabilis (Corolla). . . ... .« 83 spectabilis (Gleba) ... , WIE 83 spinifera (Creseis). . . ... Alphabetisches Register » » 2.28 striata (Hyalaea) .... . + ee striata (Hyalocylis) - 2% zz 28 striata (Hyalocylix) . . ».. 2... 28 striate (Styliola) - 2... 725 28 Styliole 0. 23, 27 Btyliola : . «U. se na 2 26 Btylivle 0 275% Korg Vo subuls (@leodore). .. 1.0. Foren 27 subula (Cleodora (Styliola)) -. - » » 27 subula (Olio (Styliola)) ». » » ».. 27 subula (Oreseis). +’. x. 2 3 u Sum 27 subule. .(Hyalaca) . = . 7 2m Ride 27 subula (Styliola): = unse 27 subulata (Cleodora) . . . 2... 27 subulata (Styliola). . .».. 2.2. 27 suleata (Oleodora) . ... .» 35 sulcata «(O110) (12. AU R Mae 35 telemus (Cavolina) . ... - 49 telemus (Monoeulus) . ». 2... 49 teniobranche (Hyale) . ...... 49 teniobranchia (Hyalaea) . .... . 49 Tihnalassopterus... 1: “ikue BiniEH 137 Thoosurybis 1% wi toRegVE 141 Thecosomata.. .:.c..... 2 Thieloa +. 0 7:0 I A . 153 TDhilear's uw. 0 0“ 1 RUNTER Bu 22 Seite pyramidata lanceolata (Clio) . . 36 pyramidata pyramidata (Clio). . 35 pyramidata var. angusta (Cleodora) . 35 pyramidata var. convexa (Cleodora) . 37 pyramidata var. lata (Cleodora) . . 86 quadridentata (Cavolina). . . . . - 41 quadridentata (Cavolinia) . . . 41 quadridentata (Cavolinia ia 4l quadridentata costata (Diacria) . 42 quadridentata (Diacria) . . . . . 41 quadridentata (Diacria) . . .» .. .» 41 quadridentata (Diacria quadri- dentata)ben.. aa ars nee 41 quadridentata (Hyalaea). .. ... 4 quadridentata quadridentata Diaeriayr un -20. der r 41 quadridentata var. costata (iyalaca) 42 quadripunctata (Cymbulia) . ARUHBGE (010) quadrispinosa (Cleodora) . . .. .» 32 Tadıata(Cymbulia) „uni ie ser 87 rangü (Atlanta) . .... a ! ran) (Limacına) =. was, RN 13) recta (Styliola) Gray . . .»..... 25 recta (Styliola) Lsr.. .. .... 927 recurva (Balantium) . . .. 2... al recurva (Olendora) "EN N). 5 3l FOLULVB (@HO).MLE a al recurvirostra (Spirialis) - » » » » . 73 reeviana (Hyalaea) SITE RER 39 retieulata (Atlanta) . „. .» . 2... 72 reticulata (Limaeina) . ..... 73 reticulata (Peracle) ». ». ....72 reticulata (Peraclis). .. .. . au; retieulata (Spirialis). -. . ..... 7 reticulata var. minor (Peraclis). .. 73 retroversa australis (Limacina) . 20 retroversa balea (Limaecina).. . . 20 retroversa (Limaeina). . .... 19 retroversa (Limaeina) . . ..... Alphabetisches Register 19 retroversa (Limacina retroversa) 19 retroversa retroversa (Limacina) 19 retroversus (Heterofusus) . » . . » 19 retroversus (Spirialis) . . ». 2... .» 19 rotnsa (Bleodora) >... „Wish 35 retune TOLo) Aa: ea EHEN, WS Be rebusa (Ole) ML UNIERT 126 Bed nur ; 42 rissoides (Peracle) . .» :. .»... 74 rissoides (Peraclis) . . ». 0... 74 rostralis (Embolus) . . .. . . 18 rostralis (Protomedea) . »..... 18 rostralig (Spirialis) KU "ra un 18 rotundata (Hyalaea). ....... 46 ruber (Pneumodermon) . . . . . 117 rugosa (Hyalaea) . . .».....x. 55 rugosum (Balantium) . ». . 2... 55 Sareopterus 4... IE We ni 153 S s s s S s s s s s s s s s S S S S S S S S s s s s s s Alphabetisches Register Seite ISITBLOGON 2 ee ae 135 Thliptodonidae ie 134 Iniıptodoentidae .- . . . 2.2. 134 Thoena Bat 153 BeHemannian ie 20 2 la 86 Beigeantha (Limaeina) : .. » » 2 2. 77 triacantha (Peracle). . . .... 7 Bneantha (Beraclis) . \ . „ern... 0.77 triacantha (Protomedea) ... ... 77 triacanthus (Embolus) . . . . . 14 Reiehoeyelus 2 nu. 2 > : 109, 119 ENT Eee I 42 msouspidata (Hyalaen). » :.. . - ...82 tridentata affinis (Cavolinia) . . 51 tridentata (Anomia). . tridentata (Cavolina) . . 2 .2....49 tridentata (Cavolinia) . . ..... 49 tridentata (Cavolinia) . . . 49 tridentata (Cavolinia Bedentaray 49 tridentata (Hyalaea) Bose... .. 49 tridentata (Hyalaea) Sow. Il... . 51 tridentata kraussi (Cavolinia). . 5l tridentata tridentata (Cavolinia). 49 tridentata var. affinis (Hyalaea) . . 51 tridentata var. truncata (Hyalaea) . 51 trifilis (Cleodora) . 2a Eee a NT Bene ee rer rl Nripterisi... 2. % a ae 1 trispinosa ers ee klae 1) trispinosa (Oavolinia Diaerie)) a trispinosa (Diacria) . . . .». ....589 keepinosa (Diserla) ».... -.. 14.2..." 39 trispinosa (Diacria ke oinosa)ı. 0239 trispinosa (Hyalaea) . trispinosa major (Diacria) ee A trispinosa trispinosa (Diacria). . 39 trispinosa var. major (Hyalaea). .. 41 trispinosa var. minor (Hyalaea). . . 39 trispinosus (Pleuropus) . . ..... 89 EAN er ee 92er DA trochiformis (Atlanta). ». ..... 21 trochiformis (Limaeina). . -... 21 trochiformis (Spirialis) . . .»....» 21 truncata (Hyalaea) Kraus. .... 51 truncata (Hyalaea) Lsr. . . ».. 54 truncata (Hyalaea tridentata var) . Alphabetisches Register 51 Turbo 2 2. ee typiea (Olione ink, eh 212 typica (Cuvierina columnella ve a typica (Limaecina helieina var). .. 17 uneinata, (Gayolina)rur en 2. .00hl uneinata ine) a ei uncinata (Hyalaea) Eon uneinata (Hyalaea) Rang ..... 51 uneinatiformis (Hyalaea). ..... 5l UNDNSS(ÜDTEREIS BE 24 urceolaris (Ouvieria) . . - 38 urceolaris (Cuvierina eoluimmenia) 38 urceolaris (Cuvierina columnella var.) 38 vaginellina (Hyalea) . a ee gr valdiviae Detstranchgen) Be ehr) valdiviae (Procymbulia). . ... 78 venftricosa, (Limaeima). „u. ventricosa (Spimalis)w En au. 2 violaceum (Pneumoderma) . .. 112 violaceum (Pneumodermon) . . 112,115 violaceum (Pneumonoderma). . . . 112 vireular (Gleodorayreee 20. ma a virgula (Clio (Creseis)) . . . ... 24 virgula coniea (Creseis). . . .. 24 vırsular (Ereseis), cr oa Ar. Ed virgula (Oreseis) . . . De ae ZA virgula (Creseis virgula) Ye varkula (Eiyalaea)ne an, en: 24 vireulanfstylaala) ee area. 24 virgula virgula (Creseis) . . . . 24 vırrea (Uymbmliopsis), 0.» ee. 85 VILrER (Styliola)r- 2 0 2.00 24 zancleus (Thalassopterus) . . . . 138 zetesios (Fowlerina) . . . . . . 180 zonata (UresBis) » u. er ntulae, 28 Nomenclator generum et subgenerum 1854. Sp.: C. krohmii. Cliopsis Troschel in: Arch. Naturg.., v.201 p. 222. 1854. Sp.: C. krohmii. Corolla W. Dall in: Amer. J. Conch., v.7 11 p. 137. 1871. Sp.: ©. spectabilis. Euromus Subgen. H.& A. Adams, Gen. Moll., v.2 p.613. 1858. Eurybia Hübner, Verz. Schmett., p. 17. 1816. Sp.: E. Nicaea, -E. Halimeda, E. Upis. Creseis Subgen. Rang in: Ann. Sei. nat., v.15 p.505. 1828. Sp.: Oleodora (Creseis) vaginella, ©. (C.) gadus, ©. (C.) spinifera, ©. (C.) subula, ©. (C.) striata, ©. (C.) virgula, ©. (C.) obtusa, ©. (C.) clava, CO. (C.) acicula. Eurybia pro: Euribia Rang 1827 A. Philippi, Handb. Conch., p. 293. 1853. Crisia pro: Creseis Rang 1828 Menke in: Z. Malak., p. 72. 1844. Fowlerina Pelseneer in: Tr. Linn. Soe, London, ser.2 v.10 p.149. 1906. Sp.: F. zetesios. Cteniurus Rafinesque, Speechio Sei., v.2 nr.11 p.155. 1814. Frocymbulia [pro: Procymbulia Meisen- heimer 1905] Meisenheimer in: Ergeb. Tiefsee-Exp., v.9 p.13. 1905. Sp.: F'. valdiviae. Cuvieria Lesueur, Voy. Terr. austr., Atl. t. 30 f.2. 1807. Sp.: ©. cariso- chroma. Acaleph. Cuvieria (Cuvieries) Peron MS. in: G. Cuvier, Regne an., v.4 p.22 nota. 1817. Echin. Gastropteron Kosse, Pterop., 2.10, 12. 1813. Gleba Forskäl, Icon. Rer. nat., t.43f.D. 1776. Sp.: G. cordata, @. kippopus. Cuvieria Rang in: Ann. Sci. nat., v.12 p. 322. 1827. Sp.: ©. columella. Gleba Tilesius in: Ann. Wetter. Ges., v.3 p. 868. 1814. Sp.: @. pseudo- hippopus, G. crispa, @G. erystallina, @. deformis, @. spiralis, @. Conus. Cuvierina pro: (wvieria Rang 1828 Boas in: Danske Selsk. Skr., ser. 6 v.4 p.131. 1886. Sp.: ©. columnella. Gleba Otto in: N. Acta Ac. Leop,, v.11 p.309. 1823. Acal. Cymkbulia Peron & Lesueur in: Ann. Mus. Paris, v.15 p.66. 1810. Halopsyche pro: Psyche Rang 1825 Bronn in: Bronn KI. Ordn, w3ı p.645. 1862. Cymbuliopsis Pelseneer in: Rep. Voy. Challenger, v.23 pars 65 p. 100. 1888. Sp.: (©. ovata, O. calceola. Heliconoides Subgen. Orbigny, Voy. Amör. merid., v.5mı p.174. 1836. Sp.: Atlanta (H.) inflata, A. (H.) rotunda, A. (H.)rangii, A. (H.) lesueurii, A.(H.) trochiformis, A. (H.) reticulata, A.(H.) bulimoides. Cymodocea Orbigny, Voy. Amör. merid., v.5ıu p.133. 1840. Sp.: C. diaphana. Desmopterus Chun in: SB. Ak. Berlin, p-540. 1889. Sp.: D. papilio. Dexiobranchaea Boas in: Zool. Anz., v.8 p.687. 1885. Sp: D. ciliata. Helicophora J. E. Gray, S#n. Brit. Mus., ed. 44 p.59, 89. 1842. Diacria J. E. Gray, Syn. Brit. Mus., ed. 42 p.151. 1840. Heterofusus Fleming in: Mem. Werner. Nomenclator generum et subgenerum Abretia Rafinesque, Speechio Sci., v.2 or. 11 p.154. 1814. [Heterop.?] Caulina pro: Cavolina Abildgaard 1791 Poli, Test. Sieil., v. 31 p.38. 1826. Cavolina Abildgaard in: Skr. Naturh. Selsk.,. v. Iıı p. 174. 1787 map natans. Aegle Oken, Lehrb. Naturg., v.1 p. 327. 1815. Sp.: A. cucullata. Agadina Aug. Gould in: U.S.expl. Exp., v.12 p.486. 1852. Kein Pteropod. Cavolina Bruguiere in: Tabl. enc. möth., v.1 t.85 f.4,5. 1792. Moll. Opistobr. AmphireaRafinesque,Anal.Nat.,p. 141. 1815. [nom. nud.] Cavolinia pro: Cavolina Abildgaard 1791 A. Philippi, Handb. Conch., p. 290. 1853. Anomia Forskäl, Deser. An., 1775. Sp.: A. tridentata. p. 124. „Celata“ Gioeni, Deser. Test., p.25. 1783. Archonta Montfort, Conchyl. syst., v.2 p.50. 1810. Sp.: A. exploratus. Cirrifer Pfeffer in: Monber. Ak. Berlin, p.246. 1879. Sp.: CO. paradoxus. Argivora Lesueur MS. in: Blainville, Man. Malaec., Planches p. 655. 1827. Sp.: A. parva. [nom. nud.] Cleodora Pöron & Lesueur in: Ann. Mus. Paris, v.15 p.66. 1810. Clerodora [pro: Oleodora Pöron & Lesueur 1810] Oken, Lehrb. Naturg., v.1 p.3%4. 1815. Atalanta [pro: Atlanta Lesueur 1817] Knocker in: P. zool. Soc. London, p-616. 1868. Clio Linn6, Syst. Nat., ed. 12 p.1094. 1767. Sp.: C. caudata, C. pyramidata, C. retusa. Atlanta Lesueur in: J. Phys. Chim. Hist. nat., ..85 p.390. 1817. Clio Subeen. Pelseneer in: Rep. Voy. Challenger, v.23 pars 65 p.47.59. 1888. Sp.: Clio (0.) politum, ©. (C.) andreae, C. (C.) balantium, CO. (C.) australis, C. (C.) sulcata, ©. (C.) pyramidata, ©. (C.) cuspidata. Atlantis [pro: Atlanta Lesueur 1817] Oken in: Isis, p.41. 1819. Aulisa Rafinesque, Anal. Nat., p. 141. 1815. [nom. nud.] \| Balantium (Children fide Gray) in: Quart. J. Sci., v.15 p.220 nota. 1823. Sp.: B. recurvum. Cliodita Quoy & Gaimard, Voy. Uranie & Physicienne, p.413. 1824. Sp.: €. caduceus, C©. fusiformis. Campylonaus Benson in: J. Asiat. Soc. Bengal, v.4 p.176. 1835. [nom. nud.]. Heterop. Cliodora pro: Cleodora Peron & Lesueur 1810 A. Philippi, Handb. Conch., p.291. 1853. Campylonaus J.E.Gray in: P. zool. Soe. London, v.15 p.149. 1847. Sp.: ©. reticulata. Clione Pallas, Spie. zool., fasc. 10 p. 28. 1774. Sp.: ©. borealis. Clionopsis pro: Cliopsis Troschel 1854 Bronn in: Bronn Kl. Ord., v.311 p.645. 1862. Carolina [pro: Cavolina Abildgaard 1791] Engelmann, Bibl. hist.-nat., p. 464. 1846. 151 Nomenelator generum et subgenerum Euribia Rang in: Ann. Sei. nat, v.12 p. 828. 1897. Sp.: E. hemispherica. Euribia Rang in: Ann. Sei. nat, v.12 p. 828. 1897. Sp.: E. hemispherica. Cliopsis Troschel in: Arch. Naturg.., v.201 p. 222. Nomenclator generum et subgenerum Soe., v.4 p.500. 1823. Sp.: H. reiro- versus. Dieroptera Rafinesque, Specchio Sci., mern: lan.‘ 1814, Heteroptera Rafinesque, Specchio Sei., v2 nr.11 p.154. 1814. [Heterop.?] Eione Rafinesque, Speechio Sei., v2 ne.11 p.1o5. 1814, Hialaeus [pro: Hyalaea Lamarck 1801] Montfort, ÜÖonchyl. syst.,, v. 2 p. 46. 1810. Embolus Jeffreys, Brit. Conch., v. 5 p-114. 1869. Sp.: E. rostralis. Embolus P. Fischer in: J. Conchyl., v.30 p.49. 1882. Sp.: E. triacanthus. „Homoderme“ J.P. Beneden in: Mem. Ac. Belgique, v.11l [nr.20] p.14. 1838. Nomenclator generum et subgenerum 152 Pelagia Lamouroux, Expos. Polyp., p.78. 1821. Sp.: P.clypeata. Actin. Hyalaea Lamarck, Syst. An. s. Vert., p.139. 1801. Sp.: H. cornea. Hyalaea Lamarck, Syst. An. s. Vert., p.139. 1801. Sp.: H. cornea. Hyalea [pro: Hyalaea Lamarck 1801] G. Cuvier, Rögne an.,v.2p.381. 1817. Pelagia Quoy & Gaimard in: Voy. Astrol., v.2 p.392. 1832. Sp.: P. alba. Hyalocylis Fol in: Arch. Zool. exper., v.4 p.177. 1875. Sp.: H. striata. Peracle Forbes in: Rep. Brit. Ass., v. 13 p- 132, 186. 1844. Sp.: P. physoides. Hyalocylix pro: Hyalocylis Fol 1875 ’P. Fiseher, Man. Oonchyl., p.436. 1883. Peraclis pro: Peracle Forbes 1844 Pelseneer in: Rep. Voy. Challenger, v.23 pars 65 p.32. 1888. Hyalus [pro: Hyalaea Lamarck 1801] Kosse, Pterop., p.8. 1813. Pleuropus Eschscholtz in: Isis, p. 735. 1825. Sp.: P. pellueidus. Hypterus Rafinesque, Speechio Sci., v.2 or.11. 1814. [Heterop.?] Pneumoderma P&ron & Lesueur in: Ann. Mus. Paris, v.15 p.65. 1810. Klio [pro: Clio Linn& 1767] Mertens in: M&m. Ac. St.-Petersb., ser.6 v.1l p.206. 1831. „Pneumo-derme“ G. Cuvier in: Ann. Mus. Paris, v.4 p.232. 1804. „Les Limaeines“ G. Cuvier, Rögne an., v.2 p.380. 1817. Pneumodermis [pro: Pneumoderma Cu- vier 1804] Oken, Lehrb. Naturg., v. 1 p: 326. 1815. Limacina Lamarck, Hist. An. s. Vert., v.6ı p.290. 1819. Sp.: L. helicialis. Pneumodermo [pro: Pneumoderma Ouvier 1804] Deshayes in: Cuvier, Rögne an., [ed. 4] Moll. t.17 Erkl. 1836—46. Limacina Hartmann in: N. Alpina, v.1 p. 201, 207,246. 1821. Sp.: L. annu- laris, L. pellucida, L. vitrea. Moll. Pneumodermon [pro: Pneumoderma Cu- vier 1804] G. Cuvier, Rögne an., v2 p. 380. 1817. Limaeina Fam. Wiegmann in: Wieg- mann & Rothe, Handb. Zool., p. 521. 1832. Pneumodermopsis Bronn in: Bronn, Kl. Ordn., v. 311 p. 645. 1862. Sp.: P. eiliatum. Monoculus Linnö, Syst. Nat., ed. 10 p. 684. 1758. Sp.: M. Polyphemus, M. foliaceus, M. Apus, M. Pulex, M. Pedi- culus, M. quadricornis, M. conchaceus, M. lenticularis, M. Telemus. Protomedea Locard in: Exp. Travailleur & Talisman, Moll. test., v.l p.27. 1897. Sp.: P. triacantha. Protomedea O.G. Costa, Microdoride, v.1 p.73. 1861. Nomenclator generum et subgenerum Pneumodermum pro: Pneumoderma Cu- vier 1804 Verany, Cat: an. invert., p.17. 1846. Notobranchaea Pelseneer in: Bull. sei. Nord, p.225. 1886. Sp.: N. macdo- naldi. Pneumonoderma pro: Pneumoderma Cu- vier 1804 L. Agassiz, Nomenel. zool., Index p.299. 1846. Orbignyia Subgen. A. Adams in: Ann. nat. Hist., ser.3 v.3 p.45. 1859. Sp.: Cavolinia, (O.) inflexa, C©. (O.) labiata. Pneumonodermum [pro: Pneumoderma Cuvier 1804] Hermannsen, Ind. Gen. Malae., v.2 p.309. 1847. Paedoclione Danforth in: P. Boston Soe., v.34 p.2. 1907. Sp.: P. dolüi- formis. Proeymbulia Meisenheimer in: Ergeb. Tiefsee-Exp., v.9 p.3, 14. 1905. Sp.: P. valdiviae. Paraclione Tesch in: Tijdschr. Neder!l. dierk. Ver., ser.2 0.8 p.117. 1903. Sp.: P. pelseneeri. Protomedea Blainville, Man. Actin,, p.121. 1834. Sp.: P.lutea. Acal. Pelagia Peron & Lesueurin: Ann. Mus. Paris, v.14 p.349. 1809. Sp.: P.pa- nopyra, P. unguiculata, P. cyanella, P. denticulata, P. noctiluca, P. purpurea, P. ?australis, P. ?americana, P. ?gui- neensis. Protomedea O.G. Costa, Microdoride, v.1 p.73. 1861. Protomedea Locard in: Exp. Travailleur & Talisman, Moll. test., v.l p.27. 1897. Sp.: P. triacantha. 155 Nomenclator generum et subgenerum Psyche Rang in: Ann. Sci. nat, v. 5 p. 284. 1825. Sp.: P. globulosa. Psyche Rang in: Ann. Sci. nat, v. 5 p. 284. 1825. Sp.: P. globulosa. „Styliole“ Lesueur MS. in: Blainville, Man. Malac., Planches p. 655. 1827. Sp.: 8. recta. Zool. 1902. Sp.: P. dia- Pteroceanis Meisenheimer in: Anz., v.26 p. 93. phana. Thalassopterus Kwietniewski in: Zool. Anz., v.36 p.271. 1910. Sp.: T. zancleus. Pteroeymodocea pro: Cymodocea Or- bieny 1840 Bronn in: Bronn, Kl. Ordn., v. 311 p. 645. 1862. Theceurybia pro: Eurybia Rang 1827 Bronn ‘in: Bronn, Kl..Ordn,, v. 3u p: 645. 1862. Pteropelagia pro: Pelagia Quoy & Gaimard 1832 Bronn in: Bronn, Kl. Ordn., v.311 p. 645. 1862. Thielea pro: Thilea Strebel 1908, Tesch in: Tierreich, v.36 p. 142. 1913. Pueumonoderma [pro: Pneumoderma G. Cuvier 1804] Pelseneer in: Rep. Voy. Challenger, v.19 pars58 p. 13. 1887. Thilea Strebelin: Ergeb. Schwed. Südp.- Rizp., v0. 00. 1. m 84, 71908252: 7. procera. Rheda [Calonne], Mus. Calonn., p. 41. 1797. Sp.: BR. bullata. [nom. nud.] Thliptodon Boas in: Danske Selsk. Skr., ser.6 v.4 p.174. 1886. Sp.: T. gegen- bauri. Sarcopterus Rafinesque, Specchio Sei., v.2 or. 11 p.154. 1814. [Heterop.?] Thoena Rafinesque, Anal. Nat., p. 141. 1815. [nom. nud.] Scaea A, Philippi, Moll. Sieil, ®.2 p-164. 1844. Sp.: $. stenogyra. Tiedemannia Leuckart in: Isis, v. 23 p- 685 nota. 1830. Sp.: T. vittata, Holothur. marmoratus, T. sarmaticus, T.Olearius, T. Pica, T.sangwineus, T. Argyrostomus, T. margaritaceus, T. Delphinus, T. dis- tortus, T. Scalaris, T. Clathrus, T.ere- natus, T.lacteus, T. striatulus, T. Uva, T. corneus, T. reflewus, T. Lineina, T. imbricatus, T.replicatus, T.acutangulus, T. duplicatus, T. exoletus, T. Terebra, T. variegatus, T. ungulinus, T.annulatus, T. bidens, T. perversus, T. muscorum, T. auriscalpium, T. politus. marmoratus, T. sarmaticus, T.Olearius, T. Pica, T.sangwineus, T. Argyrostomus, T. margaritaceus, T. Delphinus, T. dis- tortus, T. Scalaris, T. Clathrus, T.ere- natus, T.lacteus, T. striatulus, T. Uva, T. corneus, T. reflewus, T. Lineina, T. imbricatus, T.replicatus, T.acutangulus, T. duplicatus, T. exoletus, T. Terebra, T. variegatus, T. ungulinus, T.annulatus, T. bidens, T. perversus, T. muscorum, T. auriscalpium, T. politus. Nomenclator generum et subgenerum Schizobrachium Meisenheimer in: Zool. Anz., v.26 p.410. 1903. Sp.: S. polycotylum. Tiedemannia Chiaje MS. in: J. P. Be- neden in: M&m. Ac. Belgique, v. 12 [nr.13] p.22. 1839. Sp.: T.napolitana. Moll. Spiratella Blainville in: Diet. Sei. nat., v.32 p.284. 1824. Sp.: 8. limacina. Spirialis Eydoux & Souleyet in: Rev. zool., v.3 p.235. 1840. Sp.: S.rostralis, S. ventricosa, S. australis, S. trochi- formis, 8. bulimoides, 8. clathrata. Tiedemannia Ehrenberg in: Monber. Ak. Berlin, p.445. 1861. Sp.: T. anti- quissima. Moll. Spongiobranchaea [pro: Spongiobranchea Orbigny 1835] S.P.Woodward, Man. Moll., p.209. 1854. Trichoeyclus Eschscholtz in: p. 735. 1825. Sp.: T. dumerilii. Isis, Spongiobranchea Orbigny, Voy. Amer. mörid., v.5ıı p.130. 1835. Sp.: S. australis, 8. elongata. Trichoceyclus A.Costa in: Annuario Mus. Napoli, v.5 p.46. 1869. Sp.: T. medi- terrameus. Spongiobranchia \|pro: Spongiobranchea Orbigny 1835] Orbigny, Pal. franc., Terr. Oret. v.2 p.4. 1842. Tricla A.J. Retzius, Diss. Test. Gen., p-8. 1788. Kein Pteropod. Triela Oken, Lehrb. Naturg., v.1 p. 327. 1815. Sp.: T. mediterranei, T. australis, T. taeniobrancha. Spongobranchia [pro: Spongiobranchea Orbigny 1855] Bronn in: Bronn Kl. .Ordn., v.311 p.645. 1862. Triptera Quoy & Gaimard in: Voy. Uranie & Physieienne, Zool. p. 416. 1824. Sp.: T. rosea. Spongobranchus [pro: Spongiobranchea Orbigny 18355] N.Wagner, Wirbell. Weiß. Meer., v.1 p.120. 1885. Tripteres [pro: Triptera Quoy & Gaimard 1824] J. E. Gray, Syn. Brit. Mus., ed.42 p.151. 1840. Styliola Subgen. Pelseneer in: Rep. Voy, Challenger, v.23 pars 65 p.47, 56. 1888. Sp.: Clio (S.) subula, Nomenelator generum et subgenerum 154 Tripteris [pro: Triptera Quoy & Gaimard 1825] Menke, Syn. Moll., ed.2 p.9, 1830. Na 2 r Trizonius Busch, Beob. wirbell. Seeth., p.112. 1851. Sp.: T. coccus. Turbo Linne, Syst. Nat., ed.10 p.761. 1758. Sp.: T. obtusatus, T. Neritoides, T. littoreus, T. muricatus, T. Cimex, T. Pullus, T. personatus, T. Petholatus, T. Cochlus, T. Chrysostomus, T. Tectum persicum, T. Pagodus, T. Calcar, T. Dr. F. Ris in Rheinau (Schweiz), Dr. R. von Ritter-Zahony in Görz, Dr. €. Fr. Roewer in Bremen, The Hon. W. Rothschild in Tring, E. H. Rübsaamen in Berlin, Dr. F. Schaudinn in Hamburg (f), E. Schenkel in Basel, Prof. P. Schiemenz in Berlin, Prof. O.Schmeil in Heidelberg, Lehrer Ad.Schmidt in Berlin, Prof. O0. Schmiedeknecht in Blankenburg, Geh.-Rat Prof. F. E. Schulze in Berlin, Dr. R. B. Sharpe in London, Kustos Siebenrock in Wien, Dr. H. Soldanski in Berlin, Kgl. Kreisarzt Dr. P.Speiser in Labes, Geh. Hofrat Prof. J. W.Spengel in Gießen, Rev. 15. Lieferung. (Aves.) 18. Lieferung. (Aves.) 9. Lieferung. (Aves.) 16. Lieferung. (Mollusca.) 10. Lieferung. (Vermes.) 2. Lieferung. (Aves.) 7. Lieferung. (Acarina.) Nomenclator generum et subgenerum T.R.R.Stebbing in Tunbridge Wells, Oberlehrer P. Stein in Genthin, Dr. Sternfeld in Berlin, H. Stichel in Schöneberg-Berlin, Dr. T. Stingelin in Olten, Lehrer E. Stitz in Berlin, Dr. J. J. Tesch in Helder, Prof. Dr. F.V. Theobald in Wye (England), Prof. J. Thiele in Berlin, Schuldirektor $. Thor in Skien (Norwegen), Dr. E.-L. Trouessart in Paris, Dr. G. Ulmer in Hamburg, Dr. B,. Wandolleck in Dresden, Prof. F. Werner in Wien, Prof. C, Zelinka in Czernowitz. 14. Lieferung. (Lepidoptera.) 6. Lieferung. (Orustacea.) 13. Lieferung. (Acarina.) Probelieferung. (Protozoa.) 1. Lieferung. (Aves.) Erschienen sind: Heliozoa. ferung. zoa.) erung. Heliozoa. Bearbeitet von F. Schaudinn (Berlin). 24 Seiten mit 10 Abbildungen. 1896. Preis Mark 1,50. Podargidae, Caprimulgidae und Macropterygidae. Bearbeitet Podargidae, Caprimulgidae und Macropterygidae. Bearbeitet von E. Hartert (Tring). VIII und 98 Seiten mit 16 Abbildungen und 1 Beilage (Terminologie des Vogelkörpers, von A. Reichenow. 4 Seiten mit 1 Abbildung). 1897 LI. Subskriptionspreis Mark 4,50. Einzelpreis Mark 7,—. Subskriptionspreis Mark 4,50. Einzelpreis Mark 7,—. Paradiseidae. Bearbeitet von The Hon. W. Rothschild. VI und 52 Seiten mit 15 Abbildungen. 1898 IV. Subskriptionspreis Mark 2,80. Einzelpreis Mark 3,60. Subskriptionspreis Mark 4,50. Einzelpreis Mark 7,—. Paradiseidae. Bearbeitet von The Hon. W. Rothschild. VI und 52 Seiten mit 15 Abbildungen. 1898 IV. Subskriptionspreis Mark 2,80. Einzelpreis Mark 3,60. Oribatidae. Bearbeitet von A. D. Michael (London). XII und 93 Seiten mit 15 Abbildungen. 1898 VII. Subskriptionspreis Mark 4,50. Einzelpreis Mark 6,80. Eriophyidae (Phytoptidae). Bearbeitet von A. Nalepa (Wien). IX und 74 Seiten mit 3 Abbildungen. 1898 VIL. Subskriptionspreis Mark 3,80. Einzelpreis Mark 5,—. Sporozoa. Bearbeitet von A. Labbe& (Paris). XX und 180 Seiten mit 196 Abbildungen. 1899 VII. Subskriptionspreis Mark 8,80. Einzelpreis Mark 12,—. Subskriptionspreis Mark 2,80. Einzelpreis Mark 3,60. Oribatidae. Bearbeitet von A. D. Michael (London). XII und 93 Seiten mit 15 Abbildungen. 1898 VII. Subskriptionspreis Mark 4,50. Einzelpreis Mark 6,80. Eriophyidae (Phytoptidae). Bearbeitet von A. Nalepa (Wien). 3. Lieferung. (Acarina.) Subskriptionspreis Mark 4,50. Einzelpreis Mark 6,80. Eriophyidae (Phytoptidae). Bearbeitet von A. Nalepa (Wien). IX und 74 Seiten mit 3 Abbildungen. 1898 VIL. Subskriptionspreis Mark 3,80. Einzelpreis Mark 5,—. Subskriptionspreis Mark 3,80. Einzelpreis Mark 5,—. Sporozoa. Bearbeitet von A. Labbe& (Paris). XX und 180 Seiten mit 196 Abbildungen. 1899 VII. Subskriptionspreis Mark 8,80. Einzelpreis Mark 12,—. Subskriptionspreis Mark 8,80. Einzelpreis Mark 12,—. Copepoda, I. Gymnoplea. Bearbeitet von W. Giesbrecht (Neapel) und ©. Schmeil (Magdeburg). XVI und 169 Seiten mit 31 Ab- bildungen. 1898 XIl. Subskriptionspreis Mark 8,40. Einzelpreis Mark 11,—. Demodicidae und Sarcoptidae. Bearbeitet von G. Uanestrini (Padua) und P. Kramer (Magdeburg). X VI und 193 Seiten mit 31 Ab- bildungen. 1899 IV. Subskriptionspreis Mark 9,20. Einzelpreis Mark 12,—. Scorpiones und Pedipalpi. Bearbeitet von K. Kraepelin (Ham- burg). XVIII und 265 Seiten mit 94 Abbildungen. 1899 III. Subskriptionspreis Mark 12,60. Einzelpreis Mark 17,—. 6. Lieferung. (Orustacea.) Demodicidae und Sarcoptidae. Bearbeitet von G. Uanestrini (Padua) und P. Kramer (Magdeburg). X VI und 193 Seiten mit 31 Ab- bildungen. 1899 IV. Subskriptionspreis Mark 9,20. Einzelpreis Mark 12,—. Scorpiones und Pedipalpi. Bearbeitet von K. 8. Lieferung. (Arachnoidea.) Erschienen sind: Heliozoa. Subskriptionspreis Mark 24,—. Einzelpreis Mark 32,—. Salpae I: Desmomyaria. Bearbeitet von J. E. W. Ihle (Utrecht). XI und 67 Seiten mit 68 Abbildungen. 1912 V. Subskriptionspreis Mark 4,50. Einzelpreis Mark 6,—. 32. Lieferung. (Tunicata.) Subskriptionspreis Mark 24,—. Einzelpreis Mark 32,—. Salpae I: Desmomyaria. Bearbeitet von J. E. W. Ihle (Utrecht). XI und 67 Seiten mit 68 Abbildungen. 1912 V. Subskriptionspreis Mark 4,50. Einzelpreis Mark 6,—. Subskriptionspreis Mark 4,50. Einzelpreis Mark 6,—. Eublepharidae, Uroplatidae, Pygopodidae. Bearbeitet von F. Werner (Wien). X und 33 Seiten mit 6 Abbildungen. 1912V. Subskriptionspreis Mark 2,40. Einzelpreis Mark 3,20. Amathusiidae. Bearbeitet von H.Stichel(Berlin). XV und 248 Seiten Amathusiidae. Bearbeitet von H.Stichel(Berlin). XV und 248 Seiten mit 42 Abbildungen. 1912 X. 36. Lieferung. (Mollusca.) Subskriptionspreis Mark 13, 60. Einzelpreis Mark 18,—. Pteropoda. Bearbeitet von J.J.Tesch (Helder). XVI und 154 Seiten mit 108 Abbildungen. 1913 VI. Subskriptionspreis Mark 10,—. Einzelpreis Mark 13,—. 36. Lieferung. (Mollusca.) Pteropoda. Bearbeitet von J.J.Tesch (Helder). XVI und 154 Seiten mit 108 Abbildungen. 1913 VI. Subskriptionspreis Mark 10,—. Einzelpreis Mark 13,—. 36. Lieferung. (Mollusca.) Pteropoda. Bearbeitet von J.J.Tesch (Helder). XVI und 154 Seiten mit 108 Abbildungen. 1913 VI. Subskriptionspreis Mark 10,—. Einzelpreis Mark 13,—. (Berlin). Subskriptionspreis Mark 10,—. Einzelpreis Mark 13,—. Gymnophiona (Amphibia apoda). Bearbeitet von Fr. Nieden X und öl Seiten mit 20 Abbildungen. 1913 V. Subskriptionspreis Mark 2,60. Einzelpreis Mark 3,50. 37. Lieferung. (- Amphibia. ‚E (Berlin). Subskriptionspreis Mark 10,—. Einzelpreis Mark 13,—. Gymnophiona (Amphibia apoda). Bearbeitet von Fr. Nieden X und öl Seiten mit 20 Abbildungen. 1913 V. Subskriptionspreis Mark 2,60. Einzelpreis Mark 3,50. 37. Lieferung. (- Amphibia. ‚E (Berlin). Subskriptionspreis Mark 10,—. Einzelpreis Mark 13,—. Gymnophiona (Amphibia apoda). Bearbeitet von Fr. Nieden X und öl Seiten mit 20 Abbildungen. 1913 V. Subskriptionspreis Mark 2,60. Einzelpreis Mark 3,50. 37. Lieferung. (- Amphibia. ‚E (Berlin). Gymnophiona (Amphibia apoda). Bearbeitet von Fr. Nieden X und öl Seiten mit 20 Abbildungen. 1913 V. Subskriptionspreis Mark 2,60. Einzelpreis Mark 3,50. (Berlin). X und öl Seiten mit 20 Abbildungen. 1913 V. Subskriptionspreis Mark 2,60. Einzelpreis Mark 3,50. Subskriptionspreis Mark 2,60. Einzelpreis Mark 3,50. Im Druck befinden sich: 35. Lieferung. Turbellaria,II. Rhabdocoelida. BearbeitetvonL. von Graff(Graz). Im Druck befinden sich: Lieferung. Turbellaria,II. R 35. Lieferung. 38. Lieferung. 39. Lieferung. Turbellaria,II. Rhabdocoelida. BearbeitetvonL. von Graff(Graz). Solenogastres. Bearbeitet von J. Thiele (Berlin). 35. Lieferung. 38. Lieferung. Turbellaria,II. Rhabdocoelida. BearbeitetvonL. von Graff(Graz). Solenogastres. Bearbeitet von J. Thiele (Berlin). 39. Lieferung. Cumacea. Bearbeitet von T. R. R. Stebbing (Tunbridge Wells). 39. Lieferung. Erschienen sind: Heliozoa. Bearbeitet von T. R. R. Stebbing (Tunbridge Wells). XXXIX und 806 Seiten mit 127 Abbildungen. 1906 IX. Subskriptionspreis Mark 36,—. Einzelpreis Mark 48,—. Subskriptionspreis Mark 7,40. Einzelpreis Mark 9,60. Amphipoda, I. Gammaridea. Bearbeitet von T. R. R. Stebbing (Tunbridge Wells). XXXIX und 806 Seiten mit 127 Abbildungen. 1906 IX. Subskriptionspreis Mark 36,—. Einzelpreis Mark 48,—. Heliconiidae. Bearbeitet von H. Stichel & H.Riffarth. XV und 290 Seiten mit 50 Abbildungen. 1905 X. 1906 IX. Subskriptionspreis Mark 36,—. Einzelpreis Mark 48,—. Heliconiidae. Bearbeitet von H. Stichel & H.Riffarth. XV und 290 Seiten mit 50 Abbildungen. 1905 X. Subskriptionspreis Mark 14,—. Einzelpreis Mark 18,—. Subskriptionspreis Mark 14,—. Einzelpreis Mark 18,—. Turbellaria, I. Acoela. Bearbeitet von Ludwig von Graff (Graz). VIII und 35 Seiten mit 8 Abbildungen. 1905 V. Subskriptionspreis Mark 2,40. Einzelpreis Mark 3,—. Subskriptionspreis Mark 2,40. Einzelpreis Mark 3,—. Cynipidae. Bearbeitet von K. W. v. Dalla Torre & J. J. Kieffer, XXXV und 891 Seiten mit 422 Abbildungen. 1910 VIII. Subskriptionspreis Mark 42,—. Einzelpreis Mark 56,—. Subskriptionspreis Mark 42,—. Einzelpreis Mark 56,—. Brassolidae. Bearbeitet von H. Stichel (Berlin). XIV und 244 Seiten mit 46 Abbildungen. .1909 V. Subskriptionspreis Mark 11,20. Einzelpreis Mark 1. Subskriptionspreis Mark 11,20. Ixodidae. Bearbeitet von L. G. Neumann (Toulouse). 169 Seiten mit 76 Abbildungen. 1911 Vl. Subskriptionspreis Mark 8,40. Einzelpreis Mark 1. XVl und Einzelpreis Mark 11,20. Subskriptionspreis Mark 8,40. Chamaeleontidae. . Bearbeitet von Franz Werner (Wien). und 5% Seiten mit 20 Abbildungen. 1911 VIII. Subskriptionspreis Mark 3,—. Einzelpreis Mark 4, -. Einzelpreis Mark 11,20. x] 27. Lieferung. (Reptilia.) Subskriptionspreis Mark 3,—. Einzelpreis Mark 4, -. Apidae I: Megachilinae. Bearbeitet von H. Friese (Schwerin). XXVI und 440 Seiten mit 132 Abbildungen. 1911 XI. Subskriptionspreis Mark 23,50. Einzelpreis Mark 32,—. 28. Lieferung. (Hymenoptera.) Subskriptionspreis Mark 23,50. Einzelpreis Mark 32,—. Chaetognathi. Bearbeitet von R. v. Ritter-Zähony (Berlin). IX und 35 Seiten mit 16 Abbildungen. 1911 VIII. Subskriptionspreis Mark 2,40. Einzelpreis Mark 3,—. 29. Lieferung. (Vermes.) Subskriptionspreis Mark 23,50. Einzelpreis Mark 32,—. Chaetognathi. Bearbeitet von R. v. Ritter-Zähony (Berlin). IX und 35 Seiten mit 16 Abbildungen. 1911 VIII. Subskriptionspreis Mark 2,40. Einzelpreis Mark 3,—. Subskriptionspreis Mark 2,40. Einzelpreis Mark 3,—. Ichneumonidea: Evaniidae. Bearbeitet von J. J. Kieffer (Bitsch). XIX und 431 Seiten mit 76 Abbildungen. 19121V. Subskriptionspreis Mark 23,20. Einzelpreis Mark 31,— Subskriptionspreis Mark 23,20. Einzelpreis Mark 31,— Ostracoda. Bearbeitet von G. W. Müller ee XXXI und 434 Seiten mit 92 Abbildungen. 1912 VII Subskriptionspreis Mark 24,—. Einzelpreis Mark 32,—. Erschienen sind: Heliozoa. Kraepelin (Ham- burg). XVIII und 265 Seiten mit 94 Abbildungen. 1899 III. Subskriptionspreis Mark 12,60. Einzelpreis Mark 17,—. 8. Lieferung. (Arachnoidea.) Subskriptionspreis Mark 12,60. Einzelpreis Mark 17,—. Trochilidae. Bearbeitet von E. Hartert (Tring). IX und 254 Seiten mit 34 Abbildungen. 1900 II. 9. Lieferung. (Aves.) Subskriptionspreis Mark 12,—. Einzelpreis Mark 16,—. Oligochaeta. Bearbeitet von W. Michaelsen (Hamburg). XXIX und 575 Seiten mit 13 Abbildungen. 1900 X. 10. Lieferung. (Vermes.) Subskriptionspreis Mark 26,60. Einzelpreis Mark 35,—. Forficulidae und Hemimeridae. Bearbeitet von A. de Bormans (Turin) und H. Krauss (Tübingen). XV und 142 Seiten mit 47 Ab- bildungen. 1900 X. Subskriptionspreis Mark 7,—. Einzelpreis Mark 9,—. Palpigradi und Solifugae. Bearbeitet von K. Kraepelin (Ham- burg). XI und 159 Seiten mit 118 Abbildungen. 1901 II. Subskriptionspreis Mark 8,—. Einzelpreis Mark 10,—. 11. Lieferung. (Orthoptera.) 12. Lieferung. (Arachnoidea.) Subskriptionspreis Mark 8,—. Einzelpreis Mark 10,—. Hydrachnidae und Halacaridae. Bearbeitet von R. Piersig (Annaberg) und H. Lohmann (Kiel). X VIII und 336 Seiten mit 87 Ab- bildungen. 1901 VI. Subskriptionspreis Mark 16,—. Einzelpreis Mark 21,—. Libytheidae. Bearbeitet von A. Pagenstecher (Wiesbaden). IX und 18 Seiten mit 4 Abbildungen. 1901 II. 13. Lieferung. (Acarina.) Subskriptionspreis Mark 1,50. Einzelpreis Mark 2,—. Zosteropidae. Bearbeitet von O.Finsch (Leiden). XIV und 55 Seiten mit 32 Abbildungen. 1901 III. Subskriptionspreis Mark 3,60. Einzelpreis Mark 4,80. mit 32 Abbildungen. 1901 III. Subskriptionspreis Mark 3,60. Einzelpreis Mark 4,80. Cycelophoridae. Bearbeitet von W.Kobelt (Schwanheim). XXXIX und 662 Seiten mit 110 Abbildungen und 1 Landkarte. 1902 VII. Subskriptionspreis Mark 32,—. Einzelpreis Mark 42,—. Callidulidae. Bearbeitet von A. Pagenstecher (Wiesbaden). IX und 25 Seiten mit 19 Abbildungen. 1902 LIT. Subskriptionspreis Mark 2,—. Einzelpreis Mark 3,—. Cycelophoridae. Bearbeitet von W.Kobelt (Schwanheim). XXXIX und 662 Seiten mit 110 Abbildungen und 1 Landkarte. 1902 VII. Subskriptionspreis Mark 32,—. Einzelpreis Mark 42,—. Callidulidae. Bearbeitet von A. Pagenstecher (Wiesbaden). IX und 25 Seiten mit 19 Abbildungen. 1902 LIT. Subskriptionspreis Mark 2,—. Einzelpreis Mark 3,—. Subskriptionspreis Mark 2,—. Einzelpreis Mark 3,—. Paridae, Sittidae und Certhiidae. Bearbeitet von C. E.Hellmayr (München). XXXI und 255 Seiten mit 76 Abbildungen. 1903ILII. Subskriptionspreis Mark 12,60. Einzelpreis Mark 16,—. Tetraxonia. Bearbeitet von R. v. Lendenfeld (Prag). XV und 168 Seiten mit 44 Abbildungen. 1903 VII, Subskriptionspreis Mark 8,40. Einzelpreis Mark 11,—. Subskriptionspreis Mark 8,40. Einzelpreis Mark 11,—. Nemertini. Bearbeitet von Otto Bürger (Santiago). XVII und 151 Seiten mit 15 Abbildungen. 1904 VAill. Subskriptionspreis Mark 7,40. Einzelpreis Mark 9,60. Subskriptionspreis Mark 7,40. Einzelpreis Mark 9,60. Amphipoda, I. Gammaridea. 19. Lieferung. (Porifera.) Erschienen sind: Heliozoa. Cumacea. Bearbeitet von T. R. R. Stebbing (Tunbridge Wells). Es wird ersucht, Subskriptions-Anmeldungen baldigst an die unter- zeichnete Verlags-Buchhandlung Buchhandlungen, direkt, oder durch Vermittlung anderer zu richten. Es wird ersucht, Subskriptions-Anmeldungen baldigst an die unter- zeichnete Verlags-Buchhandlung Buchhandlungen, direkt, oder durch Vermittlung anderer zu richten. Berlin, Juni 1913. NW 6, Karlstr. 11. Berlin, Juni 1913. NW 6, Karlstr. 11. R. Friedländer & Sohn. BL IWETTL LT VIPPPPL IE EEE UT TU PTR ALTLITEeR LE das u, Rei T NEE EITTRRTIITNN Ui 5 i j +4 ’ pe alu u.“ n.man Bley, aut nuyur (; / I ARAAARA IE a a AÄRTTET NT hl N A Pr w) ausähti Mlinn,, hi var! ‚a? rare! LE ER \| aaahjr KERRIBRPFNTORTIT UI DEE DL gaanı, ERTL f ut HrtTiTLLITer aareen u‘ = La ‘2 de; I ag ALTE a DIDZSALATZRigEa Benin RN Bar IE nn EEE PORN TY' \| MN Ir urnilrue TIleiErHlllze e 111 11 Pe n N N RER, DDiciiEE Dn in, hi R Bene 9,8 + Ir c 4 000 Aya ne u nm BRALRAAA N All: N ra We h ».. ,.,.@ ’tr ‚r USE x ALE \ THAT 177 j »22— ® > FONAN Aa - Aa Ban Rn IN Alkaa i TR „f Ay Fe Deren BRAIN ‘ nm A; $ > G Arn A ah mr IM rare ER al 1 rw PerrT Ir LI 84 hun AR A dnieieintarne YA AS RAM Yır Ion. as BELLE LEFT re Pr Sa,,18 1 Y% 8 DEE UT Ps Naar Pan, N x hi, an ra‘ ver. 111,11 111711 11117 TOMTOM 111 e # N r “mu goAm,_ nam, ya DELTE s £ rw. N a» WB PARL WR ‚ rrr T AA er RPLLEr ' - Aahak, gpa-nan- < PrP\ Abd = phneertree ß Il ‚Anal U F wa lla" $ u { N 4111 A dloisichl £. 2) a Er N ENT Pr Dinkpet % „süße: rn DL INDODENERE Nie Papa Dan Karı aAı Iran, DREhar: Na rasen. aan N , N Mi Sy: amp ,.PFre warten N‘ 1 Te stegllnte urfartvVy/t il „Ya, Tor Add IE E \| r \ Top ef ll LUIN ine IA AAN. „HOD m, aD Sant ARFLLER iönnkpkh: \| I 9 a} AunBAnaAuE TLAABLAE Ed u lanpprfhın. N, Ra, A & An D i een Y, BE E 7 ere pap apA “a. Erschienen sind: Heliozoa. Kie a nn an .® - m ._ A Ann Supn Ne eat vi ik.o Anaa >. N RN gi. Naut TRT DOR PR ar un Sti Rn, AURRh EIN aan na BAssY ar AA TER \| { Pf Ort in aaa NRER rn... vr EN N ur, ie FR TI nah a7 [\| dan Irrr obinu., .- v Van Bi Ala -.u\nfture a urn Ma Na 8 Pa Pr DR et hnrerit INN 2 >. AHRKARAR “ “ ER HITLER KIHaLITTTHT RERHNFERE 2 ; Rr RA a) »s fi nn LER Li rT pP we L } Yıyyı « am NyaR, Da FOR Bass , 2 RR NR . u « i h Sence/ Muh. Sur, I « : rec, ERDE lerne una a ne K HIHI ' in! IREREHRHNHFIENE Aa 3 Se .N,. ‚Aral or Ma ie \| AKA TIL E 07 \| an \| 14 N. ae ERROR, AA HRR An er!» u \| Pr Wi am, Re L Ir tE4 ERLEL A, et Ne a 47 B j . w SI 7; m Destnn,.ro0--0re ir Pu} Bam A Rn B Pas yır EEE ERS h are een v oe Ib om An £ Adi Pa ] - h An aaa, FELLLETTRE PoR 2. Wr a ı PT AT em Fun HORHEREE ETIET EIER ER EIE EEE EEE fa) NN ALLEN 1 I irn Ns 1 o PETE ANOTOO SE % B 4 er ua Ran “aıhS, Inmeacr street: 64. - we am Peru fi . b; 2 \| 4 IBa\| An”, See LT nnpent., nf pt ‚ne n en DIALETTG ea eengunn OU unes TIIRHTeT An. TE TEENT N ie ce = a epsyer20 8 IH \| \| f \| \| N \| Ei a2 r” smtaaPPıw Rs „ag \| "ER 0 r ‘Bl, r Ir £ An, N \| \| Pr 2 : m: upreesartıt Banıba An n irt f7 LE BB rer ar REsSzERe Yt Io » E RE E re, .} "e PO I EIER BUN: RR TR ' DS VL > ha gıh “erue, in De Ken vn SE NEROEN - es en nn - (vr VTTTT——— ae ah: Be 1 3 Nana 1 den Eu u rel En Prrzn, waLT m gan nz I au’ it jwy AR AA ee Ri’ Arne II u“ ‚aAaA, Ms. a a Baiy! Erschienen sind: Heliozoa. sap ER PETT ARASE N ret DELIEIS mpfter H re TYY WE 17, W) ‚oh! tasaWlaahädt \| aunsiä.. IH RPRNTI SH AT a 5 A: sArıı, BUN N REBHE INH HI Fr PT 1.) =.‘ 4 Fans ur\ k f PLA Mag, Da Swan; Re Br \| Gm IP i Pan Mäßs - Sl “,. 1 u a. Hart TI Fi f ä 4 Be Aa ß IM, tr nu aan, II4 344 “29° % Er IMArTrPR f\ ; Alan FTIR dm ar Anprı LEN “_. af x 4) u” ERIK E Bas Try ayAsı B > Me AN Ar uall, Bee ERLERNT 3 IE IT #. u \ } » N a Sr BI ER ER a 4 r MAAn i \ DORF ar \ ..'‘ A EN nW ee EST AT ı l, \| an a "es g TR ar yo ee ne rn KEN MM, ar nn ] AN) yar FAR IE A) f ü m u Ar u” 4 N 2 x Nav [) f} dan. won i uM at Rheine * PERRTTO Re III \| yo vu N, 1! N sie IN onn : 2 Il WR 7 BEI Kr \| RT „Pr. MER] ! INERAAAEER ELLKLRRIEH RINA. L \| NT Ida u TIrrITHT PPEFFFUN BRRT FH une, DLIASNLBRASLT Te Werne Maya Ba aA es® P 11 um HTIRRIT Won ıs Wuasanaı Na BE-= “. y: NER un Ill BANNER MOner 111-1] MEILE 1 Aaron ku mus 1 IT PPRETT U ni ar, ana, ' ar u 2. Ba a En #7 is NETTE ag ch uBif HEhBE E { & Inn . IH H \| L uu0 „u ir Bam j = Dit LA, j ’ t 'y\ 41 „. 4 ARCHE “rg Yo zug a nt . r0e*t ham, N vRl N et -.. (Hanau in, ara N Yrr An Br y RANDE MARS Ya EBzESIBE > So Re Ih #a ur f L i pen U a \| IBTERRARSERA EEE „© T znallilihl SSR HBBAUBRAAUHÄRERRGnn, za ALLE AAN, x P- an’, Pia, A\ u Raasnananı JRAIREEMER DRAN aaı ta an ae S ers a % AAN & pn «ar } Tas % - UN . n v ur. ; surRt: LASEEEhht. Fi menperP Por‘ I nl A N Ki Ih N. IE EPPPSPERT I 10 ; S u un 5 IL Lt Fan A „ran Sana R Au, 4 RI es
https://openalex.org/W2561945037	https://figshare.com/articles/journal_contribution/Behavioral_Problems_in_Taiwanese_Children_of_Adolescent_and_Adult_Mothers/10764893/1/files/19277273.pdf	English	null	Behavioral Problems in Taiwanese Children of Adolescent and Adult Mothers	Journal of child and family studies	2,016	cc-by	6,685	Behavioral Problems in Taiwanese Children of Adolescent and Adult Mothers Chia-Kuei Lee1 • Colleen Corte2 • Shan-Tair Wang3 Running head: TAIWANESE CHILD BEHAVIORAL PROBLEMS Correspondence: S.T. Wang National Cheng Kung University Institute of Gerontology 1 University Road, Tainan 70101, Taiwan Work: 886-6-2353535 ext. 5760 Fax: 886-6-302-8175 Email: wifetz5564@hotmail.com Authors: C.K. Lee 1School of Nursing, University of Rochester, Rochester, NY, USA C. Corte 2College of Nursing, University of Illinois at Chicago, Chicago, IL, USA S.T. Wang 3Institute of Gerontology, College of Medicine, National Cheng Kung University, Tainan, Taiwan Behavioral Problems in Taiwanese Children of Adolescent and Adult Mothers Authors: C.K. Lee 1School of Nursing, University of Rochester, Rochester, NY, USA C. Corte 2College of Nursing, University of Illinois at Chicago, Chicago, IL, USA S.T. Wang 3Institute of Gerontology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 3Institute of Gerontology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 1 Behavioral Problems in Taiwanese Children of Adolescent and Adult Mothers Behavioral Problems in Taiwanese Children of Adolescent and Adult Mothers Abstract The purpose of this study was to estimate child behavioral problems over time and determine gender differences in behavioral problems between children born to adolescent and adult mothers in Taiwan. The consistency between parent’s and teacher’s reports was also examined. Secondary analysis of a longitudinal dataset from Taiwan was conducted. A matched sample of 107 children born to adolescent mothers and 111 children born to adult mothers were recruited. Child behavioral problems were assessed by parents at Time 1 (1st and 2nd grades) and by teachers at both Time 1 and Time 2 (5th and 6th grades). Generalized estimating equations and paired t-test were used. At Time 1, compared to children of adult mothers, children of adolescent mothers had had more behavioral problems by both parental and teacher’s reports. Both parents and teachers reported that boys had more behavioral problems than girls. Moreover, according to teacher reports, children of adolescent mothers and boys had more behavioral problems and these differences persisted over time, even controlling for sociodemographic characteristics. In addition, parents reported higher scores of behavioral problems than teachers. In conclusion, child behavioral problems in Taiwan are associated with maternal age at child birth and child’s gender. Interventions may profitably focus on determining the mechanisms that lead to behavior problems in children of adolescent mothers, and/or reducing adolescent pregnancy as a way of decreasing child behavioral problems. Screening and preventive interventions for child behavior problems may need to be gender-specific. Key words: Longitudinal study, gender differences, maternal influences, children of teen mothers, child behavioral problems 2 Introduction Introduction Children born to adolescent mothers have adverse developmental outcomes, such as more cognitive, behavioral, intellectual, and psychosocial problems and low school achievement compared to children born to adult mothers (Brooks-Gunn & Furstenberg, 1986; Hofferth & Reid, 2002; Jaffee, Caspi, Moffitt, Belsky, & Silva, 2001; Sommer et al., 2000; Whitman, Borkowski, Keogh, & Weed, 2001). Studies have shown that compared to preschoolers born to adult mothers, preschoolers born to adolescent mothers have more behavioral, social, and emotional problems, e.g. conduct problem, inattention, impulse, hyperactivity, and communication problems (Sommer, Whitman, Borkowski, & Gondoli, 2000; Whitman et al., 2001). During school age, children born to adolescent mothers have lower academic achievement and more behavioral problems, and thus, they are more likely to break school regulations, become a detained student, or drop out of school under these long-term disadvantages (Brooks-Gunn & Furstenberg, 1986; Sommer et al., 2000). A few longitudinal studies that were conducted in western countries also indicated that the differences in child behavioral problems between adolescent and adult mothers increase over time (Brooks-Gunn & Furstenberg, 1986; Dahinten, Shapka, & Willms, 2007; Whitman et al., 2001). Although numerous studies in western societies have documented differences in the developmental abilities of children born to adolescent mothers and adult mothers, relatively less research in Taiwan has explored the relationship between adolescent mothers and the development of behavioral problems among their offspring. Boys and girls have different behavioral developmental trajectories after age four and express behavioral problems differently, with boys’ behavioral problems emerging earlier than girls’ (Keenan & Shaw, 1997). School-age boys are more likely to have externalizing behavioral problems than school-age girls (Bongers, Koot, Van Der Ende, & Verhulst, 2004; Keenan & Shaw, 1997), whereas girls tend to develop internalizing behavioral problems from school-age and that increase during adolescence (Keenan & Shaw, 1997; Lewinsohn, Gotlib, Lewinsohn, Seeley, & Allen, 1998). Studies conducted in western countries with predominantly unmarried mothers indicate that, in addition to maternal age at child birth, the risk factors for child behavioral problems also include other maternal characteristics (e.g. educational level, maternal behavioral problems such as antisocial behavior, substance abuse, or delinquency, and mental health problems such as depression/anxiety symptoms) or parental and social 3 characteristics, e.g. education, employment, or parental involvement, parenting style, marital status, socieconomic status, and family environment (Christ et al., 1990; Jaffee et al., 2001; López Turley, 2003; Wakschlag, Gordon, Lahey, Green, & Leventhal, 2000). Introduction Young maternal age at child birth was found to be associated with conduct disorder in boys at age 7 to 12 (Wakschlag et al., 2000). Other studies found that child behavioral problems were associated with family environment factors and parental characteristics rather than young maternal age at child birth (Christ et al., 1990; López Turley, 2003). Maternal and paternal antisocial behaviors and socieconomic status influenced the number of conduct disorder behaviors (e.g. violation of major rules, bullying, assault, fighting, and cruelty to animals) in boys born to adolescent mothers at age 6 to 13 (Christ et al., 1990). Fathers of children born to adolescent mothers are more likely than fathers of children born to adult mothers to have low socioeconomic status and to have substance abuse and behavioral problems ‒ factors that may affect parenting (Tan & Quinlivan, 2006; Ekéus & Christensson, 2003). In Western countries, most biological fathers of children born to adolescent mothers do not live with their children (Gee & Rhodes, 2003). Father absence not only had a negative influence on adolescent mothers’ psychological adjustment but also on children’s behavioral development (Gee & Rhodes, 2003). Children of single mothers tend to have more behavioral problems and poorer school performance compared to children who reside with both parents (Unger & Cooley, 1992; Gee & Rhodes, 2003). Higher levels of positive paternal involvement, consistent father contact, or longer time of co-residing partners were associated with less child behavioral problems, better school performance, and higher reading achievement over time (Chang, Halpern, & Kaufman, 2007; Howard, Lefever, Borkowski, & Whitman, 2006; Smith, Grau, Duran, & Castellanos, 2013), particularly in children with a high-risk mother (e.g. depression, low cognitive readiness, high internalizing and externalizing problems). In addition, grandmother support may complement the insufficient paternal support for the adolescent mothers and supplement the child care for adolescent mothers. However, studies showed that children born to adolescent mothers had more behavioral problems in school age when grandmothers were co-residents compared to those who did not have co-resident grandmothers, especially when they experienced abuse and mothers’ depression symptoms (Black et al., 2002; Gee & Rhodes, 2003). 4 The majority of studies of children born to adolescent mothers were conducted in western countries and in the African American population. Introduction In 2012, the national statistics of teen childbearing in United State showed that 89% of adolescent mothers aged 15 to 19 with teen births were not married (Child Trends, 2014). In contrast, in Taiwan, most adolescent mothers married their children’s biologic fathers who were older, and had low educational levels, high unemployment, and less independent economic status (Chen et al., 2005; Kuo et al., 2010). Approximately one half of adolescent mothers still lived with their biological parents and around two-thirds of adolescent mothers received financial support from their parents (Chen et al., 2005). Hence, the studies about behavioral problems of children born to adolescent mothers in western societies may not generalize to Taiwan given the very different characteristics of Taiwanese adolescent mothers and Taiwanese social/cultural system. The purpose of this study is to estimate child behavioral problems over time in boys and girls born to adolescent or adult mothers in Taiwan. In the present study, our research questions were: (1) Do children’s behavioral problems in grades 1–2 vary by maternal age at child birth and child’s gender, controlling for sociodemographic characteristics? (2) Do differences in behavioral problems between children born to adolescent and adult mothers remain significant over time controlling for socioeconomic characteristics? (3) Do parents and teachers reports of child behavior problems differ? M th d Participants This study is part of a longitudinal research project about children born to adolescent and adult mothers, which examines behavioral problems and academic performance from elementary school to middle school. Participants for the parent study were initially recruited through 12 elementary schools in one school district in Taiwan. Inclusion criteria were: 1) child in first or second grade, 2) child born when mother was less than 20 years of age, 3) child had no cognitive impairment, and 4) parent(s) who are child’s primary caregivers agree to participate. After recruitment of the children of adolescent mothers was complete, a matched (on gender and classroom) comparison sample of children born to adult mothers was recruited. Parents (at Time 1 and Time 3) and teachers (at every time-point) of children who participated in this study were also recruited to provide data on children’s behavior problems. Time 1 data was collected in 2002 when children were grade 1 and 2; Time 2 was conducted in 2006 when children 5 were grade 5 and 6; and Time 3 was conducted in 2009 when children were grade 8 and 9. The relevant data for this study was measured at Time 1 and Time 2. We excluded children with incomplete information on behavioral problems or sociodemographic characteristics at Time 1 or Time 2. There were 107 children born to adolescent mothers and 111 children born to adult mothers with a total of 218 parents as well as 81 teachers at Time 1 and 102 teachers at Time 2 in the current data analyses. A total of 60 boys (56.1%) and 47 girls that were born to adolescent mothers (adolescent group) and 54 boys (48.6%) and 57 girls that were born to adult mothers (adult group) had complete data on relevant study variables at both Time 1 and Time 2. The sample characteristics are shown in Table 1. The mean age at child’s birth for mothers in the adolescent group was 18.3 (SD = 1.5) years and for mothers in the adult group was 27.1 (SD = 3.3) years. Mean age at child’s birth for fathers of children born to adolescent mothers was 23.6 (SD = 4.7) years and for fathers born of children born to adult mothers was 29.5 (SD = 4.1) years. Maternal and paternal education and occupations were lower for those in the group of adolescent mothers compared to the group of adult mothers. Participants The adolescent group was also more likely to be separated or divorced. Adolescent mothers’ family had a higher percentage of grandparents or other relatives involved in the child’s care at both Time 1 and Time 2. Procedure At both time 1 and time 2, the researchers invited children, teachers, and parents (at Time 1) who met the inclusion criteria to participate in the study. Before conducting the study activities, informed consent was obtained from all individual participants included in the parent study. Researchers distributed the questionnaires for teachers and parents in schools. Because the researchers were unable to contact the parents directly, children were asked to take the parental questionnaires home to their parents. Parents completed the questionnaires on child behavioral problems and sociodemographic characteristics at home. Teachers helped to remind children to bring completed parental questionnaires back to school. This secondary analysis of data has been approved by the University Institutional Review Boards. Measures Child behavioral problems were measured with the Chinese version of Conners Parental and Teacher Rating Scale (Wang, Chong, Chou, & Yang, 1993). The Chinese version of Conners Parental 6 and Teacher Rating Scale have been translated from the revised Conners Parental Rating Scale (CPRS- 48) and the revised Conners Teachers Rating Scale (CTRS-28) as well as reworded by pediatricians, psychiatrist, and elementary school teachers for this cohort study. The revised Conners Rating Scales, including Chinese version, have been demonstrated to have acceptable internal consistency, test-retest reliability, and construct validity (Gianarris, Golden, & Greene, 2001; Goyette, Conners, & Ulrich, 1978; Wang et al., 1993). Parent Ratings of Child Behavioral Problems Parent Ratings of Child Behavioral Problems Conners Parental Rating Scale (CPRS-48) is not a direct measure of child’s behavior, but a reflection of parental perception (Gianarris et al., 2001). It consisted of 48 items and 6 subscales, including conduct problems, learning problems, psychosomatic problems, impulsivity-hyperactivity, anxiety, and hyperactivity index (Gianarris et al., 2001; Goyette et al., 1978). Items were scored on a 4- point scale from (0) not at all present through to (3) very much present, and then the sum of score for items in each subscale was calculated. Higher scores indicated that parents perceived more child behavioral problems. Cronbach’s alpha coefficients for the subscales ranged from .72 to .83 in this study. Teacher Ratings of Child Behavioral Problems Conners Teacher Rating Scale (CTRS-28) was a teacher-reported behavior rating scale to screen for child’s psychological adjustment problems (Goyette et al., 1978). It consisted of 28 items measuring 4 subscales, including conduct problems, hyperactivity, inattention-passivity, and hyperactivity index. Each item was rated on a 0 (not at all present) to 3 (very much present) scale. Scale scores were computed by calculating the sum of scores for items in each subscale, and higher scores represented more behavioral problems. The internal consistency coefficients (Cronbach’s alpha) in the present study for subscales ranged from .85 to .92 at Time 1 and .88 to .90 at Time 2. The hyperactivity index subscale in both CPRS and CTRS was a global measure of psychopathology that reflects items from each of the other scales, and not as a specific indicator of the diagnosis of attention deficit hyperactivity disorder (Gianarris et al., 2001). The scores of CPRS had high correlation with Achenbach’s Child Behavior Checklist, and the scores of CTRS had high correlation with Teacher’s Report Form (Costenbader & Keller, 1990; Roussos et al., 1999). Sociodemographic Characteristics 7 Parental age at child birth, child’s gender, parental educational levels, parental occupation, marital status, and family structure (family members residing with child) were measured. In this study, parental education was coded into three levels: below junior high school, senior high school, college and above. Parental occupation was classified into three categories by skill and expertise: unskilled, semi- skilled, and professional. Data Analysis Generalized estimating equations was used to determine (1) if child behavioral problems differed by maternal age at child birth and child’s gender according to parent’s reports and teacher’s reports; and (2) whether teachers reports of behavior problems persisted over time. Maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure were entered as covariates. The interaction effects of time with maternal age at child birth and child’s gender were also estimated. The consistency between CPRS and CTRS was also evaluated by paired t-test and Pearson correlation. Results The means and standard deviations of child behavior problems from parental and teachers’ reports at Time 1 as well as group comparisons of child behavior problems by maternal age at child birth and child’s gender are presented in Table 2. According to parent reports (the top half of Table 2), children born to adult mothers had significantly higher scores for anxiety compared to those born to adolescent mothers; boys had higher conduct problems, learning problems, impulsivity-hyperactivity, and The means and standard deviations of child behavior problems from parental and teachers’ reports at Time 1 as well as group comparisons of child behavior problems by maternal age at child birth and child’s gender are presented in Table 2. According to parent reports (the top half of Table 2), children born to adult mothers had significantly higher scores for anxiety compared to those born to adolescent mothers; boys had higher conduct problems, learning problems, impulsivity-hyperactivity, and hyperactivity index scores than girls. However, after controlling for maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure, anxiety from parental reports no longer differed by maternal age at child birth. In contrast, children born to adolescent mothers had higher learning problems and hyperactivity index scores compared to those born to adult mothers after controlling for those sociodemographic characteristics. According to teacher reports (the bottom half of Table 2), children born to adolescent mothers and boys had higher conduct problems, hyperactivity, inattention-passivity, and hyperactivity index scores compared to those born to adult mothers and girls. These differences persisted after controlling for maternal education. 8 Next, we estimated child behavior problems by maternal age at child birth and child’s gender over time. According to teacher’s reports, the differences in behavioral problems between children born to adolescent and adult mothers and between children’s genders persisted from Time 1 to Time 2 (see Table 3). There was no interaction effect of time with maternal age at child birth and child’s gender; therefor, the interaction terms were excluded from the models. Children born to adolescent mothers and boys had higher conduct problems, hyperactivity, inattention-passivity, and hyperactivity index scores than those children born to adult mothers and girls. These differences persisted after controlling for maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure. There was no significant time effect, which indicates that the behavioral patterns were stable over time. Results Next, we compared the difference of the average scores for conduct problems and hyperactivity index between parent- and teacher-reports at Time 1 (see Table 4). Parents rated significantly higher conduct problem (Mean difference: 2.4, t(217) = 6.8, p < .001) and hyperactivity index scores (Mean difference: 2.2, t(217) = 5.2, p < .001) compared to teachers’ ratings. The conduct problem and hyperactivity index scores were also significantly higher from parent-reports than teacher-reports in each subgroup (girls of adolescent mothers, girls of adult mothers, and boys of adult mothers) with the exception of boys of adolescent mothers (p = .41). Overall, parent-reports were significantly but modestly positively correlated with teacher-reports for conduct problems (r(216) = .27, p < .001) and hyperactivity index (r(216) = .27, p < .001) for the whole sample. Both conduct problems and hyperactivity index scores had significant correlations between parent and teacher reports among the children born to adolescent mothers (r(105) = .21, p = .03; r(105) = .24, p = .01), the those born to adult mothers (r(109) = .34, p < .001; r(109) = .28, p = .003), and boys (r(115) = .26, p = .004; r(115) = .23, p = .01), but not girls (r(109) = .15, p = .15; r(109) = .17, p = .08). For the subgroups, only boys of adult mothers (conduct problem: r(55) = .37, p = .005; hyperactivity index: r(55) = .32, p = .02) had significant correlations between parent- and teacher-reports. There were no significant correlations between parent and teacher reports of conduct problems and hyperactivity for boys born to adolescent mothers (conduct problem: r(58) = .18, p = .17; hyperactivity index: r(58) = .12, p = .36), girls born to adult mothers (conduct problem: r(52) = .14, p = .31; hyperactivity index: r(52) = .08, p 9 = .55), and girls born to adolescent mothers (conduct problem: r(45) = .14, p = .37; hyperactivity index: r(45) = .24, p = .11). Discussion The purpose of this study was to examine child behavioral problems over time in boys and girls born to adolescent or adult mothers in Taiwan. Overall, our findings suggest that controlling for socioeconomic characteristics, (a) children of adolescent mothers had more behavior problems than children of adult mothers, (b) boys had more behavior problems than girls, (c) these differences in behavior problems from teacher reports persisted over time (from grades 1-2 until grades 5-6). These results suggest that maternal age at child birth vary child behavioral problems over time even after taking the sociodemongraphic characteristics into account. Our findings are also consistent with previous studies in the west that have shown gender differences in behavioral problems (Rescorla et al., 2007) – girls tended to score higher on internalizing behavioral problems, especially at ages 12 to 16, whereas boys tended to score higher on externalizing behavioral problems, especially at ages 6 to 11. There was no significant time effect on child behavioral problems from teacher’s reports. Children born to adolescent mothers and boys had more persistent behavioral problems (i.e. conduct problems, hyperactivity, and inattention-passivity) during the elementary school period compared to those born to adult mothers and girls. Accordingly, early detection or prevention for child behavioral problems in very early childhood is essential. Moreover, it is also important to continue to examine behavioral patterns through adolescence to determine whether behaviors persist. The sample characteristics were different from samples of previous studies conducted in western countries. In the western societies, grandparents assistance is typically from the maternal side for adolescent mothers, and the children’s father may be less involved in their children’s lives, particularly for if the father did not marry or keep a positive/romantic relationship with the children’s mothers (Gavin et al., 2002; Krishnakumar & Black, 2003). In contrast, children born to adolescent mothers in Taiwan were more likely to marry the child’s biological fathers and to live with grandparents in our study. As such, Fathers are more likely to be involved in the child’s life and both maternal and paternal grandparents may be more likely to be involved in the child’s life. 10 There were some differences between parent-reports and teacher-reports in this study. The parent-reports did not show many differences between children born to adolescent and adult mothers, but the teacher-reports showed that children born to adolescent mothers had more behavioral problems than children born to adult mothers. Discussion Past studies have found that teacher-reports are more objective and had stronger predictive power of the adverse outcomes of child behavioral problems than parent-reports (Cheramie, 1994; Reid et al., 1987). They suggested that parent-reports may be influenced by low educational levels, maternal depression, parental mood and ability. The discrepancy between parent- and teacher-reports may also be due to children behaving differently across different settings (i.e. home and school) or parents and teachers noticing different types of children’s problem behaviors (Youngstrom, Loeber, & Stouthamer-Loeber, 2000). Teachers may be more aware of disruptive or attentional problems than internalizing problems compared to parents and vice versa (Youngstrom et al., 2000). While parents rated conduct problems and hyperactivity scores significantly higher than teachers, for boy children of adolescent mothers, parent and teacher scores were not significantly different. This might be due to the influence of Chinese culture in which boys usually receive more attention than girls in Chinese families (Wang et al., 1993). Some parents may have underrated their boys’ levels of behavioral problems because they tend to rationalize boys' problematic behaviors. However, teachers may tend to overrate child problematic behaviors according to their students' academic achievements, family characteristics, or defiant and disobedient behavior. Thus, the results also support the premise that it is import to collect information from multiple sources to have complementary information and compare between mother and father or between parents and teachers in order to obtain an integrated evaluation of the child's problematic behavior (Tassé & Lecavalier, 2000; Wang et al., 1993). The study findings need to be considered in light of important limitations. The relatively small sample size in each subgroup (maternal age at child birth and child’s gender) may have limited internal validity and generalizability of the study results. Although we adjusted for several sociodemographic characteristics in our analyses, other factors may also potentially influence child behavior problems and the parental/teachers’ reports. For example, child’s physical/psychological trauma, parental problem behaviors and mental disorders, teacher’s characteristics (e.g. years teaching, awareness of the child's family status), and family/neighborhood environments were not measured in the parent project. Thus, we 11 could not estimate their effects on child behavioral problems. Despite these limitations, the longitudinal data with matched samples is a strength of our study. The findings of our study provide a fundamental understanding of the behavior patterns of elementary school aged-children of adolescent mothers. Discussion Child behavioral problems in Taiwan are associated with maternal age at child birth and child’s gender. Children’s behavioral patterns did not change significantly during the 4-year period of elementary school. This suggests that health care providers and teachers need to be aware that boys and children born to adolescent mothers may be more likely to have behavioral problems that persist at least throughout early childhood. Accordingly, maternal age at child birth and child’s gender should be taken into account in a risk assessment for child development. Studies to determine the mechanisms that lead to child behavior problems in children of adolescent mothers and boys are needed to inform prevention and early intervention programs. Moreover, interventions to reduce teen pregnancy may be another way to decrease and/or prevent child behavioral problems. Therefore, health care providers working with adolescents should emphasize the importance of pregnancy prevention. In addition to prevention, early intervention and continuous support/follow-up for adolescent mothers and their offspring may prevent or reduce behavioral problems in their children. Lastly, further studies are needed to follow Taiwanese adolescent mothers’ offspring until later in adolescence in order to determine behavioral trajectories and their outcomes throughout adolescence as well as identifying underlying mechanisms to target for intervention and prevention. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. However, for this type of study formal consent is not required. References Black, M. M., Papas, M. A., Hussey, J. M., Hunter, W., Dubowitz, H., Kotch, J. B., ... & Schneider, M. (2002). Behavior and development of preschool children born to adolescent mothers: Risk and 3- generation households. Pediatrics, 109, 573-580. Bongers, I. L., Koot, H. M., Van Der Ende, J., & Verhulst, F. C. (2004). Developmental trajectories of externalizing behaviors in childhood and adolescence. Child Development, 75, 1523-1537. doi: 10.1111/j.1467-8624.2004.00755.x Brooks-Gunn, J., & Furstenberg, F. F. (1986). The children of adolescent mothers: Physical, academic, and psychological outcomes. Developmental Review, 6, 224-251. doi:10.1016/0273- 2297(86)90013-4 Chang, J. J., Halpern, C. T., & Kaufman, J. S. (2007). Maternal depressive symptoms, father's involvement, and the trajectories of child problem behaviors in a US national sample. Archives of Pediatrics & Adolescent Medicine, 161, 697-703. doi:10.1001/archpedi.161.7.697. Chen, M. Y., James, K., Hsu, L. L., Chang, S. W., Huang, L. H., & Wang, E. K. (2005). Health-related behavior and adolescent mothers. Public Health Nursing, 22, 280-288. doi: 10.1111/j.0737- 1209.2005.220403.x Cheramie, G. M. (1994). The AAMD Adaptive Behavior Scale-School edition, Part two: Test-retest reliability and parent-teacher agreement in a behavior disordered sample. Perceptual and Motor Skills, 79, 275-283. Child Trends. (2014). Teen births. Retrieved from http://www.childtrends.org/wp- content/uploads/2012/11/13_Teen_Birth.pdf. Accessed 26 May 2016. Christ, M. A. G., Lahey, B. B., Frick, P. J., Russo, M. F., McBurnett, K., Loeber, R., ... & Green, S. (1990). Serious conduct problems in the children of adolescent mothers: disentangling confounded correlations. Journal of Consulting and Clinical Psychology, 58, 840-844. doi: 10.1037/0022- 006X.58.6.840 Costenbader, V. K., & Keller, H. R. (1990). Behavioral ratings of emotionally handicapped, learning disabled, and nonreferred children: Scale and source consistency. Journal of Psychoeducational Assessment, 8, 485-496. doi: 10.1177/073428299000800404 13 Dahinten, V. S., Shapka, J. D., & Willms, J. D. (2007). Adolescent children of adolescent mothers: The impact of family functioning on trajectories of development. Journal of Youth and Adolescence, 36, 195-212. doi: 10.1007/s10964-006-9140-8 Ekéus, C., & Christensson, K. (2003). Socioeconomic characteristics of fathers of children born to teenage mothers in Stockholm, Sweden.Scandinavian Journal of Public Health, 31, 73-76. doi: 10.1080/14034940210134176 Gavin, L. E., Black, M. M., Minor, S., Abel, Y., Papas, M. A., & Bentley, M. E. (2002). Young, disadvantaged fathers’ involvement with their infants: An ecological perspective. Journal of Adolescent Health, 31, 266-276. doi:10.1016/S1054-139X(02)00366-X Gee, C. B., & Rhodes, J. E. (2003). Adolescent mothers' relationship with their children's biological fathers: Social support, social strain and relationship continuity. References Journal of Family Psychology, 17, 370-383. doi: 10.1037/0893-3200.17.3.370 Gianarris, W. J., Golden, C. J., & Greene, L. (2001). The Conners'parent Rating Scales: A critical review of the literature. Clinical Psychology Review, 21, 1061-1093. doi:10.1016/S0272-7358(00)00085- 4 Goyette, C. H., Conners, C. K., & Ulrich, R. F. (1978). Normative data on revised Conners Parent and Teacher Rating Scales. Journal of Abnormal Child Psychology, 6, 221-236. doi: 10.1007/BF00919127 Hofferth, S. L., & Reid, L. (2002). Early childbearing and children's achievement and behavior over time. Perspectives on Sexual and Reproductive Health, 34, 41-49. doi: 10.2307/3030231 Howard, K. S., Lefever, J. E. B., Borkowski, J. G., & Whitman, T. L. (2006). Fathers' influence in the lives of children with adolescent mothers. Journal of Family Psychology, 20, 468-476. doi: 10.1037/0893-3200.20.3.468 Jaffee, S., Caspi, A., Moffitt, T. E., Belsky, J. A. Y., & Silva, P. (2001). Why are children born to teen mothers at risk for adverse outcomes in young adulthood? Results from a 20-year longitudinal study. Development and Psychopathology, 13, 377-397. doi: 10.1017/s0954579401002103 Keenan, K., & Shaw, D. (1997). Developmental and social influences on young girls' early problem behavior. Psychological Bulletin, 121, 95-113. doi: 10.1037/0033-2909.121.1.95 14 Krishnakumar, A., & Black, M. M. (2003). Family processes within three-generation households and adolescent mothers' satisfaction with father involvement. Journal of Family Psychology, 17(4), 488-498. doi: 10.1037/0893-3200.17.4.488 Krishnakumar, A., & Black, M. M. (2003). Family processes within three-generation households and adolescent mothers' satisfaction with father involvement. Journal of Family Psychology, 17(4), 488-498. doi: 10.1037/0893-3200.17.4.488 Kuo, C. P., Lee, S. H., Wu, W. Y., Liao, W. C., Lin, S. J., & Lee, M. C. (2010). Birth outcomes and risk factors in adolescent pregnancies: Results of a Taiwanese national survey. Pediatrics International, 52, 447-452. doi: 10.1111/j.1442-200X.2009.02979.x Lewinsohn, P. M., Gotlib, I. H., Lewinsohn, M., Seeley, J. R., & Allen, N. B. (1998). Gender differences in anxiety disorders and anxiety symptoms in adolescents. Journal of Abnormal Psychology, 107, 109-117. doi: 10.1037/0021-843X.107.1.109 Reid, J. B., Kavanagh, K., & Baldwin, D. V. (1987). Abusive parents' perceptions of child problem behaviors: An example of parental bias. Journal of Abnormal Child Psychology, 15, 457-466. doi: Reid, J. B., Kavanagh, K., & Baldwin, D. V. (1987). Abusive parents' perceptions of child problem Reid, J. B., Kavanagh, K., & Baldwin, D. V. (1987). Abusive parents' perceptions of child problem behaviors: An example of parental bias. Journal of Abnormal Child Psychology, 15, 457-466. References doi: 10.1007/BF00916461 Rescorla, L., Achenbach, T., Ivanova, M. Y., Dumenci, L., Almqvist, F., Bilenberg, N., ... & Erol, N. (2007). Behavioral and emotional problems reported by parents of children ages 6 to 16 in 31 societies. Journal of Emotional and Behavioral Disorders, 15, 130-142. doi: 10.1177/10634266070150030101 Roussos, A., Richardson, C., Politikou, K., Marketos, S., Kyprianos, S., Karajianni, S., ... & Nicolara, R. (1999). The Conners-28 teacher questionnaire in clinical and nonclinical samples of Greek children 6–12 years old. European Child & Adolescent Psychiatry, 8, 260-267. doi: 10.1007/s007870050100 Smith, E. N., Grau, J. M., Duran, P. A., & Castellanos, P. (2013). Maternal depressive symptoms and child behavior problems among Latina adolescent mothers: The buffering effect of mother-reported partner child care involvement. Merrill-Palmer Quarterly, 59, 304-329. doi: 10.1353/mpq.2013.0014 Sommer, K. S., Whitman, T. L., Borkowski, J. G., & Gondoli, D. M. (2000). Prenatal maternal predictors of cognitive and emotional delays in children of adolescent mothers. Adolescence, 35, 87-112. Sommer, K. S., Whitman, T. L., Borkowski, J. G., & Gondoli, D. M. (2000). Prenatal maternal predictors of cognitive and emotional delays in children of adolescent mothers. Adolescence, 35, 87-112. 15 Tan, L. H., & Quinlivan, J. A. (2006). Domestic violence, single parenthood, and fathers in the setting of teenage pregnancy. Journal of Adolescent Health, 38, 201-207. d i 10 1016/j j d h lth 2004 10 014 teenage pregnancy. Journal of Adolescent Health, 38, 201-207. doi:10.1016/j.jadohealth.2004.10.014 doi:10.1016/j.jadohealth.2004.10.014 Tassé, M. J., & Lecavalier, L. (2000). Comparing parent and teacher ratings of social competence and problem behaviors. American Journal on Mental Retardation, 105, 252-259. López Turley, R. N. (2003). Are children of young mothers disadvantaged because of their mother's age or family background?. Child Development,74, 465-474. doi: 10.1111/1467-8624.7402010 Unger, D. G., & Cooley, M. (1992). Partner and grandmother contact in black and white teen parent families. Journal of Adolescent Health, 13, 546-552. doi:10.1016/1054-139X(92)90367-K López Turley, R. N. (2003). Are children of young mothers disadvantaged because of their mother's age or family background?. Child Development,74, 465-474. doi: 10.1111/1467-8624.7402010 or family background?. Child Development,74, 465-474. doi: 10.1111/1467-8624.7402010 Unger, D. G., & Cooley, M. (1992). Partner and grandmother contact in black and white teen parent families. Journal of Adolescent Health, 13, 546-552. doi:10.1016/1054-139X(92)90367-K Unger, D. G., & Cooley, M. (1992). Partner and grandmother contact in black and white teen parent families. Journal of Adolescent Health, 13, 546-552. doi:10.1016/1054-139X(92)90367-K Wakschlag, L. S., Gordon, R. A., Lahey, B. References B., Loeber, R., Green, S. M., & Leventhal, B. L. (2000). Maternal age at first birth and boys' risk for conduct disorder. Journal of Research on Adolescence, 10, 417-441. doi: 10.1207/SJRA1004_03 Wang, Y. C., Chong, M. Y., Chou, W. J., & Yang, J. L. (1993). Prevalence of attention deficit hyperactivity disorder in primary school children in Taiwan.Journal of the Formosan Medical Association, 92, 133-138. Whitman, T. L., Borkowski, J. G., Keogh, D., & Weed, K. (2001). Interwoven lives: Adolescent mothers and their children. Mahwah, NJ: Lawrence Erlbaum Associates. Whitman, T. L., Borkowski, J. G., Keogh, D., & Weed, K. (2001). Interwoven lives: Adolescent mothers and their children. Mahwah, NJ: Lawrence Erlbaum Associates. Youngstrom, E., Loeber, R., & Stouthamer-Loeber, M. (2000). Patterns and correlates of agreement between parent, teacher, and male adolescent ratings of externalizing and internalizing problems. Journal of Consulting and Clinical Psychology, 68, 1038-1050. doi: 10.1037/0022- 006X.68.6.1038 16 16 Table 1 Sample characteristics Table 1 Sample characteristics Table 1 Sample characteristics Variables Adolescenta Adultb χ2 n (%) n (%) Maternal educational level 45.36c Below junior high school 58 (56.3) 25 (22.5) Senior high school 45 (43.7) 62 (55.9) College and above 0 (0.0) 24 (21.6) Paternal educational level 41.51c Below junior high school 64 (60.4) 29 (26.1) Senior high school 38 (35.8) 47 (42.3) College and above 4 (3.8) 35 (31.5) Maternal occupation 16.24 Unskilled 61 (62.2) 65 (59.6) Semi-skilled 32 (32.7) 19 (17.4) Professional 5 (5.1) 25 (22.9) Paternal occupation 24.65 Unskilled 29 (27.6) 21 (19.4) Semi-skilled 61 (58.1) 38 (35.2) Professional 15 (14.3) 49 (45.4) Family structure 1.53 Parents only 57 (54.8) 69 (62.2) Parents and Grandparents 24 (23.1) 24 (21.6) Parents and other relatives 23 (22.1) 18 (16.2) Marital Status 34.21c Single 2 (1.9) 0 (0.0) Married 82 (76.6) 111 (100.0) Separated or Divorced 23 (21.5) 0 (0.0) M (SD) M (SD) t Maternal age at child birth 18.3 (1.5) 27.1 (3.3) 25.50 Paternal age at child birth 23.6 (4.7) 29.5 (4.1) 9.48 a Children born to adolescent mothers, n = 107 bChildren born to adult mothers, n = 111 cFisher's Exact Test; M (SD) = Mean (Standard Deviation) p < 01 17 Table 2 Comparison of the CPRS and CTRS by maternal age at child birth and child’s gender at Time 1 Variables Adolescent Adult Coef. Coef. † Boys Girls Coef. Coef. References † M (SD) M (SD) M (SD) M (SD) Parent Rating (n = 218) Conduct 7.13 (5.1) 6.41 (4.1) 0.62 0.88 7.73 (4.7) 5.65 (4.3) 2.04 2.34 Learning Problem 3.95 (2.5) 3.36 (2.3) 0.56 0.95* 4.02 (2.5) 3.23 (2.2) 0.76* 0.86** Psychosomatic 1.01 (1.8) 1.11 (1.6) -0.11 0.04 1.26 (2.0) 0.83 (1.2) 0.43 0.42 Impulsivity-Hyperactivity 2.93 (2.6) 2.77 (2.1) 0.12 0.38 3.19 (2.4) 2.45 (2.2) 0.74* 0.85** Anxiety 3.46 (1.6) 3.92 (1.7) -0.47* -0.23 3.73 (1.8) 3.65 (1.5) 0.10 0.04 Hyperactivity Index 7.86 (5.5) 6.95 (4.7) 0.81 1.76* 8.42 (5.2) 6.21 (4.7) 2.17 2.37 Teacher Rating (n = 81) Conduct 5.06 (4.4) 3.66 (3.6) 1.28* 1.36* 5.50 (4.5) 3.01 (3.1) 2.42 2.09 Hyperactivity 4.73 (4.4) 3.04 (3.7) 1.55 1.53 5.34 (4.5) 2.16 (2.9) 3.11 3.06 Inattention-Passivity 7.21 (5.5) 4.16 (4.2) 2.91 2.61 7.10 (5.3) 3.99 (4.2) 2.97 3.20 Hyperactivity Index 6.56 (5.5) 3.88 (4.5) 2.51 2.54 6.85 (5.6) 3.28 (3.8) 3.46 3.39 †After controlling for maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure rison of the CPRS and CTRS by maternal age at child birth and child’s gender at Time 1 18 Table 3 CTRS scores by maternal age at child birth and child’s gender over time Variables Adolescent group Adult group Coef.a Coef.b Coef.c Boys Girls Boys Girls M (SD) M (SD) M (SD) M (SD) Conduct problem Time 1 6.32 (4.7) 3.45 (3.3) 4.63 (4.0) 2.63 (2.8) Time 2 6.32 (5.0) 4.11 (3.8) 5.04 (4.9) 2.78 (3.4) Wald X2(3) = 38.88, p < .0001 1.29 2.33 0.28 †Wald X2(9) = 42.36, p < .0001 1.17* 2.13 0.46 Hyperactivity Time 1 6.30 (4.7) 2.72 (2.9) 4.33 (4.1) 1.67 (2.8) Time 2 5.87 (4.5) 2.89 (3.1) 4.39 (4.7) 1.93 (2.4) Wald X2(3) = 63.68, p < .0001 1.39 2.91 -0.00 †Wald X2(9) = 71.80, p < .0001 1.23 2.85 0.19 Inattention-passivity Time 1 8.58 (5.4) 5.47 (5.1) 5.54 (4.8) 2.70 (2.8) Time 2 8.93 (5.2) 5.81 (5.0) 5.39 (4.7) 3.65 (3.3) Wald X2(3) = 59.30, p < .0001 2.90 2.69 0.36 †Wald X2(9) = 67.28, p < .0001 2.39 2.85 0.35 Hyperactivity index Time 1 8.28 (5.6) 4.36 (4.6) 5.35 (5.3) 2.33 (2.7) Time 2 8.45 (5.4) 4.89 (4.7) 5.68 (5.9) 2.98 (3.5) Wald X2(3) = 62.12, p < .0001 2.44 3.29 0.41 †Wald X2(9) = 66.27, p < .0001 2.31 3.23 0.60 aMaternal age; bChild’s gender; cOverall time †After controlling for maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure M (SD) = Mean (Standard Deviation); Coef. References = Coefficient 18 Table 3 CTRS scores by maternal age at child birth and child’s gender over time Variables Adolescent group Adult group Coef.a Coef.b Coef.c Boys Girls Boys Girls M (SD) M (SD) M (SD) M (SD) Conduct problem Time 1 6.32 (4.7) 3.45 (3.3) 4.63 (4.0) 2.63 (2.8) Time 2 6.32 (5.0) 4.11 (3.8) 5.04 (4.9) 2.78 (3.4) Wald X2(3) = 38.88, p < .0001 1.29 2.33** 0.28 †Wald X2(9) = 42.36, p < .0001 1.17* 2.13 0.46 Hyperactivity Time 1 6.30 (4.7) 2.72 (2.9) 4.33 (4.1) 1.67 (2.8) Time 2 5.87 (4.5) 2.89 (3.1) 4.39 (4.7) 1.93 (2.4) Wald X2(3) = 63.68, p < .0001 1.39 2.91 -0.00 †Wald X2(9) = 71.80, p < .0001 1.23 2.85 0.19 Inattention-passivity Time 1 8.58 (5.4) 5.47 (5.1) 5.54 (4.8) 2.70 (2.8) Time 2 8.93 (5.2) 5.81 (5.0) 5.39 (4.7) 3.65 (3.3) Wald X2(3) = 59.30, p < .0001 2.90 2.69 0.36 †Wald X2(9) = 67.28, p < .0001 2.39 2.85 0.35 Hyperactivity index Time 1 8.28 (5.6) 4.36 (4.6) 5.35 (5.3) 2.33 (2.7) Time 2 8.45 (5.4) 4.89 (4.7) 5.68 (5.9) 2.98 (3.5) Wald X2(3) = 62.12, p < .0001 2.44 3.29 0.41 †Wald X2(9) = 66.27, p < .0001 2.31 3.23 0.60 aMaternal age; bChild’s gender; cOverall time †After controlling for maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure M (SD) Mean (Standard Deviation); Coef Coefficient Table 3 CTRS scores by maternal age at child birth and child’s gender over time †After controlling for maternal education, paternal education, maternal occupation, paternal occupation, marital status, and family structure M (SD) = Mean (Standard Deviation); Coef. = Coefficient p < 01 19 Table 4 Comparing parent (CPRS) and teacher (CTRS) reports of child behavior problems at Time 1 Variables Conduct problem t Hyperactivity index t ∆Mean† ∆Mean† Maternal age at child birth Adolescent group 2.07 3.58** 1.30 1.98* Adult group 2.76 6.49 3.06 5.88 Child’s gender Boys 2.23 4.31** 1.56 2.51* Girls 2.64 5.44 2.93 5.37 Adolescent group Boys 1.70 2.10* 0.80 0.84 Girls 2.55 3.11** 1.94 2.24* Adult group Boys 2.79 4.40 2.37 3.03 Girls 2.72 4.80 3.80 5.62 †Mean difference between CPRS and CTRS (CPRS - CTRS) p< 05; *p< 01 Comparing parent (CPRS) and teacher (CTRS) reports of child behavior problems at Time
https://openalex.org/W3045701411	https://link.springer.com/content/pdf/10.1007/JHEP11(2020)116.pdf	English	null	Building bases of loop integrands	The Journal of high energy physics/The journal of high energy physics	2,020	cc-by	35,809	Published for SISSA by Springer Received: August 27, 2020 Revised: October 3, 2020 Accepted: October 10, 2020 Published: November 23, 2020 Received: August 27, 2020 Revised: October 3, 2020 Accepted: October 10, 2020 Published: November 23, 2020 Building bases of loop integrands JHEP11(2020)116 Open Access, c⃝The Authors. Article funded by SCOAP3. 2 Review (and redux): bases of integrands at one loop JHEP11(2020)116 3 Building bases of integrands at two loops 3.1 Two-loop integrand bases: basic building blocks 24 3.1.1 Loop-dependent denominators: vacuum/skeleton graphs 24 3.1.2 Loop-dependent numerators: notation and biases for bases 25 3.2 Integrand bases with ‘0-gon’ power-counting in d dimensions 26 3.2.1 Vector-spaces of loop-dependent numerators 26 3.2.2 Organizing loop-dependent numerators by contact topologies 27 3.3 Deﬁning ‘p-gon power-counting’ at two loops 30 3.3.1 ‘Scalar’ p-gon integrands at two loops 31 3.3.2 Two-loop numerators relative to scalar p-gon integrands 32 3.4 Two-loop integrand bases in various dimensions 34 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) 34 Jacob L. Bourjaily,a,b Enrico Herrmann,c Cameron Langera,d and Jaroslav Trnkad Jacob L. Bourjaily,a,b Enrico Herrmann,c Cameron Langera,d and Jaroslav Trnkad aInstitute for Gravitation and the Cosmos, Department of Physics, Pennsylvania State University, University Park, PA 16892, U.S.A. bNiels Bohr International Academy and Discovery Center, Niels Bohr Institute, University of Copenhagen, Blegdamsvej 17, DK-2100, Copenhagen Ø, Denmark cSLAC National Accelerator Laboratory, Stanford University, Stanford, CA 94039, U.S.A. dCenter for Quantum Mathematics and Physics (QMAP), Department of Physics, University of California, Davis, CA 95616, U.S.A. E-mail: bourjaily@psu.edu, eh10@stanford.edu, cklanger@ucdavis.edu, trnka@ucdavis.edu cSLAC National Accelerator Laboratory, Stanford University, Stanford, CA 94039, U.S.A. dCenter for Quantum Mathematics and Physics (QMAP), Department of Physics, University of California, Davis, CA 95616, U.S.A. Department of Physics, University of California, Davis, CA 95616, U.S.A. Department of Physics, University of California, Davis, CA 95616, U.S.A. Abstract: We describe a systematic approach to the construction of loop-integrand bases at arbitrary loop-order, suﬃcient for the representation of general quantum ﬁeld theories. We provide a graph-theoretic deﬁnition of ‘power-counting’ for multi-loop integrands be- yond the planar limit, and show how this can be used to organize bases according to ultraviolet behavior. This allows amplitude integrands to be constructed iteratively. We illustrate these ideas with concrete applications. In particular, we describe complete inte- grand bases at two loops suﬃcient to represent arbitrary-multiplicity amplitudes in four (or fewer) dimensions in any massless quantum ﬁeld theory with the ultraviolet behavior of the Standard Model or better. We also comment on possible extensions of our framework to arbitrary (including regulated) numbers of dimensions, and to theories with arbitrary mass spectra and charges. At three loops, we describe a basis suﬃcient to capture all ‘leading-(transcendental-)weight’ contributions of any four-dimensional quantum theory; for maximally supersymmetric Yang-Mills theory, this basis should be suﬃcient to repre- sent all scattering amplitude integrands in the theory — for generic helicities and arbitrary multiplicity. Keywords: Scattering Amplitudes, 1/N Expansion, Gauge Symmetry ArXiv ePrint: 2007.13905 ArXiv ePrint: 2007.13905 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP11(2020)116 Contents 1 Introduction and overview 1 1.1 Organization and outline 7 2 Review (and redux): bases of integrands at one loop 7 2.1 One-loop integrand bases: basic building blocks 8 2.1.1 Loop-dependent denominators: ‘scalar’ p-gon integrands 8 2.1.2 Loop-dependent numerators: notational biases for bases 10 2.2 Integrand bases with ‘p-gon’ power-counting 11 2.2.1 Vector-spaces of loop-dependent numerators 12 2.2.2 Organizing loop-dependent numerators by contact-terms 13 2.2.3 Stratifying bases of one-loop integrands 16 2.2.4 The vices and virtues of conformality (‘d-gon’ power-counting in d dimensions) 16 2.3 One-loop integrands for theories in various dimensions 18 2.3.1 Bases for theories deﬁned in four spacetime dimensions 18 2.3.2 Bases for theories deﬁned in various spacetime dimensions 20 2.3.3 Bases for theories deﬁned within ϵ of an integer dimension 21 3 Building bases of integrands at two loops 23 3.1 Two-loop integrand bases: basic building blocks 24 3.1.1 Loop-dependent denominators: vacuum/skeleton graphs 24 3.1.2 Loop-dependent numerators: notation and biases for bases 25 3.2 Integrand bases with ‘0-gon’ power-counting in d dimensions 26 3.2.1 Vector-spaces of loop-dependent numerators 26 3.2.2 Organizing loop-dependent numerators by contact topologies 27 3.3 Deﬁning ‘p-gon power-counting’ at two loops 30 3.3.1 ‘Scalar’ p-gon integrands at two loops 31 3.3.2 Two-loop numerators relative to scalar p-gon integrands 32 3.4 Two-loop integrand bases in various dimensions 34 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) 34 4 Building bases of integrands at three loops 37 4.1 Three-loop integrand bases: basic building blocks 38 4.1.1 Loop-dependent denominators: ladders and wheels 38 4.1.2 Loop-dependent numerators: open problems 39 4.2 The three-loop triangle power-counting basis for four dimensions 39 1 Introduction and overview 1 1.1 Organization and outline 7 2 Review (and redux): bases of integrands at one loop 7 2.1 One-loop integrand bases: basic building blocks 8 2.1.1 Loop-dependent denominators: ‘scalar’ p-gon integrands 8 2.1.2 Loop-dependent numerators: notational biases for bases 10 2.2 Integrand bases with ‘p-gon’ power-counting 11 2.2.1 Vector-spaces of loop-dependent numerators 12 2.2.2 Organizing loop-dependent numerators by contact-terms 13 2.2.3 Stratifying bases of one-loop integrands 16 2.2.4 The vices and virtues of conformality (‘d-gon’ power-counting in d dimensions) 16 2.3 One-loop integrands for theories in various dimensions 18 2.3.1 Bases for theories deﬁned in four spacetime dimensions 18 2.3.2 Bases for theories deﬁned in various spacetime dimensions 20 2.3.3 Bases for theories deﬁned within ϵ of an integer dimension 21 1 Introduction and overview 1 1.1 Organization and outline 7 1 Introduction and overview 1.1 Organization and outline 2 Review (and redux): bases of integrands at one loop 7 2.1 One-loop integrand bases: basic building blocks 8 2.1.1 Loop-dependent denominators: ‘scalar’ p-gon integrands 8 2.1.2 Loop-dependent numerators: notational biases for bases 10 2.2 Integrand bases with ‘p-gon’ power-counting 11 2.2.1 Vector-spaces of loop-dependent numerators 12 2.2.2 Organizing loop-dependent numerators by contact-terms 13 2.2.3 Stratifying bases of one-loop integrands 16 2.2.4 The vices and virtues of conformality (‘d-gon’ power-counting in d dimensions) 16 2.3 One-loop integrands for theories in various dimensions 18 2.3.1 Bases for theories deﬁned in four spacetime dimensions 18 2.3.2 Bases for theories deﬁned in various spacetime dimensions 20 2 Review (and redux): bases of integrands at one loop 4 Building bases of integrands at three loops 4.1 Three-loop integrand bases: basic building blocks 38 4.1.1 Loop-dependent denominators: ladders and wheels 38 4.1.2 Loop-dependent numerators: open problems 39 4.2 The three-loop triangle power-counting basis for four dimensions 39 – i – 5 Open problems and future directions 42 5.1 The importance of choosing bases wisely 42 5.2 Choosing bases according to analytic properties of integrals 47 5.3 An alternative proposal: classiﬁcation by poles at inﬁnity 53 5.4 Building amplitudes: beyond the bases of integrands 56 5.5 Concluding remarks 59 A Graph theory for integrand basis building 60 5.5 Concluding remarks A Graph theory for integrand basis building JHEP11(2020)116 1 Introduction and overview What we mean by this is that we can stratify an integrand basis B into subspaces according to some notion of ‘power-counting’ p — schematically, one partitions a basis B into parts according to ∞ B = ∞ M p=0 Bp (1.2) (1.2) from which we can construct the coeﬃcients of amplitude integrands iteratively: from which we can construct the coeﬃcients of amplitude integrands iteratively: A = X p Ap with Ap:= A∩Bp:= X bip∈Bp ai bi p . (1.3) (1.3) JHEP11(2020)116 This work is principally concerned with the systematic enumeration and construction of integrand bases suitable for representing amplitudes in generic quantum ﬁeld theories, and to deﬁne for non-planar theories how integrand bases can be carved up into subspaces as in (1.2) according to some proxy for their ultraviolet (‘UV’) behavior. An example of such a partitioning at one loop is the division of integrands by naïve power-counting — that is, the leading polynomial degree in ℓµ as ℓ→∞. We will elaborate on this in section 2, but roughly speaking, we may say that a one-loop integrand behaves ‘like a p-gon [at inﬁnity]’ if lim ℓ→∞ I(ℓ) = 1 (ℓ2)p h 1 + O(1/ℓ2) i . (1.4) (1.4) As we will see later, the construction of a basis of loop integrands large enough to represent all amplitudes in the Standard Model, for example, to all-multiplicity and at any loop order turns out to be relatively easy. What is considerably less trivial is the fact that in any integer (or within ϵ of an integer) number of dimensions, this basis is ﬁnite-dimensional and upper-triangular in cuts and hence, can be made ‘prescriptive’ in the sense of [26]. A much more important result, however, is the fact that the integrands in a basis constructed in this way can be partitioned into non-overlapping sets according to their ‘power-counting’. This is a concept that requires considerable care in its deﬁnition — and it will occupy most of our attention below. The real problem — and the source of subtlety and potential confusion — is that most measures of power-counting have relied explicitly on aspects of loop integrands such as how the loop momenta are ‘routed’ which, rather than being intrinsically graph-theoretic, are instead subject to the whims of the physicist who wrote down some particular list of rational loop integrands. 1 Introduction and overview Recent decades have been witness to breathtaking progress in our understanding of and our ability to represent and evaluate scattering amplitudes in perturbative quantum ﬁeld theory. Much of this progress can be traced to the development of unitarity-based methods at the end of the last century [1–3]. Indeed, the heroic work of e.g. [4–9] at one-loop level would lead to the discovery of tree-level recursion [10, 11] among many other things (see e.g. [12–17]). Although not originally described in this language, one way to characterize the princi- pal insight of generalized unitarity is that loop integrands — roughly ‘the sum of Feynman diagrams’ prior to loop integration — are meaningful quantities of interest when viewed as diﬀerential forms on the space of loop momenta. In any suﬃciently well-behaved local and unitary quantum ﬁeld theory, these integrands should be determined by their ‘cuts’ (residues) in terms of lower-loop, and ultimately tree-level information. Provided a suﬃ- ciently large basis of loop integrands B (viewed as a vector-space of rational functions), the coeﬃcients ai of bi ∈B of any loop amplitude integrand A representable within this basis, A = X bi∈B ai bi , (1.1) (1.1) can be determined by linear algebra — by matching all cuts. How and under which conditions this works in detail is beyond the scope of this present work, but we refer the reader to e.g. [18–20] for more detailed discussions. We have been fairly schematic in (1.1) for an important reason. Fixing any particular scattering amplitude in any particular theory, the amplitude integrand itself (however it is found) could be viewed as a single basis element whose coeﬃcient would be 1. This is tautological, and not especially useful or interesting. What is extremely interesting is that for a very wide class of quantum ﬁeld theories (including all renormalizable ones), there exists a ﬁnite-dimensional basis B at any loop order in which all scattering amplitudes of that theory can be represented — involving arbitrary numbers and species of external states. Examples of such bases suitable for representing all amplitudes in the Standard Model at one and two loops can be found in [21–24] and [25], respectively. – 1 – Importantly, it is often possible to build a basis (and therefore represent amplitudes) iteratively. 1 Introduction and overview We will propose one such partitioning deﬁned in purely graph-theoretic terms and discuss the limitations of this deﬁnition in the conclusions. In this work, we will be interested in integrands of perturbative scattering amplitudes — the rational diﬀerential forms over the space of loop momenta obtained by the Feynman expansion. Careful readers should object to our use of ‘the’ in the previous sentence, as this implies a certain degree of uniqueness which does not exist for many quantum ﬁeld theories. This non-uniqueness comes in at least two forms. The ﬁrst source of non-uniqueness of ‘the’ integrand arises from the fact that terms in the Feynman expansion do not come pre-equipped with any preferential choice of origins for the loop momenta, or how the loop momenta should be routed through the Feynman graph. – 2 – For planar ﬁeld theories (such as Yang-Mills in the planar limit), Feynman diagrams do come pre-equipped with an arguably preferential routing associated with the planar-dual of each Feynman graph (provided the labels of these dual variables are symmetrized across all loop momenta in all graphs). But such a choice is immediately absent for non-planar theories: for example, there is no natural sense in which the following Feynman diagrams encode any particular rational function of any single loop momentum variable: K1 K2 K4 K3 + K1 K2 K4 K3 + K1 K2 K4 K3 . (1.5) (1.5) JHEP11(2020)116 There is no natural way to align the origins (in loop-momentum space) of the loop momenta across the three diagrams above. This non-uniqueness has historically been a source of much confusion. But the resolution is fairly obvious: there is simply no particular integrand to discuss. When we speak of the loop integrand, we merely mean a representative integrand of the equivalence class of rational functions generated by translations of all the internal loop momenta. Indeed, we will almost never have any need to choose any particular representative (equivalently, any choice of loop-momentum origins): our integrands will (almost) always be deﬁned and discussed graph-theoretically.1 It is worth mentioning that there has been a lot of progress in deﬁning the non-planar loop integrand more rigorously [27, 28], at least in the context of certain quantum ﬁeld theories. Furthermore, it was observed in [29–31] that even without a properly deﬁned integrand there are very non-trivial properties of e.g. 1In the representation of amplitudes, however, it is worth bearing in mind that the coeﬃcients of in- tegrands (for the representation of a particular amplitude) may themselves require that particular loop- momentum routings be chosen for the elements in the basis. 1 Introduction and overview gravity amplitudes which have an imprint at the ‘integrand’ level — as peculiar cancellations between diagrams — and which give a strong hint that a satisfactory deﬁnition of non-planar integrands should indeed exist. However, these observations are indirect and not immediately useful for deﬁning a single rational integrand function. The second source of non-uniqueness of loop integrands is considerably more important and surprisingly subtle: ‘the sum of Feynman diagrams’ is not, in fact, guaranteed to be physically meaningful. Indeed, loop integrands of pure Yang-Mills theory need not be gauge invariant. This ambiguity is not hard to understand and is closely related to the fact that the forward limits of tree-amplitudes in non-supersymmetric gauge theories are ill-deﬁned as not all diagrams that would be included in the forward limit of trees should be included in loops (as they become divergent). This partitioning of diagrams — to ‘throw out’ those tree-diagrams which will diverge in the forward limit — is not gauge invariant. This is a real problem, and one that requires considerably more discussion than we will have room for here. The standard solution to this problem is to use (e.g. dimensionally-)regulated Feynman rules. In dimensional regularization [32], for example, the loop integrand would be gauge-invariant as the problematic terms described above can be meaningfully said to vanish; but then the integrand is not regulator independent. This is a wholly acceptable – 3 – viewpoint, and arguably a very powerful one considering the state of our tools for doing loop integration in dimensional regularization. However, it is not one that we insist upon. Of course, loop integrands should be integrated. One consequence of the claims above is that for any ﬁxed loop-order, the number of integrands we need to integrate is ﬁnite. We should be clear that this is a very diﬀerent statement compared to the ﬁniteness of the basis of master integrals [33] in dimensional regularization. In some ways, the ﬁniteness we are describing is much stronger: as the statement about master integrals is dimensionally agnostic, the number of Feynman integrand topologies that must be considered will grow arbitrarily with multiplicity. In another sense, however, the ﬁniteness of loop integrands is fairly weak; the number of independent integrals is always less than the number of indepen- dent integrands. 1 Introduction and overview We will not say more about integral-level identities, but emphasize that an independent set of integrals can always be chosen as a subset of independent integrands. JHEP11(2020)116 We close this introduction with one small provocative comment. It is an empirical fact (often encountered in the evaluation of master integrals via diﬀerential equations [4, 34–36]) that the diﬀerence between an ‘easy’ integral and a ‘hard’ integral is immeasurable: that hard integrals are so hard that they are essentially technically intractable using current algorithms/technology; on the other hand, there are many ‘easy’ integrands which are near-trivial to integrate. The cost of choosing a ‘wrong’ basis of master integrals often far exceeds any savings from IBP reductions. As such, it is useful to consider the integrand- level representation of amplitudes very carefully. The stratiﬁcation of perturbative quantum ﬁeld theories. Quantum ﬁeld theories can be partially ordered according to the variety of loop integrands required to represent their amplitudes — that is, by the size of the smallest basis in which all their amplitudes can expressed according to (1.1). Speciﬁcally, we will say that [theory A] ≻[theory B] if a suitable basis for theory B exists which is a subset of such a basis for theory A. To illustrate this hierarchy of theories consider, for example, that (1.6) [Standard Model]≻[(Standard Model\Higgs)]≻[QCD]≻[Yang-Mills] . . . . (1.6) This partial ordering is not hard to understand: each successive theory above involves a strict subset of the preceding theory’s Feynman diagrams. What is much less trivial — and considerably more interesting — is that this partial ordering need not have anything to do with the number or kinds of Feynman diagrams for a theory. For example, there is a strict sense in which (1.7) [(pure) Yang-Mills]≻[N = 2 super-Yang-Mills]≻[N = 4 super-Yang-Mills] . (1.7) From a Feynman-diagrammatic point of view, adding supersymmetry to pure Yang-Mills means more ﬁelds and more Feynman diagrams. And so in what sense can it be that supersymmetric Yang-Mills theory (sYM) is ‘smaller’ than non-supersymmetric (‘pure’) Yang-Mills? The answer is that for any multiplicity and any helicity conﬁguration there exists bases of loop integrands for these theories such that From a Feynman-diagrammatic point of view, adding supersymmetry to pure Yang-Mills means more ﬁelds and more Feynman diagrams. And so in what sense can it be that supersymmetric Yang-Mills theory (sYM) is ‘smaller’ than non-supersymmetric (‘pure’) Yang-Mills? 1 Introduction and overview The answer is that for any multiplicity and any helicity conﬁguration there exists bases of loop integrands for these theories such that BYM ⊋BsYM . (1.8) (1.8) – 4 – – 4 – The fact that amplitudes in sYM are simpler than those in pure YM is closely related to the ameliorating eﬀects of supersymmetry in the ultraviolet. The exceptionally good ultraviolet behavior of amplitudes in maximally supersymmetric (N =4) Yang-Mills theory has been the subject of a great deal of interest. In the planar limit, this good behavior is tied to the dual-conformal symmetry of the theory [12–14], but there is an increasingly sharp sense in which amplitudes outside the planar limit are expected [37–39] or are known to have similarly good behavior at ‘inﬁnite loop momenta’ [40]. Problems with (labeling) non-planar Feynman integrals. For planar2 theories, there is a natural way to generalize the naïve power-counting deﬁned at one loop by (1.4): namely, one can assign internal loop momenta according to each plane Feynman diagram’s dual graph; symmetrizing with respect to these variables allows us to deﬁne an unambiguous rational loop integrand with loop-momentum variables that agree over all contributions. In terms of such labeling, one can discuss subsets integrands that behave ‘like a p-gon at inﬁnity’ with respect to all of the (now unambiguous) loop momenta by direct analogy to one loop. JHEP11(2020)116 For non-planar loop integrands, there is no preferential way to route the loop momenta through a graph, making any deﬁnition analogous to (1.4) highly suspect. For example, consider the three-loop ‘wheel’ integral in scalar ϕ4-theory: (1.9) This Feynman integral has the topology of a tetrahedron, with loop momentum ﬂowing around its edges There are two seemingly natural (or at least highly symmetric) choices (1.9) (1.9) This Feynman integral has the topology of a tetrahedron, with loop momentum ﬂowing around its edges. There are two seemingly natural (or at least, highly symmetric) choices for how to represent the dependence of this Feynman integrand on its three internal loop momenta: This Feynman integral has the topology of a tetrahedron, with loop momentum ﬂowing around its edges. There are two seemingly natural (or at least, highly symmetric) choices for how to represent the dependence of this Feynman integrand on its three internal loop momenta: or . (1.10) or . (1.10) or . (1.10) or . 2For the more mathematically minded reader, we should perhaps deﬁne precisely what we mean by a planar loop integrand. This is a loop integrand which admits a plane embedding once all ‘external’ propagators (those attached to monovalent vertices) are attached together at a single node ‘at inﬁnity’ on the compactiﬁed plane. 1 Introduction and overview (1.10) (1.10) In the ﬁrst case, each loop momentum ﬂows through three propagators, suggesting that the integral behaves like a ‘triangle’ at inﬁnity; in the second case, each momentum ﬂows through four propagators, suggesting that we view the integral as behaving like three ‘boxes’ at inﬁnity. Why is one choice preferred over the other? 2For the more mathematically minded reader, we should perhaps deﬁne precisely what we mean by a planar loop integrand. This is a loop integrand which admits a plane embedding once all ‘external’ propagators (those attached to monovalent vertices) are attached together at a single node ‘at inﬁnity’ on the compactiﬁed plane. – 5 – One may immediately think of several convincing reasons to prefer one routing loop momenta over another; but we predict that this rule would not lead to a unique choice of labeling for complicated, generic graphs. Indeed, we strongly suspect that no preferential choice exists. As this example illustrates, not having some preferential way to assign loop momentum variables to the propagators of a graph is already a problem for the description of integrands consisting exclusively of scalar propagators. The problem is compounded when integrands with loop-dependent numerators (required for theories with higher spin ﬁelds) are considered. One way to deal with the problems raised by this example would be to simply assign loop momentum labels to all propagators and organize integrands in some way similar to the naïve power-counting deﬁned for one loop integrals. This will indeed lead to some notional hierarchy of integrands, and even allow one to discover that amplitude integrands in some theories are simpler than others (being expressible within better-behaved strata of bases). However pragmatic this approach may be, it nevertheless relies heavily on an obviously artiﬁcial choice of loop-momentum labeling. To avoid such burdensome complications, we must think more graph theoretically. JHEP11(2020)116 Overview of our main results. The lack of any canonical ‘routing’ of loop momenta through a non-planar Feynman graph forces us to construct loop integrand bases more graph theoretically. Among the principal results of this work is a graph-(and representation- )theoretic description of loop integrands and a description of how integrand bases can be divided up according to (some notion of) ‘power-counting’ similar to (1.2), such that this partitioning of the basis (loosely) tracks ultraviolet behavior, (some notion of) transcen- dentality, etc. 1 Introduction and overview A crucial ingredient in these results is a translationally-invariant description of vector- spaces of loop-dependent numerators of integrands. The formalism we describe is valid in any number of spacetime dimensions (including those of dimensional regularization); but there is much to be learned by specializing to a particular number of spacetime dimensions. In four dimensions, we reproduce the one-loop basis described by OPP in [21], and its two loop extension discussed in [22–24]. What is more interesting, perhaps, is that we reproduce these examples in a way that renders them essentially representation-theoretic and naturally generalizes to arbitrary numbers of spacetime dimensions. Constructing a ﬁnite-dimensional L-loop integrand basis suitable for representing all amplitudes in the Standard Model in 4 −2ϵ dimensions turns out to be surprisingly easy. What is much more subtle and interesting is how such a basis may be divided up into strata like in (1.2) in any meaningful or useful way. By this we mean that it is not terribly diﬃcult to deﬁne a graph-theoretic stratiﬁcation of integrand bases, but it turns out to be surprisingly diﬃcult to deﬁne a non-trivial substratum in which the amplitudes of maximally supersymmetric Yang-Mills may be represented, say. The stratiﬁcation we propose here will not achieve this seemingly simple goal beyond some relatively high loop order (beyond at least 7 loops). In addition to these general ideas, we provide a complete description of two-loop in- tegrand bases suﬃcient for representing all amplitudes in renormalizable quantum ﬁeld – 6 – theories in four or fewer spacetime dimensions. At three loops, we describe a complete basis of integrands suﬃcient to represent the leading transcendental weight part of any amplitude in four dimensions — which should suﬃce to represent all amplitudes in fully color-dressed maximally supersymmetric Yang-Mills theory. 1.1 Organization and outline This work is organized as follows: in section 2 we introduce all necessary ingredients and tools using the example of one-loop amplitudes for the sake of familiarity and concreteness. This includes the discussion of basic notational ideas, functional building blocks and a fa- miliar (but generalizable) deﬁnition of power-counting. The ﬁnal result of this section will be the list of topologies and the number of degrees of freedom for a given power-counting. Our main results lie in section 3, where we discuss in detail how to construct and strat- ify bases of integrands suitable for general two-loop amplitudes; we give a combinatorial rule for constructing and counting the numerator degrees of freedom for any integrand topology and power-counting. In section 4, we apply these ideas to three loops, and il- lustrate the results by providing a complete basis of triangle power-counting integrands in four dimensions. Section 5 is an extended discussion of caveats, open problems, and di- rections for further research. Several graph-theoretic deﬁnitions and notation is relegated to appendix A. Finally, in the supplementary material attached to this paper, we give a Mathematica notebook which encapsulates our results for three-loop integrand bases in four spacetime dimensions and with triangle power-counting. JHEP11(2020)116 2.1 One-loop integrand bases: basic building blocks The basic idea of integrand reduction at one loop is very easy to understand. Every Feyn- man diagram in any quantum ﬁeld theory will give rise to a rational function of the (un- ambiguous up to translation) loop momentum ℓ, external kinematics and possibly internal quantum numbers (such as color factors in gauge theory). For any particular theory, these rational functions span a ﬁnite-dimensional vector-space. We are going to say much more about the precise meaning of these statements momentarily, but let us start by considering the kinds of rational functions that arise. In the following subsections 2.1.1 and 2.1.2, we review the denominator and numerator structure of one-loop Feynman integrands, respec- tively, introducing along the way terminology and notation which proliferates through the rest of this work. JHEP11(2020)116 2 Review (and redux): bases of integrands at one loop The construction of integrand bases at one loop is well-trodden territory, with a rich history of developments. The original ideas (in a modern form) go at least as far back as to Melrose, Passarino and Veltman [41, 42], and include the more recent ideas of Bern, Dixon, Dunbar and Kosower [1, 2, 43], Ossola, Papadopoulos and Pittau (OPP) [21, 44], among many others (see for example [3, 45–53]). The insights gained from these developments include the discovery of tree-level recursion relations for amplitudes [10, 11], dual-conformal symmetry [12–14], the Yangian [54] of N = 4 sYM, and were put to use in many powerful practical applications (see e.g. [55, 56]). Although this material is quite well established (see e.g. the reviews [20, 57] and ref- erences therein), there are two key reasons for us oﬀering yet another exposition here. The ﬁrst is purely pragmatic: it will allow us to introduce critical concepts, notation, and illustrative examples that will prove important in our work ahead. The second reason why we feel this material will be useful for us to review, however, is to distinguish integrand reduction (discussed in this work) from integral reduction. The latter makes critical use of integration-by-parts relations [58, 59] about which we will have nothing to say here. A great example of the distinction between the two is the (ir-)reducibility of a pentagon integrand/integral in four spacetime dimensions — a case that we hope will be made more clear through our discussions. – 7 – We begin our review of integrands with basic notational ideas, making the case for how loop-dependent numerators should be described algebraically. We will introduce the meaning of bases of integrands with ﬁxed, ‘p-gon’ power-counting (something which involves no subtleties at one loop), and then apply these ideas to re-derive many familiar facts about loop integrand bases in four dimensions. Subsequently, we describe how this can be generalized to arbitrary integer dimensions, and also comment on extending our integer- dimensional construction to the realm of dimensional regularization [32]. 4For some recent discussions on eﬃciently reconstructing the loop-independent functions that appear in given basis, see e.g. [60–62]. 3We do not explicitly allow for eikonal propagators 1/(ℓ· Q) here, even though an extension to this case should be straight forward. 2.1.1 Loop-dependent denominators: ‘scalar’ p-gon integrands At one loop, the Feynman rules of a theory give us a map of the form3 Feynman diagram 7→ N(ℓ) ((ℓ−Q1)2 −m2 1)((ℓ−Q2)2 −m2 2) · · · ((ℓ−Qp)2 −m2p), (2.1) (2.1) where ℓis the ‘loop momentum’ variable to be integrated over, each Qi is some constant momentum oﬀset, each mi is the mass of a particle through which the loop momentum ‘propagates’, N(ℓ) is some polynomial in ℓwith coeﬃcients that depend rationally on the external particles’ momenta, color labels, and polarizations. To be clear, we consider N(ℓ) ≃N′(ℓ) if N(ℓ) = f(ext)N′(ℓ) for any function f(ext) independent of ℓ. In particu- lar, f(ext) can involve many propagators: namely, all those ﬁxed to be ℓ-independent by momentum-conservation at each vertex. Thus, for example ∼ (2.2) (2.2) should be understood as equivalent as far as their loop-dependence is concerned. In this work, we will mostly be interested in the loop-momentum-dependent parts of Feynman inte- grands — avoiding much (if any) discussion of the loop-independent factors4 and therefore should be understood as equivalent as far as their loop-dependence is concerned. In this work, we will mostly be interested in the loop-momentum-dependent parts of Feynman inte- grands — avoiding much (if any) discussion of the loop-independent factors4 and therefore 3We do not explicitly allow for eikonal propagators 1/(ℓ· Q) here, even though an extension to this case should be straight forward. 4For some recent discussions on eﬃciently reconstructing the loop-independent functions that appear in a given basis, see e.g. [60–62]. – 8 – have no reason to consider loop-momentum-independent propagators that may be involved in any actual Feynman diagram. At one loop, we call an integrand with p loop-dependent propagators a ‘p-gon’. The propagators of any p-gon have a natural ordering: namely, so that the oﬀset momenta Qi appearing in the propagators of (2.1) always diﬀer by the momentum ﬂowing into each of the vertices. Speciﬁcally, we can order the factors so that for each index i (with cyclic labeling understood), Pi =:Qi+1 −Qi is the sum of some subset of external momenta (which ‘ﬂows into’ the ith vertex). For example, P1 P2 P4 P3 Q1 Q3 Q2 Q4 . (2.3) JHEP11(2020)116 (2.3) Notice that in (2.3) we have used wedges to denote external momenta ﬂowing into the graph. 2.1.1 Loop-dependent denominators: ‘scalar’ p-gon integrands We use this notation to make it clear that we do not care how many external legs are ﬂowing into each vertex, whether they are massive or massless, or what other quantum numbers the external states may carry. Further details concerning our notation can also be found in appendix A. In the following, we often suppress the external edge labels and write Q1 Q3 Q2 Q4 ↔ 1 (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4) , (2.4) (2.4) where we have introduced the convenient shorthand notation where we have introduced the convenient shorthand notation where we have introduced the convenient shorthand notation (ℓ\|Q):= (ℓ−Q)2 , (2.5) (2.5) and where Q denotes any momentum — including ⃗0. The Feynman iϵ-prescription will be left implicit throughout our work. The propagator deﬁnition (2.5) supposes a massless spectrum. Indeed, in this work we show explicit results for massless theories only, even though a generalization to the massive case does not pose any conceptual diﬃculties. If we were to include massive propagators, we could deﬁne (ℓ\|Q)m:= (ℓ−Q)2 −m2 , with (ℓ\|Q)0 =:(ℓ\|Q) , (2.6) (2.6) but we would need to consider all graphs such as (2.4) to carry additional labels to account for all the possible distribution of internal edges’ particle masses. In eq. (2.4), the Qi’s should be understood to conserve momentum. Why have we not written them explicitly? The reason is that any explicit formula for the Qi’s would require that we eliminate or trivialize the translational invariance of the loop momentum ℓ. This could be done by going to dual-momentum coordinates, or more simply by choosing any one of the Qi’s to be ⃗0 (that is, picking an origin for ℓ). We are leaving such a choice to the – 9 – reader, because we do not want to give the appearance that anything we say in what follows depends on whether or how translational invariance is eliminated (or otherwise trivialized). At one loop, momentum conservation requires that all propagators involving the in- ternal loop momentum ℓmust form a single, closed cycle. We deﬁne an integrand with exactly p propagators involving the loop momentum ℓto be a ‘p-gon’ integrand. These will be denoted diagrammatically by a polygon with p sides with p ‘external’ legs denoting inﬂowing momentum; for example, 1, , , , , , , ,... (2.7) (2.7) JHEP11(2020)116 would be described as 0-gon, 1-gon, . . ., 7-gon integrands, respectively. 2.1.1 Loop-dependent denominators: ‘scalar’ p-gon integrands In practice (and even here), some of these are rarely named in this way: the ﬁrst integrands in (2.7) are more often referred to as ‘constant’, ‘tadpole’, ‘bubble’, ‘triangle’, and ‘box’ integrands, respectively. 5For practitioners, using inverse ‘propagators’ not necessarily appearing in a Feynman graph should not seem so strange: such vector-spaces are also needed for integral reduction, as implemented in public codes such as FIRE [64]. [ ] 6Throughout this work, we will take d ∈N+; when discussing dimensional regularization, we consider be−2ϵ to be an additional basis element used for translations. 2.1.2 Loop-dependent numerators: notational biases for bases (2.11) (2.11) JHEP11(2020)116 A key reason for using translates of inverse propagators to describe polynomial degrees of freedom in ℓis that the deﬁnition (2.8) is translationally invariant: A key reason for using translates of inverse propagators to describe polynomial degrees of freedom in ℓis that the deﬁnition (2.8) is translationally invariant: [ℓ] ≃[ℓ+Q]; for any Q . (2.12) (2.12) Thus, any propagators of a Feynman graph which diﬀer by incoming external momenta will correspond to the same vector-space [ℓ]. Moreover, these spaces are identical for massive and massless propagators: Thus, any propagators of a Feynman graph which diﬀer by incoming external momenta will correspond to the same vector-space [ℓ]. Moreover, these spaces are identical for massive and massless propagators: spanQ{(ℓ\|Q)} ≃spanQ{(ℓ\|Q)m} . (2.13) (2.13) Although [ℓ] includes the degree-two-in-components element ℓ2, it is important to empha- size that no other degree-two-in-components monomials are spanned by [ℓ]. In particular, ℓiℓj /∈[ℓ]; a corollary of this is that if we were to write ℓ2 =: bℓ2 −µ2, then neither bℓ2 nor µ2 would be in [ℓ] — although, the combination ℓ2(=: bℓ2 −µ2) ∈[ℓ]. We will have more to say about how degrees of freedom should be encoded for dimensional regularization below in section 2.3.3. 2.1.2 Loop-dependent numerators: notational biases for bases The real motivation for introducing notation such as (2.5) for propagators is that inverse propagators should be viewed as fundamental, irreducible objects. They are the right lan- guage in which to describe loop-dependent numerators exactly because of their ubiquitous role in the construction of loop-dependent denominators of any Feynman integral. Our attitude here is somewhat at odds with the more familiar approach to integrand reduction which treats Lorentz invariant scalar products as the primary monomials of consideration. For integrand reduction, one then distinguishes monomials that can be written in terms of inverse propagators and external kinematic invariants from irreducible scalar products (ISP)’s [63] (starting at two loops). Thus, it is worthwhile to compare and contrast these frameworks in detail. We suggest that all polynomials involving loop momenta should be expressed in the space of sums of products of (generalized) inverse propagators.5 In particular, we use [ℓ]d:= spanQ{(ℓ\|Q)} for Q ∈Rd (2.8) (2.8) to denote the vector-space generated by the d-dimensional translates of inverse propagators involving momentum ℓ. To be clear, 1/(ℓ\|Q) need not be a propagator in any Feynman graph which relates to our description as ‘generalized’ inverse propagators (that we occa- sionally drop in our discussions below). Provided that ℓ∈Rd ,6 then it is not hard to see that [ℓ]d ≃span n ‘1’, ℓ·be1, . . . , ℓ·bed \| {z } components ‘ℓi’:=ℓ·bei , ℓ2o , (2.9) (2.9) 5For practitioners, using inverse ‘propagators’ not necessarily appearing in a Feynman graph should not seem so strange: such vector-spaces are also needed for integral reduction, as implemented in public codes such as FIRE [64]. 6Throughout this work, we will take d ∈N+; when discussing dimensional regularization, we consider be−2ϵ to be an additional basis element used for translations. – 10 – where bei are d-dimensional basis vectors for Rd and ‘1’ signiﬁes any ℓ-independent mono- mial which should be understand to carry the scaling dimension of mass-squared. We will soon stop ﬂagging this fact with scare quotes when writing ‘1’. To see the equivalence between (2.8) and (2.9), one can conﬁrm that ‘1’ ≃Q2 = 1 2 (ℓ\|Q)+(ℓ\|−Q)−2(ℓ\|0) , ℓ·bei = 1 4 (ℓ\|−bei)−(ℓ\|bei) , ℓ2 = (ℓ\|0) . (2.10) (2.10) From (2.9), it is easy to determine the dimensionality of [ℓ]d rank [ℓ]d = d+2 . 2.2.1 Vector-spaces of loop-dependent numerators 2.2.1 Vector-spaces of loop-dependent numerators To allow for higher polynomial degrees in ℓ, we may simply consider spaces constructed from monomials built from products of inverse propagators as straight forward generalization of our basic building blocks deﬁned in eq. (2.8). Let [ℓ]p d:= span ⊕iQi n (ℓ\|Q1) · · · (ℓ\|Qp) o for Qi ∈Rd . (2.15) (2.15) From the embedding space perspective [65, 66], it is easy to see that the vector-space [ℓ]p d is a p-fold symmetric, traceless product of (d+2)-dimensional vectors of sod+2. As such, From the embedding space perspective [65, 66], it is easy to see that the vector-space [ℓ]p d is a p-fold symmetric, traceless product of (d+2)-dimensional vectors of sod+2. As such, JHEP11(2020)116 rank [ℓ]p d = d + p d ! + d + p −1 d ! . (2.16) (2.16) To better understand these vector-spaces, consider the ﬁrst non-trivial case of [ℓ]2. Fol- lowing the deﬁnition above, we could start with an over-complete vector-space spanned by To better understand these vector-spaces, consider the ﬁrst non-trivial case of [ℓ]2. Fol- lowing the deﬁnition above, we could start with an over-complete vector-space spanned by [ℓ]2 d = span n (ℓ\|Q1)2, . . . , (ℓ\|Qd+2)2, (ℓ\|Q1)(ℓ\|Q2), . . . , (ℓ\|Qd+1)(ℓ\|Qd+2) o , (2.17) (2.17) for some set of displacements Qi ∈Rd; from this one may at ﬁrst expect the rank to be (d+2) + d+2 2 ; but this over-counts the dimension of the space by (in this case) one non- trivial relation. To see this one relation most simply, consider the naïve basis of monomials as we did in (2.9); simply taking all pairs, we’d ﬁnd the (still over-complete) generators: for some set of displacements Qi ∈Rd; from this one may at ﬁrst expect the rank to be (d+2) + d+2 2 ; but this over-counts the dimension of the space by (in this case) one non- trivial relation. To see this one relation most simply, consider the naïve basis of monomials as we did in (2.9); simply taking all pairs, we’d ﬁnd the (still over-complete) generators: [ℓ]2 ≃span n 1·1, 1·ℓi, 1·ℓ2, (ℓi)2, ℓi·ℓj, ℓi·ℓ2, (ℓ2)2o . (2.18) (2.18) Here, the over-completeness is more manifest: 1·ℓ2 ∈span i (ℓi)2 . Eliminating this over- completeness would result in a non-redundant basis for [ℓ]2 of the form [ℓ]2 ≃span n 1·1, 1·ℓi, (ℓi)2, ℓi·ℓj, ℓi·ℓ2, (ℓ2)2o . 2.2 Integrand bases with ‘p-gon’ power-counting Let us now describe how we may construct and stratify bases of one-loop integrands ac- cording to their ultraviolet behavior — or, more colloquially, their ‘power-counting’. As we will see in the next section, it turns out that one loop is a deceptively simple case in this regard. Roughly speaking, we say that I has ‘p-gon power-counting’ if it scales like p or more propagators as ℓ→∞: lim ℓ→∞ I = 1 (ℓ2)p 1+O(1/ℓ2) . (2.14) (2.14) As we will argue in 2.2.2, this deﬁnition turns out to not be entirely satisfactory for our purposes. To see why, we ﬁrst introduce the relevant language to discuss numerators with multiple loop-momentum insertions of the fundamental building blocks deﬁned in section 2.1.2. As we will argue in 2.2.2, this deﬁnition turns out to not be entirely satisfactory for our purposes. To see why, we ﬁrst introduce the relevant language to discuss numerators with multiple loop-momentum insertions of the fundamental building blocks deﬁned in section 2.1.2. – 11 – 2.2.1 Vector-spaces of loop-dependent numerators (2.19) (2.19) Although we are not including the monomial ℓ2 as a basis vector in (2.19), it is important to never forget that ℓ2 ∈[ℓ]2; and moreover, more generally, [ℓ]1 ⊂[ℓ]2. Although we are not including the monomial ℓ2 as a basis vector in (2.19), it is important to never forget that ℓ2 ∈[ℓ]2; and moreover, more generally, [ℓ]1 ⊂[ℓ]2. This is part of a more general observation: because multiplying by the loop independent monomial ‘1’∈[ℓ] is an injective map from [ℓ]p ,→[ℓ]p+1, we always have [ℓ]a ⊂[ℓ]b ∀a<b . (2.20) (2.20) Whenever we have such sequences of inclusions, it is natural to stratify these vector-spaces according to their complements. Speciﬁcally, let Whenever we have such sequences of inclusions, it is natural to stratify these vector-spaces according to their complements. Speciﬁcally, let c [ℓ]p:= [ℓ]p\[ℓ]p−1 so that [ℓ]p = c [ℓ]0⊕c [ℓ]1⊕· · ·⊕c [ℓ]p ; (2.21) (2.21) that is, c [ℓ]p is the part of [ℓ]p not spanned by [ℓ]p−1. The rank of c [ℓ]p is easy to compute from (2.16): that is, c [ℓ]p is the part of [ℓ]p not spanned by [ℓ]p−1. The rank of c [ℓ]p is easy to compute from (2.16): rank c [ℓ]p d = rank [ℓ]p d −rank [ℓ]p−1 d = d+p p ! − d+p−2 p−2 ! . (2.22) (2.22) – 12 – – 12 – Such a grading of loop-dependent polynomial degrees of freedom is closer in spirit to the way that scalar products are often organized in the literature, e.g. [67, 68]. An important distinction, however, is that what we call c [ℓ]p consists of some (but not all) polynomials in the components of ℓof degree 2p or degree 2p−1. We will see below that this grading of vector-spaces plays an important role in the ‘power-counting’ stratiﬁcation of loop- dependent degrees of freedom in section 2.2.3. 2.2.2 Organizing loop-dependent numerators by contact-terms We gave a vague diagnostic for ‘p-gon’ power-counting integrands in eq. (2.14) above. However, this deﬁnition turns out to be somewhat inadequate, because we would like to diﬀerentiate between numerators such as ℓ2 ∈[ℓ] from those such as ℓiℓj /∈[ℓ]. Recall that ℓiℓj ∈[ℓ]2. Our choice to write ℓiℓj in terms of two-fold products of inverse propagators would seem to unnecessarily worsen what we consider the UV behavior of an integrand with such a numerator. However, this is perfectly okay: because 1 ∈[ℓ], it is always possible to express an integrand with some UV behavior in a space of integrands with worse UV behavior. At the end of the day, the question of how to carve up integrand bases (and how UV behavior should even be deﬁned) can only be answered by how useful the resulting basis is for representing amplitudes. JHEP11(2020)116 One major advantage of choosing to write all numerator degrees of freedom directly in terms of products of generalized inverse propagators is that it trivializes the determination of the scaling of eq. (2.14). Let bp q := [ℓ]q−p (ℓ\|Q1) · · · (ℓ\|Qq) for q ≥p , (2.23) (2.23) be the vector-space of all q-gon integrals with (q −p)-fold products of inverse propagators in their numerators. It is trivial to see that lim ℓ→∞ I = 1 (ℓ2)p for all I ∈bp q . (2.24) (2.24) Indeed, starting from the space bp p — so-called ‘scalar’ p-gon integrands, with loop- independent numerators — one may also consider (p+1)-gon integrals with numerators chosen from [ℓ], bp p+1; (p+2)-gons with numerators chosen from [ℓ]2, bp p+2; and so on. All these integrands trivially scale ‘like a p-gon’ at inﬁnite loop momentum. Graphical rules for denoting integrand vector-spaces. To better discuss these vector-spaces of integrands, it may be useful to introduce some graphical/diagrammatic notation. Let us denote the vector-space of translates of inverse propagators over a prop- agator as ⃗ℓ := [ℓ] ℓ2 . (2.25) (2.25) This vector-space includes the Feynman-rule-propagators of scalar ﬁelds, Fermions, and spin-1 bosons (in any gauge). In particular, this means that (2.25) includes as elements all the propagators involved in the Feynman expansion for the Standard Model. And because ℓ2 ∈[ℓ], it also includes so-called contact-terms. 2.2.2 Organizing loop-dependent numerators by contact-terms – 13 – Consider, for example, the space b3 4, which is represented diagrammatically by Consider, for example, the space b3 4, which is represented diagrammatically by b3 4 ⇔ . (2.26) (2.26) Because all the propagators of any one-loop graph involve the same undetermined loop momentum up to translation, it does not matter where we put the decoration: Because all the propagators of any one-loop graph involve the same undetermined loop momentum up to translation, it does not matter where we put the decoration: ∼ ∼ ∼ . (2.27) (2.27) The statement about contact-terms being included can be written graphically as, for example, The statement about contact-terms being included can be written graphically as, for example, JHEP11(2020)116 D A B C ⊃ D A B C , A B C D , B C D A , C D A B . (2.28) (2.28) Notice that this shows that b3 3 ⊂b3 4. Indeed, for any ﬁxed spacetime dimension, the rank of Bp is bounded. The proof of this statement is originally due to Passarino and Veltman in ref. [42]; let us see how we can understand this fact in our present formalism. Let B(d) p denote the space Bp in spacetime dimension d∈N+. The easiest way to see that B(d) p is ﬁnite-dimensional is to notice that the nested sequence of inclusions bp p ⊂bp p+1 ⊂bp p+2⊂· · · , (2.32) (2.32) turns around on itself eventually: bp q⊃bp q+1 for some q in any dimension d and power- counting p. This happens as soon as rank c bp q = 0 — that is, when the space of contact- terms spans everything (as we saw above for d b3 4 when d ≤2). When does this happen? There are three cases to consider: p>d, p=d, and p<d, which we discuss in turn. turns around on itself eventually: bp q⊃bp q+1 for some q in any dimension d and power- counting p. This happens as soon as rank c bp q = 0 — that is, when the space of contact- terms spans everything (as we saw above for d b3 4 when d ≤2). When does this happen? There are three cases to consider: p>d, p=d, and p<d, which we discuss in turn. JHEP11(2020)116 First, consider the case p > d; we will show that bp p ⊃bp p+1, which implies that rank d bp p+1 =0 and that B(d<p) p = bp p. To prove this, we merely need to note that rank d bp p+1 = rank bp p+1\bp p (2 33) rank d bp p+1 = rank bp p+1\bp p = rank [ℓ]d −rank span (ℓ\|Q1)bp p, . . . , (ℓ\|Qp+1)bp p = 0 . (2.33) = rank bp p+1\bp p = rank [ℓ]d −rank span (ℓ\|Q1)bp p, . . . , (ℓ\|Qp+1)bp p = 0 . (2.33) (2.33) p+1\ p = rank [ℓ]d −rank span (ℓ\|Q1)bp p, . . . , (ℓ\|Qp+1)bp p = 0 . (2.33) The ﬁrst term above is d+2 = rank [ℓ]d , and the second term has rank at least d+2 as p+1≥d+2. Thus, q=p. The next case to consider is when p = d. Notice that this shows that b3 3 ⊂b3 4. Notice that this shows that b3 3 ⊂b3 4. More generally, it is easy to see that bp q−1 ⊂bp q. It will be useful to talk about the complements of these inclusions, as we did for c [ℓ]p deﬁned in (2.21). Let d bp q := bp q\bp q−1 , (2.29) (2.29) denote the ‘top-level’ sub-space of bp q — the space not spanned by so-called contact-terms. Returning to the example of b3 4, it is natural to wonder the rank of d b3 4 . Consider the case of d = 1, for which rank b3 4 = rank [ℓ]1 d=1 = 3 and rank d b3 4 = 0; the statement (2.28) is still true, but not all of the four triangle contact-terms can be independent. For d = 2, we similarly conclude that rank d b3 4 = 0, but now the four scalar triangles in (2.28) are independent. For d ≥3, we ﬁnd that rank d b3 4 =d−2. denote the ‘top-level’ sub-space of bp q — the space not spanned by so-called contact-terms. Returning to the example of b3 4, it is natural to wonder the rank of d b3 4 . Consider the case of d = 1, for which rank b3 4 = rank [ℓ]1 d=1 = 3 and rank d b3 4 = 0; the statement (2.28) is still true, but not all of the four triangle contact-terms can be independent. For d = 2, we similarly conclude that rank d b3 4 = 0, but now the four scalar triangles in (2.28) are independent. For d ≥3, we ﬁnd that rank d b3 4 =d−2. Let us deﬁne the ‘p-gon power-counting basis’ of integrands at one loop to be Bp:= bp p ∪bp p+1 ∪bp p+2∪· · · = bp p ⊕d bp p+1⊕d bp p+2⊕· · · . (2.30) (2.30) Graphically, we could write Graphically, we could write B0 :=span 1, , , , , , , ,... B1 :=span , , , , , , ,... B2 :=span , , , , , ,... B3 :=span , , , , ,... B4 :=span , , , ,... (2.31) (2.31) – 14 – and so on. Although each of these spaces may appear to be inﬁnite-dimensional, this is never the case. 2.2.3 Stratifying bases of one-loop integrands There is one more extremely useful set of inclusions to consider related to these integrand bases: Bp ⊂Bp−1 for all p. Moreover, we can see that this holds true topology-by-topology: bp q ⊂bp−1 q in general. This follows trivially from the fact that [ℓ]a ⊂[ℓ]a+1 (see eq. (2.20)), and has the interpretation that we are always able to express any integrand with good ultraviolet behavior (high p) in terms of those with worse ultraviolet behavior (lower p). JHEP11(2020)116 To discuss the new degrees of freedom as the power-counting of the basis worsens (as p decreases), we may deﬁne c c bp q := c bp q \ d bp+1 q , (2.38) (2.38) — the vector-space of (contact-free) degrees of freedom with p-gon power-counting not expressible by numerators with (p+1)-gon power-counting. Similarly, we can discuss the ranks of these spaces by deﬁning c cdp d[q]:= rank c c bp q = bdp d[q]−bdp+1 d [q] (2.39) (2.39) where the last equality follows from (2.37) and requires that p ≤q < d, or p < q ≤d. Interestingly, it is not hard to see that where the last equality follows from (2.37) and requires that p ≤q < d, or p < q ≤d. Interestingly, it is not hard to see that c cdp d[q] = bdp d−1[q] , (2.40) (2.40) which illustrates how integrand bases can be constructed by iteratively shifting the space- time dimension upward. Notice that this shows that b3 3 ⊂b3 4. For this, it is not hard to show (by direct construction) that bd d+2 ⊂bd d+1, so that rank d bd d+2 =0. As such, the d-gon power-counting basis would consist of B(d) d = bd d⊕d bd d+1, with each of these vector-spaces carrying a single degree of freedom in their numerators. Thus, q = p+1. Actually, this is a bit of an overstatement: as we will discuss at greater length in section 2.2.4 below, the span of (d+1)-gons in d bd d+1 is over-complete: any choice of (d+2) of these integrands whose union consists of d+2 propagators will satisfy a single relation. The ﬁnal case to consider is that of p < d, for which we will show that rank c bp q = 0 for all q>d, and hence q=d. From this, it follows that B(d) p = bp p ⊕d bp p+1⊕· · ·⊕d bp d−1⊕d bp d for p<d . (2.34) (2.34) We can prove this claim by simply constructing a general formula for rank c bp q . an prove this claim by simply constructing a general formula for rank c bp q . dp d[q]:= rank bp q = rank [ℓ]q−p d , =: bdp d[q] \| {z } top rank + X i>0 q i ! bdp d[q −i] \| {z } contact-term rank , with bdp d[p]:= 1 , bdp d[q<p]:= 0 , (2.35) (2.35) {z contact-term rank {z contact-term rank {z top rank where we have recursively deﬁned where we have recursively deﬁned where we have recursively deﬁned bdp d[q]:= rank d bp q . (2.36) (2.36) This formula can be viewed as one for constructing particular bases for subspaces of bp q for its contact-terms — elements involving some subsets of the q propagators of the q-gon – 15 – – 15 – — assuming that these subspaces are all independent, which ensures the veracity of (2.35) as a statement about ranks. It turns out to be relatively straight forward to solve the recurrence relation (2.35) for bdp d[q]; doing so, we ﬁnd bdp d[q] = d−p d−q ! + d−p−1 d−q ! = d+q −2p q −p d−p−1 d−q ! for p≤q<d or p<q≤d. (2.37) (2.37) 2.2.4 The vices and virtues of conformality (‘d-gon’ power-counting in d dimensions) In section 2.2.2, we have alluded to the fact that something special happens in the decompo- sition of integrand bases into contact-terms when the power-counting parameter p coincides with the spacetime dimension — when p=d. In this case, the loop integrands (when com- bined with the measure ddℓ) become invariant under the rescaling of loop momenta ℓ→α ℓ and we refer to this property as ‘conformality’. From the integrand perspective, we would like to explain in a little more detail why we now have to include basis elements with d+1 loop dependent propagators which were completely reducible when p<d. Before discussing the general case for arbitrary integer d, it is perhaps instructive to consider the concrete case of d = 4 spacetime dimensions. Following our one-loop discus- sion for the vector-space of q-gon integrals with (q −p) numerator insertions of generalized inverse propagators in eq. (2.23), box (or 4-gon) power-counting implies that all integrands – 16 – with ﬁve propagators can have a single loop-momentum dependent numerator insertion schematically written as b4 5 = [ℓ]4 (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4)(ℓ\|Q5) , (2.41) [ℓ]4 b4 5 = [ℓ]4 (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4)(ℓ\|Q5) , (2.41) (2.41) 5 = (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4)(ℓ\|Q5) , where the numerator [ℓ]4 contains 6 total degrees of freedom. Of course, any choice of this six dimensional basis is naïvely as good as any other. However, as suggested in eq. (2.29), we ﬁnd it most desirable to decompose the basis according to propagator topologies. Such a basis decomposition is extremely natural in conjunction with generalized unitarity and is also employed in the OPP setup [21, 44]. We are led to the following basis choice JHEP11(2020)116 [ℓ]4 = span ϵ(ℓ, Q1, Q2, Q3, Q4, Q5), (ℓ\|Q1), (ℓ\|Q2), (ℓ\|Q3), (ℓ\|Q4), (ℓ\|Q5) (2.42) (2.42) which contains the ﬁve inverse propagators (ℓ\|Qi) together with the dual of these ﬁve generated by the six-dimensional epsilon symbol. Even though the explicit form of this numerator is not relevant for us here, note that ϵ(ℓ, Q1, Q2, Q3, Q4, Q5) can either be con- veniently evaluated by going to embedding space [65, 66] or by writing an expansion in inverse propagators with additional (complex) momenta. The numerators that are pro- portional to the inverse propagators (ℓ\|Qi) give rise to scalar box contact term topologies, i.e. integrands that can be obtained from the pentagon by pinching one of the propaga- tors. 2.2.4 The vices and virtues of conformality (‘d-gon’ power-counting in d dimensions) One advantage of the basis choice in (2.42) is that spacetime parity of each element is manifest: the scalar boxes give rise to parity-even integrands and the pentagon with ϵ-insertion is parity-odd. Since the parity-odd pentagon vanishes upon integration over the parity-even Feynman contour, this element is often neglected if one is interested in properties of integrated scattering amplitudes. There are a few advantages of the box power-counting basis in four dimensions at one loop. In the context of maximally supersymmetric (N =4) Yang-Mills theory (sYM) [69, 70] in the planar limit (taking N →∞for an suN gauge theory), box power-counting makes dual conformal invariance [12–14] manifest at the integrand-level and displays the inherently good UV behavior of the theory. At one loop, there is a similar statement about the surprisingly good UV behavior of supergravity theories (dubbed the ‘no-triangle hypothesis’ [71–73]) which, however, fails at high enough loop-order or for suﬃciently many external particles (starting at two loops [40]). From an integration perspective, box power-counting implies a low numerator polynomial rank which is advantageous for integral reduction and direct integration as well (see e.g. [74–78]). On the other hand, a substantial downside of the box power-counting basis in (2.42) is that it is over-complete. For details of the argument, see section 2 of [26]; but the bottom line is that the parity-odd pentagons are not all independent, but satisfy integrand-level relations that must be eliminated in order to specify a complete and not over-complete basis. There are many ways to see this redundancy among pentagon integrands. One way would be analogous to what we saw for the decomposition of 3-gon power-counting box integrands for d=1 below eq. (2.29) above. Speciﬁcally, we may consider the decomposition of a hexagon integrand with box power-counting in four dimensions. Our analysis above (see eqs. (2.35) and (2.16)) shows that the total rank of the numerators for an integrand – 17 – in the space b4 6 is d4 4[6] = rank([ℓ]2 4) = 20. Decomposing this space into contact-terms would give us 6 5 ×1 top-level degrees of freedom for contact-term pentagons, and 6 2 ×1 top-level degrees of freedom for contact-term boxes; if these contact-term contributions to the hexagon numerators were all independent, they would span a 6+15 = 21-dimensional space. 2.3 One-loop integrands for theories in various dimensions In this part of our work, we would like to take a moment to illustrate the more abstract deﬁnitions of subsections 2.1 and 2.2 with some concrete examples. In particular, we ﬁrst discuss four-dimensional integrand bases at one loop in subsection 2.3.1 and generalize this setup in two ways. First, we extend the four-dimensional analysis to arbitrary integer spacetime dimension d in 2.3.2. Second, more nontrivially, we extend our discussion to the realm of dimensional regularization — to build integrand bases within ϵ of an integer dimen- sion d in 2.3.3 where it becomes important how we deﬁne the relevant extra-dimensional components of the loop momenta (i.e. the precise deﬁnition of so-called µ-terms). 2.2.4 The vices and virtues of conformality (‘d-gon’ power-counting in d dimensions) Thus, there must be one redundancy (and it is easy to see that the scalar box integrands are always independent in d = 4). Thus, the hexagon’s six contact-term, box- power-counting-pentagon integrands must satisfy one algebraic relation (in order to span a merely ﬁve-dimensional space). Besides eliminating the redundancy by picking an arbitrary subset of independent parity-odd pentagons, there is another way to proceed that is also most relevant to represent more general quantum ﬁeld theories at one-loop and has already led to numerous fruitful results for higher-loop amplitudes even in sYM [79, 80]. As we have argued in section 2.2.2, increasing the power-counting from box (4-gon) to triangle (3-gon) scaling is suﬃcient to eliminate (at integrand level) the parity-odd pentagon entirely. In these cases, one is left with triangle-power-counting, chiral boxes and scalar triangle integrands. JHEP11(2020)116 Our four-dimensional discussion can be easily generalized to any integer dimension d where we run into exactly the same issue. In d dimensions, the d-gon power-counting numerator of a (d+1)-gon involves exactly one insertion of generalized inverse propagators. By eq. (2.11), this space is (d+2)-dimensional and can be spanned by the (d+1) inverse propagators of the (d+1)-gon together with one parity-odd integrand dual to these. At higher points, these parity-odd integrands always satisfy linear relations analogous to what we described above. To specify a complete and non-redundant basis would require us to eliminate certain permutations of the parity-odd integrands, which is undesirable from a symmetry point of view. 7In the four dimensions, there are a number of alternate ways (such as momentum twistors [81] or the embedding space formalism [65, 66]) to represent this setup; but we prefer to use the general notation introduced in this section. where ℓ∗ i are the two solutions to the quadruple-cut equations (ℓ\|Q1)=· · ·=(ℓ\|Q4)=0. 2.3.1 Bases for theories deﬁned in four spacetime dimensions In this subsection, we brieﬂy discuss the speciﬁc and particularly relevant case of d = 4- dimensional integrands to make some of the general statements above more concrete. As mentioned above, all these results are well-known [21, 42, 44] and have long been put to use in the generalized unitarity program [1–3]. Nonetheless, we ﬁnd it valuable to review here. (Our discussion here follows an earlier exposition in sections 2 and 4 of ref. [26].) We – 18 – start by specializing eqs. (2.8) and (2.9) to d = 47 eqs. (2.8) and (2.9) to d = 47 start by specializing eqs. (2.8) and (2.9) to d = 47 [ℓ]4 =span Q ∈R4{(ℓ\|Q)} ≃span{1, ℓ·be1, . . . , ℓ·be4, ℓ2} , (2.43) (2.43) which forms a six-dimensional vector-space spanned by (generalized) inverse propagators. When discussing four-dimensional numerators, we often drop the explicit indication of dimensionality. One consequence of (2.43) — in line with the general discussion above — is that any monomial {1, ℓ·be1, . . . , ℓ·be4, ℓ2} can be expanded in the six-dimensional space of inverse propagators. Importantly, this implies that (independent of the ﬁeld-theory power- counting) any integrand with six and more propagators is expressible in terms of integrands with ﬁve and fewer propagators. JHEP11(2020)116 Organizing four-dimensional integrand bases by contact-terms. To illustrate f h h b l f d f – 19 – 1 ··· B(4) 0 b0 p [ ]0=b0 0 [ ]1=b0 1⊕··· [ ]2=b0 2⊕··· [ ]3=b0 3⊕··· [ ]4=b0 4⊕··· [ ]5=b0 5⊕··· d0 4[p] 1=1 6=5+1 20=9+11 50=7+43 105=2+103 196=0+196 B(4) 1 b1 p [ ]0=b1 1 [ ]1=b1 2⊕··· [ ]2=b1 3⊕··· [ ]3=b1 4⊕··· [ ]4=b1 5⊕··· d1 4[p] 1=1 6=4+2 20=5+15 50=2+48 105=0+105 B(4) 2 b2 p [ ]0=b2 2 [ ]1=b2 3⊕··· [ ]2=b2 4⊕··· [ ]3=b2 5⊕··· d2 4[p] 1=1 6=3+3 20=2+18 50=0+50 B(4) 3 b3 p [ ]0=b3 3 [ ]1=b3 4⊕··· [ ]2=b3 5⊕··· d3 4[p] 1=1 6=2+4 20=0+20 B(4) 4 b4 p [ ]0=b4 4 [ ]1=b4 5⊕··· d4 4[p] 1=1 6=1+5 Table 1. One-loop integrand bases in 4 dimensions with various p-gon power-counting. JHEP11(2020)116 Table 1. One-loop integrand bases in 4 dimensions with various p-gon power-counting. Having discussed the 3-gon power-counting basis, we can easily extend the analy- sis to higher numerator ranks, including 0-gon power-counting. Since bases with lower power-counting are always subsets of bases with higher power-counting (in the notation of section 2.2.3; Bp+1 ⊂Bp for any p) we now know that for any p < 4-gon power-counting, the bases of one-loop integrands are spanned by box-, triangle-, bubble-, tadpole topolo- gies and potentially polynomial terms without propagators. (Polynomial terms do not play a role for integrated amplitudes in dimensional regularization as they would give rise to power-divergences which are set to zero, see e.g. [82].) We can therefore simply list the dimensionality of the bases of integrands for a given power-counting in strictly four spacetime dimensions; this can be found in table 1. Note that all numbers in table 1 are dictated by the general formula for the rank of symmetric traceless tensors of so6, consistent with eq. (2.16). As indicated in the table, we can split the overall ranks associated to a given basis topology according to ‘parent’ and ‘daughter’ (or ‘contact-term’) degrees of freedom as has been advocated for in eq. (2.35). In this split, by parent degrees of freedom, we mean integrand basis elements that can be ﬁxed in a generalized unitarity setup at the level of maximal cuts [83] of the corresponding topology. Likewise, ‘daughter’ or ‘contact-term’ degrees of freedom correspond to integrand basis elements associated to certain pinched topologies whose coeﬃcients can be ﬁxed using unitarity cuts of the pinched topology. Organizing four-dimensional integrand bases by contact-terms. To illustrate f h h b l f d f Organizing four-dimensional integrand bases by contact-terms. To illustrate the structure of higher power-counting bases at one loop in four spacetime dimensions, we follow the discussion of section 4 of [26]. The simplest extension of the box power-counting basis is to allow one additional loop-dependent numerator insertion so that all basis elements scale as ∼1/(ℓ2)3 at large ℓ. Thus, we may start with the following three structures B(4) 3 =span b3 3⊕b3 4⊕b3 5 ; in the notation of eq. (2.23): ( [ℓ]0 4 (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3) , [ℓ]1 4 (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4) , [ℓ]2 4 (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4)(ℓ\|Q5) ) . As explained above (see also [26]), it turns out that the pentagon integrands are not independent of the box- and triangle integrands and one is able to eliminate all topolo- gies with ﬁve or more propagators. This is due to the fact that in four dimensions, the rank of the space of two loop-momentum insertions according to (2.16) and (2.35) is d3 4[5]=rank [ℓ]2 4 =20; this 20-dimensional space can be fully spanned by contact-terms — namely, 5 1 ×2 degrees of freedom from contact-term box integrands and 5 2 ×1 degrees of freedom from contact-term triangles. The full triangle-power-counting basis can there- fore be spanned by box integrands (with non-trivial numerators) as well as scalar triangle integrands. A particularly convenient choice of basis for the 3-gon power-counting boxes are the so-called ‘chiral’ numerators (which turn out to be incredibly useful for matching unitarity cuts). Choosing this particular basis, we attribute the 2 top-level degrees of freedom to each box integrand according to: b3 4 = [ℓ] (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4) , with [ℓ]4 = span (ℓ\|ℓ∗ 1), (ℓ\|ℓ∗ 2), (ℓ\|Q1), (ℓ\|Q2), (ℓ\|Q3), (ℓ\|Q4) , (2.44) (2.44) where ℓ∗ i are the two solutions to the quadruple-cut equations (ℓ\|Q1)=· · · 7In the four dimensions, there are a number of alternate ways (such as momentum twistors [81] or the embedding space formalism [65, 66]) to represent this setup; but we prefer to use the general notation introduced in this section. Organizing four-dimensional integrand bases by contact-terms. To illustrate f h h b l f d f For more details, we refer the interested reader to our original exposition of the split of integrand degrees of freedom in section 4 of [26]. 2.3.2 Bases for theories deﬁned in various spacetime dimensions Based on our 4-dimensional discussion in 2.3.1, it should be quite clear how to extend the basis construction to arbitrary integer d spacetime dimensions. Let us therefore only brieﬂy summarize the corresponding results. Having ﬁxed the spacetime dimension, there is one additional ﬁgure of merit required to deﬁne a basis of integrands, namely the desired power-counting. – 20 – total rank = top rank+contact terms numerator Np d = 2 d = 3 d = 4 d = 5 d = 6 • N0 1 1=1+0 1=1+0 1=1+0 1=1+0 1=1+0 N1 1 1=1+0 1=1+0 1=1+0 1=1+0 1=1+0 N0 [ ] 4=3+1 5=4+1 6=5+1 7=6+1 8=7+1 N2 1 1=1+0 1=1+0 1=1+0 1=1+0 1=1+0 N1 [ ] 4=2+2 5=3+2 6=4+2 7=5+2 8=6+2 N0 [ ]2 9=2+7 14=5+9 20=9+11 27=14+13 35=20+15 N3 1 1=1+0 1=1+0 1=1+0 1=1+0 1=1+0 N2 [ ] 4=1+3 5=2+3 6=3+3 7=4+3 8=5+3 N1 [ ]2 9=0+9 14=2+12 20=5+15 27=9+18 35=14+21 N0 [ ]3 16=0+16 30=2+28 50=7+43 77=16+61 112=30+82 N4 1 1=1+0 1=1+0 1=1+0 1=1+0 1=1+0 N3 [ ] 4=0+4 5=1+4 6=2+4 7=3+4 8=4+4 N2 [ ]2 9=0+9 14=0+14 20=2+18 27=5+22 35=9+26 N1 [ ]3 16=0+16 30=0+30 50=2+48 77=7+70 112=16+96 N0 [ ]4 25=0+25 55=0+55 105=2+103 182=9+173 294=25+269 N5 1 1=1+0 1=1+0 1=1+0 1=1+0 1=1+0 N4 [ ] 4=0+4 5=0+5 6=1+5 7=2+5 8=3+5 N3 [ ]2 9=0+9 14=0+14 20=0+20 27=2+25 35=5+30 N2 [ ]3 16=0+16 30=0+30 50=0+50 77=2+75 112=7+105 N1 [ ]4 25=0+25 55=0+55 105=0+105 182=2+180 294=9+285 N0 [ ]5 36=0+36 91=0+91 196=0+196 378=2+376 672=11+661 Table 2. One-loop degrees of freedom for p-gon power-counting in 2–6 dimensions. JHEP11(2020)116 Table 2. One-loop degrees of freedom for p-gon power-counting in 2–6 dimensions. We can then look at diﬀerent power-counting bases. Similar to the 4-dimensional case with 4-gon power-counting (see eq. (2.42) we discussed in section 2.2.4, there are irreducible (d+1)-gon integrands that can be chosen to be parity-odd together with parity-even d-gon integrands). Again, naïvely this forms an over-complete basis and one is forced to eliminate linear relations between diﬀerent parity-odd (d+1)-gons. Exactly like the 4-dimensional case, boosting the power-counting to (d−1)-gon power- counting and higher (more loop-momentum dependence in the numerator) allows us to eliminate this redundancy and one is left with a complete (and not over-complete) basis of integrands where each integrand basis element involves up to d propagators. 2.3.2 Bases for theories deﬁned in various spacetime dimensions We can equally well split the resulting integrand degrees of freedom into ‘parent’ and ‘daughter’ basis elements associated to the maximal number of propagator topologies and various pinched topologies. The relevant data of the size of the d-dimensional integrand bases of p-gon power-counting is dictated by eq. (2.16) and we summarize the relevant results in table 2. 2.3.3 Bases for theories deﬁned within ϵ of an integer dimension So far we have been discussing one-loop integrand bases in integer dimensions, d ∈N+. Even though this integer-dimensional basis counting is interesting in its own right, from a practical point of view it may be desirable to have an analogous construction in the context – 21 – of dimensional regularization.8 To this end, the ﬁrst goal will be to upgrade the deﬁni- tions of eqs. (2.8) and (2.9) to include the extra-dimensional loop-momentum components. In particular, we are interested in a situation, where loop momenta are deﬁned in d−2ϵ spacetime dimensions and all external particle momenta and polarizations are deﬁned in strictly-integer-d spacetime dimensions (see e.g. [86]). Therefore, we schematically decom- pose each loop momentum ℓi (with an eye towards possible generalizations to higher loops) according to ℓi =: bℓi + ⃗µi with bℓi ∈Rd and ⃗µi ∈R−2ϵ (2.45) (2.45) where the extra-dimensional components are orthogonal to the integer-dimensional loop momentum, bℓi·⃗µj = 0 for all i, j. Moreover, requiring that the external states live in the integer-dimensional space, we have that ⃗µi·pa =0 for all external momenta (and similarly for external polarizations). Thus, the only new, extra-dimensional Lorentz invariants that appear in integrand construction would be the so-called ‘µ-terms’ deﬁned via JHEP11(2020)116 ⃗µi·⃗µj =: −µ2 ij . (2.46) (2.46) (When discussing one loop, we will drop these indices — leaving only µ2.) (When discussing one loop, we will drop these indices — leaving only µ2.) In the context of generalized unitarity, it has been known for some time (see e.g. the discussion in [68]) that the extra-dimensional pieces of amplitude integrands can be ob- tained from unitarity cuts in higher, integer-dimensional spacetimes. This of course raises the question of how many extra dimensions are required; but as we will see, it also raises important questions about how to organize integrands by power-counting in order to rep- resent integrands in various quantum ﬁeld theories. To make the stakes clear, we may ask: if it were known that a theory’s unregulated amplitude integrands are representable in terms of p-gon power-counting integrands in d di- mensions, is it true that, when regulated in dim-reg, this theory’s amplitude integrands are still representable in a p-gon power-counting basis? For example, it is known that unregu- lated amplitude integrands in (for the present argument, say planar) sYM are representable in terms of integrands with box power-counting; once regulated, is this still true? 8Note that we work in a dimensional regularization scheme where the external states must lie in strictly- integer-d spacetime dimensions. Examples of such schemes are the ‘t Hooft Veltman scheme [32], the dimensional reduction scheme [84], and the four-dimensional helicity scheme [85], but not the conventional dimensional regularization scheme [82], where all momenta and states are continued away from an integer dimension. 2.3.3 Bases for theories deﬁned within ϵ of an integer dimension For the sake of concreteness and illustration, let us ﬁrst restrict ourselves to the case of one loop. For integrands in d−2ϵ dimensions the µ-terms can be obtained from a (d+1)- dimensional analysis by noting that ⃗µ must span a single-dimensional space spanned by the basis element ‘be−2ϵ’: for one loop, we may identify ⃗µ:= ℓ· be−2ϵ. Notice that this is structurally identical to merely assuming that be−2ϵ spans one additional spacetime direc- tion. Thus, we know that any d-dimensional theory’s amplitudes may be represented in dimensional regularization in terms of a basis of (d+1)-dimensional loop integrands. Are the regulated amplitudes of sYM representable in terms of a p=4 power-counting basis in 5-dimensions? – 22 – If we were to use the naïve deﬁnition of power-counting of eq. (2.14), then the Lorentz- invariant monomial µ2 would scale identically as ℓ2 = bℓ2 −µ2. However, as argued at the end of section 2.1.2, our space would treat these two cases diﬀerently — as µ2 /∈[ℓ] while (bℓ2 −µ2) ∈[ℓ]. In our setup, µ2 ∈[ℓ]2, so that we would declare integrands involving this bare monomial to have worse power-counting. The fact that µ2 should be considered to have worse power-counting than (bℓ2 −µ2) is not merely semantic: the space [ℓ]d+1 involves µ2, but only in a very speciﬁc (and very precise) way: in combination with bℓ2. All existing representations of dimensionally-regulated one-loop amplitude integrands in sYM speciﬁcally make use of the bare monomial µ2 (see e.g. [4, 5, 7]) times pentagons (and higher). As such, these representations do not satisfy our notion of having p = 4 power-counting in 5 dimensions. It would be highly non-trivial (and somewhat surprising) if our more restrictive deﬁnition of box power-counting could still be used to represent these amplitudes. In a forthcoming work [87], it will be shown that this is in fact the case: all regulated amplitude integrands of sYM can be represented in the more restrictive space of 4-gon power-counting integrands in 5 dimensions, as we have deﬁned them here. L+1 JHEP11(2020)116 Beyond one loop, the new Lorentz invariants (2.46) would appear to require some L+1 2 new degrees of freedom introduced into loop integrands. This could be achieved by simply constructing integrands in d+L+1 2 spacetime dimensions. But surely this is overkill! Is it obvious that we cannot do better? 2.3.3 Bases for theories deﬁned within ϵ of an integer dimension It remains an important open — and directly answerable — question of what spacetime dimension is required to represent dimensionally-regulated amplitudes in any given theory. It would be very worthwhile to address this question using some of the known expressions for regulated amplitude integrands that exist in the literature for sYM [88–94], supergravity [88, 90–93, 95, 96], or even the recently studied two-loop four-graviton amplitude in pure GR [97]. 9Recall that the routing of loop momenta corresponds to a particular solution to momentum conservation at every vertex, expressed as a function of L loop momentum variables ℓi. 3.1 Two-loop integrand bases: basic building blocks In this subsection, we ﬁrst introduce the labeling of two-loop graphs in terms of their propagator structure, before examining the basic numerator building blocks in 3.1.2, both of which generalize our one-loop discussions above. 3 Building bases of integrands at two loops The construction of loop integrand bases at two loops turns out to be considerably more subtle and interesting than at one loop. The principal reason for this is that there are fundamentally non-planar integrands, and hence no obviously preferential ‘routing’ of loop momenta.9 Moreover, diﬀerent choices of loop momentum routing can have a severe eﬀect on how integrand bases would be stratiﬁed by more pragmatic approaches. We will see how this works in detail below. In this section, we clarify our general basis-building strategy and apply our ideas to the case of two-loop bases built for theories deﬁned in various spacetime dimensions. We will start in section 3.1 with a generalization of our loop integrand formalism to two loops; in particular, we introduce notation for describing vector-spaces of generic two-loop Feynman integrals with loop-dependent numerators constructed from inverse propagators. In section 3.2 we apply these ideas to describe the ‘0-gon power-counting basis’ of two loop integrands in d dimensions, and we will show how to better understand and generalize the results of [25]. – 23 – 3.1.1 Loop-dependent denominators: vacuum/skeleton graphs The topology of any two-loop Feynman graph can be characterized by three numbers: Γ[a,b,c] ⇔ (3.1) Γ[a,b,c] ⇔ (3.1) JHEP11(2020)116 (3.1) The numbers {a, b, c} indicate how many (loop-momentum-dependent-)propagators diﬀer only by external momentum ﬂowing into the graph. Just as there is no preferential routing for the undetermined loop momenta, these labels have no natural ordering. That being said, we choose to write representative graphs using the convention that b ≤c ≤a, where the indices have been colored merely to direct the eye. None of the counting we perform in this section depends on this choice, it just gives us a convenient way to label graphs. It is not hard to see that these indices fully characterize the loop-dependent propagator structure of any two loop Feynman diagram, and similar conventions for such graphs have been used by others, see e.g. [68, 98]. To be clear, we allow any of these indices to vanish — corresponding to product topologies. Some simple examples of these include Γ[1,1,1] ⇔ Γ[4,0,4] ⇔ Γ[4,1,3] ⇔ (3.2) (3.2) which have been called elsewhere ‘sunrise’, ‘kissing-boxes’, and ‘penta-box’ integrands, respectively. In order to write a rational expression for Γ[a,b,c], we would require either (modest) redundancy or some (ephemeral) choice of loop momentum routing. Redundantly, we could choose to write Γ[a,b,c] ⇔ 1 (ℓA\|Q1) · · · (ℓA\|Qa)(ℓB\|R1) · · · (ℓB\|Rb)(ℓC\|S1) · · · (ℓC\|Sc) (3.3) (3.3) subject to the constraint that ℓA + ℓB + ℓC ∈Rd; or we may solve this condition of momentum conservation and eliminate one of the three classes of loop momenta. When required in the following, we choose to solve momentum conservation and associate ℓA = ℓ1 with a-type propagators, ℓC = ℓ2 with c-type propagators, and ℓB = ℓ1−ℓ2 with b-type propagators. Furthermore, unless otherwise speciﬁed, we assume all Qi, Rj and Sk in eq. (3.3) to be distinct; in a later discussion, however, we will drop this requirement and also allow for ‘doubled-propagator’ graphs which can be relevant for two-loop amplitudes — depending on renormalization scheme (see e.g. [99] for the absence of certain residues for integrals with doubled-propagators in the on-shell scheme). 3.1.1 Loop-dependent denominators: vacuum/skeleton graphs – 24 – In d spacetime dimensions, it should be clear from our one-loop discussion that we mostly need to consider integrand topologies with d+1 propagators of either a, b, or c type, as any topology with more propagators can be trivially reduced by one-loop methods (at the cost of worsening the power-counting). As we will see shortly, for p<d-gon power- counting we can reduce the number of topologies relevant for two-loop integrands even further. In order to make this discussion more transparent, let us ﬁrst mention the relevant building blocks for the numerator structures. 3.1.2 Loop-dependent numerators: notation and biases for bases However, the rank of the combination (3.8) is a little less trivial. One might think that the linear span of the two spaces would add independently resulting in a combined rank of 2(d+2) for (3.8); however, this would double- count the constant ‘1’ shared by both pieces. Eliminating this over-counting, one can easily verify that the above rank ([ℓi]d ⊕[ℓj]d) = 2(d+2)−1. Each space individually has rank[ℓi]d = (d+2). However, the rank of the combination (3.8) is a little less trivial. One might think that the linear span of the two spaces would add independently resulting in a combined rank of 2(d+2) for (3.8); however, this would double- count the constant ‘1’ shared by both pieces. Eliminating this over-counting, one can easily verify that the above rank ([ℓi]d ⊕[ℓj]d) = 2(d+2)−1. Going one step further, we can consider the combined rank of all three numerator factors, rank [ℓA]d⊕[ℓB]d⊕[ℓC]d = ? (3.9) (3.9) Besides over-counting the constant term, we now encounter a subtlety with momentum conservation. As mentioned above, the three loop-momenta ℓA, ℓB, and ℓC are not all in- dependent but satisfy a d-dimensional momentum conservation constraint. In fact, adding this third numerator factor only adds a single basis element: JHEP11(2020)116 rank [ℓA]d⊕[ℓB]d⊕[ℓC]d = 2(d + 2) . (3.10) (3.10) In the particular choice of routing introduced above, this counting can be understood as the combination of ℓ2 i for i = 1, 2, the 2×d angles of the two independent loop momenta ℓ1·be1, . . . , ℓ1·bed, ℓ2·be1, . . . , ℓ2·bed, the constant term ‘1’, as well as the angle between the two loop-momenta, which is proportional to ℓ1·ℓ2. Note that, just as in our one-loop discussion, besides ℓ2 i , the only new degree-two-in-components element spanned by [ℓ1]⊕[ℓ1 −ℓ2]⊕[ℓ2] is ℓ1·ℓ2; all other linear combination of terms (ℓ1·bei)(ℓ2·bej) would be assigned to numerators with higher power-counting. In the particular choice of routing introduced above, this counting can be understood as the combination of ℓ2 i for i = 1, 2, the 2×d angles of the two independent loop momenta ℓ1·be1, . . . , ℓ1·bed, ℓ2·be1, . . . , ℓ2·bed, the constant term ‘1’, as well as the angle between the two loop-momenta, which is proportional to ℓ1·ℓ2. 3.1.2 Loop-dependent numerators: notation and biases for bases Note that, just as in our one-loop discussion, besides ℓ2 i , the only new degree-two-in-components element spanned by [ℓ1]⊕[ℓ1 −ℓ2]⊕[ℓ2] is ℓ1·ℓ2; all other linear combination of terms (ℓ1·bei)(ℓ2·bej) would be assigned to numerators with higher power-counting. 3.2 Integrand bases with ‘0-gon’ power-counting in d dimensions Before diving into the general discussion and some of the subtleties with deﬁning the two- loop version of p-gon power-counting in the following subsection 3.3, we would like to discuss the conceptually straightforward case of 0-gon power-counting that is relevant for theories such as the Standard Model. Here, we restrict our discussion to an integer-dimensional setup, but we expect that a suitable generalization of our one-loop implementation of a dimensional regularization friendly basis including µ-terms should be possible as well. 3.1.2 Loop-dependent numerators: notation and biases for bases In order to discuss the general structure of two-loop numerators, we generalize our initial discussion of the fundamental one-loop numerator objects from section 2.1.2. We have argued that it is most natural to express loop-dependent numerators in terms of generalized inverse propagators JHEP11(2020)116 [ℓ]d = spanQ {(ℓ\|Q)} , Q ∈Rd (3.4) (3.4) with loop-momentum independent coeﬃcients. Following our discussion of the two-loop propagator structure in the previous subsection, it is natural to deﬁne the associated two- loop numerator building blocks with loop-momentum independent coeﬃcients. Following our discussion of the two-loop propagator structure in the previous subsection, it is natural to deﬁne the associated two- loop numerator building blocks [ℓA]d:= spanQ{(ℓA\|Q)} (≃span{1, ℓA·be1, . . . , ℓA·bed, ℓ2 A}) , [ℓB]d:= spanR {(ℓB\|R)} (≃span{1, ℓB·be1, . . . , ℓB·bed, ℓ2 B}) , [ℓC]d:= spanS {(ℓC\|S)} (≃span{1, ℓC ·be1, . . . , ℓC ·bed, ℓ2 C}) . (3.5) (3.5) Since individual numerator polynomials [ℓi]k d are basically one-loop objects, we may refer back to our one-loop discussions for more details on their properties. In the following section, we will discuss integrand bases (with ‘0-gon power-counting’) consisting of graphs Γ[a,b,c] with numerator-spaces constructed directly as products of these factors — for example, Γ[3,1,3] ⇔ with numerator N0 Γ[3,1,3] = [ℓA]3[ℓB]1[ℓC]3 . (3.6) (3.6) However, one novelty that will arise when we consider bases with better power-counting is that we must also consider vector-spaces of numerators constructed as sums of these objects. For example, we may consider an integrand However, one novelty that will arise when we consider bases with better power-counting is that we must also consider vector-spaces of numerators constructed as sums of these objects. For example, we may consider an integrand Γ[3,1,3] ⇔ Γ[3,1,3] ⇔ with numerator N3 Γ[3,1,3] = [ℓA][ℓC]⊕[ℓB] (3.7) (3.7) — which would be appropriate for 3-gon power-counting, as we will deﬁne below. Such sums of vector-spaces never appear at one loop, for the simple reason that [ℓ]j⊕[ℓ]k ⊂[ℓ]max(j,k). Consider for example the sum of two vector-spaces — which would be appropriate for 3-gon power-counting, as we will deﬁne below. Such sums of vector-spaces never appear at one loop, for the simple reason that [ℓ]j⊕[ℓ]k ⊂[ℓ]max(j,k). Consider for example the sum of two vector-spaces rank ([ℓi]d⊕[ℓj]d) , for i ̸= j, i, j ∈{A, B, C} . (3.8) (3.8) – 25 – Each space individually has rank[ℓi]d = (d+2). 3.2.1 Vector-spaces of loop-dependent numerators As alluded to above, the rule for writing down the relevant numerator space for 0-gon power-counting is extremely simple to state: for any propagator structure characterized by the three indices a, b, and c in Γ[a,b,c], we write exactly the same number of generalized inverse propagators in the numerator. As such, the numerator is given by a monomial in terms of [ℓA][ℓB][ℓC] Γ[a,b,c] ↔N0 Γ[a,b,c] := [ℓA]a[ℓB]b[ℓC]c . (3.11) (3.11) Let us give one concrete example to make the above deﬁnition abundantly clear, Γ[4,2,2] ⇔ ↔N0 Γ[4,2,2] := [ℓA]4 d[ℓB]2 d[ℓC]2 d . (3.12) (3.12) – 26 – The logic behind this rule should be apparent: as any propagator involving ℓi ap- proaches inﬁnity, every numerator in the vector-space (3.11) will cancel all the propagators involving ℓi. Thus, at inﬁnite loop momentum, these integrands scale like a constant — or, a 0-gon. A key advantage of discussing 0-gon power-counting is the extremely simple graph- theoretic rule on how to construct the associated numerator space to start with. The next non-trivial part of this analysis is to determine how many of the resulting numerators are linearly independent. As we have seen in our simple linear example in the previous subsection 3.1.2, there are intricate dependencies that only become more involved due to various completeness relations. Of course, computing the rank of linearly independent basis vectors is a ‘simple’ linear algebra problem that can, however, become prohibitive for suﬃciently large graphs in higher dimensions as the ranks of the associated matrices grow. 10We thank Andrew McLeod for help with ﬁnding these formulae. 3.2.1 Vector-spaces of loop-dependent numerators JHEP11(2020)116 In the particular case of 0-gon power-counting, where the numerator ansatz is a simple monomial, we were able to ﬁnd closed form formulae for the ranks f0 d(a, b, c):= rank [ℓA]a d[ℓB]b d[ℓC]c d of the integrand spaces in integer dimensions d ≤4 for arbitrary values of a, b and c.10 f0 1(a, b, c) = 1+2s1 +4s2 , f0 2(a, b, c) = (1+s1 +s2)2, f0 3(a, b, c) = 1 36 h 36+12s3 1 +29s2 2 +4s3 2 +s2 1(54+26s2)+3s3(9+4s3) +s2(61+18s3)+s1(78+9s2(9+2s2)+22s3) i , (3.13) (3.13) i f0 4(a, b, c) = 1 144 h (1+s1 +s2)(4+2s1 +s2)2(9+3s1 +s2) +2(4+2s1 +s2)(7s1 +4(3+s2))s3 +(15+8s1 +4s2)s2 3 i , f0 4(a, b, c) = 1 144 h (1+s1 +s2)(4+2s1 +s2)2(9+3s1 +s2) ( )( ( )) ( +2(4+2s1 +s2)(7s1 +4(3+s2))s3 +(15+8s1 +4s2)s2 3 i , written in terms of symmetric polynomials written in terms of symmetric polynomials s1:= (a+b+c) , s2:= (ab+bc+ca) , s3:= abc . (3.14) (3.14) These expressions were obtained by writing a polynomial ansatz in a, b, c and matching this with the boundary one-loop-square cases when one of the indices is zero. Furthermore to ﬁx the remaining ambiguity, we matched the ansatz with explicitly calculated oﬀ-shell ranks. Leftover rank ‘data’ was then used as a nontrivial cross-check of eqs. (3.13). These expressions were obtained by writing a polynomial ansatz in a, b, c and matching this with the boundary one-loop-square cases when one of the indices is zero. Furthermore to ﬁx the remaining ambiguity, we matched the ansatz with explicitly calculated oﬀ-shell ranks. Leftover rank ‘data’ was then used as a nontrivial cross-check of eqs. (3.13). As written, it is not clear that the ranks eqs. (3.13) are integer, but it turns out that they indeed are. It would be interesting to ﬁnd a group-theoretic interpretation of these formulae analogous to the interpretation of the one-loop ranks as the dimensions of symmetric traceless tensors of sod+2. 3.2.2 Organizing loop-dependent numerators by contact topologies Having counted the dimensionality of the relevant numerator spaces, it is straightforward to write down an arbitrary representative basis that ﬁlls up the full rank space. In the – 27 – following, however, we will argue that there is again a natural organization of the numer- ators in terms of contact-terms and top-level degrees of freedom analogous to eqs. (2.29) and (2.35): following, however, we will argue that there is again a natural organization of the numer- ators in terms of contact-terms and top-level degrees of freedom analogous to eqs. (2.29) and (2.35): f p d (a, b, c):= rank h Np Γ[a,b,c] i =: bf p d (a, b, c) \| {z } top rank + X (i,j,k)>⃗0 a i ! b j ! c k ! bf p d (a −i, b −j, c −k) \| {z } contact-term rank . (3.15) (3.15) {z contact-term rank JHEP11(2020)116 This is very similar to the case at one loop. And as with one loop, this formula requires boundary data to solve — namely, which integrands are given numerators ‘1’. As we will see, this boundary data is provided by our deﬁnition of ‘scalar’ p-gon power-counting integrands discussed in the following subsection. The recursive rank formula (3.15) can be interpreted directly as giving us a rule for constructing the corresponding vector-spaces of numerators Np Γ[a,b,c] =: bNp Γ[a,b,c] \| {z } top-level numerators (3.16) M (i,j,k)>⃗0 (ℓA\|Qa1) · · · (ℓA\|Qai) (ℓB\|Qb1) · · · (ℓB\|Qbj) (ℓC\|Sc1) · · · (ℓC\|Sck) bNp Γ[a−i,b−j,c−k] \| {z } contact-term numerators Np Γ[a,b,c] =: bNp Γ[a,b,c] \| {z } top-level numerators (3.16) Np Γ[a,b,c] =: bNp Γ[a,b,c] \| {z } top-level numerators (3.16) M (i,j,k)>⃗0 (ℓA\|Qa1) · · · (ℓA\|Qai) (ℓB\|Qb1) · · · (ℓB\|Qbj) (ℓC\|Sc1) · · · (ℓC\|Sck) bNp Γ[a−i,b−j,c−k] \| {z } contact-term numerators (3.16) {z contact-term numerators This makes it clear that (3.15) requires that the vector-spaces appearing in (3.16) are all mutually independent. This will be true whenever p<d. This makes it clear that (3.15) requires that the vector-spaces appearing in (3.16) are all mutually independent. This will be true whenever p<d. Γ[0,0,0] ⇔• with N0 Γ[0,0,0] := 1 3.2.2 Organizing loop-dependent numerators by contact topologies Next we consider the tadpole×tadpole graph whose numerator is given by the two one-loop numerator factors JHEP11(2020)116 Γ[1,0,1] ⇔ with N0 Γ[1,0,1] := [ℓA]1[ℓC]1 (3.19) (3.19) In d=4, this numerator has f0 4(1, 0, 1)=36 total degrees of freedom that can be decomposed into 36 = 25 +2×5+1: 25 top-level degrees of freedom together with 11 contact-terms that we have identiﬁed with degrees of freedom for its daughters’ Γ[1,0,0] and Γ[0,0,0]. With these arguably trivial one-loop type examples in hand, we can now discuss an honest irreducible two-loop graph Γ[1,1,1] ⇔ with N0 Γ[1,1,1] := [ℓA]1[ℓB]1[ℓC]1 . (3.20) (3.20) In four dimensions, Γ[1,1,1] has f0 4(1, 1, 1)=181 total degrees of freedom that are decomposable into 3 × 25 degrees of freedom of Γ[1,0,1] topologies, 3 × 5 Γ[1,0,0] and 1 degree of freedom obtained by pinching all three propagators leading to a single Γ[0,0,0]. Adding up all the contact degrees of freedom constitutes 91 of the 181 degrees of freedom in Γ[1,1,1], leaving 90 top-level degrees of freedom for the sunrise integral: In four dimensions, Γ[1,1,1] has f0 4(1, 1, 1)=181 total degrees of freedom that are decomposable into 3 × 25 degrees of freedom of Γ[1,0,1] topologies, 3 × 5 Γ[1,0,0] and 1 degree of freedom obtained by pinching all three propagators leading to a single Γ[0,0,0]. Adding up all the contact degrees of freedom constitutes 91 of the 181 degrees of freedom in Γ[1,1,1], leaving 90 top-level degrees of freedom for the sunrise integral: f0 4(1, 1, 1) = rank d=4 h N0 Γ[1,1,1] i = rank n [ℓA]1 4[ℓB]1 4[ℓC]1 4 o = 181 = 90+91 . (3.21) (3.21) As mentioned above, the same number of top-level degrees of freedom can alternatively be obtained by evaluating the numerators N[1,1,1] on the triple-cut surface ℓ2 A = ℓ2 B = ℓ2 C = 0 and checking the remaining matrix rank. Proceeding in a similar fashion, we can explicitly stratify the bases of integrands for all other two-loop topologies. As one might guess from our iterative description, this algorithm is extremely suitable for automation in available computer-algebra systems to allow for an eﬃcient rank counting. We will not give the complete answer to the counting problem here, but defer a detailed presentation of our results to table 3, which also includes similar results for diﬀerent degrees of power-counting in various dimensions. 3.2.2 Organizing loop-dependent numerators by contact topologies In order to stratify the relevant loop-dependent numerators for all two-loop integrand topologies, we propose a bottom-up strategy: ﬁrst identify graphs with ‘scalar’ numerators — those with the fewest propagators for a given p — and work our way upwards. In doing so, we have full control over the relevant numerator spaces of all contact-terms of more complicated graphs. Together with our general counting formulae in eq. (3.13) it is then easy to compute the top-level degrees of freedom of a given graph. Alternatively, one can also compute the top-level rank of a given numerator by evaluating the span of numerators on the maximal cut [83] surface of a given topology. In d ≤4, we have pursued both strategies and independently conﬁrmed the various numerator ranks, which also serves as a nontrivial cross-check of our closed form expressions (3.13). After these general considerations, it is perhaps instructive to return to 0-gon power- counting and demonstrate how integrand decomposition works for a few concrete examples. Recall that for our deﬁnition of 0-gon power-counting, the basic integrand topology has no propagator and is solely given by a loop-momentum independent normalization. (Of course, in dimensional regularization all these topologies correspond to power-divergent integrals that integrate to zero and therefore are usually not considered. However, for building bases of integrands, these topologies are relevant.) Therefore, we assign Γ[0,0,0] ⇔• with N0 Γ[0,0,0] := 1 (3.17) (3.17) – 28 – a single degree of freedom to this topology. Going up in the number of propagators, the next topology to consider is a tadpole, where one loop is completely pinched and the other loop has a single propagator. In this case, we write down a single numerator factor a single degree of freedom to this topology. Going up in the number of propagators, the next topology to consider is a tadpole, where one loop is completely pinched and the other loop has a single propagator. In this case, we write down a single numerator factor Γ[1,0,0] ⇔ with N0 Γ[1,0,0] := [ℓA]1 . (3.18) (3.18) Specifying to d = 4 for concreteness, we have f0 4(1, 0, 0) = 6. Γ[1,0,0] is of course a one-loop graph and we know how to decompose its 6 total numerator degrees of freedom: 6 = 5+1 represents 5 top-level degrees of freedom and 1 contact term. 3.2.2 Organizing loop-dependent numerators by contact topologies Like in our one-loop discussion, where we had found that all pentagon integrands (and integrands with more propagators) become reducible in d=4 beyond 4-gon power-counting, we ﬁnd that the basis of integrands of two-loop topologies is completely spanned by contact- terms beyond some number of propagators for any given power-counting. Whenever this – 29 – happens, we deem such a topology reducible and we do not discuss it further. In particular, for the 0-gon power-counting under consideration in this section, this appears in d=4 for integrands with more than 8 propagators or whenever a single loop-momentum a, b, or c appears in more than 4 propagators. More generally, we ﬁnd that (for p < d-gon power- counting), all graphs with more than d×L propagators are reducible. (Notice that the number of propagators at two loops is simply a+b+c.) There is one notable exception to this rule involving propagator-renormalization graphs that we discuss separately in 3.4.1. 11Recall that a planar graph is one which admits a plane embedding. A plane graph is one endowed with a particular embedding. If a graph is planar, its plane embedding is unique provided the graph’s edge- connectivity is 3 or greater. The easiest example of a planar graph which admits multiple plane embeddings is a (2-edge-connected) graph which includes as a sub-diagram a loop-correction to a propagator. 3.3 Deﬁning ‘p-gon power-counting’ at two loops JHEP11(2020)116 As with one loop, we will start our analysis of two-loop integrands without any restriction to the dimension of spacetime. One striking diﬀerence between one and two (or more) loops is that it will no longer be so obvious to describe the ‘power-counting’ of an integrand beyond p = 0: due to the lack of a natural origin in loop momentum space, the naïve deﬁnition given for one loop is no longer suﬃcient. Consider, for example, the two-loop graph . (3.22) (3.22) For this integrand, how many propagators involve the loop momenta ℓ1, ℓ2? Possible an- swers include {4, 3}, {5, 4}, or {5, 3}, as can be seen by re-drawing the graph in three ways: or . (3.23) (3.23) This problem seems artiﬁcial in the planar case, if only because there is always a (seem- ingly) natural prescription for how to route the loop momenta of a planar graph — namely, according to the faces of the plane11 graph. To be clear: we deﬁne the routing of loop mo- menta of an L loop diagram to be a choice of L simple cycles whose union encompasses all edges of the graph. When we consider diagrams that are not planar, we are forced to reckon with the fact that there is no intrinsic (or even obviously preferential) choice of routing. We are using the example (3.22) in order to emphasize that this problem aﬀects planar diagrams as well. Minimally, this indicates that more care is required to discuss the ‘power-counting’ of an integrand. Another example which will help illustrate our point would be the following: ↔ . (3.24) ↔ . (3.24) (3.24) 11Recall that a planar graph is one which admits a plane embedding. A plane graph is one endowed with a particular embedding. If a graph is planar, its plane embedding is unique provided the graph’s edge- connectivity is 3 or greater. The easiest example of a planar graph which admits multiple plane embeddings is a (2-edge-connected) graph which includes as a sub-diagram a loop-correction to a propagator. – 30 – While this example could be interpreted as four propagators involving ℓ1 and four propa- gators involving ℓ2, no one would overlook the fact that this integral contains a bubble! As such, it would seem absurd to assign this integral a power-counting of two box-integrals. 3.3 Deﬁning ‘p-gon power-counting’ at two loops One solution to this problem (especially to combat the example in (3.24)) would be to declare that any routing must consist of cycles of ‘minimal’ length — by some metric of ordering on cycle sets. This feels like a dangerous approach to us, as at asymptotically large loop order, there would seem to be no natural way to ordering the ‘minimality’ of large collections of loops (and very little reason to believe that, choosing some way to order choices for routing, that degeneracies of choice would behave similarly). Thus, we are forced to face the problem that there would seem to be no intrinsically obvious way to assign ‘power-counting’ to a multi-loop (especially non-planar) Feynman integrand. However, in the following, we will put forward one suggestion of an intrinsically graph-theoretic way to deﬁne power-counting. JHEP11(2020)116 3.3.1 ‘Scalar’ p-gon integrands at two loops Our basic strategy for deﬁning integrands p-gon power-counting follows from the recursive deﬁnition of how integrand numerators get stratiﬁed by contact-terms according to (3.16). That is, we will deﬁne a vector-space of numerators for a given graph relative to its contact- terms. For example, suppose that we wanted to construct a space of integrands that all behave exactly like the ‘scalar’ integrand . (3.25) (3.25) Adding any new propagators together with each new propagator’s vector-space of inverse propagators in the numerator will clearly result in an integrand that behaves like (3.25). Graphically, integrands that ‘scale like Γ[2,0,2] (at inﬁnity)’ would include      , , , , , . . .      . (3.26) (3.26) It is easy to see that this deﬁnes numerators for all integrands that contain Γ[2,0,2] as a contact-term. This may or may not be a useful vector-space of integrands to deﬁne, as there may be no interesting quantum ﬁeld theories whose amplitudes are expressible in this space. It is easy to see that this deﬁnes numerators for all integrands that contain Γ[2,0,2] as a contact-term. This may or may not be a useful vector-space of integrands to deﬁne, as there may be no interesting quantum ﬁeld theories whose amplitudes are expressible in this space. This rule can easily be generalized to construct numerators for integrands that scale like one or more of a list of ‘scalar’ integrands (at inﬁnity): we simply add the vector- spaces of loop-dependent monomials assigned to each integrand as dictated by each of a given set of the scalars (which are obtainable as edge contractions relative to the graph) from a given list. Thus, we may take any subset of graphs to be given ‘scalar’ numerators ‘1’, and thereby deﬁne an inﬁnite set of integrands with more propagators which scale like one or more of the graphs from this list. The only missing ingredient is to deﬁne the space of integrands to be taken to be scalar. It should also be clear that this rule generalizes to arbitrary loop order. – 31 – Our proposal for integrands assigned scalar numerators for p-gon power-counting is as follows. Deﬁnition. A scalar p-gon is any integrand having girth p, such that all its daughters — graphs obtained by single edge-contractions — have girth <p. 3.3.1 ‘Scalar’ p-gon integrands at two loops We denote the space of p-gon power-counting scalars at L loops by SL p . Recall that the girth of a graph is the length of its shortest cycle. This deﬁnition clearly generalizes to any loop-order. At two loops, it is quite easy to list the scalar p-gons for any p. For example, JHEP11(2020)116 1-gon power-counting scalars S2 1:=           2-gon power-counting scalars S2 2:=      ,      3-gon power-counting scalars S2 3:=      ,      4-gon power-counting scalars S2 4:=      , ,      5-gon power-counting scalars S2 5:=      , ,      (3.27) (3.27) and so on. Assigning ‘scalar’ numerators ‘1’ to each of these integrands allows us to deﬁne spaces of integrands that behave asymptotically like one or more of these. and so on. Assigning ‘scalar’ numerators ‘1’ to each of these integrands allows us to deﬁne spaces of integrands that behave asymptotically like one or more of these. It is clear that this deﬁnition is purely graph theoretic, and provides us with a precise rule for assigning vector-spaces of numerators to every graph that contains one or more of the scalar graphs as contact-terms. Moreover, it is clear that these vector-spaces are deﬁned without respect to the dimension of spacetime (although the sizes of these vector- spaces, and the degree to which these spaces are spanned by contact-terms will, of course, depend strongly on the dimension of spacetime). 3.3.2 Two-loop numerators relative to scalar p-gon integrands Given this deﬁnition of scalar p-gon integrands, we may follow the rule described above to construct the vector-space of p-gon power-counting integrands — those integrands which are constructed explicitly to scale like one or more of the scalar p-gons at inﬁnite loop momentum. That is, we assign any graph that is a parent of a scalar graph, Γ≻Σ∈SL p , a numerator consisting of the inverse propagators associated with the edge-set in the quotient of Γ relative to Σ; if there are multiple Σ∈SL p , we add the vector-spaces of loop-dependent numerators for each. Let us illustrate this rule with a number of concrete examples. – 32 – Consider the graph Γ[2,1,2]; what numerator would be assigned to this Feynman inte- grand for various power-countings? Because Γ[2,1,2] ∈S2 3, it would be assigned the numerator ‘1’ for 3-gon power-counting. For 2-gon (or ‘bubble’) power-counting, it is easy to see that Γ[2,1,2] ≻Γ[2,0,2] ∈S2 2 and also Γ[2,1,2] ≻Γ[1,1,1] ∈S2 2. Thus, N2 Γ[2,1,2] = span ( ⊕ = span [ℓB] ⊕ \| {z } ≺ [ℓA][ℓC] . \| {z } ≺ ) (3.28) N2 Γ[2,1,2] = span ( ⊕ = span [ℓB] ⊕ \| {z } ≺ [ℓA][ℓC] . \| {z } ≺ ) (3.28) (3.28) JHEP11(2020)116 Now, the total rank of this space varies with dimension, as does the breakdown of this vector-space into top-level degrees of freedom and contact-terms. Speciﬁcally, we ﬁnd Now, the total rank of this space varies with dimension, as does the breakdown of this vector-space into top-level degrees of freedom and contact-terms. Speciﬁcally, we ﬁnd in d=2 : rank [ℓB]2⊕[ℓA]2[ℓC]2 = 16 = 3+13 ; in d=3 : rank [ℓB]3⊕[ℓA]3[ℓC]3 = 25 = 8+17 ; in d=4 : rank [ℓB]4⊕[ℓA]4[ℓC]4 = 36 = 15+21 ; (3.29) (3.29) and so-on. To be clear, the breakdown of N2 Γ[2,1,2] into top-level degrees of freedom and contact-terms in any number of dimensions follows (recursively) via the deﬁnitions (3.15) and (3.16). and so-on. To be clear, the breakdown of N2 Γ[2,1,2] into top-level degrees of freedom and contact-terms in any number of dimensions follows (recursively) via the deﬁnitions (3.15) and (3.16). For 1-gon power-counting, the numerators assigned to Γ[2,1,2] would be given as the sum of three monomials: N1 Γ[2,1,2] = span ( ⊕ = span [ℓA][ℓB][ℓC]⊕ ⊕ [ℓA]2[ℓC] ⊕ [ℓA][ℓC]2 . 3.3.2 Two-loop numerators relative to scalar p-gon integrands \| {z } ≺ ) (3.30) (3.30) As before, we can easily decompose the ranks (and breakdowns) of these vector-spaces in various dimensions: As before, we can easily decompose the ranks (and breakdowns) of these vector-spaces in various dimensions: in d=2 : rank [ℓA]2[ℓB]2[ℓC]2⊕[ℓA]2 2[ℓC]1 2⊕[ℓA]1 2[ℓC]2 2 = 63 = 0+63 ; in d=3 : rank [ℓA]3[ℓB]3[ℓC]3⊕[ℓA]2 3[ℓC]1 3⊕[ℓA]1 3[ℓC]2 3 = 131 = 16+115 ; in d=4 : rank [ℓA]4[ℓB]4[ℓC]4⊕[ℓA]2 4[ℓC]1 4⊕[ℓA]1 4[ℓC]2 4 = 229 = 49+180 . (3.31) in d=2 : rank [ℓA]2[ℓB]2[ℓC]2⊕[ℓA]2 2[ℓC]1 2⊕[ℓA]1 2[ℓC]2 2 = 63 = 0+63 ; in d=3 : rank [ℓA]3[ℓB]3[ℓC]3⊕[ℓA]2 3[ℓC]1 3⊕[ℓA]1 3[ℓC]2 3 = 131 = 16+115 ; in d=4 : rank [ℓA]4[ℓB]4[ℓC]4⊕[ℓA]2 4[ℓC]1 4⊕[ℓA]1 4[ℓC]2 4 = 229 = 49+180 . (3.31) in d=2 : rank [ℓA]2[ℓB]2[ℓC]2⊕[ℓA]2 2[ℓC]1 2⊕[ℓA]1 2[ℓC]2 2 = 63 = 0+63 ; in d=3 : rank [ℓA]3[ℓB]3[ℓC]3⊕[ℓA]2 3[ℓC]1 3⊕[ℓA]1 3[ℓC]2 3 = 131 = 16+115 ; in d=4 : rank [ℓA]4[ℓB]4[ℓC]4⊕[ℓA]2 4[ℓC]1 4⊕[ℓA]1 4[ℓC]2 4 = 229 = 49+180 . (3.31) (3.31) – 33 – For one further illustration of how this works, consider the case of Γ[3,2,3]. For p-gon power-counting with p=0, . . . , 4, we would have integrands built according to B0 ⊃ ( ) B1 ⊃ ( ⊕ ⊕ ) B2 ⊃ ( ⊕ ⊕ ⊕ ) B3 ⊃ ( ⊕ ⊕ ⊕ ) B4 ⊃ ( ⊕ ) . (3.32) B0 ⊃ ( ) B1 ⊃ ( ⊕ ⊕ ) B2 ⊃ ( ⊕ ⊕ ⊕ ) B3 ⊃ ( ⊕ ⊕ ⊕ ) B4 ⊃ ( ⊕ ) . (3.32) JHEP11(2020)116 (3.32) These examples illustrate how for any power-counting p we may decorate all graphs that are parents of subsets of SL p with vector-spaces of loop-dependent numerators. These examples illustrate how for any power-counting p we may decorate all graphs that are parents of subsets of SL p with vector-spaces of loop-dependent numerators. 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) In table 3 we have summarized the bases for non-product topologies relevant for d=2, 3, 4 dimensions for p-gon power-counting through p = 4. In table 3, we have written ‘[1]’, ‘[1−2]’, and ‘[2]’ for [ℓA], [ℓB], and [ℓC], respectively. (We do not list product topologies, as their numerator-spaces are entirely dictated by the breakdown of one-loop degrees of freedom.) As the reader will recall from one loop, the case of p = d is a non-typical case. Thus, table 3 does not include the complete list of topologies relevant for 4-gon power-counting in 4 dimensions. In addition to the product topologies — Γ[4,0,5] and Γ[5,0,5] — whose degrees of freedom (1 top-level degree of freedom for each) follow from one loop results, there are three additional integrand structures that need to be included to make the basis complete. Table 3. Two-loop integrands’ loop-dependent numerators Np for p-gon power-counting, and the breakdown of their ranks into top rank and contact-terms in various dimensions. 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) These integrands are g N4: [ℓA][ℓC]⊕[ℓB] [ℓA]2[ℓC]⊕[ℓA][ℓB] [ℓA][ℓB][ℓC]⊕[ℓA]2⊕[ℓB]2⊕[ℓC]2 ranks: d=4 36=3+33 120=2+118 181=1+180 (3.33) (In each of these examples it is interesting to notice that the total numerator space is spanned by the ﬁrst monomial: [ℓB]⊂[ℓA][ℓC], [ℓA][ℓB]⊂[ℓA]2[ℓC], [ℓA]2⊂[ℓA][ℓB][ℓC], and so-on.) N4: [ℓA][ℓC]⊕[ℓB] ranks: d=4 36=3+33 (3.33) (In each of these examples it is interesting to notice that the total numerator space is spanned by the ﬁrst monomial: [ℓB]⊂[ℓA][ℓC], [ℓA][ℓB]⊂[ℓA]2[ℓC], [ℓA]2⊂[ℓA][ℓB][ℓC], and so-on.) – 34 – – 34 – tensor numerator structure Np total numerator rank = top rank contact terms for p-gon power-counting d = 2 d = 3 d = 4 Γ[1,1,1] N2 1 1=1+0 1=1+0 1=1+0 N1 [1]⊕[2]⊕[1−2] 8=5+3 10=7+3 12=9+3 N0 [1][1−2][2] 49=12+37 103=42+61 181=90+91 Γ[2,1,1] N2 [1] 4=2+2 5=3+2 6=4+2 N1 [1]2⊕[1][2]⊕[1][1−2] 24=4+20 38=13+25 55=24+31 N0 [1]2[1−2][2] 100=4+96 263=37+226 552=127+425 Γ[3,1,1] N2 [1]2 9=0+9 14=2+12 20=5+15 N1 [1]3⊕[1]2[2]⊕[1]2[1−2] 48=0+48 95=6+89 164=24+140 N0 [1]3[1−2][2] 169=0+169 533=10+523 1305=79+1226 Γ[2,1,2] N3 1 1=1+0 1=1+0 1=1+0 N2 [1−2]⊕[1][2] 16=3+13 25=8+17 36=15+21 N1 [1]2[2]⊕[1][2]2⊕[1][1−2][2] 63=0+63 131=16+115 229=49+180 N0 [1]2[1−2][2]2 196=0+196 644=24+620 1612=149+1463 Γ[3,1,2] N3 [1] 4=1+3 5=2+3 6=3+3 N2 [1]2[2]⊕[1][1−2] 36=0+36 70=4+66 120=17+103 N1 [1]3[2]⊕[1]2[2]2⊕[1]2[1−2][2] 120=0+120 312=4+308 650=39+611 N0 [1]3[1−2][2]2 324=0+324 1273=4+1269 3710=77+3633 Γ[2,2,2] N4 1 1=1+0 1=1+0 1=1+0 N3 [1]⊕[2]⊕[1−2] 8=2+6 10=4+6 12=6+6 N2 [1]2⊕[2]2⊕[1−2]2⊕[1][1−2][2] 49=0+49 103=8+95 181=32+149 N1 [1]2[1−2][2]⊕[1][1−2][2]2⊕[1][1−2]2[2] 143=0+143 391=8+383 822=60+762 N0 [1]2[1−2]2[2]2 361=0+361 1479=8+1471 4401=122+4279 Γ[4,1,1] N2 [1]3 16=0+16 30=0+30 50=2+48 N1 [1]4⊕[1]3[2]⊕[1]3[1−2] 80=0+80 191=0+191 385=8+377 N0 [1]4[1−2][2] 256=0+256 941=0+941 2636=18+2618 Γ[4,1,2] N3 [1]2 9=0+9 14=0+14 20=2+18 N2 [1]3[2]⊕[1]2[1−2] 64=0+64 150=0+150 300=6+294 N1 [1]4[2]⊕[1]3[2]2⊕[1]3[1−2][2] 195=0+195 609=0+609 1480=10+1470 N0 [1]4[1−2][2]2 484=0+484 2210=0+2210 7356=14+7342 Γ[3,1,3] N4 1 1=1+0 1=1+0 1=1+0 N3 [1−2]⊕[1][2] 16=0+16 25=3+22 36=8+28 N2 [1]2[2]2⊕[1][1−2][2] 81=0+81 196=0+196 400=16+384 N1 [1]3[2]2⊕[1]2[2]3⊕[1]2[1−2][2]2 224=0+224 725=0+725 1796=24+1772 N0 [1]3[1−2][2]3 529=0+529 2480=0+2480 8400=32+8368 Γ[3,2,2] N4 [1] 4=1+3 5=2+3 6=3+3 N3 [1]2⊕[1][2]⊕[1][1−2] 24=0+24 38=3+35 55=10+45 N2 [1]3⊕[1][2]2⊕[1][1−2]2⊕[1]2[1−2][2] 100=0+100 263=0+263 552=22+530 N1 [1]3[1−2][2]⊕[1]2[1−2][2]2⊕[1]2[1−2]2[2] 255=0+255 865=0+865 2157=30+2127 N0 [1]3[1−2]2[2]2 576=0+576 2811=0+2811 9706=42+9664 Γ[4,1,3] N4 [1] 4=0+4 5=1+4 6=2+4 N3 [1]2[2]⊕[1][1−2] 36=0+36 70=0+70 120=4+116 N2 [1]3[2]2⊕[1]2[1−2][2] 144=0+144 420=0+420 1000=4+996 N1 [1]4[2]2⊕[1]3[2]3⊕[1]3[1−2][2]2 360=0+360 1394=0+1394 4020=4+4016 N0 [1]4[1−2][2]3 784=0+784 4264=0+4264 16470=4+16466 Γ[4,2,2] N4 [1]2 9=0+9 14=0+14 20=2+18 N3 [1]3⊕[1]2[2]⊕[1]2[1−2] 48=0+48 95=0+95 164=4+160 N2 [1]4⊕[1]2[2]2⊕[1]2[1−2]2⊕[1]3[1−2][2] 169=0+169 533=0+533 1305=4+1301 N1 [1]4[1−2][2]⊕[1]3[1−2][2]2⊕[1]3[1−2]2[2] 399=0+399 1613=0+1613 4676=4+4672 N0 [1]4[1−2]2[2]2 841=0+841 4750=0+4750 18676=4+18672 Γ[3,2,3] N4 [1−2]⊕[1][2] 16=0+16 25=2+23 36=7+29 N3 [1−2]2⊕[1]2[2]⊕[1][2]2⊕[1][1−2][2] 63=0+63 131=0+131 229=8+221 N2 [1]3[2]⊕[1][2]3⊕[1]2[1−2][2]2⊕[1][1−2]2[2] 196=0+196 644=0+644 1612=8+1604 N1 [1]3[1−2][2]2⊕[1]2[1−2][2]3⊕[1]2[1−2]2[2]2 440=0+440 1839=0+1839 5412=8+5404 N0 [1]3[1−2]2[2]3 900=0+900 5216=0+5216 20836=8+20828 Table 3. Two-loop integrands’ loop-dependent numerators Np for p-gon power-counting, and the JHEP11(2020)116 Table 3. 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) Two-loop integrands’ loop-dependent numerators Np for p-gon power-counting, and the breakdown of their ranks into top rank and contact-terms in various dimensions. Table 3. Two-loop integrands’ loop-dependent numerators Np for p-gon power-counting, and the breakdown of their ranks into top rank and contact-terms in various dimensions. able 3. Two-loop integrands’ loop-dependent numerators Np for p-gon power-counting, and the eakdown of their ranks into top rank and contact-terms in various dimensions. – 35 – These additional topologies required for 4-gon power-counting in 4 dimensions suﬀer from the same problem we saw for the 4-gon power-counting pentagons at one loop: they are topologically over-complete. By this we mean that when we include all graphs of these topologies we ﬁnd that these integrands satisfy non-trivial relations among themselves. It is interesting to note how this over-completeness manifests itself (and can be cleverly avoided) in the case of the planar integrands at two loops. The degrees of freedom associated with the product topologies Γ[4,0,4], Γ[4,0,5], and Γ[5,0,5] can all be re-cast as non-contact degrees of freedom attached to Γ[4,1,4]. This can easily be understood from the viewpoint of cuts: these topologies can all be seen as necessary to match the 4 chiral solutions to the kissing-box cuts, and it is clear that we can match all four using just Γ[4,1,4], if this integrand were assigned 4 top-level degrees of freedom. This is precisely what was done in refs. [100–102] at two loops: by excluding the product topologies from the basis, a topologically complete — and importantly, not over-complete — basis of planar, two-loop integrands with 4-gon power-counting was constructed (and used to represent all two-loop amplitude integrands of planar sYM). We do not know of any similarly clever choice of assigning non-planar degrees of freedom to two-loop integrands in (3.33). JHEP11(2020)116 One ﬁnal comment worth mentioning is that the breakdown of integrand numerators into top-level degrees of freedom and contact-terms depends on there being generic (and non-zero) momentum ﬂowing into every necessary external edge indicated (by a solid wedge attached to the vertex of a graph). When there are conspiracies amongst the momenta, or when some momenta vanish, some of the propagators can become doubled, and their corresponding degrees of freedom must change slightly. Dealing with this subtlety requires a small aside. Si opus sit: exceptional cases requiring modest reﬁnement. 12Applying unitarity-based ideas to determine the coeﬃcients of such integrands can be subtle, but some technology does exist to deal with this case; see for example refs. [103] or [104]. 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) In many renormal- ization schemes, amplitudes require integrands that involve loop-corrections to propagators — or otherwise integrands involving doubled propagators12 (see e.g. [99] for possible ex- ceptions). These integrands must be handled with some care, as our combinatorial rules discussed above relied upon an assumption of generality among the propagators of a given integrand. This rule is enforced by our conventions requiring that solid wedges of momenta ﬂowing into a graph are all generic and non-vanishing, while dashed wedges of external mo- menta can be taken to be zero. Thus, all the propagators in , (3.34) (3.34) for example, are distinct. This is true even if the momentum ﬂowing into the bottom, ‘optional’ external edge of the graph were taken to be zero. However, if the momentum ﬂowing into the top of the graph were to become zero as well as in (3.35) (3.35) 12Applying unitarity-based ideas to determine the coeﬃcients of such integrands can be subtle, but some technology does exist to deal with this case; see for example refs. [103] or [104]. – 36 – then, as a rational function of loop momentum, the integrand would be indistinguishable from then, as a rational function of loop momentum, the integrand would be indistinguishable from ∼ . (3.36) ∼ . (3.36) (3.36) This example may seem like a purely academic concern, but it aﬀects the breakdown of total numerator degrees of freedom into top-level and contact-terms — and has a knock-on eﬀect for many graphs that include (3.35) among their contact-terms. Moreover, this can render otherwise reducible integrands suddenly irreducible. This example may seem like a purely academic concern, but it aﬀects the breakdown of total numerator degrees of freedom into top-level and contact-terms — and has a knock-on eﬀect for many graphs that include (3.35) among their contact-terms. Moreover, this can render otherwise reducible integrands suddenly irreducible. Consider for example case of bubble (2-gon) power-counting in d=4. In the generic case of Γ[2,1,1] shown in (3.34), it is easy to see that its 6 total numerator degrees of freedom from [ℓA]4 decompose into 4 top-level degrees of freedom and 2 contact-terms. In the degenerate case, there is only a single contact term, leaving us now with 5 top-level numerators. JHEP11(2020)116 Consider now the degenerate case of Γ[3,1,1] — also for bubble power-counting in d=4. It would be assigned a numerator of [ℓA]2 4 with 20 total degrees of freedom. 3.4.1 Exempli gratia: p-gon power-counting bases in d-dimensions (d≤4) In this case, some of its contact-terms would be the non-degenerate (3.34), while others would be the degenerate case. A simple exercise shows that rank d=4 " N2 !# = 20 = 9+11 , (3.37) (3.37) instead of the usual breakdown of 20 = 5+15 of the generic case of Γ[3,1,1]. Continuing in this manner, we would discover that instead of the usual breakdown of 20 = 5+15 of the generic case of Γ[3,1,1]. Continuing in this manner, we would discover that rank d=4 " N2 !# = 105 = 0+105 while rank d=4 " N2 !# = 105 = 2+103 . 4 Building bases of integrands at three loops Following such detailed one- and two-loop discussions above, we will be more telegraphic in our description of our main results at three loops. From what we have seen previously, it is fairly straightforward to generate a basis of integrands at higher loops guaranteed to be big enough for quantum ﬁeld theories such as the Standard Model in any ﬁxed number of dimensions. Amplitudes in such (renormalizable) theories can be represented in a basis with 0-gon power-counting, as this basis will include literally every Feynman diagram. The much harder — and more subtle (and interesting) — problem is how to con- struct and organize bases of integrands with better-than-0-gon power-counting such that the amplitudes of interesting quantum ﬁeld theories can be represented. Take for exam- ple maximally supersymmetric Yang-Mills theory (sYM). Amplitudes in this theory are widely expected to be representable in terms of integrands with ‘box power-counting’ in 4 dimensions; but until now, there has been no precise deﬁnition of such an integrand basis beyond the planar limit. In this section, we describe the basic ingredients required to describe integrand bases at three loops, and summarize what is found in the case of 3-gon power-counting in 4 – 37 – dimensions. This particular case is interesting because this basis should span all max- imally transcendental (another poorly deﬁned notion) functions — as integrands with bubble power-counting are expected to universally have less than maximal transcenden- tal weights.13 Thus, the basis we describe here should suﬃce to represent all scattering amplitudes in sYM beyond the planar limit. 4.1.1 Loop-dependent denominators: ladders and wheels At three loops, the Feynman propagator structures of all integrands can be classiﬁed as ‘wheel’ or ‘ladder’ type topologies. Speciﬁcally, let us deﬁne JHEP11(2020)116 W (a1,a2,a3) (b1,b2,b3)⇔ , L (a1,a2)(b1,b2) (c1,c2) ⇔ . (4.1) )⇔ . (4.1) (4.1) The color-coding above is merely to help guide the eye toward notational meaning and conventions. To be clear, these two classes overlap for degenerate conﬁgurations. To disambiguate such cases, we conventionally require that none of the indices of a wheel integral vanish. For example, one can easily see that W (a,b,c) (d,e,0) ≃L (a,e)(d,b) (0,c) is a graph isomorphism. Because of this, we choose to identify all degenerate wheels as instances of (degenerate) ladders. The color-coding above is merely to help guide the eye toward notational meaning and conventions. To be clear, these two classes overlap for degenerate conﬁgurations. To disambiguate such cases, we conventionally require that none of the indices of a wheel integral vanish. For example, one can easily see that W (a,b,c) (d,e,0) ≃L (a,e)(d,b) (0,c) is a graph isomorphism. Because of this, we choose to identify all degenerate wheels as instances of (degenerate) ladders. In addition to the overlap in name-space for the degenerate wheels and ladders, there are additional redundancies among the labels associated with standard graph isomorphisms. These are the analogues of the permutation-invariance among the indices {a, b, c} labeling the two-loop graphs Γ[a,b,c]. When no indices vanish, the wheel integrands enjoy 24 sym- metric relabelings, and the ladders enjoy 16 relabelings corresponding to the sizes of the automorphism groups of the graphs drawn in (4.1), respectively. (There are more symme- tries for certain degenerate conﬁgurations; for example: L (a,b)(c,d) (0,0) is permutation-invariant in its four non-zero labels.) As before, we could in principle solve the momentum conserva- tion constraints and write all topologies in terms of three independent loop momenta, e.g. ℓ1 ℓ2 ℓ3 ℓ2−ℓ1 ℓ1−ℓ3 ℓ3−ℓ2 and ℓ1 ℓ2 ℓ3 ℓ1−ℓ2 ℓ3−ℓ2 . (4.2) 13Providing a precise deﬁnition of transcendental weight is complicated by the need for non- polylogarithmic integrands in general. See e.g. [105–109]. (4.2) 13Providing a precise deﬁnition of transcendental weight is complicated by the need for non- polylogarithmic integrands in general. See e.g. [105–109]. 4.1.2 Loop-dependent numerators: open problems By dressing each of the propagators of each type (4.1) with the corresponding space of generalized inverse-propagators, and specifying the dimension of spacetime, one may con- struct a complete basis of loop integrands suﬃcient to reproduce scattering amplitudes (to arbitrary multiplicity) in many theories. The total ranks of these spaces of numerators, however, grow very large; and we have not found a closed formula for them as we did at two loops (for dimensions less than ﬁve) in eq. (3.13). Analogous three-loop formulae would involve six indices and could be written JHEP11(2020)116 w0 d(a1, . . . , b3) = rank [ℓ1]a1[ℓ2]a2[ℓ3]a3[ℓ3−ℓ2]b1[ℓ1−ℓ3]b2[ℓ2−ℓ1]b3 , l0 d(a1, . . . , c2) = rank [ℓ1]a1[ℓ1−ℓ2]a2[ℓ2]c1+c2 [ℓ3]b1[ℓ3−ℓ2]b2 . (4.3) Again, it would be desirable to ﬁnd a group-theoretic expression for the relevant numerator ranks similar to the simple one loop expression in (2 16) w0 d(a1, . . . , b3) = rank [ℓ1]a1[ℓ2]a2[ℓ3]a3[ℓ3−ℓ2]b1[ℓ1−ℓ3]b2[ℓ2−ℓ1]b3 , l0 d(a1, . . . , c2) = rank [ℓ1]a1[ℓ1−ℓ2]a2[ℓ2]c1+c2 [ℓ3]b1[ℓ3−ℓ2]b2 . (4.3) (4.3) Again, it would be desirable to ﬁnd a group-theoretic expression for the relevant numerator ranks similar to the simple one-loop expression in (2.16). 4.1.1 Loop-dependent denominators: ladders and wheels – 38 – Often, we will only make use of graph theoretic notions of the relevant numerator spaces; however, when we actually compute the ranks of various numerator spaces, we do solve momentum conservation explicitly as indicated above. 4.2 The three-loop triangle power-counting basis for four dimensions Despite lacking a general rank count at three loops, however, it seems like a good idea to tackle this general problem in stages, starting with an integrand basis suitable for a theory with ‘good’ power-counting, such as sYM. In four dimensions, the best-case would probably correspond to ‘box’ power-counting p=4. But as with lower loops, there are good reasons to consider instead the space of integrands with next-to-optimal power-counting. In four dimensions, this corresponds to triangles. Why should we be interested in three loop integrands with triangle power-counting? After all, we expect that the best quantum ﬁeld theories (in terms of ultraviolet behavior) should be expressible in terms of boxes. The answer is the same as at lower loops: insisting on integrands with box power-counting forces us to include topologies with more than 4L propagators, and such integrands generically satisfy relations that must be eliminated. In the best case scenario, these redundancies can be excluded by throwing out entire topological classes of integrands — as was (accidentally) the case for two loops in the planar limit. We suspect that such a strategy is doomed in general; but as with the concrete examples provided in refs. [79, 80], we suspect that nice integrand formulae exist for sYM beyond the planar limit even if we use a basis of integrands with next-to-optimal (namely, p=3) power-counting. Recall that our deﬁnition of p-gon power-counting (in any number dimensions, and any loop-order) starts with a deﬁnition of scalar integrands SL p . Recall that this consists of all vacuum graphs with girth p, such that all single-edge quotients have lower girth. – 39 – At three loops and 3-gon power-counting, the set of scalars S3 3 is given by             W (1,1,1) (1,1,1) , L (3,0)(0,3) (1,2) , L (3,0)(0,3) (0,3) , L (3,0)(1,2) (1,1) , L (3,0)(2,2) (0,1) , L (2,1)(1,2) (0,1) , L (2,1)(2,2) (0,0)              . (4.4) Notice that all but the ﬁrst, sixth, and seventh of these are product-topologies. As always, there are multiple ways to label each of these graphs; the labeling we have chosen should be viewed as representative. H d ﬁ d b l 3 l d Notice that all but the ﬁrst, sixth, and seventh of these are product-topologies. 4.2 The three-loop triangle power-counting basis for four dimensions As always, there are multiple ways to label each of these graphs; the labeling we have chosen should be viewed as representative. Notice that all but the ﬁrst, sixth, and seventh of these are product-topologies. As always, there are multiple ways to label each of these graphs; the labeling we have chosen should be viewed as representative. JHEP11(2020)116 Having deﬁned our basic scalar 3-gon power-counting topologies, we proceed to con- struct the numerator spaces for integrands with more propagators. The basic setup is almost identical to our more detailed two-loop discussion, which is why we are going to be relatively brief here. We need not dwell on the numerator decomposition of any product topologies, as their decomposition will follow trivially from our one- and two-loop discus- sions above. As before, we ﬁnd that all (generic) integrands with more than 12 = 3×4 propagators are entirely decomposable; the ranks for these numerator spaces quoted below were obtained mostly from direct construction. Although the construction of p-gon power-counting numerator spaces follows directly from our discussion at two loops, it may be helpful to illustrate the non-triviality of this construction with a couple examples. Consider the ladder and wheel integrands, L (3,1)(2,2) (1,2) = , W (3,2,1) (2,1,1) = . (4.5) (4.5) For each of these topologies, the numerator space is deﬁned as the product of translated inverse propagators for all sets of edges that, upon their collapse, would lead to an element of S3 3 in (4.4). From these spaces, the total rank may be computed (by brute force) in any number of dimensions, and the breakdown into top-level degrees of freedom and contact-terms follows recursively by analogy with (3.15) at two loops. For each of these topologies, the numerator space is deﬁned as the product of translated inverse propagators for all sets of edges that, upon their collapse, would lead to an element of S3 3 in (4.4). From these spaces, the total rank may be computed (by brute force) in any number of dimensions, and the breakdown into top-level degrees of freedom and contact-terms follows recursively by analogy with (3.15) at two loops. 4.2 The three-loop triangle power-counting basis for four dimensions For the ladder example in (4.5), we would ﬁnd the total numerator vector-space to be given by [1−2][2][2−3] ⊕[1−2][2][3] ⊕ \| {z } ≺ [1−2][2][2−3] ⊕[1−2][2][3] ⊕ [1−2][2]2 ⊕ \| {z } ≺ [1][2]2[2−3] ⊕ [1][2]2[3] ⊕ \| {z } ≺ [1][2]3 \| {z } ≺ resulting in a loop-dependent numerator of N3 L (3,1)(2,2) (1,2) := [1−2][2][2−3]⊕[1−2][2][3]⊕[1−2][2]2⊕[1][2]2[2−3]⊕[1][2]2[3]⊕[1][2]3. (4.6) – 40 – This vector-space is the same in any number of dimensions, but its size and breakdown into contact-terms depends strongly on d. In four dimensions, it can readily be conﬁrmed that This vector-space is the same in any number of dimensions, but its size and breakdown into contact-terms depends strongly on d. In four dimensions, it can readily be conﬁrmed that N3 L (3,1)(2,2) (1,2) = rank d=4 [1−2][2][2−3]⊕[1−2][2][3]⊕[1−2][2]2⊕[1][2]2[2−3]⊕[1][2]2[3]⊕[1][2]3 = 984 = 32+952 . (4.7) (4.7) Thus, even though the total rank of the numerator space is 984, the number of degrees of freedom that are honestly associated to the L (3,1)(2,2) (1,2) topology is 32 and therefore rela- tively small. Thus, even though the total rank of the numerator space is 984, the number of degrees of freedom that are honestly associated to the L (3,1)(2,2) (1,2) topology is 32 and therefore rela- tively small. For the wheel example in (4.5), we would ﬁnd its numerator constructed according to the scalar contact-term topologies, JHEP11(2020)116 [1]2[2][2−3] ⊕ \| {z } ≺ [1−2][1−3] ⊕ \| {z } ≺ [1][1−2][2−3]⊕[1][2][1−3] ⊕[1][2][1−2] ⊕[1][2][3] ⊕ [1][2]2 ⊕ [1]2[3] ⊕ \| {z } ≺ [1][3][1−2] \| {z } ≺ [1]2[2][2−3] ⊕ \| {z } ≺ [1−2][1−3] ⊕ \| {z } ≺ [1][1−2][2−3]⊕[1][2][1−3] ⊕[1][2][1−2] ⊕[1][2][3] ⊕ [1][2]2 ⊕ [1]2[3] ⊕ \| {z } ≺ [1][3][1−2] \| {z } ≺ resulting in a loop-dependent vector-space of numerators given by resulting in a loop-dependent vector-space of numerators given by N3 W (3,2,1) (2,1,1) := [1]2[2][2−3] ⊕[1−2][1−3] ⊕[1][1−2][2−3] ⊕[1][2][1−3] ⊕[1][2][1−2] ⊕[1][2][3] ⊕[1][2]2 ⊕[1]2[3] ⊕[1][3][1−2]. (4.8) (4.8) As always, this vector-space for 3-gon power-counting is the same for all spacetime dimen- sions. In d=4, As always, this vector-space for 3-gon power-counting is the same for all spacetime dimen- sions. In d=4, N3 W (3,2,1) (2,1,1) = rank d=4 [1]2[2][2−3] ⊕[1−2][1−3] ⊕[1][1−2][2−3] ⊕[1][2][1−3] ⊕[1][2][1−2] ⊕[1][2][3] ⊕[1][2]2 ⊕[1]2[3] ⊕[1][3][1−2] = 864 = 64+800 . 4.2 The three-loop triangle power-counting basis for four dimensions (4.9) (4.9) As with the ladder example, we ﬁnd that, although the total numerator space is quite large, the number of top-level degrees of freedom is comparatively small. For both of these examples — and all the other topologies relevant to triangle power- counting in four dimensions at three loops — these ranks were computed in two diﬀerent ways. First, we computed the total rank by literally constructing the vector-spaces deﬁned as in (4.6) and (4.8), for example, and determining the rank by brute force and determining the break-down into contact-terms according to the recursive deﬁnition analogous to (3.15) at two loops. Secondly, we determined the top-level degrees of freedom of each topology by constructing the total vector-spaces and determining the rank spanned on the maximal cut of the graph; and we used this data for all subtopologies of each graph to (recursively) infer the total rank of the space of numerators. That these two strategies produced the same rank counts gives us conﬁdence in their correctness. – 41 – Following this procedure, we were able to construct numerator spaces and their break- downs into top-level degrees of freedom an contact-terms for all graphs with as many as 12(=d×L) propagators at three loops. We also veriﬁed that all graphs with more than 12 propagators are entirely decomposable into contact-terms. The results of our analysis are summarized in tables 4, 5, and 6 and also attached to this paper as Mathematica note- book in the supplementary material. Similarly to our two-loop discussion in section 3.4.1 there can be exceptional cases of highly degenerate leg ranges where additional topologies are required (that we do not list explicitly here). As mentioned above, this basis of integrands should be suﬃcient to match the four- dimensional integrand of all-multiplicity scattering amplitudes in maximally supersym- metric (N =4) sYM at three loops beyond the planar limit. As discussed in the following section, the brute-force construction of these vector-spaces is still quite far from providing us with a ‘nice’ set of loop-integrand numerators suitable for the eﬃcient representation of scattering amplitudes. Below, we describe the features desirable in a nice choice of basis elements — which goes well beyond the scope of our present analysis. JHEP11(2020)116 5.1 The importance of choosing bases wisely The primary objective of this paper has been the enumeration and stratiﬁcation of integrand bases from purely graph-theoretic considerations. Speciﬁcally, we have shown how one can determine (by direct construction) the number of independent integrands for all relevant topologies necessary to express large classes of scattering amplitudes at one, two, and three loops in various spacetime dimensions. However, to construct a particular representation of an amplitude requires a choice of basis and therefore one is required to select speciﬁc loop-momentum dependent numerators for each topology. Of course, we require those numerators to ﬁll up the full numerator rank associated to a given integrand topology for a given power-counting and spacetime dimension. Implementing this procedure in an eﬃcient (and elegant) way is an important open question with no obvious or unique answer. For the particular case of sYM at two loops in four spacetime dimensions, one arguably nice representation has been the subject of our related work [79, 80]. In order to give the reader another example of the substantial beneﬁts gained from our basis partitioning described in this work, we consider some simple examples of an explicit basis construction. Furthermore, we comment on some statements (though not completely general) regarding ‘desirable’ basis choices in this section. One strategy for basis construction is to follow the analysis done for three loops. Namely, construct arbitrary representatives of all basis elements, and use a computer alge- bra package (such as Mathematica) to construct the independent vector-spaces by brute force. This method is not far from what was followed in traditional strategies of basis construction such as OPP [21, 44]; it does furnish us with a valid and complete basis of loop integrands in which scattering amplitudes can be expressed, and these can be further reﬁned according to certain criteria, see e.g. [52, 53]. 5.1 The importance of choosing bases wisely – 42 – JHEP11(2020)116 W (2,2,2) (2,2,2) 23471=52+23419 W (3,2,1) (3,2,1) 15534=40+15494 W (3,2,2) (2,1,2) 17254=36+17218 W (3,3,1) (2,2,1) 14703=32+14671 W (3,2,2) (3,1,1) 13230=32+13198 W (3,2,2) (1,2,2) 15589=28+15561 W (3,3,2) (1,2,1) 11873=24+11849 W (3,3,1) (1,3,1) 10045=24+10021 W (3,3,3) (1,1,1) 8000=16+7984 W (4,2,2) (2,1,1) 10290=16+10274 W (4,2,1) (2,2,1) 11486=16+11470 W (4,2,1) (3,1,1) 8670=16+8654 W (3,3,2) (1,1,2) 9959=16+9943 W (3,2,2) (1,1,3) 10492=16+10476 W (4,3,2) (1,1,1) 6000=8+5992 W (4,3,1) (1,2,1) 6730=8+6722 W (4,3,1) (2,1,1) 7146=8+7138 W (4,1,1) (4,1,1) 5060=8+5052 W (4,2,2) (1,1,2) 8104=8+8096 W (4,2,1) (2,1,2) 9046=8+9038 W (2,2,2) (1,2,2) 5645=118+5527 W (3,2,1) (2,2,1) 4589=92+4497 W (3,2,2) (2,1,1) 4008=80+3928 W (2,3,1) (1,3,1) 3229=68+3161 W (2,2,2) (1,1,3) 3696=60+3636 W (3,2,2) (1,1,2) 3459=56+3403 W (3,3,1) (1,2,1) 2759=44+2715 W (3,3,2) (1,1,1) 2400=40+2360 W (4,2,1) (2,1,1) 2264=20+2244 W (4,1,1) (3,1,1) 1784=20+1764 W (4,2,2) (1,1,1) 1800=16+1784 W (4,2,1) (1,2,1) 1929=16+1913 W (3,3,1) (1,1,2) 1612=8+1604 W (4,3,1) (1,1,1) 1000=4+996 W (4,2,1) (1,1,2) 1305=4+1301 W (2,2,1) (2,2,1) 1381=132+1249 W (2,2,2) (1,1,2) 1154=100+1054 W (3,2,1) (2,1,1) 864=64+800 W (3,2,1) (1,2,1) 783=54+729 W (3,2,2) (1,1,1) 720=52+668 W (3,1,1) (3,1,1) 640=46+594 W (3,2,1) (1,1,2) 552=22+530 W (3,3,1) (1,1,1) 400=16+384 W (4,1,1) (2,1,1) 434=12+422 W (4,2,1) (1,1,1) 300=6+294 W (2,2,1) (1,2,1) 241=57+184 W (2,2,2) (1,1,1) 216=52+164 W (2,2,1) (1,1,2) 181=32+149 W (3,1,1) (2,1,1) 161=28+133 W (3,2,1) (1,1,1) 120=17+103 W (4,1,1) (1,1,1) 50=2+48 W (2,1,1) (2,1,1) 43=19+24 W (2,2,1) (1,1,1) 36=15+21 W (3,1,1) (1,1,1) 20=5+15 W (2,1,1) (1,1,1) 6=4+2 Table 4. Three-loop wheel integrand topologies consistent with triangle power-counting, and the decomposition of their numerators into top-level and contact-term ranks. JHEP11(2020)116 Table 4. Three-loop wheel integrand topologies consistent with triangle power-counting, and the decomposition of their numerators into top-level and contact-term ranks. 5.1 The importance of choosing bases wisely – 43 – JHEP11(2020)116 L (3,2)(2,3) (1,1) 7951=32+7919 L (3,2)(1,3) (1,2) 3900=16+3884 L (3,2)(2,2) (1,2) 5146=16+5130 L (3,3)(1,3) (1,1) 4932=16+4916 L (3,3)(2,2) (1,1) 6835=16+6819 L (4,1)(2,3) (1,1) 4580=16+4564 L (2,2)(2,2) (1,3) 2921=8+2913 L (2,2)(2,2) (2,2) 2921=8+2913 L (3,1)(1,3) (1,3) 1800=8+1792 L (3,1)(1,3) (2,2) 1800=8+1792 L (3,1)(2,2) (1,3) 2310=8+2302 L (3,1)(2,2) (2,2) 2310=8+2302 L (4,1)(1,3) (1,2) 2400=8+2392 L (4,1)(1,4) (1,1) 2400=8+2392 L (4,1)(2,2) (1,2) 3280=8+3272 L (4,2)(1,3) (1,1) 3900=8+3892 L (4,2)(2,2) (1,1) 5518=8+5510 L (3,2)(2,3) (0,2) 7951=32+7919 L (3,3)(2,3) (0,1) 8978=32+8946 L (3,2)(1,3) (0,3) 3900=16+3884 L (3,2)(2,2) (0,3) 5146=16+5130 L (3,3)(1,3) (0,2) 4932=16+4916 L (3,3)(2,2) (0,2) 6835=16+6819 L (4,1)(2,3) (0,2) 4580=16+4564 L (4,1)(3,3) (0,1) 4580=16+4564 L (4,2)(2,3) (0,1) 6846=16+6830 L (2,2)(2,2) (0,4) 2921=8+2913 L (3,1)(1,3) (0,4) 1800=8+1792 L (3,1)(2,2) (0,4) 2310=8+2302 L (4,1)(1,3) (0,3) 2400=8+2392 L (4,1)(1,4) (0,2) 2400=8+2392 L (4,1)(2,2) (0,3) 3280=8+3272 L (4,2)(1,3) (0,2) 3900=8+3892 L (4,2)(1,4) (0,1) 3280=8+3272 L (4,2)(2,2) (0,2) 5518=8+5510 L (4,3)(1,3) (0,1) 3900=8+3892 L (4,3)(2,2) (0,1) 5867=8+5859 L (3,3)(3,3) (0,0) 8117=16+8101 L (4,2)(3,3) (0,0) 5800=16+5784 L (4,1)(3,4) (0,0) 2400=8+2392 L (4,2)(2,4) (0,0) 3964=8+3956 L (3,2)(2,2) (1,1) 1964=52+1912 L (3,2)(1,3) (1,1) 1374=44+1330 L (2,2)(2,2) (1,2) 1313=36+1277 L (3,1)(2,2) (1,2) 984=32+952 L (3,1)(1,3) (1,2) 720=28+692 L (4,1)(2,2) (1,1) 1100=20+1080 L (4,1)(1,3) (1,1) 720=16+704 L (3,2)(1,2) (1,2) 650=8+642 L (3,3)(1,2) (1,1) 822=8+814 L (2,2)(1,2) (1,3) 385=4+381 L (2,2)(1,2) (2,2) 385=4+381 L (3,1)(1,2) (1,3) 300=4+296 L (3,1)(1,2) (2,2) 300=4+296 L (4,1)(1,2) (1,2) 400=4+396 L (4,2)(1,2) (1,1) 650=4+646 L (3,2)(2,3) (0,1) 2364=64+2300 L (3,2)(2,2) (0,2) 1964=52+1912 L (3,3)(2,2) (0,1) 2135=48+2087 L (3,2)(1,3) (0,2) 1374=44+1330 L (3,3)(1,3) (0,1) 1374=40+1334 L (2,2)(2,2) (0,3) 1313=36+1277 L (3,1)(2,2) (0,3) 984=32+952 L (3,1)(1,3) (0,3) 720=28+692 L (4,1)(2,2) (0,2) 1100=20+1080 Table 5. (1/2) Three-loop ladder integrand topologies consistent with triangle power-counting, and the decomposition of their numerators into top-level and contact-term ranks. JHEP11(2020)116 Table 5. (1/2) Three-loop ladder integrand topologies consistent with triangle power-counting, and the decomposition of their numerators into top-level and contact-term ranks. 5.1 The importance of choosing bases wisely – 44 – JHEP11(2020)116 L (4,1)(2,3) (0,1) 1100=20+1080 L (4,2)(2,2) (0,1) 1600=20+1580 L (4,1)(1,3) (0,2) 720=16+704 L (4,2)(1,3) (0,1) 984=16+968 L (3,2)(1,2) (0,3) 650=8+642 L (3,3)(1,2) (0,2) 822=8+814 L (2,2)(1,2) (0,4) 385=4+381 L (3,1)(1,2) (0,4) 300=4+296 L (4,1)(1,2) (0,3) 400=4+396 L (4,1)(1,4) (0,1) 400=4+396 L (4,2)(1,2) (0,2) 650=4+646 L (4,3)(1,2) (0,1) 650=4+646 L (3,2)(3,3) (0,0) 1928=48+1880 L (4,2)(2,3) (0,0) 1294=20+1274 L (4,1)(3,3) (0,0) 720=16+704 L (4,1)(2,4) (0,0) 400=4+396 L (2,2)(2,2) (1,1) 481=56+425 L (3,1)(2,2) (1,1) 330=42+288 L (3,1)(1,3) (1,1) 216=30+186 L (3,2)(1,2) (1,1) 229=18+211 L (2,2)(1,2) (1,2) 164=14+150 L (3,1)(1,2) (1,2) 120=12+108 L (4,1)(1,2) (1,1) 120=6+114 L (2,1)(1,2) (1,3) 50=2+48 L (2,1)(1,2) (2,2) 50=2+48 L (2,2)(2,2) (0,2) 481=56+425 L (3,2)(2,2) (0,1) 548=54+494 L (3,1)(2,2) (0,2) 330=42+288 L (3,2)(1,3) (0,1) 330=36+294 L (3,1)(1,3) (0,2) 216=30+186 L (3,2)(1,2) (0,2) 229=18+211 L (3,3)(1,2) (0,1) 229=16+213 L (2,2)(1,2) (0,3) 164=14+150 L (3,1)(1,2) (0,3) 120=12+108 L (4,1)(2,2) (0,1) 240=12+228 L (4,1)(1,2) (0,2) 120=6+114 L (4,1)(1,3) (0,1) 120=6+114 L (4,2)(1,2) (0,1) 164=6+158 L (2,1)(1,2) (0,4) 50=2+48 L (3,2)(2,3) (0,0) 419=38+381 L (3,1)(3,3) (0,0) 216=26+190 L (4,2)(2,2) (0,0) 270=12+258 L (4,1)(2,3) (0,0) 120=6+114 L (2,2)(1,2) (1,1) 55=16+39 L (3,1)(1,2) (1,1) 36=11+25 L (2,1)(1,2) (1,2) 20=5+15 L (2,2)(2,2) (0,1) 125=36+89 L (3,1)(2,2) (0,1) 72=20+52 L (2,2)(1,2) (0,2) 55=16+39 L (3,2)(1,2) (0,1) 55=13+42 L (3,1)(1,2) (0,2) 36=11+25 L (3,1)(1,3) (0,1) 36=9+27 L (2,1)(1,2) (0,3) 20=5+15 L (4,1)(1,2) (0,1) 20=2+18 L (3,2)(2,2) (0,0) 86=20+66 L (3,1)(2,3) (0,0) 36=9+27 L (4,1)(2,2) (0,0) 20=2+18 L (2,1)(1,2) (1,1) 6=4+2 L (2,2)(1,2) (0,1) 12=7+5 L (2,1)(1,2) (0,2) 6=4+2 L (3,1)(1,2) (0,1) 6=3+3 L (2,2)(2,2) (0,0) 17=9+8 L (3,1)(2,2) (0,0) 6=3+3 L (2,1)(1,2) (0,1) 1=1+0 L (2,1)(2,2) (0,0) 1=1+0 Table 6. (2/2) Three-loop ladder integrand topologies consistent with triangle power-counting, and the decomposition of their numerators into top-level and contact-term ranks. JHEP11(2020)116 Table 6. (2/2) Three-loop ladder integrand topologies consistent with triangle power-counting, and the decomposition of their numerators into top-level and contact-term ranks. – 45 – The rank tables included in our present work provide the structure of the numerators consistent with a given power-counting and therefore allow the straightforward construction of such numerators. As an example, consider the integral topology Γ[2,2,2] at two loops in d = 4 spacetime dimensions, with triangle (i.e. 3-gon) power-counting with ﬁve massless external legs: p1 p2 p3 p4 p5 ℓ1 ℓ2 . (5.1) (5.1) JHEP11(2020)116 For triangle power-counting, we would have a loop-momentum-dependent numerator given by [1]⊕[1−2]⊕[2]. 14For 4-gon power-counting, this integrand topology has only a single numerator and appears in the context of sYM with a special (loop-momentum independent) normalization [39]. It was later shown that this integrand satisﬁes a generalized ‘directional dual-conformal invariance’ [110] which has further implications on the analytic structure of the result after integration [111]. 5.1 The importance of choosing bases wisely According to table 3, the dimension of this total numerator space is 12, which can easily be seen to split into 6 top-level (non-contact) numerators and 6 additional contact-term numerators — those that are proportional to one of the six inverse propaga- tors of Γ[2,2,2] leading to Γ[2,1,2] topologies. Concretely, the space is spanned by numerators of the form N3 Γ[2,2,2] = span d=4 [ℓ1]⊕[ℓ1−ℓ2]⊕[ℓ2] . (5.2) (5.2) As described earlier, there are many ways in which to choose representative bases of nu- merators, although the rank counting obviously does not depend on any particular choice. For the particular loop-momentum routing shown in (5.1), one fairly arbitrary choice for the 6 non-contact numerators would be14 As described earlier, there are many ways in which to choose representative bases of nu- merators, although the rank counting obviously does not depend on any particular choice. For the particular loop-momentum routing shown in (5.1), one fairly arbitrary choice for the 6 non-contact numerators would be14 bN3 Γ[2,2,2] = span 1, (ℓ1\|p2), (ℓ1\|p4), (ℓ2\|p2), (ℓ2\|p4), (ℓ1−ℓ2\|p2+p4) . (5.3) (5.3) (Notice that none of these generalized inverse propagators are propagators of the graph in (5.1).) To determine the coeﬃcients of these six integrands in the expansion of some amplitude under consideration requires the solution of a 6 × 6 linear system. If one is not careful in deﬁning integrands that have Γ[2,2,2] as subtopology, the linear system can become even larger since one has to take integrand topologies with more propagators into account. However, in the method of maximal cuts (see e.g. [83]) their coeﬃcients are taken to be ﬁxed by appropriate unitarity cuts so that one is still left with only 6 unknowns. While this example is of course fairly trivial, analogous linear algebra problems quickly become prohibitive as the sizes of integrand bases grow. Therefore, it is clearly desirable to choose ‘nice’ numerators — which partially pre-diagonalize the linear system we need to solve to represent amplitudes. These could be numerators designed to vanish at partic- ular points in loop-momentum space, making the calculation of their coeﬃcients (often) completely trivial. There is a considerable amount of literature on this subject — see – 46 – e.g. [26, 79, 80, 102] — although many open questions remain. 5.1 The importance of choosing bases wisely In the context of sYM am- plitudes, we studied some of these integrands extensively and constructed special numera- tors which diagonalized the system completely [79, 80] by demanding that the numerators vanished at certain special kinematic points, thus implementing the prescriptive unitarity method of [26]. While we are not yet able to extend this approach to all integrand topologies for arbitrary power-counting, there are cases for which particular numerators help make various properties of amplitudes more manifest. For example, for the Γ[2,2,2] topology, there exist certain chiral numerators [101] which provide a diﬀerent (not necessarily better) basis choice for the 6 non-contact degrees of freedom in bN3 Γ[2,2,2] : JHEP11(2020)116 bN3 Γ[2,2,2] = span (ℓ1\|ℓ∗ 1,1), (ℓ1\|ℓ∗ 1,2), (ℓ2\|ℓ∗ 2,1), (ℓ2\|ℓ∗ 2,2), (ℓ1−ℓ2\|ℓ∗ 12,1), (ℓ1−ℓ2\|ℓ∗ 12,2) (5.4) (5.4) where ℓ∗ x,i can be chosen as the i=1, 2 cut-solutions to the following cut equations ℓ∗ 1,i ↔ ℓ2 1 = (ℓ1 + p1)2 = (ℓ1 + p1 + p3)2 = (ℓ1 −p5)2 = 0 , ℓ∗ 2,i ↔ ℓ2 2 = (ℓ2 + p3)2 = (ℓ2 + p3 + p5)2 = (ℓ2 −p1)2 = 0 , (5.5) ℓ∗ 12,i ↔ (ℓ1−ℓ2−p3−p4)2 = (ℓ1−ℓ2+p1+p2)2 = (ℓ1−ℓ2+p2)2 = (ℓ1−ℓ2−p4)2 = 0 . (5.5) In spinor-helicity notation, they may be written as ℓ∗ 1,1 = ⟨13⟩ ⟨35⟩λ5eλ1, ℓ∗ 2,1 = ⟨35⟩ ⟨51⟩λ1eλ3, and ℓ∗ 12,1 = ⟨15⟩ ⟨13⟩λ3eλ5. The ℓ∗ x,i=2 solutions are obtained from these by replacing λi ↔eλi and ⟨· ⟩↔[ · ]. Of course, our choice of chiral numerators is also somewhat arbitrary and diﬀerent deﬁnitions of ℓ∗ x,i would have worked just as well. As we will explain shortly, these chiral numerators are suitable to ameliorate certain IR singularities that can be present otherwise, and therefore represent better choices than the arbitrary choices in (5.3). In the following subsection, we discuss some of the criteria by which good integrand bases could be chosen more wisely. 5.2 Choosing bases according to analytic properties of integrals It would be desirable to establish a link between the choice of numerators to the an- alytic properties of the results of integration. In particular, the connections between IR divergences, transcendentality, and the behavior of integrands (and their numera- tors) on generalized unitarity cuts has been studied extensively from several perspec- tives [16, 38, 39, 100, 102, 112–114], and we have learned some important lessons. Moreover, there is mounting evidence that good integrands — those with wisely chosen numerators — are much easier to integrate directly (see e.g. [74–78]). For example, it is well understood that IR divergences arise from soft and collinear regions in loop-momentum space (see e.g. [115]). Therefore, it is desirable to choose as many numerators as possible to vanish fast enough in precisely these regions so that the resulting integral is IR ﬁnite. In fact, a signiﬁcant fraction of the chiral numerators introduced in [101] satisfy this property, which will undoubtedly render them useful in determining analytic expressions for amplitudes. – 47 – Another desirable aspect of loop integrands regards their diﬀerential structure. Indeed, many loop integrands can be chosen such that they may be expressed directly in dlog- form [37, 38]; speciﬁcally, this is the case when an integrand only has logarithmic dx/x poles throughout its cut structure. When this is the case, it is widely expected that — at least in most cases15 — the result of loop integration will be a polylogarithmic function of maximal transcendentality. On the other hand, if there are double (or higher) poles in the cut structure, then the post-integration result is expected to contain terms of lower transcendentality; see e.g. the discussion of the one-loop bubble integral in d=4 in [38]. In the remainder of this subsection, we elaborate on these general features and discuss their connection to the results of this work. JHEP11(2020)116 Manifesting infrared divergences and infrared ﬁniteness. The example given above for the integrand topology Γ[2,2,2] illustrates an important point: we can often ex- plicitly construct numerators for a given integral topology which eliminate IR poles — provided we are using a basis with suﬃciently high power-counting. If the power-counting is limited, we can nonetheless use whatever freedom we have to attempt to eliminate at least a subset of IR singularities. Let us return brieﬂy to the example of the integrand topology Γ[2,2,2] described above. 15Counterexamples exist that involve certain square roots in the arguments of the dlog forms which can lead to more complicated functions [116]. We thank Claude Duhr for interesting discussions on this point. 5.2 Choosing bases according to analytic properties of integrals JHEP11(2020)116 Simple examples of integrals with only logarithmic singularities include the scalar box and triangle integrals at one loop in four dimensions: ( , ) . (5.7) (5.7) It is quite easy to see that taking any sequence of residues of the above integrands will always result in simple poles. This agrees with the well-known fact that both integrands integrate to functions of maximal transcendental weight — namely, Li2 and log2 functions. Moreover, there turns out to be a simple change of variables which makes this structure manifest. For the scalar box with propagators (ℓ\|Qi), the integrand can be re-written as It is quite easy to see that taking any sequence of residues of the above integrands will always result in simple poles. This agrees with the well-known fact that both integrands integrate to functions of maximal transcendental weight — namely, Li2 and log2 functions. Moreover, there turns out to be a simple change of variables which makes this structure manifest. For the scalar box with propagators (ℓ\|Qi), the integrand can be re-written as d4ℓ (Q1\|Q3)(Q2\|Q4) (ℓ\|Q1)(ℓ\|Q2)(ℓ\|Q3)(ℓ\|Q4) = 1 2dlog (ℓ\|Q1) (ℓ\|Q2)dlog(ℓ\|Q2) (ℓ\|Q3)dlog(ℓ\|Q3) (ℓ\|Q4)dlog(ℓ\|ℓ∗ 1) (ℓ\|ℓ∗ 2) , (5.8) (5.8) where ℓ∗ 1,2 are the two solutions to the cut equations (ℓ\|Qi) = 0 , i ∈{1, 2, 3, 4}. Although checking whether or not the poles of any given integrand are logarithmic on all cuts is in principle straightforward (see [127, 128] for possible practical subtleties), ﬁnding an explicit (or compact) dlog-form for such an integrand remains something of an art, and no general procedure for obtaining this transformation is currently available (but see [127, 128] for a partial-fraction strategy — which, unfortunately, often yields rather unwieldy expressions for the dlog forms). If an integrand involves higher-order poles like dx/x2 in its cut structure, it is empiri- cally the case that the degree of transcendentality of the resulting integral after integration drops. In particular, this may mean it is a mixture of pieces with diﬀerent transcendental weights. These higher-order poles can be located either in the UV region, corresponding to large loop momenta, ℓ→∞, or in the IR region, corresponding to poles of higher degree in the denominator. The simplest example of the former type of pole is the scalar bubble integral at one loop in d=4. 5.2 Choosing bases according to analytic properties of integrals For this example, there are potential collinear divergences in three regions: where ℓ1 ∼p1, where ℓ2 ∼p3, or where ℓ1−ℓ2−p3−p4 ∼p5. It is easy to see that the numerators we chose in (5.5) were each designed to eliminate one of the three IR divergent regions. However, with only 3-gon power-counting, we would only be allowed to insert a single inverse propagator, making it impossible to make this integral IR ﬁnite in all regions. For example, the chiral numerator (ℓ1\|ℓ∗ 1,1) = ℓ1 −⟨13⟩ ⟨35⟩λ5eλ1 2 →0 as ℓ1 →αλ1eλ1 (5.6) (5.6) vanishes in the collinear region ℓ1 ∼p1 but does not do anything to help the other two IR- divergent regions. Similar statements hold for the other ﬁve numerators given in (5.5). An interesting open problem is to count, for a given integral topology, spacetime dimension and power-counting, the number of independent numerators that give rise to IR-ﬁnite integrals; and moreover, to partition the remaining (IR divergent) part of the basis into subspaces according to particular degrees of divergence after integration. For example, one could imagine constructing integrands that give rise to divergences of a particular degree 1/ϵk in dimensional regularization. This partitioning was resolved in the planar sector at two loops by some of the authors [102], and each IR divergent integral was linked directly to a particular soft or collinear residue of a scattering amplitude’s integrand. Why might such a decomposition be useful? Physically, the sorting of integrands according to their IR structure can help organize and recognize the expected intricate divergence structure in non-abelian gauge theory [117–125] from the start. On the other hand, from an integration point of view, it has already been pointed out why one might prefer an IR ﬁnite basis of master integrals, see e.g. [126]. Instead of shifting dimensions – 48 – or doubling propagators, we would construct such a basis by designing appropriate loop- momentum-dependent numerators that eliminate all IR-divergences. Of course, actually achieving this goal must be left to future work. Polylogarithmic poles and integral transcendentality. There is a close connection between the types of singularities of integrands and the degree of transcendental functions which result from integration. As mentioned above, integrands with only logarithmic sin- gularities which can always in principle be written in d log form, are expected to yield maximally transcendental functions, while the presence of multiple poles indicates a drop in transcendentality. 5.2 Choosing bases according to analytic properties of integrals While the oﬀ-shell integrand does not have any poles of degree greater than one, when evaluated on the unitarity cut ℓ2 = (ℓ+p1 +p2)2 = 0, the integral becomes p12 p34 ℓ cut = Z dz dw z , where ℓ:= (wλ1 + zλ2) eλ1 −w + 1 z eλ2 , (5.9) (5.9) – 49 – which has a double-pole at inﬁnity when w →∞(the double-pole is exposed by the usual inversion w→1/u) which sends ℓ→∞. As a result, we can expect a transcendental weight- drop in the post-integration result; this expectation is indeed realized in its expression from dimensional regularization [129, 130]. Z d4−2ϵℓ h i ∝1 ϵ + log −s µ2 + 2 + O(ϵ) , (5.10) (5.10) where 4−2ϵ is the dimensionally-regulated spacetime dimension, µ2 is the usual renormal- ization scale, and s=:(p1 +p2)2. The UV divergence of the bubble integral around d=4 is encoded in the presence of the 1/ϵ pole in the result. Often, there is a close link between higher poles at inﬁnity of the integrand and UV divergences of the integrated answer based on simple power-counting arguments. However, in certain situations there are cases of higher poles at inﬁnity that are not necessarily associated to the UV divergences; for a more detailed discussion of this subtle issue, see e.g. [31, 40]. JHEP11(2020)116 JHEP11(2020)116 As mentioned above, the other common source of higher-degree poles can be found in the IR region, where loop momenta become soft and/or collinear. If we consider only integrals with simple propagators, these double-poles arise either from Jacobians generated when the integral is evaluated on cuts, or by the factorization of uncut propagators when evaluated on cuts. The simplest example where this occurs is for the topology Γ[2,2,2] for four massless external particles (in four dimensions): I∗ [2,2,2] = p1 p2 p4 p3 ℓ1 ℓ2 (5.11) (5.11) We ﬁrst cut ℓ12 = 0 which sets ℓ1 = λℓ1eλℓ1 on-shell. On the support of this cut, the propagator involving p1 factorizes (ℓ1 +p1)2 = ⟨ℓ11⟩[ℓ11], and sending both factors to zero sets ℓ1 = αλ1eλ1 — i.e. ℓ1 becomes proportional to p1. Of course, this cut has merely localized ℓ1 to the collinear region of p1. Similarly, on the other side of the diagram by cutting ℓ22 and both factors in (ℓ2 +p4)2 we set ℓ2 = βλ4eλ4. 5.2 Choosing bases according to analytic properties of integrals On this cut, the two central propagators become (ℓ1 + ℓ2)2 = αβs14 , (ℓ1 + ℓ2 −p3)2 = αβs14 −αs13 −βs34 . (5.12) (5.12) If we consider this integrand topology with scalar numerator s2 14, the residue on this cut evaluates to If we consider this integrand topology with scalar numerator s2 14, the residue on this cut evaluates to Res (5.12) h I∗ [2,2,2] i = Z dα dβ s2 14 αβ(αβs14 + αs13 + βs34) β=0 −−→ Z dα α2 , (5.13) (5.13) which exposes the double-pole at α = 0. According to general wisdom, this double-pole should be reﬂected in the structure of the integrated result. Indeed, performing the integral yields lower transcendental terms as can be seen by evaluating this integral in dimensional regularization and expanding in ϵ, [131]. – 50 – If we work with box power-counting, the above scalar numerator is the only possibility (up to rescaling by external kinematic dependent factors that do not change the analysis). Therefore, this double-pole of the integrand and the associated transcendentality drop is unavoidable. However, for triangle (and higher) power-counting, we can choose a numerator for (5.11) which cancels this pole — for example, (ℓ2 +p1)2 — and write down an integrand which is of uniform transcendentality. However, this procedure requires a bit of care, as loop-dependent numerators can accidentally introduce additional double-poles at inﬁnity (which would in turn re-introduce a drop in transcendentality). Establishing a hierarchy for integrand basis elements in accordance with their highest degree poles anywhere in their cut structure serves as an integrand-level proxy for the transcendental weight of the resulting integrated answers and is an important open prob- lem which we leave for future work. Besides higher degree poles, there is at least one further essential part of the story we have neglected: namely, the presence of new types of singularities which are not simply poles of some rational function. JHEP11(2020)116 Non-polylogarithmic singularities: elliptics and beyond. It is now a well-known fact that — at suﬃciently high multiplicity and/or loop-order — almost all scattering amplitudes in almost all quantum ﬁeld theories involve non-polylogarithmic structures. The appearance of such structures can be avoided at low multiplicity and loop-order, but these additional structures eventually appear necessary (even if only in local integrand representations). 5.2 Choosing bases according to analytic properties of integrals Recently, the analysis of simple examples of such non-polylogarithmic pieces have attracted considerable interest in the high energy physics community, for both practical reasons and formal motivations alike (see, e.g. [105–109]). From an integrand perspective, there are many diﬀerent types of singularities of Feyn- man integrals beyond single and multiple poles, and the complete list is not known in general. Among the best known examples in four dimensions is the two-loop double-box integral with a scalar numerator (which corresponds to Γ[3,1,3] in the notation of this paper), which is known to be elliptic [105, 132–134]: I[3,1,3] = (5.14) (5.14) Evaluating the integrand on one of the two solutions to the maximal (hepta-)cut when all propagators in the graph are put on-shell yields an integral over one remaining parameter, Res I[3,1,3] = Z dx p x4 + αx3 + βx2 + γx + δ , (5.15) (5.15) where x parameterizes the last unﬁxed degree of freedom in ℓ1, ℓ2 and the coeﬃcients α, . . . , δ only depend on external kinematics. There are no poles in this expression, in- cluding the pole at x →∞. The nature of the singularity obtained here is aptly called elliptic as the integration over x ∈R would give an elliptic function. Indeed, evaluating the integral (5.14), one ﬁnds that the result is not a polylogarithm, but rather an elliptic function [105]. – 51 – In general, there is an obvious connection between post-integration results and the types of singularities which appear in the associated integrand. If the only singularities are logarithmic, the integral is expected to evaluate to a sum of (generalized) polylogarithms. However, we do not currently have suﬃcient knowledge about the space of functions for Feynman integrals with non-logarithmic singularities in their unitarity cut structure. Try- ing to make progress on this very diﬃcult question is an active area of research in both physics and mathematics. Transcendental ﬁltration and integrand stratiﬁcation. An important question from the integrand perspective is whether or not we can establish a hierarchy of numerators for a given integrand topology based on the type of singularities which appear in their cuts. Such a classiﬁcation would imply that we can take any integrand topology and (for a given power-counting) divide the numerator basis into groups classiﬁed by the presence of single poles (logarithmic singularities), higher-degree poles, elliptic singularities, etc. 5.3 An alternative proposal: classiﬁcation by poles at inﬁnity Our discussion of integrand bases has been most detailed for two and three loops. While a similar analysis can be extended to higher loops, our deﬁnition of p-gon power-counting suﬀers from some unfortunate features. This is easiest to see in the case of planar inte- grands, where a more familiar (and powerful) deﬁnition of planar-power-counting exists. Recall that a deﬁnition of power-counting for plane integrands can be given by demanding that every loop momentum variable (encoded by the dual of the plane graph) scales like a p-gon at inﬁnity. JHEP11(2020)116 Starting at four loops, there exist planar integrands which we would classify as ‘scalar’ p-gons, but which would admit non-trivial loop-dependent numerators while still satisfying planar p-gon power-counting. The simplest examples of these are graphs with a (p+1)-gon surrounded by p-gons: , , , , . . . (5.19) , , , , . . . (5.19) (5.19) All of these integrands meet our deﬁnition of being ‘scalar’ p-gon integrands, as all daughter topologies have girth strictly less than p. Nevertheless, as planar integrands, we may use the preferential loop-momentum routing of the dual graph to see that each of these may admit a non-trivial numerator while remaining ‘p-gons’ according to planar-power-counting. The third and fourth examples above are particularly poignant as they would be rendered dual- conformal with such a numerator, while if given the numerator ‘1’, they would not be. As these examples illustrate, our deﬁnition of power-counting — while applicable to all graphs — is more restrictive than what is traditionally used in the planar limit. This is particularly problematic because it is clear, for example, that our deﬁnition of p = 4 power-counting fails to include dual-conformal integrands at suﬃciently high loop order (namely, 5). It is well-known that amplitudes in planar sYM may be expressed in terms of dual-conformal integrands; moreover, a complete basis of such integrands requires non- zero coeﬃcients for 4-particle amplitudes, as seen in [138–140]. But our deﬁnition of ‘box’ power-counting fails to include all such integrands — and, in fact, therefore, all amplitudes in planar sYM at suﬃciently high loop orders. Thus, our stratiﬁcation, while being well deﬁned for all graphs, does not have the property that amplitudes in sYM can be expressed in terms of integrands with box power-counting beyond 4 loops or triangle power-counting beyond 7 loops. 5.2 Choosing bases according to analytic properties of integrals JHEP11(2020)116 As an example, let us take the Γ[4,1,3] topology, the so-called penta-box integral: (5.16) (5.16) With box power-counting, the Γ[4,1,3] topology is allowed to carry numerators drawn from the basis structure [ℓA]. According to our basis counting summarized in table 3, in four spacetime dimensions these numerators consist of 2 top-level and 4 contact-term degrees of freedom (each of which cancels one propagator of the left loop leading to Γ[3,1,3] topologies). Speciﬁcally, we can span the full-rank basis by two (familiar) chiral numerators and four double box contact-terms, [ℓA] = span (ℓA\|ℓ∗ A,1), (ℓA\|ℓ∗ A,2), (ℓA\|Q1), (ℓA\|Q2), (ℓA\|Q3), (ℓA\|Q4) , (5.17) (5.17) [ℓA] = span (ℓA\|ℓ∗ A,1), (ℓA\|ℓ∗ A,2), (ℓA\|Q1), (ℓA\|Q2), (ℓA\|Q3), (ℓA\|Q4) , (5.17) where the special external kinematic points ℓ∗ A,i are again taken as the two solutions to the cut equations (ℓA\|Q1) = (ℓA\|Q2) = (ℓA\|Q3) = (ℓA\|Q4) = 0. In the generic case of massive external momenta, all double-box integrals are of the form (5.14) and therefore have elliptic maximal cuts (5.15). A priori, the two top-level integrands have support on four diﬀerent elliptic cuts associated to the various pinchings of propagators to yield Γ[3,1,3] topologies. On one of the heptacuts, the numerators take the schematic form, Res h I(5.16) i = Z dx N(x) (x −x1)(x −x2) p x4 + αx3 + βx2 + γx + δ, (5.18) (5.18) where x1 and x2 are the two positions of leading singularities associated with cutting the last propagator, and the numerator N(x) is a quadratic function of x. It is easy to see that no choice of numerator N(x) can remove the elliptic singularity from the heptacut, and it remains an important open question whether or not there is any choice of numerator for Γ[4,1,3] for which the ﬁnal result is polylogarithmic (without elliptic contributions). While we cannot remove the elliptic cut completely, it might be possible that there is some special choice of numerator which removes all elliptic eﬀects from the integrated result. – 52 – We should also note that for special cases when some of the corners in Γ[4,1,3] or Γ[3,1,3] are massless (or vanishing) momenta, all singularities are logarithmic and the integrated result does indeed evaluate to generalized polylogarithms [135–137]. 5.3 An alternative proposal: classiﬁcation by poles at inﬁnity To be clear: it would not be hard to alter our deﬁnition of the box-power-counting basis to include dual-conformal integrands in the planar limit. Our rule for generating numerators according to graph inclusions would permit us to deﬁne a basis that ‘scales like’ any set of speciﬁed graphs with any pre-chosen, loop-dependent numerators. The real problem is that we do not know any clear rule for adding such numerators into the space – 53 – we use to recursively deﬁne a basis of integrands. This remains an important open prob- lem that must be left to future work. Speciﬁcally: Is there a non-planar, graph-theoretic deﬁnition of a basis of integrands with ‘4-gon power-counting’ such that all amplitudes in sYM are inside this basis? More generally, it remains an open problem to deﬁne any well-behaved integrand basis of integrands guaranteed to include the amplitudes of ‘nice’ quantum ﬁeld theories to all loop-orders. In the planar limit, we have the basis of integrands dictated by dual-conformal invariance — which is in fact stronger than mere planar-box-power-counting; and there is a great deal of evidence that amplitudes in planar sYM can be represented in such a basis. We know of no non-planar analogue of box-power-counting (let alone dual-conformal invariance) that should suﬃce for amplitudes in sYM beyond the planar limit to all orders of perturbation theory. JHEP11(2020)116 One promising strategy — at least, formally — would be to deﬁne power-counting in terms of a hierarchy of poles ‘at inﬁnity’. The real challenge here is to make this deﬁnition useful without speciﬁc reference to loop momentum routing or to a brute force survey of potential singularities of integrands. Nevertheless, the poles at inﬁnite loop-momentum have been extensively studied in many papers [37–40], mainly in the context of particular theories. For example, it was conjectured and later veriﬁed [40] up to three loops that sYM integrands are free of any poles at inﬁnity to all orders of perturbation theory, suggesting something like a non-planar analogue of dual-conformal symmetry. (In contrast, amplitudes in N =8 supergravity have higher-degree poles at inﬁnity that grow with multiplicity and loop-order.) To make this more precise, we may organize loop integrands by the maximal degree of singularity encountered as the momentum ﬂowing through any edge goes to inﬁnity via any sequence of residues. This maximal degree is formally well-deﬁned, if hard to detect in practice. 5.3 An alternative proposal: classiﬁcation by poles at inﬁnity Speciﬁcally, this requires that one perform all possible cuts, and list the degrees of all singularities which send ℓ→∞: max cuts n Cut I −−−→ ℓ→∞O(ℓs) o . (5.20) (5.20) The maximal degree s over the set of all cuts for the momentum ℓﬂowing through any edge is then deﬁned to be the degree of a given integrand’s pole(s) at inﬁnity. Obviously, from a practical point of view, this is not a very constructive approach, as it requires that many checks be made to decide s for a given integrand. Despite these practical limitations, it certainly gives us a unique answer. This deﬁnition extends to all loops and provides a hierarchy of loop integrands based on this degree. We may illustrate this at one loop in d=4. In fact, our deﬁnition above overlaps with the standard deﬁnition of power-counting with the following identiﬁcations: power-counting degree of pole p = 4 (box) s = −2 (no pole) p = 3 (triangle) s = −1 (single pole) p = 2 (bubble) s = 0 (double-pole) p = 1 (tadpole) s = 1 (triple pole) p = 0 (constant) s = 2 (quadruple pole) – 54 – As an example, in eq. (5.9) we computed a double-cut of the scalar bubble integral and identiﬁed the double-pole at inﬁnity for w→∞. One may readily verify that a double-pole at inﬁnity is indeed the worst possible singularity on any cut of the bubble integral. The analogous procedure now extends to higher loops without any conceptional problem: the (maximal) degree of poles at inﬁnity is always well deﬁned as the supremum of degrees we encounter over the space of all sequences of cuts. At two loops, for the scalar planar double-box Γ[3,1,3] and the non-planar double-box Γ[3,2,2], the degree at inﬁnity is s = −2 — i.e., no pole at inﬁnity — which is in agreement with the deﬁnition of the power-counting we have used throughout this paper. However, there is a diﬀerence for certain Γ[2,1,2] topologies, where our graph-theoretic power-counting deﬁnition allows for one degree of freedom (the scalar numerator) within triangle power- counting p=3. 5.3 An alternative proposal: classiﬁcation by poles at inﬁnity From the pole at inﬁnity perspective, we see that a special degeneration of this integrand in fact has not just a single pole, but a double-pole at inﬁnity: JHEP11(2020)116 I∗∗ [2,1,2] = p12 p34 ℓ1 ℓ2 (5.21) (5.21) An intuitive way to understand this fact for this particular diagram is to realize that we generate a bubble when we remove the central propagator. This can be exposed by ﬁrst cutting all propagators An intuitive way to understand this fact for this particular diagram is to realize that we generate a bubble when we remove the central propagator. This can be exposed by ﬁrst cutting all propagators ℓ12 = (ℓ1 + ℓ2)2 = (ℓ1+p1+p2)2 = ℓ22 = (ℓ2+p3+p4)2 = 0 , (5.22) (5.22) and further localizing ℓ1 on the composite leading singularity, which double-cuts (ℓ1 +ℓ2)2 =0 and sets ℓ1 = −ℓ2. The residue on this cut has Jacobian 1/p2 12, and the resulting expression is just a one-loop cut bubble integral in ℓ2, Cut h I∗∗ [2,1,2] i ≃Cut "Z d4ℓ2 ℓ22(ℓ2+p3+p4)2 # (5.23) (5.23) which has a double-pole at inﬁnity, as discussed in (5.9). Note that while it is necessary to choose some parameterization for this test and express the degrees of freedom in loop momenta in a particular way (in addition to an arbitrariness of how the loop momenta are to be routed) for performing cuts, the degree of poles at inﬁnity is unambiguous and does not depend on the choices made. We suspect that tracking the degree of poles at inﬁnity will be the best — and morally (more) correct way — to deﬁne the power-counting at arbitrary loop-order. Moreover, we conjecture that amplitudes in sYM may be represented by a basis that meets this deﬁnition of ‘box power-counting’ to all orders. Another natural question is whether or not it is desirable to expand a four-dimensional integrand with overall box power-counting — however this basis may be deﬁned — in terms of a basis of integrands with the same property. We have argued in previous pa- pers where we explicitly constructed all multiplicity two-loop supersymmetric Yang-Mills amplitudes [79, 80] that the answer is: not necessarily. 5.3 An alternative proposal: classiﬁcation by poles at inﬁnity For example, we have found that – 55 – amplitudes in N =8 supergravity require arbitrarily bad power-counting bases (the degree of which grows with multiplicity) starting at two loop despite the fact that amplitudes in this theory are known to have bounded behavior in the ultraviolet. The reader may wonder why we have not used the proposed ‘poles at inﬁnity’ criterion for our two- and three-loop bases described in sections 3 and 4, but rather resorted to a more purely graph-theoretic deﬁnition. The answer was already alluded to above: the pole at inﬁnity check is not a constructive approach and it can be computationally prohibitive. We do not a priori know the degree of poles at inﬁnity of a given integrand, and to check this we would have to perform all possible cuts, which is extremely laborious. Moreover, the degree can diﬀer between the general case and boundary cases. For Γ[2,1,2], for example, the general case with external momenta ﬂowing into the middle vertices has a single pole at inﬁnity s = −1; in contrast, the boundary case with three-point vertices in the middle has a double-pole at inﬁnity (s = 0) as discussed below (5.21). This makes it harder to do the bookkeeping of various integrand topologies. While this all is just a technical inconvenience, it makes it hard to implement our conjecture systematically at present. Minimally, our graph-theoretic deﬁnition of power-counting should suﬃce to construct box-power-counting bases big enough for sYM through at least four loops — with 3-gon power-counting through at least seven loops. JHEP11(2020)116 In the future, we would like to use the results, technology, and the counting of basis elements from the current work and transfer this knowledge to a new framework which uses poles at inﬁnity as the primary condition. This almost certainly requires that we build new tools to systematically detect the degree of the poles without performing all possible cuts. 5.4 Building amplitudes: beyond the bases of integrands It is suggestive that each double-pentagon matches one kissing-box leading-singularity of the amplitude, and indeed the numerator counting works along these lines: provided that we exclude from our basis the Γ[4,0,4], Γ[5,0,4], and Γ[5,0,5] integrands, we would ﬁnd 4 (as opposed to 3) independent top-level numerators for box power-counting Γ[4,1,4] = ⇔N4 Γ[4,1,4] = [ℓA][ℓC] , rank d=4 N4 Γ[4,1,4] = 36 = 4+32 (5.25) JHEP11(2020)116 (5.25) matching exactly the four kissing-box leading singularities, matching exactly the four kissing-box leading singularities, . (5.26) (5.26) The special numerator that is used in (5.24) was chosen to only have support on the MHV-compatible solution to these cuts. So far, this all looks reasonably natural but there is an important subtlety. Apart from the kissing-box leading singularity there are many more leading singularities of the amplitude which also must be matched. Moreover, by using these ‘chiral’ double-pentagon integrands, we introduce a number of unphysical singularities (term-by-term) which only cancelled in the sum. None of that was used in [100, 101] to ﬁnd the formula (5.24), but it is post-facto-guaranteed by global residue theorems (GRTs) which connect various singularities of the amplitude together in a consistent framework. The diﬃculty is that it is very hard to write down (or guess) expressions such as (5.24) and verify that they give the correct amplitude exactly because not all singularities are matched correctly by construction. This is in stark contrast to the generalized unitarity approach [1–3] or its prescriptive reﬁnement [26], where we do match each physical cut separately and we can be conﬁdent that the resulting expression is correct. However, the prescriptive approach here is most straightforward to use within boosted p < d-gon power-counting. For d = 4, we would ﬁnd that the nine-propagator graphs such as Γ[4,1,4] would be fully decomposable; but we would be able to meet all the eight-propagator cuts distinctly and directly. With boosted power-counting, it is therefore easy to match one leading singularity at a time while integrands such as Γ[4,1,4] (required for box power-counting in d = 4) would contribute to many leading singularities in violation of the prescriptive philosophy. Therefore, we would be hard pressed to recover an expression like (5.24) from the prescriptive approach. 5.4 Building amplitudes: beyond the bases of integrands As was discussed above, integrands with more than d×L propagators can always be re- duced to simpler topologies for an integrand basis with p < d power-counting. However, there are certain situations when integrands with more propagators are convenient and useful for the representation of amplitudes. For example, they may be more ﬁnite in the ultraviolet/infrared than their decomposition into daughters would suggest. The best example is perhaps the two-loop MHV integrand in planar sYM, which was written in [100–102]: A2-loops MHV, planar = X 1≤a<b<c b<c<d<a+n a b c d n(ℓA) n(ℓC) (5.24) A2-loops MHV, planar = X 1≤a<b<c b<c<d<a+n a b c d n(ℓA) n(ℓC) (5.24) (5.24) where n(ℓi) represents a particular choice of loop-dependent numerators for the graph. In this work’s terminology, this formula involves particular choices for integrands of the Γ[4,1,4] topology. In particular, the numerators are chosen to match all cuts in ﬁeld theory of a diﬀerent topology — namely, Γ[4,0,4], a.k.a. the kissing-boxes depicted in eq. (5.26). In contrast to the integrand Γ[4,0,4], which would have a scalar numerator in box power- counting, the double pentagon integrand Γ[4,1,4] can be assigned numerators to match all where n(ℓi) represents a particular choice of loop-dependent numerators for the graph. In this work’s terminology, this formula involves particular choices for integrands of the Γ[4,1,4] topology. In particular, the numerators are chosen to match all cuts in ﬁeld theory of a diﬀerent topology — namely, Γ[4,0,4], a.k.a. the kissing-boxes depicted in eq. (5.26). In contrast to the integrand Γ[4,0,4], which would have a scalar numerator in box power- counting, the double pentagon integrand Γ[4,1,4] can be assigned numerators to match all – 56 – four kissing-box cuts separately. However, it comes at the cost of introducing spurious cuts, which cancel in the sum over terms in (5.24). The representation (5.24) is a miraculously simple expression, and it is natural to ask about its origin and if we can use the underlying principle in diﬀerent cases too. The answer is a bit complicated. 5.4 Building amplitudes: beyond the bases of integrands In [26], we showed how to use prescriptive unitarity to reconstruct general two and three-loop n-point amplitudes in planar sYM preserving the box power-counting in the planar limit, but these constructions required very judicious choices (and some magic) to work. Today, we do not know how such tricks and ‘magic’ can be (or even could be) generalized to higher loop-orders. – 57 – We know that if we go beyond the planar limit, for which we generated integrand bases in this paper, the prescriptive approach to constructing amplitudes works without any problems (other than increasing complexity); but the search for simple representations of amplitudes with lower power-counting become increasingly diﬃcult to ﬁnd because of the presence of color factors. In [80], we used the prescriptive approach to determine the integrand for the full (non-planar) two-loop n-point MHV amplitude in sYM by carefully constructing the basis of integrands with triangle power-counting (while making some con- venient choices) and matching all of the relevant leading singularities, A2-loops MHV, non-planar = X inequivalent leading singularities f f × If . (5.27) A2-loops MHV, non-planar = X inequivalent leading singularities f f × If . (5.27) (5.27) JHEP11(2020)116 As discussed above, this representation (as it involves integrands with 3-gon power- counting) involves poles at inﬁnity in virtually every integrand basis element, while it is known (at two loops) that the ﬁnal amplitude is free of these poles. The absence of poles at inﬁnity for non-planar amplitudes suggested an extension of dual-conformal symmetry beyond the planar sector in sYM and was partially explored in [38, 39]. From the perspec- tive of our work here, the lack of poles at inﬁnity in the ﬁnal amplitude raises the question whether or not we can write the amplitude using integrands with box power-counting only to make the behavior at inﬁnity manifest term-by-term. This would necessarily introduce integrals with 9 or more propagators such as Γ[3,3,3], Γ[4,2,3], or Γ[4,2,4]: (5.28) (5.28) (5.28) Such integrands would have support on many leading singularities at the same time — and could only be chosen to match particular subsets. Thus, they must conspire to match everything at once in some spectacular way to give the correct expression for a scattering amplitude. This would have to follow from GRTs (including color information) that link various color-dressed on-shell functions together into a bigger interrelated network. 5.4 Building amplitudes: beyond the bases of integrands Using these relations practically requires one to enumerate all the independent GRTs, which is hard to do systematically. Naïvely, one can just use the traditional version of generalized unitarity with some deﬁnition of box power-counting, write an ansatz for numerators and color factors which multiply a given topology and check all cuts. Apart from being a rather involved exercise for high multiplicity (let alone general n-point), the resulting solution would suﬀer from a major problem: the coeﬃcients would generically be some complicated sums of products of kinematics and color factors (with diﬀerent graph structures), where the latter do not necessarily correspond to any particular color-dressed on-shell function. The color factors would appear as essentially ad hoc expressions — appearing merely to satisfy the constraints of generalized unitarity. This makes perfect sense because the color factors are tied to the leading singularity pictures (with 8 or fewer propagators) while our integrands can have 9 or more propagators, and can not be translated to color factors in this way. On the hand, the prescriptive unitarity approach treatment of color factors is very straightforward: each – 58 – integrand is multiplied by a single color-dressed on-shell function directly and the color factor can be read oﬀfrom the graph directly; for further details and powerful illustrations, see [79, 80]. Of course, there can in principle exist a fundamentally new approach that would allow us to use a minimal set of building block integrals, such as Γ[3,3,3], Γ[4,2,3], or even Γ[4,2,4] with some magic numerators. Similar to the planar amplitude written in terms of the chiral pentagon expansion in eq. (5.24), each integrand would match many leading singularities at the same time. It would be great to ﬁnd some way to constructively ﬁnd such numerators based on some underlying principles. We know that such an approach must combine both color and kinematics in a non-trivial way, and at the same time must exploit the richness of the GRTs. We would love to pursue this path in the future. JHEP11(2020)116 5.5 Concluding remarks In this work, we have discussed a systematic approach to the construction of loop-integrand bases for general, non-planar scattering amplitudes of general quantum ﬁeld theories. We provided a graph-theoretic deﬁnition of ‘power-counting’ and used it as constructive tool to organize and stratify integrand bases according to their UV behavior. We illustrated our approach by explicitly enumerating all diagram topologies and their associated numerator degrees of freedom for various power-counting at one, two, and three loops. These results provide the number of independent basis elements required to express scattering amplitudes in diﬀerent quantum ﬁeld theories. The same graph-theoretic implementation is in principle applicable to any loop order where the size of the integrand basis grows. We also discussed the limitations and open problems in our approach. In particular, we compared our choices of power-counting with a diﬀerent, and in our opinion ultimately better, framework of poles at inﬁnity. Following this program should lead to elegant new representations of amplitude integrands in a variety of quantum ﬁeld theories. Moreover, since ﬁnding explicit expressions for amplitude integrands has proven to be an essential stepping stone to discovering unexpected properties of integrated amplitudes, we expect that ‘wise’ choices of integrand bases will be ideal tools in further probing the structure of scattering amplitudes. The further exploration along these lines at higher loops is the main open question for the future. Acknowledgments The authors gratefully acknowledge fruitful conversations with Bo Feng, Franz Herzog, Andrew McLeod, Ben Page, J.J. Stankowicz, and Ellis Yuan. This work was performed in part at the Aspen Center for Physics, which is supported by National Science Foundation grant PHY-1607611, and the Harvard Center of Mathematical Sciences and Applications. This project has been supported by an ERC Starting Grant (No. 757978), a grant from the Villum Fonden (No. 15369), and by a grant from the Simons Foundation (341344, LA) (JLB). The research of J.T. is supported in part by U.S. Department of Energy grant DE-SC0009999 and by the funds of University of California. E.H. is supported by the U.S. Department of Energy under contract DE-AC02-76SF00515. – 59 – 16These ‘external’ monovalent vertices are never drawn in our ﬁgures. A Graph theory for integrand basis building In this appendix, we introduce a few formal deﬁnitions that should be useful to better understand the results described in this work. Beyond clarifying terms and their usage, we hope that this list of deﬁnitions and concepts are useful for the sake of precision. In what follows, we describe the main ingredients (mostly graph-theoretic) required in our work. Deﬁnitions are organized loosely by narrative and logical ﬂow. • Deﬁnition: internal edges (‘propagators’) — edges eint ∈Γ of a graph Γ (of edge- connectivity at least 2) which connect pairs of vertices of valency at least two. They are indicated diagrammatically as lines with constant, medium stroke and represent standard Feynman propagators of a scalar ﬁeld theory. Most often, internal edges are colored black, unless subsets of edges are being highlighted to emphasize some additional structure. JHEP11(2020)116 • Deﬁnition: external edges (‘legs’) — edges eext ∈Γ of a graph Γ which connect at least one monovalent vertex.16 To make these edges visually distinct, and to avoid confusion with internal edges deﬁned above, external edges are drawn as wedges. There are two types of external edges, drawn diﬀerently as wedges: There are two types of external edges, drawn diﬀerently as wedges: necessary external edges — drawn as ‘ ’ — those external edges connected to a vertex of valency exactly three. These legs are called ‘necessary’ because they allow us to diﬀerentiate the momentum ﬂowing through pairs of internal edges separated by the 3-valent vertex. These edges are always drawn in our ﬁgures as solid wedges, (with slightly gray coloring) to distinguish them from internal edges. Physically, necessary edges denote any non-empty subset of external particles. As these subsets can be of arbitrary (but never empty) size, we consider graphs necessary external edges — drawn as ‘ ’ — those external edges connected to a vertex of valency exactly three. These legs are called ‘necessary’ because they allow us to diﬀerentiate the momentum ﬂowing through pairs of internal edges separated by the 3-valent vertex. These edges are always drawn in our ﬁgures as solid wedges, (with slightly gray coloring) to distinguish them from internal edges. These edges are always drawn in our ﬁgures as solid wedges, (with slightly gray coloring) to distinguish them from internal edges. Physically, necessary edges denote any non-empty subset of external particles. A Graph theory for integrand basis building The qualiﬁcation in the previous sentence is to clarify that — very occasionally — we will have reason to discuss loop integrands that involve no propa- gators for some or all of the internal loop momenta; for example, we may speak of the ‘L loop integrand’ dL⃗ℓ× 1. In such cases, there are no loop-dependent propagators to draw; but we hope that the ‘loop order’ of such integrands is always clear from the surrounding context. • Deﬁnition: (number of) loops — the ﬁrst Betti number of the graph, unless oth- erwise speciﬁed. The qualiﬁcation in the previous sentence is to clarify that — very occasionally — we will have reason to discuss loop integrands that involve no propa- gators for some or all of the internal loop momenta; for example, we may speak of the ‘L loop integrand’ dL⃗ℓ× 1. In such cases, there are no loop-dependent propagators to draw; but we hope that the ‘loop order’ of such integrands is always clear from the surrounding context. • Deﬁnition: (simple) cycle — a connected subgraph of a given graph with loop number 1. Throughout this work, the term ‘cycle’ should always be understand to mean (what is more formally described as) a simple cycle. • Deﬁnition: (simple) cycle — a connected subgraph of a given graph with loop number 1. Throughout this work, the term ‘cycle’ should always be understand to mean (what is more formally described as) a simple cycle. • Deﬁnition: girth — the length of the shortest cycle of a given graph. • Deﬁnition: cycle basis — for a graph with L loops, a choice of L simple cycles such that every internal edge is an element of at least one cycle. • Deﬁnition: cycle basis — for a graph with L loops, a choice of L simple cycles such that every internal edge is an element of at least one cycle. • Deﬁnition: (loop momentum) routing — a choice of cycle basis for a given graph. We usually take these cycles to be oriented (even though the internal edges do not have any intrinsic orientation). A routing for a graph is equivalent to a choice of loop- momentum variables up to translation (associated with internal degrees of freedom not ﬁxed by momentum conservation at every vertex in the graph). A Graph theory for integrand basis building As these subsets can be of arbitrary (but never empty) size, we consider graphs Physically, necessary edges denote any non-empty subset of external particles. As these subsets can be of arbitrary (but never empty) size, we consider graphs ≃ ≃ (A.1) ≃ ≃ (A.1) (A.1) optional external edges — drawn as ‘ ’ — those external edges connected to (internal) vertices of valency at least four. These are called ‘optional’ because they need not carry any momentum to allow us to diﬀerentiate the momentum ﬂowing through the multiples of internal edges to which they connect. These edges are always drawn in our ﬁgures as ‘sliced’ greyscale wedges to emphasize their optionality. Ph i ll i l d h ld b d d f i b f optional external edges — drawn as ‘ ’ — those external edges connected to (internal) vertices of valency at least four. These are called ‘optional’ because they need not carry any momentum to allow us to diﬀerentiate the momentum ﬂowing through the multiples of internal edges to which they connect. These edges are always drawn in our ﬁgures as ‘sliced’ greyscale wedges to emphasize their optionality. These edges are always drawn in our ﬁgures as ‘sliced’ greyscale wedges to emphasize their optionality. Physically, optional edges should be understood of representing any subset of external particles’ momenta — including empty subsets of external particles. Physically, optional edges should be understood of representing any subset of external particles’ momenta — including empty subsets of external particles. – 60 – • Deﬁnition: skeleton graph eΓ — for a given graph Γ, we deﬁne its skeleton eΓ to be the subgraph obtained by deleting all external edges and vertices. For example, =:Γ 7→eΓ =: . (A.2) (A.2) This concept is useful in the discussion of the parent-daughter covering relations in which we are interested as external edges never carry internal loop momenta and therefore never play any role in numerator decompositions. This concept is useful in the discussion of the parent-daughter covering relations in which we are interested as external edges never carry internal loop momenta and therefore never play any role in numerator decompositions. JHEP11(2020)116 • Deﬁnition: (number of) loops — the ﬁrst Betti number of the graph, unless oth- erwise speciﬁed. A Graph theory for integrand basis building One point that is worth clarifying is that our deﬁnition of loop-momentum routing is slightly broader than what is more common in the physics literature. Frequently physi- cists discuss (especially non-planar, multiloop) integrands’ loop momentum dependence by choosing a subset of L (oriented) edges of the graph whose graph-complement is a 1-forest (a tree graph). Such a choice of edges is always possible, and is understood as dictating that ‘the momentum ﬂowing through edge i of the graph to be loop momentum ℓi’. This convention is considerably more restrictive than what we mean by routing here: not only does the more familiar prescription eliminate the all translational invariance of each loop – 61 – omentum, but it also prevents us from choosing routings such as momentum, but it also prevents us from choosing routings such as with cycle basis          , ,          . (A.3) Covering relations: ‘parents’ and ‘daughters’ (a.k.a. ‘contact-terms’). Graphs may be partially ordered according to quotients generated by internal edge contractions. Thus, we say that Γd ≺Γp if the daughter — Γd can be obtained by contracting some number of (exclusively) internal edges of the parent — graph Γp. To be perhaps overly pedantic, we always use the freedom of (2.2) to delete excess external edges. Alternatively, we could speak exclusively of skeleton graphs. JHEP11(2020)116 This partial ordering on the set of Feynman graphs provides us with covering relations which prove extremely useful. In particular, we use the symbol ∂to denote the set of daughters of a given parent obtained by a single edge-contraction. A skeleton graph Γ with ne internal edges will have ne daughters — that is, \|∂(Γ)\|=ne. In the colloquial vernacular of physicists, daughter graphs are often called contact- terms of their parents. This is because a suﬃciently general space of loop-dependent numerators can include terms proportional to the inverse of any propagator (which are represented by the internal edges of the graphs). Thus, the vector-space of rational diﬀer- ential forms on the space of loop momenta with denominators ﬁxed as Feynman propagators associated with a particular graph include all daughter integrals unless bounds are speciﬁed for loop-dependent factors in the numerators of these integrands. Open Access. A Graph theory for integrand basis building This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] Z. Bern, L.J. Dixon, D.C. Dunbar and D.A. Kosower, One loop n-point gauge theory amplitudes, unitarity and collinear limits, Nucl. Phys. B 425 (1994) 217 [hep-ph/9403226] [INSPIRE]. [2] Z. Bern, L.J. Dixon, D.C. Dunbar and D.A. Kosower, Fusing gauge theory tree amplitudes into loop amplitudes, Nucl. Phys. B 435 (1995) 59 [hep-ph/9409265] [INSPIRE]. [3] R. Britto, F. Cachazo and B. Feng, Generalized unitarity and one-loop amplitudes in N = 4 super-Yang-Mills, Nucl. Phys. B 725 (2005) 275 [hep-th/0412103] [INSPIRE]. [4] Z. Bern, L.J. Dixon and D.A. Kosower, Dimensionally regulated one loop integrals, Phys. Lett. B 302 (1993) 299 [Erratum ibid. 318 (1993) 649] [hep-ph/9212308] [INSPIRE]. [5] Z. Bern, L.J. Dixon and D.A. Kosower, Progress in one loop QCD computations, Ann. Rev. Nucl. Part. Sci. 46 (1996) 109 [hep-ph/9602280] [INSPIRE]. – 62 – [6] Z. Bern, L.J. Dixon and D.A. Kosower, Unitarity based techniques for one loop calculations in QCD, Nucl. Phys. B Proc. Suppl. 51 (1996) 243 [hep-ph/9606378] [INSPIRE]. [7] Z. Bern, L.J. Dixon, D.C. Dunbar and D.A. Kosower, One loop selfdual and N = 4 superYang-Mills, Phys. Lett. B 394 (1997) 105 [hep-th/9611127] [INSPIRE]. [8] Z. Bern, L.J. Dixon, M. Perelstein and J.S. Rozowsky, One loop n-point helicity amplitudes in (selfdual) gravity, Phys. Lett. B 444 (1998) 273 [hep-th/9809160] [INSPIRE]. [9] S.J. Bidder, N.E.J. Bjerrum-Bohr, D.C. Dunbar and W.B. Perkins, One-loop gluon scattering amplitudes in theories with N < 4 supersymmetries, Phys. Lett. B 612 (2005) 75 [hep-th/0502028] [INSPIRE]. JHEP11(2020)116 [10] R. Britto, F. Cachazo and B. Feng, New recursion relations for tree amplitudes of gluons, Nucl. Phys. B 715 (2005) 499 [hep-th/0412308] [INSPIRE]. [11] R. Britto, F. Cachazo, B. Feng and E. Witten, Direct proof of tree-level recursion relation in Yang-Mills theory, Phys. Rev. Lett. 94 (2005) 181602 [hep-th/0501052] [INSPIRE]. [12] J.M. Drummond, J. Henn, V.A. Smirnov and E. Sokatchev, Magic identities for conformal four-point integrals, JHEP 01 (2007) 064 [hep-th/0607160] [INSPIRE]. [13] L.F. Alday and J.M. Maldacena, Gluon scattering amplitudes at strong coupling, JHEP 06 (2007) 064 [arXiv:0705.0303] [INSPIRE]. [14] J.M. Drummond, J. Henn, G.P. Korchemsky and E. Sokatchev, Dual superconformal symmetry of scattering amplitudes in N = 4 super-Yang-Mills theory, Nucl. Phys. B 828 (2010) 317 [arXiv:0807.1095] [INSPIRE]. [15] N. Beisert et al., Review of AdS/CFT Integrability: An Overview, Lett. Math. Phys. 99 (2012) 3 [arXiv:1012.3982] [INSPIRE]. [16] N. Arkani-Hamed, J.L. Bourjaily, F. Cachazo, A.B. Goncharov, A. Postnikov and J. References Trnka, Grassmannian Geometry of Scattering Amplitudes, Cambridge University Press (2016) [DOI] [arXiv:1212.5605] [INSPIRE]. [17] N. Arkani-Hamed and J. Trnka, The Amplituhedron, JHEP 10 (2014) 030 [arXiv:1312.2007] [INSPIRE]. [18] Z. Bern and A.G. Morgan, Massive loop amplitudes from unitarity, Nucl. Phys. B 467 (1996) 479 [hep-ph/9511336] [INSPIRE]. [19] Z. Bern and Y.-t. Huang, Basics of Generalized Unitarity, J. Phys. A 44 (2011) 454003 [arXiv:1103.1869] [INSPIRE]. [20] R. Ellis, Z. Kunszt, K. Melnikov and G. Zanderighi, One-loop calculations in quantum ﬁeld theory: from Feynman diagrams to unitarity cuts, Phys. Rept. 518 (2012) 141 [arXiv:1105.4319] [INSPIRE]. [21] G. Ossola, C.G. Papadopoulos and R. Pittau, Reducing full one-loop amplitudes to scalar integrals at the integrand level, Nucl. Phys. B 763 (2007) 147 [hep-ph/0609007] [INSPIRE]. [22] P. Mastrolia, E. Mirabella and T. Peraro, Integrand reduction of one-loop scattering amplitudes through Laurent series expansion, JHEP 06 (2012) 095 [Erratum ibid. 11 (2012) 128] [arXiv:1203.0291] [INSPIRE]. [23] P. Mastrolia, E. Mirabella, G. Ossola and T. Peraro, Multiloop Integrand Reduction for Dimensionally Regulated Amplitudes, Phys. Lett. B 727 (2013) 532 [arXiv:1307.5832] [INSPIRE]. – 63 – [24] R.H.P. Kleiss, I. Malamos, C.G. Papadopoulos and R. Verheyen, Counting to One: Reducibility of One- and Two-Loop Amplitudes at the Integrand Level, JHEP 12 (2012) 038 [arXiv:1206.4180] [INSPIRE]. [25] B. Feng and R. Huang, The classiﬁcation of two-loop integrand basis in pure four-dimension, JHEP 02 (2013) 117 [arXiv:1209.3747] [INSPIRE]. [26] J.L. Bourjaily, E. Herrmann and J. Trnka, Prescriptive Unitarity, JHEP 06 (2017) 059 [arXiv:1704.05460] [INSPIRE]. [27] P. Tourkine, Integrands and loop momentum in string and ﬁeld theory, Phys. Rev. D 102 (2020) 026006 [arXiv:1901.02432] [INSPIRE]. JHEP11(2020)116 [28] R. Ben-Israel, A.G. Tumanov and A. Sever, Scattering amplitudes — Wilson loops duality for the ﬁrst non-planar correction, JHEP 08 (2018) 122 [arXiv:1802.09395] [INSPIRE]. [29] E. Herrmann and J. Trnka, Gravity On-shell Diagrams, JHEP 11 (2016) 136 [arXiv:1604.03479] [INSPIRE]. [30] E. Herrmann and J. Trnka, UV cancellations in gravity loop integrands, JHEP 02 (2019) 084 [arXiv:1808.10446] [INSPIRE]. [31] A. Edison, E. Herrmann, J. Parra-Martinez and J. Trnka, Gravity loop integrands from the ultraviolet, arXiv:1909.02003 [INSPIRE]. [32] G. ’t Hooft and M.J.G. Veltman, Regularization and Renormalization of Gauge Fields, Nucl. Phys. B 44 (1972) 189 [INSPIRE]. [33] A.V. Smirnov and A.V. Petukhov, The Number of Master Integrals is Finite, Lett. Math. Phys. 97 (2011) 37 [arXiv:1004.4199] [INSPIRE]. [34] A.V. Kotikov, Diﬀerential equations method: New technique for massive Feynman diagrams calculation, Phys. Lett. References B 254 (1991) 158 [INSPIRE]. [35] T. Gehrmann and E. Remiddi, Diﬀerential equations for two loop four point functions, Nucl. Phys. B 580 (2000) 485 [hep-ph/9912329] [INSPIRE]. [36] J.M. Henn, Multiloop integrals in dimensional regularization made simple, Phys. Rev. Lett. 110 (2013) 251601 [arXiv:1304.1806] [INSPIRE]. [37] N. Arkani-Hamed, J.L. Bourjaily, F. Cachazo and J. Trnka, Singularity Structure of Maximally Supersymmetric Scattering Amplitudes, Phys. Rev. Lett. 113 (2014) 261603 [arXiv:1410.0354] [INSPIRE]. [38] Z. Bern, E. Herrmann, S. Litsey, J. Stankowicz and J. Trnka, Logarithmic Singularities and Maximally Supersymmetric Amplitudes, JHEP 06 (2015) 202 [arXiv:1412.8584] [INSPIRE]. [39] Z. Bern, E. Herrmann, S. Litsey, J. Stankowicz and J. Trnka, Evidence for a Nonplanar Amplituhedron, JHEP 06 (2016) 098 [arXiv:1512.08591] [INSPIRE]. [40] J.L. Bourjaily, E. Herrmann and J. Trnka, Maximally supersymmetric amplitudes at inﬁnite loop momentum, Phys. Rev. D 99 (2019) 066006 [arXiv:1812.11185] [INSPIRE]. [41] D.B. Melrose, Reduction of Feynman Diagrams, Nuovo Cim. 40 (1965) 181. [42] G. Passarino and M.J.G. Veltman, One Loop Corrections for e+e−Annihilation Into µ+µ− in the Weinberg Model, Nucl. Phys. B 160 (1979) 151 [INSPIRE]. [43] Z. Bern, L.J. Dixon and D.A. Kosower, One loop amplitudes for e+e−to four partons, Nucl. Phys. B 513 (1998) 3 [hep-ph/9708239] [INSPIRE]. – 64 – [44] P. Mastrolia, G. Ossola, T. Reiter and F. Tramontano, Scattering AMplitudes from Unitarity-based Reduction Algorithm at the Integrand-level, JHEP 08 (2010) 080 [arXiv:1006.0710] [INSPIRE]. [45] W.L. van Neerven and J.A.M. Vermaseren, Large loop integrals, Phys. Lett. B 137 (1984) 241 [INSPIRE]. [46] A. Denner and S. Dittmaier, Reduction of one loop tensor ﬁve point integrals, Nucl. Phys. B 658 (2003) 175 [hep-ph/0212259] [INSPIRE]. [47] A. Denner and S. Dittmaier, Reduction schemes for one-loop tensor integrals, Nucl. Phys. B 734 (2006) 62 [hep-ph/0509141] [INSPIRE]. [48] F. Cachazo, Sharpening The Leading Singularity, arXiv:0803.1988 [INSPIRE]. JHEP11(2020)116 [49] S. Badger, H. Frellesvig and Y. Zhang, An Integrand Reconstruction Method for Three-Lo Amplitudes, JHEP 08 (2012) 065 [arXiv:1207.2976] [INSPIRE]. [50] P. Mastrolia, E. Mirabella, G. Ossola and T. Peraro, Scattering Amplitudes from Multivariate Polynomial Division, Phys. Lett. B 718 (2012) 173 [arXiv:1205.7087] [INSPIRE]. [51] S. Badger, H. Frellesvig and Y. Zhang, Multi-loop Integrand Reduction with Computational Algebraic Geometry, J. Phys. Conf. Ser. 523 (2014) 012061 [arXiv:1310.4445] [INSPIRE]. [52] P. Mastrolia, T. Peraro and A. Primo, Adaptive Integrand Decomposition in parallel and orthogonal space, JHEP 08 (2016) 164 [arXiv:1605.03157] [INSPIRE]. [53] H. Ita, Two-loop Integrand Decomposition into Master Integrals and Surface Terms, Phys. References Rev. D 94 (2016) 116015 [arXiv:1510.05626] [INSPIRE]. [54] J.M. Drummond, J.M. Henn and J. Plefka, Yangian symmetry of scattering amplitudes in N = 4 super Yang-Mills theory, JHEP 05 (2009) 046 [arXiv:0902.2987] [INSPIRE]. [55] C.F. Berger et al., An Automated Implementation of On-Shell Methods for One-Loop Amplitudes, Phys. Rev. D 78 (2008) 036003 [arXiv:0803.4180] [INSPIRE]. [56] J.L. Bourjaily, Eﬃcient Tree-Amplitudes in N = 4: Automatic BCFW Recursion in Mathematica, arXiv:1011.2447 [INSPIRE]. [57] Y. Zhang, Lecture Notes on Multi-loop Integral Reduction and Applied Algebraic Geometry, 12, 2016 [arXiv:1612.02249] [INSPIRE]. [58] K.G. Chetyrkin and F.V. Tkachov, Integration by Parts: The Algorithm to Calculate β-functions in 4 Loops, Nucl. Phys. B 192 (1981) 159 [INSPIRE]. [59] F.V. Tkachov, A Theorem on Analytical Calculability of Four Loop Renormalization Group Functions, Phys. Lett. B 100 (1981) 65 [INSPIRE]. [60] T. Peraro, Scattering amplitudes over ﬁnite ﬁelds and multivariate functional reconstruction, JHEP 12 (2016) 030 [arXiv:1608.01902] [INSPIRE]. [61] S. Abreu, J. Dormans, F. Febres Cordero, H. Ita, B. Page and V. Sotnikov, Analytic Form of the Planar Two-Loop Five-Parton Scattering Amplitudes in QCD, JHEP 05 (2019) 084 [arXiv:1904.00945] [INSPIRE]. [62] T. Peraro, FiniteFlow: multivariate functional reconstruction using ﬁnite ﬁelds and dataﬂow graphs, JHEP 07 (2019) 031 [arXiv:1905.08019] [INSPIRE]. [63] H. Johansson, D.A. Kosower and K.J. Larsen, Maximal Unitarity for the Four-Mass Double Box, Phys. Rev. D 89 (2014) 125010 [arXiv:1308.4632] [INSPIRE]. – 65 – [64] A.V. Smirnov, Algorithm FIRE — Feynman Integral REduction, JHEP 10 (2008) 107 [arXiv:0807.3243] [INSPIRE]. [65] S. Weinberg, Six-dimensional Methods for Four-dimensional Conformal Field Theories, Phys. Rev. D 82 (2010) 045031 [arXiv:1006.3480] [INSPIRE]. [66] D. Simmons-Duﬃn, Projectors, Shadows, and Conformal Blocks, JHEP 04 (2014) 146 [arXiv:1204.3894] [INSPIRE]. [67] D.A. Kosower and K.J. Larsen, Maximal Unitarity at Two Loops, Phys. Rev. D 85 (2012) 045017 [arXiv:1108.1180] [INSPIRE]. [68] S. Badger, H. Frellesvig and Y. Zhang, A Two-Loop Five-Gluon Helicity Amplitude in QCD, JHEP 12 (2013) 045 [arXiv:1310.1051] [INSPIRE]. JHEP11(2020)116 JHEP11(2020)116 [69] L. Brink, J.H. Schwarz and J. Scherk, Supersymmetric Yang-Mills Theories, Nucl. Phys. B 121 (1977) 77 [INSPIRE]. [70] F. Gliozzi, J. Scherk and D.I. Olive, Supersymmetry, Supergravity Theories and the Dual Spinor Model, Nucl. Phys. B 122 (1977) 253 [INSPIRE]. [71] Z. Bern, L.J. Dixon and R. Roiban, Is N = 8 supergravity ultraviolet ﬁnite?, Phys. Lett. B 644 (2007) 265 [hep-th/0611086] [INSPIRE]. [72] N.E.J. Bjerrum-Bohr, D.C. Dunbar, H. Ita, W.B. Perkins and K. References Risager, The No-Triangle Hypothesis for N = 8 Supergravity, JHEP 12 (2006) 072 [hep-th/0610043] [INSPIRE]. [73] N.E.J. Bjerrum-Bohr and P. Vanhove, Absence of Triangles in Maximal Supergravity Amplitudes, JHEP 10 (2008) 006 [arXiv:0805.3682] [INSPIRE]. [74] J.L. Bourjaily, S. Caron-Huot and J. Trnka, Dual-Conformal Regularization of Infrared Loop Divergences and the Chiral Box Expansion, JHEP 01 (2015) 001 [arXiv:1303.4734] [INSPIRE]. [75] J.L. Bourjaily, A.J. McLeod, M. von Hippel and M. Wilhelm, Rationalizing Loop Integration, JHEP 08 (2018) 184 [arXiv:1805.10281] [INSPIRE]. [76] J.L. Bourjaily, A.J. McLeod, C. Vergu, M. Volk, M. Von Hippel and M. Wilhelm, Rooting Out Letters: Octagonal Symbol Alphabets and Algebraic Number Theory, JHEP 02 (2020) 025 [arXiv:1910.14224] [INSPIRE]. [77] J.L. Bourjaily, F. Dulat and E. Panzer, Manifestly Dual-Conformal Loop Integration, Nucl. Phys. B 942 (2019) 251 [arXiv:1901.02887] [INSPIRE]. [78] J.L. Bourjaily, M. Volk and M. Von Hippel, Conformally Regulated Direct Integration of the Two-Loop Heptagon Remainder, JHEP 02 (2020) 095 [arXiv:1912.05690] [INSPIRE]. [79] J.L. Bourjaily, E. Herrmann, C. Langer, A.J. McLeod and J. Trnka, Prescriptive Unitarity for Non-Planar Six-Particle Amplitudes at Two Loops, JHEP 12 (2019) 073 [arXiv:1909.09131] [INSPIRE]. [80] J.L. Bourjaily, E. Herrmann, C. Langer, A.J. McLeod and J. Trnka, All-Multiplicity Nonplanar Amplitude Integrands in Maximally Supersymmetric Yang-Mills Theory at Two Loops, Phys. Rev. Lett. 124 (2020) 111603 [arXiv:1911.09106] [INSPIRE]. [81] A. Hodges, Eliminating spurious poles from gauge-theoretic amplitudes, JHEP 05 (2013) 135 [arXiv:0905.1473] [INSPIRE]. [82] J.C. Collins, Renormalization: An Introduction to Renormalization, The Renormalization Group, and the Operator Product Expansion, vol. 26 of Cambridge Monographs on Mathematical Physics, Cambridge University Press, Cambridge (1986) [DOI] [INSPIRE]. – 66 – [83] Z. Bern, J.J.M. Carrasco, H. Johansson and D.A. Kosower, Maximally supersymmetric planar Yang-Mills amplitudes at ﬁve loops, Phys. Rev. D 76 (2007) 125020 [arXiv:0705.1864] [INSPIRE]. [84] W. Siegel, Supersymmetric Dimensional Regularization via Dimensional Reduction, Phys. Lett. B 84 (1979) 193 [INSPIRE]. [85] Z. Bern, A. De Freitas, L.J. Dixon and H.L. Wong, Supersymmetric regularization, two loop QCD amplitudes and coupling shifts, Phys. Rev. D 66 (2002) 085002 [hep-ph/0202271] [INSPIRE]. [86] Z. Bern, L.J. Dixon and D.A. Kosower, A Two loop four gluon helicity amplitude in QCD, JHEP 01 (2000) 027 [hep-ph/0001001] [INSPIRE]. JHEP11(2020)116 [87] J.L. Bourjaily and K. Patatoukos, On Dimensionally Regulated One Loop Integrands, in progress. [88] Z. Bern, J.S. Rozowsky and B. Yan, Two loop four gluon amplitudes in N = 4 superYang-Mills, Phys. Lett. B 401 (1997) 273 [hep-ph/9702424] [INSPIRE]. [89] Z. Bern, L.J. References Dixon and V.A. Smirnov, Iteration of planar amplitudes in maximally supersymmetric Yang-Mills theory at three loops and beyond, Phys. Rev. D 72 (2005) 085001 [hep-th/0505205] [INSPIRE]. [90] Z. Bern, J.J. Carrasco, L.J. Dixon, H. Johansson, D.A. Kosower and R. Roiban, Three-Loop Superﬁniteness of N = 8 Supergravity, Phys. Rev. Lett. 98 (2007) 161303 [hep-th/0702112] [INSPIRE]. [91] Z. Bern, J.J.M. Carrasco, L.J. Dixon, H. Johansson and R. Roiban, Manifest Ultraviolet Behavior for the Three-Loop Four-Point Amplitude of N = 8 Supergravity, Phys. Rev. D 78 (2008) 105019 [arXiv:0808.4112] [INSPIRE]. [92] Z. Bern, J.J.M. Carrasco, L.J. Dixon, H. Johansson and R. Roiban, The Complete Four-Loop Four-Point Amplitude in N = 4 Super-Yang-Mills Theory, Phys. Rev. D 82 (2010) 125040 [arXiv:1008.3327] [INSPIRE]. [93] J.J.M. Carrasco and H. Johansson, Five-Point Amplitudes in N = 4 Super-Yang-Mills Theory and N = 8 Supergravity, Phys. Rev. D 85 (2012) 025006 [arXiv:1106.4711] [INSPIRE]. [94] Z. Bern, J.J.M. Carrasco, H. Johansson and R. Roiban, The Five-Loop Four-Point Amplitude of N = 4 super-Yang-Mills Theory, Phys. Rev. Lett. 109 (2012) 241602 [arXiv:1207.6666] [INSPIRE]. [95] Z. Bern, J.J.M. Carrasco, W.-M. Chen, H. Johansson, R. Roiban and M. Zeng, Five-loop four-point integrand of N = 8 supergravity as a generalized double copy, Phys. Rev. D 96 (2017) 126012 [arXiv:1708.06807] [INSPIRE]. [96] Z. Bern et al., Ultraviolet Properties of N = 8 Supergravity at Five Loops, Phys. Rev. D 98 (2018) 086021 [arXiv:1804.09311] [INSPIRE]. [97] S. Abreu et al., Two-Loop Four-Graviton Scattering Amplitudes, Phys. Rev. Lett. 124 (2020) 211601 [arXiv:2002.12374] [INSPIRE]. [98] J. Gluza, K. Kajda and D.A. Kosower, Towards a Basis for Planar Two-Loop Integrals, Phys. Rev. D 83 (2011) 045012 [arXiv:1009.0472] [INSPIRE]. [99] R. Baumeister, D. Mediger, J. Pečovnik and S. Weinzierl, Vanishing of certain cuts or residues of loop integrals with higher powers of the propagators, Phys. Rev. D 99 (2019) 096023 [arXiv:1903.02286] [INSPIRE]. – 67 – [100] N. Arkani-Hamed, J.L. Bourjaily, F. Cachazo, S. Caron-Huot and J. Trnka, The All-Loop Integrand For Scattering Amplitudes in Planar N = 4 SYM, JHEP 01 (2011) 041 [arXiv:1008.2958] [INSPIRE]. [101] N. Arkani-Hamed, J.L. Bourjaily, F. Cachazo and J. Trnka, Local Integrals for Planar Scattering Amplitudes, JHEP 06 (2012) 125 [arXiv:1012.6032] [INSPIRE]. [102] J.L. Bourjaily and J. Trnka, Local Integrand Representations of All Two-Loop Amplitudes in Planar SYM, JHEP 08 (2015) 119 [arXiv:1505.05886] [INSPIRE]. [103] M. Sogaard and Y. References Zhang, Unitarity Cuts of Integrals with Doubled Propagators, JHEP 07 (2014) 112 [arXiv:1403.2463] [INSPIRE]. [104] S. Abreu, F. Febres Cordero, H. Ita, M. Jaquier, B. Page and M. Zeng, Two-Loop Four-Gluon Amplitudes from Numerical Unitarity, Phys. Rev. Lett. 119 (2017) 142001 [arXiv:1703.05273] [INSPIRE]. JHEP11(2020)116 JHEP11(2020)116 [105] J.L. Bourjaily, A.J. McLeod, M. Spradlin, M. von Hippel and M. Wilhelm, Elliptic Double-Box Integrals: Massless Scattering Amplitudes beyond Polylogarithms, Phys. Rev. Lett. 120 (2018) 121603 [arXiv:1712.02785] [INSPIRE]. [106] J.L. Bourjaily, Y.-H. He, A.J. Mcleod, M. Von Hippel and M. Wilhelm, Traintracks through Calabi-Yau Manifolds: Scattering Amplitudes beyond Elliptic Polylogarithms, Phys. Rev. Lett. 121 (2018) 071603 [arXiv:1805.09326] [INSPIRE]. [107] J.L. Bourjaily, A.J. McLeod, M. von Hippel and M. Wilhelm, Bounded Collection of Feynman Integral Calabi-Yau Geometries, Phys. Rev. Lett. 122 (2019) 031601 [arXiv:1810.07689] [INSPIRE]. [108] J.L. Bourjaily, A.J. McLeod, C. Vergu, M. Volk, M. Von Hippel and M. Wilhelm, Embedding Feynman Integral (Calabi-Yau) Geometries in Weighted Projective Space, JHEP 01 (2020) 078 [arXiv:1910.01534] [INSPIRE]. [109] C. Vergu and M. Volk, Traintrack Calabi-Yaus from Twistor Geometry, JHEP 07 (2020) 160 [arXiv:2005.08771] [INSPIRE]. [110] Z. Bern, M. Enciso, C.-H. Shen and M. Zeng, Dual Conformal Structure Beyond the Planar Limit, Phys. Rev. Lett. 121 (2018) 121603 [arXiv:1806.06509] [INSPIRE]. [111] D. Chicherin, J.M. Henn and E. Sokatchev, Implications of nonplanar dual conformal symmetry, JHEP 09 (2018) 012 [arXiv:1807.06321] [INSPIRE]. [112] J.L. Bourjaily, Positroids, Plabic Graphs, and Scattering Amplitudes in Mathematica, arXiv:1212.6974 [INSPIRE]. [113] N. Arkani-Hamed, J.L. Bourjaily, F. Cachazo, A. Postnikov and J. Trnka, On-Shell Structures of MHV Amplitudes Beyond the Planar Limit, JHEP 06 (2015) 179 [arXiv:1412.8475] [INSPIRE]. [114] J.L. Bourjaily, S. Franco, D. Galloni and C. Wen, Stratifying On-Shell Cluster Varieties: the Geometry of Non-Planar On-Shell Diagrams, JHEP 10 (2016) 003 [arXiv:1607.01781] [INSPIRE]. [115] G.F. Sterman, An Introduction to Quantum Field Theory, Cambridge University Press (1993). [116] F. Brown and C. Duhr, A double integral of d log forms which is not polylogarithmic, 6, 2020 [arXiv:2006.09413] [INSPIRE]. [117] J.G.M. Gatheral, Exponentiation of Eikonal Cross-sections in Nonabelian Gauge Theories, Phys. Lett. B 133 (1983) 90 [INSPIRE]. – 68 – [118] J. Frenkel and J.C. Taylor, Nonabelian eikonal exponentiation, Nucl. Phys. B 246 (1984) 231 [INSPIRE]. [119] S. Catani, The Singular behavior of QCD amplitudes at two loop order, Phys. Lett. B 427 (1998) 161 [hep-ph/9802439] [INSPIRE]. [120] G.F. Sterman and M.E. Tejeda-Yeomans, Multiloop amplitudes and resummation, Phys. Lett. B 552 (2003) 48 [hep-ph/0210130] [INSPIRE]. [121] S. References Aybat, L.J. Dixon and G.F. Sterman, The Two-loop soft anomalous dimension matrix and resummation at next-to-next-to leading pole, Phys. Rev. D 74 (2006) 074004 [hep-ph/0607309] [INSPIRE]. [122] L.J. Dixon, L. Magnea and G.F. Sterman, Universal structure of subleading infrared poles in gauge theory amplitudes, JHEP 08 (2008) 022 [arXiv:0805.3515] [INSPIRE]. JHEP11(2020)116 [123] E. Gardi and L. Magnea, Factorization constraints for soft anomalous dimensions in QCD scattering amplitudes, JHEP 03 (2009) 079 [arXiv:0901.1091] [INSPIRE]. [124] T. Becher and M. Neubert, Infrared singularities of scattering amplitudes in perturbative QCD, Phys. Rev. Lett. 102 (2009) 162001 [Erratum ibid. 111 (2013) 199905] [arXiv:0901.0722] [INSPIRE]. [125] O. Almelid, C. Duhr and E. Gardi, Three-loop corrections to the soft anomalous dimension in multileg scattering, Phys. Rev. Lett. 117 (2016) 172002 [arXiv:1507.00047] [INSPIRE]. [126] A. von Manteuﬀel, E. Panzer and R.M. Schabinger, A quasi-ﬁnite basis for multi-loop Feynman integrals, JHEP 02 (2015) 120 [arXiv:1411.7392] [INSPIRE]. [127] P. Wasser, Analytic Properties of Feynman Integrals for Scattering Amplitudes, Ph.D. Thesis, Mainz U. (2018) [INSPIRE]. [128] J. Henn, B. Mistlberger, V.A. Smirnov and P. Wasser, Constructing d log integrands and computing master integrals for three-loop four-particle scattering, JHEP 04 (2020) 167 [arXiv:2002.09492] [INSPIRE]. [129] Z. Bern, L.J. Dixon and D.A. Kosower, Dimensionally regulated pentagon integrals, Nucl. Phys. B 412 (1994) 751 [hep-ph/9306240] [INSPIRE]. [130] Z. Bern and G. Chalmers, Factorization in one loop gauge theory, Nucl. Phys. B 447 (1995) 465 [hep-ph/9503236] [INSPIRE]. [131] J.B. Tausk, Nonplanar massless two loop Feynman diagrams with four on-shell legs, Phys. Lett. B 469 (1999) 225 [hep-ph/9909506] [INSPIRE]. [132] S. Caron-Huot and K.J. Larsen, Uniqueness of two-loop master contours, JHEP 10 (2012) 026 [arXiv:1205.0801] [INSPIRE]. [133] M.F. Paulos, M. Spradlin and A. Volovich, Mellin Amplitudes for Dual Conformal Integrals, JHEP 08 (2012) 072 [arXiv:1203.6362] [INSPIRE]. [134] D. Nandan, M.F. Paulos, M. Spradlin and A. Volovich, Star Integrals, Convolutions and Simplices, JHEP 05 (2013) 105 [arXiv:1301.2500] [INSPIRE]. [135] J.M. Drummond, J.M. Henn and J. Trnka, New diﬀerential equations for on-shell loop integrals, JHEP 04 (2011) 083 [arXiv:1010.3679] [INSPIRE]. [136] C.G. Papadopoulos, D. Tommasini and C. Wever, The Pentabox Master Integrals with the Simpliﬁed Diﬀerential Equations approach, JHEP 04 (2016) 078 [arXiv:1511.09404] [INSPIRE]. – 69 – [137] T. Gehrmann, J.M. Henn and N.A. Lo Presti, Pentagon functions for massless planar scattering amplitudes, JHEP 10 (2018) 103 [arXiv:1807.09812] [INSPIRE]. [138] J.L. Bourjaily, A. DiRe, A. Shaikh, M. Spradlin and A. References Volovich, The Soft-Collinear Bootstrap: N = 4 Yang-Mills Amplitudes at Six and Seven Loops, JHEP 03 (2012) 032 [arXiv:1112.6432] [INSPIRE]. [139] J.L. Bourjaily, P. Heslop and V.-V. Tran, Perturbation Theory at Eight Loops: Novel Structures and the Breakdown of Manifest Conformality in N = 4 Supersymmetric Yang-Mills Theory, Phys. Rev. Lett. 116 (2016) 191602 [arXiv:1512.07912] [INSPIRE]. [140] J.L. Bourjaily, P. Heslop and V.-V. Tran, Amplitudes and Correlators to Ten Loops Using Simple, Graphical Bootstraps, JHEP 11 (2016) 125 [arXiv:1609.00007] [INSPIRE]. JHEP11(2020)116 – 70 –
https://openalex.org/W2740805769	http://periodicos.uem.br/ojs/index.php/ActaSciTechnol/article/download/29672/pdf	Latin	null	<b>Mechanical analysis of a shear-cracked RC beam	Acta Scientiarum. Technology/Acta scientiarum. Technology	2,017	cc-by	4,133	Acta Scientiarum Acta Scientiarum http://www.uem.br/acta ISSN printed: 1806-2563 ISSN on-line: 1807-8664 Doi: 10.4025/actascitechnol.v39i3.29672 http://www.uem.br/acta ISSN printed: 1806-2563 ISSN on-line: 1807-8664 Doi: 10 4025/actascitechno Análise mecânica de uma viga de concreto armado rompida por cisalhamento RESUMO. A análise de cisalhamento é essencial para que os engenheiros estimem o trabalho de reparo em estruturas de concreto. Até agora, análises teóricas e numéricas convencionais em mecânica da fratura foram aplicadas para estudar vigas de flexão de concreto, mas ainda há pouco conhecimento sobre a capacidade de cisalhamento de vigas com um entalhe inicial em diagonal. Este estudo apresenta uma análise teórica para obter resistência à fratura em uma viga de concreto armado de quatro pontos com dois entalhes iniciais inclinados em suportes. Aqui, adota-se uma abordagem de fissura fictícia para estimar o efeito equivalente da zona de processo de fratura (ZPF) do concreto em cisalhamento. Com base na equação de equilíbrio na secção transversal do entalhe da viga, a força de cisalhamento foi expressa em termos de comprimento da zona de processo de fratura. Em seguida, numa viga de quatro pontos com entalhe duplo, determina-se o modo II do fator de intensidade de tensão devido à carga externa. Finalmente, este processo apresenta a relação entre a capacidade de cisalhamento e o comprimento da zona de processo de fratura e expressa a resistência à fratura em função do comprimento da zona de processo de fratura. Os resultados previstos das capacidades de carga são então apresentados. Palavras-chave: ZPF, fratura, cisalhamento, intensidade de tensão. Shahriar Shahbazpanahi Shahriar Shahbazpanahi Department of Civil Engineering, Sanandaj Branch, Islamic Azad University, 66169 Pasdaran St. Sanandaj, Kurdistan, Iran. Em sh.shahbazpanahi@gmail.com; sh.shahbazpanahi@iausdj.ac.ir ABSTRACT. Shear crack analysis is essential for engineers to estimate repair work in concrete structures. So far, conventional theoretical and numerical analyses in fracture mechanics have been applied to study concrete flexural beams, but there is still little knowledge regarding the shear capacity for beams with a diagonal initial notch. In this study, a theoretical analysis is presented to obtain fracture resistance in a four- point RC beam with two inclined initial notch on supports. Here, a fictitious crack approach is adopted for estimating the equivalent effect of the fracture process zone of concrete in shear-cracked. Based on equilibrium equation in the beam notch cross section, shear force was expressed in terms of fracture process zone length. Then, in a double notch four-point beam, Mode II of the stress intensity factor due to the external load is determined. Finally, this process presents the relationship between the shear capacity and the fracture process zone length and expresses the fracture resistance as a function of fracture process zone length.The predicted results of loading capacities are then shown. Introduction has the ability to transfer normal and shear stresses to close the crack (Shahbazpanahi, Ali, Aznieta, Kamgar, & Farzadnia, 2013b; Dong, Wu, Zhou, Dong, & Kastiukas, 2017). The closure stress associated with cracks is a maximum at the tip of the FPZ and decreases to zero at the continuous crack tip where the crack opening reaches its critical value, beyond which an open crack forms (Dong et al., 2016). Since a significant amount of energy is stored in the FPZ, a crack can have stable growth before the peak load (Simon & Kishen, 2017). The energy consumed in this process is the fracture energy required for creating a new crack surface (Brake & Chatti, 2013; Dai & Ng, 2014; Ohno, Uhi, Ueno, & Ohtsu, 2014). Fracture mechanics is a branch of solid mechanics, which deals with the behavior of the material and conditions in the vicinity of a crack (Shahbazpanahi, Ali, Aznieta, Kamgar, & Farzadnia, 2013a).The concept of fracture mechanics was first used for pre-cracked concrete structures in the early 1960s (Shahbazpanahi, Abang, Kamgar, & Farzadnia, 2014). Concrete structures are prone to crack propagation. As of now, the fracture process has become a fundamental concept in fracture mechanics of concrete. The first investigation of concrete structure based on fracture mechanics was conducted proposed by Hillerborg, Modeer, & Petersson (1976). This study introduced a region, often termed fracture process zone (FPZ), which Based on the theory of linear elastic fracture mechanics (LEFM), a fairly high coefficient is Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Acta Scientiarum. Technology 286 Shahbazpanahi applied to the stress in the vicinity of the crack tip. This coefficient is called stress intensity factor. The LEFM converts stress to a unique form of distribution. The stress intensity factor depends on the material properties, on the size of the crack, on the load, and on the geometry of the structure. This factor presents a relationship between the material and the reaction of the structure. Numerous researchers have aimed to calculate stress intensity factor in concrete structure using experimental, analytical and numerical methods (Dong, He, & Wu, 2011; Ooi & Yang, 2011; Ray & Kishen, 2014). mechanics theory, this beam is called four-point loading RC beam. Shear cracks appear along the shear span in an intermediate position between the loading point and the support depending on the amount of longitudinal reinforcement. Introduction Initially, a shear crack follows a vertical trajectory and then turns toward the loading point. However, in the present study, shear crack is induced using an initial notch in the support. Given that the shear force is large and the flexural moment in the support is small, this moment can be ignored when analysing shear cracks in Mode II. The shear crack angle has an important effect on the shear capacity and on the fracture resistance. Actual shear crack angles are seldom reported, but an angle of 45° was used to design RC be arms. Thus, the assumption that the shear crack is at 45° and starts from the support is not far from reality. Figure 1 shows a beam with two inclined initial notches at the shear crack angle of 45º on the support. The four- point loading RC beam has a rectangular cross- section, and the length of the initial notch is a0. Figure 2 indicates that crack grows to ‘a’ due to the extended FPZ if load is increased, where lp, a(σ=0) and ‘a’ are the FPZ length, the stress-free region length and the effective crack length, respectively. The shear strength Vn of the beam is given by (Equation 1): There have also been numerous studies on predicting the influence of FPZ (Xu, Wu, Zheng, Zhao, & Liu, 2011; Wu, Rong, Zheng, & Xu, 2011; Guo, Su, & Young, 2012) and on flexural crack using different methods. However, the effect of FPZ on shear crack has not been studied. Studying stress intensity factors in shear cracks is particularly significant as concrete is usually weak in shear. The present theoretical study focuses on stress intensity factor in RC beam with double incline notches to determine the critical crack length and the fracture toughness in a shear crack. Here, the objective is to develop of an analytical method to calculate the effect of FPZ on shear cracks in RC beam based on the fictitious crack approach. In the present study, a four-point load RC beam with two incline initial notches on supports is analysed to obtain shear capacity.In linear fracture mechanics, the FPZ at the tip of the crack is considered as the base of a fictitious crack to obtain shear stress distribution in the crack cross section. Material and methods ‘fc’ is the compressive strength, ‘fc’ is the compressive strength, ‘fc’ is the compressive strength, Acta Scientiarum. Technology Introduction Interaction between the shear stress distribution and the normal stress in damage zone is used to estimate the cohesive stress of the fracture. Based on the equilibrium equation in the notch cross-section, the change of the shear force versus the FPZ length are determined. Then, Mode II of the stress intensity factor in a double notch four-point beam due to the external load is determined. The fracture resistance of the material is expressed in terms of the fracture toughness of the plain concrete and the shear stress due to the FPZ. Finally, the relationship between the shear capacity and the FPZ length is used to express the fracture resistance as a function of the FPZ length. VN = VC + VFPZ (1) (1) where: VC and VFPZ are the shear strength contributions of the concrete; FPZ, respectively. where: VC and VFPZ are the shear strength contributions of the concrete; FPZ, respectively. Figure 1. Four-point loading RC beam with two notch’s. Figure 1. Four-point loading RC beam with two notch’s. For members under flexure and shear load, the following simple formula for shear strength contribution of the concrete, (American Concrete Institute, 2007) is used (Equation 2): Vc=0.1ටfc ' bh (2) (2) where: Fracture resistance of crack Figure 2. Across section at the cracked and stress distribution. Since shear force prevails the flexure moment in the shear span, a sliding mode (Mode II) is developed. In the present study, Mode II is used to formulate the fracture resistance of the crack. A crack will grow if the stress intensity factor, due to the external load, ܭ௣಺಺ reaches the fracture resistance of the material, ܭோ಺಺, i.e., (Equation 7) The normal force due to the cohesive fracture zone is expressed as: ܨ= න ݍ൫ݓ(௫)൯ܾ ݀ݔ ௔ ௔ି௟೛ (3) (3) Where: q(w(x)) is the distribution of the stress in the FPZ and w(x) is one-half of the crack opening where the origin of x lies (Figure 2). This means that the normal stress depends on the opening of the crack and the damage zone is still capable to sustain load. Although several complex distributions of stress are available, an simple linear curve is used in the present study. However, an exact expressions of the FPZ stress is especially not easy and the use of these functions are limited. Where: q(w(x)) is the distribution of the stress in the FPZ and w(x) is one-half of the crack opening where the origin of x lies (Figure 2). This means that the normal stress depends on the opening of the crack and the damage zone is still capable to sustain load. Although several complex distributions of stress are available, an simple linear curve is used in the present study. However, an exact expressions of the FPZ stress is especially not easy and the use of these functions are limited. ܭ௣಺಺= ܭோ಺಺ (7) (7) ܭ௣಺಺= ܭோ಺಺ The stress intensity factor of Mode II for the double notch four-point beam due to the external load P, is given by (Guryao, 1996) (Equation 8): ܭ௣಺಺= ܲ ܾℎଵଶ ⁄ ݂(ܽℎ ⁄ ) (8) (8) Where ‘b’ and f (a/h) are the thickness of the beam and the geometric shape function, respectively. In which (Equation 9) On the other hand, the interaction between the shear stress distribution and the normal stress in the damage zone was derived by Jefferson (2002) and Koutromanos and Shing (2012). Equilibrium equation of the shear-cracked beam ‘h’ and ‘b’ are the depth and the thickness of the beam, respectively. Equation (2) is the expression for concrete shear strength with reinforcement. Let us consider a simply supported RC beam under two concentrated load. In the fracture Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Acta Scientiarum. Technology Analysis of a shear-cracked beam 287 the beam is obtained based on the shear strength of concrete, and the shear and normal stresses provided by the cohesive zone. Fracture resistance of the crack will be obtained in the next section by applying the Equation (6). With the assumption that the cross-section in mid-span is un-cracked, the deflection can be obtained by conventional formula for a four-point load beam with simple support. Figure 2. Across section at the cracked and stress distribution. Acta Scientiarum. Technology Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Fracture resistance of crack ݂ቀܽℎ ൗቁ= ൤ −3.40(ܽℎ ⁄ )ସ+ 15.78 (ܽℎ ⁄ )ଷ −16.04(ܽℎ ⁄ )ଶ+ 9.70(ܽℎ ⁄ ) −0.85൨ (9) (9) ߬= ݎቀ݂௧−ݍ൫ݓ(௫)൯ቁ ଶ ଷ (4) (4) The material fracture resistance is given by (Equation 10): Where: ft is tensile strength of concrete and the factor ‘r’ is given by (Equation 5): ܭோ಺಺= ܭ஼಺಺+ ܭ்಺಺ (10) (10) ݎ= 0.78݂௖ ᇱଵ/ଷ (5) ݎ= 0.78݂௖ ᇱଵ/ଷ (5) where ܭ஼಺಺ and ܭ்಺಺ are the plain concrete fracture toughness for Mode II and the toughness due to the shear stress of the fracture process zone, respectively. The shear stress toughness is given by (Zheng, Dai, & Fan, 2016) (Equation 11): Equation (4) was obtained by computing the strength envelope of the consecutive Mohr’s circles of stress. The load ‘P’ applied to the beam with equilibrium equation is expressed as: ܲ= 0.1ඥ݂௖ ᇱܾℎ + 0.707[න ݍ൫ݓ(௫)൯ܾ݀ݔ ௔ ௔ି௟೛ +ݎܾන ቀ݂௧− ݍ൫ݓ(௫)൯ቁ ଶ ଷ ௔ ௔ି௟೛ ݀ݔ ] (6) ܭ்ூூ= ߬√ߨܽܩ(ܽℎ ൗ) (11) (11) (6) ܩ(ܽℎ ⁄ ) = 1.122 −0.561(ܽℎ ⁄ ) + 0.085(ܽℎ ⁄ )ଶ+ 0.18(ܽℎ ⁄ )ଷ ට1 −ܽℎ ൗ (12) Using Equation (6), the shear capacity can be determined when ݈௣is known. The shear capacity of (12) Acta Scientiarum. Technology 288 288 Shahbazpanahi with 6882 C3DBR S4R (average size of 15 X 15 mm) elements. The fracture toughness is the fracture resistance at a special crack length. Thus, the critical crack length is defined numerically by (Equation 13): mm) elements. Figure 3. Comparison load vs. deflection in experimental (Walraven, 1978), ABAQUS software and present model. Figure 4. Crack paths of the beam by ABAQUS software. Figures 5 and 6 show the effects of various parameters, such as the FPZ length and the compressive strength of concrete, on the fracture resistance. Figure 5 shows the relationship between fracture resistance and the effective crack length Figure 3. Comparison load vs. deflection in experimental (Walraven, 1978), ABAQUS software and present model. ߲ܭ௣಺಺ ߲ܽ = ߲ܭோ಺಺ ߲ܽ (13) (13) Substituting Equation (8 and 10) into Equation (7 and 13) and then solving the equation, the critical crack length can be obtained. The fracture toughness can be determined using Equation (10) or (11) if the critical crack length is known. Results and discussions A simply supported beam without shear reinforcement under four-point bending is considered to validate the proposed theoretical method (Walraven, 1978). This shear beam has since become a benchmark for crack propagation analysis using FEM. The length, the depth, the width, the concrete modulus of elasticity, and the compressive strength are 3520 mm, 450 mm, 200 mm, 28 Gpa and 34.2 Mpa , respectively. The steel bars have 2100 GPa and 600 mm2 elastic modulus and cross- sectional area, respectively. The plain concrete fracture toughness for shear is 5.5 Mpa mm0.5. Figure 3. Comparison load vs. deflection in experimental (Walraven, 1978), ABAQUS software and present model. Figure 4. Crack paths of the beam by ABAQUS software. Figure 3 shows a comparison of the experimental and the proposed theoretical method between the load ‘P’ and the deflection at mid-span with the assumption that a0 = 0. To calculate the deflection at mid-span, the section is assumed to be un- cracked. The deflection at mid-span of the beam is calculated by the basic principles of mechanics of elastic structures. Therefore, deflection at mid-span is: Figure 4. Crack paths of the beam by ABAQUS software. Figures 5 and 6 show the effects of various parameters, such as the FPZ length and the compressive strength of concrete, on the fracture resistance. Figure 5 shows the relationship between fracture resistance and the effective crack length with three FPZ lengths. The fracture resistance increases with the effective crack length and the FPZ length. The increase in the fracture resistance was slow at the initial stage. Fracture resistance increases quickly as the effective crack length increases because extra load was needed to prevent shear crack growth. Crack propagation was arrested after its growth to a certain position. This finding indicates that, when the effective crack length turns to the beam depth, the fracture resistance approaches infinity. Moreover, the FPZ has a significant role in the fracture toughness of the shear crack. Fracture resistance increases as the FPZ length increases. The FPZ has a significant role in arresting the shear crack similar to the flexural crack. δ= 23PL2 72EI (14) (14) where: ‘I’ is the moment of inertia of the transformed section. The result of the present study was slightly higher than the experimental observation (approximately 8%). Acta Scientiarum. Technology Results and discussions This error was acceptable because crushing, nonlinear behavior of the bulk concrete, yielding of the reinforcement, the bond slip between the concrete and steel, and the plastic deformation were neglected in the theoretical method. The result of the ABAQUS FEA software was considerably higher than the experimental observation (16 to 27%). In the early stage, yielding occurs at approximately 20 k N in the experimental result, but not in the present model. Figure 4 illustrates finite element mesh the crack path modelled by the ABAQUS software for the beam where: ‘I’ is the moment of inertia of the transformed section. The result of the present study was slightly higher than the experimental observation (approximately 8%). This error was acceptable because crushing, nonlinear behavior of the bulk concrete, yielding of the reinforcement, the bond slip between the concrete and steel, and the plastic deformation were neglected in the theoretical method. The result of the ABAQUS FEA software was considerably higher than the experimental observation (16 to 27%). In the early stage, yielding occurs at approximately 20 k N in the experimental result, but not in the present model. Figure 4 illustrates finite element mesh the crack path modelled by the ABAQUS software for the beam Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Acta Scientiarum. Technology Analysis of a shear-cracked beam 289 Figure 5. Fracture resistance versus effective crack length and influence FPZ length. in preventing the propagation of the shear crack because it prevents the propagation of flexural crack. Figure 7 shows the variation of the load, which is given as a the stress-free region length in the current study with different concrete compressive strengths at ݈௣= 270 mm. The load increases with the decrease in the compressive strength when the FPZ length fully develops; this finding is similar to that in the flexure crack. Moreover, when FPZ length fully develops (݈௣= 270 mm), the length of the stress-free region appears ahead of the notch tip and expands to the top of the beam (Wu et al., 2011). Figure 7. Load vs. stress-free region length with different concrete compressive strength. Figure 5. Fracture resistance versus effective crack length and influence FPZ length. Figure 6 shows the fracture resistance as the FPZ length varied with different compressive strengths of the concrete for the shear crack. Fracture toughness increases with compressive strength concrete based on the FPZ length. Results and discussions Figure 6 Fracture resistance versus FPZ length with different compressive strength Figure 7. Load vs. stress-free region length with different concrete compressive strength. Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Acta Scientiarum. Technology References American Concrete Institute [ACI]. (2007). Building code requirements for structural concrete (ACI 318). Armington Hills, MI: ACI. Shahbazpanahi, S., Abang, A., Kamgar, A., & Farzadnia, N. (2014). Fracture mechanic modeling of fiber reinforced polymer shear-strengthened reinforced concrete beam. Composites Part B Engineering, 68(1), 113-120. Brake, N. A., & Chatti, K. (2013). Prediction of size effect and non-linear crack growth in plain concrete under fatigue loading. Engineering Fracture Mechanics, 109(1), 169-185. Dai, Q., & Ng, K. (2014). 2D cohesive zone modeling of crack development in cementitious digital samples with microstructure characterization. Construction and Building Materials, 54(1), 584-595. Shahbazpanahi, S., Ali, A. A., Aznieta, F. N., Kamgar, A., & Farzadnia, N. (2013a). A simple and practical model for FRP-reinforced cracked beam. European Journal of Environmental and Civil Engineering, 19(1), p. 293-306. Dong, W., He, H. N., & Wu, Z. M. (2011). Experimental and numerical study on mode I-II crack propagation for small size specimens of concrete. Advanced Materials Research. 163-167(1), 908-912. Shahbazpanahi, S., Ali, A. A., Aznieta, F. N., Kamgar, A., & Farzadnia, N. (2013b). Modelling of the fracture process zone to improve the crack propagation criterion in concrete. Journal of the South African Institution of Civil Engineering, 55(3), 2-9. Dong, W., Wu, Z., Zhou, X., Dong , L., & Kastiukas, G. (2017). FPZ evolution of mixed mode fracture in concrete: Experimental and numerical. Engineering Failure Analysis. 75, 54-70. Simon, K. M., & Kishen, J. M. (2017). A multiscale approach for modeling fatigue crack growth in concrete. International Journal of Fatigue, 98, 1-13. Dong, W., Yang, D., Kastiukas, G., & Zhang, B. (2016). Experimental and numerical investigations on fracture process zone of rock–concrete interface. Fatigue and Fracture of Engineering Materials and Structures. 40(5), 820-835. Walraven, J. (1978). The infuence of depth on the shear strength of lightweight concrete beams without shear reinforcement. Delf, NL: Delft University of Technology. Wu, Z., Rong, H., Zheng, J., & Xu, F. (2011). An experimental investigation on the FPZ properties in concrete using digital image correlation technique. Engineering Fracture Mechanics, 78(17), 2978-2990. Guo, X., Su, R. K., & Young, B. (2012). Numerical investigation of the bilinear softening law in the cohesive crack model for normal-strength and high- strength concrete. Advances in Structural Engineering, 15(3), 373-388. Xu, F., Wu, Z., Zheng, J., Zhao, Y., & Liu, K. (2011). Crack extension resistance curve of concrete considering variation of FPZ length. Journal of Materials in Civil Engineering, 23(5), 703-710. License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received on October 30, 2015. Accepted on August 5, 2016. Conclusion A new theoretical procedure is proposed for determining the shear capacity and the fracture resistance of shear–cracked concrete beams. Based on the equilibrium equation in the notch cross- section, the shear force is expressed in terms of the FPZ length. The fracture toughness of the RC beam is calculated based on the linear fracture mechanics. Based on the equilibrium equation in the notch cross-section, shear force is expressed in terms of the FPZ length. Then, Mode II of the stress intensity factor in double notch four-point beam due to the external load is determined. The fracture resistance of the materials is explained by the plain concrete fracture resistance and the shear stress due to the FPZ. Finally, the relationship between the shear capacity and the FPZ length is used to express the fracture resistance as a function of the FPZ length. It is observed that the results in the present study are slightly (approximately 8%) higher than those of previous experimental observations. It is also observed that the FPZ had a positive and significant influence on the fracture toughness and Figure 6 Fracture resistance versus FPZ length with different compressive strength The growth was 8.5 to 22%. For example, if the compressive strength of the concrete increases from 34.2 to 40 MPa, then the fracture toughness will increase from 157.1 MPa mm1/2 to 191.8 MPa mm1/2. When the compressive strength increases to 40 MPa, the fracture resistance initially increases insignificantly and then increases rapidly. This finding was partially verified by a theoretical method in the fracture for the flexure crack. Thus, the FPZ played a significant role in increasing the fracture resistance of shear crack. The FPZ plays a major role Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 Acta Scientiarum. Technology 290 Shahbazpanahi under three-point bending by acoustic emission. Construction and Building Materials, 67(1), 139-145. in preventing the propagation of shear cracks. The proposed method considers the effect of the geometric parameters and shows acceptable similarity to the experimental data ABAQS software data. Ooi, E. T., & Yang, Z. J. (2011). Modelling crack propagation in reinforced concrete using a hybrid finite element–scaled boundary finite element method. Engineering Fracture Mechanics, 78(2), 252-273. Ray, S., & Kishen, J. M. (2014). Analysis of fatigue crack growth in reinforced concrete beams. Materials and Structures/Materiaux et Constructions, 47 (1), 183-198. Maringá, v. 39, n. 3, p. 285-290, July-Sept., 2017 References Guryao, W. (1996). Study of mode II fracture of rock and its engineering application. Changsha, CN: Centeral South Univercity of Technology . Zheng, J., J., Dai, J., G. & Fan, X. L. (2016). Fracture analysis of FRP-plated notched concrete beams subjected to three-point bending. Journal of Engineering Mechanics, 142(3). Doi: 10.1061/(ASCE)EM.1943- 7889.0001021 Hillerborg, A., Modeer, M., & Petersson, P. (1976 ). Analysis of crack formation and crack growth in concrete by means of mechanics and finite element. Cement and Concrete Research, 6(1), 773-782. Jefferson, D. (2002). Tripartite cohesive crack model. Journal of Engineering Mechanics, 128 (6), 644-653. Koutromanos, I., & Shing, P. (2012). Cohesive crack model to simulate cyclic response of concrete and masonry structures. ACI Structural Journal, 109 (3), 349-358. Ohno, K., Uji, K., Ueno, A., & Ohtsu, M. (2014 ). Fracture process zone in notched concrete beam Acta Scientiarum. Technology
https://openalex.org/W2619794261	https://link.springer.com/content/pdf/10.1007%2Fs00415-017-8518-8.pdf	English	null	Iodinated contrast agents in patients with myasthenia gravis: a retrospective cohort study	Journal of neurology	2,017	cc-by	5,733	& Fritz Zimprich friedrich.zimprich@meduniwien.ac.at J Neurol (2017) 264:1209–1217 DOI 10.1007/s00415-017-8518-8 J Neurol (2017) 264:1209–1217 DOI 10.1007/s00415-017-8518-8 ORIGINAL COMMUNICATION Iodinated contrast agents in patients with myasthenia gravis: a retrospective cohort study Jakob Rath1 • Matthias Mauritz1 • Gudrun Zulehner1 • Eva Hilger1 • Hakan Cetin1 • Gregor Kasprian2 • Eduard Auff1 • Fritz Zimprich1 Received: 11 March 2017 / Revised: 14 May 2017 / Accepted: 15 May 2017 / Published online: 26 May 2017 The Author(s) 2017. This article is an open access publication Abstract Currently, it has not been satisfactorily estab- lished, whether modern low-osmolality iodinated contrast agents (ICAs) used in computed tomography (CT) studies are a risk factor for exacerbation of myasthenic symptoms. The rate of acute adverse events as well as delayed clinical worsening up to 30 days were analyzed in 73 patients with conﬁrmed myasthenia gravis (MG) who underwent con- trast-enhanced CT studies and compared to 52 patients who underwent unenhanced CT studies. One acute adverse event was documented. 12.3% of MG patients experienced a delayed exacerbation of symptoms after ICA adminis- tration. The rate of delayed severe exacerbation was higher in the contrast-enhanced group. Alternative causes for the exacerbation of MG-related symptoms were more likely than ICA administration in all cases. ICA administration for CT studies in MG patients should not be withheld if indicated, but patients particularly those with concomitant acute diseases should be carefully monitored for exacer- bation of symptoms. Abbreviations CI Conﬁdence interval CT Computed tomography ICA Iodinated contrast agent MG Myasthenia gravis MGFA Myasthenia Gravis Foundation of America MuSK Muscle-speciﬁc tyrosine kinase OR Odds ratio SD Standard deviation 1 Department of Neurology, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, 1090 Vienna, Austria 2 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, 1090 Vienna, Austria & Fritz Zimprich friedrich.zimprich@meduniwien.ac.at 1 Department of Neurology, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, 1090 Vienna, Austria 2 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Wa¨hringer Gu¨rtel 18-20, 1090 Vienna, Austria Data analysis In summary, there are still too few data available to conﬁdently judge the risk that modern ICAs pose for patients with myasthenia gravis, especially with regard to a delayed exacerbation of myasthenic symptoms. Since ICAs are frequently required in many different indications in modern day medicine, there is an urgent need to better evaluate this risk. This point was recently stressed by a report on this issue by the Myasthenia Gravis Foundation of America (MGFA), which called for more data to be generated on this topic [2]. Baseline demographic and all clinical characteristics were retrospectively extracted from medical records (Table 1). Baseline MGFA state was deﬁned as MGFA class at the time of CT study. The primary endpoint was deﬁned as a clinically relevant deterioration of myasthenic symptoms within 30 days of the CT study, deﬁned as clinical wors- ening by at least one MGFA class. Subgroup analysis was performed for patients with an increase of the MGFA state by at least one class but not fulﬁlling the criteria of myasthenic crisis (i.e., MGFA class V) and for patients with a severe worsening deﬁned by the occurrence of a myasthenic crisis with respiratory insufﬁciency and intu- bation or death within the post-scan observation period. Secondary endpoints were (a) the occurrence of an immediate, acute adverse reaction as documented in the radiological report (b) in the case of reaching the primary endpoint the time (in days) to clinical deterioration after ICA administration. The aim of this study was to add more information on this question by retrospectively examining adverse event rates and delayed exacerbations of myasthenic symptoms after administration of modern low-osmolality ICAs in patients with conﬁrmed myasthenia gravis. Introduction Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disease of the neuromuscular junction char- acterized by muscle weakness and abnormal fatigability. Prevalence rates of around 16 per 100,000 [1] mean all branches of medicine care for patients with myasthenia gravis. One feared complication of myasthenia gravis is a clinical deterioration upon a wide range of reported drugs. While the risk posed by some medications (e.g., quino- lones) is well documented [2], conﬂicting results have been published regarding the exacerbation of myasthenic symptoms following the administration of iodinated con- trast agents (ICAs) used for computed tomography (CT) [3, 4]. Keywords Iodinated contrast agent Myasthenia gravis Computed tomography Adverse events Myasthenic crisis Anaphylaxis ICAs can be divided by their osmolality. Older sub- stances were of very high osmolality (above 1500 mOsm/ L) and are no longer used routinely because of their comparatively high toxicity. Modern ICAs, in contrast, are low-osmolality agents (290–860 mOsm/L) and generally better tolerated [5]. MG-unrelated acute adverse events (e.g., anaphylactoid reactions, contrast-induced nephropa- thy, or extravasation of contrast agent) were estimated to occur in up to 3% of all patients receiving low-osmolality 123 12 3 3 1210 J Neurol (2017) 264:1209–1217 By reviewing the medical charts of these patients, we next identiﬁed 73 (31 male, 42 female, and median age 62) who underwent CT scans with the administration of low- osmolality ICAs at the Department of Biomedical Imaging and Image-guided Therapy within our hospital between 2005 and 2015. A non-overlapping control group of 52 patients (25 male, 27 female, and median age 64) consisted of patients with myasthenia gravis who underwent only unenhanced CT studies during the same period (Fig. 1). Only one (i.e., the ﬁrst) CT scan was considered for each patient to avoid double inclusions into any of the study groups. CT studies were, furthermore, only included if a sufﬁcient clinical documentation was available to assess the clinical state of patients before and for 30 days after the CT study. Exclusion criteria were congenital myasthenia gravis, concomitant serious renal disease, and an age of less than 18 years. Ethical approval was obtained from the institutional ethics committee and the requirement to obtain patient consent was waived for this retrospective study. contrast agents, though only a small fraction of these are classiﬁed as severe [6]. Introduction Delayed adverse events (predomi- nately cutaneous reactions appearing up to 1 week after administration) are estimated by some to be as high as 14% [7]. Myasthenic crisis following the administration of old, high-osmolality ICAs was initially reported in the 1980s in a number of case reports [8–13]. One systematic retro- spective study on acute adverse events (within 24 h) associated with high-osmolality agents presented ﬁve patients with a subsequent exacerbation of their myasthenic weakness, although in each case, alternative explanations for the deterioration other than ICA administration were possible [14]. Immediate adverse reactions including myasthenic exacerbations were reported in less than 1% of patients in one recent study on low-osmolality ICAs [4]. Another study on low-osmolality agents observed an exacerbation rate of 12.5% in MG patients within 45 days of ICA administration [3]. Patients Statistical analysis was performed with the SPSS 22 software package (IBM Corp. Released 2013. IBM SPSS Statistics for Macintosh, Version 22.0. Armonk, NY: IBM Corp). Baseline variables between the two groups of patients were compared using the Mann–Whitney U or Student’s t test (for continuous variables) and the Fisher’s exact test or Chi-square test (for categorical variables). Post hoc power calculations were calculated using an online tool [15]. 426 consecutive patients (186 male, 240 female) with conﬁrmed myasthenia gravis who were treated at the Department of Neurology of the Medical University of Vienna were retrospectively identiﬁed and considered for inclusion into this study. All patients were required to meet the standard diagnostic criteria consisting of the typical clinical symptoms in combination with either a positive test for myasthenia gravis-speciﬁc autoantibod- ies [acetylcholine receptor or muscle-speciﬁc kinase (MuSK)], a typical decrement ([10%) shown by repeti- tive nerve stimulation or a positive edrophonium chloride test. Univariate and multivariate logistic regression analyses were used to compare the cumulative primary endpoint between the groups. Covariates were selected if judged to be clinically meaningful. For the multivariate analysis, these were: age, disease duration, MGFA class, indication 123 1211 J Neurol (2017) 264:1209–1217 J Neurol (2017) 264:1209–1217 Fig. 1 Flowchart of study population. CT computed tomography Fig. 1 Flowchart of study population. CT computed tomography for CT scan, and concomitant acute disease. Subgroups of patients reaching the primary endpoint were compared using the Fisher’s exact test. Time to primary endpoint was compared using the log-rank test and Kaplan–Meier curves were calculated with censoring after 30 days. Binominal 95% conﬁdence intervals were calculated using Clopper– Pearson intervals. A P value of B0.05 was considered statistically signiﬁcant. Nine patients (12.3%) in the contrast-enhanced and two patients (3.8%) in the unenhanced CT group reached the primary endpoint of worsening of myasthenic symptoms within 30 days of the scans (Table 2). We did not ﬁnd a statistically signiﬁcant difference for the chances of this event between the two study groups (Table 2), though it should be noted that the sample size only afforded us a power of 0.37 for the detection of an 8.5% difference between the groups at a signiﬁcance level of 0.05. Multivariate analysis revealed signiﬁcant effects of age and MGFA class for the occurrence of the primary end- point. The administration of ICAs had no effect in this analysis (Table 2). Results The baseline characteristics (Table 1) for most variables were well matched between patients with contrast-en- hanced CT studies and unenhanced CT studies. The two groups differed, however, in the indication for the CT study and the body region scanned as well in the presence of an acute concomitant disease. The mean time to worsening within 30 days did not differ signiﬁcantly between the two study groups and was 11.1 days for patients with contrast-enhanced CT studies and 13 days in the control group (Table 2; Fig. 2). We detected only one patient who reached the secondary endpoint of an acute worsening of symptoms immediately after administration of a contrast agent documented in the radiological report. The event was described as a transient increase of preexisting dyspnea for which CT scanning was performed. Scans of chest and abdomen were performed more often with contrast agents in comparison with scans of the head and other regions. Indications for the scans in the contrast-enhanced group were more often dyspnea and other, non-neurological, acute symptoms, and concomi- tant non-neurological acute diseases were accordingly more frequent in the contrast-enhanced group. The type of contrast agents could not be extracted retrospectively from the available data in 61.6% of patients, Jopamiro 300 was used in 24.7%, Jopamiro 370 in 2.7%, and Iomeron 400 in 11% of patients. The mean dose of administered ICA in the contrast-enhanced group was 101.7 mL (SD 22.4 mL) and 103.3 mL (SD 18.7 mL) in patients reaching the primary endpoint. The volume could not be extracted in one patient. The subgroup analysis of patients having reached the primary endpoint was limited because of the small absolute numbers. Still, a comparison between the two groups regarding the proportion of patients experiencing a severe worsening (i.e., myasthenic crisis or death) reached nominal signiﬁcance suggesting a greater risk for patients in the ICAs group. In fact, a severe exacerbation of symptoms was observed for six patients in the contrast-enhanced CT group but not at all in the control group (Table 2). Of these six patients, two died and four developed a myasthenic crisis. Results 12 3 123 J Neurol (2017) 264:1209–1217 1212 Table 1 Baseline characteristics CT study All patients Patients reaching primary endpoint Contrast-enhanced CT scans, N = 73 Controls (unenhanced CT scans), N = 52 P value* Contrast-enhanced CT scans, N = 9 Controls (unenhanced scans), N = 2 Sex 0.534 Male 31 (42.5%) 25 (48.1%) 2 (22.2%) 1 (50%) Female 42 (57.5%) 27 (51.9%) 7 (78.8%) 1 (50%) Age (median) 62 (range 79) 64 (range 77) 0.389 69 (range 21) 64 (range 0) Antibodies 0.679 AChR 60 (82.2%) 40 (76.9%) 9 (100%) 2 (100%) MuSK 2 (2.7%) 2 (3.8%) 0 0 Negative 10 (13.7%) 10 (19.2%) 0 0 No data 1 (1.4%) 0 0 0 Disease duration (median) 18 months (range 486) 24 months (range 528) 0.755 5 months (range 486) 1 months (range 2) MGFA class 0.404 CSR/PR/MM 11 (15.1%) 13 (25%) 1 (11.1%) 0 1 14 (19.2%) 12 (23.1%) 0 0 2 23 (31.5%) 17 (32.7%) 1 (11.1%) 1 (50%) 3 20 (27.4%) 7 (13.5%) 5 (55.6%) 1 (50%) 4 3 (4.1%) 1 (1.9%) 1 (11.1%) 0 5 2 (2.7%) 2 (3.8% 1 (11.1% 0 Thymectomy 37 (50.7%) 19 (36.5%) 0.117 3 (33.3%) 0 Thymic pathology 0.442 Normal 10 (13.7% 3 (5.8%) 2 0 Hyperplasia 9 (12.3%) 6 (11.5%) 0 0 Thymoma 12 (16.4% 8 (15.4%) 1 0 No histology 6 (8.2%) 2 (3.8%) 0 0 Therapy No Therapy 12 (16.4%) 10 (19.2%) 0.686 3 (33.3%) 0 Cholinesterase inhibitor 58 (79.5%) 38 (73.1%) 0.405 6 (66.7%) 2 (100%) Cortisone 29 (39.7%) 21 (40.44%) 0.941 0 0 Immunosuppressant 20 (27.4%) 8 (15.4%) 0.112 1 (11.1%) 0 IVIG 1 (1.4%) 4 (7.7%) 0.159 1 (11.1%) 1 (50%) Plasma exchange 8 (11%) 4 (7.7%) 0.76 1 (11.1%) 1 (50%) Repetitive nerve stimulation 0.814 Positive 33 (45.2%) 22 (42.3%) 5 (55.6%) 1 (50%) Negative 20 (27.4%) 13 (25%) 2 (20.2%) 1 (50%) No data 20 (27.4%) 17 (32.7%) 2 (20.2%) 0 Edrophonium test 0.915 Positive 37 (50.7%) 28 (53.8%) 5 (55.6%) 1 (50%) Negative 10 (13.7%) 6 (11.5%) 1 (11.1%) 1 (50%) No data 26 (35.6%) 18 (34.6%) 3 (33.3%) 0 Concomitant acute diseases at CT 0.038 None 46 (63%) 44 (84.6%) 3 (33.3%) 2 (100%) Cardiac 2 (2.7%) 2 (3.8%) 1 (11.1%) Pulmonary (non- neuromuscular) 6 (8.2%) 0 2 (22.2%) 1 Table 1 Baseline characteristics 12 1213 J Neurol (2017) 264:1209–1217 Table 1 continued CT study All patients Patients reaching primary endpoint Contrast-enhanced CT scans, N = 73 Controls (unenhanced CT scans), N = 52 P value* Contrast-enhanced CT scans, N = 9 Controls (unenhanced CT scans), N = 2 Neurological (not MG related) 1 (1.4%) 1 (1.9%) Other 18 (24.7%) 5 (9.6%) 3 (33.3%) Indication \0.000 Thymus evaluation 31 (42.5%) 19 (36.5%) 2 (22.2%) 2 (100%) Focal neurological symptoms 7 (9.6%) 12 (23.1%) 1 (11.1%) 0 Dyspnea 12 (16.4%) 0 1 (11.1%) 0 Acute non- neurological symptoms 21 (28.8%) 4 (7.7%) 5 (55.6%) 0 Chronic disease/ symptoms 2 (2.7%) 8 (15.4%) 0 Trauma 0 9 (17.3%) 0 0 Region \0.000 Chest 41 (56.2%) 22 (42.3%) 6 (66.7%) 2 (100%) Abdomen 11 (15.1%) 3 (5.8%) 1 (11.1%) 0 Chest/abdomen 11 (15.1%) 0 2 (22.2%) 0 Head/CT angiography 5 (6.8%) 17 (32.7%) 0 0 Other 5 (6.8%) 10 (19.2%) 0 0 B li h t i ti f ll ti t d f th h h d th i d i t AChR acetylcholine receptor, MuSK muscle-speciﬁc tyrosine kinase, IVIG intravenous immunoglobulin, SD standard deviation, ICA iodinated contrast agent, CSR complete stable remission, PR pharmacologic remission, MM minimal manifestation, NA not applicable * P values were obtained with the Mann–Whitney U or Student’s t test (for continuous variables) and with the Fisher’s exact test or Chi-square test (for categorical variables) as appropriate St ti ti ll i iﬁ t Statistically signiﬁcant Baseline characteristics of all patients and of those who reached the primary endpoint Case summaries of patients reaching the primary endpoint Chest CT was performed for evaluation of thymic pathol- ogy and revealed no pathological ﬁndings. Aggravation of symptoms occurred 23 days after CT scan, most likely reﬂecting the natural course of the disease with insufﬁcient therapy (only cholinesterase inhibitors at the time). After repeated plasmaphereses (PLEX) treatment and initiation of prednisolone therapy, the patient subsequently improved. Chest CT was performed for evaluation of thymic pathol- ogy and revealed no pathological ﬁndings. Aggravation of symptoms occurred 23 days after CT scan, most likely reﬂecting the natural course of the disease with insufﬁcient therapy (only cholinesterase inhibitors at the time). After repeated plasmaphereses (PLEX) treatment and initiation of prednisolone therapy, the patient subsequently improved. In the control group, two patients showed clinical wors- ening 14 and 12 days after unenhanced CT of the chest was performed for thymus evaluation in new onset MG. Clin- ical deterioration was attributed to the natural course of the disease under insufﬁcient therapy. To differentiate between ICA being a possible cause of the clinical worsening or just an innocent, merely asso- ciated factor in a developing deterioration for other rea- sons, a detailed clinical analysis of the nine patients in the contrast-enhanced group was performed (Table 3). All nine patients had antibodies against acetylcholine recep- tor. None of these patients was the contrast agent con- sidered, on clinical grounds, a likely cause of the deterioration: Patient 2 Chest CT was performed in this 68-year-old man with new onset MG for thymus evaluation and was without relevant pathological ﬁndings. The patient initially presented with progressive weakness of limb and ocular and bulbar muscles. Cholinesterase inhibitors and repeated PLEX were started, but myasthenic crisis still developed (7 days after the CT scan), presumably as part of the nat- ural course of the disease compounded by aspiration pneumonia. After high prednisolone treatment and subse- quent intravenous immunoglobulins and eventually thymectomy, the patient improved. Patient 1 MG was diagnosed in this 77-year-old woman with initially mild weakness of oropharyngeal muscles. Case summaries of patients reaching the primary endpoint 12 3 J Neurol (2017) 264:1209–1217 1214 Table 2 Results Contrast-enhanced CT scans (N = 73) Unenhanced CT scans (N = 52) P value** Acute reaction 1 (1.4%) NA NA Primary endpoint 9 (12.3%); 95% CI 5.8–– 22.1%* 2 (3.8%); 95% CI 0.5–13.2%* Univariate analyses: P = 0.12 (OR 3.52, 95% CI 0.73–17.0) Subtypes of endpoint Severe (death or myasthenic crisis) 6 (8.2%) (4 myasthenic crisis, 2 deaths) 0 0.04 C1 increase in MGFA class but not myasthenic crisis or death) 3 (4.1%) 2 (3.8%) 1.00 Time to primary endpoint 11.1 days (SD 8.6) 13 days (SD 1.4) 0.10 Primary and secondary endpoints, as well as time to primary endpoint MGFA Myasthenia Gravis Foundation of America, OR odds ratio, CI conﬁdence interval, SD standard deviatio * Binominal 95% conﬁdence intervals were calculated using Clopper–Pearson intervals * Binominal 95% conﬁdence intervals were calculated using Clopper–Pearson intervals ** P values were obtained with univariate or multivariate (adjusted for age, disease duration, MGFA class, indication, and concomitant acute di ) l i i i l f h i d i i h h Fi h f b i d h l k f i * Binominal 95% conﬁdence intervals were calculated using Clopper–Pearson intervals ** P values were obtained with univariate or multivariate (adjusted for age, disease duration, MGFA class, indication, and concomitant acute disease) logistic regression analyses for the primary endpoint, with the Fisher exact test for subgroups comparison and the log-rank test for time to primary endpoint Patient 3 MG was newly diagnosed in this 73-year-old man with initially moderate weakness of oropharyngeal muscles. CT of the neck and chest was performed because of dysphagia and for evaluation of thymic pathology (without relevant pathological ﬁndings). Myasthenic crisis occurred 11 days after CT scan, most likely reﬂecting the natural course of the disease with insufﬁcient therapy (only cholinesterase inhibitors at the time). The patient improved subsequently after repeated PLEX was performed and therapy with prednisolone as well as azathioprine was started. Patient 4 Chest CT was performed because of progres- sive dyspnea in this 69-year-old woman who additionally suffered from severe oropharyngeal and to a lesser extent limb weakness. She had received high-dose prednisolone (1000 mg) 2 days and diazepam the day before because of Fig. Case summaries of patients reaching the primary endpoint 2 Kaplan–Meier curves for the primary endpoint of patients undergoing contrast- enhanced and unenhanced CT scans Patient 3 MG was newly diagnosed in this 73-year-old man with initially moderate weakness of oropharyngeal muscles. CT of the neck and chest was performed because of dysphagia and for evaluation of thymic pathology (without relevant pathological ﬁndings). Myasthenic crisis occurred 11 days after CT scan, most likely reﬂecting the natural course of the disease with insufﬁcient therapy (only cholinesterase inhibitors at the time). The patient improved Patient 3 MG was newly diagnosed in this 73-year-old man with initially moderate weakness of oropharyngeal muscles. CT of the neck and chest was performed because of dysphagia and for evaluation of thymic pathology (without relevant pathological ﬁndings). Myasthenic crisis occurred 11 days after CT scan, most likely reﬂecting the natural course of the disease with insufﬁcient therapy (only cholinesterase inhibitors at the time). The patient improved Patient 3 MG was newly diagnosed in this 73-year-old man with initially moderate weakness of oropharyngeal muscles. CT of the neck and chest was performed because of dysphagia and for evaluation of thymic pathology (without relevant pathological ﬁndings). Myasthenic crisis occurred 11 days after CT scan, most likely reﬂecting the natural course of the disease with insufﬁcient therapy (only cholinesterase inhibitors at the time). The patient improved subsequently after repeated PLEX was performed and therapy with prednisolone as well as azathioprine was started. subsequently after repeated PLEX was performed and therapy with prednisolone as well as azathioprine was started. Patient 4 Chest CT was performed because of progres- sive dyspnea in this 69-year-old woman who additionally suffered from severe oropharyngeal and to a lesser extent limb weakness. Case summaries of patients reaching the primary endpoint Pneumonia and pleural effusions 10 64; m IIA IVA Chest, unenhanced New onset MG, thymus evaluation 14 Progressive muscular exhaustion within the natural course of disease 11 64; f IIIB IVB Chest, unenhanced New onset MG, thymus evaluation 12 Natural course of the disease under insufﬁcient therapy (only cholinesterase inhibitors) Characteristics of patients with delayed clinical worsening within 30 days after contrast-enhanced CT studies (number 1–9) and after unenhanced CT studies (number 10–11) MG th i i MGFA M th i G i F d ti f A i ERCP d i t d h l i t h MM Table 3 Detailed description of patients with exacerbation of myasthenic symptoms Characteristics of patients with delayed clinical worsening within 30 days after contrast-enhanced CT studies (number 1–9) and after unenhanced CT studies (number 10–11) ( ) MG myasthenia gravis, MGFA Myasthenia Gravis Foundation of America, ERCP endoscopic retrograde cholangiopancreatography, MM minimal manifestation, ICU intensive care unit ( ) MG myasthenia gravis, MGFA Myasthenia Gravis Foundation of America, ERCP endoscopic retrograde cholangiopancreatography, MM minimal manifestation, ICU intensive care unit MG myasthenia gravis, MGFA Myasthenia Gravis Foundation of America, ERCP endoscopic retrograde cholangiopancreatography, MM minimal manifestation, ICU intensive care unit suspected cervical spine pathology at an orthopedic ward. One day after CT scanning, the patient had to be intubated because of respiratory insufﬁciency. MG was subsequently diagnosed and the patients improved after adequate therapy (cholinesterase inhibitors and prednisolone). Patient 6 A CT scan of the chest and abdomen was performed in this 72-year-old woman with a known MG after intubation had become necessary because of pro- gressive dyspnea. The CT revealed a small cell lung cancer encompassing nearly the entire right lung. After extuba- tion, the patient died 14 days after the CT scan because of respiratory insufﬁciency due to pulmonary edema. Patient 5 CT of the chest and abdomen was performed in an 80-year-old woman with a known MG (moderate limb weakness at the time of CT) because of weight loss and a reduced general state of health. CT showed a pre- viously unknown central lung carcinoma (stage 4) with widespread metastasis. Because of the extensive disease, no escalation of therapy was performed and the patients died due to respiratory insufﬁciency caused by the tumor 2 days after CT scan. Patient 7 This 66-year-old woman underwent a CT study of the head and chest for staging of a hepatic tumor. Case summaries of patients reaching the primary endpoint She had received high-dose prednisolone (1000 mg) 2 days and diazepam the day before because of 123 123 1215 J Neurol (2017) 264:1209–1217 Table 3 Detailed description of patients with exacerbation of myasthenic symptoms Age; sex MGFA pre CT MGFA after CT Region; ICA dose Indication Time to endpoint Clinically likely cause of endpoint 1 77; f IIB IVB Chest; 90 ml New onset MG; thymus evaluation 23 days Insufﬁcient therapy (only cholinesterase inhibitors) 2 68; m IIIA V Chest; 90 ml New onset MG; thymus evaluation 7 days Nightly aspiration and respiratory worsening during plasma exchange the following day 3 73; m IIIB V Chest and neck; 90 ml New onset MG; dysphagia 11 days Respiratory insufﬁciency due to insufﬁcient therapy (only cholinesterase inhibitors) 4 69; f IVB V Chest; 120 ml New onset MG; dyspnea 1 day 1000 mg Prednisolone 2 days and Diazepam the day before CT scanning 5 80; f IIIA Death Chest and abdomen; 140 ml Weight loss, reduced general state of health 2 days Previously unknown metastasized central lung carcinoma; death due to respiratory insufﬁciency 6 72; f V Death Chest and abdomen; 90 ml Dyspnea 14 days Small cell lung cancer encompassing nearly the entire right lung; death after extubation because of respiratory insufﬁciency due to pulmonary edema 7 66; f MM-3 IIIB Head and chest: 100 ml Staging, hepatic tumor 21 days Azathioprine discontinued. Infection of unknown etiology with necessity of different antibiotic therapies (vancomycin, meropenem, piperacillin/tazobactam) 8 58; f IIIA IVB Abdomen; 120 ml Perforation of common bile duct after ERCP 2 days Perforation of the common bile duct during ERCP with necessity of surgical treatment 9 60; f IIIA V Chest; 90 ml Suspected tumor in X-ray 10 days Previously unknown mediastinal diffuse large B-cell lymphoma. MG myasthenia gravis, MGFA Myasthenia Gravis Foundation of America, ERCP endoscopic retrograde cholangiopancreatography, MM minimal manifestation, ICU intensive care unit Discussion In this study, we retrospectively investigated the occur- rence of adverse events after administration of modern, low-osmolality CT-contrast agents in patients with myas- thenia gravis. We ascertained only a single patient (1.4%) with an acute, transient probably anaphylactic reaction (dyspnea) occurring immediately after application of the contrast agent. This rate is within the range of the previous studies in patients with or without myasthenia gravis [4, 6]. Finally, the exact characteristics of the used contrast agents could not be extracted retrospectively from the available data in all patients; therefore, we could not compare the potential side effects of different ICAs with each other. Our ﬁgures are comparable to the only other literature reference reporting delayed exacerbation of myasthenic symptoms after low-osmolality intravenous contrast agents. Somashekar et al. [3] observed MG-related symp- tom exacerbation in 12.5% of patients within 45 days, though the median delay was only 2.5 days, shorter than the 11 days in our study. The authors could not separate any direct effect of the contrast agent versus that mediated by any concurrent diseases on the worsening of myasthenia gravis, a causal relationship between ICA and MG wors- ening remained, therefore, undetermined. The other main ﬁnding of the study was that 9 of 73 patients (12.3%) experienced a delayed worsening of myasthenic symptoms, i.e., they reached the primary end- point of progressing by at least one grade in the MGFA classiﬁcation within 30 days. This worsening can be said to have occurred in a temporal association with the ICA administration with a median delay of 11 days. The rate was higher in comparison with the control group of patients receiving CT scans without ICAs (3.8%), but the difference did not reach statistical signiﬁcance in a low powered comparison. In a subgroup analysis, six of these nine patients (8.2% of all patients) developed a severe deterio- ration, i.e., a myasthenic crisis or died in comparison with none in the control group. Summarizing our data and interpreting those of other authors, we conclude that an acute, non-MG-related adverse reaction is a rare event with a risk comparable to other patients. A delayed worsening of myasthenia gravis-related symptoms might occur in approximately 12% of patients after ICA administration. In most cases, this delayed reaction seems to be a purely temporal rather than a causative asso- ciation. Case summaries of patients reaching the primary endpoint At the time of the CT scan, the patient showed only min- imal manifestation of myasthenic symptoms. 21 days after the CT scan, the patient developed moderate weakness of oropharyngeal and limb muscles presumably due to the discontinuation of azathioprine and a concomitant severe 12 3 3 J Neurol (2017) 264:1209–1217 1216 Patients receiving a contrast-enhanced CT scan more often suffered from acute concomitant (not neuromuscular) dis- eases, which was the reason for the contrast-enhanced CT study in the ﬁrst place. An analysis of the individual patient charts conﬁrms the above point in that, by clinical judg- ment, the worsening could be attributed in all patients with more likelihood to other causes than to the administration of the contrast agent. These other causes included an insufﬁcient MG-speciﬁc therapy in a newly developing myasthenia gravis, changes in the immunosuppressive therapy, or rapidly emerging systemic diseases such as cancer. In support of this view, a multivariate analysis only identiﬁed older age and a higher MGFA class at baseline as signiﬁcant risk factors for clinical exacerbation after CT studies but not the administration of contrast agents. septic infection of unknown origin that required different antibiotic therapies (vancomycin, meropenem, and piperacillin/tazobactam). The patient improved after ther- apy with IVIG and prednisolone. Patient 8 CT scan of the abdomen was performed in this 58-year-old woman with a known MG because of a per- foration of the common bile duct during an endoscopic retrograde cholangiopancreatography procedure. Surgical treatment was necessary and the patient exhibited a tran- sient worsening of myasthenic symptoms with severe weakness of oropharyngeal muscles 2 days after the CT scan most likely as a consequence of the abdominal com- plication and/or surgical intervention. Patient 9 This 60-year-old woman with established MG underwent a CT scan of the chest because of a suspected tumor. CT showed a previously unknown mediastinal dif- fuse large B-cell lymphoma. The patient was intubated 10 days after the CT scan because of respiratory failure due to pneumonia and pleural effusions. Limitations of this study are the mentioned selection bias for the enhanced and unenhanced CT scans and the relatively low patient numbers. The retrospective nature of this investigation entails the possibility that some adverse events might have been missed in some patients as we had to rely on electronic medical records. To minimize this effect, we only included patients with a sufﬁcient clinical information available. 123 Compliance with ethical standards Compliance with ethical standards 175:621–628. doi:10.1148/radiology.175.3.2343107 7. Loh S, Bagheri S, Katzberg RW et al (2010) Delayed adverse reaction to contrast-enhanced CT: a prospective single-center study comparison to control group without enhancement. Radi- ology 255:764–771. doi:10.1148/radiol.10091848 Conﬂicts of interest None of the authors has any conﬂict of interest to disclose. 8. Eliashiv S, Wirguin I, Brenner T, Argov Z (1990) Aggravation of human and experimental myasthenia gravis by contrast media. Neurology 40:1623–1625 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. gy 9. de Rocha MS, Bacheschi LA (1994) Exacerbation of myasthenia gravis by contrast media. AJR Am J Roentgenol 162:997. doi:10. 2214/ajr.162.4.8141034 10. Bonmarchand G, Weiss P, Clavier E et al (1987) Myasthenic crisis following the injection of an iodinated contrast medium. Intensive Care Med 13:365 11. Anzola GP, Capra R, Magoni M, Vignolo LA (1986) Myasthenic crisis during intravenous iodinated contrast medium injection. Ital J Neurol Sci 7:273 Discussion However, given the inevitable uncertainty regarding this analysis, a causative relationship cannot be excluded in all cases, a view which was only recently exempliﬁed by the case report of a patient developing a myasthenic crisis hours after injection of a low-osmolality ICA [16]. Temporal association does not necessarily imply the presence of a causal relationship. The ﬁgure of patients reaching the formal endpoints could include those in whom the clinical worsening was induced by ICA administration and others who were set to deteriorate independently of the contrast agent. The comparison with the control group (patients receiving no ICA) is not very informative for this purpose because of the inherent underlying selection bias. Balancing risks and beneﬁts, we argue that with justiﬁed indications for contrast-enhanced CT scans, ICAs should 123 1217 J Neurol (2017) 264:1209–1217 not be generally withheld from patients with myasthenia gravis. Further prospective studies are clearly necessary to evaluate the true risk of ICAs in patients with myasthenia gravis. not be generally withheld from patients with myasthenia gravis. Further prospective studies are clearly necessary to evaluate the true risk of ICAs in patients with myasthenia gravis. 4. Mehrizi M, Pascuzzi RM (2014) Complications of radiologic contrast in patients with myasthenia gravis. Muscle Nerve 50:443–444. doi:10.1002/mus.24254 5. Pasternak JJ, Williamson EE (2012) Clinical pharmacology, uses, and adverse reactions of iodinated contrast agents: a primer for the non-radiologist. Mayo Clin Proc 87:390–402. doi:10.1016/j. mayocp.2012.01.012 Acknowledgements Open access funding provided by Medical University of Vienna. This study received no speciﬁc grant from any funding agency. 6. Katayama H, Yamaguchi K, Kozuka T et al (1990) Adverse reactions to ionic and nonionic contrast media. A report from the Japanese Committee on the Safety of Contrast Media. Radiology 175:621–628. doi:10.1148/radiology.175.3.2343107 References 1. Cetin H, Fu¨lo¨p G, Zach H et al (2012) Epidemiology of myas- thenia gravis in Austria: rising prevalence in an ageing society. Wien Klin Wochenschr 124:763–768. doi:10.1007/s00508-012- 0258-2 12. Chagnac Y, Hadani M, Goldhammer Y (1985) Myasthenic crisis after intravenous administration of iodinated contrast agent. Neurology 35:1219–1220 13. Canal N, Franceschi M (1983) Myasthenic crisis precipitated by iothalamic acid. Lancet 1:1288 2. Mehrizi M, Fontem RF, Gearhart TR, Pascuzzi RM (2012) Medications and myasthenia gravis (A Reference for Health Care Professionals). Department of Neurology, Indiana University School of Medicine, USA 14. Frank JH, Cooper GW, Black WC, Phillips LH (1987) Iodinated contrast agents in myasthenia gravis. Neurology 37:1400–1402 15. Post-hoc power calculator provided by ClinCalc LLC http://clin calc.com/stats/Power.aspx#1. Accessed Sept 2016 3. Somashekar DK, Davenport MS, Cohan RH et al (2013) Effect of intravenous low-osmolality iodinated contrast media on patients with myasthenia gravis. Radiology 267:727–734. doi:10.1148/ radiol.12121508 3. Somashekar DK, Davenport MS, Cohan RH et al (2013) Effect of intravenous low-osmolality iodinated contrast media on patients with myasthenia gravis. Radiology 267:727–734. doi:10.1148/ radiol.12121508 16. Bonanni L, Dalla Vestra M, Zancanaro A, Presotto F (2014) Myasthenia gravis following low-osmolality iodinated contrast media. Case Rep Radiol 2014:963461. doi:10.1155/2014/963461 123 123
https://openalex.org/W2981170486	https://digibug.ugr.es/bitstream/10481/57668/1/Marin%20Aguilar_Autophagy.pdf	English	null	NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice	Aging Cell	2,019	cc-by	10,665	Fabiola Marín‐Aguilar1 \| Ana V. Lechuga‐Vieco2,3 \| Elísabet Alcocer‐Gómez4 \| Beatriz Castejón‐Vega1 \| Javier Lucas2 \| Carlos Garrido2 \| Alejandro Peralta‐Garcia5 \| Antonio J. Pérez‐Pulido5 \| Alfonso Varela‐López6 \| José L. Quiles6 \| Bernhard Ryffel7,8 \| Ignacio Flores2 \| Pedro Bullón1 \| Jesús Ruiz‐Cabello3,9,10,11 \| Mario D. Cordero6 Fabiola Marín‐Aguilar1 \| Ana V. Lechuga‐Vieco2,3 \| Elísabet Alcocer‐Gómez4 \| Beatriz Castejón‐Vega1 \| Javier Lucas2 \| Carlos Garrido2 \| Alejandro Peralta‐Garcia5 \| Antonio J. Pérez‐Pulido5 \| Alfonso Varela‐López6 \| José L. Quiles6 \| Bernhard Ryffel7,8 \| Ignacio Flores2 \| Pedro Bullón1 \| Jesús Ruiz‐Cabello3,9,10,11 \| Mario D. Cordero6 1Research Laboratory, Oral Medicine Department, University of Sevilla, Sevilla, Spain 2Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain 3CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain 4Departamento de Psicología Experimental, Facultad de Psicología, Universidad de Sevilla, Seville, Spain 5Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo de Olavide‐CSIC‐Junta de Andalucía, Sevilla, Spa 6Institute of Nutrition and Food Technology "José Mataix Verdú", Department of Physiology, Biomedical Research Cente Spain 7Laboratory of Experimental and Molecular Immunology and Neurogenetics (INEM), UMR 7355, CNRS‐University of Orleans, Orléans, France 8IDM, University of Cape Town, Cape Town, South Africa 9CIC biomaGUNE, San Sebastian‐Donostia, Spain 10IKERBASQUE, Basque Foundation for Science, Bilbao, Spain 11U i id d C l t M d id M d id S i 10IKERBASQUE, Basque Foundation for Science, Bilbao, Spain 11Universidad Complutense Madrid, Madrid, Spain 11Universidad Complutense Madrid, Madrid, Spain Correspondence Mario D. Cordero, Department of Physiology, Biomedical Research Center, Institute of Nutrition and Food Technology "José Mataix Verdú", University of Granada, 18100 Granada, Spain. Email: mdcormor@ugr.es Funding information Andalusian regional government; Consejería de Salud de la Junta de Andalucia, Grant/ Award Number: PI‐0036‐2014; Ministerio de economía y competitividad, Grant/Award Number: SAF2017‐84494‐C2‐1‐R Correspondence Mario D. Cordero, Department of Physiology, Biomedical Research Center, Institute of Nutrition and Food Technology "José Mataix Verdú", University of Granada, 18100 Granada, Spain. Email: mdcormor@ugr.es Abstract While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐ dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD+ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 Th A h A i C ll bli h d b h A i l S i d J h Wil & S L d O R I G I N A L A R T I C L E NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice Fabiola Marín‐Aguilar1 \| Ana V. Lechuga‐Vieco2,3 \| Elísabet Alcocer‐Gómez4 \| Beatriz Castejón‐Vega1 \| Javier Lucas2 \| Carlos Garrido2 \| Alejandro Peralta‐Garcia5 \| Antonio J. Pérez‐Pulido5 \| Alfonso Varela‐López6 \| José L. Quiles6 \| Bernhard Ryffel7,8 \| Ignacio Flores2 \| Pedro Bullón1 \| Jesús Ruiz‐Cabello3,9,10,11 \| Mario D. Cordero6 1Research Laboratory, Oral Medicine Department, University of Sevilla, Sevilla, Spain 2Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain 3CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain 4Departamento de Psicología Experimental, Facultad de Psicología, Universidad de Sevilla, Seville, Spain 5Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo de Olavide‐CSIC‐Junta de Andalucía, Sevilla, Spain 6Institute of Nutrition and Food Technology "José Mataix Verdú", Department of Physiology, Biomedical Research Center, University of Granada, Granada, Spain 7Laboratory of Experimental and Molecular Immunology and Neurogenetics (INEM), UMR 7355, CNRS‐University of Orleans, Orléans, France 8IDM, University of Cape Town, Cape Town, South Africa 9CIC biomaGUNE, San Sebastian‐Donostia, Spain 10IKERBASQUE B F d i f S i Bilb S i O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Received: 1 March 2019 \| Revised: 19 September 2019 \| Accepted: 23 September 2019 Received: 1 March 2019 \| Revised: 19 September 2019 \| Accepted: 23 September 2019 Received: 1 March 2019 \| Revised: 19 September 2019 \| Accepted: 23 September 2019 DOI: 10.1111/acel.13050 1 \| INTRODUCTION using a Kaplan–Meier survival curve was augmented with an increase in mean lifespan of 34% and in maximum lifespan of 29% (Figure 1a), while body weights and food intake did not differ between the two groups during the entire observation period (Figure 1b,c). Twenty‐ four‐month‐old WT animals displayed increased age‐related alopecia than their coveal NLRP3 knockout mice (Figure 1d). Old NLRP3−/− mice exhibited a significant decrease in glucose at the OGTT peak (>15 min), compared with old WT mice (Figure 1e,f), indicating a higher glucose tolerance as measured as a trend toward lower values of the area under the curve (AUC) of the glucose tolerance test (in‐ sert of Figure 1f). Fasting blood glucose and circulating IGF‐1 levels were reduced in young and old NLRP3−/− mice, indicating that the insulin sensitivity of these animals was considerably higher than sham controls during aging (Table S1). Reduced levels of glucose and IGF‐1 have been associated with stress resistance and an antiaging effect (Brandhorst et al., 2015). Furthermore, leptin is an established regu‐ lator of body weight, and leptin/adiponectin dysregulation has been associated with cardiovascular disease, metabolic syndrome, and non‐ alcoholic fatty liver disease (DiNicolantonio, Lucan, & O'Keefe, 2016). Young and old NLRP3−/− mice showed similar serum levels of leptin compared to young and old WT, but a reduced leptin/adiponectin ratio with increased levels of adiponectin was observed in old NLRP3−/− mice (Figure 1g–i). Plasma lipid levels were reduced in NLRP3−/− old mice, accompanied by a significant reduction in hepatic transami‐ nases, creatine phosphokinase, and lactate dehydrogenase (Table S1). However, plasma IL‐1β levels were not detected in old mice, but in‐ creased protein levels of active caspase 1 and IL‐1β were observed in old WT, when compared to NLRP3−/− mice (Figure S1), and increased levels of TNF‐α, IL‐6, and IL‐8 were observed in WT similar old mice and NLRP3−/− mice. This shows that the loss of NLRP3 did not affect the age‐related increase of other inflammatory pathways and confers an important role on inflammasome in cardiac aging (Table S1). Cardiovascular diseases (CVD) constitute the leading cause of death worldwide, with high prevalence in industrialized and low‐ to mid‐ dle‐income countries (Ford, Li, Zhao, Pearson, & Capewell, 2009). Several risk factors have been identified, including genetics, hyper‐ tension, obesity, smoking, and physical inactivity. Secondary risk factors correlated with cardiovascular risk include insulin resistance and lipid profile (Gómez‐Pardo et al., 2016). 1 \| INTRODUCTION However, aging, a nat‐ ural process, poses the largest risk factor for cardiovascular disease (Fontana, 2008). During aging, we suffer a progressive impairment of several metabolic pathways that define body composition, in‐ sulin resistance, mitochondrial and autophagy dysfunction, and in‐ flammation (Finkel, 2015). Many of these alterations are implicated in cardiac aging and age‐related cardiovascular diseases (North & Sinclair, 2012). Markers of inflammation have been associated with cardiovas‐ cular diseases and proposed as other cardiovascular risk factors (Bullón et al., 2017). Recently, the role of the NLR family pyrin do‐ main containing 3 protein (NLRP3) inflammasome has been studied in cardiovascular diseases. NLRP3 inflammasome is upregulated after myocardial infarction, atherosclerosis, ischemic heart disease, diabetic cardiomyopathy, chronic heart failure, and hypertension, and recently, NLRP3 and IL‐1β have also been proposed as new car‐ diovascular risk biomarkers (Bullón et al., 2017; Liu, Zeng, Li, Mehta, & Wang, 2017). Previous studies have suggested a role for NLRP3 inflammasome in several events associated with aging. Genetic de‐ letion of NLRP3 in mice has been shown to improve healthspan by attenuation of multiple age‐related degenerative changes, such as glycemic control, bone loss, cognitive function, and motor perfor‐ mance (Youm et al., 2013). Furthermore, the deletion of NLRP3 in old mice increased muscle strength and endurance and prevented from age‐related increase in the number of myopathic fibers (McBride et al., 2017). However, the role of the NLRP3 inflammasome in lifespan and cardiac aging has not been studied. Hence, we sought to deter‐ mine whether or not genetic deletion of NLRP3 may have effect on lifespan and potentially prevent cardiac aging. 2.2 \| NLRP3 deletion preserved cardiac integrity Heart weight normalized to body weight was increased in old mice in comparison with young mice, and heart weight was higher in WT in comparison with NLRP3−/− (p < .05) (Figure 2a and Table S2). Cardiac hypertrophy measured by the left ventricular wall thickness was sig‐ nificantly increased in elderly WT when compared to NLRP3−/− mice which was corroborated by LV mass measured by echocardiography (Figure 2b). To assess the impact of aging and cardiac hypertrophy on myocardial histology, the cardiomyocyte cross‐sectional area and fibrosis were quantified. In the hematoxylin‐and‐eosin–stained sec‐ tions, aged WT mice showed an increased cardiomyocyte transverse cross‐sectional area unlike NLRP3−/− mice (Figure 2c,d), which was corroborated by wheat germ agglutinin staining (Figure S2). Further 2 of 14 \| 2 of 14 2 of 14 MARÍN‐AGUILAR et al. Funding information \| 1 of 14 wileyonlinelibrary.com/journal/acel Aging Cell. 2019;00:e13050. https://doi.org/10.1111/acel.13050 2 of 14 \| 2.1 \| NLRP3 deletion improves lifespan and metabolic aging To evaluate the impact of NLRP3 deletion on survival and metabolic changes during aging, we followed NLRP3 deficient (NLRP3 −/−) and NLRP3 +/+ littermate control (WT) mice throughout the entire lifes‐ pan. The survival of NLRP3 −/− mice compared to littermate controls MARÍN‐AGUILAR et al. \| 3 of 14 MARÍN‐AGUILAR et al. examination with Masson trichrome and Sirius red staining revealed overt interstitial and perivascular fibrosis in the aged WT group with damage in aged WT myocardium, including mitochondrial disarray, degeneration fragmentation reduction of mitochondrial area and FI G U R E 1 NLRP3 signaling suppression in mice extend lifespan and improve metabolic homeostasis. (a) Kaplan–Meier graph showing a significant increment of the máximum lifespan in WT mice (blue) compared with NLRP3 −/− mice (red). (b, c) Body weights and average daily oral food intake normalized to body weight and to mouse of the groups over time. Images of representative mice to illustrate phenotypic body mass of the groups at 20 months of age. (d) Representative photographs of 24 months of age mice. (e, f) Oral glucose tolerance test with area under the curve (inset). (g–i) Levels of leptin, adiponectin, and ratio in plasma. Blood samples were collected after overnight fasting. All data are presented as means ± SEM, n = 10 mice; p < .05, p < .005, *p < .001 young vs. old mice. aap < .005, WT vs. NLRP3 −/− mice FI G U R E 1 NLRP3 signaling suppression in mice extend lifespan and improve metabolic homeostasis. (a) Kaplan–Meier graph showing a significant increment of the máximum lifespan in WT mice (blue) compared with NLRP3 −/− mice (red). (b, c) Body weights and average daily oral food intake normalized to body weight and to mouse of the groups over time. Images of representative mice to illustrate phenotypic body mass of the groups at 20 months of age. (d) Representative photographs of 24 months of age mice. (e, f) Oral glucose tolerance test with area under the curve (inset). (g–i) Levels of leptin, adiponectin, and ratio in plasma. Blood samples were collected after overnight fasting. All data are presented as means ± SEM, n = 10 mice; p < .05, p < .005, p < .001 young vs. old mice. aap < .005, WT vs. 2.1 \| NLRP3 deletion improves lifespan and metabolic aging NLRP3 −/− mice examination with Masson trichrome and Sirius red staining revealed overt interstitial and perivascular fibrosis in the aged WT group, with no significant changes in the aged NLRP3−/− group (Figure 2c,d). Ultrastructural analysis of the left ventricle in WT and NLRP3−/− showed mitochondrial abnormalities. We compared electron micro‐ scopic images of cardiac tissues from young and old WT mice and NLRP3−/− mice. TEM studies revealed evidence of mitochondrial damage in aged WT myocardium, including mitochondrial disarray, degeneration, fragmentation, reduction of mitochondrial area, and cristae disorganization, that is,. pointing in varying oblong and oblique directions in the matrix (Figures S3a,b and S4a,b). Upon electrocardiographic examination, the mean QRS complex was not significantly wider in old WT mice; however, we observed a significant prolongation of the age‐dependent PR interval, which is 4 of 14 \| MARÍN‐AGUILAR et al. 4 of 14 \| MARÍN‐AGUILAR et al. 4 of 14 \| MARÍN‐AGUILAR et al. FI G U R E 2 NLRP3 signaling suppression in mice induces cardiac protection. (a) Heart weight normalized to body weight. (b) Representative image of centripetal concentric LV hypertrophy (c) Representative Hematoxylin and eosin (H&E)‐stained section (left) of cardiac tissues from young and old WT and NLRP3 −/− mice. Representative Masson trichrome‐stained section (center) and Sirius red‐stained section (right). (d) Quantitative analysis of cardiomyocyte cross‐sectional (transverse) area with measurements of ≈100 cardiomyocytes and fibrotic areas from 3 to 6 mice per group. *p < .001 young vs. old mice. aaap < .001, WT vs. NLRP3 −/− mice. (e) Summary of differences in PR and QRS intervals in young and aged WT vs. NLRP3 −/− mice. All data are presented as means ± SEM, n = 10 mice; p < .05 FI G U R E 2 NLRP3 signaling suppression in mice induces cardiac protection. (a) Heart weight normalized to body weight. (b) Representative image of centripetal concentric LV hypertrophy (c) Representative Hematoxylin and eosin (H&E)‐stained section (left) of cardiac tissues from young and old WT and NLRP3 −/− mice. Representative Masson trichrome‐stained section (center) and Sirius red‐stained section (right). (d) Quantitative analysis of cardiomyocyte cross‐sectional (transverse) area with measurements of ≈100 cardiomyocytes and fibrotic areas from 3 to 6 mice per group. **p < .001 young vs. old mice. aaap < .001, WT vs. NLRP3 −/− mice. (e) Summary of differences in PR and QRS intervals in young and aged WT vs. NLRP3 −/− mice. 2.1 \| NLRP3 deletion improves lifespan and metabolic aging All data are presented as means ± SEM, n = 10 mice; p < .05 \| 5 of 14 MARÍN‐AGUILAR et al. FI G U R E 3 Telomere length diferentes in cardiac cells from WT and NLRP3 −/− mice. (a) Representative confocal images of telomere Q‐ FISH and PCM1 immunofluorescence of cardiac tissue from young and old WT and NLRP3−/− mice. Orange arrowheads indicate PCM1+ cardiomyocytes. White arrowheads indicate PCM1− noncardiomyocytes. Dashed lines encircle telomere signals in those cardiac cells. Bars, 10 µm. (b) Telomere length distribution in total cardiac cells (left) and cardiomyocytes (right) of young and old WT and NLRP3−/− mice. Vertical lines indicate mean length (*p < .001, Wilconxon's rank‐sum test) MARÍN‐AGUILAR et al. \| FI G U R E 3 Telomere length diferentes in cardiac cells from WT and NLRP3 −/− mice. (a) Representative confocal images of telomere Q‐ FISH and PCM1 immunofluorescence of cardiac tissue from young and old WT and NLRP3−/− mice. Orange arrowheads indicate PCM1+ cardiomyocytes. White arrowheads indicate PCM1− noncardiomyocytes. Dashed lines encircle telomere signals in those cardiac cells. Bars, 10 µm. (b) Telomere length distribution in total cardiac cells (left) and cardiomyocytes (right) of young and old WT and NLRP3−/− mice. Vertical lines indicate mean length (p < .001, Wilconxon's rank‐sum test) in two‐year‐old animals, NLRP3 −/− mice showed slightly longer telomeres (Figure 3a,b). This is due to an increased telomere length reduction rate in WT mice compared with NLRP3 −/− mice (Figure S5a). Furthermore, less lipofuscin accumulation in the heart of NLRP3−/− mice was shown after a qualitative observation (Figure S5b). Additionally, we explored classical senescence biomarkers such as IL‐6, p21, and p53. Similar to serum levels of IL‐6, cardiac tissues showed increased IL‐6 protein levels in old mice, accompanied by increased p21 and phospho‐p53 protein levels compared with young mice in WT and KO mice; however, there was a higher increase in old KO mice than WT mice (Figure S6). associated with atrial fibrillation by cardiovascular aging (Figure 2e) (Magnani et al., 2013). Due to the improvement in the cardiac func‐ tion of old KO mice and the low incidence of cancer in these mice (WT showed an increased rate of hepatocarcinoma and adenocar‐ cinoma at death), the cause of death was unknown and will require further study. 2.3 \| Age‐associated metabolic changes were prevented in NLRP3−/− In order to evaluate the role of NLRP3 during aging of the heart, several markers and pathways associated with aging were studied in hearts from young and aged WT and NLRP3−/− mice. Telomeres in young animals were similar in WT and NLRP3 −/− mice, whereas To gain insight into metabolic “longevity regulatory” path‐ ways, we investigated IGF‐1, PI3K, mTOR in the heart. Since NLRP3−/− mice showed low levels of IGF‐1 in young and old 6 of 14 \| MARÍN‐AGUILAR et al. 6 of 14 \| MARÍN‐AGUIL 6 of 14 \| MARÍN‐AGUILAR et al. 6 of 14 \| MARÍN‐AGUILAR et al. FI G U R E 4 Changes in the Pi3K/mTOR pathways and autophagy observed in cardiac tissues from young and old mice. (a) Western blot analysis showing reduced levels in the Pi3K/mTOR pathway in the heart of NLRP3 −/− mice compared with WT. Densitometric analysis are presented as means ± SEM, n = 10 mice; *p < .001 young vs. old mice. aaap < .001, WT vs. NLRP3 −/− mice. (b) Western blot analysis with representative blot including ATG12, Beclin 1, LC3, Parkin, and p62 level in the heart of Young and old mice. Densitometric analysis are presented as means ± SEM, n = 10 mice; p < .05, p < .005, and p < .001 young vs. old mice. (c) Cardiac tissues showing typical ultrastructure with several lamellar bodies and autophagosome (black arrows) present in cardiac tissues from old mice and (white arrows). Scale bar 2 µm (low magnification) and 1 µm (high magnification) FI G U R E 4 Changes in the Pi3K/mTOR pathways and autophagy observed in cardiac tissues from young and old mice. (a) Western blot analysis showing reduced levels in the Pi3K/mTOR pathway in the heart of NLRP3 −/− mice compared with WT. Densitometric analysis are presented as means ± SEM, n = 10 mice; *p < .001 young vs. old mice. aaap < .001, WT vs. NLRP3 −/− mice. (b) Western blot analysis with representative blot including ATG12, Beclin 1, LC3, Parkin, and p62 level in the heart of Young and old mice. Densitometric analysis are presented as means ± SEM, n = 10 mice; p < .05, p < .005, and p < .001 young vs. old mice. (c) Cardiac tissues showing typical ultrastructure with several lamellar bodies and autophagosome (black arrows) present in cardiac tissues from old mice and (white arrows). 2.3 \| Age‐associated metabolic changes were prevented in NLRP3−/− Scale bar 2 µm (low magnification) and 1 µm (high magnification) FI G U R E 4 Ch i th Pi3K/ TOR th d t h b d i di ti f d ld i ( ) W t bl t FI G U R E 4 Changes in the Pi3K/mTOR pathways and autophagy observed in cardiac tissues from young and old mice. (a) Western blot analysis showing reduced levels in the Pi3K/mTOR pathway in the heart of NLRP3 −/− mice compared with WT. Densitometric analysis are presented as means ± SEM, n = 10 mice; *p < .001 young vs. old mice. aaap < .001, WT vs. NLRP3 −/− mice. (b) Western blot analysis with representative blot including ATG12, Beclin 1, LC3, Parkin, and p62 level in the heart of Young and old mice. Densitometric analysis are presented as means ± SEM, n = 10 mice; p < .05, p < .005, and p < .001 young vs. old mice. (c) Cardiac tissues showing typical ultrastructure with several lamellar bodies and autophagosome (black arrows) present in cardiac tissues from old mice and (white arrows). Scale bar 2 µm (low magnification) and 1 µm (high magnification) \| 7 of 14 MARÍN‐AGUILAR et al. \| 7 of 14 MARÍN‐AGUILAR et al. FI G U R E 5 Transcriptional changes in heart from Young and old WT and NLRP3 −/− mice. Gene Ontology enrichment analysis in “Biological Process” in WT young vs. old mice (a), NLRP3 −/− Young vs. old mice (b), and WT old vs. NLRP3 −/− old mice (c). (d) Heatmap clustering of enrichment (z‐scores) of the functions defined by the DAVID in set of coding genes differentially expressed between WT old vs. NLRP3 −/− old mice (n = 3 per treatment) FI G U R E 5 Transcriptional changes in heart from Young and old WT and NLRP3 −/− mice. Gene Ontology enrichment analysis in “Biological Process” in WT young vs. old mice (a), NLRP3 −/− Young vs. old mice (b), and WT old vs. NLRP3 −/− old mice (c). (d) Heatmap clustering of enrichment (z‐scores) of the functions defined by the DAVID in set of coding genes differentially expressed between WT old vs. NLRP3 −/− old mice (n = 3 per treatment) microscopic analysis, we corroborated that the numbers of accu‐ mulated autophagosomes were reduced in hearts from NLRP3−/− old mice (Figure 4c). 2.3 \| Age‐associated metabolic changes were prevented in NLRP3−/− This could be explained by where NLRP3 inhibition induced improved autophagy quality in the heart during aging. mice, we examined signaling changes through these pathways in the heart. Despite no significant differences in phosphorylation of PI3K (p110α), mTOR (Ser2448) was decreased in the heart of aged NLRP3−/− mice (Figure 4a). These data are consistent with the previous observations that cardiac aging is retarded and that healthspan is increased by mTOR inhibition (Inuzuka et al., 2009; Wu et al., 2013). mTOR inhibition is associated with the important physiological process of lysosomal‐dependent recycling, known as autophagy, which is involved in cellular homeostasis through pro‐ tein degradation and removal of damaged intracellular organelles (Pyo et al., 2013). Autophagic disfunction has also been linked to aging with blocked autophagic flux and accumulation of nondegra‐ dated substrates in the form of autophagosome (Pyo et al., 2013). Interestingly, NLRP3−/− mice showed increased levels of ATG12, beclin 1 expression, and LC3II protein expression in NLRP3−/− old mice, with a reduction of p62/SQSTM1 (Figure 4b). From electron In this respect, NLRP3−/− mice showed increased NAD+ levels in both cases (HFD, HSD, and aging) and increased SIRT‐1 protein ex‐ pression (Figure S9A,B). These findings could explain the improved metabolic status and autophagic flux observed in NLRP3−/− mice during aging (Fang et al., 2017). Rad (Ras associated with diabetes) GTPase has been established as an endogenous regulator of cardiac excitation‐contraction (Wang et al., 2010). Rrad gene expression was increased in WT, but not in old NLRP3−/− mice, which could explain the increased cardiac pathology. Moreover, upregulation of Thbs1 was also observed in old WT mice when compared with NLRP3−/− mice, which is associ‐ ated with a disturbed flow from arterial stiffening (Kim et al., 2017). Tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) and tripartite motif containing 72 (TRIM72) overexpression have been associated with atherosclerosis and diabetic cardiomyopathy, respectively (Liu et al., 2015; Lyu et al., 2018). Accordingly, these genes were upregulated in old WT mice compared with NLRP3−/− mice, supporting the enhancing role of NLRP3 inhibition in the cardiovascular aging process. Furthermore, the expression of transglutaminase 2 (TGM2), an arterial calcifica‐ tion‐related protein that is positively associated with hypertension and atherosclerosis (Mattison et al., 2014), and Collagen type IV alpha1 (COL4A1) and alpha2 (COL4A2) linked to the pathogenesis of vascular lesions were increased in WT but not in NLRP3−/− mice (Jeanne, Jorgensen, & Gould, 2015). Collectively, these data sup‐ port significant protection imparted by NLRP3 inhibition on car‐ diac aging and age‐induced stress and vascular changes that occur during aging. Furthermore, this microarray study showed upreg‐ ulation of genes associated with the mTOR pathway (Arntl, Akt1 and Ddit4) from old WT mice and changes associated with the neg‐ ative regulation of autophagy processes (Nampt, Stat3, Fez2 and Akt1), which are associated with our findings of an inhibited mTOR pathway and increased autophagy in NLRP3 −/− mice during aging (Table S2 and S3). Several of the metabolic changes presented in this study corrobo‐ rated a special protection for cardiac changes by NLRP3−/− deletion. The metabolic hallmarks related to aging such as glucose tolerance and lipid metabolism are potentially corrected in NLRP3−/− mice, probably related to the reduced IGF‐1 signaling and the PI3K/AKT/ mTOR pathway. Notably, adiponectin was also increased in these mice during aging. Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin recep‐ tors, AdipoR1 and AdipoR2 (Lindgren et al., 2013). WT mice when compared with old NLRP3−/− mice. Recently, Acta1 has been associated with cardiac hypertrophy through increased levels of IGF‐1, so the reduced levels of Acta1 in NLRP3−/− mice could be associated with the reduced serum levels of IGF‐1 shown in this study (Bisping et al., 2012). The insulin receptor substrate protein 1 (IRS‐1) was also upregulated in old WT mice when com‐ pared to old NLRP3−/− mice. IRS‐1 may contribute to longevity (Selman, Partridge, & Withers, 2011). than 2, and a p‐value equal to or lower than 0.05 (Figure S7a). The most significant changes common to aging in WT and NLRP3−/− mice are available at http://www.ncbi.nlm.nih.gov/geo/ with code GSE124483. All the gene expression data were loaded into DAVID for gene ontology (GO) enrichment analysis. The enrichment anal‐ ysis in WT young‐to‐old mice showed that for the biological pro‐ cess, most of the genes were enriched in the response to stress and organic substances. However, these and other important bio‐ logical and molecular enriched processes were not significantly different from those in NLRP3−/− young and old mice. NLRP3 deletion conferred protection related to aging, and significant dif‐ ferences were found between WT and NLRP3−/− (Figure 5a–c). To examine the differences in gene expression profiling, gene coding pathways were represented in a heatmap (Figure 5d). Our analysis indicated that 43 pathways were significantly altered be‐ tween old WT and old NLRP3−/− mice. For a deeper analysis, the downregulated changes in protein coding are presented in Table S3 and the upregulated changes are summarized in Table S4. A subset of expression changes was verified by polymerase chain reaction with reverse transcription (RT‐qPCR) (Figure S8). Notably, the most significant downregulated gene expression in old WT mice compared with old NLRP3 was nicotinamide phosphoribosyl‐ transferase (Nampt), the rate‐limiting enzyme in mammalian NAD+ biosynthesis (Table S2). NAD+ deficiency is suggested to be a com‐ mon central pathological factor in a number of diseases, including cardiovascular diseases and aging (North & Sinclair, 2012; Zhang & Ying, 2018). Interestingly, Nampt‐mediated NAD+ deficiency is severely associated with glucose intolerance and insulin resist‐ ance in metabolic dysfunction by a high‐fat diet (HFD) and aging (Yoshino, Mills, Yoon, & Imai, 2011). Accordingly, we determined NAD+ levels from the heart during HFD and a high sucrose diet (HSD) (exposed 15 weeks) and aging in WT and NLRP3−/− mice. 2.4 \| Age‐associated change of the cardiac gene expression profile was prevented in NLRP3 −/− mice To better define the molecular basis of improved cardiac health in the absence of NLRP3, a microarray expression profiling was per‐ formed on cardiac tissues obtained from 22‐month‐old animals. In old WT mice, 202 transcripts (from the 65,956 transcripts ex‐ amined) changed significantly when compared with old NLRP3−/− mice: 142 transcripts were upregulated and 60 transcripts were downregulated, as those with a fold change equal to or higher MARÍN‐AGUILAR et al. 8 of 14 3 \| DISCUSSION Aging is the principal pathological process of cardiovascular dis‐ eases in healthy people. The principal age‐dependent changes in cardiac structure and function in the heart during normal aging are not well understood and, if defined, could provide new clues for protection from aging‐specific cardiac functional decline. This study showed that NLRP3 is associated with aging by an improved lifespan and healthspan via the modification of several hallmarks of aging. Little has been studied about the role of NLRP3 inhibition during aging, and nothing has been studied about longevity; our data, such as glucose tolerance, are consistent with previous stud‐ ies on the effect of NLRP3 ablation on aging (Youm et al., 2013). Inflammation is highly associated with aging and age‐related dis‐ eases and many rejuvenation strategies adopt anti‐inflammatory diets (Finkel, 2015; Fontana et al., 2012). Increased systemic in‐ flammation is commonly concomitant with metabolic alterations and the deterioration of metabolic health, including the appear‐ ance of increased adiposity, insulin resistance, and dyslipidemia, which could prove to be a key determinant of a shortening lifespan and healthspan (Finkel, 2015). According to this, one should an‐ ticipate that an experimental manipulation of a specific inflamma‐ tory pathway would entail systemic and metabolic effects with an improvement in life expectancy and health. Our results provide convincing evidence that the NLRP3 ablation causes an increase in longevity that could be due to several of the metabolic changes induced by this manipulation. In this study, we have observed an increase in glucose tolerance, a reduction and an increase, re‐ spectively, in lectin and adiponectin levels, and a regulation of dyslipemia. All these changes are associated with common path‐ ways, such as IGF‐1, PI3K/AKT/mTOR, autophagy, and intracellu‐ lar NAD+ levels (Finkel, 2015). According to our data, the ablation of NLRP3 showed low serum levels of IGF‐1 in old mice. The role of the protective pathological effects of IGF‐1 is contradictory, but our data suggest that low serum levels of IGF‐1 are the end product of decreased insulin/IGF‐1 signaling, which is known to prolong life, both in invertebrates and in vertebrates (Finkel, 2015; Fontana et al., 2012). Thus, low levels of insulin and/or IGF‐1 sign‐ aling, along with a high sensitivity to insulin and IGF‐1, are physio‐ logical characteristics that support the prolonged lifespan of Ames dwarf mice (Finkel, 2015) in which the levels of IRS‐1 associated with longevity were reduced (Papaconstantinou & Hsieh, 2015). We also found that protein levels of senescence/DNA damage markers, such as p21 or p53, measured by Western blot increase with age in WT hearts, but their expression does not vary with age in NLRP3 −/− hearts, which could be related to a reduced DNA damage response activated by dysfunctional telomeres in these animals. Interestingly, DNA damage in dysfunctional telomeres is a key hallmark of cardiomyocytes with a senescent‐like phenotype (Anderson et al., 2019), and clearance of senescence cells in mice alleviates cardiac deterioration with aging (Anderson et al., 2019; Lewis‐McDougall et al., 2019). Similarly, inhibi‐ tion or deletion of NLRP3 would improve the detrimental effect of se‐ nescence cells in the heart, opening the door to a new line of research. In this sense, the NLRP3 inflammasome activation has been shown to promote the aging of the thymus and lead to immunosenescence (Spadaro et al., 2016). Moreover, the term of inflammaging has been nurtured and associated with a low‐grade proinflammatory pheno‐ type that accompanies aging (Latz & Duewell, 2018). The different im‐ plications of NLRP3 in metabolism during aging and the protective role of the inhibition of NLRP3 show a relevant role of this in inflammaging. In this respect, NLRP3 −/− mice showed increased inflammatory levels during aging and, despite this, cardiac aging was prevented by NLRP3 deletion. Senescence influences the cellular environment through the secretion of proinflammatory cytokines, proteases, and chemokines called senescence‐associated secretory phenotype (SASP). Since ac‐ tivation of the NLRP3 system is a probable driver of SASP (Latz & AdipoR2 in WT mice in aging could be interpreted as an attempt to contribute to the optimization of their metabolic environment. This has a negative effect, increasing the leptin/adiponectin ratio. In this sense, the NLRP3 inflammasome activation has been shown to promote the aging of the thymus and lead to immunosenescence (Spadaro et al., 2016). Moreover, the term of inflammaging has been nurtured and associated with a low‐grade proinflammatory pheno‐ type that accompanies aging (Latz & Duewell, 2018). The different im‐ plications of NLRP3 in metabolism during aging and the protective role of the inhibition of NLRP3 show a relevant role of this in inflammaging. In this respect, NLRP3 −/− mice showed increased inflammatory levels during aging and, despite this, cardiac aging was prevented by NLRP3 deletion. Senescence influences the cellular environment through the Ryffel, 2018; Wu et al., 2013). Since it is known that mTOR induces autophagy, the ablation of NLRP3 also showed, consistent with previous data showing the effect of inhibition of NLRP3 with other stressors, such as a hypercaloric diet, an increase in autophagy during aging (Pavillard et al., 2017). Cardiac aging is characterized by the presence of hypertrophy, fibrosis, and the accumulation of misfolded proteins and dysfunctional mitochondria. Therefore, autophagy and autophagic fluxes generally were reduced in cardiac tissues during aging, and models of loss of murine autophagy function models show an increase in cardiac dysfunction associated with the accumulation of misfolded proteins and dysfunctional organelles. Accordingly, it has been shown that the stimulation of autophagy improves cardiac function by eliminating accumulated cellular content, thus relieving different aging‐associated pathologies in the heart (Shirakabe, Ikeda, Sciarretta, Zablocki, & Sadoshima, 2016). This mechanism could be key to the improvement of longevity and health induced by the inhibition of NLRP3 and the support of many of the strategies to improve the extension of lifespan and healthspan through the use of rapamycin, caloric restriction with metformin or resveratrol, which have two com‐ mon mechanisms: an improvement in autophagy and NLRP3 inhibition of inflammasome (Cordero et al., 2018). Furthermore, we found a re‐ duced telomere shortening rate in WT mice when compared to NLRP3 −/− mice. Interestingly, reductions in telomere shortening rate, rather than the initial telomere length, have been suggested as a critical vari‐ able that determines a species’ lifespan in a wide variety of species, including mice and humans (Canela, Vera, Klatt, & Blasco, 2007; Vera, Bernardes de Jesus, Foronda, Flores, & Blasco, 2012; Whittemore, Vera, Martínez‐Nevado, Sanpera, & Blasco, 2019). Ryffel, 2018; Wu et al., 2013). Since it is known that mTOR induces autophagy, the ablation of NLRP3 also showed, consistent with previous data showing the effect of inhibition of NLRP3 with other stressors, such as a hypercaloric diet, an increase in autophagy during aging (Pavillard et al., 2017). Cardiac aging is characterized by the presence of hypertrophy, fibrosis, and the accumulation of misfolded proteins and dysfunctional mitochondria. Therefore, autophagy and autophagic fluxes generally were reduced in cardiac tissues during aging, and models of loss of murine autophagy function models show an increase in cardiac dysfunction associated with the accumulation of misfolded proteins and dysfunctional organelles. Accordingly, it has been shown that the stimulation of autophagy improves cardiac function by eliminating accumulated cellular content, thus relieving different aging‐associated pathologies in the heart (Shirakabe, Ikeda, Sciarretta, Zablocki, & Sadoshima, 2016). This mechanism could be key to the improvement of longevity and health induced by the inhibition of NLRP3 and the support of many of the strategies to improve the extension of lifespan and healthspan through the use of rapamycin, caloric restriction with metformin or resveratrol, which have two com‐ mon mechanisms: an improvement in autophagy and NLRP3 inhibition of inflammasome (Cordero et al., 2018). Furthermore, we found a re‐ duced telomere shortening rate in WT mice when compared to NLRP3 −/− mice. Interestingly, reductions in telomere shortening rate, rather than the initial telomere length, have been suggested as a critical vari‐ able that determines a species’ lifespan in a wide variety of species, including mice and humans (Canela, Vera, Klatt, & Blasco, 2007; Vera, Bernardes de Jesus, Foronda, Flores, & Blasco, 2012; Whittemore, Vera, Martínez‐Nevado, Sanpera, & Blasco, 2019). We also found that protein levels of senescence/DNA damage markers, such as p21 or p53, measured by Western blot increase with age in WT hearts, but their expression does not vary with age in NLRP3 −/− hearts, which could be related to a reduced DNA damage response activated by dysfunctional telomeres in these animals. Interestingly, DNA damage in dysfunctional telomeres is a key hallmark of cardiomyocytes with a senescent‐like phenotype (Anderson et al., 2019), and clearance of senescence cells in mice alleviates cardiac deterioration with aging (Anderson et al., 2019; Lewis‐McDougall et al., 2019). Similarly, inhibi‐ tion or deletion of NLRP3 would improve the detrimental effect of se‐ nescence cells in the heart, opening the door to a new line of research. AdipoR2−/− mice were resistant to obesity induced by a high‐fat diet and exhibited improved glucose tolerance and decreased plasma cholesterol levels (Fontana, Vinciguerra, & Longo, 2012). Increased levels of AdipoR2 were observed in old WT mice when compared with NLRP3−/−, which could be associated with AdipoR2 deficiency‐dependent pro‐ tection of atherosclerosis (Lindgren et al., 2013). Furthermore, the reduced levels of adiponectin associated with increased levels of The homocysteine inducible ER protein with ubiquitin‐like domain 1 (Herpud1), which has been proposed as necessary for adequate insulin‐induced glucose uptake (Navarro‐Marquez et al., 2018), was also shown to be downregulated in old WT when compared to old NLRP3 −/− mice (Table S2). Herpud1 inhibition has recently been associated with induced pathological cardiac hypertrophy, which could explain the reduced hypertrophy ob‐ served in NLRP3 −/− mice (Torrealba et al., 2017). Similarly, our transcriptomic study showed upregulated gene expression of the cardiac hypertrophy‐related gene. Expression of established bio‐ markers atrial natriuretic peptide A (Nppa) and B (Nppb), which are associated with cardiac hypertrophy and strongly upregulated in the ventricular myocardium during cardiac stress (Man, Barnett, & Christoffels, 2018; Newman, Nguyen, Watson, Hull, & Yu, 2017), significantly increased in old WT mice when compared with old NLRP3 mice. Acta1 was the second most upregulated gene in old MARÍN‐AGUILAR et al. 9 of 14 4.5 Glucose tolerance tests were performed by fasting the mice over‐ night for 16 hr and then injecting glucose (1 g/kg), intraperitoneally. Glucose measurements were performed using a Bayer Contour blood glucose meter and test strips. for the use of laboratory animals in chronic experiments (RD 53/2013 on the care of experimental animals). All experiments were approved by the local institutional animal care committee. Duewell, 2018), our findings could suggest a role of NLRP3 in the se‐ nescence phenotype during inflammaging. Another age‐related mechanism linked to autophagy impairment is the intracellular reduction of NAD+. NAD+ is an electron acceptor in the mitochondrial electron transport chain that is also an essential substrate for NAD+‐dependent enzymes, such as sirtuins and poly ADP ribose polymerase (Rajman, Chwalek, & Sinclair, 2018). NAD+ levels decrease with age due to Nampt downregulation, oxidative stress, inflammation, defective circadian rhythm, and accumulation of DNA damage. Nampt, a key enzyme in the salvage pathway of NAD+ biosynthesis, is downregulated in the heart in response to ischemia, which induces a decrease in NAD+ levels in the heart, in‐ hibition of autophagic flux, and cell death (Shirakabe et al., 2016). Therefore, restoring NAD+ content by overexpressing Nampt or adding NAD+ supplements restores the level of autophagy during ischemia and reduces the extent of myocardial infarction (Rajman et al., 2018). We observed an increased level of Nampt in NLRP3−/− old mice by transcriptomic analysis, which was corroborated by real‐ time PCR. This is probably connected to the high levels of NAD+ observed in hearts of elderly NLRP3−/− mice fed with hypercaloric diets. 4.3 Young and old C57/BL6/J and NLRP3−/− transgenic mice (C57BL/6J background), weighing 25–30 g, were maintained on a regular 12 hr light/dark cycle. Mice were housed in groups of four to eight same‐ sex littermates under specific pathogen‐free conditions. Individuals were monitored daily and weighed monthly, but were otherwise left undisturbed until they died. Survival was assessed using male and female mice, and all animals were dead by the time of this report. Kaplan–Meier survival curves were constructed using known birth and death dates, and differences between groups were evaluated using the logrank test. We acknowledge the limitations of using male mice only. On the other hand, we have studied the effect of the NLRP3 ablation in the expression of several markers of senescence by Western blots; how‐ ever, our results must be extended to detect the specific cell type using, among others, gH2AX‐telomere immuno FISH or gH2AX‐PML colocalization in Immunofluorescence and IHC. Thus, future investi‐ gations should account for the implication of other inflammasomes in aging and their modulation. 4.2 \| Animals For all experiments, only male mice were used. Young and old NLRP3−/− transgenic mice (C57BL/6J background) and WT/ NLRP3+/+ littermate controls, weighing 25–30 g, were main‐ tained on a regular 12 hr light/dark cycle. All groups had ad li‐ bitum access to their prescribed diet and water throughout the whole study. Body weight and food intake were monitored weekly. Animal rooms were maintained at 20–22°C with 30%–70% relative humidity. 4.4 \| Reagents In conclusion, our findings suggest that NLRP3 inhibition atten‐ uates the harmful effects of cardiac aging and extends the lifespan in male mice. NLRP3 ablation improves metabolic characteristics related to aging, such as glucose tolerance, lipid metabolism, and leptin/adiponectin. These results could be associated with reduced IGF‐1 signaling and the PI3K/AKT/mTOR pathway and with auto‐ phagy activation. Our data associate the inhibition of NLRP3 with previous interventions against aging, such as caloric restriction, metformin, resveratrol or protein restriction, and involve levels of Nampt‐dependent NAD+ and SIRT1 (Cordero et al., 2018). In addi‐ tion, our transcriptomic results show a profile related to metabolic improvement and an anti‐hypertrophic effect of cardiac protection. Finally, NLRP3 inhibition could be associated with a specific inflam‐ masome‐dependent inflammaging. Therefore, prevention of the aging process through multiple mechanisms by NLRP3 inhibition is likely to attenuate the associated decrease in cardiac function. Thus, it offers a promising goal for the prevention of cardiac aging. Monoclonal antibodies specific for Beclin‐1 and p62 were pur‐ chased from Sigma‐Aldrich. The anti‐GAPDH monoclonal an‐ tibody was acquired from Calbiochem‐Merck Chemicals Ltd. Similarly, anti‐active caspase‐3, anti‐SIRT‐1, p‐PI3K and PI3K, p‐mTOR and mTOR, and anti‐Parkin were obtained from Cell Signaling Technology. Finally, anti‐Bcl‐2, anti‐Bax, anti‐ATG12, and anti‐MAP‐LC3 antibodies were obtained from Santa Cruz Biotechnology. A cocktail of protease inhibitors (Complete™ Protease Inhibitor Cocktail) was purchased from Boehringer Mannheim. The Immun Star HRP substrate kit was obtained from Bio‐Rad Laboratories Inc. 3 \| DISCUSSION Interestingly, our transcriptomic analysis showed reduced IRS‐1 expression in old NLRP3 mice when compared to old WT mice. NLRP3 ablation also showed inhibition of PI3K/AKT/mTOR. mTOR is a serine‐threonine kinase that functions as an intracellular energy NLRP3 ablation also showed inhibition of PI3K/AKT/mTOR. mTOR is a serine‐threonine kinase that functions as an intracellular energy sensor whose genetic and pharmacological inhibition has been shown to extend life in a wide range of organisms (Cordero, Williams, & 10 of 14 MARÍN‐AGUILAR et al. 4.12 Transmission electron microscopy (TEM). Mice were euthanized by cervical dislocation and the left ventricle apex was immediately dis‐ sected (3 months old, n = 3 per genotype and 20 months old, n = 3 per genotype). Heart samples were fixed in 2.5% glutaraldehyde and 4% formaldehyde in 0.1 M HEPES buffer for 4–5 hr. After buffer washes, samples were postfixed for 1 hr at room temperature in a 1:1 solution of 1% osmium tetroxide and 3% aqueous potassium fer‐ rocyanide. Samples were rinsed in distilled H2O. Tissues were de‐ hydrated through a graded acetone series and embedded in Spurr's low viscosity embedding mixture (Electron Microscopy Sciences). 4.9 Mice were anesthetized with 1.5%–2% isoflurane in oxygen, inhaled through a facial mask. To avoid night–day circadian variations, ECG was performed in the morning. ECG electrodes were inserted sub‐ cutaneously in the four limbs, and sequential ECG recordings were acquired at 2 KHz sweep‐speed using a MP36R data acquisition workstation (Biopac Systems). Data were stored for off‐line analysis using custom MatLab scripts. In 3 and 20 months of age/ death of the animal, NLRP3−/− and WT mice were given a weekly β‐adrener‐ gic challenge with isoproterenol (i.v. bolus 0.34 mg/kg). ECG traces were recorded at baseline, after challenge, and during recovery (10–25 min). (1.5%–2% isoflurane in a mixture with oxygen), and the analysis was carried out using a Vevo770 system (Vevo 2100, Visualsonics Inc.) equipped with a 30‐MHz linear transducer probe. To avoid night–day circadian variations, echocardiographs were performed in the mornings. Before echocardiography, animal fur was removed with a depilatory agent and animals were warmed to maintain body temperature. The heart was imaged in the 2D parasternal long‐ and short‐axis projections with guided M‐mode recordings at the midventricular level in both views. Images were recorded and transferred to a computer for posterior blinded analysis using the Vevo 2100 Workstation software. LV end‐diastolic diameter (LVEDD), LV‐systolic diameter (LVESD), end‐diastolic LV anterior wall thickness (LVAW), and LV posterior wall thickness (LVPW) were measured from images obtained with M‐mode echocardi‐ ography. LV fractional shortening (FS) and aortic cardiac output (AA.CO) was calculated. LV ejection fraction (EF) by the Teichholz formula was assessed by using the M‐mode images displayed over time and obtained from a single line in the middle of LV. The LV mass was calculated from the same M‐mode images by using diastolic LV diameters of LV internal diameter (LVID), posterior wall (PW), and interventricular septum (IVS) as follow: LV Mass (mg) = 1.05 (Lid + LVPWd + IVSd3 – LVIDd3) 0.8. Corrected LV Mass = (LV Mass) 0.8. 4.8 Western blotting was performed using standard methods. After protein transfer, the membrane was incubated with various primary antibodies diluted at 1:1,000 and then with the corresponding sec‐ ondary antibodies coupled to horseradish peroxidase at a 1:10,000 dilution. Specific protein complexes were identified using the Immun Star HRP substrate kit (Biorad Laboratories Inc.). 4.11 \| Histological study After anesthesia of mice, hearts were excised and placed in a 16 mM KCl solution to arrest the heart in diastole prior to fixation and imme‐ diately placed in 10% neutral‐buffered formalin at room temperature for 24 hr after a brief rinse with PBS. The specimens were embedded in paraffin, cut into 5‐μm sections, and stained with hematoxylin and eosin. To detect fibrosis in heart sections, consecutive formalin‐fixed, paraffin‐embedded, 4‐µm sections were stained with Masson tri‐ chrome or with Sirius Red. Masson trichrome staining was performed following the manufacturer's instructions (Accustain HT15, Sigma‐ Aldrich). Sirius Red staining was performed by incubating slides in 0.1% Sirius Red F3B for 1 hr, rinsing twice in acidified water, dehydrating thrice in 100% ethanol, and then clearing in xylene. Manually delimited cardiomyocyte or fibrotic areas were also calculated on a digital micro‐ scope (×400) with ImageJ (version ImageJ 1.49) software. ECG recordings were analyzed offline using custom scripts for preprocessing, visualization and quantification of electrophysiologic intervals and heart rate variability markers. After band‐pass filtering between 0.5 and 250 Hz, baseline wander was removed using a bi‐ directional filtering strategy. Baseline drift removal is essential for morphological analysis of T waves. Specifically: (a) PR intervals were measured from the beginning or the P wave to the peak of the R wave; (b) QRS intervals were measured from the beginning of the Q wave until the point where the S wave crosses the baseline; and (c) QT inter‐ vals were measured from the beginning of the Q wave until the point where the T wave declined to 90% (T90) from the peak. Finite differ‐ ential methods and wavelet transform were used for fiducial point estimation. R‐peak detection was robustly estimated by parabolic fit‐ ting of the coiflet wavelet transform and detection of the maximum magnitude point. All R detections were supervised to validate accuracy of ECG segmentations. After QRS detection, P and T wave segments were extracted using adaptive windowing, depending upon beat‐to‐ beat RR changes. After segmentation using differential methods, both waves were low‐pass filtered at 20 Hz using a Kaiser window FIR filter. 4.7 \| Serum biomarkers Serum levels of glucose, triglycerides, cholesterol, uric acid, aspar‐ tate aminotransferase, alanine aminotransferase, and creatine ki‐ nase were assayed using commercial kits (Randox Laboratories). 4.6 Animal studies were performed in accordance with European Union guidelines (2010/63/EU) and the corresponding Spanish regulations Serum levels of leptin, adiponectin, and IGF‐1 were assayed in dupli‐ cate using commercial ELISA kits (R&D Systems). MARÍN‐AGUILAR et al. 11 of 14 ACKNOWLEDGMENTS This study was supported by a grant from the Andalusian regional government (Grupo de Investigacion Junta de Andalucia CTS113), Consejería de Salud de la Junta de Andalucia: PI‐0036‐2014 and Ministerio de economía y competitividad: SAF2017‐84494‐C2‐1‐R. FMA has the benefit of a FPU Fellowship (FPU 13/03173) from The Ministry of Education, Science and Sport. IF laboratory was funded by grants from Ministerio de Ciencia, Innovación y Universidades (SAF2016‐80406‐R) and Comunidad de Madrid (S2017/ BMD‐3875). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV‐2015‐0505). Authors are indebted to Mr. Kastin Mattern for extensive editing of the article. 4.15 Total NAD+ concentrations were determined independently in whole cardiac extracts using the NAD+ cycling assay (Abcam ab65348, Inc.), as described previously. NAD+ levels were expressed as pmol/ mgprotein. For telomere length analysis, DAPI, Cy3 (telomeres), and im‐ munofluorescence signals were acquired sequentially in separate channels of a confocal microscope (SP5) fitted with a 63× objec‐ tive and linked to LasAF and MatrixScreener software. All slides were stained at the same time, and all images were acquired at the same laser intensity. For image quantification, Nikon NIS Elements software was used to subtract Cy3 channel background and to generate maximum‐intensity projections from 16‐bit image stacks (15 sections at steps of 1.0 µm). Quantitative image anal‐ ysis was performed using the Metamorph platform as previously described (Flores et al., 2008). The DAPI images were signal‐in‐ tensity thresholded. After conversion to a 1‐bit binary mask, the DAPI image was used to define the nuclear area and the Cy3 image to quantify telomere fluorescence. The binary DAPI mask was ap‐ plied to the Cy3 image to obtain a combined image with the telo‐ mere fluorescence information for each nucleus. Cy3 fluorescence intensity was measured as the mean gray value in each nucleus in arbitrary units of fluorescence (auf). Telomere intensity values were exported to Excel. For the determination of telomere length in cardiomyocytes, cardiomyocyte nuclei were manually selected with Metamorph using the PCM1 immunofluorescence images. Only cells clearly identifiable as cardiomyocytes were considered for the analysis. 4.13 \| Telomere length analysis The telomere length of cardiac cells was measured by quantita‐ tive fluorescence in situ hybridization (Q‐FISH). Hearts were fixed in 4% PFA, and paraffin‐embedded tissue sections of the same thickness (4 μm) were first immunostained for PCM1 (1.300). Slides were then fixed in 4% formaldehyde for 2 min, treated with 0.28 mM pepsin for 10 min at 37°C and then hybridized with a Cy3‐labeled peptide nucleic acid probe targeting the telomere repeat sequence (Flores et al., 2008). The intensity of the fluo‐ rescent signal for a given telomere is directly proportional to tel‐ omere length, providing a quantitative measure of telomere length (Lansdorp et al., 1996). 4.16 \| Statistics All data are expressed as means ± SEM. After, evaluation of normality using the Shapiro–Wilk test, statistical differences among the differ‐ ent groups were measured using either an unpaired Student t test or 1‐way analysis of variance (ANOVA) when appropriate with Tukeys post hoc test. A Wilcoxon's ram sum test was used to calculate the statistical significance between telomere length distributions. A p‐ value of ≤.05 was considered statistically significant. Statistical anal‐ yses were performed using Prism software version 5.0a (GraphPad). Asterisks in the figures represent the following: : p ≤ .05; : p ≤ .01; and *: p ≤ .001. Microarray Analysis; Irizarry et al., 2003), and the mutant mice were compared vs. WT, using the 3 replicates for every analysis. Then, a fold change and p‐value were calculated for every gene by an un‐ paired test one‐way (single factor) using the NMATH package. A gene was considered as differentially expressed when it had a fold change equal to or higher than 1.5, and a p‐value equal to or lower than 0.05 using Fisher's exact test. Then, a functional enrichment was performed with the differentially expressed genes using the functional annotation tool from the DAVID web site (https://www. ncbi.nlm.nih.gov/pmc/articles/PMC3381967/). As such, we found a number of annotation categories. To analyze the expression profile for the genes included in these categories, a heatmap was created for every annotation category, using the heatmap.2 function in the gplots R package, and considering normalized values by z‐scores, and making a clustering based on the expression profiles and the average method. Finally, a mean of the expression values in every annotation category was calculated and it was again plotted in a heatmap. Ultra‐thin sections (60 nm) were then mounted on copper grids and stained with lead citrate. Samples were examined on a JEOL 10‐10 electron microscope through 1,500×, 5,000×, 40,000×, and 80,000× objectives. Mitochondrial morphometry, cristae area (2 im‐ ages, 5,000×, per animal), and lipid droplets (50 images, 5,000×, per animal) were segmented manually and analyzed using Fiji (http://fiji. sc/Fiji) and ImageJ 1.48v software. The investigator was blinded to the group allocation when assessing the outcome. 4.10 \| E Two‐dimensional and M‐mode echocardiography longitudi‐ nal studies were performed in 3‐month intervals throughout the mice's entire lifespan. Males (n = 10–15) were anesthetized MARÍN‐AGUILAR et al. 12 of 14 REFERENCES Latz, E., & Duewell, P. (2018). NLRP3 inflammasome activation in inflam‐ maging. Seminars in Immunology, 40, 61–73. Anderson, R., Lagnado, A., Maggiorani, D., Walaszczyk, A., Dookun, E., Chapman, J., … Passos, J. F.. (2019). Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence. The EMBO Journal, 38. pii: e100492. https://doi.org/10.15252/embj.20181 00492 Lewis‐McDougall, F. C., Ruchaya, P. J., Domenjo‐Vila, E., Shin Teoh, T., Prata, L., Cottle, B. J., … Ellison‐Hughes, G. M. (2019). Aged‐senes‐ cent cells contribute to impaired heart regeneration. Aging Cell, 18, e12931. Bisping, E., Ikeda, S., Sedej, M., Wakula, P., McMullen, J. R., Tarnavski, O., … Pieske, B. (2012). Transcription factor GATA4 is activated but not required for insulin‐like growth factor 1 (IGF1)‐induced cardiac hypertrophy. Biological Chemistry, 287, 9827–9834. Lindgren, A., Levin, M., Rodrigo Blomqvist, S., Wikström, J., Ahnmark, A., Mogensen, C., … Linden, D. (2013). Adiponectin receptor 2 defi‐ ciency results in reduced atherosclerosis in the brachiocephalic ar‐ tery in apolipoprotein E deficient mice. PLoS ONE, 8, e80330. Brandhorst, S., Choi, I. Y., Wei, M., Cheng, C. W., Sedrakyan, S., Navarrete, G., … Longo, V. D. (2015). A periodic diet that mimics fasting pro‐ motes multi‐system regeneration, enhanced cognitive performance, and healthspan. Cell Metabolism, 22, 86–99. Liu, D., Zeng, X., Li, X., Mehta, J. L., & Wang, X. (2017). Role of NLRP3 inflammasome in the pathogenesis of cardiovascular diseases. Basic Research in Cardiology, 113, 5. Liu, F., Song, R., Feng, Y., Guo, J., Chen, Y., Zhang, Y., … Xiao, R. P. (2015). Upregulation of MG53 induces diabetic cardiomyopathy through transcriptional activation of peroxisome proliferation‐activated re‐ ceptor α. Circulation, 131, 795–804. Bullón, P., Cano‐García, F. J., Alcocer‐Gómez, E., Varela‐López, A., Roman‐ Malo, L., Ruiz‐Salmerón, R. J., … Cordero, M. D. (2017). Could NLRP3‐ inflammasome be a cardiovascular risk biomarker in acute myocardial infarction patients? Antioxidants & Redox Signaling, 27, 269–275. Lyu, M., Cui, Y., Zhao, T., Ning, Z., Ren, J., Jin, X., … Zhu, Y. (2018). Tnfrsf12a‐mediated atherosclerosis signaling and inflammatory re‐ sponse as a common protection mechanism of shuxuening injection against both myocardial and cerebral ischemia‐reperfusion injuries. Frontiers in Pharmacology, 9, 312. Canela, A., Vera, E., Klatt, P., & Blasco, M. A. (2007). High‐throughput telomere length quantification by FISH and its application to human population studies. Proceedings of the National Academy of Sciences of the United States of America, 104, 5300–5305. Cordero, M. D., Williams, M. R., & Ryffel, B. (2018). CONFLICT OF INTERESTS Ford, E. S., Li, C., Zhao, G., Pearson, W. S., & Capewell, S. (2009). Trends in the prevalence of low risk factor burden for cardiovascular disease among United States adults. Circulation, 120, 1181–1208. MAC currently holds a fractional Professorial Research Fellow ap‐ pointment at the University of Queensland with his remaining time as CEO of Inflazome Ltd., a company headquartered in Dublin, Ireland that is developing drugs to address unmet clinical needs in inflammatory disease by targeting the inflammasome. AR is an in‐ ventor on inflammasome related patents (WO2017140778 and WO2016131098). Gómez‐Pardo, E., Fernández‐Alvira, J. M., Vilanova, M., Haro, D., Martínez, R., Carvajal, I., … Fuster, V. (2016). A comprehensive lifestyle peer group‐based intervention on cardiovascular risk fac‐ tors: The randomized controlled fifty‐fifty program. Journal of the American College of Cardiology, 67, 476–485. Inuzuka, Y., Okuda, J., Kawashima, T., Kato, T., Niizuma, S., Tamaki, Y., … Shioi, T. (2009). Suppression of phosphoinositide 3‐kinase prevents cardiac aging in mice. Circulation, 120, 1695–1703. Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B., & Speed, T. P. (2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Research, 31(4), e15. Mario D. Cordero https://orcid.org/0000-0003-0151-3644 Lansdorp, P. M., Verwoerd, N. P., van de Rijke, F. M., Dragowska, V., Little, M. T., Dirks, R. W., … Tanke, H. J. (1996). Heterogeneity in telo‐ mere length of human chromosomes. Human Molecular Genetics, 5, 685–691. DATA AVAILABILITY STATEMENT Jeanne, M., Jorgensen, J., & Gould, D. B. (2015). Molecular and genetic analyses of collagen type IV mutant mouse models of spontaneous intracerebral hemorrhage identify mechanisms for stroke preven‐ tion. Circulation, 131, 1555–1565. Results of transcriptomic changes by microarray and most significant changes common to aging in WT and NLRP3−/− mice are available at http://www.ncbi.nlm.nih.gov/geo/ with code GSE124483. Kim, C. W., Pokutta‐Paskaleva, A., Kumar, S., Timmins, L. H., Morris, A. D., Kang, D. W., … Brewster, L. P. (2017). Disturbed flow promotes arterial stiffening through thrombospondin‐1. Circulation, 136, 1217–1232. 4.14 \| Transcriptome analysis We used the Affymetrix clariomTM D assay mouse, which includes 65,956 genes. The raw data were analyzed by the Affymetrix software Trancriptome Analysis Console. Expression values were normalized by SST (Signal Space Transformation)‐RMA (Robust MARÍN‐AGUILAR et al. 13 of 14 Wang, G., Zhu, X., Xie, W., Han, P., Li, K., Sun, Z., … Cheng, H. (2010). Rad as a novel regulator of excitation‐contraction coupling and beta‐ad‐ renergic signaling in heart. Circulation Research, 106, 317–327. Navarro‐Marquez, M., Torrealba, N., Troncoso, R., Vásquez‐Trincado, C., Rodriguez, M., Morales, P. E., … Lavandero, S. (2018). Herpud1 im‐ pacts insulin‐dependent glucose uptake in skeletal muscle cells by controlling the Ca2+‐calcineurin‐Akt axis. Biochimica Et Biophysica Acta, 1864, 1653–1662. Whittemore, K., Vera, E., Martínez‐Nevado, E., Sanpera, C., & Blasco, M. A. (2019). Telomere shortening rate predicts species life span. Proceedings of the National Academy of Sciences of the United States of America, 116, 15122–15127. Newman, M. S., Nguyen, T., Watson, M. J., Hull, R. W., & Yu, H. G. (2017). Transcriptome profiling reveals novel BMI‐ and sex‐specific gene expression signatures for human cardiac hypertrophy. Physiological Genomics, 49, 355–367. Wu, J. J., Liu, J., Chen, E. B., Wang, J. J., Cao, L., Narayan, N., … Finkel, T. (2013). Increased mammalian lifespan and a segmental and tissue‐ specific slowing of aging after genetic reduction of mTOR expres‐ sion. Cell Reports, 4, 913–920. North, B. J., & Sinclair, D. A. (2012). The intersection between aging and cardiovascular disease. Circulation Research, 110, 1097–1108. Yoshino, J., Mills, K. F., Yoon, M. J., & Imai, S. (2011). Nicotinamide mono‐ nucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet‐ and age‐induced diabetes in mice. Cell Metabolism, 14, 528–536. Papaconstantinou, J., & Hsieh, C. C. (2015). IGF‐1 mediated phosphor‐ ylation of specific IRS‐1 serines in Ames dwarf fibroblasts is associ‐ ated with longevity. Oncotarget., 6, 35315–35323. Pavillard, L. E., Cañadas‐Lozano, D., Alcocer‐Gómez, E., Marín‐Aguilar, F., Pereira, S., Robertson, A. A. B., … Cordero, M. D. (2017). NLRP3‐ inflammasome inhibition prevents high fat and high sugar diets‐in‐ duced heart damage through autophagy induction. Oncotarget, 8, 99740–99756. Youm, Y. H., Grant, R. W., McCabe, L. R., Albarado, D. C., Nguyen, K. Y., Ravussin, A., … Dixit, V. D. (2013). Canonical Nlrp3 inflammasome links systemic low‐grade inflammation to functional decline in aging. Cell Metabolism, 18, 519–532. Zhang, M., & Ying, W. (2018). NAD+ deficiency is a common central pathological factor of a number of diseases and aging: Mechanisms and therapeutic implications. Antioxidants & Redox Signaling, 30, 890–905. Pyo, J. O., Yoo, S. M., Ahn, H. H., Nah, J., Hong, S. H., Kam, T. I., … Jung, Y. K. (2013). REFERENCES AMP‐activated pro‐ tein kinase regulation of the NLRP3 inflammasome during aging. Trends in Endocrinology and Metabolism, 29, 8–17. Magnani, J. W., Wang, N., Nelson, K. P., Connelly, S., Deo, R., Rodondi, N., … Benjamin, E. J. (2013). Health, Aging, and body composition study. Electrocardiographic PR interval and adverse outcomes in older adults: The health, aging, and body composition study. Circulation. Arrhythmia and Electrophysiology, 6, 84–90. DiNicolantonio, J. J., Lucan, S. C., & O'Keefe, J. H. (2016). The evidence for saturated fat and for sugar related to coronary heart disease. Progress in Cardiovascular Diseases, 58, 464–472. Man, J., Barnett, P., & Christoffels, V. M. (2018). Structure and function of the Nppa‐Nppb cluster locus during heart development and disease. Cellular and Molecular Life Sciences, 75, 1435–1444. Fang, E. F., Lautrup, S., Hou, Y., Demarest, T. G., Croteau, D. L., Mattson, M. P., & Bohr, V. A. (2017). NAD+ in aging: Molecular mechanisms and translational implications. Trends in Molecular Medicine, 23, 899–916. Mattison, J. A., Wang, M., Bernier, M., Zhang, J., Park, S. S., Maudsley, S., … de Cabo, R. (2014). Resveratrol prevents high fat/sucrose diet‐in‐ duced central arterial wall inflammation and stiffening in nonhuman primates. Cell Metabolism, 20, 183–190. Finkel, T. (2015). The metabolic regulation of aging. Nature Medicine, 21, 1416–1423. Flores, I., Canela, A., Vera, E., Tejera, A., Cotsarelis, G., & Blasco, M. A. (2008). The longest telomeres: A general signature of adult stem cell compartments. Genes & Development, 22, 654–667. McBride, M. J., Foley, K. P., D'Souza, D. M., Li, Y. E., Lau, T. C., Hawke, T. J., & Schertzer, J. D. (2017). The NLRP3 inflammasome contrib‐ utes to sarcopenia and lower muscle glycolytic potential in old mice. American Journal of Physiology. Endocrinology and Metabolism, 313, E222–E232. Fontana, L., Vinciguerra, M., & Longo, V. D. (2012). Growth factors, nu‐ trient signaling, and cardiovascular aging. Circulation Research, 110, 1139–1150. MARÍN‐AGUILAR et al. 14 of 14 14 of 14 Overexpression of Atg5 in mice activates autophagy and extends lifespan. Nature Communications, 4, 2300. Rajman, L., Chwalek, K., & Sinclair, D. A. (2018). Therapeutic potential of NAD‐boosting molecules: The in vivo evidence. Cell Metabolism, 27, 529–547. SUPPORTING INFORMATION Selman, C., Partridge, L., & Withers, D. J. (2011). Replication of extended lifespan phenotype in mice with deletion of insulin receptor sub‐ strate 1. PLoS ONE, 6, e16144. Additional supporting information may be found online in the Supporting Information section at the end of the article. Shirakabe, A., Ikeda, Y., Sciarretta, S., Zablocki, D. K., & Sadoshima, J. (2016). Aging and autophagy in the heart. Circulation Research, 118, 1563–1576. How to cite this article: Marín‐Aguilar F, Lechuga‐Vieco AV, Alcocer‐Gómez E, et al. NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice. Aging Cell. 2019;00:e13050. https://doi.org/10.1111/ acel.13050 How to cite this article: Marín‐Aguilar F, Lechuga‐Vieco AV, Alcocer‐Gómez E, et al. NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice. Aging Cell. 2019;00:e13050. https://doi.org/10.1111/ acel.13050 Spadaro, O., Goldberg, E. L., Camell, C. D., Youm, Y. H., Kopchick, J. J., Nguyen, K. Y., … Dixit, V. D. (2016). Growth hormone receptor defi‐ ciency protects against age‐related NLRP3 inflammasome activation and immune senescence. Cell Reports, 14, 1571–1580. Torrealba, N., Navarro‐Marquez, M., Garrido, V., Pedrozo, Z., Romero, D., Eura, Y., … Lavandero, S. (2017). Herpud1 negatively regulates pathological cardiac hypertrophy by inducing IP3 receptor degrada‐ tion. Scientific Reports, 7, 13402. Vera, E., Bernardes de Jesus, B., Foronda, M., Flores, J. M., & Blasco, M. A. (2012). The rate of increase of short telomeres predicts longevity in mammals. Cell Reports, 2, 732–737.
https://openalex.org/W4289545428	https://www.annalsofgeophysics.eu/index.php/annals/article/download/8741/7509	English	null	Estimation of Curie-Point Depths and Heat Flow from Spectral Analysis of EMAG2 Magnetic Data in Cyprus Island	Annals of geophysics	2,022	cc-by	10,058	Abstract This study aims to determine Curie point depth (CPD), Heat flow and the boundaries of geological structures of Cyprus Island using EMAG2 magnetic data. CPD values were calculated by applying spectral analysis technique to magnetic anomaly map divided into 60 blocks (60×60 km2). Then, dif‑ ferent thermal conductivity values (K = 1, 1.5 and 2.5 Wm–1K–1) and heat flow values were calculated using CPD values. CPD values ranged from 12.4 km to 28.18 km, and heat flow values were calculated between 20 and 50 mW/m2 for K = 1. Shallow CPD values (CPD < 15 km) were calculated in Polis, Morphou Bay, in the area between Larnaca and Famagusta and north of Kyrenia. Heat flow values are relatively high in these areas which can be researched in detail in terms of potential geothermal. In the final phase of the study, the boundaries of buried geological structures were determined by Analytic Signal (AS), Total Horizontal Derivative (THDR) and Tilt angle (TA) methods. Moho depth, Moho‑Curie difference, 2D cross‑correlation map of Moho and Curie depths and Earthquake dis‑ tribution map are used for interpretation of the tectonic regime. Moho‑Curie difference is roughly 0 and the 2D cross‑correlation map produces higher (0.60‑0.75) values in the southern part of the study area which might be evaluated as a passive crust. It is possible to say that few earthquakes are observed where the difference is around 0. Keywords: Cyprus Island; Curie Point Depth; Heat Flow; Moho Depth; Edge Detection Article history: received October 21, 2021; accepted April 5, 2022 Article history: received October 21, 2021; accepted April 5, 2022 Article history: received October 21, 2021; accepted April 5, 2022 ANNALS OF GEOPHYSICS, 65, 4, GT427, 2022; doi:10.4401/ag-8741 ANNALS OF GEOPHYSICS, 65, 4, GT427, 2022; doi:10.4401/ag-8741 ANNALS OF GEOPHYSICS, 65, 4, GT427, 2022; doi:10.4401/ag-8741 OPEN ACCESS ANNALS OF GEOPHYSICS, 65, 4, GT427, 2022; doi:10.4401/ag-8741 OPEN ACCESS Estimation of Curie‑Point Depths and Heat Flow from Spectral Analysis of EMAG2 Magnetic Data in Cyprus Island Eren Pamuk,1, İlkin Özsöz2 (1) Department of Geophysical Research, General Directorate of the Mineral Research & Exploration of Turkey, 06800, Ankara, Turkey (2) Department of Marine Research, General Directorate of the Mineral Research & Exploration of Turkey, 06800, Ankara, Turkey 1. Introduction Curie point is defined as the temperature at which the magnetization disappears. The Curie point depth (CPD) is known as the depth at which magnetic minerals in the crust move from a ferromagnetic state to a paramagnetic state under the influence of increasing temperature [Nagata, 1961; Mohammed et al., 2019]. CPD data is often used in determining the thermal structure of the crust and estimating potential geothermal areas. The method, developed by Okubo et al. [1985], was used for CPD prediction in this study. 1 Curie-Point Depth Estimation in Cyprus Island As it can be seen from previous studies, global magnetic maps such as the EMAG2 are quite advantageous in CPD value estimation due to the wide coverage. The EMAG2 stands for Earth Magnetic Anomaly Grid and it comprises ship, airborne and satellite magnetic measurements. In this study, the EMAG2 has been used to estimate depth to the Curie isotherm in and around Cyprus Island. Numerous studies were carried out in Cyprus in terms of earth sciences. We can list some of them as follows. Cagnan and Tanircan [2010] conducted probabilistic seismic hazard analysis for Cyprus in their study. Asim et al. [2020] conducted a seismicity analysis for Cyprus. Ulutas [2020] analyzed the spread, altitude and arrival times of a possible tsunami for Cyprus in his study. Zissimos et al. [2019] investigated the spatial distribution of soil inorganic carbon (SIC) and soil organic carbon (SOC) across Cyprus. McPhee and Hinsbergen [2019] examined the structural and tectonic history of Cyprus. Elmas [2018] determined the structural discontinuities of Cyprus Island by applying total horizontal derivative and tilt angle techniques to satellite gravity and vertical first derivative data. i Cyprus Island can be considered a natural laboratory in terms of its geological importance. The convergent plate margin in the study area provides essential information about young mountain belts which can be related to the oceanic crust. Cyprus Island is located at the intersection of the platform of the African and Eurasian plates approaching each other. The margin between African and Anatolian plates can be defined by the Cyprus Arc where complex bathymetric structural trends are observed [Ergün et al., 2003]. Makris et al., [1983] and Robertson [1998] showed that the northern edge of the plate margin has fairly complex characteristics. The western part of the margin, Herodotus abyssal plain, is characterised by deep water which indicates stable crust whereas the eastern part of the boundary has a more variable crust. The subduction regime in the Cyprus Arc is in the transition phase to collision regime due to the thicker and less dense crust, in the northern boundary of the African plate [Kempler and Ben‑Avraham, 1987; Robertson, 1998]. Destructive earthquakes were observed in Cyprus Island, which can be characterised as active and complex neotectonic history [Harrison et al., 2004]. The Troodos Massif in Cyprus Island is one of the largest ophiolite complexes in the region. Curie-Point Depth Estimation in Cyprus Island There are numerous studies on the Troodos Massif [Harrison et al., 2004]. The formation and development of Troodos Ophiolite covers the Cenomanian‑Turonian (92‑90My) time interval [Mukasa and Ludden, 1987; Staudigel et al., 1986; Blome and Irwin, 1985; Hakyemez, 2014]. This ophiolite consists of serpentinite, harzburgite, dunite, gabbro, diabase and basalts [Kinnaird 2008, Symeou 2018]. Mamonia Complex, located in the southernmost part of Cyprus Island, consists of deformed sedimentary and volcanic rocks. The ages of these units vary from Triassic to Lower Cretaceous [Swarbrick and Robertson 1980; McPhee et. al. 2019]. The Kyrenia range, consisting of massif limestone bands, recrystallized limestones, dolomites and flysch, is between the Permian and Lower Cretaceous ages [Montadert et al., 2014; Symeou, 2018]. The Kalavasos Formation is the upper Miocene‑aged sediments and has been characterised with gypsum deposits alternating with chalky marls and marly chalks [Symeou, 2018] (Figure 1). In this paper, it is attempted to focus on estimating Curie Point Depth (CPD) and heat flow values for Cyprus Island via spectral analysis of the EMAG2 data set. Furthermore, boundaries of the subsurface geological structures and tectonic activity are interpreted by TA (tilt angle), AS (analytic signal), THDR (total horizontal derivative) and Moho‑Curie comparison and the spatial distribution of earthquakes (M > 2.5). 2. Data and Methods 2.1 EMAG2 Magnetic Data Eren Pamuk and İlkin Özsöz There are many studies about CPD estimation. Njeudjang et al. [2020] estimated Curie point depth, heat flow and geothermal gradient parameters for the Adamawa volcanic region (northern Cameroon) using EMAG2 data. Kumar et al. [2020] used satellite‑based remote sensing, gravity and magnetic data to determine potential Kimberlite regions in their study. Li et al. [2017] obtained the first global model of Curie‑point depth (GCDM) from EMAG2 magnetic data. Arnaiz‑Rodríguez and Orihuela [2013] conducted CPD calculations for Venezuela and the Eastern Caribbean using the Enhanced Magnetic Model (EMM2010). For Eastern and South‑eastern Asia Li and Wang [2016] investigated magnetic, heat flow and gravity data and their implications on deep crustal and uppermost mantle structures. Xu et al. [2017] determined CPD values from the magnetic anomaly data of EMAG2 in North China. Pamuk [2019] calculated CPD and heat flow values for the northern part of Eastern Anatolia, Turkey using EMAG2 magnetic data in his study. Özsöz [2021] estimated depth to the Curie isotherm in the Eastern Mediterranean Region with 39 sub‑blocks using the World Digital Magnetic Anomaly Map (WDMAM) to reveal differences in thermal characteristics of the northern and southern part of the study area. Özer et al. [2022] investigated tectonic properties of Erzurum (Eastern Turkey) using CPD, heat flow and seismological data. Figure 1. Simplified tectonic and geological map of Cyprus Island [modified from Symeou, 2018; the faults obtained from Symeou, 2018; Cagnan and Tanircan, 2010]. Figure 1. Simplified tectonic and geological map of Cyprus Island [modified from Symeou, 2018; the faults obtained from Symeou, 2018; Cagnan and Tanircan, 2010]. 2 Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island 2.2 Earthquake Data Spatial distribution of earthquakes in the study area is obtained from USGS Earthquake Catalogue [USGS, 2021]. During the earthquake selection, the study area is slightly expanded to evaluate the region on large scale. Additionally, all earthquakes between 1900 and 2020 whose magnitudes are higher than 2.5 are plotted. Focal depths of the earthquake events are not constrained. 2.1 EMAG2 Magnetic Data In this study, the magnetic data used in CPD, heat flow and boundary analysis is EMAG2 (global earth magnetic anomaly grid (2‑arc‑minute)) data, which is a compilation of measurements collected from satellite, sea and air [Maus et al., 2009]. The approximate resolution of EMAG2 data is 2 arc minutes. The magnetic anomaly grid is given for an altitude of about 4 km above sea level [Maus et al., 2009]. The processing sequence of the EMAG2 includes 5 steps: (1) grid merging, (2) airborne and ship magnetic data processing, (3) line levelling, (4) using an anisotropic correlation model over the oceans and (5) replacing the longest wavelength data (≥ 330 km) with MF 6 model [Maus et al., 2008]. EMAG2 provides wide opportunities such as testing tectonic hypotheses, investigating tectonic- structural relationships, generating plate reconstruction models and estimating CPD. The EMAG2 magnetic anomaly map obtained for the study area is given in Figure 2a. Reduced to the north magnetic pole (RTP) was applied to the total magnetic anomaly values to eliminate bipolarity, and the obtained magnetic anomaly map is shown in Figure 2b. 3 Eren Pamuk and İlkin Özsöz Eren Pamuk and İlkin Özsöz b) a) Figure 2. a) EMAG2 total field magnetic anomaly map [compiled from Maus et al., 2009], b) RTP total magnetic anomaly map with major tectonic structures in the study area (the black plus show the block centers used in the CPD calculation). b) a) a) b) b)i Figure 2. a) EMAG2 total field magnetic anomaly map [compiled from Maus et al., 2009], b) RTP total magnetic anomaly map with major tectonic structures in the study area (the black plus show the block centers used in the CPD calculation). Curie-Point Depth Estimation in Cyprus Island 2.3 Calculation of Curie Point Depth and Heat Flow The radially averaged power spectrum procedure which provides depth to the top of the magnetic layer was in‑ troduced by Spector and Grant [1970]. Additionally, this method was improved by Treitel et al. [1971], Bhattacharyya and Leu [1975], Connard et al. [1983], Okubo et al. [1985], Blakely [1995], Tanaka et al. [1999], Ravat [2004] and Ross et al. [2006]. These methods allow the thermal structure of a region to be obtained by using magnetic anomaly values. A simplified definition of the radially averaged spectrum can be given as: (1) (1) where 𝐴 is constant, 𝑘 is wavenumber and 𝑧𝑏 and 𝑧𝑡 are depth to the top and the bottom of the magnetic source. Applying the natural logarithm to both sides of equation 1 leads to: (2) (2) For high and medium values of wavenumber components, the exponential part in equation 2 can be ignored. Consequently, equation 2 is re‑written as: (3) (3) In equation 3 slope equals to 2𝑧𝑡(𝑘). If equation 2 is divided by 2, the linear estimation model can be obtained. (4) (4) where 𝐶 is constant. The slope of equation 4 provides depth to the top of the magnetic layer (𝑧𝑡(𝑘)). Multiplying equation 1 by 𝑒−\|𝑘\|(𝑧0−𝑧0) and dividing by 2 presents: where 𝐶 is constant. The slope of equation 4 provides depth to the top of the magnetic layer (𝑧𝑡(𝑘)). Multiplying equation 1 by 𝑒−\|𝑘\|(𝑧0−𝑧0) and dividing by 2 presents: (5) (5) where 𝑧0 is depth to the centroid of the magnetic source and it is calculated as (𝑧𝑏−𝑧𝑡)/2. In order to compute 𝑧𝑏 (CPD), 𝑧0 must be calculated accurately and reliably. Replacing the exponential term in equation 5 by first terms of their Taylor series expansion produces approximated equation for 𝑧0. (6) (6) 5 Eren Pamuk and İlkin Özsöz (𝑧𝑏−𝑧𝑡) is defined as the thickness of the magnetic crust [Buddington and Lindsley, 1964; Gasparini et al., 1979; Hunt et al., 1995; Nishitani and Kono, 1983; Salazar et al., 2017]. Substituting and rearranging thickness of the magnetic crust by 𝑧0 yields: (7) (7) In equation 7, D is constant and 𝑧0 is computed from the slope for the low wavenumber components. Table 1. Curie Point Depths (CPD) and heat flow for the study area with block center coordinates and 𝑧0, 𝑧𝑡,values. 2.3 Calculation of Curie Point Depth and Heat Flow Centroid depth of the magnetic layer is associated with the bottom of the magnetic layer [Okubo et al., 1985; Tanaka et al., 1999]: (8) (8) The magnetic anomaly map for CPD calculation is divided into 60 blocks with a size of 60 km×60 km. Each block is overlapped with an adjacent block by 50%. In other words, the distance of the centres of the two blocks to each other is 30 km. The centres of the blocks are shown with a black plus (Figure 2b). The power spectrum method of The magnetic anomaly map for CPD calculation is divided into 60 blocks with a size of 60 km×60 km. Each block is overlapped with an adjacent block by 50%. In other words, the distance of the centres of the two blocks to each other is 30 km. The centres of the blocks are shown with a black plus (Figure 2b). The power spectrum method of Block No blok center‑X blok center‑Y 𝒛𝟬 𝒛𝒕 CPD (km) Surface_heat Flow (mW/m2) K = 2.5 K = 1.5 K = 1 b1 390790 3786850 10.37 1.8 18.94 76.6 45.9 30.6 b2 420790 3786850 11.19 2.48 19.9 72.9 43.7 29.1 b3 450790 3786850 9.04 1.8 16.28 89.1 53.4 35.6 b4 480790 3786850 13.26 1.73 24.79 58.5 35.1 23.4 b5 510790 3786850 8.95 1.51 16.39 88.5 53.1 35.4 b6 540790 3786850 13.36 2.18 24.54 59.1 35.5 23.6 b7 570790 3786850 14.39 1.86 26.92 53.9 32.3 21.5 b8 600790 3786850 13.92 2.02 25.82 56.2 33.7 22.5 b9 630790 3786850 9.06 2.13 15.99 90.7 54.4 36.3 b10 660790 3786850 13.26 1.69 24.83 58.4 35.0 23.4 b11 390790 3816850 11.11 1.98 20.24 71.6 43.0 28.7 b12 420790 3816850 7.73 1.72 13.74 105.5 63.3 42.2 b13 450790 3816850 7.73 1.54 13.92 104.2 62.5 41.7 b14 480790 3816850 10.97 1.7 20.24 71.6 43.0 28.7 b15 510790 3816850 10.77 1.9 19.64 73.8 44.3 29.5 b16 540790 3816850 15.33 2.48 28.18 51.5 30.9 20.6 b17 570790 3816850 12.43 2.02 22.84 63.5 38.1 25.4 b18 600790 3816850 8.74 1.98 15.5 93.5 56.1 37.4 b19 630790 3816850 12.62 1.8 23.44 61.9 37.1 24.7 b20 660790 3816850 13.62 1.88 25.36 57.2 34.3 22.9 b21 390790 3846850 14.58 3.36 25.8 56.2 33.7 22.5 b22 420790 3846850 13.97 3.16 24.78 58.5 35.1 23.4 b23 450790 3846850 8.49 1.7 15.28 94.9 56.9 38.0 6 Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island Block No blok center‑X blok center‑Y 𝒛𝟬 𝒛𝒕 CPD (km) Surface_heat Flow (mW/m2) K = 2.5 K = 1.5 K = 1 b24 480790 3846850 11.51 1.91 21.11 68.7 41.2 27.5 b25 510790 3846850 10.89 1.92 19.86 73.0 43.8 29.2 b26 540790 3846850 11.36 2.41 20.31 71.4 42.8 28.6 b27 570790 3846850 10.37 1.88 18.86 76.9 46.1 30.8 b28 600790 3846850 8.2 1.92 14.48 100.1 60.1 40.1 b29 630790 3846850 10.54 2.29 18.79 77.2 46.3 30.9 b30 660790 3846850 12.98 2.4 23.56 61.5 36.9 24.6 b31 390790 3876850 15.58 2.89 28.27 51.3 30.8 20.5 b32 420790 3876850 9.94 2.22 17.66 82.1 49.3 32.8 b33 450790 3876850 7.46 1.65 13.27 109.3 65.6 43.7 b34 480790 3876850 10.13 1.57 18.69 77.6 46.5 31.0 b35 510790 3876850 8.75 2.04 15.46 93.8 56.3 37.5 b36 540790 3876850 9.02 1.72 16.32 88.8 53.3 35.5 b37 570790 3876850 7.77 1.65 13.89 104.4 62.6 41.8 b38 600790 3876850 8.7 2.02 15.38 94.3 56.6 37.7 b39 630790 3876850 11.1 2.63 19.57 74.1 44.5 29.6 b40 660790 3876850 9.42 1.94 16.9 85.8 51.5 34.3 b41 390790 3906850 12.52 2.11 22.93 63.2 37.9 25.3 b42 420790 3906850 9.17 1.96 16.38 88.5 53.1 35.4 b43 450790 3906850 9.37 1.91 16.83 86.2 51.7 34.5 b44 480790 3906850 8.02 2.22 13.82 104.9 63.0 42.0 b45 510790 3906850 9.28 2.06 16.5 87.9 52.7 35.2 b46 540790 3906850 9.76 2.13 17.39 83.4 50.0 33.4 b47 570790 3906850 10.52 1.77 19.27 75.2 45.1 30.1 b48 600790 3906850 9.3 1.76 16.84 86.1 51.7 34.4 b49 630790 3906850 12.43 1.74 23.12 62.7 37.6 25.1 b50 660790 3906850 10.17 1.77 18.57 78.1 46.8 31.2 b51 390790 3936850 8.84 1.69 15.99 90.7 54.4 36.3 b52 420790 3936850 9.94 1.76 18.12 80.0 48.0 32.0 b53 450790 3936850 9.36 1.86 16.86 86.0 51.6 34.4 b54 480790 3936850 8.13 1.81 14.45 100.3 60.2 40.1 b55 510790 3936850 8.98 1.78 16.18 89.6 53.8 35.8 b56 540790 3936850 7.14 1.88 12.4 116.9 70.2 46.8 b57 570790 3936850 8.78 1.82 15.74 92.1 55.3 36.8 b58 600790 3936850 11.67 1.7 21.64 67.0 40.2 26.8 b59 630790 3936850 13.92 2.11 25.73 56.4 33.8 22.5 b60 660790 3936850 14.04 2.21 25.87 56.0 33.6 22.4 Table 1 Curie Point Depths (CPD) and heat flow for the study area with block center coordinates and 𝑧 𝑧values 7 Curie-Point Depth Estimation in Cyprus Island where 𝑞 is heat flow, is thermal gradient, 𝑘 is the coefficient of thermal conductivity. According to Tanaka et al. [1999], Curie temperature (θ) can be determined as (Eq 10): , (10) , (10) where 𝑧𝑏 is CPD. The heat flow values were obtained by combining Eqs. (9) and (10): where 𝑧𝑏 is CPD. The heat flow values were obtained by combining Eqs. (9) and (10): , (11) , (11) where 𝑞 is heat flow, 𝑘 is the coefficient of thermal conductivity, 𝑧𝑏 is CPD. In heat flow calculation, it is assumed that thermal conductivity is 2.5 Wm–1K–1, Curie point temperature of 580 °C [Bektaş et al., 2007; Nwankwo et al. 2011; Pamuk 2019]. In addition, heat flow computation was made for K value 1 and 1.5 Wm–1K–1 in this study. where 𝑞 is heat flow, 𝑘 is the coefficient of thermal conductivity, 𝑧𝑏 is CPD. In heat flow calculation, it is assumed that thermal conductivity is 2.5 Wm–1K–1, Curie point temperature of 580 °C [Bektaş et al., 2007; Nwankwo et al. 2011; Pamuk 2019]. In addition, heat flow computation was made for K value 1 and 1.5 Wm–1K–1 in this study. 2.3.1 Wavenumber Ranges for the Estimated CPD The computed 𝑧𝑏 and 𝑧𝑡 values should be validated by comparing theoretical approximation and linear estima‑ tion. Most of the authors ignore this comparison and select the wavenumber ranges manually. Núñez Demarco et al. [2021] evaluated 72 articles in terms of the wavenumber ranges. Núñez Demarco et al. [2021] suggested mathematical validity of computation can be tested by comparing the results of equations (2) and (3) for 𝑧𝑡 and (5) and (6) for 𝑧𝑏. Comparison can be conducted by slope difference between linear estimation model and theoretical curve. , (12) (12) where Δ𝑚 represents slope difference, 𝑡(𝑘) is the theoretical curve and 𝑙(𝑘) is the linear estimation. As Δ𝑚 converges to 0, it is possible to say that the estimated 𝑧0 and 𝑧𝑡 are valid. In other words, the wavenumber ranges, where the difference between linear approximation and the theoretical curve is less than 5%, are valid for 𝑧0 and 𝑧𝑡 estimation. Broadly speaking, valid regions of 𝑧𝑡 is larger than that of 𝑧0. As the thickness of the magnetic layer (Δ𝑍) increases, valid regions tend to be wider for 𝑧𝑡 and narrower for 𝑧0. Moreover, if 𝑧𝑡 is deeper for the constant Δ𝑍, the confidence region becomes broader. Eren Pamuk and İlkin Özsöz Spector and Grant [1970] was applied to each block. Through the power spectra, 𝑧𝑡 and 𝑧0 were determined by the least‑squares method. Calculated depths are given in Table 1. As an example, the power spectrum of the magnetic anomaly of block b16 is given in Figure 3. After the centre depth (𝑧0) of the deepest magnetic source is obtained, the upper boundary depth (𝑧𝑡) of the magnetic source is estimated from the slope of the second‑longest wavelength part of the spectrum. (Fig. 3a, 3b). Centre (𝑧0) and top (𝑧𝑡) depths were computed as 15.33 km and 2.48 km for block b16. CPD was obtained as 28.18 km using Equation 8. The calculation of heat flow and thermal gradient based on Fourier Law [1955] is given in equation 2. In this equation, it is assumed that the direction of the heat flow is vertical and the heat gradient 𝑑𝑇/𝑑𝑍 is constant. , (9) , (9) 8 a) b) Figure 3. Examples of power spectrum of the block 16 for estimation of the CPD, a) the determining of the centroid depth 𝑧0 b) the determining of the top depth, 𝑧𝑡. a) a) b) b) Figure 3. Examples of power spectrum of the block 16 for estimation of the CPD, a) the determining of the centroid depth, 𝑧0 b) the determining of the top depth, 𝑧𝑡. 8 Curie-Point Depth Estimation in Cyprus Island 2.5 Computation of Moho Depth Airy‑Heiskanen Isostasy theory is used for the estimation of Moho depth in the study area. Fundamentally, the theory assumes that topographic features are compensated by subsurface variations [Kirby, 2019]. According to Airy [1855] and Heiskanen [1931], crust‑mantle boundary fluctuates regarding to significant undulations on the surface. It is worth noting that it is assumed that uniform densities are observed in both crust and mantle. The compensation column can be described as the depth at which lithostatic pressures are uniform. The com‑ pensation depth is at the bottom of the lithospheric block, which is floating in the asthenosphere [Mukherjee, 2017]. For Airy‑Heiskanen Isostasy theory, load lies directly under the topographic features and is not affected by neighbouring topographic loads [Liu et al., 2017]. Haxby and Turcotte [1978] suggested that the standard thickness of the crust with zero topographic elevation is usually taken as 30 km. The equation of the Airy‑Heiskanen Isostasy model is different for land and sea. For sea and land, the model can be described as: , (16) , (17) , (16) (16) , (17) (17) where 𝑡𝑠𝑒𝑎 and 𝑡𝑙𝑎𝑛𝑑 are crust thickness for land and sea. Density in crust 𝜌𝑐, water (𝜌𝑤) and Moho (𝜌𝑀) are assumed as 2.67 g/cc, 1.03 g/cc and 3.30 g/cc respectively. T denotes compensation column from mean sea level and presumed as 30 km. where 𝑡𝑠𝑒𝑎 and 𝑡𝑙𝑎𝑛𝑑 are crust thickness for land and sea. Density in crust 𝜌𝑐, water (𝜌𝑤) and Moho (𝜌𝑀) are assumed as 2.67 g/cc, 1.03 g/cc and 3.30 g/cc respectively. T denotes compensation column from mean sea level and presumed as 30 km. Eren Pamuk and İlkin Özsöz Eren Pamuk and İlkin Özsöz The second method used to determine the boundaries of geological structures is THDR developed by Cordell and Grauch [1985]. THDR can be determined using equation 14: , (14) (14) The last method used for boundary analysis is the tilt angle developed by Miller and Singh [1994]. TA can be easily determined using equation 15. , (15) (15) where TA is tilt angle, 𝜕𝑀/𝜕𝑧 is the vertical derivative of the magnetic anomaly, THDR is the total horizontal derivative. Tilt angle varies from –1.570 to 1.570 (–𝜋/2 to +𝜋/2). The TA values are negative outside the source while TA values are zero at the boundary location of the source in the vertical position. 2.4 Edge Detection in Magnetic Data An attempt was made to determine the boundaries of the source causing the magnetic anomaly using Analytic Signal (AS), Total Horizontal Derivative (TDHR) and Tilt angle (TA) methods, which are the function of derivatives in the x, y or z directions of the potential field data. First, the approximate boundaries of the structures that cause the magnetic anomaly are determined by the Analytic Signal (AS) given in equation 13 [Nabighian, 1972; MacLeod et al., 1993; Bilim et al., 2017]. , (13) (13) where 𝑀 is the magnitude of the magnetic anomaly and 𝜕𝑀/𝜕𝑥 and 𝜕𝑀/𝜕𝑦 are the horizontal derivatives, ∂M/∂z is the vertical derivative of the magnetic anomaly. where 𝑀 is the magnitude of the magnetic anomaly and 𝜕𝑀/𝜕𝑥 and 𝜕𝑀/𝜕𝑦 are the horizontal derivatives, ∂M/∂z is the vertical derivative of the magnetic anomaly. 9 Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island , (18) (18) where and are mean values of two gridded datasets, 𝑑 and 𝑢 are lags. If the output of the normalised cross‑correlation is 1 or –1, it is likely to say that the two datasets are positively or negatively correlated. Never‑ theless, 0 or approximately 0 cross‑correlation value represents uncorrelated datasets. where and are mean values of two gridded datasets, 𝑑 and 𝑢 are lags. If the output of the normalised cross‑correlation is 1 or –1, it is likely to say that the two datasets are positively or negatively correlated. Never‑ theless, 0 or approximately 0 cross‑correlation value represents uncorrelated datasets. 2.6 Cross‑Correlation Cross‑correlation compares two series and provides the quantitative representation that indicates the degree of match [Bourke, 1996]. In this study, normalised 2D cross‑correlation is preferred to compare Moho and Curie depths. Therefore, two gridded data are compared instead of series. Since the cross‑correlation equation is normalised, the output will range from –1 to 1. The empirical description [Bourke, 1996] of cross‑correlation for two gridded data (𝑥(𝑖,𝑗) and 𝑦(𝑖,𝑗)) is presented as: 10 Eren Pamuk and İlkin Özsöz the southeast of Akrotiri Bay, west of the study area, around Rizokarpaso (Dipkarpaz). CPD values in these areas are deeper than 25 km. The shallowest CPD values were obtained around Polis, Morphou Bay, in the area between Larnaca and Famagusta and north of Kyrenia. CPD values in these areas are shallower than 15 km. CPD values range from 18 to 20 km in Limassol, and range from 15 to 18 km in Nicosia (Lefkoşa) and south of it, around Kyrenia, and west of Famagusta (Figure 4). Heat Flow values were calculated based on CPD values, and these calculated values were given in Figure 5 and Table 1. Heat flow values (for K = 2.5 Wm–1K–1) were calculated between 51.3 and 116.0 mW/m2 for the study area (Figure 5). Minimum heat flow values were obtained to the west of the study area, southeast of Akrotiri Bay, around Rizokarpaso (Dipkarpaz), and southeast of the study area. Heat flow values are less than 65 mW/m2 in these areas. The highest heat flow values were obtained around Polis, Morphou Bay, in the area between Larnaca and Famagusta and north of Kyrenia. Heat flow values are more than 100 mW/m2 in these areas. Moreover, Heat flow values range from 65 to 75 mW/m2 around Limassol, and range from 85 to 95 mW/m2 in Nicosia and south of it, around Kyrenia, west of Famagusta (Figure 5). Figure 5. Heat‑flow map of Cyprus Island for thermal conductivity 2.5 Wm–1K–1; 1.5 Wm–1K–1 and 1 Wm–1K–1 [with heat flow values from boreholes, Morgan, 1979]. Figure 5. Heat‑flow map of Cyprus Island for thermal conductivity 2.5 Wm–1K–1; 1.5 Wm–1K–1 and 1 Wm–1K–1 [with heat flow values from boreholes, Morgan, 1979]. In this study, AS, THDR and TA methods were used to detect the boundaries (geological contacts, faults, etc.) of the structures that cause magnetic anomalies in and around the study area. Maximum and minimum values of AS and THDR ranges from 0.0002 to 0.0537 and 0 to 0.0394 respectively. The AS and THDR maps present higher values in the NW part of the study area while lower values are observed in the SE part. TA values vary between +1.57 rad (90°) and –1.57 rad (–90°). The higher TA values are noted in the NW, NE and S part of the study area whilst SE and E parts have lower TA values. 3. Results RTP magnetic anomaly map of the study area was investigated. The RTP anomalies are centred over magnetic sources which provide a more interpretable map in terms of causative bodies. RTP only take the phase of the magnetic field into account instead of the amplitude of the field. RTP has required for edge detection and depth estimation methods except for analytic signal and local wavenumber [Fairhead et al., 2011]. It was observed that magnetic values ranged from –202 to 413 nT. The lowest magnetic values (negative anom‑ alies) were observed in the east of the Polis and Paphos regions, and the highest magnetic values were obtained in the west and east of the Rizokarpaso region in the east of Nicosia and Kyrenia. Values in the west of the Rizokarpaso (Dipkarpaz) region range from 140 to 400 nT (Figure 2b). It is possible to say that lower magnetic values in the RTP map may correspond to the thinner magnetic crust where lower CPD or high heat flow are values observed. However, this assumption is not valid in all areas since the primary factor that effect the strength field is magnetic mineral content in the subsurface. The distribution of other CPD values obtained for Cyprus Island and its surroundings is given in Table 1 and Figure 4. CPD values range from 12.4 km to 28.18 km for the study area. Maximum CPD values were obtained at Figure 4. Curie Point Depth (CPD) map of Cyprus Island. Figure 4. Curie Point Depth (CPD) map of Cyprus Island. 11 11 Curie-Point Depth Estimation in Cyprus Island data, and then generated magnetic anomaly map was divided into 60 parts for CPD calculation. CPD and heat flow values were calculated for each block and mapped. CPD values ranged from 12.4 km to 28.18 km, and heat flow values were calculated between 20 and 50 mW/m2 for K = 1. Low CPD values and high heat flow values can potentially be associated with geothermal fields. Therefore, areas with high heat flow and low CPD value may be recommended for further geothermal research. Structure boundaries obtained by boundary analysis methods (AS, THDR, TA) are generally compatible with each other. data, and then generated magnetic anomaly map was divided into 60 parts for CPD calculation. CPD and heat flow values were calculated for each block and mapped. CPD values ranged from 12.4 km to 28.18 km, and heat flow values were calculated between 20 and 50 mW/m2 for K = 1. Low CPD values and high heat flow values can potentially be associated with geothermal fields. Therefore, areas with high heat flow and low CPD value may be recommended for further geothermal research. Structure boundaries obtained by boundary analysis methods (AS, THDR, TA) are generally compatible with each other. The estimated CPD values are justified by comparing theoretical and linear approximation models for different magnetic thicknesses. The confidence region corresponds to areas where slope difference < 5%. The range of the estimated 𝑧𝑡 is from 1.51 km to 3.36 km. Furthermore, the average thickness of the magnetic layer is 25 km. Since the minimum 𝑧𝑡 for the constant Δ𝑍 corresponds to the narrowest confidence region, 1.51 km is chosen to test the validity of the estimations (Figure 6). For 𝑧𝑡 = 1.51 km and Δ𝑍 = 25 km, the valid region covers k > 0.72 km–1 is valid for 𝑧𝑡 estimation whereas confidence region for 𝑧0 is k < 0.71 km–1. During the estimation process wavenumber range for 𝑧𝑡 is from 0.79 km–1 to 2.50 km–1 while the same range for 𝑧0 is between 0.13 km–1 and 0.69 km–1. It is possible to say that 𝑧𝑡 and 𝑧0 values are computed within the mathematically appropriate region. a) c) b) d) Figure 6. 4. Discussion and Conclusion In this study, CPD and heat flow were obtained for Cyprus Island by spectral analysis of EMAG2 magnetic data. In addition, the boundaries of geological structures, which play an important role in interpreting potential field data, are defined by different edge detection techniques. First, reduction to the pole was applied to the magnetic 12 Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island In their study, estimated thermal conductivity values range from 0.6 to 4.5 Wm–1K–1. In his study, Morgan [1979] reported that thermal conductivity is lower than 2 Wm–1K–1 in general terms for Cyprus. Since the K value is very variable for Cyprus, the heat flow calculation was calculated for the cases where the thermal conductivity is 2.5, 1.5 and 1 Wm–1K–1 and shown in Figure 5. Morgan [1979] also performed heat flow calculations for Cyprus with the help of 33 boreholes. These heat flow calculations were also given in Figure 5 and compared with the heat flow values calculated in this study. Morgan [1979] observed the best fit value as K = 1 Wm–1K–1 amongst the heat flow values he calculated and the heat flow values obtained in this study. For K = 1, the heat flow values ranged from 20 to 50 mW/m2 in this study, and the heat flow values ranged from 5 to 46 mW/m2 in Morgan’s study [1979]. Morgan [1979] calculated between 28‑32 mW/m2 in Larnaca, and it was 35 mW/m2 in this study. The heat flow values measured in the wells between Polis and Paphos were between 5 and 46 mW/m2, and the heat flow in this region ranges from 35‑40 mW/m2 in this study. The heat flow was between 28‑46 mW/m2 in the well measurements in the old study at the northeast of the Troodos mountains, and in this study, the heat flow in this area was about 35 mW/m2. Heat flow measurements in wells at the southwest of Limassol were between 5 and 28 mW/m2, and in this study, these values in the same area are around 25 mW/m2. It can be said that the results of this study are consistent with older [Morgan, 1979] well studies in general terms for these areas mentioned. The heat flow was between 5‑12 mW/m2 in the wells at the east of Kyrenia, and The heat flow in this area was obtained as 30 mW/m2 in this study. The results are incompatible with older studies for this region (Figure 5). In addition, Kalogirou [2014] prepared a heat flow map for Cyprus Island using artificial neural networks in their study. In this study, the map they created was consistent with areas with high heat flow (Polis and its surroundings, Morphou Bay and its surroundings, Paphas and its surroundings). Figure 7. Curie-Point Depth Estimation in Cyprus Island Comparison of theoretical curve and linear approximation: a) Slope difference (%) map for 𝑧𝑡 estimation, b) Correlation between theoretical and linear approximation model for 𝑧𝑡 computation, c) Slope difference (%) map for 𝑧0 estimation, d) Correlation between theoretical and linear approximation model for 𝑧0 computation. a) b) b) b) a) c) d) d) c) d) Figure 6. Comparison of theoretical curve and linear approximation: a) Slope difference (%) map for 𝑧𝑡 estimation, b) Correlation between theoretical and linear approximation model for 𝑧𝑡 computation, c) Slope difference (%) map for 𝑧0 estimation, d) Correlation between theoretical and linear approximation model for 𝑧0 computation. Losif Stylianou et al. [2017] focused on a methodology to measure and analyze the thermal properties of lithology in their study. They obtained thermal conductivity between 0.5 and 1.5 Wm–1K–1 for Nicosia (Lefkoşa). Florides et al. [2014] presented information about the density, thermal conductivity, specific heat, and thermal diffusivity of various lithologies in Cyprus Island. They calculated thermal conductivity at different values for different samples. They noted that the specific weight of the rocks is the cause of this. For example, they measured 0.50 Wm–1K–1 for Nicosia marl, and 2.29 Wm–1K–1 for Serpentinite, and 0.82 Wm–1K–1 for Upper pillow Lava. In another study, Florides et al. [2010] calculated thermal conductivity values as follows; 1.42 to 1.97 Wm–1K–1, for Lakatamia 1.68 Wm–1K–1, Agia Napa 1.58 Wm–1K–1, Meneou 1.4 to 1.72 Wm–1K–1, and Prodromi 1.87 Wm–1K–1. Losif Stylianou et al. [2016] presented a thermal conductivity map for Cyprus Island in the studies they conducted 13 Eren Pamuk and İlkin Özsöz in 2016. In their study, estimated thermal conductivity values range from 0.6 to 4.5 Wm–1K–1. In his study, Morgan [1979] reported that thermal conductivity is lower than 2 Wm–1K–1 in general terms for Cyprus. Since the K value is very variable for Cyprus, the heat flow calculation was calculated for the cases where the thermal conductivity is 2.5, 1.5 and 1 Wm–1K–1 and shown in Figure 5. Morgan [1979] also performed heat flow calculations for Cyprus with the help of 33 boreholes. These heat flow calculations were also given in Figure 5 and compared with the heat flow values calculated in this study. Morgan [1979] observed the best fit value as K = 1 Wm–1K–1 amongst the heat flow values he calculated and the heat flow values obtained in this study. in 2016. Curie-Point Depth Estimation in Cyprus Island Analytic signal (AS) map of the magnetic anomaly of Cyprus Island. Figure 7. Analytic signal (AS) map of the magnetic anomaly of Cyprus Island. In the AS map, maximum amplitudes were obtained at the west of Nicosia (Lefkoşa) and Kyrenia, and north of Famagusta (Figure 6). Regarding to the AS map, the structures that cause magnetic anomaly generally draw an arc between Rizokarpaso, Kyrenia, and Morphou Bay (Figure 7). THDR map was examined, and it showed similar results 14 Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island It is probable to say that if Moho and Curie considerably match, 15 Eren Pamuk and İlkin Özsöz Figure 9. TA (Tilt Angle) map of the magnetic anomaly of Cyprus Island (red line shows 0 contour in TA map). Figure 9. TA (Tilt Angle) map of the magnetic anomaly of Cyprus Island (red line shows 0 contour in TA map). the region might be interpreted as a passive crust. On the other hand, the mismatch or notable difference between Moho and Curie is likely to indicate an active tectonic regime. Illustration of Curie depth, Moho depth, Moho‑Curie and cross‑correlation of Curie and Moho depths are given in Figure 10. There are two reasons for obtaining shallower Moho depth than CPD. The first reason is the Moho‑Curie differ‑ ence is higher than 0 unless upper mantle magnetic phases are observed [Haggerty, 1978; Roberts, 2006; Ferré et al., 2013]. In that case, it is possible to say that the temperature of the magnetised mantle is lower than Curie tempera‑ ture [Guimarães et al., 2014]. The second reason is that Curie depth estimation using spectral analysis is a fairly subjective process. Additionally, the Moho depth calculation approach includes assumptions that affect the result significantly. Hence, the unrealistic negative values may appear in the Moho‑Curie difference due to subjectivity and the presumptions. As it can be seen from Figure 10, Moho depth values are remarkably high (around 35‑38 km) in Troodos Moun‑ tains due to the isostatic compensation theory. Curie depth values are relatively deeper (28‑30 km) in the S‑SE part of the study area. Regarding to cross‑correlation results, a higher correlation coefficient (0.60‑0.75) in the southern part of Cyprus Island is observed. Moho‑Curie difference produced approximately 0 contours in the southern part of the study area. It is possible to say that the tectonically passive regime becomes dominant towards the southern part of the study area due to high cross‑correlation values and low difference in that region. It is crucial to note that earthquakes are generally occurring in the brittle part of the crust. Therefore, it is fair to say that both rigidity of the crust and Curie depth are dependent on temperature in the subsurface. Consequently, including the focal depth and distribution of the seismic activity would enhance the interpretation stage. Curie-Point Depth Estimation in Cyprus Island to the AS map. In the west and east of Nicosia and Kyrenia, and north of Famagusta, the direction of the structure causing the magnetic anomaly was determined as N‑S (Figure 8). As known in the TA distribution, zero contours are directly related to the structure boundary. The boundaries of the structure that caused magnetic anomaly were obtained as N‑S, such as THDR in the west of Nicosia and Kyrenia, and north of Famagusta. The N‑S structures was indicated by dashed circles. The structure that causes the anomaly in this area extends to the land boundary in the area between Larnaca and Famagusta. The structure boundaries were obtained by different orientations around the Troodos Mountains. The structure boundaries that start at the Episkoi and Akrotiti bays and extend to the southeast of the study area are NW‑SE directional. In the NE of the study area, the structures are generally NE‑SW directional (Figure 9). Figure 8. THDR (total horizontal derivative) map of the magnetic anomaly of Cyprus Island. Figure 8. THDR (total horizontal derivative) map of the magnetic anomaly of Cyprus Island. Moho depth and CPD are two physical situations that reflect temperature and discontinuity layer. However, both physical situations are indicating a boundary. CPD demonstrates the boundary between magnetic and nonmagnetic crust whereas Moho depth illustrates crust‑mantle boundary. Moho depth can be considered as a boundary at which compositional change occurs. In some cases, especially in stable tectonic zones, magnetic rocks are substituted by non‑magnetic rocks at these transition zones [Ravat et al., 2007]. As a result of this, the difference between Moho and Curie depths becomes close to 0 [Idárraga‑García and Vargas, 2008; Özsöz, 2021]. Karabulut et al. [2019] was computed crustal thickness from receiver function analysis. According to their estimations, the crustal thickness in Cyprus Island is between 20 and 30 km. Furthermore, thicker crust is observed in the northern part of Cyprus Island which is compatible with Moho depth estimation results in this study. Calculated Moho depth through the Airy‑Heiskanen theory is compared to Curie depth to enhance interpretation of the tectonic activity. In order to analyse the tectonic regime of the area, correlation and difference between Moho and Curie depths are used. Additionally, cross‑correlation of Moho and Curie is computed to evaluate the areas where Moho and Curie depths match remarkably. Curie-Point Depth Estimation in Cyprus Island Regarding the quantitative analysis of the earthquakes, mean focal depth and magnitude are 25.86 ± 16.06 km and 3.42 ± 0.64 respectively. The earthquake distribution map and Moho‑Curie should be associated since 0 contours of Moho‑Curie represent passive tectonic activity. Spatial distribution of earthquake data indicates that the majority of tectonic activity occurs in the south‑western part of Cyprus Island where the Moho‑Curie difference is approximately 10 km. Few earthquakes are noted in the southern and the western part of the study area where Moho‑Curie is around 0. 16 Curie-Point Depth Estimation in Cyprus Island a) c) b) d) e) Figure 10. Interpretation of tectonic activity in the study area: a) Curie depth map, b) Moho depth map, c) cross‑correla‑ tion between Moho and Curie depth maps, d) Map that obtained by subtracting Curie depth values from Moho depth values, e) Earthquake (M > 2.5) distribution map. Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island b) b) a) c) b) d) a) a) d) d) d) c) d) e) Figure 10. Interpretation of tectonic activity in the study area: a) Curie depth map, b) Moho depth map, c) cross‑correla‑ tion between Moho and Curie depth maps, d) Map that obtained by subtracting Curie depth values from Moho depth values, e) Earthquake (M > 2.5) distribution map. Conversely, if Moho‑Curie is relatively high with respect to the surrounding region such as the south‑western part of Cyprus Island, an abundant number of earthquakes are observed. The deeper earthquake activity (> 30 km) dominantly occurred in the southwestern part of the study area where Curie and Moho depths are relatively shallow. Moreover, this area corresponds to the convergent plate boundary in which deep tectonic activity tends to occur. This work presents a tectonic interpretation of Cyprus Island by CPD estimation, edge detection methods, Moho‑Curie comparison and spatial earthquake distribution. The findings of the study shed light on stable‑unstable crustal structure and orientation of the magnetic sources in the study area. The AS, THDR and TA results showed that N‑S oriented structure is observed in the west and east of Kyrenia (Girne) and Nicosia (Lefkoşa). However, This work presents a tectonic interpretation of Cyprus Island by CPD estimation, edge detection methods, Moho‑Curie comparison and spatial earthquake distribution. The findings of the study shed light on stable‑unstable crustal structure and orientation of the magnetic sources in the study area. References Airy, G.B. (1855). III. On the computation of the effect of the attraction of mountain‑masses, as disturbing the apparent astronomical latitude of stations in geodetic surveys, Philosophical Trans. Royal Soc. Lon., 145, 101‑104. Arnaiz‑Rodríguez, M.S., N. Orihuela (2013). Curie point depth in Venezuela and the Eastern Caribbean, Tectono‑ physics, 590, 38‑51. Asim, K. M., S. S. Moustafa, I.A. Niaz, E.A. Elawadi, T. Iqbal, F. Martinez‑Alvarez (2020). Seismicity analysis and machine learning models for short‑term low magnitude seismic activity predictions in Cyprus, Soil Dyn. Earthq. Engin., 130, 105932. Bektaş, Ö., D. Ravat, A. Büyüksaraç, F. Bilim, A. Ateş (2007). Regional geothermal characterisation of East Anatolia from aeromagnetic, heat flow and gravity dataì, Pure Appl. Geophys., 164, 5, 975‑998. Bilim, F., S. Kosaroglu, A. Aydemir, A. Buyuksarac (2017). Thermal investigation in the Cappadocia region, Central Anatolia‑Turkey, analyzing curie point depth, geothermal gradient, and heat‑flow maps from the aeromagnetic data, Pure Appl. Geophys. 174, 12, 4445‑4458. Bhattacharyya, B.K. and L.K. Leu (1975). Spectral analysis of gravity and magnetic anomalies due to two‑dimensional structures, Geophysics, 40, 6, 993‑1013. Blakely, R.J. (1995). Potential theory in gravity and magnetic applications, Cambridge University Press. Blome, C.D. and W.P. Irwin (1985). Equivalent radiolarian ages from ophiolitic terranes of Cyprus and Oman, Geology, 13, 401‑404. Bourke, P., (1996). Cross correlation, autocorrelation, 2d pattern identification. Research Gate, 2019. Bourke, P., (1996). Cross correlation, autocorrelation, 2d pattern identification. Research Gate, 2019. Buddington, A.F. and D.H. Lindsley (1964). Iron‑titanium oxide minerals and synthetic equivalents, J. Petrol., 5, 2, 310‑357. Buddington, A.F. and D.H. Lindsley (1964). Iron‑titanium oxide minerals and synthetic equivalents, J. Pe 310‑357. Cagnan, Z. and G.B. Tanircan (2010). Seismic hazard assessment for Cyprus, J. Seismol., 14, 2, 225‑246. Chukwu, C.G., E.E. Udensi, E.M. Abraham, A.C. Ekwe, A.O. Selemo (2018). Geothermal energy potential from analysis of aeromagnetic data of part of the Niger‑delta basin, southern Nigeria, Energy, 143, 846‑853. Connard, G., R. Couch and M. Gemperle (1983). Analysis of aeromagnetic measurements from the Cascade Range in central Oregon, Geophysics, 48, 3, 259‑401. Cordell, L. and V.J.S. Grauch (1985). Mapping basement magnetization zones from aeromagnetic data in the San Juan Basin, New Mexico, In The utility of regional gravity and magnetic anomaly maps, 181‑197, Society of Exploration Geophysicists (SEG). Elmas, A. (2018). Determination of Structural Discontinuities in Cyprus Island by Using EGM08 Gravity Data, J. Geol. Engin., 42, 1, 17‑32 (in turkish). Ergün, M., S. Okay, C. Sari and E.Z. Oral (2003). Curie-Point Depth Estimation in Cyprus Island The AS, THDR and TA results showed that N‑S oriented structure is observed in the west and east of Kyrenia (Girne) and Nicosia (Lefkoşa). However, 17 Eren Pamuk and İlkin Özsöz the orientation of the magnetic anomaly is not identical around the Troodos Mountains for these edge detection methods. Combined analysis of Moho and Curie depths showed that stable crustal characteristics are observed in the southern part of the study area. There is a large number of earthquakes are noted in the south‑western part of the study area where the Moho‑Curie difference relatively high. Curie-Point Depth Estimation in Cyprus Island Artificial Intel., 95‑99, ISBN: 978‑960‑474‑379‑7 Karabulut, H., A. Paul, A.D. Özbakır, T. Ergün, and S. Şentürk (2019). A new crustal model of the Anatolia‑Aegean domain: evidence for the dominant role of isostasy in the support of the Anatolian plateau, Geophys. J. Int., 218, 1, 57‑73. Kempler, D. and Z. Ben‑Avraham (1987). The tectonic evolution of the Cyprean Arc, Ann. Tecton., 1, 58‑71. Kinnaird, T.C. (2008). Tectonic and sedimentary response to oblique and incipient continental collision the east‑ ernmost Mediterranean (Cyprus), unpublished Phd thesis, University of Edinburgh. Kirby, J.F. (2019). On the pitfalls of Airy isostasy and the isostatic gravity anomaly in general, Geophys. J. Int., 216, 1, 103‑122. Kumar, S., S.K. Pal, A. Guha, S.D. Sahoo, A. Mukherjee (2020). New insights on Kimberlite emplacement around the Bundelkhand Craton using integrated satellite‑based remote sensing, gravity and magnetic data, Geocarto Int., 1‑23. Li, C.F., J. Wang (2016). Variations in Moho and Curie depths and heat flow in Eastern and Southeastern Asia, Marine Geophys. Res., 37, 1, 1‑20. Li, C.F., Y. Lu, J. Wang (2017). A global reference model of Curie‑point depths based on EMAG2, Scientific Rep. 7, 1, 1‑9. Liu, B., Y. Zhou, and G. Yang (2017). Characteristics of isostatic gravity anomaly in Sichuan‑Yunnan region, China, Geod. Geodyn., 8, 4, 238‑245. , , ,J g ( ) g p p ,i p , , Liu, B., Y. Zhou, and G. Yang (2017). Characteristics of isostatic gravity anomaly in Sichuan‑Yunnan region, China, Geod. Geodyn., 8, 4, 238‑245. MacLeod, I.N., K. Jones and T.F. Dai (1993). 3‑D analytic signal in the interpretation of total magnetic field data at low magnetic latitudes, Explor. Geophys., 24, 679‑688. Makris, J., Z.B. Abraham, A. Behle, A. Ginzburg, P. Giese, L. Steinmetz, R.B. Whitmarsh and S. Eleftheriou (1983). Seismic refraction profiles between Cyprus and Israel and their interpretation, Geophys. J. Int., 75, 3, 575‑591. Maus, S., F. Yin, H. Lühr, C. Manoj, M. Rother, J. Rauberg, I. Michaelis, C. Stolle, and R.D. Müller (2008). Resolution of direction of oceanic magnetic lineations by the sixth‑generation lithospheric magnetic field model from CHAMP satellite magnetic measurements, Geochem., Geophys., Geosys., 9, 7, https://doi. org/10.1029/2008GC001949. Maus, S., U. Barckhausen, H. Berkenbosch, N. Bournas, J. Brozena, V. Childers, F. Dostaler, J.D. Fairhead, C. Finn, R.R.B von Frese, C. Gaina, S. Golynsky, R. Kucks, H. Lühr, P. Milligan, S. Mogren, R.D. Muller, O. Olesen, M. Pilkington, R. Saltus, B. Schreckenberger, E. Thebault and F.C. Tontini (2009). Curie-Point Depth Estimation in Cyprus Island Fourier, J. (1955). Analytical Theory of Heat, Dover Publications, New York. Fourier, J. (1955). Analytical Theory of Heat, Dover Publications, New York. Gasparini, P., M.S.M. Mantovani, G. Corrado and A. Rapolla (1979). Depth of Curie temperature in continental shields: a compositional boundary?. Nature, 278, 5707, 845‑846. Guimarães, S.N.P., D. Ravat and V.M. Hamza (2014). Combined use of the centroid and matched filtering spectral magnetic methods in determining thermomagnetic characteristics of the crust in the structural provinces of Central Brazil, Tectonophysics, 624, 87‑99. Central Brazil, Tectonophysics, 624, 87‑99. Haggerty S.E. (1978). Mineralogical constraints on Curie isotherms in deep crustal magnetic anomalies, Geophys. Res. Lett., 5, 105e108, https://doi.org/ 10.1029/GL005i002p00105. Hakyemez, H.Y. (2014). Main geological characterıstıcs of Northern Cyprus, TAPG Bulletin, 26, 2, 7‑46, (in turkish). Heiskanen, W. (1931). Isostatic tables for the reduction of gravimetric observations calculated on the basis of Airy’s hypothesis, Bulletin Géodésique, 30, 1, 110‑153. Harrison, R. W., W. L. Newell, H. Batıhanlı, I. Panayides, J. P. McGeehin, S. A. Mahan, A. Ozhur E. Tsiolaki, M. Necdet (2004). Tectonic framework and Late Cenozoic tectonic history of the northern part of Cyprus: implications for earthquake hazards and regional tectonics, J. Asian Earth Sci., 23, 2, 191‑210. Haxby, W.F. and D.L. Turcotte (1978). On isostatic geoid anomalies, J. Geophys. Res.: Solid Earth, 83, B11, 5473‑5478. Hunt, C.P., B.M. Moskowitz and S.K. Banerjee (1995). Magnetic properties of rocks and minerals. Rock physics and Haxby, W.F. and D.L. Turcotte (1978). On isostatic geoid anomalies, J. Geophys. Res.: Solid Earth, 83, B11, 5473‑5478. Haxby, W.F. and D.L. Turcotte (1978). On isostatic geoid anomalies, J. Geophys. Res.: Solid Earth, 83, B11, 5473‑5478. Hunt, C.P., B.M. Moskowitz and S.K. Banerjee (1995). Magnetic properties of rocks and minerals. Rock physics and phase relations: A handbook of physical constants, 3, 189‑204. Haxby, W.F. and D.L. Turcotte (1978). On isostatic geoid anomalies, J. Geophys. Res.: Solid Earth, 83, Hunt, C.P., B.M. Moskowitz and S.K. Banerjee (1995). Magnetic properties of rocks and minerals. Rock physics and phase relations: A handbook of physical constants, 3, 189‑204. Idárraga‑García, J. and C.A. Vargas (2018). Depth to the bottom of magnetic layer in South America and its relationship to Curie isotherm, Moho depth and seismicity behavior, Geod. Geodyn., 9, 1, 93‑107. Kalogirou, S. A., P. Christodoulides, G. A. Florides, P. Pouloupatis, I. Iosif‑Stylianou (2014). Using Artificial Neural Networks for the construction of contour maps of thermal conductivity, Adv. Neural Net., Fuzzy Sys. References April. Gravity and magnetic anomalies of the Cyprus arc and tectonic implications, In EGS‑AGU‑EUG Joint Assembly, 1663. Fairhead, J.D., A. Salem, L. Cascone, M. Hammill, S. Masterton and E. Samson (2011). New developments of the magnetic tilt‑depth method to improve structural mapping of sedimentary basins, Geophys. Prosp., 59, Advances in Electromagnetic, Gravity and Magnetic Methods for Exploration, 1072‑1086. Ferré, E.C., S.A. Friedman, F. Martín‑Hernández, J.M. Feinberg, J.A. Conder, and D.A. Ionov (2013). The magnetism of mantle xenoliths and potential implications for sub‑Moho magnetic sources, Geophys. Res. Lett., 40, 1, 105‑110. Florides, G. A., I. Iosif‑Stylianou, Z. Zomeni, E. Tsiolakis, P. Pouloupatis, S. A. Kalogirou, V. Messarites, Z. Zomeni, E. Tsiolakis, P.D. Pouloupatis, E. Theofanous (2014). Thermal properties of the ground in Cyprus and their correlations and effect on the efficiency of ground heat exchangers, Int. J. Environ., Ecol., Geol. Marine Engin., 8, 2, 112‑116. Florides, G.A., P. Pouloupatis, S.A. Kalogirou, V. Messaritis, I. Panayides, Z. Zomeni, G. Partasides, A. Lizides E. Eleni Sophocleous, K. Koutsoumpas (2010). Temperature profiles and thermal properties of the ground in Cyprus, for use in the design of ground heat exchangers, In Euro Sun 2010. 18 Curie-Point Depth Estimation in Cyprus Island Curie-Point Depth Estimation in Cyprus Island Özer, Ç., S. Öztürk, E. Pamuk (2022). Tectonic and structural characteristics of Erzurum and its surroundings (Eastern Turkey): a detailed comparison between different geophysical parameters. Turkish J. Earth Sci., 31, 85‑108. Özsöz, İ. (2021). Combined qualitative and quantitative regional interpretation of the thermal results of magnetic data in the Eastern Mediterranean Region, Turkish J. Earth Sci., 30, 5, 665‑680. Pamuk, E. (2019). Investigating edge detection, Curie point depth, and heat flow using EMAG2 magnetic and EGM08 gravity data in the northern part of Eastern Anatolia, Turkey, Turkish J. Earth Sci., 28, 6, 805‑821. Ravat, D., A. Pignatelli, I. Nicolosi and M. Chiappini (2007). A study of spectral methods of estimating the depth to the bottom of magnetic sources from near‑surface magnetic anomaly data, Geophys. J. Int., 169, 2, 421‑434. Ravat, D., A. Pignatelli, I. Nicolosi and M. Chiappini (2007). A study of spectral methods of estimating the depth to the bottom of magnetic sources from near‑surface magnetic anomaly data, Geophys. J. Int., 169, 2, 421‑434. Roberts A P (2006) High resolution magnetic analysis of sediment cores: strengths limitations and strategies for Roberts, A.P. (2006). High‑resolution magnetic analysis of sediment cores: strengths, limitations and strategies for maximizing the value of long‑core magnetic data, Phys. Earth Planet. Int., 156, 3‑4, 162‑178. , ( ) g g y g , g maximizing the value of long‑core magnetic data, Phys. Earth Planet. Int., 156, 3‑4, 162‑178. Robertson, A.H. (1998). Mesozoic‑Tertiary tectonic evolution of the easternmost Mediterranean area: integration o marine and land evidence, In Proceedings of the Ocean Drilling Program, Scientific Results, 160, 54. marine and land evidence, In Proceedings of the Ocean Drilling Program, Scientific Results, 160, 54. Ross, H.E., R.J. Blakely and M.D. Zoback (2006). Testing the use of aeromagnetic data for the determination of Curie depth in California, Geophysics, 71, 5, L51‑L59. Salazar, J.M., C.A. Vargas and H. Leon (2017). Curie point depth in the SW Caribbean using the radially averaged spectra of magnetic anomalies, Tectonophysics, 694, 400‑413. or, A. and F.S. Grant (1970). Statistical models for interpreting aeromagnetic data, Geophysics, 35, 2, 293‑30 p , ( ) p g g , p y , , , Staudigel, H., K.M. Gillis, R. Duncan (1986). K/Ar and Rb/Sr ages of celadonites from the Troodos ophiolite, Cyprus, Geology 14, 72‑75. Stylianou, I.I., G. Florides, S. Tassou, E. Tsiolakis, P. Christodoulides (2017). Methodology for estimating the ground heat absorption rate of Ground Heat Exchangers. Curie-Point Depth Estimation in Cyprus Island EMAG2: A 2‑arc min resolution Earth Magnetic Anomaly Grid compiled from satellite, airborne, and marine magnetic measurements. Geochemistry, Geophysics, Geosystems 10, 8, https://doi.org/10.1029/2009GC002471 McPhee, P.J. and D.J. van Hinsbergen (2019). Tectonic reconstruction of Cyprus reveals Late Miocene continental collision of Africa and Anatolia, Gondwana Res., 68, 158‑173. 19 Eren Pamuk and İlkin Özsöz Miller, H.G. and V. Singh (1994). Potential field tilt ‑ a new concept for location of potential field sources., J. Appl. Geophys., 32, 213‑217. Mohammed, A., T. Adewumi, S. A. Kazeem, R. Abdulwaheed, A.A. Adetona, A. Usman (2019). Assessment of geother‑ mal potentials in some parts of upper Benue Trough northeast Nigeria using aeromagnetic data, J. Geosci., Engin., Env., Technol., 4, 1, 7‑15. Montadert, L., S. Nicolaides, H.P. Semb, O. Lie (2014). Petroleum Systems Offshore Cyprus, in Petroleum systems of the Tethyan region, edited by L. Marlow, C. Kendall and L. Yose, AAPG memoir 106, 301‑334. Morgan, P. (1979). Cyprus heat flow with comments on the thermal regime of the eastern Mediterranean. In Terrestrial heat flow in Europe (Springer, Berlin, Heidelberg, 144‑151. Mukasa, S.B., J.N. Ludden (1987). Uranium‑lead isotopic ages of plagiogranites from the Troodos ophiolite, Cyprus, and their tectonic significance, Geology, 15, 9, 825‑828. Mukherjee, S. (2017). Airy’s isostatic model: a proposal for a realistic case, Arabian J. Geosci., 10, 12, 1‑7. Nabighian, M.N. (1972). The analytic signal of two‑dimensional magnetic bodies with polygonal cross‑section: its properties and use for automated anomaly interpretation, Geophysics, 37, 507‑517. Nagata, T. (1961). Rock magnetism, Maruzen, Tokyo. Nnange, J. Mining, Poudjom Djomani 350. Nishitani, T. and M. Kono (1983). Curie temperature and lattice constant of oxidized titanomagnetite, Geophys. J.Int., 74, 2, 585‑600. Njeudjang, K., J. D. Kana, A. Tom, J.M.A. Essi, N. Djongyang, R. Tchinda (2020). Curie point depth and heat flow deduced from spectral analysis of magnetic data over Adamawa volcanic region (Northern Cameroon): geothermal implications, SN Appl. Sci., 2, 8, 1‑16. Núñez Demarco, P., C. Prezzi, and L. Sánchez Bettucci (2021). Review of Curie point depth determination through different spectral methods applied to magnetic data, Geophys. J. Int., 224, 1, 17‑39. Nwankwo, L. I., P.I. Olasehinde and C.O. Akoshile (2011). Heat flow anomalies from the spectral analysis of airborne magnetic data of Nupe Basin, Nigeria, Asian J. Earth Sci., 4, 1, 20‑28. Okubo, Y., R.J. Graf, R.O. Hansen, K. Ogawa, H. Tsu (1985). Curie‑point depths of the Island of Kyushu and surrounding areas, Japan, Geophysics, 50, 481‑494. CORRESPONDING AUTHOR: Eren PAMUK, Department of Geophysical Research, General Directorate of the Mineral Research & Exploration of Turkey, 06800, Ankara, Turkey, e‑mail: eren.pamuk@mta.gov.tr Curie-Point Depth Estimation in Cyprus Island Energy 127, 258‑270. Stylianou, I.I., S. Tassou, P. Christodoulides, I. Panayides, G. Florides (2016). Measurement and analysis of thermal properties of rocks for the compilation of geothermal maps of Cyprus, Renew. Ener. 88, 418‑429. Swarbrick, R.E., A.H.F. Robertson (1980). Revised stratigraphy of the Mesozoic rocks of southern Cyprus, Geol. Mag., 117, 06, 547, https://doi.org/10.1017/S0016756800028892. Symeou, V. (2018). Transition from compression to strike‑slip tectonic styles along the northern margin of the Levant Basin Doctoral dissertation, Sorbonne université. 20 Curie-Point Depth Estimation in Cyprus Island Tanaka, A., Y. Okubo, O. Matsubayashi (1999). Curie point depth based on spectrum analysis of the magnetic anomaly data in East and Southeast Asia, Tectonophysics, 306, 3‑4, 461‑470. Treitel, S., W.G. Clement, and R.K. Kaul (1971). The spectral determination of depths to buried magnetic basement rocks, Geophys. J. Int., 24, 4, 415‑428. Treitel, S., W.G. Clement, and R.K. Kaul (1971). The spectral determination of depths to buried magnetic basement rocks, Geophys. J. Int., 24, 4, 415‑428. Ulutaş, E. (2020). The May 11 Paphos, Cyprus, earthquake: implications for stress regime and tsunami modelling for the Eastern Mediterranean shorelines, Arabian J. Geosci., 13, 18, 1‑13. Ulutaş, E. (2020). The May 11 Paphos, Cyprus, earthquake: implications for stress regime and tsunami modelling for the Eastern Mediterranean shorelines, Arabian J. Geosci., 13, 18, 1‑13. USGS (2021). Earthquake Catalogue [online]. Website https://earthquake.usgs.gov/earthquakes/search/ [accessed 25 August 2021]. USGS (2021). Earthquake Catalogue [online]. Website https://earthquake.usgs.gov/earthquakes/search/ [accessed 25 August 2021]. Xu, Y., T. Hao, H. Zeyen, F. Nan (2017). Curie point depths in North China Craton based on spectral analysis of magnetic anomalies, Pure Appl. Geophys., 174, 1, 339‑347. Xu, Y., T. Hao, H. Zeyen, F. Nan (2017). Curie point depths in North China Craton based on spectral analysis of magnetic anomalies, Pure Appl. Geophys., 174, 1, 339‑347. Zissimos, A.M., I.C. Christoforou, D.R. Cohen, S.D. Mooney, N.F. Rutherford (2019). Spatial distribution and controls on organic and inorganic carbon in the soils of Cyprus, J. Geochem. Expl., 196, 95‑104. 21
https://openalex.org/W3193606476	https://mediatum.ub.tum.de/doc/1715318/document.pdf	English	null	Partitioning and Translocation of Dry Matter and Nitrogen During Grain Filling in Spring Barley Varieties and Their Roles in Determining Malting Quality	Frontiers in plant science	2,021	cc-by	11,397	Partitioning and Translocation of Dry Matter and Nitrogen During Grain Filling in Spring Barley Varieties and Their Roles in Determining Malting Quality To meet the strict requirements for the malting quality of both grain size and protein content for malting barley, a better understanding of the partitioning and remobilization of dry matter (DM) and nitrogen (N) from individual vegetative organs during grain ﬁlling may contribute to adjusting a balance in both quality parameters to satisfy the malting criteria of the brewing industry. A 2-year experiment that included 23 spring malting barley varieties was carried out to determine the DM and N partitioning in different organs at anthesis and maturity and to estimate their remobilization to grains. In contrast to the genetic variation of the 23 barley varieties, year effect was the most important single factor inﬂuencing the DM and N accumulation at pre-anthesis, and the DM and N translocation from their reserves at pre-anthesis. Post-anthesis assimilates accounted for 71–94% of the total grain yield among the barley varieties in 2014 and 53–81% in 2015. In contrast, the N reserved in vegetative tissues at anthesis contributed to barley grain N from 67% in the variety Union to 91% in the variety Marthe in 2014, and 71% in the variety Grace to 97% in the variety Shakira in 2015. The results concluded that photosynthetically derived assimilates at post-anthesis played an important role in determining grain size, whereas N reserves at pre-anthesis and N remobilization at post-anthesis probably determined the grain protein content of the malting barley. To achieve a high quality of malting barley grains in both grain size and protein content simultaneously, balancing photosynthetic assimilates at post-anthesis and N reserves at pre-anthesis and N remobilization should be considered as strategies for the combination of the selection of spring malting barley varieties together with agronomic N management. Edited by: Hans-Peter Kaul, University of Natural Resources and Life Sciences Vienna, Austria Edited by: Hans-Peter Kaul, University of Natural Resources and Life Sciences Vienna, Austria Reviewed by: Jens Leon, University of Bonn, Germany Othmane Merah, Université de Toulouse, France Correspondence: Yuncai Hu hu@wzw.tum.de Reviewed by: Jens Leon, University of Bonn, Germany Othmane Merah, Université de Toulouse, France Correspondence: Yuncai Hu hu@wzw.tum.de Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Received: 09 June 2021 Accepted: 23 July 2021 Published: 23 August 2021 Keywords: dry matter, genotype, malting quality, nitrogen, partitioning, remobilization, spring barley ORIGINAL RESEARCH published: 23 August 2021 doi: 10.3389/fpls.2021.722871 Citation: Barmeier G, Hu Y and Schmidhalter U (2021) Partitioning and Translocation of Dry Matter and Nitrogen During Grain Filling in Spring Barley Varieties and Their Roles in Determining Malting Quality. Front. Plant Sci. 12:722871. doi: 10.3389/fpls.2021.722871 Barley is unique among crop plants and is the fourth most important crop globally among cereals after maize, rice, and wheat (Newton et al., 2011). Barley grain, in the form of malt, is a nutritional source for yeast, which is very important for the brewing industry. In addition to achieving high yield, both grain protein content and size that are speciﬁc quality criteria for malting barley must be met to maximize the eﬃciency of the malting process and the quality of the products. August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org Partitioning and Translocation in Malting Barley Varieties Barmeier et al. The requirements for grain protein content and size range from 9.5 to 12.5% and >2.38 mm in North America (Shrestha and Lindsey, 2019), while the grain protein content is from 10 to 12%, and the requirement for the grain size is >2.5 mm (>70%) and <2.2 mm (<5%) in Australia (Fox et al., 2003). In France and Germany, the malting barley grain protein content must be in the range of 9.5 to 11.5% of the dry weight, and the retention fraction (proportion of grains larger than 2.5 mm) must be >90% (Incograin, 2014; Bundessortenamt, 2016; Beillouin et al., 2018). If they do not meet these requirements, the grains can be downgraded to feed barley, resulting in a much lower price paid to farmers (Incograin, 2014; Bundessortenamt, 2016). The grain quality of malting barley is very variable because of genotypic and environmental eﬀects (Carreck and Christian, 1991; Atanassov et al., 1999; Savin et al., 2006; Vahamidis et al., 2021), making it challenging to meet quality standards. Since variation in annual weather conditions leads to diﬀerent inﬂuences on grain retention fraction and grain protein content, the same varieties may show a large variation in the annual quality parameters (Savin et al., 2006; Hu et al., 2021). Field Experiments p A 2-year ﬁeld experiment with 23 spring malting barley varieties in 2014 and 2015 (Table 1) was conducted at the Technical University of Munich’s experimental station at Dürnast in Germany (11◦41′60′′E, 48◦23′60′′ N). Barley seeds were sown in mid-March with a seed density of 330 seeds m−2, and the ﬁnal harvest was carried out at the end of July. A randomized complete block design with four replicates was used for the experiments. Plots consisted of 12 rows and were 10.9 m in length, i.e., 16.35 m² plot−1. The soil is characterized as a mostly homogeneous Cambisol of silty clay loam. The residual soil mineral nitrogen (soil Nmin) to a 60-cm depth before sowing was 65 kg ha−1 in 2014 and 40 kg ha−1 in 2015. Nitrogen fertilization was applied as a dressing at 70 kg N ha−1 at sowing in both years based on local N recommendation. The ﬁnal total N supply was 135 kg N ha−1 in 2014 and 110 kg N ha−1 in 2015. Post-anthesis stages of barley play a crucial role in balancing grain protein content and grain size (Borras et al., 2004). During grain ﬁlling of spring barley, the supply to grains may originate from both post-anthesis current photosynthetic assimilation or N uptake and remobilization of their reserves at pre-anthesis in the vegetative parts of the plant, such as leaves, stems, sheaths, and chaﬀ(Austin et al., 1980; van Sanford and Mackown, 1987; Borras et al., 2004). Grain DM is sink-limited (Borras et al., 2004) and mostly acquired by photosynthesis during grain ﬁlling, while DM remobilization from the reserves in vegetative organs to grains during grain ﬁlling only reaches approximately 10% of barley grain yield (Austin et al., 1980; Przulj and Momcilovic, 2001a,b; Dordas, 2012). In contrast, pre-anthesis-accumulated N seems to be the predominant source of N during grain ﬁlling (van Sanford and Mackown, 1987). The amount of N at anthesis in the aboveground parts of cereal crops can be as high as >90% of the total plant N at maturity (Clarke et al., 1990; Heitholt et al., 1990). Although studies have reported N remobilization during grain ﬁlling in barley, this has invariably been at a coarse level, and included remobilization from vegetative tissues or combined vegetative organs together to barley and wheat grain (Przulj and Momcilovic, 2001b; Abeledo et al., 2003; Sylvester-Bradley and Kindred, 2009; Dordas, 2012). Citation: Therefore, to meet the strict requirements for both quality parameters, it is necessary to understand the partitioning and remobilization of dry matter (DM) and nitrogen (N) from vegetative organs of malting barley at pre-anthesis and post-anthesis photosynthetic assimilation and N uptake, which may regulate the balance between the grain size and protein content in diﬀerent varieties of malting barley. Therefore, the objectives of this study were (i) to quantify both the genetic variation in the partitioning and remobilization of DM and N reserves from vegetative organs to the grain and post-anthesis current photosynthetic assimilation or N uptake in a selection of 23 spring malting barley varieties under recommended N fertilization conditions; (ii) to provide a benchmark audit of barley DM and N relationships in a 2-year study; and (iii) to assess the relationships among DM and N partitioning and remobilization, and grain size and protein content. Field Experiments The studies (Przulj and Momcilovic, 2001a,b; Dordas, 2012) have also shown a genetic variation in DM and N immobilization for barley. However, N partitioning in diﬀerent plant parts has not been determined in barley, and very few studies have provided a complete N audit during grain ﬁlling in diﬀerent varieties of spring malting barley; that is, how overall plant N uptake was partitioned to individual organs and subsequently remobilized to the grain to determine their roles in inﬂuencing the characteristics of malting quality. Weather Conditions The average annual precipitation in this region is ∼800 mm, and the average annual temperature is 7.8◦C. The data derived from the weather station of the German Meteorological Service (DWD) next to the experimental site in 2014 and 2015 are shown in Figure 1. The year 2014 had favorable growing conditions in March with a higher temperature and more radiation than 2015 (Figure 1). However, the air temperature in both years was similar in April. In contrast to some drought periods in April in both years, there was more precipitation in May 2014 and 2015. In 2015, strong precipitation in May caused ﬂooding in some plots. In June, there was less precipitation in 2014 than in 2015, i.e., the grain ﬁlling phase in 2014 beneﬁted from a high radiation budget in June. Physiological maturity occurred mid-July in both years; the plants were ﬁnally harvested at the end of July. Frontiers in Plant Science \| www.frontiersin.org Measurements and Analysis Growth stages, such as anthesis, dough ripening, and maturity, among the barley varieties were recorded (Figure 1). Among the 23 barley varieties, the anthesis stage diﬀered by only 1–2 days in 2014 and 1 day in 2015, even though there was diﬀerent precocity among the varieties reported in the literature (Table 1). Biomass sampling was performed at anthesis (ZS65) and maturity (ZS92) (Zadoks et al., 1974) by randomly harvesting 30 representative plants across each plot by hand-cutting. At anthesis, the plants were separated into leaves, sheaths, and (stems + ears) (STER) in 2014 and 2015. The plants were August 2021 \| Volume 12 \| Article 722871 2 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. TABLE 1 \| Spring malting barley varieties in 2014 and 2015 showing the name, year of release, precocity, genetic origin, breeder and country of origin (G-Germany, Aus-Australia). Precocity* No. Variety Name Listed Head emergence Maturity Genetic origin Breeder Country 1 Aspen 1999 5 5 Vintage x Chariot Nickerson G 2 Barke 1996 5 5 Libelle x Alexis Breun G 3 Baronesse 1989 4 5 (343/6 × J-427) × (Oriol × 6153 P40) Nordsaat G 4 Braemar 2002 5 5 NFC 5563/NFC 94-20 NFC/Cebeco G 5 Carina 1973 5 4 (Union x W 16 WV) x Volla Ackermann G 6 Grace 2008 4 5 (Xanadu x Simba) x Marnie Ackermann G 7 IPZ 24727 – – – (Br.3546OmegaTrumpf)Maresi LfL G 8 Irina 2012 5 6 – SKW G 9 Mackay 2003 – – – – AUS 10 Marthe 2005 5 5 Neruda/Recept Nordsaat G 11 Melius 2012 5 5 Conchita Tamtam Syngenta G 12 Power 1998 5 5 Saloon/(Colada/(Lux/Annabell)) – G 13 Quench 2006 6 6 Sebastian × Drum Syngenta/NFC G 14 Salome 2011 5 5 (Publican × Beatrix) × Auriga Nordsaat G 15 Scarlett 1995 5 5 (Amazone × Br. 2730e) × Kym Breun G 16 Shakira 2004 – 5 – – G 17 Sissy 1990 – – (Frankengold × Mona) × Trumpf Streng G 18 Solist 2012 5 5 S03F049(MarnieSimba)S99G264 Streng G 19 Trumpf 2003 – – (Diamant × 14029/64/6 (Alsa × S3170/Abyss) × 11719/59) × Union Hadmersleben G 20 Union 1955 – – (Weihenst. MR II × Donaria) × Firl. Measurements and Analysis 621 Firlbeck G 21 Ursa 2002 5 6 (Thuringia × Hanka) × Annabell Nordsaat G 22 Volla 1957 – – Wisa × Haisa I Breun G 23 Wiebke 1998 – – – – G Numbers 4, 5, 6, and 7 indicate “early to medium,” “medium,” “medium to late,” and “late” head emergence or maturity, respectively. TABLE 1 \| Spring malting barley varieties in 2014 and 2015 showing the name, year of release, precocity, genetic origin, breeder and country of origin (G-Germany, Aus-Australia). Numbers 4, 5, 6, and 7 indicate “early to medium,” “medium,” “medium to late,” and “late” head emergence or maturity, respectively. (Gebbing et al., 1998; Przulj and Momcilovic, 2001a,b; Dordas, 2012). DM and N translocation within the barley plants were calculated as follows: (Gebbing et al., 1998; Przulj and Momcilovic, 2001a,b; Dordas, 2012). DM and N translocation within the barley plants were calculated as follows: separated into ears, leaves, sheaths, and stems at plant maturity, and then the barley ears were threshed into grain and chaﬀ. The plant materials were oven-dried at 60◦C for 2 days to achieve constant dry weight, and then the dry weight for individual parameters was determined. The N content of each measured DM parameter was detected by mass spectrometry using an isotope ratio mass spectrometer with an ANCA SL 20–20 preparation unit (Europe Scientiﬁc, Crewe, United Kingdom). (1) DM translocation (DMT) (kg ha−1) = DMi at anthesis – DMi at maturity, where i is leaf, sheath, STER at anthesis or (stem + chaﬀ) (STCH) at maturity, and shoot total (leaf + sheath + STER) at anthesis or total aboveground vegetative organs (leaf + sheath + STCH) at maturity; (1) DM translocation (DMT) (kg ha−1) = DMi at anthesis – DMi at maturity, where i is leaf, sheath, STER at anthesis or (stem + chaﬀ) (STCH) at maturity, and shoot total (leaf + sheath + STER) at anthesis or total aboveground vegetative organs (leaf + sheath + STCH) at maturity; (2) total DM translocation eﬃciency (DMTE) (%) = (total DMT/total DM at anthesis) ∗100, which is presented in Supplementary Tables 1 and 3; Calculations of DM and N Translocation Within the Barley Plants FIGURE 1 \| (A) Daily air temperature, (B) global radiation, and (C) precipitation during the growing season from March to July in dwd.de/). Global radiation is presented as smoothed by a 10-day moving average. Calculations of DM and N Translocation Within the Barley Plants y (3) DM accumulation at post-anthesis (t ha−1 DM at maturity – total DM at anthesis; (3) DM accumulation at post-anthesis (t ha−1) (DMPA) = total DM at maturity – total DM at anthesis; Remobilization or translocation of reserves of carbon assimilates and N originating from pre-anthesis assimilation, which is the net loss of DM and N of the vegetative organs from anthesis to maturity, has been calculated by diﬀerent authors (e.g., Cox et al., 1985a,b; Papakosta and Gagianas, 1991). This approach assumes that decreases in aboveground vegetative biomass and N accumulation between anthesis and maturity are exclusively due to the mobilization of reserves, but respiratory losses involved with maintenance and redistribution or reserve photosynthates are not considered, and the role of the roots as a source of pre-anthesis C is ignored. The shedding of dead leaves that occurs during grain ﬁlling may overestimate the mobilization of both DM and N (4) N translocation (NT) (kg ha−1) = Ni at anthesis – Ni at maturity, where i is leaf, sheath, STER at anthesis or STCH at maturity, and shoot total (leaf + sheath + STER) at anthesis or total aboveground vegetative organs (leaf + sheath + STCH) at maturity; (4) N translocation (NT) (kg ha−1) = Ni at anthesis – Ni at maturity, where i is leaf, sheath, STER at anthesis or STCH at maturity, and shoot total (leaf + sheath + STER) at anthesis or total aboveground vegetative organs (leaf + sheath + STCH) at maturity; (5) N translocation eﬃciency (NTE) (%) = (NT/N accumulation at anthesis) ∗100, which is presented in Supplementary Tables 5 and 7; and (6) N uptake at post-anthesis (kg ha−1) (NPA) = total aboveground N accumulation at maturity – total N uptake at pre-anthesis. Frontiers in Plant Science \| www.frontiersin.org August 2021 \| Volume 12 \| Article 722871 3 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. FIGURE 1 \| (A) Daily air temperature, (B) global radiation, and (C) precipitation during the growing season from March to July in 2014 and 2015 (DWD, https://www. dwd.de/). Global radiation is presented as smoothed by a 10-day moving average. FIGURE 1 \| (A) Daily air temperature, (B) global radiation, and (C) precipitation during the growing season from March to July in 2014 and 2015 (DWD, https://www. dwd.de/). Global radiation is presented as smoothed by a 10-day moving average. Dry Matter Accumulation, Partitioning, and Translocation Analysis of variance revealed that, except for the DM of the sheath at ZS65 and STCH at ZS92, there was no signiﬁcant diﬀerence in the measured DM parameters at anthesis (ZS65) and maturity (ZS92) among the varieties (Table 2). Although diﬀerences between the 2 years were generally signiﬁcant for the DM of leaves, sheaths, and STER at anthesis, there was no signiﬁcant diﬀerence in the measured DM parameters at maturity except for leaves. The year × variety interaction was not signiﬁcant for all the measured DM parameters at either anthesis or maturity. The PES values shown in Table 2 indicate that the year eﬀect is the most important single factor inﬂuencing the performance of DM at pre-anthesis. The analysis of variance for the amount of DM translocation from vegetative organs during grain ﬁlling (DM in ZS 65 minus DM in ZS 92) (Table 3) showed that the variety eﬀect was not signiﬁcant for DM remobilization traits. The year eﬀect was signiﬁcant for DM translocation from all vegetative organs, while the V×Y interactive eﬀect was only signiﬁcant for DM translocation of the DM reserves of leaves at anthesis. The DM accumulation from post-anthesis was signiﬁcant between the 2 years, whereas there were no signiﬁcant eﬀects of genotypes and interactive eﬀects of genotypes × years for post-anthesis DM contributing to grain yield (Table 3). The PES values shown in Table 3 indicate that the year eﬀect is the most important single factor inﬂuencing the performance of DM translocation from their reserves at pre-anthesis. p The partitioning of DM in individual organs (leaf blades, sheaths, STER) of the fertile shoots of the 23 barley varieties at anthesis and maturity is shown in Figure 2. The total shoot DM at anthesis, averaged over all varieties, was 5.4 t ha−1 in 2014 and 6.5 t ha−1 in 2015. This was distributed in the order: STER (69%) > leaves (23%) > sheaths (8%) in 2014 and STER (75%) > leaves (14%) > sheaths (11%) in 2015. Total shoot DM for diﬀerent varieties ranged from 4.5 t ha−1 (Shakira) to 6.9 t ha−1 (Marthe) in 2014, and from 4.9 (Power) to 8 t ha−1 (Carina) in 2015. Leaf DM for the diﬀerent varieties ranged from 1 (Shakira) to 1.6 t ha−1 (Marthe) in 2014, and from 0.7 (Scarlett) to 1.2 t ha−1 (IPZ 24727) in 2015. Statistical Analysis of variance components (Tables 2 and 3). Compared with the variety eﬀect, the year eﬀect was signiﬁcant for most traits of the DM and N partitioning and translocation (Tables 2 and 3). The PES values also indicate that the year eﬀect is the most important single factor inﬂuencing the performance of DM and N accumulation at pre-anthesis, and DM and N translocation from their reserves at pre- anthesis. The results of Tukey’s tests are presented in Supplementary Tables 1–8. The degree of association between Statistical analysis was performed using the SPSS software (SPSS ver. 26, IBM, Armonk, NY, United States). An ANOVA was carried out using a general linear model (GLM) in SPSS to compare the eﬀects of variety (V) as ﬁxed factor, year (Y) as random factor, and their interactions on partitioning and translocation traits of DM and N. The results of the ANOVA are reported to show the mean square, P-value, and partial Eta squared (PES) that is considered as a measure August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 4 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. variables and malting quality parameters was estimated by Pearson correlation analysis. TABLE 2 \| ANOVA (mean squares) of dry matter (DM) and nitrogen (N) accumulation in leaves, sheaths, STER or STCH at anthesis (ZS65) and maturity (ZS92), and grain and total aboveground organs of the 23 spring malting barley varieties cultivated for 2 years at the same site under recommended N fertilization conditions. Statistical Analysis Source of variance DF Anthesis (ZS65) Maturity (ZS92) Leaves Sheaths STER Shoot total Leaves Sheaths STCH Grain Above-ground organs Mean square P PES Mean square P PES Mean square P PES Mean square P PES Mean square P PES Mean square P PES Mean square P PES Mean square P PES Mean square P PES Dry matter (DM) Year (Y) 1 4.8 * 0.78 3.7 * 0.90 55.7 * 0.74 52.0 * 0.62 0.14 * 0.22 0.02 ns 0.02 0.43 ns 0.06 2.21 ns 0.07 2.61 ns 0.03 Variety (V) 22 0.07 ns 0.55 0.05 * 0.73 1.15 ns 0.56 1.57 ns 0.52 0.03 ns 0.58 0.07 ns 0.59 0.98 0.75 1.18 ns 0.46 2.89 ns 0.44 V × Y 22 0.06 ns 0.15 0.02 ns 0.16 0.91 ns 0.16 1.43 ns 0.14 0.02 ns 0.12 0.05 ns 0.11 0.33 ns 0.12 1.39 ns 0.15 3.69 ns 0.13 Nitrogen (N) Year (Y) 1 1,643 * 0.61 3,157 * 0.95 29,480 * 0.91 35,104 * 0.84 2 ns 0.07 0.07 ns 0.09 14 ns 0.08 27,000 * 0.84 28,568 *** 0.81 Variety (V) 22 52 ns 0.52 12 ns 0.62 212 ns 0.60 459 ns 0.60 0.7 ns 0.42 0.91 * 0.72 7.1 ns 0.50 403 ns 0.64 513 ns 0.63 V × Y 22 48 ns 0.14 7 * 0.21 141 ns 0.18 307 ns 0.14 1.0 ns 0.17 0.36 ns 0.11 7.1 ns 0.16 227 ns 0.11 305 ns 0.11 Statistically signiﬁcant differences are indicated: P < 0 05; P < 0 01 P < 0 001 and ns not signiﬁcant The partial Eta squared (PES) is considered as a measure of variance components Frontiers in Plant Science \| www.frontiersin.org Nitrogen (N) Accumulation, Partitioning, and Translocation Analysis of variance reveals that at anthesis, there is no signiﬁcant diﬀerence in N partitioning among the genotypes except for sheath N at maturity (Table 2). The year eﬀect was signiﬁcant for N reserves in all measured vegetative organs at anthesis, whereas the signiﬁcant year eﬀect was found only for grain N and total N at maturity. The variety × year interaction was only found for sheath N at anthesis. The PES values shown in Table 2 indicate that the year eﬀect is the most important single factor inﬂuencing the performance of N accumulation at pre-anthesis. The analysis of variance for the amount of N translocation from vegetative organs during grain ﬁlling (N reserves at ZS 65 minus at ZS 92) (Table 3) showed that there was no signiﬁcant diﬀerence in the N remobilization from the sheaths (stems + ears) and shoot total at anthesis among the genotypes. The year eﬀect showed a signiﬁcant diﬀerence in N from all individual organs. The variety × year interactive eﬀects were only signiﬁcant for sheath N translocation. The N uptake for grains post-anthesis is not signiﬁcant between the 2 years, among the varieties or for the variety × year interaction (Table 3). The PES values shown in Table 3 indicate that the year eﬀect is the most important single factor inﬂuencing the performance of N translocation from their reserves at pre-anthesis. The partitioning of nitrogen (N) in individual organs (leaf blades, sheaths, STER) of the fertile shoots of the 23 barley varieties at anthesis and maturity is shown in Figure 4. The total shoot N at anthesis, averaged over all the varieties, was 83 kg ha−1 in 2014 and 111 kg ha−1 in 2015. This was distributed in the order: STER (59%) > leaves The amount of DM translocation from vegetative organs during grain ﬁlling (ZS 65 minus ZS 92) from the diﬀerent varieties ranges from 0.35 (Carina) to 1.9 t ha−1 (Scarlett) in 2014, and from 1.1 (Power) to 2.8 t ha−1 (Salome) in 2015 (Figure 3). This calculation assumed that all post-anthesis photosynthetic assimilates went directly to the grains. Total translocation from the DM reserves in vegetative organs at pre-anthesis accounts for 6–29% of the contribution to grain DM among the varieties in 2014 and 19–47% in 2015 (Figure 3). STER at ZS 65 and STCH at ZS 92 were the dominant contributors to this transfer in 2015. Dry Matter Accumulation, Partitioning, and Translocation TABLE 3 \| Analysis of variance (mean squares) of translocation of leaves, sheaths, STER from pre-anthesis and DM accumulation or N uptake of grains from post-anthesis of the 23 spring malting barley varieties cultivated for 2 years at the same site under recommended N fertilization conditions. This was distributed in the order: grain (59%) > STCH (27%) > sheaths (9%) > leaves (5%) in 2014, and grain (60%) > STCH (27%) > sheaths (8%) > leaves (5%) in 2015. Total shoot DM for diﬀerent varieties ranged from 8.8 (Shakira) to 12.7 t ha−1 (Union) in 2014, and from 9.4 (Salome) to 13.3 t ha−1 (Carina) in 2015. The leaf DM of the diﬀerent varieties ranged from 0.45 (Shakira) to 0.84 t ha−1 (Union) in 2014, and from 0.38 (Scarlett) to 0.68 t ha−1 (Shakira) in 2015. The sheath DM of the diﬀerent varieties ranged from 0.7 (Shakira) to 1.2 t ha−1 (Marthe) in 2014, and from 0.6 (Power) to 1.2 t ha−1 (Carina) in 2015. STCH DM for diﬀerent varieties ranged from 2.2 (Scarlett) to 3.9 t ha−1 (Union) in 2014, and from 2.2 (Salome) to 4.1 t ha−1 (Carina) in 2015. Grain DM for diﬀerent varieties ranged from 5.1 (Shakira) to 7.4 t ha−1 (Marthe) in 2014, and from 5.9 (Salome) to 7.6 t ha−1 (Baronesse) in 2015. There was no signiﬁcant diﬀerence in DM accumulation of all the organs measured among the varieties either in 2014 or 2015 (Supplementary Tables 1 and 3). (Figure 3). The post-anthesis assimilates accounted for 71–94% of the total grain yield among the barley varieties in 2014 and 53– 81% in 2015. However, there was no signiﬁcant diﬀerence in post- anthesis DM assimilation in either year (Supplementary Tables 2 and 4). Frontiers in Plant Science \| www.frontiersin.org Dry Matter Accumulation, Partitioning, and Translocation Sheath DM for the diﬀerent varieties ranged from 0.3 (Shakira) to 0.59 t ha−1 (IPZ 24727) in 2014, and from 0.5 (Power) to 0.9 t ha−1 (IPZ 24727) in 2015. STER DM for diﬀerent varieties ranged from 3.2 (IPZ 24727) to 4.9 t ha−1 (Marthe) in 2014, and from 3.5 (Power) to 6.2 t ha−1 (Carina) in 2015. In addition to sheath DM accumulation, there is no signiﬁcant diﬀerence in leaf and STER DM accumulation among the varieties in 2014 (Supplementary Table 1), and no signiﬁcant diﬀerence among the varieties is found in leaf DM accumulation in 2015 (Supplementary Table 3). The total shoot DM at maturity, averaged over all varieties, was 10.9 t ha−1 in 2014 and 11.1 t ha−1 in 2015 (Figure 2). August 2021 \| Volume 12 \| Article 722871 5 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. TABLE 3 \| Analysis of variance (mean squares) of translocation of leaves, sheaths, STER from pre-anthesis and DM accumulation or N uptake of grains from post-anthesis of the 23 spring malting barley varieties cultivated for 2 years at the same site under recommended N fertilization conditions. Translocation DM or N accumulation at post-anthesis Source of variance DF Leaves Sheaths STER Shoot total Mean square P PES Mean square P PES Mean square P PES Mean square P PES Mean square P PES Dry matter (DM) Year (Y) 1 3.29 * 0.76 4.34 * 0.80 46.4 * 0.81 50.2 * 0.80 31.4 * 0.52 Variety (V) 22 0.03 ns 0.37 0.07 ns 0.57 0.76 ns 0.60 0.74 ns 0.57 1.35 ns 0.51 V x Y 22 0.05 * 0.22 0.05 ns 0.15 0.51 ns 0.20 0.56 ns 0.18 1.30 ns 0.15 Nitrogen (N) Year (Y) 1 1,746 * 0.67 3,187 * 0.96 28,202 * 0.92 33,339 * 0.87 336 ns 0.11 Variety (V) 22 45 ns 0.53 9.0 ns 0.57 176 ns 0.60 352 ns 0.61 207 ns 0.62 V x Y 22 40 ns 0.14 6.7 * 0.23 119 ns 0.19 228 ns 0.14 129 ns 0.19 Statistically signiﬁcant differences are indicated: P < 0.05; P < 0.001 and ns, not signiﬁcant. The partial eta squared (PES) is considered as a measure of variance components. Nitrogen (N) Accumulation, Partitioning, and Translocation In contrast, the reserve from leaf DM contributed similarly to that from STER at pre-anthesis for most of the varieties in 2014. The diﬀerence in the total shoot translocation among the barley varieties in 2014 was signiﬁcant, whereas no signiﬁcant diﬀerence was found in 2015 (Supplementary Tables 2 and 4). The amount of post-anthesis DM accumulation in grains from diﬀerent varieties ranged from 4.3 (Shakira) to 6.7 t ha−1 (Union) in 2014, and from 3.1 (Salome) to 5.3 t ha−1 (Carina) in 2015 August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 6 Barmeier et al. Partitioning and Translocation in Malting Barley Varieties IGURE 2 \| Dry matter partitioning in different organs: dry matter (DM) accumulation (t ha−1) of different plant organs of the 23 spring malting barley varieties at nthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on STER DM at anthesis or STCH DM at maturity. Mean comparison mong the varieties from Tukey’s honestly signiﬁcant difference (HSD) test are shown in Supplementary Tables 1 and 3. FIGURE 2 \| Dry matter partitioning in different organs: dry matter (DM) accumulation (t ha−1) of different plant organs of the 23 spring malting barley varieties at anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on STER DM at anthesis or STCH DM at maturity. Mean comparisons among the varieties from Tukey’s honestly signiﬁcant difference (HSD) test are shown in Supplementary Tables 1 and 3. FIGURE 2 \| Dry matter partitioning in different organs: dry matter (DM) accumulation (t ha−1) of different plant organs of the 23 spring malting barley varieties at anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on STER DM at anthesis or STCH DM at maturity. Mean comparisons among the varieties from Tukey’s honestly signiﬁcant difference (HSD) test are shown in Supplementary Tables 1 and 3. August 2021 \| Volume 12 \| Article 722871 7 Frontiers in Plant Science \| www.frontiersin.org Barmeier et al. Partitioning and Translocation in Malting Barley V FIGURE 3 \| Dry matter translocation from different organs: DM translocation (t ha−1) of different plant organs of the 23 spring malting barley varieties to grain from pre-anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on DM translocation of STER. Nitrogen (N) Accumulation, Partitioning, and Translocation Mean comparisons among the varieties from Tukey’s HSD test are shown in Supplementary Tables 2 and 4. Partitioning and Translocation in Malting Barley Varieties Barmeier et al. FIGURE 3 \| Dry matter translocation from different organs: DM translocation (t ha−1) of different plant organs of the 23 spring malting barley varieties to grain from pre-anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on DM translocation of STER. Mean comparisons among the varieties from Tukey’s HSD test are shown in Supplementary Tables 2 and 4. FIGURE 3 \| Dry matter translocation from different organs: DM translocation (t ha−1) of different plant organs of the 23 spring malting barley varieties to grain from pre-anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on DM translocation of STER. Mean comparisons among the varieties from Tukey’s HSD test are shown in Supplementary Tables 2 and 4. (36%) > sheaths (5%) in 2014, and STER (67%) > leaves (22%) > sheaths (11%) in 2015. The total shoot N for the diﬀerent varieties ranged from 63 (Sharika) to 111 kg ha−1 (Marthe) in 2014, and from 96 (Power) to 136 kg ha−1 (IPZ 24727) in 2015. Leaf N for the diﬀerent varieties ranged from 24 (Sharika) to 40 kg ha−1 (Marthe) in 2014, and from 20 (Salome) to 30 kg ha−1 (Carina) in 2015. Sheath N for the diﬀerent varieties ranged from 2 (Sharika) to 7 kg ha−1 (Wiebke) in 2014, and from 9 (Power) to 18 kg ha−1 (IPZ 24727) in 2015. STER N for the diﬀerent varieties ranged from 37 (Sharika) to 67 kg ha−1 (Marthe) in 2014, and from 63 (Mackay) to 92 kg ha−1 (IPZ 24727) in 2015. Besides leaf DM accumulation, there was a signiﬁcant diﬀerence in sheaths and STER N accumulation between the varieties in 2014 (Supplementary Table 5), while no signiﬁcant diﬀerence between the varieties was found in leaf and STER N accumulation in 2015 (Supplementary Table 7). The total shoot N at maturity, averaged over all the varieties, was 100 kg ha−1 in 2014 and 125 kg ha−1 in 2015 (Figure 4). This August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 8 Barmeier et al. Nitrogen (N) Accumulation, Partitioning, and Translocation Grain N for the diﬀerent varieties ranged from 63 (Sharika) to 111 kg ha−1 (Union) in 2014, and from 98 (Power) to 127 kg ha−1 (IPZ 42727) in 2015. In addition to the sheath N accumulation in 2014, there was no signiﬁcant diﬀerence in measured plant organs at ZS 92 among the varieties in 2014 and 2015 (Supplementary Tables 5 and 7). parameters, most parameter diﬀerences were not statistically signiﬁcant (Supplementary Tables 1–8). A similar tendency was also found for the grain quality properties of the grain protein content and the grain retention fraction of grain size > 2.5 mm (Hu et al., 2021). Most importantly, because the N partitioning in the diﬀerent plant parts in barley has not been reported in the literature, the results in this study can provide a benchmark audit of the DM and N accumulation in diﬀerent organs at pre- and post-anthesis in a 2-year study, which allows us to ﬁrst discuss the physiological basis of genetic variation in DM and N partitioning, and DM and N remobilization, and shows source- sink relationships and their responses to the year eﬀect. Second, a better understanding of the roles of DM and N partitioning and remobilization in determining the quality properties of spring malting barley may help to identify their roles in determining malting quality, which will allow us to develop strategies for trait selection and agronomic management to obtain suitable grain size and protein content that can ensure meeting the requirements from the malting and brewing industry. The amount of N translocation from vegetative organs during grain ﬁlling (N accumulation at ZS 65 minus at ZS 92) of diﬀerent varieties ranged from 52 (Shakira) to 94 kg ha−1 (Marthe) in 2014, and from 82 (Power) to 117 kg ha−1 (IPZ 42727) in 2015 (Figure 5). The contribution of N translocation from pre- anthesis to grain N varied, ranging from 67 (Union) to 91% (Marthe) in 2014, and 71% (Grace) to 97% (Sharika) in 2015. However, a signiﬁcant diﬀerence among the varieties was only found in 2014 (Supplementary Tables 6 and 8). Similar to DM translocation, the N reserves from STER were the dominant contributor to this transfer. The amount of post-anthesis N uptake of grains among the 23 varieties ranged from 9.7 (Volla) to 37 kg N ha−1 (Union) in 2014, and from 2.9 (Sharika) to 34 kg N ha−1 (Grace) in 2015 (Figure 5). Nitrogen (N) Accumulation, Partitioning, and Translocation Partitioning and Translocation in Malting Barley Varieties URE 4 \| Nitrogen partitioning in different organs: N accumulation (kg ha−1) of different plant organs of the 23 spring malting barley varieties at anthesis and rity under recommended N fertilization conditions in 2014 and 2015, ranked on STER N at anthesis or STCH N at maturity. Mean comparisons among the ties from Tukey’s HSD test are shown in Supplementary Tables 5 and 7. FIGURE 4 \| Nitrogen partitioning in different organs: N accumulation (kg ha−1) of different plant organs of the 23 spring malting barley varieties at anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on STER N at anthesis or STCH N at maturity. Mean comparisons among the varieties from Tukey’s HSD test are shown in Supplementary Tables 5 and 7. Nitroge er reco FIGURE 4 \| Nitrogen partitioning in different organs: N accumulation (kg ha−1) of different plant organs of the 23 spring malting barley varieties at anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on STER N at anthesis or STCH N at maturity. Mean comparisons among the varieties from Tukey’s HSD test are shown in Supplementary Tables 5 and 7. August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 9 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. was distributed in the order: grain (86%) > STCH (8.5%) > leaves (2.7%) > sheaths (2.6%) in 2014, and grain (88%) > STCH (7.4%) > leaves (2.3%) > sheaths (2%) in 2015. The total shoot N for the diﬀerent varieties ranged from 75 (Sharika) to 131 kg ha−1 (Union) in 2014, and from 111 (Mackay) to 145 kg ha−1 (IPZ 42727) in 2015. Leaf N for the diﬀerent varieties ranged from 2 (Baronesse) to 4 kg ha−1 (Union) in 2014, and from 2 (Scarlett) to 4 kg ha−1 (IPZ 42727) in 2015. Sheath N for the diﬀerent varieties ranged from 2 (Shakira) to 4 kg ha−1 (Marthe) in 2014, and from 1.8 (Wiebke) to 3.4 kg ha−1 (IPZ 42727) in 2015. STCH N for the diﬀerent varieties ranged from 7 (Power) to 13 kg ha−1 (Union) in 2014, and from 7 (Salome) to 12 kg ha−1 (Carina) in 2015. Nitrogen (N) Accumulation, Partitioning, and Translocation The post-anthesis N uptake accounted for 9–33% of the total grain N among the barley varieties in 2014, and for 3–29% in 2015. A signiﬁcant diﬀerence in the post-anthesis N uptake of grains was found in both years (Supplementary Tables 6 and 8). Partitioning and Translocation of Dry Matter and Their Roles in Determining Malting Quality g y Earlier studies on barley have reported that DM remobilization from reserves in vegetative organs at pre-anthesis to grains reached ∼4–24 (Przulj and Momcilovic, 2001a,b) and 12–28% (Dordas, 2012). In this study, the DM stored in vegetative tissues at anthesis contributed to grain yield from 6 in the variety Union to 29% in the variety Scarlett in 2014, and from 19 in the variety Power to 47% in the variety Salome in 2015 (Figure 2). The values in 2014 were similar to those reported in the literature, whereas the values in 2015 were much higher than those in this study from 2014 and the other studies cited above, indicating that fewer photosynthetic assimilates were produced during grain ﬁlling in 2015, probably because of faster senescence. Figure 3 demonstrates a higher DM assimilate at post-anthesis in 2014 than that in 2015. More interestingly, the varieties with a longer period from heading and maturity (Table 1) such as Baronesse, Grace, Irina, and Ursa showed a higher rank among the 23 varieties based on the DM translocation in 2015 than in 2014. With regard to DM translocation, DM reserves in leaves and stems at pre-anthesis represent the major contribution to barley grain yield (Przulj and Momcilovic, 2001a). In wheat, the cultivars with more assimilates stored in the stem and greater assimilative capacity of ears, especially a greater contribution of ear assimilates, are expected to increase the grain yield (Sun et al., 2021). Elazab et al. (2021) suggested that to develop winter wheat (Triticumaestivum L.) breeding strategies, the increase in ear size plays an important role in photosynthetic assimilates for grain ﬁlling. In this study, the average DM translocation from leaves among all the barley varieties at pre-anthesis was similar to that from stems and ears in 2014, whereas this was 5-fold higher than that from leaves in 2015. Merah et al. (2018) reported that the contribution of ear photosynthesis and re-mobilization from the stem in durum wheat increased with post-anthesis water stress. Furthermore, this study found that the DM translocation from DISCUSSION In this study, the most important single factor inﬂuencing the performance of dry matter and nitrogen accumulation at pre-anthesis and dry matter and nitrogen partitioning and translocation from their reserves at pre-anthesis was the year (Tables 2 and 3). The year eﬀect could be due to the variation in weather conditions, such as precipitation and temperature, between the 2 years (Figure 1). Figures 2–5 show that the ranking of the 23 barley varieties based on DM and N parameters is inconsistent with the DM and N parameters for a given year and varied with the year for the same barley variety. Abeledo et al. (2008) reported that modern malting barley cultivars tended to have a higher N content in ears at pre-anthesis than old cultivars. However, this tendency was not observed in this study. For example, Figure 4 shows that cultivars registered after the 2000s had both higher and lower N contents (kg ha−1) in plant organs at pre-anthesis in 2014 and 2015 (Table 1). Furthermore, although Figures 2–5 demonstrate genetic variation for most August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 10 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. FIGURE 5 \| Nitrogen translocation from different organs: N translocation (kg ha−1) of different plant organs of the 23 spring malting barley varieties to grain from pre-anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on N translocation of STER. Mean comparisons among varie from Tukey’s HSD test are shown in Supplementary Tables 6 and 8. FIGURE 5 \| Nitrogen translocation from different organs: N translocation (kg ha−1) of different plant organs of the 23 spring malting barley varieties to grain from pre-anthesis and maturity under recommended N fertilization conditions in 2014 and 2015, ranked on N translocation of STER. Mean comparisons among varieties from Tukey’s HSD test are shown in Supplementary Tables 6 and 8. sheaths at pre-anthesis was negative in both years (Figure 3). In contrast, less negative values were found in 2015 than in 2014. This may be due to the barley’s earlier ﬂowering in 2014 than in 2015 (Figure 1). source and sink capacity during grain ﬁlling varies depending on environmental conditions and season (Grashoﬀand d’Antuono, 1997; Borras et al., 2004). Frontiers in Plant Science \| www.frontiersin.org DISCUSSION This study showed that the post- anthesis assimilates accounted for 71–94% of the total grain yield among the barley varieties in 2014 and 53–81% in 2015, which is in agreement with more sink- than source-limiting for grain ﬁlling (Borras et al., 2004). Hu et al. (2021) reported that the grain number per ha in 2015 was higher than that in 2014, indicating a higher sink potential in 2015 for carbon assimilates. However, the grain retention fraction >2.5 mm from the 23 barley varieties was <90% (Hu et al., 2021), which could not meet Barley growth and development before anthesis determine the grain sink capacity, which is a function of the number of grains per unit land area and their potential size (individual storage capacity). Post-anthesis photosynthetic activity and the remobilization of soluble carbohydrate reserves stored from pre- anthesis supply carbon assimilates during grain ﬁlling (Kennedy et al., 2017; Bingham et al., 2019). The balance between the August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 11 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. TABLE 4 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with DM and N accumulation in leaves, sheaths, STER or STCH, and total aboveground organs at anthesis and maturity and grain yield among the 23 spring malting barley varieties under the recommended N fertilization conditions across 2 years. Grain protein content Grain size Anthesis Maturity Anthesis Maturity Dry matter of Leaves −0.62 −0.17 ns 0.64 0.20 ns Sheaths 0.80 0.07 ns −0.78 0.04 ns STER or STCH 0.70 0.34 * −0.73 −0.24 ns Grains – 0.04 ns – −0.19 ns Shoot total or above-ground organs 0.66 0.17 ns −0.68 −0.20 ns N accumulation in Leaves −0.49 0.31 * 0.54 −0.26 ns Sheaths 0.87 0.12 ns −0.86 0.05 ns STER or STCH 0.83 0.38 −0.83 −0.29 * Grains – 0.83 – −0.74 Shoot total or above-ground organs 0.80 0.83 −0.78 −0.73 Statistically signiﬁcant differences are indicated: P < 0.05; P < 0.01 and ns, not signiﬁcant. DISCUSSION G i t i t t G i i TABLE 4 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with DM and N accumulation in leaves, sheaths, STER or STCH, and total aboveground organs at anthesis and maturity and grain yield among the 23 spring malting barley varieties under the recommended N fertilization conditions across 2 years. TABLE 4 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with DM and N accumulation in leaves, sheaths, STER or STCH, and total aboveground organs at anthesis and maturity and grain yield among the 23 spring malting barley varieties under the recommended N fertilization conditions across 2 years. TABLE 5 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with the translocation of DM and N accumulation in leaves, sheaths and STER from pre-anthesis, and assimilation and N accumulation from post-anthesis among the 23 spring malting barley varieties under recommended N fertilization conditions across 2 years. Grain protein content Grain size TABLE 5 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with the translocation of DM and N accumulation in leaves, sheaths and STER from pre-anthesis, and assimilation and N accumulation from post-anthesis among the 23 spring malting barley varieties under recommended N fertilization conditions across 2 years. Grain protein content Grain size Dry matter (DM) Nitrogen (N) Dry matter (DM) Nitrogen (N) Leaves −0.69 * −0.54 0.70 0.58 Sheaths 0.66 0.86 −0.71 −0.87 STER 0.60 0.82 −0.70 −0.83 Shoot total 0.61 0.80 −0.71 −0.80 Post-anthesis DM accumulation or N uptake −0.54 −0.04 ns 0.51 0.24 ns Statistically signiﬁcant differences are indicated: P < 0.01 and ns, not signiﬁcant. even better correlated with grain than stem and leaf. According to the studies on wheat by Merah and Monneveux (2015) and Elazab et al. (2021), carbon isotope discrimination is a rapid and non-destructive technique for the estimation of variations in the contribution of diﬀerent organs to grain ﬁlling. To gain a better understanding of increased ear photosynthesis, manipulation techniques such as carbon isotope discrimination are needed. the malting barley quality requirement of the brewing industry in the West European market. In contrast, the grain retention fraction >2.5 mm was > 90% for almost all the barley varieties tested in 2014. DISCUSSION This may suggest that the photosynthetic activity in 2015 was a limiting factor during grain ﬁlling, i.e., source was limited. The results of correlations among the grain retention fraction and DM accumulation at pre- and post-anthesis and translocation across the 2 years shown in Tables 4, 5 demonstrate that the grain retention fraction was signiﬁcantly associated with leaf DM accumulation at pre-anthesis and photosynthetic assimilates for grains during grain ﬁlling, while there was a negative correlation among the grain retention fraction and DM accumulation in sheaths and STER at pre-anthesis and translocation from sheaths and STER. This further suggests that photosynthetic activity for high photosynthetic assimilates at post-anthesis may determine barley grain size, suggesting that this is an important factor in improving malting quality. The awns of the ear have been reported as important sources of assimilates in wheat. Studies by Maydup et al. (2014) and Merah and Monneveux (2015) have shown that there is a positive relationship between awn size and contribution of the ear to grain ﬁlling under stress conditions, and that chaﬀand awns were DISCUSSION TABLE 5 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with the translocation of DM and N accumulation in leaves, sheaths and STER from pre-anthesis, and assimilation and N accumulation from post-anthesis among the 23 spring malting barley varieties under recommended N fertilization conditions across 2 years. Grain protein content Grain size Dry matter (DM) Nitrogen (N) Dry matter (DM) Nitrogen (N) Leaves −0.69 −0.54 0.70 0.58 Sheaths 0.66 0.86 −0.71 −0.87 STER 0.60 0.82 −0.70 −0.83 Shoot total 0.61 0.80 −0.71 −0.80 Post-anthesis DM accumulation or N uptake −0.54 −0.04 ns 0.51 0.24 ns Statistically signiﬁcant differences are indicated: P < 0.01 and ns, not signiﬁcant. TABLE 4 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with DM and N accumulation in leaves, sheaths, STER or STCH, and total aboveground organs at anthesis and maturity and grain yield among the 23 spring malting barley varieties under the recommended N fertilization conditions across 2 years. Grain protein content Grain size Anthesis Maturity Anthesis Maturity Dry matter of Leaves −0.62 −0.17 ns 0.64 0.20 ns Sheaths 0.80 0.07 ns −0.78 0.04 ns STER or STCH 0.70 0.34 * −0.73 −0.24 ns Grains – 0.04 ns – −0.19 ns Shoot total or above-ground organs 0.66 0.17 ns −0.68 −0.20 ns N accumulation in Leaves −0.49 0.31 * 0.54 −0.26 ns Sheaths 0.87 0.12 ns −0.86 0.05 ns STER or STCH 0.83 0.38 −0.83 −0.29 * Grains – 0.83 – −0.74 Shoot total or above-ground organs 0.80 0.83 −0.78 −0.73 Statistically signiﬁcant differences are indicated: P < 0.05; *P < 0.01 and ns, not signiﬁcant. TABLE 5 \| Pearson’s correlation coefﬁcient of grain protein content and grain size with the translocation of DM and N accumulation in leaves, sheaths and STER from pre-anthesis, and assimilation and N accumulation from post-anthesis among the 23 spring malting barley varieties under recommended N fertilization conditions across 2 years. Frontiers in Plant Science \| www.frontiersin.org Nitrogen Partitioning and Translocation, and Their Roles in Determining Malting Quality y Nitrogen uptake and translocation play a major role in determining grain protein content. Several studies have shown that the amount of N uptake in the aboveground parts of barley and wheat crops from pre-anthesis accounts for up to 90% of the total grain N at maturity, depending on the variety and environment (e.g., Clarke et al., 1990; Heitholt et al., 1990; Przulj and Momcilovic, 2001a,b; Dordas, 2012). In this study, the N reserved in vegetative tissues at anthesis contributed to barley grain N from 67 in the variety Union to 91% in the variety Marthe in 2014, and from 71 in the variety Grace to 97% in August 2021 \| Volume 12 \| Article 722871 12 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. Eagles et al. (1995) and Laidig et al. (2017). Therrien et al. (1994) showed that, compared with genetic eﬀects, environmental eﬀects were a dominant factor in determining malting quality, and suggested the use of management practices to optimize malting barley quality, i.e., nitrogen had the greatest eﬀect on malting quality traits. Laidig et al. (2017) reported that barley genetic variation accounted for only 3% of the total variation; thus, the environment and crop management, particularly nitrogen supply, are mainly responsible for the variation of the protein level in barley grain quality. In a simpliﬁed way, rapid on-farm tests of mineral nitrogen content at the beginning of the season will allow for the determination of residual mineral nitrogen content (Schmidhalter, 2005). To further optimize N management, sensing technology is currently available for in- season N fertilization of ﬁeld crops, since this not only allows the detection of the actual growth and N status but can also be used to estimate soil nitrogen mineralization (Schmidhalter et al., 2006). Spectral sensing techniques can be used for a more targeted N application in spring barley (Barmeier and Schmidhalter, 2017; Barmeier et al., 2017). the variety Shakira in 2015. The results of correlations among the grain retention fraction and DM accumulation at pre- and post-anthesis and translocation across the 2 years in Tables 4, 5 show that the grain protein content was signiﬁcantly associated with sheath and STER N accumulation at pre-anthesis and translocation from sheaths and STER, suggesting that N uptake before anthesis may play an important role in determining the barley grain protein content. CONCLUSIONS This study showed that the year eﬀect was the most important single factor inﬂuencing the performance of DM and N accumulation at pre-anthesis and DM and N translocation from their reserves at pre-anthesis (Tables 2 and 3), which may be due to the variation in weather conditions such as precipitation and temperature between the 2 years. Post-anthesis assimilates contributed to grain yield, accounting for 71–94% among the barley varieties in 2014 and 53–81% in 2015. In contrast, the contribution of N reserved in vegetative tissues from pre-anthesis to grain N accounted for 67–91% in 2014 and 71–97% in 2015. The positive correlations among post-anthesis assimilation, grain size and N reserves at pre-anthesis, and N remobilization and grain protein content of malting barley suggest that DM and N partitioning and remobilization played an important role in determining the quality properties of spring malting barley. To achieve a high quality of malting barley grains in both grain size and protein content simultaneously, parallel strategies of optimized trait selection of spring malting barley and improved agronomic N management for higher photosynthetic activity at post-anthesis and an increase in N reserves at pre-anthesis have to be developed. Improved management strategies should include the detection of residual soil mineral nitrogen at the beginning of the season as well as the detection of soil N mineralization to optimize the production of spring malting barley. Figures 4, 5 show that at anthesis, most plant N is present in stems and ears, and then in leaf blades and leaf sheaths. Stems have long been identiﬁed as a major N pool for mobilization (e.g., Gregory et al., 2013), although in most studies, “stems” also include sheaths. Thus, the results indicate that an important target to increase N reserves in stems at pre-anthesis as a selection trait for malting barley varieties can lead to enhanced N translocation from non-photosynthetic organs. Consequently, this also results in an increase in N use eﬃciency. In contrast, maintenance of the N level in green leaves, i.e., a delay in leaf senescence during grain ﬁlling, may contribute to more photosynthetic assimilates at post-anthesis. Nitrogen Partitioning and Translocation, and Their Roles in Determining Malting Quality The contribution of translocated N from the N reverses in vegetative tissue at pre-anthesis to grain N can indicate growing conditions and N availability in the soil during vegetation, i.e., higher N translocation indicates good growing conditions and the availability of N from the sources before anthesis. Hu et al. (2021) reported that grain protein content varied signiﬁcantly within a year for the same barley varieties, and that average protein content was lower in 2014 than in 2015. For example, in 2015, among the 23 barley varieties, 22 obtained a protein content between 9.5 and 11.5% despite a lower N supply rate in; while in 2014, the protein content among the 23 varieties ranged from 8.3 to 9.8%, and only two varieties reached a value higher than 9.5%, which is the critical level for malting quality requirement. The lower protein content of barley grain in 2014 may reﬂect a lower N supply rate that may be derived from less N mineralization in 2014 during the vegetative growth because the N fertilization rate was the same for both years. Studies have shown that N level increases N concentration in plant tissue and aﬀects dry matter and N accumulation, partitioning, and translocation (Dordas, 2012). Thus, barley needs to receive an adequate amount of N, as this aﬀects dry matter and N translocation. Furthermore, N uptake is inﬂuenced by the available water (Clarke et al., 1990), degree of association between the roots and soil, and supply of nitrate (Cox et al., 1985a,b; Papakosta and Gagianas, 1991). This may indicate unfavorable growth conditions before anthesis in 2014 compared with 2015. Furthermore, because of the negative relationships between grain yield (and/or grain size) and protein content, protein grain content concentration in the grain depends not only on the N amount in the grain but also on the level of photosynthetic activity at post-anthesis during grain ﬁlling (Cox et al., 1986). Higher photosynthetic assimilates at post-anthesis that caused a dilution eﬀect on grain protein content in 2014 may also be a reason for lower grain protein content (Figure 3). DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Frontiers in Plant Science \| www.frontiersin.org REFERENCES High-throughput ﬁeld phenotyping of leaves, leaf sheaths, culms and ears of spring barley cultivars at anthesis and dough ripeness. Front. Plant Sci. 8:1920. doi: 10.3389/fpls.2017.01920 Grashoﬀ, C., and d’Antuono, L. F. (1997). Eﬀect of shading and nitrogen application on yield, grain size distribution and concentrations of nitrogen and water soluble carbohydrates in malting spring barley (Hordeum vulgare L.). Eur. J. Agron. 6, 275–293. doi: 10.1016/S1161-0301(97)00001-4 J. Agron. 6, 275–293. doi: 10.1016/S1161-0301(97)00001-4 Beillouin, D., Lecl‘ere, M., Barbu, C. M., B’en’ezit, M., Tr’epos, R., Gauﬀreteau, A., et al. (2018). Azodyn-Barley, a winter-barley crop model for predicting and ranking genotypic yield, grain protein and grain size in contrasting pedoclimatic conditions. Agric. For. Meteorol. 262, 237–248. doi: 10.1016/j.agrformet.2018.06.002 Gregory, P. J., Atkinson, C. J., Bengough, A. G., Else, M. A., Fernandez-Fernandez, F., Harrison, R. J., et al. (2013). Contributions of roots and rootstocks to sustainable, intensiﬁed crop production. J. Exp. Bot. 64, 1209–1222. doi: 10.1093/jxb/ers385 Bingham, I. J., Young, C., Bounds, P., and Paveley, N. D. (2019). In sink-limited spring barley crops, light interception by green canopy does not need protection against foliar disease for the entire duration of grain ﬁlling. Field Crop. Res. 239, 124–134. doi: 10.1016/j.fcr.2019.04.020 Heitholt, J. J., Croy, L. I., Maness, N. O., and Nguyen, H. T. (1990). Nitrogen partitioning in genotypes of winter-wheat diﬀering in grain n-concentration. Field Crop. Res. 23, 133–144. doi: 10.1016/0378-4290(90)90108-N Hu, Y., Barmeier, G., and Schmidhalter, U. (2021). Genetic variation in grain yield and quality traits of spring malting barley by year under optimal N application. Agronmics 11, 1177. doi: 10.3390/agronomy11061177 Borras, L., Slafer, G. A., and Otegui, M. E. (2004). Seed dry weight response to source-sink manipulations in wheat, maize and soybean: a quantitative reappraisal. Field Crop Res. 86, 131–146. doi: 10.1016/j.fcr.2003.08.002 Incograin (2014). Addendum technique N◦IV pour la vente des orges de brasseries. Incograin Bundessortenamt (2016). Beschreibende Sortenliste 2016: Getreide, Mais, Öl- und Faserpﬂanzen, Leguminosen, Rüben, Zwischenfrüchte. Bundessortenamt, Hannover. (Accessed October 23, 2020) Kennedy, S. P., Bingham, I. J., and Spink, J. H. (2017). Determinants of spring barley yield in a high-yield potential environment. J. Agric. Sci. 155, 60–80. doi: 10.1017/S0021859616000289 Carreck, N. L., and Christian, D. G. (1991). Studies on the patterns of nitrogen uptake and translocation to the grain of winter barley intended for malting. Ann. Appl. Biol. 119, 549–559. doi: 10.1111/j.1744-7348.1991.tb04894.x Laidig, F., Piepho, H. P., Rentel, D., Drobek, T., and Meyer, U. (2017). REFERENCES assimilation in relation to grain yield and protein. Crop Sci. 26, 737–740. doi: 10.2135/cropsci1986.0011183X002600040022x assimilation in relation to grain yield and protein. Crop Sci. 26, 737–740. doi: 10.2135/cropsci1986.0011183X002600040022x Abeledo, L. G., Calderini, D. F., and Slafer, G. A. (2003). Genetic improvement of barley yield potential and its physiological determinants in Argentina (1944–1998). Euphytica 130, 325–334. doi: 10.1023/A:1023089421426 Dordas, C. (2012). Variation in dry matter and nitrogen accumulation and remobilization in barley as aﬀected by fertilization, cultivar, and source-sink Dordas, C. (2012). Variation in dry matter and nitrogen accumulation and remobilization in barley as aﬀected by fertilization, cultivar, and source-sink relations. Eur. J. Agron. 37, 31–42. doi: 10.1016/j.eja.2011.10.002 remobilization in barley as aﬀected by fertilization, cultivar, and relations. Eur. J. Agron. 37, 31–42. doi: 10.1016/j.eja.2011.10.002 Abeledo, L. G., Calderini, D. F., and Slafer, G. A. (2008). Nitrogen economy in old and modern malting barleys. Field Crop Res. 106, 171–178. doi: 10.1016/j.fcr.2007.11.006 Eagles, H. A., Bedggood, A. G., Panozzo, J. F., and Martin, P. J. (1995). Cultivar and environmental eﬀects on malting quality in barley. Aust. J. Agric. Res. 46, 831–844. doi: 10.1071/AR9950831 Elazab, A., Moraga, F., and del Pozo, A. (2021). Photosynthetic organs contributions to grain yield genetic gains in Chilean winter wheat. Agron. J. 2021, 1–21. doi: 10.1002/agj2.20718 Atanassov, P., Zaharieva, M., Merah, O., and Monneveux, P. (1999). Genetic and environmental variation of useful traits in a collation of naked barley I. Yield components and water use eﬃciency. Cereal Res. Commun. 27, 315–322. doi: 10.1007/BF03543542 Fox, G. P., Panozzo, J. F., Li, C. D., Lance, R. C. M., Inkerman, P. A., and Henry, R. (2003). Molecular basis of barley quality. Aust. J. Agric. Res. 54, 1081–1101. doi: 10.1071/AR02237 Austin, R. B., Morgan, C. L., Ford, M. A., and Blackwell, R. D. (1980). Contributions to grain yield from pre-anthesis assimilation in tall and dwarf barley phenotypes in two contrasting seasons. Ann. Bot. 45, 309–319. doi: 10.1093/oxfordjournals.aob.a085826 Gebbing, T., Schnyder, H., and Kuhbauch, W. (1998). Carbon mobilization in shoot parts and roots of wheat during grain ﬁlling: assessment by C-13/C-12 steady-state labelling, growth analysis and balance sheets of reserves. Plant Cell Environ. 21, 301–313. doi: 10.1046/j.1365-3040.1998. 00286.x Barmeier, G., Hofer, K., and Schmidhalter, U. (2017). Mid-season prediction of grain yield and protein content of spring barley cultivars using high-throughput spectral sensing. Euro. J. Agron. 90, 108–116. doi: 10.1016/j.eja.2017.07.005 Barmeier, G., and Schmidhalter, U. (2017). AUTHOR CONTRIBUTIONS In this study, the ﬁndings of the most important eﬀect of the year as an environmental factor on the performance of barley DM and N accumulation and translocation (Tables 2 and 3) is in agreement with studies by Therrien et al. (1994), US and GB conceived and designed the experiments. GB performed the experiments. GB and YH August 2021 \| Volume 12 \| Article 722871 13 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. analyzed the data. YH and US wrote the article. All authors contributed to the article and approved the submitted version. research was supported by funds of the German Research Foundation (DFG) for project No. SCHM 1456/8-1. research was supported by funds of the German Research Foundation (DFG) for project No. SCHM 1456/8-1. FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. 722871/full#supplementary-material We thank Dr. M. Herz from LfL who provided the cultivar-speciﬁc information on spring malting barley. This REFERENCES Breeding progress, genotypic and environmental variation and correlation of quality traits in malting barley in German oﬃcial variety trials between 1983 and 2015. Theor. Appl. Gen. 130, 2411–2429. doi: 10.1007/s00122-017-2967-4 Clarke, J. M., Campbell, C. A., Cutforth, H. W., Depauw, R. M., and Winkleman, G. E. (1990). Nitrogen and phosphorus uptake, translocation, and utilization eﬃciency of wheat in relation to environment and cultivar yield and protein- levels. Can. J. Plant Sci. 70, 965–977. doi: 10.4141/cjps90-119 Maydup, M. L., Antonietta, M., Graciano, C., Guiamet, J. J., and Tambussi, E. A. (2014). The contribution of the awns of bread wheat (Triticum aestivum L.) to grain ﬁlling: responses to water deﬁcit and the eﬀects of awns on ear temperature and hydraulic conductance. Field Crop Res. 167, 102–111. doi: 10.1016/j.fcr.2014.07.012 Cox, M. C., Qualset, C. O., and Rains, D. W. (1985a). Genetic variation for nitrogen assimilation and translocation in wheat. I. Dry matter and nitrogen accumulation. Crop Sci. 25, 430–435. doi: 10.2135/cropsci1985.0011183X002500030002x Merah, O., Evon, P., and Monneveux, P. (2018). Participation of green organs to grain ﬁlling in triticum turgidum var durum grown under mediterranean conditions. Int. J. Mol. Sci. 19, 56. doi: 10.3390/ijms19010056 Cox, M. C., Qualset, C. O., and Rains, D. W. (1985b). Genetic variation for nitrogen assimilation and translocation in wheat. II. Nitrogen assimilation in relation to grain yield and protein. Crop Sci. 25, 435–440. doi: 10.2135/cropsci1985.0011183X002500030003x Merah, O., and Monneveux, P. (2015). Contribution of diﬀerent organs to grain ﬁlling in durum wheat under mediterranean conditions I. Contribution of post- anthesis photosynthesis and remobilization. J. Agron. Crop Sci. 20, 344–352. doi: 10.1111/jac.12109 Cox, M. C., Qualset, C. O., and Rains, D. W. (1986). Genetic variation for nitrogen assimilation and translocation in wheat. III. Nitrogen August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 14 Partitioning and Translocation in Malting Barley Varieties Barmeier et al. Therrien, M. C., Carmichael, C. A., Noll, J. S., and Grant, C. A. (1994). Eﬀect of fertilizer management, genotype, and environmental factors on some malting quality characteristics in barley. Can. J. Plant Sci. 74, 545–547. doi: 10.4141/cjps94-098 Newton, A. C., Flavell, A. J., George, T. S., leat, P., Mullholland, B., Ramsay, L., et al. (2011). Crops that feed the world 4. Barley: a resilient crop? Strengths and weaknesses in the context of food security. Food Security 3, 141–178. REFERENCES doi: 10.1007/s12571-011-0126-3 Vahamidis, P., Stefopoulou, A., Kotoulas, V., Voloudakis, D., Dercas, N., and Economou, G. (2021). A further insight into the environmental factors determining potential grain size in malt barley under Mediterranean conditions. Eur. J. Agron. 122:126184. doi: 10.1016/j.eja.2020.126184 Papakosta, D. K., and Gagianas, A. A. (1991). Nitrogen and dry-matter accumulation, remobilization, and losses for Mediterranean wheat during grain ﬁlling. Agron. J. 83, 864–870. doi: 10.2134/agronj1991.0002196200830005 0018x Przulj, N., and Momcilovic, V. (2001a). Genetic variation for dry matter and nitrogen accumulation and translocation in two-rowed spring barley I. Dry matter translocation. Eur. J. Agron. 15, 241–254. doi: 10.1016/S1161-0301(01)00109-5 van Sanford, D. A., and Mackown, C. T. (1987). Cultivar diﬀerences in nitrogen remobilization during grain ﬁll in soft red winter wheat. Crop Sci. 27, 295–300. doi: 10.2135/cropsci1987.0011183X002700020035x Zadoks, J. C., Chang, T. T., and Konzak, C. F. (1974). Decimal code for growth stages of cereals. Weed Res. 14, 415–421. doi: 10.1111/j.1365-3180.1974.tb01084.x Przulj, N., and Momcilovic, V. (2001b). Genetic variation for dry matter and nitrogen accumulation and translocation in two-rowed spring barley II. Nitrogen translocation. Eur. J. Agron. 15, 255–265. doi: 10.1016/S1161-0301(01)00108-3 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Savin, R., Prystupa, P., and Araus, J. L. (2006). Hordein composition as aﬀected by post-anthesis source-sink ratio under diﬀerent nitrogen availabilities. J. Cereal Sci. 44, 113–116. doi: 10.1016/j.jcs.2006.01.003 Schmidhalter, U. (2005). Development of a quick on-farm test to determine nitrate levels in soil. J. Plant Nutr. Soil Sci. 168, 432–438. doi: 10.1002/jpln.200520521 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Schmidhalter, U., Bredemeier, C., Geesing, D., Mistele, B., Selige, T., and Jungert, S. (2006). Precision agriculture: spatial and temporal variability of soil water, soil nitrogen and plant crop response. Bib. Frag. Agron. 11, 97–105. Shrestha, R. K., and Lindsey, L. E. (2019). Agronomic management of malting barley and research needs to meet demand by the craft brew industry. Agron. J. 111, 1–11. doi: 10.2134/agronj2018.12.0787 Copyright © 2021 Barmeier, Hu and Schmidhalter. Frontiers in Plant Science \| www.frontiersin.org August 2021 \| Volume 12 \| Article 722871 REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Sun, Y., Zhang, S., and Yan, J. (2021). Contribution of green organs to grain weight in dryland wheat from the 1940s to the 2010s in Shaanxi Province, China. Sci. Rep. 11:3377. doi: 10.1038/s41598-021-82718-y Sylvester-Bradley, R., and Kindred, D. R. (2009). Analysing nitrogen responses of cereals to prioritize routes to the improvement of nitrogen use eﬃciency. J. Exp. Bot. 60, 1939–1951. doi: 10.1093/jxb/erp116 August 2021 \| Volume 12 \| Article 722871 Frontiers in Plant Science \| www.frontiersin.org 15
https://openalex.org/W3165134059	https://europepmc.org/articles/pmc8447518?pdf=render	English	null	Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition	Molecular brain	2,021	cc-by	17,433	Abstract Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense muta- tion (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobi- ology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Den- dritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA- type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the muta- tion results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N muta- tion in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurode- generation, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro. Keywords: Parkinson’s disease, Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), NMDA, Dopamine Receptor Glutamate Synapses Synaptic transmission Knock in mouse Retromer Protein traffic Keywords: Parkinson’s disease, Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), NMDA, Dopamine Receptor, Glutamate, Synapses, Synaptic transmission, Knock-in mouse, Retromer, Protein traffic Kadgien et al. Mol Brain (2021) 14:143 https://doi.org/10.1186/s13041-021-00848-w Kadgien et al. Mol Brain (2021) 14:143 https://doi.org/10.1186/s13041-021-00848-w Open Access Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock‑in mouse neurons, and resistant to LRRK2 kinase inhibition Chelsie A. Kadgien1,2, Anusha Kamesh2 and Austen J. Milnerwood1,2* © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction Correspondence: austen.milnerwood@mcgill.ca 1 Graduate Program in Neuroscience and Centre for Applied Neurogenetics, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, Canada Full list of author information is available at the end of the article Correspondence: austen.milnerwood@mcgill.ca 1 Graduate Program in Neuroscience and Centre for Applied Neurogenetics, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, Canada Full list of author information is available at the end of the article Vacuolar protein sorting 35 (VPS35) is a core component of the retromer complex, which recycles transmembrane cargo from endosomes to the trans-Golgi network (TGN) In neurons, changes to LRRK2 levels, and LRRK2 mutations, dysregulate synaptic vesicle (SV) recy- cling and release [30–34], post-synaptic receptor traffick- ing, and dendritic spine development [35–38]. Inoshita and colleagues [25] recently reported that LRRK2 local- izes with VPS35 at Drosophila neuromuscular junctions where they are proposed to work together regulate the SV cycle. PD-associated mutations in LRRK2 increase its kinase activity (reviewed in: 39), which hyperphosphorylates multiple LRRK2 substrates (reviewed in: 40). The best- characterized LRRK2 substrates are several Rab-GTPases [41,42]. Increased autophosphorylation of LRRK2 and phosphorylation of Rab10 have been observed in post- mortem substantia nigra from individuals with idiopathic PD [43], monocytes from humans with the D620N muta- tion, and tissues from VPS35 D620N knock-in mice [27]. This provides evidence that VPS35 and LRRK2 muta- tions converge on LRRK2 kinase activity, and that aber- rant phosphorylation of Rab proteins involved in synaptic transmission may be related to the several synaptic phe- notypes observed in PD models (reviewed in: 40). yp Here we probe the neurobiological function of VPS35 in developing neural networks, potentially pathophysio- logical dysfunction, and drug responses in neurons from VKI mice. This may uncover factors that combine with age and other environmental stresses to eventually trig- ger transition to pathology. We examined synapse devel- opment, structure, and function, in addition to protein levels, phosphorylation state, and binding relationships in brain tissue and cultured cortical neurons. The D620N mutation reduced retromer complex association with its regulatory proteins by coIP, increased dendritic cluster- ing of proteins involved in surface protein recycling, and augmented glutamate transmission. We assayed Rab10 phosphorylation in brain and cultured cortical neurons and the effects of LRRK2 kinase inhibition on gluta- matergic transmission phenotypes. LRRK2 kinase inhi- bition did not rescue mutant synaptic phenotypes. In wild-type cells, LRRK2 kinase inhibition produced effects consistent with LRRK2 negatively regulating the delivery of AMPARs to developing/silent synapses. In neurons, retromer clusters are highly motile in soma, axons, dendrites, and dendritic spines [14,15]. We, and others, demonstrated by overexpression and knock-out studies that VPS35 participates in surface delivery of GluA1 subunit-containing alpha-amino-3-hy- droxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors, supports synapse development, maturation, and activity-dependent AMPA receptor (AMPAR) delivery required for the expression of long- term potentiation (LTP; 14–22). We found that exog- enous expression of D620N mutant VPS35 impairs its motility in dendrites and trafficking into spines [15]. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kadgien et al. Mol Brain (2021) 14:143 Kadgien et al. Mol Brain (2021) 14:143 Page 2 of 20 or surface plasma membranes, circumventing lysosomal degradation (reviewed in: 1–4). A missense mutation in VPS35 (D620N) causes late-onset autosomal Parkinson’s disease (PD) that is clinically indistinguishable from idi- opathic PD [5–7]. PD is classically thought of as a motor disorder caused by degeneration of dopamine neurons in the substantia nigra; however, disease progression con- tinues after functional loss of nigral projections to the striatum [8]. PD is accompanied by non-motor symp- toms that can precede motor onset by decades, and that are not responsive to dopamine replacement therapies (reviewed in: 9,10). The disease is also characterized by impaired cortical synaptic plasticity occurring prior to motor symptom onset and cortical neuron loss at later stages (reviewed in: 11). Such observations highlight the involvement of the glutamatergic system early in the disease process, and throughout its progression. VPS35 D620N knock-in (VKI) mice eventually develop nigral pathology [12,13] validating their relevance as models of PD, and here we investigate whether the VPS35 mutation impacts glutamatergic function in cortical neurons. We identify early alterations that might be used as biomark- ers of mutant effects and targeted to prevent transition to later disease states. risk, the most common mutation being the G2019S sub- stitution [29]. Altered protein binding relationships and LRRK2 kinase activity in VKI brainh The D620N mutation does not impair VPS35 binding to other retromer complex members (vacuolar protein sort- ing 26 or vacuolar protein sorting 29; VPS26 & VPS29, respectively) by semi-quantitative co-immunoprecipita- tion (coIP) in overexpression systems [15, 44–46] or brain lysate from VKI mice [12]. We assayed subunit assembly by coIP in brain lysate from 3-month-old VKI mice and confirmed no genotype effect on the amount of VPS26 pulled by VPS35 (Fig. 1A.i–ii; 1-way ANOVA p = 0.44). In cell lines, the D620N mutation impairs VPS35 asso- ciation with the Wiskott–Aldrich syndrome protein and SCAR homolog (WASH) complex member family with sequence similarity 21 (FAM21) by coIP [44, 45, 47]. We found the level of FAM21 pulled by VPS35 was similarly reduced in coIP of whole-brain lysates from mutant mice (Fig. 1A.i & iii; 1-way ANOVA p < 0.003; Uncorrected Fisher’s LSD Het p < 0.01; Ho p < 0.01) and we success- fully pulled VPS35 by FAM21 in the reverse coIP (Addi- tional file 1: Fig. S1A). In agreement with our previous report and with others [12, 48], we found no genotype effect on the level of VPS35, VPS26, or WASH complex member FAM21 protein relative to β-tubulin in whole brain lysate from 3 month old VKI mice, quantified by WES capillary-based western blot (Additional file 1: Fig. S1B.i–iv). We performed coIP of striatal lysates pulling for VPS35 and blotting for LRRK2 and for a panel of possible neu- rotransmitter cargoes relevant to corticostriatal syn- apses (Additional file 1: Fig. S2A.i). We replicated our previous finding that GluA1 associates with retromer [15] and found two previously unreported putative ret- romer cargoes; D2-type dopamine receptors (D2R) and GluN1-containing N-methyl-D-aspartate (NMDA)-type glutamate receptors (Additional file 1: Fig. S2A.i). There was no mutation effect on the levels of VPS35, LRRK2, GluN1, D2R, or GluA1 in striatal lysate, nor on their association with VPS35 by coIP (Additional file 1: Fig. S2 A.ii–xi). i In summary, physiological expression of VPS35 D620N does not alter protein levels of core retromer compo- nents, the known interactor FAM21, or LRRK2 and its substrate Rab10, but does result in hyperphosphorylation of Rab10 which is reversed by acute in vivo LRRK2 kinase inhibition. While retromer complex assembly and neu- ronal cargo binding appear unaltered, we found the asso- ciation of VPS35 with FAM21 is reduced in VKI brain. 1C.i & iii; 2-way ANOVA genotype p = 0.04; treat- ment p < 0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p = 0.82; Het-HetMLi2 p = 0.10; Ho-HoMLi2 p = 0.007) but had little effect on WT brain pRab10 levels, in agree- ment with Mir et al. [27]. This occurred in the absence of any change to levels of LRRK2, GluA1, VPS35, VGluT1, or Rab10 protein (Additional file 1: Fig. S3B.ii–vi). The solubilizing agent sulfobutylether-β-cyclodextrin (Cap- tisol®) had no effect on pLRRK2 or pRab10 compared to saline in western blots of whole-brain lysate from injected mice (Fig. 1D.i–iii; Mann–Whitney p = 0.89 & p = 0.69, respectively), nor on levels of LRRK2, GluA1, VPS35, VGluT1, or Rab10 (Additional file 1: Fig. S4). neurobiology, and have important implications for inter- pretation of mutation effects induced by exogenous pro- tein expression and the potential utility of LRRK2 kinase inhibitors in the treatment of non-LRRK2 PD. 1C.i–ii; 2-way ANOVA treatment p < 0.0001; Uncorrected Fish- er’s LSD WT-WTMLi2 p < 0.0001; Het-HetMLi2 p < 0.0002; Ho-HoMLi2 *p < 0.0001) demonstrating successful target engagement. MLi-2 treatment reversed pRab10 increases in whole brain lysate from VKI mice (Fig. 1C.i & iii; 2-way ANOVA genotype p = 0.04; treat- ment p < 0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p = 0.82; Het-HetMLi2 p = 0.10; Ho-HoMLi2 p = 0.007) but had little effect on WT brain pRab10 levels, in agree- ment with Mir et al. [27]. This occurred in the absence of any change to levels of LRRK2, GluA1, VPS35, VGluT1, or Rab10 protein (Additional file 1: Fig. S3B.ii–vi). The solubilizing agent sulfobutylether-β-cyclodextrin (Cap- tisol®) had no effect on pLRRK2 or pRab10 compared to saline in western blots of whole-brain lysate from injected mice (Fig. 1D.i–iii; Mann–Whitney p = 0.89 & p = 0.69, respectively), nor on levels of LRRK2, GluA1, VPS35, VGluT1, or Rab10 (Additional file 1: Fig. S4). Dunn’s Het p < 0.003, Ho p < 0.003). We found no dif- ference in LRRK2 protein levels in any genotype (Addi- tional file 1: Fig. S3A.i–iii) and no difference in LRRK2 phosphorylation at the serine 935 residue (pS935, herein pLRRK2) in untreated heterozygous brain. We did find a significant increase of basal pLRRK2 in untreated homozygotes (Additional file 1: Fig. S3A.i–iii), but this was not observed in the larger cohort comparison of vehicle vs. inhibitor treated animals (Fig. 1C). This resi- due, considered constitutive, is required for LRRK2 kinase activity but is not an autophosphorylation site and its phosphorylation level does not correlate with LRRK2 kinase enzymatic activity levels [49]; however, S935 is dephosphorylated when LRRK2 is inactivated, and it is widely accepted as indicative of effective LRRK2 kinase inhibition [50]. In mice injected with the highly selective LRRK2 kinase inhibitor MLi-2 [27, 41, 49, 50], pLRRK2 was significantly reduced after 2 h (~ 90%) in all geno- types, relative to vehicle injected controls (Fig. 1C.i–ii; 2-way ANOVA treatment p < 0.0001; Uncorrected Fish- er’s LSD WT-WTMLi2 **p < 0.0001; Het-HetMLi2 p < 0.0002; Ho-HoMLi2 *p < 0.0001) demonstrating successful target engagement. MLi-2 treatment reversed pRab10 increases in whole brain lysate from VKI mice (Fig. Fur- thermore, we previously showed mutant VPS35 expres- sion altered excitatory synaptic current amplitudes in mouse neurons, and AMPA receptor cluster intensities in both mouse neurons and dopamine neuron-like cells derived from human mutation carrier induced pluri- potent stem cells [15]. Together, this argues retromer is important in synapse development, maintenance, and functional connectivity. However, it was previously unclear the extent to which overexpression artefacts might impact physiological retromer function, and how endogenous mutations in VPS35 might alter its role at the synapse. VKI mice provide a model system in which to develop insights into the molecular and cellular effects of VPS35 mutations, and potentially the etiology PD. Dysregulation of glutamate transmission in VKI mice resembles that reported in LRRK2 G2019S knock-in mouse models of parkinsonism; however, the failure of LRRK2 kinase inhi- bition to reverse glutamate phenotypes in VKI mice sug- gests VPS35 D620N mutation effects do not result from ongoing LRRK2 kinase activity or are not rapidly revers- ible. Our findings extend our understanding of VPS35 VPS35 interacts with leucine-rich repeat kinase 2 (LRRK2), another PD-implicated protein, both physically and functionally [23–28]. LRRK2 is a large multi-domain protein implicated in ~ 5% of all familial Parkinson’s dis- ease through autosomal-dominant mutations and genetic Kadgien et al. Mol Brain (2021) 14:143 Kadgien et al. Mol Brain (2021) 14:143 Page 3 of 20 Dunn’s Het p < 0.003, Ho p < 0.003). We found no dif- ference in LRRK2 protein levels in any genotype (Addi- tional file 1: Fig. S3A.i–iii) and no difference in LRRK2 phosphorylation at the serine 935 residue (pS935, herein pLRRK2) in untreated heterozygous brain. We did find a significant increase of basal pLRRK2 in untreated homozygotes (Additional file 1: Fig. S3A.i–iii), but this was not observed in the larger cohort comparison of vehicle vs. inhibitor treated animals (Fig. 1C). This resi- due, considered constitutive, is required for LRRK2 kinase activity but is not an autophosphorylation site and its phosphorylation level does not correlate with LRRK2 kinase enzymatic activity levels [49]; however, S935 is dephosphorylated when LRRK2 is inactivated, and it is widely accepted as indicative of effective LRRK2 kinase inhibition [50]. In mice injected with the highly selective LRRK2 kinase inhibitor MLi-2 [27, 41, 49, 50], pLRRK2 was significantly reduced after 2 h (~ 90%) in all geno- types, relative to vehicle injected controls (Fig. Increased colocalization and density of endosomal recycling proteinsh For C.ii–iii, WTCap n = 5, WTMLi2 n = 6, HetCap n = 6, HetMLi2 n = 6, HoCap n = 5, HoMLi2 n = 5 Fig. 1 Altered FAM21 binding and LRRK2 kinase inhibitor-reversible Rab-GTPase phosphorylation in VKI mouse brain. A Co-immunoprecipitations of VPS26 and FAM21 with VPS35 from VKI whole-brain lysate run on a WES capillary-based western blot system (i) uncovered no effect on VPS26 pulled by VPS35 (ii, 1-way ANOVA p = 0.44) but a significant reduction in FAM21 pulled by mutant VPS35 (iii, 1-way ANOVA p < 0.003; Uncorrected Fisher’s LSD Het p < 0.01; Ho p < 0.01). B Western blot of Rab10, Rab10 pT73, and β-actin in VKI whole brain lysate (i) revealed no significant genotype effect on Rab10 levels (ii, Kruskal–Wallis p = 0.16), but a significant increase in Rab10 pT73 in VKI (iii, Kruskal–Wallis p < 0.0001; Uncorrected Dunn’s Het p < 0.003, Ho p < 0.003). C Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in VKI whole brain lysate (i) revealed that MLi2 treatment significantly reduced LRRK2 pS935 in all genotypes (ii, 2-way ANOVA treatment p < 0.0001; Uncorrected Fisher’s LSD WT-WTMLi2 p < 0.0001; Het-HetMLi2 p < 0.0002; Ho-HoMLi2 *p < 0.0001) and had significant genotype and treatment effects on Rab10 pT73 due to significant reductions in homozygous cells (iii, 2-way ANOVA genotype p = 0.04; treatment p < 0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p = 0.82; Het-HetMLi2 p = 0.10; Ho-HoMLi2 p = 0.007). D Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in whole brain lysate revealed no significant effect of the solubilizing agent Captisol on LRRK2 pS935 (ii, Mann–Whitney p = 0.89) or Rab10 pT73 (iii, Mann–Whitney p = 0.69). For C.ii–iii, WTCap n = 5, WTMLi2 n = 6, HetCap n = 6, HetMLi2 n = 6, HoCap n = 5, HoMLi2 n = 5 trimer (Fig. 2A.i; Pearson’s ~ 0.6). Dendritic VPS35 cluster density was unaffected by genotype (Fig. 2A.ii; Kruskal–Wallis p = 0.30); however, the density of VPS26 clusters was reduced in heterozygous and homozygous neurons (Fig. 2 A.iii; Kruskal–Wallis p < 0.04; Uncor- rected Dunn’s Het p < 0.04 & Ho p < 0.02). There was no genotype effect on colocalized cluster density or Pearson’s correlation coefficient (Fig. 2A.iv–v; Kruskal– Wallis p = 0.45 & 0.10, respectively). Increased colocalization and density of endosomal recycling proteinsh Others have reported increased LRRK2 kinase-depend- ent phosphorylation of Rab10 at threonine 73 (pRab10) in VKI mouse brain in the absence of changes to Rab10 levels [27], a finding we replicated here using fluores- cence western blot with knock-out validated phospho- specific antibodies (Fig. 1B.i–iii; Rab10 Kruskal–Wallis p = 0.16; pRab10 Kruskal–Wallis p < 0.0001; Uncorrected The retromer complex requires the recruitment of the WASH complex to drive the rescue of receptors out of maturing endosomes for recycling to the plasma mem- brane [51–53]. Since the mutation alters VPS35 interac- tion with FAM21, we studied the localization of VPS35, Kadgien et al. Mol Brain (2021) 14:143 Page 4 of 20 Fig. 1 Altered FAM21 binding and LRRK2 kinase inhibitor-reversible Rab-GTPase phosphorylation in VKI mouse brain. A Co-immunoprecipitations of VPS26 and FAM21 with VPS35 from VKI whole-brain lysate run on a WES capillary-based western blot system (i) uncovered no effect on VPS26 pulled by VPS35 (ii, 1-way ANOVA p = 0.44) but a significant reduction in FAM21 pulled by mutant VPS35 (iii, 1-way ANOVA p < 0.003; Uncorrected Fisher’s LSD Het p < 0.01; Ho p < 0.01). B Western blot of Rab10, Rab10 pT73, and β-actin in VKI whole brain lysate (i) revealed no significant genotype effect on Rab10 levels (ii, Kruskal–Wallis p = 0.16), but a significant increase in Rab10 pT73 in VKI (iii, Kruskal–Wallis p < 0.0001; Uncorrected Dunn’s Het p < 0.003, Ho p < 0.003). C Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in VKI whole brain lysate (i) revealed that MLi2 treatment significantly reduced LRRK2 pS935 in all genotypes (ii, 2-way ANOVA treatment p < 0.0001; Uncorrected Fisher’s LSD WT-WTMLi2 **p < 0.0001; Het-HetMLi2 p < 0.0002; Ho-HoMLi2 *p < 0.0001) and had significant genotype and treatment effects on Rab10 pT73 due to significant reductions in homozygous cells (iii, 2-way ANOVA genotype p = 0.04; treatment p < 0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p = 0.82; Het-HetMLi2 p = 0.10; Ho-HoMLi2 p = 0.007). D Western blot of LRRK2, LRRK2 pS935, GAPDH, Rab10, and Rab10 pT73 in whole brain lysate revealed no significant effect of the solubilizing agent Captisol on LRRK2 pS935 (ii, Mann–Whitney p = 0.89) or Rab10 pT73 (iii, Mann–Whitney p = 0.69). Increased colocalization and density of endosomal recycling proteinsh In summary, the D620N mutation increased clustering of proteins found on structures involved in surface deliv- ery (Rab11) and VPS35 co-clustered with complexes that drive surface recycling (FAM21). Conversely, overlap of VPS35 signal with a marker of the degradative pathway (NEEP21) was reduced. Together the observations sug- gest the mutation increases capacity for surface recycling; alternatively it may halt recycling traffic, resulting in the accumulation of surface bound carriers (VPS35-FAM21 & Rab11). There was no genotype effect on the cluster den- sity of the WASH complex member FAM21 (Fig. 2 B.ii; Kruskal–Wallis p = 0.24). VPS35 and FAM21 were robustly colocalized (Fig. 2B.i; Pearson’s ~ 0.6) with no genotype effect on Pearson’s correlation coefficient (Fig. 2B.iv; 1-way ANOVA p = 0.24). Contrary to the observed reduction in coIP with FAM21, the density of VPS35-FAM21 co-clusters was increased in mutants in a gene dose-dependent manner, with a significant post hoc increase in homozygous cells (Fig. 2B.iii; Kruskal–Wallis p < 0.03; Uncorrected Dunn’s Ho p < 0.02). p < 0.003). A reduction in Pearson’s coefficients in this instance may be reflective of an accumulation of recy- cling endosomes downstream of retromer tubulation. In summary, the D620N mutation increased clustering of proteins found on structures involved in surface deliv- ery (Rab11) and VPS35 co-clustered with complexes that drive surface recycling (FAM21). Conversely, overlap of VPS35 signal with a marker of the degradative pathway (NEEP21) was reduced. Together the observations sug- gest the mutation increases capacity for surface recycling; alternatively it may halt recycling traffic, resulting in the accumulation of surface bound carriers (VPS35-FAM21 & Rab11). Neuronal endosomal enriched protein 21 (NEEP21 or NSG1) is itinerant in dendritic endosomes and rapidly degraded with little to no recycling [54]. We therefore used NEEP21 as a marker of the non-recycling endolys- osomal pathway (Fig. 2C.i). We observed no genotype effect on the density of NEEP21 clusters (Fig. 2C.ii; Kruskal–Wallis p = 0.37), nor VPS35-NEEP21 co-clus- ter densities (Fig. 2C.iii; Kruskal–Wallis p = 0.38), sug- gesting no change in VPS35 localization at early or late endosomes. VPS35 and NEEP21 were tightly apposed with little overlap (Fig. 2C.i; Pearson’s coefficient ~ 0.2), in accordance with retromer-coated tubules extending from endosomes to drive recycling, not lysosomal degra- dation. Consistent with this, there was a decrease in the Pearson’s coefficient in VKI dendrites (Fig. 2C.iv; Welch’s ANOVA p < 0.03; Unpaired t with Welch’s correction Ho p < 0.02). Increased colocalization and density of endosomal recycling proteinsh its known interactors, and endosomal markers in corti- cal neuron dendrites by immunostaining at 21 days- in-vitro (DIV21) to detect VPS35 colocalization with VPS26 (retromer complex), FAM21 (WASH complex), NEEP21 (early and late endosomes), or Rab11 (recycling endosomes; Fig. 2A–D). We observed robust colocalization of VPS35 signal with VPS26, a member of the retromer complex core Kadgien et al. Mol Brain (2021) 14:143 Page 5 of 20 (See figure on next page.) Fig. 2 Accumulation of VPS35- FAM21 co-clusters and Rab 11 clusters in VKI dendrites. A–D Cultured cortical neurons were immunostained for MAP2 (blue), VPS35 (cyan), and VPS26, FAM21, NEEP21, or Rab11 (magenta) A VPS35 and VPS26 (i); There was no genotype effect on dendritic cluster density of VPS35 (ii, Kruskal–Wallis p = 0.30). VPS26 cluster density was significantly reduced in both mutants (iii, Kruskal–Wallis p < 0.04; Uncorrected Dunn’s Het p < 0.04 & Ho p < 0.02), yet there was no genotype effect on co-cluster density or Pearson’s coefficient (iv & v, Kruskal– Wallis p = 0.45 & 0.10, respectively). B VPS35 and FAM21 (i); there was no genotype effect on FAM21 cluster density (ii, Kruskal–Wallis p = 0.24) or Pearson’s coefficients (iv, 1-way ANOVA p = 0.24). There was a significant genotype effect on co-cluster density due to increases in homozygous cells (iii, Kruskal–Wallis p < 0.03; Uncorrected Dunn’s Ho p < 0.02). C NEEP21 and VPS35 (i); there was no genotype effect on NEEP21 cluster (ii, Kruskal– Wallis p = 0.37) or co-cluster density (iii, Kruskal–Wallis p = 0.38), but a significant genotype effect on Pearson’s coefficient due to a significant reduction in homozygous VKI dendrites (iv, Welch’s ANOVA p < 0.03; Unpaired t with Welch’s correction Ho p < 0.02). D VPS35 and Rab11 (i); Rab11 cluster density was increased in both genotypes (ii, 1-way ANOVA p < 0.03; Uncorrected Fisher’s LSD p < 0.02 & p < 0.03). There was no genotype effect on co-cluster density (iii, 1-way ANOVA p = 0.92), but a significant genotype effect in the Pearson’s coefficient due to reduced correlation in heterozygous dendrites (iv, Welch’s ANOVA p < 0.009; Unpaired t with Welch’s correction Het p < 0.003) p < 0.003). A reduction in Pearson’s coefficients in this instance may be reflective of an accumulation of recy- cling endosomes downstream of retromer tubulation. Glutamate transmission is increased in VKI and not reversed by LRRK2 kinase inhibitionf Glutamate synaptic phenotypes in LRRK2 G2019S knock-in mice are reversed by acute LRRK2 kinase inhibition [35]. Thus, we hypothesized that in light of increased LRRK2 kinase activity in VKI brain (Fig. 1), LRRK2 inhibition by MLi-2 would reverse glutamater- gic phenotypes in VKI primary cortical cultures. We treated DIV21 primary cortical cultures for 2 h with 500 nM MLi-2. LRRK2 and pLRRK2 were detected in culture lysates by fluorescent western blot, and pLRRK2 appeared reduced after MLi2 in all genotypes, although we deemed bands were too close to background to quan- tify reliably (Additional file 1: Fig. S7). Due to its large size (286 kDa), LRRK2 is difficult to assay by western blot, usually requiring a high concentration of total protein for detection with currently available antibodies. As a proxy for LRRK2 activity in vitro, we assayed pRab10 and found a mutation dose-dependent increase in pRab10 (as seen in brain in Fig. 1). MLi-2 treatment reduced pRab10 in all genotypes and significantly reduced VKI pRab10 hyper- phosphorylation levels to that of WT (Fig. 3 C.i–ii; 2-way ANOVA genotype x treatment p < 0.08; genotype p < 0.03; treatment p < 0.0004; Uncorrected Fisher’s LSD WT- WTMLi2 p = 0.99; Het-HetMLi2 p = 0.26; Ho-HoMLi2 *p < 0.005). Thus, D620N results in increased pRab10 in cultured cortical neurons at DIV21 (as in brain Fig. 1), and is reversed by LRRK2 inhibition with MLi-2.hf lf To assess glutamate synapse function, we performed whole-cell patch clamp recording of mEPSCs in VKI cultures (Fig. 3B.i). Inter-event interval cumulative prob- abilities showed that event frequency is higher in mutant neurons (Fig. 3B.ii; 2-way RM ANOVA WT-Het inter- action p < 0.0001; genotype p < 0.03; 2-way RM ANOVA WT-Ho interaction p < 0.0001; genotype p < 0.11). Increased mEPSC frequencies on a background of equiv- alent synapse density is usually interpreted as increased probability of vesicular release (Pr), or fewer silent (AMPAR-lacking) synapses. In our previous report, we found mutant overex- pression resulted in larger mEPSC amplitudes than WT; however, neither were significantly different from control making it difficult to determine whether this represented a gain- or loss-of-function upon surface AMPAR trafficking [15]. Here we found the amplitude of mEPSCs was increased in mutant neurons (Fig. 3B. iii; 2-way RM ANOVA WT-Het interaction p < 0.0001; genotype p < 0.03; 2-way RM ANOVA WT-Ho interac- tion p < 0.002; genotype p < 0.09). Glutamate transmission is increased in VKI and not reversed by LRRK2 kinase inhibitionf To help determine if mutation effects reported in Fig. 2 reflect an increase in, or backlog of, dendritic surface traffic, we examined glutamatergic synapse function in cortical cultures from VKI mice. In light of reports that VPS35 knock-down and over- expression both result in impaired neurite outgrowth [17, 19], we performed Sholl analysis of cultured cortical neurons from VKI expressing green fluorescent protein (pAAV-CAG-GFP) at DIV21 and found no mutant effects on cell density or neurite morphology (Additional file 1: Fig. S5). We previously reported that VPS35 wild-type and mutant overexpression reduced synapse number and miniature excitatory post-synaptic current (mEPSC) frequency in cultured cortical neurons [15]. Here excita- tory synapses were quantified by immunostaining for presynaptic and postsynaptic markers, vesicular gluta- mate transporter 1 (VGluT1) and postsynaptic density protein 95 (PSD95; Fig. 3A.i), respectively. There was no genotype effect on density of synapses, as evidenced by equal numbers of VGluT1-PSD95 co-clusters (Fig. 3A.ii; 1-way ANOVA p = 0.57) and individual cluster densities of PSD95 and VGluT1 (Additional file 1: Fig. S6A.i–ii). In concert with equivalent neurite morphology (Additional Rab11 decorates recycling endosomes (REs) and par- ticipates in AMPAR surface trafficking [55–60], thus we used Rab11 as a marker of REs (Fig. 2D.i). Rab11 clus- ter density was increased in both VKI mutants (Fig. 2D. ii; 1-way ANOVA p < 0.03; Uncorrected Fisher’s LSD p < 0.02 & p < 0.03). Despite increased Rab11 cluster abundance, there was no genotype effect on the colo- calization density of VPS35 and Rab11 (Fig. 2D.iii; 1-way ANOVA p = 0.92) and a decrease in Pearson’s coeffi- cient in heterozygous cells (Fig. 2D.iv; Welch’s ANOVA p < 0.009; Unpaired t with Welch’s correction Het Kadgien et al. Mol Brain (2021) 14:143 Page 6 of 20 Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Kadgien et al. Mol Brain (2021) 14:143 Page 7 of 20 Fig. 3 Synaptic transmission is increased in VKI and altered by acute LRRK2 kinase inhibition. A Cortical neurons immunostained for PSD95 (cyan) and VGluT1 (magenta) (i). There was no genotype effect on VGluT1-PSD95 co-clusters (ii, 1-way ANOVA p = 0.57). B Whole-cell patch voltage clamp recording of mEPSCs in cortical neurons (i). Glutamate transmission is increased in VKI and not reversed by LRRK2 kinase inhibitionf There were significant shifts in frequency cumulative probabilities due to significantly smaller inter-event intervals in cells from both genotypes (ii, 2-way RM ANOVA WT-Het interaction p < 0.0001; genotype p < 0.03; 2-way RM ANOVA WT-Ho interaction p < 0.0001; genotype p < 0.11; Uncorrected Fisher’s LSD p < 0.001; p < 0.007; p < 0.05). Significant shifts were seen in amplitude cumulative probabilities due to increases in heterozygous and homozygous cells (iii, 2-way RM ANOVA WT-Het interaction p < 0.0001; genotype p < 0.03; 2-way RM ANOVA WT-Ho interaction p < 0.002; genotype p < 0.09; Uncorrected Fisher’s LSD *p < 0.0001; p < 0.004; p < 0.01; p < 0.05). C Western blot of MLi-2 or vehicle treated cortical culture lysates probed for Rab10, Rab10 pT73, and GAPDH (i). There were significant genotype and treatment effects on Rab10 pT73 due to significant increases in vehicle-treated homozygous neurons over WT, which are significantly reduced by treatment (ii, 2-way ANOVA genotype x treatment p < 0.08; genotype p < 0.03; treatment p < 0.0004; Uncorrected Fisher’s LSD WT-Ho p < 0.01; WT-WTMLi2 p = 0.99; Het-HetMLi2 p = 0.26; Ho-HoMLi2 p < 0.005). D Cortical neurons immunostained as in A) plus MAP2 (blue) following MLi-2 or vehicle treatment (i). There were no significant effects on PSD95-VGluT1 co-cluster density (ii, 2-way ANOVA genotype x treatment p = 0.08; genotype p = 0.52; treatment p = 0.68; All groups n = 40[4]). E Whole-cell patch voltage clamp recording of mEPSCs in cortical neurons following MLi-2 treatment (i). Change in mEPSC frequency following treatment revealed a significant genotype effect due to a ~ two-fold increase in mEPSC frequency in only WT cells following treatment (ii, Kruskal–Wallis p < 0.04; Uncorrected Dunn’s WT-Ho p < 0.01). Change in mEPSC amplitude following treatment revealed a significant genotype effect due to mildly opposing effects in heterozygous and homozygous cultures (iii, Kruskal– Wallis p < 0.03; Uncorrected Dunn’s Het-Ho p < 0.01) (See figure on next page.) S6B.i–iii). Thus, we conclude increased mEPSC ampli- tude is due to increased AMPAR surface levels. file 1: Fig. S5), the data suggest no difference in the den- sity of glutamatergic synapses in VKI cultures. That said, in homozygous neurons, VGluT1 cluster intensity was decreased (Additional file 1: Fig. S6 A.iii), possibly reflecting a presynaptic effect in the homozygous mutant. Glutamate transmission is increased in VKI and not reversed by LRRK2 kinase inhibitionf Changes in amplitude are usually taken to reflect increased surface expression of AMPARs, or altered receptor subtype expression i.e., a higher percentage of GluA2-lacking AMPARs with a higher channel conductance and faster decay kinetics (reviewed in: 61, 62). Thus, we examined mEPSC decay tau and single-channel conductance by peak-scaled non- stationary fluctuation analysis (NSFA) but found no significant genotype effect on either, suggesting a simi- lar AMPAR subunit composition (Additional file 1: Fig. The effect of LRRK2 kinase inhibition on excita- tory synapses was first examined by immunostaining for PSD95 and VGluT1 to determine synapse density (Fig. 3D.i). There were no significant effects of genotype or treatment on excitatory synapse density (Fig. 3D.ii; 2-way ANOVA genotype × treatment p = 0.08; genotype p = 0.52; treatment p = 0.68), despite a trend to increases Kadgien et al. Mol Brain (2021) 14:143 Page 8 of 20 Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Fig. 3 (See legend on previous page.) Kadgien et al. Mol Brain (2021) 14:143 Page 9 of 20 in MLi-2 treated WT neurons relative to vehicle-treated control. neurons in multiple comparisons, mirroring the increases in mEPSC amplitude (Fig. 4A.iii; Kruskal–Wal- lis p < 0.007; Uncorrected Dunn’s Het p < 0.02). The mutation does not affect GluA1 protein expression or association with VPS35 by coIP in cortical lysate (Addi- tional file 1: Fig. S8A–B) or striatal lysate (Additional file 1: Fig. S2), nor does it affect colocalization density of VPS35 with GluA1 in cultured cortical neuron den- drites by immunocytochemistry (Additional file 1: Fig. S8 C.i,iii,iv). We did find a significant reduction in total dendritic GluA1 clusters that were not colocalized with VPS35 in homozygous neurons, possibly revealing an additional postsynaptic phenotype similar to the pre- synaptic reduced VGluT1 intensity in the homozygous mutant (Additional file 1: Fig. S8C.ii). To assay the effects of MLi-2 treatment on synapse function, we conducted whole-cell patch clamp record- ing of mEPSCs (Fig. 3E.i). We observed an unexpected near twofold increase in mEPSC frequency in treated WT cells and little effect in heterozygous or homozygous neurons (Fig. 3E.ii; Kruskal–Wallis p < 0.04; Uncorrected Dunn’s WT-Ho p < 0.01). MLi-2 increased mEPSC amplitude slightly in WT and heterozygous cells and produced a significantly different effect (a reduction) in homozygous cells relative to heterozygous neurons (Fig. Glutamate transmission is increased in VKI and not reversed by LRRK2 kinase inhibitionf 3E.iii; Kruskal–Wallis p < 0.03; Uncorrected Dunn’s Het-Ho p < 0.01). p Together the data demonstrate the D620N muta- tion results in increased mEPSC frequency and ampli- tude, in the absence of changes to synapse density or neurite complexity. This suggests an increase in surface expression of synaptic AMPARs (mEPSC amplitude) and either an increase in the probability of quantal glu- tamate release, or an increase the number of excitatory synapses with functional surface AMPARs (mEPSC fre- quency). Acute treatment with MLi-2 reduced LRRK2 substrate phosphorylation with little or no effect on syn- apse density and had minor effects on mEPSC amplitude. MLi-2 did not rescue elevated frequency in mutants, but resulted in a near two-fold increase in mEPSC frequency in wild-type cells that could reflect either an increase in glutamate release, or increased delivery of AMPARs to AMPAR-lacking (silent) synapses. i We also performed surface GluA1 staining on non- permeabilized cultured cortical neurons from WT mice at DIV21 following 2 h of treatment with saline or Cap- tisol® (Fig. 4B.i) to ensure no vehicle effects on AMPAR expression. We found no effect of Captisol® on either the density (Fig. 4B.ii; Unpaired t-test p = 0.87) or intensity (Fig. 4B.iii; Mann–Whitney p = 0.73) of clusters, sug- gesting that the vehicle does not affect GluA1 surface expression. Surface GluA1 staining was then performed on cul- tured VKI cortical neurons following acute treatment with MLi-2 or vehicle (Fig. 4C.i). Surface GluA1 cluster density revealed MLi-2 produced a ~ 50% increase in WT cluster density, and little effect in mutants (Fig. 4C.i&ii; Welch’s ANOVA p < 0.0002; Unpaired t with Welch’s cor- rection WT-Het p < 0.003; WT-Ho **p < 0.0001). The effect of MLi-2 on surface GluA1 cluster density and mEPSC frequency in WT cells (with a slight increase in excitatory synapse density) suggests LRRK2 kinase inhi- bition drives delivery of AMPARs to a small number of new synapses and more so to existing AMPAR-lacking synapses. MLi-2 treatment resulted in different effects on surface GluA1 cluster intensity in heterozygous neu- rons (increase) relative to a small decrease observed in WT and homozygous cells (Fig. 4C.iii; Kruskal–Wallis p < 0.02; WT-Het p < 0.02; Het-Ho p < 0.02). The data suggest a genotype-dependent effect of LRRK2 kinase inhibition on AMPAR surface expression, which is simi- lar to MLi-2 effects on mEPSC amplitude.h Surface GluA1 is increased basally in VKI and increased in WT by LRRK2 kinase inhibition In the face of similar synapse numbers, increased mEPSC frequency can result from increased quantal glutamate release, or unsilencing of AMPA-silent synapses through surface delivery of AMPARs. To disambiguate the source of the observed mEPSC frequency changes, and to con- firm that changes to mEPSC amplitude are the result of changes to synaptic AMPAR expression, we performed immunostaining against an extracellular epitope of the AMPAR subunit GluA1 in non-permeabilized neurons (Fig. 4A.i). We targeted GluA1 as we previously dem- onstrated that VPS35 preferentially associates with this AMPAR subunit by coIP in mouse brain lysate [15]. Neither VKI culture had surface GluA1 cluster densities significantly different from WT; however, heterozygous neurons had significantly fewer clusters than homozy- gous (Fig. 4A.ii; Kruskal–Wallis p < 0.009). Thus mEPSC frequency changes, at least in heterozygous neurons, likely reflect an increase in Pr of glutamate at excitatory synapses. Surface GluA1 cluster intensity was higher in both mutants, and significantly so for heterozygous f The LRRK2 kinase substrate Rab10 is involved in actin dynamics at recycling endosomes [63], transport of ret- romer cargoes including GLR-1 [64–68], and can be co- purified with synaptic vesicles [69]. Given that pRab10 was increased in mutant cultures (Fig. 3) and that MLi-2 affected surface AMPARs (Fig. 4), we analyzed dendritic Rab10 signals and colocalization with GluA1 in cultured cortical neurons at DIV21 by immunocytochemistry (Additional file 1: Fig. S9A.i). We found Rab10 clusters Kadgien et al. Mol Brain (2021) 14:143 Page 10 of 20 Fig. 4 Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p < 0.009; Uncorrected Dunn’s Het-Ho p < 0.05). There was a significant genotype effect on surface GluA1 cluster intensity (synaptic GluA1) due to significant increases in heterozygous cells (iii, Kruskal–Wallis p < 0.007; Uncorrected Dunn’s Het *p < 0.02). B GFP-filled cultured cortical neurons from WT mice treated with saline or Captisol and immunostained for MAP2 (blue), GFP (cyan), and extracellular GluA1 (magenta) without permeabilization. Surface GluA1 is increased basally in VKI and increased in WT by LRRK2 kinase inhibition 4 Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p < 0.009; Uncorrected Dunn’s Het-Ho p < 0.05). There was a significant genotype effect on surface GluA1 cluster intensity (synaptic GluA1) due to significant increases in heterozygous cells (iii, Kruskal–Wallis p < 0.007; Uncorrected Dunn’s Het p < 0.02). B GFP-filled cultured cortical neurons from WT mice treated with saline or Captisol and immunostained for MAP2 (blue), GFP (cyan), and extracellular GluA1 (magenta) without permeabilization. There were no effects of Captisol treatment on surface GluA1 cluster density (ii, Unpaired t-test p = 0.87) or intensity (iii, Mann–Whitney p = 0.73). C Non-permeabilized cultured cortical cells immunostained for MAP2 (blue), and surface GluA1 (magenta) following acute MLi-2 or vehicle treatment (i). Changes to GluA1 surface density after treatment showed a significant genotype effect due to increases in WT cells not observed in heterozygous and homozygous mutant cells (ii, Welch’s ANOVA p < 0.0002; Unpaired t with Welch’s correction WT-Het p < 0.003; WT-Ho ***p < 0.0001). There was a significant genotype effect on change in surface GluA1 intensity following treatment, due to opposing increases in heterozygous cells and decreases in WT and homozygous cells (iii, Kruskal–Wallis p < 0.02; WT-Het p < 0.02; Het-Ho p < 0.02) GluA1 cluster density that may account for the less-pro- nounced increase to synaptic transmission we observed. MLi-2 treatment had the greatest effect on post-synaptic AMPAR expression in WT cells, through elevated deliv- ery of GluA1-containing AMPARs to AMPAR-lacking silent synapses (increased mEPSC frequency and sur- face GluA1 cluster density with minimal changes to synapse density). This effect was absent in mutant cells. The abundance of GluA1 in existing AMPAR-containing synapses, as measured by surface GluA1 cluster intensity, was altered by MLi-2 treatment in a genotype-dependent manner. The clearest change was an increase in synaptic GluA1 in heterozygous neurons, which are genetically the most relevant to human VPS35 mutation carriers. were present along dendrites, but sparse compared to other dendritic endosome markers. Surface GluA1 is increased basally in VKI and increased in WT by LRRK2 kinase inhibition We saw increased Rab10 cluster density in both mutants that strongly trended to significance (Additional file 1: Fig. S9A.ii); however, GluA1-Rab10 were poorly colocalized (Addi- tional file 1: Fig. S9A.i; Pearson’s 0.17) and there were no significant genotype effects on co-cluster density or Pear- son’s coefficients (Additional file 1: Fig. S9A.ii–iii). The data demonstrate that Rab10 clusters may be increased along VKI dendrites, but do not provide any evidence that Rab10 is involved in local surface delivery or recy- cling of AMPARs within dendrites. Altogether the data demonstrate an endogenous VPS35 D620N mutation produces a gain-of-function in pre- and post-synaptic glutamate transmission. Synaptic surface AMPAR expression (increased surface GluA1 cluster intensity and mEPSC amplitude) and the Pr of glutamate at excitatory synapses (increased mEPSC frequency with no change in synapse or surface GluA1 cluster density) are increased. Homozygous neurons had an additional phenotype of reduced VGluT1 intensity and dendritic Surface GluA1 is increased basally in VKI and increased in WT by LRRK2 kinase inhibition There were no effects of Captisol treatment on surface GluA1 cluster density (ii, Unpaired t-test p = 0.87) or intensity (iii, Mann–Whitney p = 0.73). C Non-permeabilized cultured cortical cells immunostained for MAP2 (blue), and surface GluA1 (magenta) following acute MLi-2 or vehicle treatment (i). Changes to GluA1 surface density after treatment showed a significant genotype effect due to increases in WT cells not observed in heterozygous and homozygous mutant cells (ii, Welch’s ANOVA p < 0.0002; Unpaired t with Welch’s correction WT-Het p < 0.003; WT-Ho **p < 0.0001). There was a significant genotype effect on change in surface GluA1 intensity following treatment, due to opposing increases in heterozygous cells and decreases in WT and homozygous cells (iii, Kruskal–Wallis p < 0.02; WT-Het p < 0.02; Het-Ho p < 0.02) Fig. 4 Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p < 0.009; Uncorrected Dunn’s Het-Ho p < 0.05). There was a significant genotype effect on surface GluA1 cluster intensity (synaptic GluA1) due to significant increases in heterozygous cells (iii, Kruskal–Wallis p < 0.007; Uncorrected Dunn’s Het p < 0.02). B GFP-filled cultured cortical neurons from WT mice treated with saline or Captisol and immunostained for MAP2 (blue), GFP (cyan), and extracellular GluA1 (magenta) without permeabilization. There were no effects of Captisol treatment on surface GluA1 cluster density (ii, Unpaired t-test p = 0.87) or intensity (iii, Mann–Whitney p = 0.73). C Non-permeabilized cultured cortical cells immunostained for MAP2 (blue), and surface GluA1 (magenta) following acute MLi-2 or vehicle treatment (i). Changes to GluA1 surface density after treatment showed a significant genotype effect due to increases in WT cells not observed in heterozygous and homozygous mutant cells (ii, Welch’s ANOVA p < 0.0002; Unpaired t with Welch’s correction WT-Het p < 0.003; WT-Ho ***p < 0.0001). There was a significant genotype effect on change in surface GluA1 intensity following treatment, due to opposing increases in heterozygous cells and decreases in WT and homozygous cells (iii, Kruskal–Wallis p < 0.02; WT-Het p < 0.02; Het-Ho p < 0.02) Fig. Discussion Previous reports of VPS35 neurobiology at glutamater- gic synapses, and the effects of the D620N mutant, have relied on exogenous protein expression or knock-out, and produced somewhat conflicting results. Studies on retrograde trafficking of the canonical retromer cargo Kadgien et al. Mol Brain (2021) 14:143 Kadgien et al. Mol Brain (2021) 14:143 Page 11 of 20 (Fig. 5). The reduced association of VPS35 with FAM21 observed in brain tissue may be relevant for other ret- romer cargoes/trafficking pathways in neuronal soma, or in non-neuronal cell types. cation-independent mannose-6-phosphate receptor (CI- MPR), for example, concluded that the mutation results in a loss-of-function [15, 44, 46, 47] or has no effect [45, 70]. This discordance may result from the variety of cell types and modes of expression used to glean insights. We previously reported that exogenous WT VPS35 expression in mouse cortical neurons resulted in a slight decrease in mEPSC amplitudes compared to control, whereas mutant expression resulted in a slight increase. The result was a significant increase in mEPSC ampli- tudes in neurons expressing mutant D620N protein [15]. The mutation also reduced motility of exogenous GFP- tagged VPS35 in dendrites and its localization to spines— results we interpreted at the time as potentially indicative of a loss-of-function [15]. The only other study to date on the effect of D620N on AMPAR trafficking found exog- enous D620N expression in hippocampal neurons from haploinsufficient mice was unable to rescue impairments in LTP [21]. cation-independent mannose-6-phosphate receptor (CI- MPR), for example, concluded that the mutation results in a loss-of-function [15, 44, 46, 47] or has no effect [45, 70]. This discordance may result from the variety of cell types and modes of expression used to glean insights. We previously reported that exogenous WT VPS35 expression in mouse cortical neurons resulted in a slight decrease in mEPSC amplitudes compared to control, whereas mutant expression resulted in a slight increase. The result was a significant increase in mEPSC ampli- tudes in neurons expressing mutant D620N protein [15]. The mutation also reduced motility of exogenous GFP- tagged VPS35 in dendrites and its localization to spines— results we interpreted at the time as potentially indicative of a loss-of-function [15]. The only other study to date on the effect of D620N on AMPAR trafficking found exog- enous D620N expression in hippocampal neurons from haploinsufficient mice was unable to rescue impairments in LTP [21]. Discussion To date, little is known about retromer in presynap- tic physiology. Retromer is highly motile in axons, and present in glutamatergic synaptic boutons in cultured murine neurons [15, 22], but retromer deficiency or acute knock-down in murine hippocampal slices has lit- tle or no effect on synaptic glutamate release [16, 21], or SV endo- or exocytosis [22]. We previously found expres- sion of both WT and mutant VPS35 in cortical neu- rons resulted in fewer excitatory synapses and reduced frequency of mEPSCs, but there were no clear mutant- specific effects on frequency, and only a trend toward synapse density differences in mutant-expressing cells [15]. It is important to note this was a fairly low expres- sion level, on the background of WT protein. Therefore, asides our observations of increased mEPSC frequency in VPS35 mutants here, there is little other evidence to date for retromer having a critical role in glutamate release per se. VPS35 associates with presynaptically expressed transmembrane proteins such as D2R (present study) and dopamine transporter (DAT; 73), and dopamine release is increased in ex vivo slices from 3-month-old VKI mice alongside changes in expression of DAT and vesicular monoamine transporter 2 (VMAT2; 48); thus it may impact upon SV release indirectly by participating in the sorting and recycling or retrograde transport of old synaptic vesicle proteins, presynaptic channels, and/or receptors (Fig. 5). Whether retromer is involved in trans- mitter release directly, or indirectly by regulating axonal transport and/or recycling of synaptic proteins that mod- ulate release, requires further study. Having found endogenous expression of VPS35 D620N increases surface GluA1 expression and mEPSC ampli- tude in cortical neurons from VKI, we conclude this may be a gain-of-function effect which is only evident with endogenous total levels of VPS35. Overexpression of VPS35 has similar effects as haploinsufficiency or knock-down on neurite outgrowth and synapse density [15–17, 19, 20, 24, 71]. This implies that overexpressing VPS35 may have a dominant-negative effect on some of its functions. An emerging theme in the literature is that changing ratios of VPS35 to its multiple binding partners can shift its function, thus it follows that its function is sensitive to both under and overexpression. Our work here underscores the importance of endogenous protein expression levels when assaying the physiological func- tion of VPS35 and any mutant effects. Discussion Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer Fi 5 W ki d l f i d f AMPAR i i D62 heterozygous mutants In wild-type cells postsynaptic VPS35 traffics Fig. 5 Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [25]; however, in mammalian neurons it localizes to only a subset of terminals [22] and retromer deficiency has no effect on SV endo- or exocytosis [22] or neurotransmitter release [16, 21]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer Previous work in LRRK2 knock-out mice has shown that LRRK2 negatively regulates protein kinase A (PKA)- mediated AMPAR insertion, can alter PKA traffick- ing in and out of spines, and that pathogenic mutations in LRRK2 increase PKA-mediated phosphorylation of GluA1 [36]. While Parisiadou and colleagues [36] found that forskolin-induced phosphorylation of GluA1 by PKA was resistant to LRRK2 kinase inhibition, they did not assay the effect of LRRK2 inhibition on endogenous GluA1 phosphorylation nor surface expression. Discussion While much remains to be discovered about a presyn- aptic role for VPS35, it is noteworthy that PD-associated mutations in LRRK2 cause similar increases in gluta- mate and dopamine release [30, 35, 74, 75] in addition to impinging upon several post-synaptic processes [36– 38]. Pathogenic mutations in LRRK2 clearly increase its kinase function [39, 41, 76], resulting in increased phos- phorylation of several LRRK2 substrates including a large subset of Rab-GTPases that have roles in the SV cycle and postsynaptic AMPAR traffic [41, 42, 77]. Previous studies have shown that acute (30 min) treatment with LRRK2 kinase inhibitors is sufficient to reverse trans- mitter release and post-synaptic phenotypes in LRRK2 G2019S mice [35, 37]. f A reduction in the association of FAM21 with VPS35 D620N has been proposed elsewhere as a mechanism for mutation effects on retrograde trafficking of CI-MPR [44, 47] and autophagy [45]. While we show here (for the first time) that this reduced association also occurs in whole brain lysates of endogenous knock-in animals, this impairment phenotype is not obviously related to GluA1 trafficking. A recent study showed that reduced association of VPS35 D620N with FAM21 can be rescued by TBC1D5 knock-down [72], thus the loss of association is likely not due to an inherent loss of VPS35’s ability to bind FAM21 in the presence of the mutation. Further- more, loss of FAM21 by knock-down has been shown to cause misdirection of surface-bound retromer cargoes to the TGN, reducing their surface expression [53]. Our results are suggestive of increased retromer association with FAM21 in dendrites, and increased surface traffick- ing of GluA1 in VKI pyramidal cortical neuron dendrites Our replication of the previous report of D620N muta- tion resulting in LRRK2 kinase-dependent hyperphos- phorylation of Rab10 [27] led us to hypothesize that glutamate transmission phenotypes in VKI would be reversed by acute LRRK2 kinase inhibition. The results Kadgien et al. Mol Brain (2021) 14:143 Page 12 of 20 Fig. 5 Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [25]; however, in mammalian neurons it localizes to only a subset of terminals [22] and retromer deficiency has no effect on SV endo- or exocytosis [22] or neurotransmitter release [16, 21]. VPS35 D620N knock‑in mice and genotyping VPS35 D620N knock in mice and genotyping Constitutive VPS35 D620N knock-in mice (VKI) were generated by Ozgene (Australia) under guidance of Dr. Matthew Farrer using gene targeting in C57Bl/6 embryo stem cells (Bruce4) as previously described [48]. The VKI strain has been deposited in Jackson Labs with open dis- tribution supported by the Michael J Fox Foundation (VPS35 knock-in: B6(Cg)-Vps35tm1.1Mjff/J). All mice were bred, housed, and handled according to Canadian Council on Animal Care regulations. All procedures were conducted in accordance with ethical approval cer- tificates from the UBC ACC (A16-0088; A15-0105) and the Neuro CNDM (2017-7888B). Animals were group- housed in single-sex cages with littermates after weaning. The procedure for creating the VKI resulted in 51 base- pair insertion in the non-coding regions of VPS35, such that PCR amplification of the WT gene using appropri- ate primers creates a product of 303 base pairs and the knock-in gene a product of 354 base pairs. The animals were genotyped by PCR amplification of VPS35, followed by confirmation of the presence of a 303 bp product (WT), a 354 bp product (Ho) or both (Het). Small tissue samples were digested in 100 uL 10% Chelex (Bio-Rad 142–1253) at 95 ℃ for 20 min and spun down to result in DNA-containing supernatant. 2uL DNA was mixed with 18uL of master mix containing taq polymerase, buffer (DNAse- and RNAse-free water, 10 × buffer, MgCl 25 mM, dNTPs 10 mM), and primers (ThermoFisher Custom DNA oligos: forward-TGGTAGTCACATTGC CTCTG; reverse-ATGAACCAACCATCAATAGGA Constitutive VPS35 D620N knock-in mice (VKI) were generated by Ozgene (Australia) under guidance of Dr. Matthew Farrer using gene targeting in C57Bl/6 embryo stem cells (Bruce4) as previously described [48]. The VKI strain has been deposited in Jackson Labs with open dis- tribution supported by the Michael J Fox Foundation (VPS35 knock-in: B6(Cg)-Vps35tm1.1Mjff/J). All mice were bred, housed, and handled according to Canadian Council on Animal Care regulations. All procedures were conducted in accordance with ethical approval cer- tificates from the UBC ACC (A16-0088; A15-0105) and the Neuro CNDM (2017-7888B). Animals were group- housed in single-sex cages with littermates after weaning.h yf p g For traditional chemiluminescence detection, 10–15 µg protein was prepared in 4× NuPage LDS sample buffer (Invitrogen NP0008) with 2.5% β-mercaptoethanol or 500 mM DTT to a total volume of 10–24 uL, and dena- tured at 70 °C for 10 min. Discussion Here we show that LRRK2 kinase activity negatively regulates traf- ficking of GluA1-containing AMPARs to silent synapses in developing neurons, yet has no effect on GluA1 surface expression in mutant neurons. This could be indicative of accelerated synapse maturation in mutant cells beyond a LRRK2-sensitive time-point. While outside the scope of the current study, further inquiry into the time course of synapse formation and maturation in VKI neurons, and suggest the mechanism of glutamate dysregulation in VKI cultures is distinct from that in LRRK2 G2019S mutant mice (in which glutamate release phenotypes are reversed within 30 min of LRRK2 kinase inhibition; 35,37). It is possible VKI phenotypes are unrelated to increased LRRK2 kinase activity or Rab10 phosphoryla- tion state; however, it remains possible that longer-term inhibition will render different results. LRRK2 localizes to endosomes [78], co-immunoprecipitates with VPS35 in brain lysates (in the present study and elsewhere [24, 28]), and has been shown to interact functionally with VPS35 in neurons [24, 25]. Thus, it remains a possibil- ity that LRRK2 is involved in the dysregulation of gluta- mate synapses in VKI through one of its other functional domains (e.g., structural scaffolding or GTPase activity). Future investigations of the contributions of the other functional domains of LRRK2 in VPS35 D620N synaptic phenotypes may prove illuminating. Kadgien et al. Mol Brain (2021) 14:143 Page 13 of 20 Page 13 of 20 the role of LRRK2 and its kinase activity in PKA-depend- ent and -independent AMPAR trafficking, is warranted. ACAC) according to the instructions for the taq poly- merase kit (Qiagen 201203), and the PCR was performed in a programmable machine (program available upon request). Agarose gel electrophoresis was used to sepa- rate the products on a 3–4% gel with fluorescent DNA dye (ZmTech LB-001G) and visualized on a Bio-Rad ultraviolet gel imager. fi To our knowledge this is the first study of gluta- matergic neuron biology in a knock-in model of VPS35 D620N parkinsonism. We found that the D620N muta- tion results in increases to glutamate release similar to LRRK2 G2019S, with an additional post-synaptic phe- notype of increased GluA1 surface expression (Fig. 5). LRRK2 kinase inhibition reversed pRab10 increases in mutant brain and cultures, but did not reduce postsyn- aptic AMPAR expression in heterozygous cells (that are most relevant to human PD), or mutant Pr; however, LRRK2 inhibition increased forward traffic of GluA1 to silent synapses in WT. Discussion This suggests caution is neces- sary in the wider application of LRRK2 kinase inhibitor treatment for PD e.g., non-LRRK2 PD. We add support to the hypothesis that synaptic transmission is augmented at early time points in PD, which potentially represents early pathophysiological processes that can be targeted to prevent transition to later pathological damage (reviewed in: 40,79,80). Western blots and co‑immunoprecipitationsh Three-month-old male mice were decapitated, and brains removed and chilled for 1 min in ice-cold carbogen-bub- bled artificial cerebrospinal fluid (ACSF; 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, pH 7.3–7.4, 300–310 mOsm). For region-specific analysis, this was followed by rapid (< 6 min) microdissection of cortex, striatum, hippocampus, dorsal midbrain, olfactory bulbs, and cerebellum, with all remaining tissue pooled as ‘rest’. Tissues were flash frozen in liquid nitrogen and either lysed for immediate use or stored at −80 ℃. For WES and chemiluminescent western blots, tissues were mechani- cally homogenized in HEPES buffer (20 mM HEPES, 50 mM KAc, 200 mM Sorbitol, 2 mM EDTA, 0.1% Triton X-100, pH 7.2; Sigma Aldrich) containing protease inhib- itor cocktail (Roche 11697498001), then incubated on ice for 45 min with occasional gentle agitation. For fluo- rescent western blots, tissue was homogenized by probe sonication at 20 kHz for 10 s in ice-cold TBS buffer (tris- buffered saline, 1% Triton X-100; pH 7.4) containing pro- tease inhibitor (Roche 11697498001) and phosphatase inhibitor (Sigma 4906845001) cocktails. Protein was quantified by Pierce BCA assay (ThermoFisher 23255) and samples were adjusted to equal concentrations in lysis buffer prior to denaturing. Primary cortical cultures Dames were euthanized by rapid decapitation at embry- onic day 16.5. Pups were microdissected in Hank’s Bal- anced Salt Solution (HBSS; Gibco 14170161) with 1 × penicillin–streptomycin (penstrep; Sigma-Aldrich P4333) in a petri dish on ice. The cortices from each pup were held in 500uL supplemented Hibernate-E medium (Gibco A1247601; 1 × GlutaMax Gibco 35050061; and 1% NeuroCult SM1 StemCell 5711) at 4 ℃ during geno- typing as described above. Genotype-pooled tissue was dissociated chemically for 10 min in 0.05% Trypsin– EDTA (Gibco 25300054), followed by deactivation with 10% FBS, then mechanically dissociated in supple- mented Neurobasal plating medium (Neurobasal Gibco 21103049; 1× GlutaMax Gibco 35050061; and 1% Neuro- Cult SM1 StemCell 5711). Images for both types of blots were background-sub- tracted and analyzed for band intensity with ImageLab software. Signals were normalized to a housekeeping protein quantified from the same gel (GAPDH, β-tubulin, β-actin). All original blots are available in Additional file 2. i Wherever possible, lysates were blotted on a Protein- Simple WES automated capillary-based size sorting sys- tem as previously described [30]. Briefly, lysates were mixed with reducing fluorescent master mix (Protein- Simple SM-001), heated (70 °C for 10 min) and loaded into manufacturer microplates containing primary anti- bodies (see below) and blocking reagent, wash buffer, HRP-conjugated secondary antibodies, and chemilu- minescent substrate (ProteinSimple DM-001/2). WES data was analyzed on manufacturer-provided Compass software. All original data files presented in the style of a western blot membrane are provided in Additional file 2. Cells were plated onto poly-D-lysine-coated plates or coverslips (Sigma P7280) and matured to 21 days in-vitro (DIV21) while incubating at 37 ℃ with 5% CO2. For bio- chemistry, cells were plated at 1 million cells/well in 2 mL on 6-well plates. For immunocytochemistry experiments, cells were plated in 1 mL medium onto no.1.5 glass cov- erslips in 24-well plates. Non-nucleofected cells were plated at 115000 cells/well. For GFP-filled neurons, 1 mil- lion cells were nucleofected with 1 µg pAAV-CAG-GFP plasmid DNA (Addgene 37825) in Ingenio electropora- tion buffer (Mirus MIR50111) using an Amaxa Nucleo- fector2b (Lonza), mixed 1:1 with non-nucleofected cells, and plated at 225 k cells/well in 1 mL medium as above. From DIV4, 10% fresh media was added to all wells every 3–4 days until use. i We used the following primary antibodies: NEEP21/ NSG1 (Genscript A01442), FAM21 (Millipore ABT79), VPS35 (Abnova H00055737-M02), VPS26 (a kind gift from J. VPS35 D620N knock‑in mice and genotyping Samples were loaded into a NuPAge 4–12% Bis–Tris gel (Invitrogen NP0322BOX) in an XCell II Blot module (Invitrogen) and run at 70 V for 30 min, followed by 110 V for 1 h. Separated proteins were transferred to methanol-activated Immobilon-P PVDF membrane (Millipore IPVH00010) for 90 min at 25 V at room temperature, then blocked with 5% milk in PBS for 1 h at room temperature. Membranes were probed by shaking with primary antibodies in pri- mary antibody solution (PBS, 2% BSA, 0.05% Tween- 20) for either 1 h at room temperature or overnight at 4 °C, washed 4 × in PBST (PBS, 0.05% Tween-20), and detected by HRP-conjugated secondary antibodies (Inv- itrogen; shaken in PBS, 5% milk, 0.05% Tween-20 for 30 min at room temperature). Chemi-luminescence was The procedure for creating the VKI resulted in 51 base- pair insertion in the non-coding regions of VPS35, such that PCR amplification of the WT gene using appropri- ate primers creates a product of 303 base pairs and the knock-in gene a product of 354 base pairs. The animals were genotyped by PCR amplification of VPS35, followed by confirmation of the presence of a 303 bp product (WT), a 354 bp product (Ho) or both (Het). Small tissue samples were digested in 100 uL 10% Chelex (Bio-Rad 142–1253) at 95 ℃ for 20 min and spun down to result in DNA-containing supernatant. 2uL DNA was mixed with 18uL of master mix containing taq polymerase, buffer (DNAse- and RNAse-free water, 10 × buffer, MgCl 25 mM, dNTPs 10 mM), and primers (ThermoFisher Custom DNA oligos: forward-TGGTAGTCACATTGC CTCTG; reverse-ATGAACCAACCATCAATAGGA Kadgien et al. Mol Brain (2021) 14:143 Page 14 of 20 Page 14 of 20 detected with Pierce ECL (ThermoFisher 32209) imaged on a Chemi-Doc imaging system (Cell-Bio). aliquots of each lysate were set aside before IP to verify the equivalence of starting concentrations. 24 h later, loaded beads were washed with ice-cold lysis buffer (×3) prior to resuspension in reducing 1× NuPage LDS sam- ple buffer (for traditional; Invitrogen NP0008) or Protein- Simple fluorescent master mix (for WES; ProteinSimple SM-001). Protein was eluted and denatured by heating at 70 °C for 10 min prior to western blotting as described above. VPS35 D620N knock‑in mice and genotyping Fluorescence western blots were performed with the following modifications to the above protocol: (1) 40 ug protein samples; (2) Bolt 4–12% Bis–Tris Plus Gels (Inv- itrogen NW04120BOX); (3) Fluorescence detection optimized membrane, Immobilon-FL PVDF (Millipore IPFL00010); (4) TBS was substituted for PBS in all buff- ers to prevent possible cross-reaction of phospho-spe- cific antibodies; (5) transfer increased to 2.5 h for larger proteins (LRRK2), and; (6) LiCor fluorescent secondary antibodies were used (LI-COR) and imaged on a LI-COR Odyssey Infrared imaging system (LI-COR). Antibody specificity was tested using lysate from LRRK2 knock-out mouse brain or Rab10 knock-out AtT20 cell lysates. The LRRK2 knock-out mice have been previously described by Hinkle and colleagues [81]. Rab10 knock-out AtT30 cells were a kind gift from Dr. Peter McPherson, and have been previously described [67]. When necessary, phosphorylated or low-abundance proteins were blotted first, and membranes were stripped using LI-COR NewBlot IR stripping buffer (LI-COR 928-40028; 30 m at a time until no signal remained from the first blot), then reblotted for non-phosphorylated or higher abundance proteins of similar sizes. Primary cortical cultures Bonifacino, NICHD), GluA1 (Millipore 05-855R), D2R (Millipore AB5084P), GluN1 (Millipore 05–432), LRRK2 (Abcam ab133474), LRRK2 phos- phoS935 (Abcam ab133450), Rab10 phosphoT73 (Abcam ab230261), Rab10 (Abcam ab104859), VGluT1 (Mil- lipore AB5905), β-tubulin (Covance MRB-435P), β-Actin (Abcam ab6276), and GAPDH (Cell Signaling 2118; ThermoFisher MA5-15738). h For co-immunoprecipitation, 500 µg of protein at 1 µg/ µL was rotary-incubated overnight at 4 °C with VPS35 antibody (Abnova H00055737-M02) or Mouse IgG2a control antibody (Abcam ab18414) coupled to M-280 Tosyl-activated Dynabeads (Invitrogen 14204). Small In vitro electrophysiology Wh l ll h l Whole-cell patch-clamp recordings were performed on cortical cells at DIV18-22. Neurons were perfused at room temperature with extra-cellular solution (167 mM NaCl, 2.4 mM KCl, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 1 mM CaCl2, 1 µM tetrodotoxin and 100 µM pic- rotoxin, pH 7.4, 290–310 mOsm). In MLi-2 experiments, ECS was supplemented with 500 nM MLi-2 in 45% Cap- tisol® PBS or an equal volume of 45% Captisol® PBS.i Pipettes were filled with intracellular solution (130 mM Cs-methanesulfonate, 5 mM CsCl, 4 mM NaCl, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 5 mM QX-314, 0.5 mM GTP, 10 mM Na2-phosphocreatine, 5 mM MgATP, and 0.1 mM spermine, at pH 7.3 and 290 mOsm). Pipette resistance was constrained to 3–7 MOhms for recording. Recordings were acquired by a Multiclamp 700B amplifier in voltage clamp mode at Vh -70 mV, signals were filtered at 2 kHz, digitized at 10 kHz. The membrane test function was used to determine intrinsic membrane properties 1–2 min after obtaining whole-cell configuration, as described previ- ously [15, 83–85]. i Cells having a pyramidal morphology—triangular or teardrop shaped cell bodies with spiny, clearly identifiable apical and basal dendrites [82]—were selected for imag- ing. All images were blinded and randomized prior to processing and analysis. Cell density counts were performed manually on DAPI and MAP2 co-labeled images acquired at 10× with an Olympus Fluoview 1000 confocal microscope. Sholl anal- ysis images were acquired at 20× with an Evos FL epif- luorescence microscope. Dendritic neurites (excluding axons) were traced and analyzed using the Simple Neur- ite Tracer plugin for FIJI ImageJ using a radial segmenta- tion of 5 µm. Tolerance for series resistance was < 28 мOhm and uncompensated, and recordings discarded if Rs changed by 10% or more. mEPSC frequency and amplitudes were analyzed with Clampfit 10 (Molecular Devices) with a detection threshold of 5pA and followed by manual con- firmation of all accepted peaks; non-unitary events were suppressed from amplitude analysis but retained for frequency.l Images for colocalization were acquired on either an Olympus Fluoview 1000 confocal microscope, with images taken at 60× with 2× optical zoom, in 0.5 µm stacks, or a Zeiss Axio Observer with Apotome.2 struc- tured illumination upon which images were taken at 63× in 0.25 um stacks. Z-stack acquisition was set to capture all MAP2 stained dendrite for unfilled cells, or GFP-filled dendrite for filled cells. Immunocytochemistry and imaging analysisi Cultured cortical neurons were fixed at DIV21 (4% PFA, 4% sucrose in PBS, 5–10 min), permeabilized where appropriate (ice-cold methanol, 3 min), and blocked (5% Kadgien et al. Mol Brain (2021) 14:143 Page 15 of 20 goat serum in PBS) prior to incubation with primary antibodies for 1 h at RT or overnight at 4 ℃. Primary antibodies were prepared in antibody solution (2% goat serum, 0.02% Tween20 in PBS). For surface labelling, the cells were not permeabilized until after primary antibody incubation and no detergent was added to primary anti- bodies. Proteins were fluorescently labeled with Alexa- conjugated secondary (Invitrogen) in antibody solution for 30 min (RT), and coverslips were slide mounted with Prolong Gold (Invitrogen P36930). automated pipelines in CellProfiler (www.cellprofiler.org; pipelines available upon request). Briefly, the pipeline first uses the dendrite mask to restrict all further analy- ses to the masked area. From there, the binary masks of clusters are used for the size, density, and colocalization densities within dendrites. Dendrite-masked greyscale images are used for Pearson’s coefficients, and greyscale images are overlaid with the cluster masks to meas- ure intensity within clusters inside the dendritic region selected for analysis. We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Milli- pore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Mil- lipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Mil- lipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009). Rab10 specificity was tested using Rab10 knock-out AtT30 cells (a kind gift from Dr. Peter McPherson; described in [67]) and cultured cortical neu- rons (Additional file 1: Fig. S8). In vitro electrophysiology Wh l ll h l Acquisition param- eters were constrained within each culture set. Z-stacks for each channel were flattened using the max projection function in FIJI. MAP2 or GFP stains were used to mask dendrites after their first branch point; primary dendrites and cell bodies were excluded from masks. Areas of the dendritic arbor with many intersecting neurites from other cells were excluded from analyses. Images were manually thresholded to create binary masks of clusters. Cluster densities, intensities, areas, colocalization densi- ties, and Pearson’s coefficients were all calculated using Peak-scaled nonstationary fluctuation analysis was performed on unitary events from each recording in the following way: events were aligned by peak ampli- tude, baselines adjusted, and all events normalized to −1 at their maximum amplitude. Event amplitudes and variance from the mean at each recording interval were calculated using the built-in NSFA plugin in Clampfit 10 (Molecular Devices), then rescaled to pre-normalization values. Mean–variance plots were made in GraphPad Prism using values from the event peak to 5pA above baseline (due to baseline noise), and fit using the least Kadgien et al. Mol Brain (2021) 14:143 Page 16 of 20 Page 16 of 20 squares method and the second order polynomial func- tion representing the following equation: and drug effect comparisons (3 groups) when the data failed the normality test (alpha < 0.05), nonparamet- ric tests were used (Kruskal–Wallis with uncorrected Dunn’s post-test). If the data passed the normality test, (alpha > 0.05), a parametric test was chosen. When the SDs were not significantly different, a one-way analy- sis of variance (ANOVA) with uncorrected Fisher’s LSD post-test was used. If the SDs were significantly differ- ent, Welch’s ANOVA and an unpaired t-test with Welch’s correction post-test was used. In the event that the n was too low for normality testing, nonparametric tests were used. For Captisol® tests (2 groups), the tests cho- sen were appropriate for two groups (Mann–Whitney for nonparametric and unpaired two-tailed t-test for para- metric). MLi-2 treatment data was analyzed using 2-way ANOVA (2-way ANOVA) with uncorrected Fisher’s LSD post-tests. δ2 = iI −I2 N + δ2 b where δ2 = variance, i = single channel current, I = mean current, δ2 b = background variance as previously described by others [86–89]. For conventional NSFA, N = number of open channels at peak current; however the process of peak-normalizing required to analyze mEPSCs renders this value arbitrary. Abbreviations AMPA: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; Capti- sol®: Sulfobutylether-β-cyclodextrin; CI-MPR: Cation-independent mannose- 6-phosphate receptor; coIP: Co-immunoprecipitation; D2R: D2-type dopamine receptor; DAT: Dopamine transporter; FAM21: Family with sequence similarity 21; LRRK2: Leucine-rich repeat kinase 2; LTP: Long-term potentiation; mEPSC: Miniature excitatory post-synaptic current; NEEP21: Neuronal endosomal enriched protein 21; NMDA: N-methyl-D-aspartate; NSFA: Non-stationary fluc- tuation analysis; PD: Parkinson’s disease; PKA: Protein kinase A; Pr: Probability of release; PSD95: Post-synaptic density protein 95; RE: Recycling endosome; SV: Synaptic vesicle; TGN: trans-Golgi network; VGluT1: Vesicular glutamate transporter 1; VKI: VPS35 D620N knock-in; VMAT2: Vesicular monoamine trans- porter 2; VPS26: Vacuolar protein sorting 26; VPS35: Vacuolar protein sorting 35; WASH: Wiskott–Aldrich syndrome protein and SCAR homolog. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13041-021-00848-w. The online version contains supplementary material available at https://doi. org/10.1186/s13041-021-00848-w. Additional file 1: Fig. S1. Retromer protein levels not altered in VKI. A) Co-immunoprecipitation performed in VKI whole brain lysate pulling with FAM21 antibody and blotted on a WES capillary-based western blotting system shows VPS35 association with FAM21 in brain tissue. B) WES capillary-based western blot of VPS35, VPS26, FAM21, and β-tubulin in VKI whole-brain lysate (i) revealed no significant genotype effects on levels of VPS35 (ii, 1-way ANOVA p = 0.97), VPS26 (iii, Kruskal–Wallis p = 0.73), or WASH complex member FAM21 (iv, 1-way ANOVA p = 0.88). Fig. S2. Neuronal cargo and LRRK2 binding are not altered i n VKI. A) Western blot of striatal lysates and co-immunoprecipitates from 3-month-old VKI mice (pulling with VPS35 antibody) were probed for VPS35, GluN1, D2R, GluA1, LRRK2, and GAPDH (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii–iii, Kruskal–Wallis p = 0.97; p = 0.13, respectively); GluN1 levels or coIP (iv–v, Kruskal–Wallis p = 0.51; p = 0.42, respectively); LRRK2 kinase inhibition with MLi2 treatment We inhibited LRRK2 kinase activity with the selective LRRK2 inhibitor MLi-2 (Tocris 5756). MLi-2 has low solubility in water, necessitating the use of a vehicle for solubilization. We chose to use the cyclodextrin Capti- sol® (Ligand RC-0C7-100) as a vehicle, due to its world- wide safety approval and use in human drug formulations (www.captisol.com/about). We bath sonicated 1 mg MLi-2 in 2 mL 45% Captisol®-PBS for ~ 2 h at room tem- perature (until complete solubilization of MLi-2). Solu- tions were filter sterilized prior to use. Primary cortical cultures were treated with 500 nM MLi-2 or Captisol®- only control (each 1 mL well treated with 0.4 uL stock in 100 mL fresh media; final Captisol® concentration 0.00016%) for 2 h prior to fixation, lysis, or electro- physiological recording. Treatment concentrations and times were selected based on the pharmacokinetic data collected by Fell and colleagues [50]. For western blot experiments in brain tissue, animals were injected intra- peritoneally with MLi-2 or Captisol®-only control at a dose of 5 mg/kg, 2 h prior to rapid decapitation without anaesthesia. All tissues were collected, frozen, and stored as described above. In vitro electrophysiology Wh l ll h l Weighted single- channel conductance was calculated by the following equation: γ = i/(Vm −Erev) where γ = weighted single-channel conductance, i = sin- gle channel current, Vm = holding potential (−70 mV), and Erev = reversal potential for AMPAR current (0 mV in our ECS). Recordings were rejected if the best-fit curve had an R2 < 0.5. Sample sizes were selected according to generally accepted standards in the field. No power analyses were conducted to predetermine sample sizes. Data is repre- sented as scatter plots of all data points with mean and standard error of the mean. Sample sizes represent bio- logical replicates. Where n = x(y), x = number of cells imaged/recorded, y = number of independent cultures. Data visualization and statistics All statistical analyses and data visualizations were con- ducted in GraphPad Prism 8. Because biological data is prone to lognormal distribution, outliers were only removed if inspection revealed that they resulted from human error. Data sets were analyzed for normality using the D’Agostino & Pearson test. In untreated experiments Kadgien et al. Mol Brain (2021) 14:143 Page 17 of 20 not colocalize strongly with GluA1 in cortical neurites. A) GFP-filled (blue) cortical neurons immunostained for Rab10 (magenta), and GluA1 (cyan) (i). Rab10 cluster density was increased in both mutant genotypes, falling just shy of statistical significance (ii, Kruskal–Wallis p < 0.06). There was no effect of genotype on co-cluster density or Pearson’s coefficient (iii–iv, 1-way ANOVA p = 0.24; Kruskal–Wallis p = 0.56, respectively). B) Knock- out testing of specificity of Rab10 antibody for immunocytochemistry. Cultured cortical neurons and Rab10 knock-out AtT30 cells were stained by immunocytochemistry for Rab10, demonstrating punctate staining in the cortical neuron that is absent from the knock-out cells. D2R levels or coIP (vi–vii Kruskal–Wallis p = 0.70; p = 0.45, respectively); GluA1 levels or coIP (viii–ix, Kruskal–Wallis p = 0.83; p = 0.44, respectively); or LRRK2 levels or coIP (x–xi, Kruskal–Wallis p > 0.99; p = 0.40, respectively). Fig. S3. Phospho-LRRK2 is increased in VKI and MLi-2 does not alter pro- tein levels. A) Western blot of LRRK2 pS935, LRRK2, and β-actin (i) revealed no genotype effect on LRRK2 expression levels (ii, Kruskal–Wallis p = 0.09). LRRK2 pS935 levels were significantly altered, due to an increase in phos- phorylation in homozygous tissue (iii, 1-way ANOVA p < 0.03; Uncorrected Fisher’s LSD p < 0.02). B.i) Whole brain lysates from VKI animals after acute LRRK2 kinase inhibition were blotted for LRRK2, GluA1, VPS35, VGluT1, Rab10, and β-actin (loading control). There were no significant effects of genotype or treatment on levels of LRRK2 (ii, 2-way ANOVA genotype x treatment p = 0.93; genotype p = 0.90; treatment p = 0.24), GluA1 (iii, 2-way ANOVA interaction p = 0.45; genotype p = 0.61; treatment p > 0.99), VPS35 (vi, 2-way ANOVA interaction p = 0.23; genotype p = 0.94; treat- ment p = 0.89), VGluT1 (vii, 2-way ANOVA interaction p = 0.55; genotype p = 0.39; treatment p = 0.69), or Rab10 (iv, 2-way ANOVA genotype x treat- ment p = 0.5258; genotype p = 0.4683; treatment p = 0.9659). Data visualization and statistics For Bii-vi, WTCap n = 5, WTMLi2 n = 6, HetCap n = 6, HetMLi2 n = 6, HoCap n = 5, HoMLi2 n = 5. Fig. S4. Captisol does not affect protein levels. A) Further analysis of blots from Fig. 1. There were no significant genotype effects on the level of LRRK2 (i; Mann–Whitney p > 0.99) or Rab10 (ii; Mann–Whitney p = 0.89). B) Western blots of WT brain lysate following acute treatment with Captisol or saline, probed for GluA1, VPS35, VGluT1, and β-actin (loading control). There were no significant effects of Captisol treat- ment on protein levels of VPS35, GluA1, or VGluT1 (ii–iv; Mann–Whitney p = 0.49; p > 0.99; p = 0.49, respectively). Fig. S5. Cell density and dendritic morphology are not altered in VKI. A) Cortical cells were nucleofected with CAG-AAV-GFP plasmids on the day of plating and fixed at DIV21. GFP signal was amplified and imaged (top panel), then 2D in silico cell reconstruction performed in ImageJ (bottom panel). B) There was no effect of genotype on neuron density, indicating equivalent survival and no cell death (1-way ANOVA p = 0.47). C) Sholl analysis revealed no significant effect of genotype upon neurite complexity (2-way RM ANOVA radial distance x genotype interaction p = 0.87; genotype p = 0.78). D-F) There were also no genotype effects on total branch number, average branch length, or maximum branch length (Kruskal–Wallis p = 0.79, 0.34 & 0.29, respectively). Fig. S6. VGluT1 cluster intensity reduced in VKI and channel kinetics not affected. A) Supplemental analysis from untreated cortical cell culture synapse staining presented in Fig. 3. PSD95 and VGluT1 densities were not altered by genotype (i-ii, Welch’s ANOVA p = 0.29 & 0.42, respectively). There was a genotype effect on VGluT1 cluster intensity, due to significant reductions in homozygous cells only (iii, Kruskal–Wallis p < 0.007; Uncorrected Dunn’s p < 0.003). B) Supplemental analysis of whole-cell patch clamp recordings from cultured cortical cells presented in Fig. 3. Mean mEPSC decay times (τ) were not affected by genotype (i, Kruskal–Wallis p = 0.74). Peak-scaled non-stationary noise analysis was performed by plotting the mean variance of traces from the recording average amplitude (ii, representative mean–variance plots); the best fit curve allows for the calculation of weighted single channel conductance of the synapses involved in each recording. Availability of data and materials All data generated or analyzed during this study are included in this published article (and its Additional files 1, 2). Funding AJM was supported by a Parkinson Canada New Investigator Award, and is an FRQS Investigator and Killam Scholar. CK held a Parkinson Canada Graduate Scholarship. The work was supported by a CIHR operating grant to AJM & Matthew J Farrer (Molecular neuroscience of Parkinson’s Disease: Retromer (VPS35) dysfunction 2012–2017) and the Canada Fund For Innovation (AJM). The funding bodies had no input to the design of the study, collection, analy- sis, and interpretation of data, or writing the manuscript. Data visualization and statistics Calculation of weighted single channel conductance from best-fit curves revealed no genotype effect on single channel conductance (iii, Kruskal Wallis p = 0.78). Fig. S7. LRRK2 is expressed and phosphorylated in cultured neurons, and MLi-2 decreases pLRRK2. Fluorscence western blot of pLRRK2, LRRK2, and GAPDH VKI cortical culture lysate revealed that the presence of LRRK2 and LRRK2 p935 in vehicle treated cultures, and absence of pLRRK2 following acute MLi-2 treatment; however, due to low stoichiometry bands were not high enough above background to be reliably quantified. Fig. S8. GluA1 levels unaltered but dendritic cluster density is reduced in VKI. A) Western blot of GluA1 and β-actin in cortical lysates of VKI mice (i) revealed no genotype effect on GluA1 protein levels (ii, Kruskal–Wallis p = 0.99). B) Co- immunoprecipitation of GluA1 with VPS35 (i) revealed no genotype effect (ii, Kruskal–Wallis p = 0.99). C) Cultured cortical neurons immunostained for MAP2 (blue), VPS35 (cyan), and GluA1 (magenta) (i). There was a sig- nificant reduction in GluA1 cluster density in homozygous VKI neurons (ii, Kruskal–Wallis p < 0.02; Uncorrected Dunn’s p < 0.005) and no genotype effect on VPS35-GluA1 co-cluster density of (iii, 1-way ANOVA p = 0.13), or Pearson’s coefficient (iv, Kruskal–Wallis p = 0.57). Fig. S9. Rab10 does Additional file 2: Western blot images. Original western blot images. Acknowledgements Th h h k h The authors thank the Fon & McPherson labs and the Neuro Microscopy Core Facility at McGill University, Liping Cao, Jaskaran Khinda, Jesse Fox, Matthew Farrer and members of The Centre for Applied Neurogenetics UBC. Dr. Kadg- ien would like to thank donors of the Parkinson Society Canada for graduate student funding support. Authors’ contributions CK: conceptualization, methodology, validation, formal analysis, investigation, data curation, writing—original draft, visualization, project administration. AK: formal analysis, investigation, data curation, writing—review & editing, visualization. AJM: conceptualization, methodology, writing—review and editing, supervision, funding acquisition. All authors read and approved the final manuscript. Author details 1 Graduate Program in Neuroscience and Centre for Applied Neurogenetics, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, Canada. 2 Montreal Neurological Institute-Hospital, McGill Univer- sity, Montreal, Canada. Received: 10 June 2021 Accepted: 27 August 2021 Received: 10 June 2021 Accepted: 27 August 2021 Received: 10 June 2021 Accepted: 27 August 2021 Ethics approval and consent to participate pp p p Husbandry and experiments received ethical approvals from UBC (breeding A16-0088; experimentation A15-0105) and McGill University (2017-7888B), in accordance with Canadian Council on Animal Care regulations. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 1. Burd C, Cullen PJ. Retromer: a master conductor of endosome sorting. Cold Spring Harb Perspect Biol. 2014;6(2):a016774. References Goldman JG, Postuma R. Premotor and nonmotor features of Parkinson’s disease. Curr Opin Neurol. 2014;27(4):434–41. 31. Beccano-Kelly DA, Volta M, Munsie LN, Paschall SA, Tatarnikov I, Co K, et al. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory. Hum Mol Genet. 2014;24:1–14. p 11. Foffani G, Obeso JA. A cortical pathogenic theory of Parkinson’s disease. Neuron. 2018;99(6):1116–28. 12. Chen X, Kordich JK, Williams ET, Levine N, Cole-Strauss A, Marshall L, et al. Parkinson’s disease-linked D620N VPS35 knock-in mice manifest tau neuropathology and dopaminergic neurodegeneration. Proc Natl Acad Sci U S A. 2019;116(12):5765–74. y 32. Cirnaru MD, Marte A, Belluzzi E, Russo I, Gabrielli M, Longo F, et al. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex. Front Mol Neurosci. 2014;7:49. 13. Niu M, Zhao F, Bondelid K, Siedlak SL, Torres S, Fujioka H, et al. VPS35 D620N knockin mice recapitulate cardinal features of Parkinson’s disease. Aging Cell. 2021;2021(e13347):1–15. 33. Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, De Astis S, et al. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool. J Neurosci. 2011;31(6):2225–37. 14. Choy RW-Y, Park M, Temkin P, Herring BE, Marley A, Nicoll RA, et al. Ret- romer mediates a discrete route of local membrane delivery to dendrites. Neuron. 2014;82(1):55–62. 34. Volta M, Cataldi S, Beccano-Kelly D, Munsie L, Tatarnikov I, Chou P, et al. Chronic and acute LRRK2 silencing has no long-term behavioral effects, whereas wild-type and mutant LRRK2 overexpression induce motor and cognitive deficits and altered regulation of dopamine release. Parkinson- ism Relat Disord. 2015;21(10):1156–63. 15. Munsie L, Milnerwood A, Seibler P, Beccano-Kelly D, Tatarnikov I, Khinda J, et al. Retromer-dependent neurotransmitter receptor trafficking to synapses is altered by the Parkinson’s disease VPS35 mutation p.D620N. Hum Mol Genet. 2015;24(6):1691–703. 35. Matikainen-Ankney BA, Kezunovic N, Mesias RE, Tian Y, Williams FM, Hunt- ley GW, et al. Altered development of synapse structure and function in striatum caused by Parkinson’s disease-linked LRRK2-G2019S mutation. J Neurosci. 2016;36(27):7128–41. 16. Tian Y, Tang F-L, Sun X, Wen L, Mei L, Tang B-S, et al. VPS35-deficiency results in an impaired AMPA receptor trafficking and decreased dendritic spine maturation. Mol Brain. 2015;8(1):70. 17. Tsika E, Glauser L, Moser R, Fiser A, Daniel G, Sheerin U-M, et al. Parkinson’s disease-linked mutations in VPS35 induce dopaminergic neurodegenera- tion. Hum Mol Genet. 2014;23(17):4621–38. 36. References Parisiadou L, Yu J, Sgobio C, Xie C, Liu G, Sun L, et al. LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity. Nat Neurosci. 2014;17(3):367–76. y 37. Sweet ES, Saunier-Rebori B, Yue Z, Blitzer RD. The Parkinson’s disease- associated mutation LRRK2-G2019S impairs synaptic plasticity in mouse hippocampus. J Neurosci. 2015;35(32):11190–5. 18. Zhang D, Isack NR, Glodowski DR, Liu J, Chen CCH, Xu XZS, et al. RAB-62 and the retromer regulate glutamate receptor recycling through a retro- grade pathway. J Cell Biol. 2012;196(1):85–101. 38. Matikainen-Ankney BA, Kezunovic N, Menard C, Flanigan ME, Zhong Y, Russo SJ, et al. Parkinson’s disease-linked lrrk2-g2019s mutation alters synaptic plasticity and promotes resilience to chronic social stress in young adulthood. J Neurosci. 2018;38(45):9700–11. 19. Wang C-L, Tang F-L, Peng Y, Shen C-Y, Mei L, Xiong W-C. VPS35 regulates developing mouse hippocampal neuronal morphogenesis by promoting retrograde trafficking of BACE1. Biol Open. 2012;1(12):1248–57. fi 20. Tang FL, Zhao L, Zhao Y, Sun D, Zhu XJ, Mei L, et al. Coupling of terminal differentiation deficit with neurodegenerative pathology in Vps35-defi- cient pyramidal neurons. Cell Death Differ. 2020;7:2099–116. 39. Taylor M, Alessi DR. Advances in elucidating the function of leucine-rich repeat protein kinase-2 in normal cells and Parkinson’s disease. Curr Opin Cell Biol. 2020;63:102–13. https://doi.org/10.1016/j.ceb.2020.01.001. 21. Temkin P, Morishita W, Goswami D, Arendt K, Chen L, Malenka R. The retromer supports AMPA receptor trafficking during LTP. Neuron. 2017;94(1):74-82.e5. https://doi.org/10.1016/j.neuron.2017.03.020. 40. Kuhlmann N, Milnerwood AJ. A critical LRRK at the synapse? The neuro- biological function and pathophysiological dysfunction of LRRK2. Front Mol Neurosci. 2020;13(August):1–19. 22. Vazquez-Sanchez S, Bobeldijk S, Dekker MP, Van Keimpema L, Van Weering JRT. VPS35 depletion does not impair presynaptic struc- ture and function. Sci Rep. 2018;8(1):1–12. https://doi.org/10.1038/ s41598-018-20448-4. 41. Steger M, Tonelli F, Ito G, Davies P, Trost M, Vetter M, et al. Phosphoprot- eomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. Elife. 2016. https://doi.org/10.7554/eLife.12813. 42. Jeong GR, Jang EH, Bae JR, Jun S, Kang HC, Park CH, et al. Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration. Mol Neurodegener. 2018;13(1):1–17. 23. Linhart R, Wong SA, Cao J, Tran M, Huynh A, Ardrey C, et al. Vacuolar pro- tein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson’s disease mutant of Leucine-Rich Repeat Kinase 2 (LRRK2). Mol Neurodegener. 2014;9:23. 43. References References 1. Burd C, Cullen PJ. Retromer: a master conductor of endosome sorting. Cold Spring Harb Perspect Biol. 2014;6(2):a016774. Page 18 of 20 Kadgien et al. Mol Brain (2021) 14:143 Kadgien et al. Mol Brain (2021) 14:143 2. Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol. 2006;7(8):568–79. through the endosomal pathway in Drosophila. Hum Mol Genet. 2017;26(15):2933–48. g 3. Cullen PJ, Korswagen HC. Sorting nexins provide diversity for retromer- dependent trafficking events. Nat Cell Biol. 2011;14(1):29–37. 26. Zhao Y, Perera G, Takahashi-Fujigasaki J, Mash DC, Vonsattel JPG, Uchino A, et al. Reduced LRRK2 in association with retromer dysfunc- tion in post-mortem brain tissue from LRRK2 mutation carriers. Brain. 2018;141(2):486–95. fi 4. Seaman MNJ, Gautreau A, Billadeau DD. Retromer-mediated endosomal protein sorting: all WASHed up! Trends Cell Biol. 2013;23(11):522–8. protein sorting: all WASHed up! Trends Cell Biol. 2013;23(11):522– 5. Struhal W, Presslauer S, Spielberger S, Zimprich A, Auff E, Bruecke T, et al. VPS35 Parkinson’s disease phenotype resembles the sporadic disease. J Neural Transm. 2014;121(7):755–9. 27. Mir R, Tonelli F, Lis P, Macartney T, Polinski NK, Martinez TN, et al. The Parkinson’s disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human. Biochem J. 2018;475(11):1861–83. 6. Vilariño-Güell C, Wider C, Ross OA, Dachsel JC, Kachergus JM, Lincoln SJ, et al. VPS35 mutations in Parkinson disease. Am J Hum Genet. 2011;89(1):162–7. 28. Vilariño-Güell C, Rajput A, Milnerwood AJ, Shah B, Szu-Tu C, Trinh J, et al. DNAJC13 mutations in Parkinson disease. Hum Mol Genet. 2013;1–8. 7. Zimprich A, Benet-Pagès A, Struhal W, Graf E, Eck SH, Offman MN, et al. A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease. Am J Hum Genet. 2011;89(1):168–75. 29. Healy DG, Falchi M, O’Sullivan SS, Bonifati V, Durr A, Bressman S, et al. Phenotype, genotype, and worldwide genetic penetrance of LRRK2- associated Parkinson’s disease: a case-control study. Lancet Neurol. 2008;7(7):583–90. 8. Kordower JH, Olanow CW, Dodiya HB, Chu Y, Beach TG, Adler CH, et al. Disease duration and the integrity of the nigrostriatal system in Parkin- son’s disease. Brain. 2013;136(8):2419–31. 30. Beccano-Kelly DA, Kuhlmann N, Tatarnikov I, Volta M, Munsie LN, Chou P, et al. Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice. Front Cell Neurosci. 2014;8:301. 9. Beitz JM. Parkinson’s disease: a review. Front Biosci. 2014;S6(3):65–74. 10. References RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent endo- cytosis pathway. Mol Biol Cell. 2007;18:4387–96. 49. Kluss JH, Mazza MC, Li Y, Manzoni C, Lewis PA, Cookson MR, et al. Preclini- cal modeling of chronic inhibition of the Parkinson’s disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo. Mol Neurodegener. 2021;16(1):1–19. 69. Pavlos NJ, Grønborg M, Riedel D, Chua JJE, Boyken J, Kloepper TH, et al. Quantitative analysis of synaptic vesicle Rabs uncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis. J Neurosci. 2010;30(40):13441–53. 50. Fell MJ, Mirescu C, Basu K, Cheewatrakoolpong B, DeMong DE, Ellis JM, et al. MLi-2, a potent, selective, and centrally active compound for explor- ing the therapeutic potential and safety of LRRK2 kinase inhibition. J Pharmacol Exp Ther. 2015;355(3):397–409. 70. Tang F-L, Erion JR, Tian Y, Liu W, Yin D-M, Ye J, et al. VPS35 in dopamine neurons is required for endosome-to-golgi retrieval of Lamp2a, a recep- tor of chaperone-mediated autophagy that is critical for -synuclein degradation and prevention of pathogenesis of Parkinson’s disease. J Neurosci. 2015;35(29):10613–28. https://doi.org/10.1523/JNEUROSCI. 0042-15.2015. p 51. Gomez TS, Billadeau DD. A FAM21-containing WASH complex regulates retromer-dependent sorting. Dev Cell. 2009;17(5):699–711. 71. Wen L, Tang F, Hong Y, Luo S, Wang C, He W, et al. VPS35 haploinsuf- ficiency increases Alzheimer’s disease neuropathology. J Cell Biol. 2011;195(5):765–79. 52. Derivery E, Sousa C, Gautier JJ, Lombard B, Loew D, Gautreau A. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. Dev Cell. 2009;17(5):712–23. 72. Seaman MNJ. Retromer and the cation-independent mannose 6-phos- phate receptor—time for a trial separation? Traffic. 2018;19(2):150–2. 53. Lee S, Chang J, Blackstone C. FAM21 directs SNX27-retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus. Nat Commun. 2016;7:10939. 73. Wang C, Niu M, Zhou Z, Zheng X, Zhang L, Tian Y, et al. VPS35 regulates cell surface recycling and signaling of dopamine receptor D1. Neurobiol Aging. 2016;46:22–31. https://doi.org/10.1016/j.neurobiolaging.2016.05. 016. 54. Yap CC, Digilio L, McMahon L, Winckler B. The endosomal neuronal proteins Nsg1/NEEP21 and Nsg2/P19 are itinerant, not resident proteins of dendritic endosomes. Nat Sci Rep. 2017;7(1):1–17. https://doi.org/10. 1038/s41598-017-07667-x. 74. Volta M, Beccano-Kelly DA, Paschall SA, Cataldi S, Macisaac SE, Kuhlmann N, et al. Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice. Elife. 2017;6. 55. References Di Maio R, Hoffman EK, Rocha EM, Keeney MT, Sanders LH, De Miranda BR, et al. LRRK2 activation in idiopathic Parkinson’s disease. Sci Transl Med. 2018;10(451):eaar5429. 24. MacLeod DA, Rhinn H, Kuwahara T, Zolin A, Di Paolo G, McCabe BD, et al. RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson’s disease risk. Neuron. 2013;77(3):425–39. 44. McGough IJ, Steinberg F, Jia D, Barbuti PA, McMillan KJ, Heesom KJ, et al. Retromer binding to FAM21 and the WASH complex is perturbed 25. Inoshita T, Arano T, Hosaka Y, Meng H, Umezaki Y, Kosugi S, et al. Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis Kadgien et al. Mol Brain (2021) 14:143 Kadgien et al. Mol Brain (2021) 14:143 Page 19 of 20 by the parkinson disease-linked VPS35(D620N) mutation. Curr Biol. 2014;24(14):1670–6. 64. Bruno J, Brumfield A, Chaudhary N, Iaea D, McGraw TE. SEC16A is a RAB10 effector required for insulinstimulated GLUT4 trafficking in adipocytes. J Cell Biol. 2016;214(1):61–76. by the parkinson disease-linked VPS35(D620N) mutation. Curr Biol. 2014;24(14):1670–6. 45. Zavodszky E, Seaman MNJ, Moreau K, Jimenez-Sanchez M, Breusegem SY, Harbour ME, et al. Mutation in VPS35 associated with Parkinson’s disease impairs WASH complex association and inhibits autophagy. Nat Com- mun. 2014;5:3828. 65. Chen Y, Wang Y, Zhang J, Deng Y, Jiang L, Song E, et al. Rab10 and myosin- va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes. J Cell Biol. 2012;198(4):545–60. 66. Pan X, Zaarur N, Singh M, Morin P, Kandror KV. Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes. Mol Biol Cell. 2017;28(12):1667–75. 46. Follett J, Norwood SJ, Hamilton NA, Mohan M, Kovtun O, Tay S, et al. The Vps35 D620N mutation linked to Parkinson’s disease disrupts the cargo sorting function of retromer. Traffic. 2013;(6). 47. Cui Y, Yang Z, Flores-Rodriguez N, Follett J, Ariotti N, Wall AA, et al. Forma- tion of retromer transport carriers is disrupted by the Parkinson disease- linked Vps35 D620N variant. Traffic. 2021;22(4):123–36. 67. Alshafie W, Francis V, Bednarz K, Pan YE, Stroh T, McPherson PS. Regulated resurfacing of a somatostatin receptor storage compartment fine-tunes pituitary secretion. J Cell Biol. 2020. https://doi.org/10.1083/jcb.20190 4054. fi 48. Cataldi S, Follett J, Fox JD, Tatarnikov I, Kadgien C, Gustavsson EK, et al. Altered dopamine release and monoamine transporters in Vps35 pD620N knock-in mice. npj Park Dis. 2018;4(1):1–11. https://doi.org/10. 1038/s41531-018-0063-3. 68. Glodowski DR, Chen CC-H, Schaefer H, Grant BD, Rongo C. References Bodrikov V, Pauschert A, Kochlamazashvili G, Stuermer CAO. Reggie-1 and reggie-2 (flotillins) participate in Rab11a-dependent cargo trafficking, spine synapse formation and LTP-related AMPA receptor (GluA1) surface exposure in mouse hippocampal neurons. Exp Neurol. 2017;289:31–45. https://doi.org/10.1016/j.expneurol.2016.12.007. 75. Yue M, Hinkle K, Davies P, Trushina E, Fiesel F, Christenson T, et al. Progres- sive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock in mice. Neurobiol Dis. 2015;78:172–95. 56. Correia SS, Bassani S, Brown TC, Lisé M-F, Backos DS, El-Husseini A, et al. Motor protein-dependent transport of AMPA receptors into spines dur- ing long-term potentiation. Nat Neurosci. 2008;11(4):457–66. https://doi. org/10.1038/nn2063. 76. West AB, Moore DJ, Biskup S, Bugayenko A, Smith WW, Ross CA, et al. Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci U S A. 2005;102(46):16842–7. 57. da Silva EM, Adrian M, Schätzle P, Lipka J, Watanabe T, Cho S, et al. Positioning of AMPA receptor-containing endosomes regulates synapse architecture. Cell Rep. 2015;13(5):933–43. 77. Steger M, Diez F, Dhekne HS, Lis P, Nirujogi RS, Karayel O, et al. Systematic proteomic analysis of LRRK2-mediated rab GTPase phosphorylation establishes a connection to ciliogenesis. Elife. 2017;6(i):1–22. 58. Jaafari N, Henley JM, Hanley JG. PICK1 mediates transient synaptic expres- sion of GluA2-lacking AMPA receptors during glycine-induced AMPA receptor trafficking. J Neurosci. 2012;32(34):11618–30. 78. Schreij AM, Chaineau M, Ruan W, Lin S, Barker PA, Fon EA, et al. LRRK2 localizes to endosomes and interacts with clathrin-light chains to limit Rac1 activation. EMBO Rep. 2015;16(1):79–86. 79. Picconi B, Piccoli G, Calabresi P. Synaptic Dysfunction in Parkinson’s Disease. In: Kreutz MR, Sala C, editors. Vienna: Springer Vienna; 2012 (cited 2013 Dec 12). Advances in Experimental Medicine and Biology; vol. 970; p. 553–72. https://doi.org/10.1007/978-3-7091-0932-8. 59. Seebohm G, Neumann S, Theiss C, Novkovic T, Hill EV, Tavaré JM, et al. Identification of a novel signaling pathway and its relevance for GluA1 recycling. PLoS One. 2012;7(3):e33889. 60. Bowen AB, Bourke AM, Hiester BG, Hanus C, Kennedy MJ. Golgi-Inde- pendent secretory trafficking through recycling endosomes in neuronal dendrites and spines. Elife. 2017;6:1–27. 80. Volta M, Milnerwood AJ, Farrer MJ. Insights from late-onset familial par- kinsonism on the pathogenesis of idiopathic Parkinson’s disease. Lancet Neurol. 2015;14(10):1054–64. 61. Benke T, Traynelis SF. AMPA-type glutamate receptor conduct- ance changes and plasticity: still a lot of noise. Neurochem Res. 2019;44(3):539–48. 81. Hinkle KM, Yue M, Behrouz B, Dächsel JC, Lincoln SJ, Bowles EE, et al. References LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors. Mol Neuro- degener. 2012;7(1):1–17. 62. Diering GH, Huganir RL. The AMPA receptor code of synaptic plasticity. Neuron. 2018;100(2):314–29. https://doi.org/10.1016/j.neuron.2018.10. 018. 82. Kriegstein AR, Dichter MA. Morphological classification of rat cortical neurons in cell culture. J Neurosci. 1983;3(8):1634–47. 63. Wang P, Liu H, Wang Y, Liu O, Zhang J, Gleason A, et al. RAB-10 promotes EHBP-1 bridging of filamentous actin and tubular recycling endosomes. PLoS Genet. 2016;12(6):1–28. 83. Kaufman AM, Milnerwood AJ, Sepers MD, Coquinco A, She K, Wang L, et al. Opposing roles of synaptic and extrasynaptic NMDA recep- tor signaling in cocultured striatal and cortical neurons. J Neurosci. 2012;32(12):3992–4003. Kadgien et al. Mol Brain (2021) 14:143 Kadgien et al. Mol Brain (2021) 14:143 Page 20 of 20 Page 20 of 20 88. Smith-Dijak AI, Nassrallah WB, Zhang LYJ, Geva M, Hayden MR, Raymond LA, et al. Impairment and restoration of homeostatic plasticity in cultured cortical neurons from a mouse model of huntington disease. Front Cell Neurosci. 2019. https://doi.org/10.3389/fncel.2019.00209. 84. Brigidi GS, Sun Y, Beccano-Kelly D, Pitman K, Mobasser M, Borgland SL, et al. Palmitoylation of δ-catenin by DHHC5 mediates activity-induced synapse plasticity. Nat Neurosci. 2014;17(4):522–32. 85. Milnerwood AJ, Kaufman AM, Sepers MD, Gladding CM, Zhang L, Wang L, et al. Mitigation of augmented extrasynaptic NMDAR signaling and apoptosis in cortico-striatal co-cultures from Huntington’s disease mice. Neurobiol Dis. 2012;48(1):40–51. 89. Benke TA, Lüthi A, Palmer MJ, Wikström MA, Anderson WW, Isaac JTR, et al. Mathematical modelling of non-stationary fluctuation analysis for studying channel properties of synaptic AMPA receptors. J Physiol. 2001;537(2):407–20. 86. Traynelis SF, Silver RA, Cull-Candy SC. Estimated conductance of G luta- mate Receptor Channels Activated during EPSCs at the Cerebellar Mossy Fiber-Granule Cell Synapse. Neuron. 1993;11:279–89. Publisher’s Note S N 87. Hartveit E, Veruki ML. Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches. Nat Protoc. 2007;2(2):434–48. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from:
https://openalex.org/W2800530796	https://europepmc.org/articles/pmc6024774?pdf=render	English	null	Clinicopathological Diversity of Canine Mammary Gland Tumors in Sri Lanka: A One-Year Survey on Cases Presented to Two Veterinary Practices	Veterinary sciences	2,018	cc-by	8,028	Received: 19 February 2018; Accepted: 24 April 2018; Published: 27 April 2018 Abstract: Mammary gland tumors (MGTs) are one of the most common neoplasms among dogs in Sri Lanka. However, the clinicopathological diversity of MGTs in Sri Lanka is largely unknown, impeding accurate diagnosis and effective treatment of the disease. The present study investigated the clinicopathological features of MGTs in 74 dogs presented to two veterinary practices in Sri Lanka treated surgically, over a one-year period. Information regarding the patient signalment, clinical presentation, and reproductive history were collected, and each neoplasm was examined histologically. Forty-one (54.4%) dogs were primarily presented for mammary neoplasia, while a MGT was an incidental ﬁnding in 33 (44.6%) dogs. The majority of tumors were histologically malignant (n = 65, 87.8%), and 18 malignant tumor sub-types were identiﬁed. A signiﬁcantly higher proportion of malignant tumors were large (>3 cm diameter) and observed in inguinal mammary glands. Nulliparous (n = 42, 55.3%) dogs predominated in the group, and the mean age of MGT diagnosis was 8.0 ± 2.41 years. The present study identiﬁed tumor location and size to be predictive of malignancy. A high histological diversity of MGTs was observed. Overall, the present ﬁndings emphasize the necessity of improving awareness of MGTs among Sri Lankan clinicians as well as dog owners. Keywords: canine; mammary gland tumors; malignant tumors; Sri Lanka Harsha Ariyarathna 1, Niranjala de Silva 2, Danielle Aberdein 1, Dayananda Kodikara 3, Manjula Jayasinghe 2, Ranjith Adikari 2 and John S. Munday 1,* h.ariyarathna@massey.ac.nz (H.A.); d.aberdein@massey.ac.nz (D.A.) 2 Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya 20400, Sri Lanka; niranjalad@yahoo.com (N.d.S.); chamivet@gmail.com (M.J.); adikari04@yahoo.com (R.A.) 3 New Animal Clinic, 132/B, S. De. S. Jayasinghe Mawatha, Kohuwala, Nugegoda 10250, Sri Lanka; dskodikara132@gmail.com y y y 2 Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya 20400, Sri Lanka niranjalad@yahoo.com (N.d.S.); chamivet@gmail.com (M.J.); adikari04@yahoo.com (R.A.) 3 New Animal Clinic, 132/B, S. De. S. Jayasinghe Mawatha, Kohuwala, Nugegoda 10250, Sri Lanka; dskodikara132@gmail.com g * Correspondence: j.munday@massey.ac.nz; Tel.: +64-6-356-9099 (ext. 85172) g * Correspondence: j.munday@massey.ac.nz; Tel.: +64-6-356-9099 (ext. 85172) veterinary sciences veterinary sciences veterinary sciences veterinary sciences veterinary sciences Keywords: canine; mammary gland tumors; malignant tumors; Sri Lanka 1. Introduction As female reproductive hormones promote mammary carcinogenesis, MGTs are the most common neoplasm among intact dogs [1,2]. Ovariohysterectomy (OHE) performed at an early age minimizes the prolonged exposure of mammary tissues to reproductive hormones, and thereby reduces the risk of mammary neoplasia [3]. Speciﬁcally, rates of MGTs in dogs that undergo OHE prior to ﬁrst estrus are around 8%, but this rate increases to 26% in dogs that undergo OHE between the ﬁrst and second estrus [4]. Consequently, the incidence of canine MGTs is decreasing in the regions of the world where OHE is routinely performed at an early age [5]. Sri Lanka is a south Asian country where spaying of dogs at an early age is not a common practice [6]. Dog spaying in Sri Lanka is mostly conducted during mass de-sexing programs that are generally done on an opportunistic basis [6,7]. Therefore, most of the dogs in Sri Lanka are either intact or have been spayed at an older age, which predisposes them to Vet. Sci. 2018, 5, 46; doi:10.3390/vetsci5020046 www.mdpi.com/journal/vetsci www.mdpi.com/journal/vetsci Vet. Sci. 2018, 5, 46 2 of 12 mammary neoplasia. In fact, MGTs are one of the most common neoplasms of dogs in Sri Lanka, and are an important cause of mortality in this population [8]. Given the importance of this disease in Sri Lanka, it is desirable to improve the diagnostic, prognostic, and therapeutic aspects of canine MGTs. To achieve this, a basic understanding of the clinicopathological diversity of MGTs in the Sri Lankan dogs is essential. Therefore, the primary aim of the present study was to determine the different clinicopathological aspects of MGTs presented to two veterinary practices in Sri Lanka during a one-year period. It was considered possible that MGTs in Sri Lankan dogs would show some features that are different to MGTs in dogs elsewhere in the world. These features could be a useful guide for Sri Lankan veterinary pathologists and clinicians to inform—and possibly modify—their current diagnostic and therapeutic approaches, allowing a more accurate diagnosis and more effective treatment of canine MGTs in Sri Lanka dog. A knowledge of possible risk factors for canine MGTs is important to help developing effective strategies to minimize the incidence of these neoplasms. Previous studies have suggested that dogs older than 7 years, small-sized dogs, obese dogs, and dogs that were spayed later in life but still nulliparous, are at increased risk for MGTs [2,9]. 1. Introduction However, the relative impact of these risk factors appears to be variable within different regions of the world. Therefore, as a secondary objective, we determined the age, breed, body condition score, reproductive status, and parity of the dogs with MGTs to identify the common proﬁle of affected dogs in Sri Lanka. The ﬁndings of the current study will provide a basic understanding about the clinicopathological diversity of MGTs in Sri Lanka. By determining the common characteristics of dogs with MGTs, it may be able to identify some of the likely risk factors of canine MGTs in Sri Lanka. To the authors’ knowledge, there are no published studies describing MGTs in dogs in Sri Lanka. 2. Materials and Methods Sample collection: Samples for the present study were obtained from the Veterinary Teaching Hospital (VTH), Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, and a private veterinary practice in Colombo (VPC), Sri Lanka. All dogs that presented to these clinics between June 2016 and June 2017, with one or more spontaneous MGTs that were treated by surgical excision, were included in the study. The primary clinicians that attended to the MGT cases kindly provided us with the patient records and surgically excised mammary tumors collected in 10% neutral buffered formalin. Full owner consent was obtained before the sample collection. The following information was determined from the provided patient records: primary complaint, breed, age, body condition score, age at neutering, parity, general clinical exam findings, and information regarding any prior investigations related to the presenting MGTs. According to the tumor diameter specified in the patient records, tumors were classified as T1 (≤3 cm in greatest dimension), T2 (tumor >3 cm but <5 cm in greatest diameter), or T3 (tumor >5 cm in greatest diameter), following the World Health Organization guidelines as applied by Sorenmo et al. (2007) [5]. Other gross pathological features of the tumors: adherence of tumor mass to the underlying tissues and ulcerations on the skin overlying the tumor, were also extracted from the patient records. Histopathology and tumor classification: The formalin-fixed MGTs received from the primary clinicians were further processed in the histopathology laboratory of the VTH. Briefly, tumors were dehydrated in a gradient of alcohol, embedded in wax, processed in to thin sections (3-µm), stained with hematoxylin and eosin, and examined microscopically. When multiple tumors were present in a single dog, only the tumor with the greatest diameter was considered for histological examination. Immunohistochemistry for smooth muscle actin (SMA) was performed to evaluate a myoepithelial origin of the neoplastic cells using an anti—α SMA antibody (Sigma-Aldrich, St. Louis, MO, USA), following the standard protocols using vascular smooth muscles as the positive control and cardiac muscle as the negative control. Mammary tumor classiﬁcation was performed according to the deﬁnitions in the 2011 classiﬁcation proposed by Goldschmidt and colleagues [10]. Histological malignancy of the tumors was determined following the criteria described in the 2011 Goldschmidt classiﬁcation: tumor 3 of 12 Vet. Sci. 2. Materials and Methods 2018, 5, 46 type, nuclear and cellular pleomorphism, mitotic index, presence of randomly distributed areas of necrosis within the neoplasm, peri-tumoral and lymphatic invasion, and presence of intra-tumoral inﬂammatory cell inﬁltration [10]. All carcinomas except inﬂammatory carcinomas were graded according to the guidelines provided by Pena et al. (2012) [11]. Brieﬂy, tubule formation, nuclear pleomorphism, and mitotic counts were considered and rated on a scale from 1 to 3. The scores for each category were added together and the total scores were used to determine the histological grades for each tumor. In heterogeneous carcinomas, tubular scoring was assessed in the most representative malignant area. In complex and mixed tumors, the percentage of tubular formation was scored considering only epithelial areas, and nuclear pleomorphism was evaluated in all the malignant components. Statistical analysis: All statistical tests were performed using add-on for Excel/XLSTAT software (Version 2017.4) (Addinsoft, New York, NY, USA). The single proportion test was used to compare the malignant tumor proportions in the right and left inguinal mammary glands respectively. Differences were considered signiﬁcant if the calculated p values were <0.05. A chi-square test was performed to determine whether the malignant mammary tumors were equally distributed among the thoracic, abdominal and inguinal mammary glands. The chi-square test was followed by the Marascuillo process, to identify the glands which had a signiﬁcantly different proportion of malignant mammary tumors compared to the other glands. The number of malignant MGTs in each anatomical location was compared with the MGTs in other locations in a pair-wise manner using a calculated absolute and critical value for each pair. The difference was considered signiﬁcant if the calculated absolute value was greater than the critical value. The same statistical methods were used to investigate differences in the distribution of malignant mammary neoplasms among T1, T2 and T3 tumor size categories. 3.1. Clinical Characterization of Dogs with Mammary Gland Tumors 3.1. Clinical Characterization of Dogs with Mammary Gland Tumors Seventy-four dogs with MGTs were included in the study. Thirty-six (48.6%) of them were from Veterinary Teaching Hospital (VTH), Peradeniya, and 38 (51.4%) were from the veterinary practice in Colombo (VPC). Out of the 74 dogs included in the study, 41 (55.4%) dogs were presented primarily seeking veterinary care for mammary neoplasia. Among these, 11 dogs had tumors that had been previously diagnosed as benign MGTs by cytology. In addition to a MGT, reduced appetite and lethargy were secondary complaints in 10 (13.5%) and 17 (23.0%) dogs respectively. In 33 (44.6%) dogs, mammary neoplasia was detected during a clinical examination when the dog was presented for an unrelated complaint. These dogs were presented for veterinary care primarily due to reduced appetite (n = 19, 25.7%) or lethargy (n = 14, 18.9%). Interestingly, the owners of 9 (12.1%) of the dogs admitted that even though they had noticed the mammary masses in their dogs prior to the clinical exams, they did not seek veterinary care speciﬁcally regarding them, assuming that the masses were harmless. During clinical examination of the 74 dogs, lymphadenopathy was identiﬁed in 40 (54.1%) dogs. According to the clinical records, lymphadenopathy was detected in inguinal lymph nodes alone in 10 (13.5%) dogs, while both inguinal and popliteal lymph nodes were concurrently enlarged in 15 (20.3%) dogs. The affected lymph nodes were not speciﬁed in 15 (20.3%) dogs. Cytological examination of ﬁne needle aspirates from the enlarged lymph nodes was performed in only 4 dogs and none of the aspirates were reported to contain neoplastic cells. Further, concurrent with lymphadenopathy, dyspnea or pyrexia was detected during clinical examination in 16 (21.6%) and 14 (18.9%) dogs respectively. Lateral thoracic radiographs had been taken in six dogs and evidence suggestive of pulmonary tumor metastasis was observed in 3 dogs. Weight loss was recorded in 7 out of the 30 dogs for which previous weight records were available. Two dogs were diagnosed with pyometra in Vet. Sci. 2018, 5, 46 4 of 12 addition to mammary neoplasia. Overall, 54 dogs, which accounted for approximately 75% of the group, were systemically ill at the time of presentation. Thirty nine of the 74 dogs presented were mixed breed dogs which represented approximately half of the group (50%). 3.1. Clinical Characterization of Dogs with Mammary Gland Tumors German shepherd was the most common pure dog breed (n = 21, 28.4%), followed by dachshund (n = 2, 2.7%). Single cases of following breeds were also observed: Boxer, Fox Terrier, Japanese Spitz, Pomeranian, Tibetan Terrier, Cocker Spaniel, English Springer Spaniel, Pekingese, Doberman, Dalmatian, Great Dane, Labrador Retriever, Rottweiler and Rhodesian Ridgeback. The overall mean and median ages of dogs with MGTs were 8.0 ± 2.41 years and 8.0 years respectively. The age of the dogs with MGTs was further analyzed separately in 4 categories, namely: 0–4 years, 5–8 years, 9–12 years and ≥13 years. The most frequently represented age category was 5–8 years, which included 41 (55.4%) dogs. There were 26 (35.1%) dogs in the 9–12 years category. The categories 0–4 years and ≥13 years, which included the youngest and oldest dogs, were less frequently represented as there were only 4 (5.4%) and 3 (4.0%) dogs in those categories respectively. The mean age of the dogs with benign and malignant MGTs were 6.7 ± 1.66 years and 7.9 ± 2.15 years respectively. There was no signiﬁcant difference between the age of the dogs with benign tumors and malignant tumors. Primary clinicians had used the 1–5 body condition scoring system described by Eastland-Jones et al. (2014) [12]. Accordingly, the body condition of the majority of dogs was BCS 3 with 42 (56.8%) dogs in this category. BCS 2 and BCS 4 categories included 15 (20.6%) and 13 (17.6%) dogs respectively, while the BCS 5 group was the least represented group of all (n = 4, 5.4%). Reproductive status of the dogs with MGTs is summarized in Table 1. The majority of dogs were intact, while the remainder had been spayed at varying ages. Table 1. Reproductive status of the dogs with mammary gland tumors. Table 1. Reproductive status of the dogs with mammary gland tumors. Reproductive Status n of Cases % Intact 46 62.2 OHE OHE: ≤3 years 8 10.8 OHE: 4–6 years 12 16.2 OHE: ≥7 years 6 8.1 Unknown 2 2.7 Forty-two dogs (55.3%) included in the study were nulliparous. Others had reportedly whelped once (n = 12, 21.1%), twice (n = 18, 23.7%) or three times (n = 4, 5.3%). Contraceptives had not been used to prevent pregnancy in any of the dogs included in the study. 3.2. 3.1. Clinical Characterization of Dogs with Mammary Gland Tumors Gross Pathological Characterization of the Mammary Gland Tumors Tumors were detected in a single mammary gland in 53 (71.6%) dogs, while 21 (28.3%) dogs had tumors in multiple mammary glands. The distribution of the mammary tumors among the mammary glands is summarized in Table 2. Inguinal mammary glands were most often affected: 46 (62.1%) dogs had tumors in these glands. Surprisingly, a signiﬁcantly higher (p < 0.001) number of dogs had tumors in the left inguinal gland (n = 42, 56.8%) than the right (n = 4, 5.4%). No such signiﬁcance was detected in MGTs in the thoracic or abdominal mammary glands. Nine inguinal MGTs were associated with another mammary tumor in a different location, while 4 inguinal MGTs were associated with multiple tumors in other glands. 5 of 12 Vet. Sci. 2018, 5, 46 Table 2. Distribution of mammary gland tumors. Table 2. Distribution of mammary gland tumors. Location Benign Malignant Total Thoracic 5 (55.6%) 9 (13.8%) a 14 (18.9%) Abdominal 2 (22.2%) 12 (18.4%) a 14 (18.9%) Inguinal 2 (22.2%) 44 (67.7%) b 46(62.2%) Total 9 (100%) 65 (100%) 74 (100%) For malignant neoplasms, uncommon superscripts (a, b) between different tumor locations indicate signiﬁcant differences. Critical and absolute values: Thoracic—Abdominal (0.13, 0.04), Thoracic—Inguinal (0.17, 0.55), Abdominal—Inguinal (0.17, 0.50). The size of the MGTs is summarized in Table 3. Overall, 31 dogs had T2 tumors, 25 had T3 tumors, and 18 had T1 tumors. Dogs which were primarily presented for mammary neoplasia had either T2 (n = 18) or T3 (n = 25) tumors, but none had T1 tumors. The MGTs which were incidentally detected in a clinical exam of a dog that was presented for unrelated complaints were either T1 (n = 18) or T2 (n = 15). Table 3. Size of mammary gland tumors. Benign Malignant Total T1 8 (88.8%) 10 (15.4%) a 18 (24.3%) T2 1 (11.1%) 30 (47.7%) b 31 (41.9%) T3 0 (0%) 25 (38.5%) b 25 (33.8%) Total 9 (100%) 65 (100%) 74 (100%) For malignant neoplasms, uncommon superscripts (a, b) between different tumor size categories indicate signiﬁcant differences. Critical and absolute values: T1–T2 (0.18, 0.32), T1–T3 (0.17, 0.22), T2–T3 (0.2, 0.1). Table 3. Size of mammary gland tumors. Ulceration of the skin overlying the tumor was observed in 20 (27.0%) dogs, while 15 (20.3%) tumors were ﬁxed to the underlying tissues. 3.3. Histological Characterization of the Mammary Gland Tumors Sixty-ﬁve (87.8%) of the tumors examined histologically were classiﬁed as malignant, while 9 (12.2%) were classiﬁed as benign. All MGTs histologically identiﬁed as malignant included at least three cellular or nuclear criteria of malignancy (Figures 1 and 2). Histological evidence of peri-tumoral (n = 6) and lymphatic (n = 4) invasion was identiﬁed in 10 malignant tumors, while randomly distributed areas of necrosis within the neoplasm were observed in 16 malignant tumors. Using the histologic classiﬁcation of malignant MGTs, tumors were identiﬁed in three categories: carcinomas, carcinomas-special types, and sarcomas (Table 4). There were 9 sub-types of carcinomas, 6 sub-types of special carcinomas and 3 sub-types of sarcomas. The carcinoma sub-types, simple carcinoma (n = 13, 17.6%) and mixed-type carcinoma (n = 10, 10.8%), were the most frequent sub-types in the carcinoma group. Thirteen simple carcinomas included 5 tubular carcinomas, 7 tubulo-papillary carcinomas, and 1 cribriform carcinoma. In addition, single cases of ductal carcinoma, anaplastic carcinoma, and carcinoma-spindle cell variant were identiﬁed in the carcinoma group. Adenosquamous carcinoma (n = 8, 10.5%) was the most frequent special type carcinoma, while hemangiosarcoma (n = 2, 2.7%) was the most frequent type of sarcoma. Simple adenoma (n = 3, 4.0%) was the most frequent benign MGT sub-type, while single cases of ﬁbroadenoma and mixed benign tumor sub-types were observed. Immunostaining using antibodies against smooth muscle actin (α-SMA) was present in 3 MGTs interpreted to be complex carcinomas and in 1 neoplasm that was classiﬁed as a malignant myoepithelioma. The absence of immunostaining was used to support a classiﬁcation of mammary gland ﬁbrosarcoma in one case. Intra-tumoral inﬂammatory cell inﬁltrates were observed in 21 malignant tumors; moderate intra-tumoral cell inﬁltration was identiﬁed in 11 tumors, and it was low and marked in 6 and 4 tumors respectively. Sixty carcinomas were graded. Of the sixty, 24 were classiﬁed as grade I, 19 were grade II, and 17 were grade III carcinomas. 46 2018, 5, x Figure 1. Tubular carcinoma (Grade III), mammary gland, canine (low power). Figure 1. Tubular carcinoma (Grade III), mammary gland, canine (low power). 2018, 5, x Figure 1. Tubular carcinoma (Grade III), mammary gland, canine (low power). 6 of 12 Vet. Sci. 2018, 5, 46 Figure 1. Tubular carcinoma (Grade III), mammary gland, canine (low power). Figure 1. Tubular carcinoma (Grade III), mammary gland, canine (low power). Figure 1 Tubular carcinoma (Grade III) mammary gland canine (low power) Figure 2. 3.3. Histological Characterization of the Mammary Gland Tumors In addition, all the tumors with surface skin ulcerations, and those which were fixed to the underlying tissues, were malignant. The tumor location was predictive of malignancy with a significantly higher proportion of malignant MGTs developing in the inguinal mammary glands than in the thoracic and abdominal mammary glands (Table 2). Regarding the tumor size, the proportions of malignant T2 or T3 tumors All the dogs that were presented primarily due to MGTs had neoplasms that were classiﬁed as malignant, and all the benign tumors included in this study had been detected incidentally during clinical examination. No clinical features allowed deﬁnitive determination between malignant and benign neoplasms. However ability to associate malignancy with a clinical feature in this study was impaired by the small number of dogs with dog benign MGTs. In all the dogs with multiple MGTs, the tumors with the greatest diameters were malignant. In addition, all the tumors with surface skin ulcerations, and those which were ﬁxed to the underlying tissues, were malignant. T3 tumors are more likely to be malignant than T1 tumors. However, there was no significant difference between the proportions of malignant T2 and T3 tumors. This indicates that the further differentiation of large MGTs in to T2 or T3 categories does not provide any additional advantage when predicting malignancy. mammary glands (Table 2). Regarding the tumor size, the proportions of malignant T2 or T3 tumors were significantly higher compared to the proportion of malignant T1 tumors (Table 3). Thus, T2 or T3 tumors are more likely to be malignant than T1 tumors. However, there was no significant difference between the proportions of malignant T2 and T3 tumors. This indicates that the further differentiation of large MGTs in to T2 or T3 categories does not provide any additional advantage when predicting malignancy. The tumor location was predictive of malignancy with a signiﬁcantly higher proportion of malignant MGTs developing in the inguinal mammary glands than in the thoracic and abdominal mammary glands (Table 2). Regarding the tumor size, the proportions of malignant T2 or T3 tumors were signiﬁcantly higher compared to the proportion of malignant T1 tumors (Table 3). Thus, T2 or T3 tumors are more likely to be malignant than T1 tumors. However, there was no signiﬁcant difference between the proportions of malignant T2 and T3 tumors. 3.3. Histological Characterization of the Mammary Gland Tumors Tubular carcinoma (Grade III), mammary gland, canine (high power). Highly pleomorphic neoplastic cells exhibiting multiple cellular and nuclear criteria of malignancy. All the dogs that were presented primarily due to MGTs had neoplasms that were classified as malignant, and all the benign tumors included in this study had been detected incidentally during Figure 2. Tubular carcinoma (Grade III), mammary gland, canine (high power). Highly pleomorphic neoplastic cells exhibiting multiple cellular and nuclear criteria of malignancy. All h d h d l d G h d l h l f d Figure 2. Tubular carcinoma (Grade III), mammary gland, canine (high power). Highly pleomorphic neoplastic cells exhibiting multiple cellular and nuclear criteria of malignancy. neoplastic cells exhibiting multiple cellular and nuclear criteria of malignancy. All the dogs that were presented primarily due to MGTs had neoplasms that were classified as malignant and all the benign tumors included in this study had been detected incidentally during Figure 2. Tubular carcinoma (Grade III), mammary gland, canine (high power). Highly pleomorphic neoplastic cells exhibiting multiple cellular and nuclear criteria of malignancy. Figure 2. Tubular carcinoma (Grade III), mammary gland, canine (high power). Highly pleomorphic neoplastic cells exhibiting multiple cellular and nuclear criteria of malignancy. clinical examination. No clinical features allowed definitive determination between malignant and benign neoplasms. However ability to associate malignancy with a clinical feature in this study was impaired by the small number of dogs with dog benign MGTs. In all the dogs with multiple MGTs, the tumors with the greatest diameters were malignant. In addition, all the tumors with surface skin ulcerations, and those which were fixed to the underlying tissues, were malignant. The tumor location was predictive of malignancy with a significantly higher proportion of malignant MGTs developing in the inguinal mammary glands than in the thoracic and abdominal mammary glands (Table 2). Regarding the tumor size, the proportions of malignant T2 or T3 tumors were significantly higher compared to the proportion of malignant T1 tumors (Table 3). Thus, T2 or malignant, and all the benign tumors included in this study had been detected incidentally during clinical examination. No clinical features allowed definitive determination between malignant and benign neoplasms. However ability to associate malignancy with a clinical feature in this study was impaired by the small number of dogs with dog benign MGTs. In all the dogs with multiple MGTs, the tumors with the greatest diameters were malignant. 3.3. Histological Characterization of the Mammary Gland Tumors This indicates that the further differentiation of large MGTs in to T2 or T3 categories does not provide any additional advantage when predicting malignancy. 7 of 12 Vet. Sci. 2018, 5, 46 Table 4. Histological sub-types of mammary gland tumors. Malignant Tumors 65 n of Cases % (a) Carcinomas 45 Carcinoma-simple 13 17.6 Carcinoma: mixed type 10 13.5 Carcinoma-solid 6 8.1 Intra-ductal papillary carcinoma 5 6.7 Comedocarcinoma 5 6.7 Carcinoma: complex 3 4.1 Ductal carcinoma 1 1.3 Carcinoma-anaplastic 1 1.3 Carcinoma in situ 1 1.3 (b) Carcinomas: special types 16 Adenosquamous carcinoma 8 10.8 Squamous cell carcinoma 3 4.1 Lipid-rich carcinoma 2 2.7 Carcinoma-spindle cell variant 1 1.3 Inﬂammatory carcinoma 1 1.3 Malignant myoepithelioma 1 1.3 (c) Sarcomas 4 Hemangiosarcoma 2 2.7 Fibrosarcoma 1 1.3 Osteosarcoma 1 1.3 Benign Tumors 9 n of Cases % Simple adenoma 3 4.1 Intra-ductal papilloma 2 2.7 Complex Adenoma 2 2.7 Fibroadenoma 1 1.3 Mixed-benign tumor 1 1.3 Table 4. Histological sub-types of mammary gland tumors. 4. Discussion g g y Nine malignant MGTs included in the present study had been previously diagnosed as benign, using cytology. The discrepancy could be either due to the limited capability of cytology to differentiate benign from malignant tumors [13] or possible benign to malignant transformation which had occurred during the lapse of time between the initial diagnosis and the second examination [20,21]. This observation suggests that pathologists should be cautious when classifying a MGT as benign solely on cytology. Additionally, it suggests that benign tumors should be carefully monitored for evidence of progression to a malignant neoplasm. It is noteworthy that nine dog owners who had observed the MGTs in their dogs had not considered them as conditions requiring veterinary care. This indicates that some Sri Lankan dog owners may not to be sufﬁciently aware of the adverse consequences of MGTs, and emphasizes the necessity of improving awareness on MGTs among the Sri Lankan dog owners. In the present study, the majority of MGTs were detected in the inguinal mammary glands. This distribution is consistent with the ﬁndings of many previous studies [22–24]. The frequent involvement of the inguinal glands is attributed to their abundant tissue mass and prolonged secretory activity, compared to other glands [20]. However, unlike in previous studies, our results show signiﬁcantly higher involvement of the left inguinal gland compared to the right. As both left and right inguinal glands have been previously reported to be affected at equal rates [16,20], the marked left gland involvement observed in the present study is difﬁcult to explain. In this study, the proportion of malignant MGTs in the inguinal glands was signiﬁcantly higher than the proportions of MGTs in the thoracic or abdominal MGTs. This has not been described in previous studies, and the reason for a higher proportion of malignant inguinal MGTs in the present study is unknown. The results of the present study suggest that tumors which have a diameter >3 cm are more likely to be malignant. This is consistent with the ﬁndings of previous studies. A retrospective study conducted by Philibert and colleagues conﬁrmed that dogs with tumors >3 cm in diameter to have decreased overall survival compared to the dogs with tumors <3 cm in diameter [25]. In another study, tumor size of >3 cm diameter was correlated with the factors indicating poor prognosis, such as loss of hormone receptors or higher proliferation index [5]. 4. Discussion The present study reports a systematic evaluation of the clinicopathological features of MGTs in 74 Sri Lankan dogs. Within these dogs, 88% of the MGTs were histologically classiﬁed as malignant. This proportion of malignant MGTs is higher than the 40–50% of MGTs reported to be malignant in studies conducted in United States of America (USA) [13], Canada [14], Japan [15], and Mexico [16]. However, the proportion of malignant tumors observed in Sri Lankan dogs was similar to the rates reported from India (83%) and Brazil (86%) [17,18]. The reasons for a higher proportion of malignant MGTs in dogs in Sri Lanka, Brazil and India are unknown. It is possible that the malignant MGTs were over-represented in these countries as a consequence of frequent exposure of the dogs to carcinogens which may not be present in countries such as the USA, Canada, Japan and Mexico. However, it is also possible that malignant MGTs were over-represented in the developing countries as a consequence of under-detection of benign MGTs. Benign MGTs are mostly incidental clinical exam ﬁndings, and not the primary concerns of the dog owners [19]. The incidental detection of benign MGTs during a clinical examination may be less likely to occur in developing countries because dog owners may seek veterinary care less frequently compared to owners in more developed countries. It appears likely that owners may not seek veterinary advice unless they observe a rapidly growing ulcerated mammary gland mass. Such masses are much more likely to be malignant tumors [20]. This is supported by the observation in the present study that all dogs that were presented for a mammary gland mass had malignant tumors, while all the benign tumors observed in this study were from dogs that had presented to the veterinarian for a reason not related to the MGT. The high percentage of MGTs that were malignant at the time of presentation in Sri Lanka suggest that veterinarians should be aware Vet. Sci. 2018, 5, 46 8 of 12 of this disease, and consider mammary neoplasia as a serious health problem among Sri Lankan dogs. In this study, when there were multiple mammary tumors in a single dog, only the tumor with the greatest diameter was considered for histological analysis due to the ﬁnancial limitations. The preferential examination of the largest neoplasm could have contributed to the higher proportion of MGTs being malignant in this study. 4. Discussion However, tumor size alone does not conﬁrm the malignancy of a mammary tumor, and histological examination is essential for conﬁrmation. In the present study, all the tumors with surface skin ulcerations, and tumors which were ﬁxed to the underlying tissues, were malignant, indicating that these features could also be predictive of malignancy. Previous studies also indicate that malignant MGTs which are usually large in size are more likely to develop ulceration, due to more frequent contact with the rough surfaces compared to small size benign tumors [20]. Since malignant neoplasms invade or inﬁltrate surrounding muscle, nerve, blood vessels, and connective tissues, they are also more likely to become ﬁxed to the underlying tissues [20]. An interesting feature regarding the reproductive histories of the dogs in the present study was that over half of the dogs were nulliparous. A recent study conducted in Switzerland in 2018 conﬁrmed that nulliparous dogs are at a signiﬁcantly higher risk for developing mammary tumors compared to multiparous dogs [26]. The elevated risk was attributed to the higher frequency of pseudopregnancy and estrus in nulliparous dogs than multiparous dogs. As both pseudopregnancy and estrus increase the production of female reproductive hormones, the mammary gland tissues of nulliparous dogs may be exposed to greater amounts of female hormones than the mammary gland tissues of multiparous dogs [26]. While the results of the present study suggest that nulliparous dogs in Sri Lanka may be at similar higher risk for MGTs, it has to be noted that the proportion of dogs in the studied populations Vet. Sci. 2018, 5, 46 9 of 12 that are nulliparous is unknown. Therefore, it is possible that the high proportion of dogs with MGTs that were nulliparous in the study was simply due to the high proportion of dogs in Sri Lanka that are nulliparous. p The histological diversity of the malignant MGTs included in the present study was high. In fact, out of the 23 malignant MGT sub-types listed in the Goldschmidt classiﬁcation, 18 were reported in the present study. A recent prospective study conducted in Italy conﬁrmed the prognostic signiﬁcance of the Goldschmidt classiﬁcation [27]. Given the high histological diversity revealed by the present results, Sri Lankan pathologists should be able to accurately differentiate tumor sub-types in order to provide reliable prognostic information. In this paper we used α-SMA to help differentiate between complex and simple carcinomas. 4. Discussion In addition, the same antibody was used to help differentiate the myoepithelial origin of a malignant myoepithelioma, and to exclude myoepithelial origin within a mammary gland ﬁbrosarcoma. However, deﬁnitive differentiation of myoepithelial cells was not possible using α-SMA alone. Instead, it is currently recommended that p63 or a panel of antibodies be used to differentiate between these tumor types. In the present study, only α-SMA was used to reproduce the likely situation in developing countries in which p63 is not often available, and clients are unlikely to be able to afford a panel of immunostains. Interestingly, one of the simple mammary carcinomas reported in the present study co-existed with a cutaneous mast cell tumor. Therefore, apart from being classiﬁed as a mammary carcinoma, it was identiﬁed as a collision tumor referring to the mixed presentation. Collision tumors are a type of a mixed tumor with 2 foci of neoplasia which develop adjacent to one another, yet remain separate [28]. These tumors are rare, and there is minimal information regarding treatment recommendations and outcome for animals [28]. A majority of affected dogs in the present study were 5–8 years old, and the mean age of the diagnosis of mammary neoplasia was 8.0 ± 2.47 years. The mean age reported from Sri Lanka is comparatively lower to the age of diagnosing mammary neoplasia in dogs reported from Sweden (9.33 years) [29], Slovenia (10 years), Turkey (10.3 years) [23], Canada (11 years) [14], Brazil (11.6 years and 12 years) [30,31], Mexico (9–12 years) [16] and Czech Republic (13 years) [32]. However, the reported ages of diagnosing mammary neoplasia in three studies from India, Bhutan and Malaysia were 7–9 years [33], 8.4 years [34] and 8.6 years [20] respectively. It is interesting to note that dogs from Asia have been reported to develop MGTs at an earlier age than dogs from North America, South America and Europe. Whether this reﬂects exposure to an external carcinogen, or a genetic predisposition in dogs in Asian counties is unknown. Other possible factors include different feeding practices, spaying practices, and immunization protocols followed in Asian countries compared to those of North American, South American and European countries [35]. Alternatively, it is possible that dogs in the Asian countries do not live long and the earlier onset of mammary neoplasia detected in these countries is simply due to fewer old dogs in these populations. 4. Discussion The proportion of dogs with multiple MGTs in this study (27.6%) was higher than the proportions of dogs reported in the majority of previous studies mainly of dogs from Western countries [33,36]. An exception was a Malaysian study in which 29.2% of dogs were reported to have multiple MGTs [20]. Multiple MGTs is well recognized in dogs [21], and is believed to be due to the concurrent exposure of all mammary glands in a single animal to circulating reproductive hormones. The resulting tumors are more likely to be at the same stage of development, and are more frequently benign than malignant [21]. However, benign to malignant transformation may occur in some tumors over time, resulting a combination of malignant and benign tumors in the same animal [21]. In the present study, mixed-breed dogs had the highest incidences of MGTs. Generally, mixed-breed dogs are considered to be comparatively less predisposed for MGTs compared to pure breeds [32]. In Sri Lanka, mixed-breed dogs are reportedly the most common pet dog breed [37]. Thus, the predominance of them in the present study is more likely to be a reﬂection of their commonality, rather than a true breed predisposition. Similarly, the over-representation of German shepherds in the present study might also be due to their high popularity in Sri Lanka, and may not necessarily Vet. Sci. 2018, 5, 46 10 of 12 indicate a breed predisposition. The minimal representation of Labradors in this study is noteworthy, considered the reportedly high popularity of this breed among Sri Lankan dog owners [35]. Most of the dogs in the present study had an ideal body condition score (BCS 3). Obesity at 1 year of age and in the year before the diagnosis of mammary neoplasia is signiﬁcantly related with a higher prevalence of MGTs [38]. In contrast, we found only 5 obese dogs in our study. While this may suggest obesity does not predispose to MGTs, this cannot be determined without the knowledge of overall proportion of obese dogs in the population. Previous studies have reported that dogs with MGTs are generally healthy at the initial presentation [35]. However, most of the dogs included in our study were systemically ill at the time of presentation, which might be due to tumor metastasis or other concurrent diseases. 4. Discussion Systemic illness due to tumor metastasis was conﬁrmed in few cases in the present study, due to the unavailability of necessary diagnostic testing. In systemically ill dogs, pre-surgical patient stabilization is important for successful surgical and post-surgical management [21]. Therefore, the results suggest that Sri Lankan veterinary surgeons should be more vigilant about the pre-surgical patient stabilization procedures to minimize the possible post-surgical complications. 5. Conclusions A majority of mammary gland tumors in Sri Lankan dogs were histologically malignant, and a considerably high histological diversity was observed. Tumor size and location were identiﬁed to be predictive of malignancy, even though they are not conﬁrmative of malignancy when considered alone. Relatively younger dogs were found to be affected; the mean age of MGT diagnosis was 8.0 ± 2.41 years. Nulliparous dogs predominated in the group. Overall, the present ﬁndings emphasize the necessity of improving awareness on MGTs among Sri Lankan clinicians, as well as dog owners. Author Contributions: H.A., D.K. and N.d.S designed the study; H.A., M.J., D.K. and R.A. collected the data; H.A., D.A. and J.S.M. analyzed the data; H.A. wrote the paper. J.S.M. and D.A. checked the manuscript before submission. Acknowledgments: We would like to thank all the primary clinician in VTH, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya for their cooperation and efforts in facilitating data collection. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 11 of 12 Vet. Sci. 2018, 5, 46 8. Soorasena, T.; Kularathne, M.; Best Care Animal Hospital, Nugegoda, Colombo, Sri Lanka. Personal communication, 2017. 9. Dhami, M.; Tank, P.H.; Karle, A.S.; Vedpathak, H.S.; Bhatia, A.S. Epidemiology of canine mammary gland tumours in Gujarat. Vet. World 2010, 3, 282–285. 10. Goldschmidt, M.; Pena, L.; Rasotto, R.; Zappulli, V. Classiﬁcation and grading of canine mammary tumors. Vet. Pathol. 2011, 48, 117–131. [CrossRef] [PubMed] 11. Pena, L.; De Andres, P.J.; Clemente, M.; Cuesta, P.; Perez-Alenza, M.D. Prognostic value in histological grading in noninﬂammatory mammary carcinomas in a prospective study with two-year follow up: Relationship with clinical and histological characteristics. Vet. Pathol. 2013, 50, 94–105. [CrossRef] [PubMed] 12. Eastland-Jones, R.C.; German, A.J.; Holden, S.L.; Biourge, V.; Pickavance, L.C. Owner misperception of canine body condition persists despite use of a body condition score chart. J. Nutr. Sci. 2014, 3, 1–5. [CrossRef] [PubMed] 13. Allen, S.W.; Prasse, K.W.; Mahaffey, E. Cytologie differentiation of benign from malignant canine mammary tumors. Vet. Pathol. 1986, 23, 649–655. [CrossRef] [PubMed] 14. Mitchell, L.; De la Iglesia, F.A.; Wenkoff, M.S.; Van Dreumel, A.A.; Lumb, G. Mammary tumors in dogs: Survey of clinical and pathological characteristics. Can. Vet. J. 1974, 15, 131–138. [PubMed] 15. Yamagami, T.; Kobayashi, T.; Takahashi, K.; Sugiyama, M. Prognosis for canine malignant mammary tumors based on TNM and histologic classiﬁcation. J. Vet. Med. Sci. 1996, 58, 1079–1083. [CrossRef] [PubMed] 16. Salas, Y.; Márquez, A.; Diaz, D.; Romero, L. Epidemiological study of mammary tumors in female dogs di d d i h i d 2002 2012 A i i l h l h bl PL S ONE 10 0127381 14. Mitchell, L.; De la Iglesia, F.A.; Wenkoff, M.S.; Van Dreumel, A.A.; Lumb, G. Mammary tumors in dogs: Survey of clinical and pathological characteristics. Can. Vet. J. 1974, 15, 131–138. [PubMed] 15. Yamagami, T.; Kobayashi, T.; Takahashi, K.; Sugiyama, M. Prognosis for canine malignant mammary tumors based on TNM and histologic classiﬁcation J Vet Med Sci 1996 58 1079 1083 [CrossRef] [PubMed] 14. Mitchell, L.; De la Iglesia, F.A.; Wenkoff, M.S.; Van Dreumel, A.A.; Lumb, G. Mammary tumors in dogs: Survey of clinical and pathological characteristics. Can. Vet. J. 1974, 15, 131–138. [PubMed] 5. Yamagami, T.; Kobayashi, T.; Takahashi, K.; Sugiyama, M. Prognosis for canine malignant mammary tum based on TNM and histologic classiﬁcation. J. Vet. Med. Sci. 1996, 58, 1079–1083. [CrossRef] [PubMed] 16. References Salas, Y.; Márquez, A.; Diaz, D.; Romero, L. Epidemiological study of mammary tumors in female dogs diagnosed during the period 2002–2012: A growing animal health problem. PLoS ONE 2015, 10, e0127381. [CrossRef] [PubMed] 17. Dileepkumar, K.; Maiti, S.K.; Kumar, N.; Zama, M.M.S. Occurrence of canine mammary tumours. Ind. J. Can. Pract. 2014, 6, 179–183. 18. Terzian, A.C.B.; de Campos, Z.; Debora, A.P.P.; Rodrigo, S.P.; Marcília, V.R.; Camila, M.; Montanari, S.; Sueiro, F.A.R.; Coelho, J. Avaliação da caspase-3 e Ki-67 como marcadores prognósticos nas neoplasias mamárias em cadelas. Braz. J. Vet. Res. An. Sci. 2007, 44, 96–102. [CrossRef] 19. Saba, C.F.; Rogers, K.S.; Newman, S.J.; Mauldin, G.E.; Vail, D.M. Mammary gland tumors in male dogs. J. Vet. Intern. Med. 2007, 21. [CrossRef] 20. Sahabi, K.; Selvarajah, G.T.; Noordin, M.M.; Sharma, R.S.K.; Dhaliwal, G. Retrospective histopathological study of canine mammary gland tumors diagnosed from 2006–2012 in University Putra Malaysia. J. Vet. Malays. 2015, 27, 1–6. y 21. Withrow, S.J.; Vail, D.; Page, R. Tumors of the mammary gland. In Withrow and MacEwen’s Small Animal Clinical Oncology, 5th ed.; Vail, D., Ed.; Elsevier: New York, NY, USA, 2012; pp. 538–552, ISBN 9781437723625. 22. Cerovšek, M.; Plavec, T.; Zrimšek, P.; Pogaˇcnik, M.; Zabavnik, J. Clinicopathological survey of 56 canine malignant mammary tumours in Slovenia—Prognostic value of clinical stage and histological grade. Slov. Vet. Res. 2013, 50, 93–102. 3. Sorenmo, K. Canine mammary gland tumors. Vet. Clin. Small Anim. Pract. 2003, 33, 573–596. [CrossRef 23. Sorenmo, K. Canine mammary gland tumors. Vet. Clin. Small Anim. Pract. 2003, 33, 573–596. [CrossRef] 24. Sontas, B.; Ozyogurtcu, H.; Gurel, A.; Ekici, H. Evaluation of clinical and pathological characteristics of 155 canines with mammary tumours: A retrospective study. Arch. Med. Vet. 2009, 41, 53–59. [CrossRef] 24. Sontas, B.; Ozyogurtcu, H.; Gurel, A.; Ekici, H. Evaluation of clinical and pathological characteristics of 155 canines with mammary tumours: A retrospective study. Arch. Med. Vet. 2009, 41, 53–59. [CrossRef] 24. Sontas, B.; Ozyogurtcu, H.; Gurel, A.; Ekici, H. Evaluation of clinical and pathological characteristics of 155 canines with mammary tumours: A retrospective study. Arch. Med. Vet. 2009, 41, 53–59. [CrossRef] 25. Philibert, J.C.; Snyder, P.W.; Glickman, N.; Glickman, L.T.; Knapp, D.W.; Waters, D.J. Inﬂuence of host factors on survival in dogs with malignant mammary gland tumors. J. Vet. Intern. Med. 2003, 17. [CrossRef] 26. Sudson, S.R.S.; Nongnapas, R.; Nattanun, U.; Chawisa, W. References 1. Gleicher, N. Why are reproductive cancers more common in nulliparous women? Reprod. Biomed. Online 2013, 26, 416–419. [CrossRef] [PubMed] 1. Gleicher, N. Why are reproductive cancers more common in nulliparous women? Reprod. Biomed. Online 2013, 26, 416–419. [CrossRef] [PubMed] 2. Benavente, M.A.; Bianchi, C.P.; Aba, M.A. Canine mammary tumors: Risk factors, prognosis and treatments. J. Vet. Adv. 2016, 6, 1291–1300. [CrossRef] 2. Benavente, M.A.; Bianchi, C.P.; Aba, M.A. Canine mammary tumors: Risk factors, prognosis and treatments. J. Vet. Adv. 2016, 6, 1291–1300. [CrossRef] 3. Spain, C.V.; Scarlett, J.M.; Houpt, K.A. Long-term risks and beneﬁts of early-age gonadectomy in dogs. J. Am. Vet. Med. Assoc. 2004, 224, 380–387. [CrossRef] [PubMed] 3. Spain, C.V.; Scarlett, J.M.; Houpt, K.A. Long-term risks and beneﬁts of early-age gonadectomy in dogs. J. Am. Vet. Med. Assoc. 2004, 224, 380–387. [CrossRef] [PubMed] 4. Kustritz, M.V.R. Determining the optimal age for gonadectomy of dogs and cats. J. Am. Vet. Med. Assoc. 2007, 231, 1665–1675. [CrossRef] [PubMed] 4. Kustritz, M.V.R. Determining the optimal age for gonadectomy of dogs and cats. J. Am. Vet. Med. Assoc. 2007, 231, 1665–1675. [CrossRef] [PubMed] 5. Sorenmo, K.U.; Kristiansen, V.M.; Cofone, M.A.; Shofer, F.S.; Breen, A.M.; Langeland, M.; Mongil, C.M.; Grondahl, A.M.; Teige, J.; Goldschmidt, M.H. Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence. Vet. Comp. Oncol. 2009, 7, 162–172. [CrossRef] [PubMed] 5. Sorenmo, K.U.; Kristiansen, V.M.; Cofone, M.A.; Shofer, F.S.; Breen, A.M.; Langeland, M.; Mongil, C.M.; Grondahl, A.M.; Teige, J.; Goldschmidt, M.H. Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence. Vet. Comp. Oncol. 2009, 7, 162–172. [CrossRef] [PubMed] 6. De Silva, G.; Nalinika, O. Sterilizing more than 80% of female dog population: Is it enough to keep the population under control? In Proceedings of the 2nd International Conference on Dog Population Management, Istanbul, Turkey, 3 March 2015. 6. De Silva, G.; Nalinika, O. Sterilizing more than 80% of female dog population: Is it enough to keep the population under control? In Proceedings of the 2nd International Conference on Dog Population Management, Istanbul, Turkey, 3 March 2015. 7. Blue Paw Trust; World Society for Animal Protection. Dog Population Management in Colombo, Sri Lanka. 2012. Available online: http://www.carodog.eu/wp-content/uploads/2014/10/Case-Study_Colombo2.pdf (accessed on 2 February 2018). 7. Blue Paw Trust; World Society for Animal Protection. Dog Population Management in Colombo, Sri Lanka. 2012. Available online: http://www.carodog.eu/wp-content/uploads/2014/10/Case-Study_Colombo2.pdf (accessed on 2 February 2018). References High Prevalence of canine mammary gland tumor y p y 25. Philibert, J.C.; Snyder, P.W.; Glickman, N.; Glickman, L.T.; Knapp, D.W.; Waters, D.J. Inﬂuence of host factors on survival in dogs with malignant mammary gland tumors. J. Vet. Intern. Med. 2003, 17. [CrossRef] 26. Sudson, S.R.S.; Nongnapas, R.; Nattanun, U.; Chawisa, W. High Prevalence of canine mammary gland tumor in nulliparous compared with multiparous female dogs. Int. J. Anim. Vet. 2018, 12, 616. [CrossRef] 27. Rasotto, R.; Berlato, D.; Goldschmidt, M.H.; Zappulli, V. Prognostic signiﬁcance of canine mammary tumor histologic subtypes: An Observational cohort study of 229 cases. Vet. Pathol. 2017. 54, 571–578. [CrossRef] 28. Scott, J.E.; Liptak, J.M.; Powers, B.E. Malignant collision tumors in two dogs. J. Am. Vet. Med. Assoc. 2017, 251, 941–945. [CrossRef] [PubMed] 29. Hellmén, E.; Ergström, R.; Holmberg, L.; Spångberg, I.B.; Hansson, K.; Lindgren, A. Prognostic factors in canine mammary tumors: A multivariate study of 202 consecutive cases. Vet. Pathol. 1993, 30, 20–27. [CrossRef] [PubMed] 30. Dias, M.L.; Andrade, J.M.L.; Castro, M.B.G.; Paula, D. Survival analysis of female dogs with mammary tumors after mastectomy: Epidemiological, clinical and morphological aspects. Pesq. Vet. Bras. 2016, 36, 181–186. [CrossRef] 12 of 12 Vet. Sci. 2018, 5, 46 31. Frehse, M.S.; Bracarense, A.P.S.; de Santis, G.W.; da Silva, E.O.; Freire, R.L.; Machado, M.A.; Matins, M.I.M. Epidemiological and histological aspects of canine mammary tumors diagnosed at the Veterinary Teaching Hospital/UEL. Braz. J. Vet. Pathol. 2014, 7, 118–122. 2. Zatloukal, J.; Lorenzova, J.; Tichý, F.; Neˇcas, A.; Kecova, H.; Kohout, P. Breed and age as risk factors canine mammary tumours. Acta Vet. Brno 2005, 74, 103–109. [CrossRef] 33. Gupta, K.; Sood, N.K.; Uppal, S.K.; Mohindroo, J.; Mahajan, S.; Raghunath, M.; Singh, K. Epidemiological studies on canine mammary tumour and its relevance for breast cancer studies. IOSR J. Pharm. Biol. Sci. 2012, 2, 322–333. [CrossRef] 34. Chang, S.C.; Chang, C.C.; Chang, T.J.; Wong, M.L. Prognostic factors associated with survival two years after surgery in dogs with malignant mammary tumors: 79 cases (1998–2002). J. Am. Vet. Med. Assoc. 2005, 227, 1625–1629. [CrossRef] [PubMed] 35. Seneviratne, M.; Subasinghe, D.W.; Watson, P.J. A survey of pet feeding practices of dog owners visiting a veterinary practice in Colombo, Sri Lanka. Vet. Med. Sci. 2016, 2, 106–116. [CrossRef] [PubMed] 36. Sleeckx, N.; De Rooster, H.; Van, G.C.; Veldhuis, E.J.B.; Van Brantegem, L. Canine mammary tumours, an overview. Reprod. Domest. Anim. 2011, 46. [CrossRef] [PubMed] 37. References Karunanayake, D.; Matsumoto, T.; Wimalaratne, O.N.; Susilakanthi, P.D.; Nishizono, A.; Ahmed, K. Twelve years of rabies surveillance in Sri Lanka, 1999–2010. PLoS Negl. Trop. Dis. 2014, 8, 3205. [CrossRef] [PubMed] 38. Alenza, D.P.; Rutteman, G.R.; Peña, L.B.; Anton, C.; Cuesta, P. Relation between habitual diet and canine mammary tumors in a case-control study. J. Vet. Intern. Med. 1998, 12, 132–139. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2939106700	https://hal.archives-ouvertes.fr/hal-02106549/file/Costa_Rican_mortality.pdf	English	null	Costa Rican mortality 1950‒2013: An evaluation of data quality and trends compared with other countries	Demographic research	2,019	cc-by	11,144	To cite this version: Dana Glei, Magali Barbieri, Carolina Santamaría-Ulloa. Costa Rican mortality 1950-2013: An eval- uation of data quality and trends compared with other countries. Demographic Research, 2019, 40 (29), pp.835-864. 10.4054/DemRes.2019.40.29. hal-02106549 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02106549 https://hal.science/hal-02106549v1 Submitted on 23 Apr 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License DEMOGRAPHIC RESEARCH © 2019 Dana A. Glei, M. Barbieri & C. Santamaría-Ulloa. This open-access work is published under the terms of the Creative Commons Attribution 3.0 Germany (CC BY 3.0 DE), which permits use, reproduction, and distribution in any medium, provided the original author(s) and source are given credit. g See https://creativecommons.org/licenses/by/3.0/de/legalcode. 1 Georgetown University, Washington, DC, USA, and University of California, Berkeley, USA. Email: dglei@sonic.net. 2 University of California, Berkeley, USA, and Institut national d’études démographiques (INED), Paris, France. 3 Instituto de Investigaciones en Salud, Universidad de Costa Rica, San Pedro, Costa Rica. Contents 1 Introduction 836 2 Data and methods 841 2.1 The Human Mortality Database protocol to construct life tables 841 2.2 Assessing data reliability through internal consistency checks 842 2.3 Assessing data reliability through external consistency checks 844 3 Evaluation of data quality 844 3.1 Age misreporting 844 3.1.1 Age heaping 844 3.1.2 Age exaggeration 847 3.2 Relative completeness 848 3.3 Comparing Costa Rica mortality estimates across all sources 850 4 Mortality trends compared with other countries 853 4.1 Life expectancy at birth (e0) 853 4.2 Life expectancy at age 80 (e80) 854 4.3 Relationship between child and older-age mortality 856 4.4 Pattern of mortality at higher ages in comparison with Sweden 857 5 Intercountry comparisons with LAMBdA life tables 857 6 Conclusion and discussion 858 7 Acknowledgements 859 References 860 Demographic Research: Volume 40, Article 29 Research Material Demographic Research: Volume 40, Article 29 Research Material BACKGROUND Mortality estimates from various sources suggest that Costa Ricans experience record- high life expectancy at birth in Latin America and higher longevity than the populations of many high-income countries, although there is some uncertainty as to the reliability of those estimates. OBJECTIVE We construct a life table series for Costa Rica to assess the quality of national demographic statistics for the period 1950–2013 and to determine whether reliable mortality estimates can be directly calculated from this data. Costa Rican mortality 1950–2013: An evaluation of data quality and trends compared with other countries Dana A. Glei1 Magali Barbieri2 Dana A. Glei1 Magali Barbieri2 Carolina Santamaría-Ulloa3 METHODS We apply the methods from the Human Mortality Database (HMD) to national statistics to construct the Costa Rica life table series without adjusting for data quality. We also validate our results through internal consistency by evaluating the plausibility of the mortality patterns and its change over time and through external consistency by comparing our results with those from other sources. http://www.demographic-research.org CONCLUSIONS Other organizations have produced mortality estimates for Costa Rica that are higher than our unadjusted estimates, but it is difficult to evaluate the accuracy of the available estimates. CONTRIBUTION This analysis provides a more thorough evaluation of data quality issues regarding Costa Rica mortality than previously available. Unadjusted life tables by sex for 1950– 2013 are included as supplemental material, together with the raw data upon which those life tables are based and with links to the detailed methods protocol implemented. RESULTS Our mortality estimates for Costa Rica tend to be lower than others, especially for the period before 1970. They also produce a suspicious age pattern of mortality, with low adult and old-age mortality relative to the infant and child mortality, casting doubt on the quality of national demographic data. 835 http://www.demographic-research.org Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 http://www.demographic-research.org 1. Introduction Costa Ricans are among the longest-lived people in Latin America. Based on the United Nations Population Division (UNPD) estimates for 2010–2015, life expectancy at birth (e0, both sexes combined) in Costa Rica was 79.2 years, far higher than for Central America as a whole (75.8) and surpassed by no other country within the Latin American and Caribbean (LAC) region (United Nations, Department of Economic and Social Affairs, Population Division 2017). The UNPD estimates suggest that e0 in Costa Rica also exceeds that of the US population (78.9). This situation would be quite remarkable if it were accurate. How much credence we can give to such comparisons depends on the reliability of the estimates and the quality of the underlying data. Our purpose in this paper is to determine the degree of reliability of the mortality indicators derived from national demographic statistics using direct estimation methods, that is without adjustment. Costa Rica has collected detailed vital registration and census data for more than 100 years. The first population census was conducted in 1864 and the vital registration system was created in 1883, when the Statistics and Census Directorate was established. The UN Statistics Division (UNSD) reports that death registration in Costa Rica was 97% complete in 2016 (United Nations Statistics Division 2017). For the 1950s, other estimates suggest that Costa Rica had higher relative completeness of death registration (92% in 1956) than most other LAC countries (with a low of 46% for Nicaragua in that same year); only Argentina and Chile fared better (Palloni, Pinto- Aguirre, and Beltrán-Sánchez 2015). After 2000, only four countries in the LAC region (Argentina, Brazil, Chile, and Uruguay) surpassed Costa Rica (98% in 2005) in terms of relative completeness. Census coverage, evaluated using indirect methods, increased from 83% in 1956 to 96% in 2000 but then declined again in the most recent census to 93% (Borges and Sacco 2016). Among the 20 Latin American countries considered, http://www.demographic-research.org 836 http://www.demographic-research.org Demographic Research: Volume 40, Article 29 only Cuba and Uruguay consistently outperformed Costa Rica in terms of census coverage throughout the period since 1950 (Borges and Sacco 2016). One indicator of data quality is the degree of uncertainty in mortality estimates. 1. Introduction Here we consider variations in mortality indicators for Costa Rica over the whole age range (i.e., estimates of life expectancy at birth, e0) and at high ages (i.e., life expectancy at age 80, e80), where many problems of data quality are concentrated (both in terms of coverage and accuracy, regarding age, for instance). We know of five different sets of life table series for Costa Rica. They have been produced by the Instituto Nacional de Estadística y Censos in collaboration with the Centro Centroamericano de Población at the Universidad de Costa Rica (INEC-CCP); the United Nations Population Division (UNPD); the Latin American Mortality Database (LAMBdA); the World Health Organization (WHO); and the Institute for Health Metrics and Evaluation-Global Burden of Disease program (GBD). While the INEC-CCP estimates are directly constructed from vital statistics and census-based population data using classic demographic techniques, the LAMBdA and GBD estimates are indirectly estimated using a complex set of methods developed by demographers at LAMBdA (Beltrán-Sánchez et al. 2018) and by statisticians at the GBD (Wang et al. 2016). The estimates for Costa Rica published by the UNDP were prepared by the Latin American and Caribbean Demographic Centre (CELADE) at the Economic Commission for Latin America and the Caribbean, which is the Latin American office of the United Nations. CELADE constructed abridged life tables by sex for all census years using the census population combined with birth and death counts from the vital statistics system. Life tables for each five-year period were estimated by linear interpolation on the probabilities of dying at each age from the life tables centered on the censuses. Postcensal life tables were built by interpolation between the last census- based mortality estimates (those for 2011 in the case of Costa Rica) and a ‘life limit table’ believed to represent the mortality pattern that will be achieved by 2050 (see Latin America and the Caribbean Demographic Observatory 2007: 239–242, for a detailed description of the methods used by CELADE for their mortality estimates and projections). LAMBdA estimates have been adjusted for relative completeness and age misreporting (Beltrán-Sánchez et al. 2018). http://www.demographic-research.org 1. Introduction More specifically, the life tables available in LAMBdA have been constructed in three separate steps: 1) Infant and child mortality were estimated by modeling (using local area regressions and splines) from a combined set of national values obtained from direct estimation based on birth histories in nationally representative surveys and indirectly estimated from the census and adjusted vital statistics; 2) Adult mortality was estimated by first adjusting census data for completeness using the General Growth Balance method, then relying on these to adjust 837 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 for completeness of death registration using the Synthetic Extinct Generation method, and, finally, correcting for age misstatement assuming a systematic pattern of overstatement; and 3) Mortality estimates for ages below 5 derived from the first step were combined with mortality estimates for ages 5 and above derived from the second step to produce a full set of age-specific death rates and from those, complete life tables. The methodological procedures followed by other producers of life table estimates for Costa Rica (namely WHO and the GBD) are poorly documented. What the documentation suggests is that all appear to have used the same source of raw data, meaning vital statistics and population estimates or census data published by INEC- CCP. The general process followed within the GBD relies on statistical modeling following the Bayesian approach, but the specific model implemented was too poorly documented for reproducibility at the time this article was prepared. The rest of this paper will thus place greater emphasis on the comparison between our own estimates and those from the UNPD and LAMBdA rather than those from WHO and the GBD. Figures 1 and 2 compare the values of e0 and e80 by sex and calendar year from these various sources. The UNPD and LAMBdA estimates of life expectancy at birth and at age 80 are much lower than the corresponding estimates from INEC-CCP and the GBD, though there is increasing convergence across all sources over time. Since 2000, the UNPD estimates for e0 differ from the INEC-CCP estimates by less than one year, compared to more than 6 years in the 1950s and early 1960s, but the LAMBdA estimates for 2005 are still more than 2.5 years lower than the official estimates. For e80, the LAMBdA estimates continue to be 1.7 (for women) to 1.8 (for men) years lower at this later date. 1. Introduction Nonetheless, taken together, these estimates suggest that Costa Rica has experienced a dramatic increase in survival over the past 65 years, particularly during the period from 1950 to 1975, but the reliability of these mortality trends depends on the quality of the underlying data. 838 http://www.demographic-research.org Demographic Research: Volume 40, Article 29 Demographic Research: Volume 40, Article 29 Figure 1: Estimates of life expectancy at birth (e0) for Costa Rica, 1950–2015, by sex Note: Estimates from the UNPD are based on life tables for 5-year periods and are plotted at the midpoint of the period. Source: UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015); GBD (Institute for Health Metrics and Evaluation 2016); WHO (World Health Organization 2014); INEC-CCP (Instituto Nacional de Estadística y Censos 2009, 2017a); and LAMBdA (Palloni, Pinto-Aguirre, and Beltrán-Sánchez 2016). 50 55 60 65 70 75 80 85 1950 1960 1970 1980 1990 2000 2010 e0 a) Males UNPD GBD WHO INEC-CCP LAMBdA 50 55 60 65 70 75 80 85 1950 1960 1970 1980 1990 2000 2010 e0 b) Females UNPD GBD WHO INEC-CCP LAMBdA Figure 1: Estimates of life expectancy at birth (e0) for Costa Rica, 1950–2015, by sex 50 55 60 65 70 75 80 85 1950 1960 1970 1980 1990 2000 2010 e0 a) Males UNPD GBD WHO INEC-CCP LAMBdA Figure 1: Estimates of life expectancy at birth (e0) for Costa Rica, 1950–2015, by sex Note: Estimates from the UNPD are based on life tables for 5-year periods and are plotted at the midpoint of the period. Source: UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015); GBD (Institute for Health Metrics and Evaluation 2016); WHO (World Health Organization 2014); INEC-CCP (Instituto Nacional de Estadística y Censos 2009, 2017a); and LAMBdA (Palloni, Pinto-Aguirre, and Beltrán-Sánchez 2016). 50 55 60 65 70 75 80 85 1950 1960 1970 1980 1990 2000 2010 e0 b) Females UNPD GBD WHO INEC-CCP LAMBdA Note: Estimates from the UNPD are based on life tables for 5-year periods and are plotted at the midpoint of the period. Source: UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015); GBD (Institute for Health Metrics and Evaluation 2016); WHO (World Health Organization 2014); INEC-CCP (Instituto Nacional de Estadística y Censos 2009, 2017a); and LAMBdA (Palloni, Pinto-Aguirre, and Beltrán-Sánchez 2016). 1. Introduction http://www.demographic-research.org 839 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 Figure 2: Estimates of life expectancy at age 80 (e80) for Costa Rica, 1950– 2015, by sex Note: Estimates from the UNPD and INEC-CCP (prior to 1995) are based on life tables for 5-year periods and are plotted at th midpoint of the period. Sources: UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015); INEC-CCP (Centr Centroamericano de Población 2017c; Instituto Nacional de Estadística y Censos 2017a); and LAMBdA (Palloni, Pinto-Aguirre, an Beltrán-Sánchez 2016). 5 6 7 8 9 10 11 1950 1960 1970 1980 1990 2000 2010 e80 a) Males UNPD INEC-CCP LAMBdA 5 6 7 8 9 10 11 1950 1960 1970 1980 1990 2000 2010 e80 b) Females UNPD INEC-CCP LAMBdA Figure 2: Estimates of life expectancy at age 80 (e80) for Costa Rica, 1950– 2015, by sex 5 6 7 8 9 10 11 1950 1960 1970 1980 1990 2000 2010 e80 a) Males UNPD INEC-CCP LAMBdA 2: Estimates of life expectancy at age 80 (e80) for Costa Rica, 1950– 2015, by sex 5 6 7 8 9 10 11 1950 1960 1970 1980 1990 2000 2010 e80 b) Females UNPD INEC-CCP LAMBdA b) Females b) Females Note: Estimates from the UNPD and INEC-CCP (prior to 1995) are based on life tables for 5-year periods and are plotted at the midpoint of the period. Sources: UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015); INEC-CCP (Centro Centroamericano de Población 2017c; Instituto Nacional de Estadística y Censos 2017a); and LAMBdA (Palloni, Pinto-Aguirre, and Beltrán-Sánchez 2016). Note: Estimates from the UNPD and INEC CCP (prior to 1995) are based on life tables for 5 year periods and are plotted at the midpoint of the period. Sources: UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015); INEC-CCP (Centro Centroamericano de Población 2017c; Instituto Nacional de Estadística y Censos 2017a); and LAMBdA (Palloni, Pinto-Aguirre, and Beltrán-Sánchez 2016). 1. Introduction http://www.demographic-research.org 840 Demographic Research: Volume 40, Article 29 The objectives of this paper are to: (1) evaluate the data quality of mortality estimates for Costa Rica 1950–2013 using standard methods applied to the official vital statistics and population counts without making further adjustments, and (2) compare the estimates with those produced by others (in some cases based on adjusted data) and with corresponding estimates for the 38 countries that were included in the Human Mortality Database (HMD) at the time the Costa Rican data series was constructed. The HMD is an international repository of mortality series for (currently 40) countries with high-quality data produced by a collaboration between the Department of Demography at the University of California, Berkeley, and the Max Planck Institute for Demographic Research (MPIDR) in Rostock (for details, see Barbieri et al. 2015). Starting in 2006, the project has also benefited from continuous support by the French Institute for Demographic Studies. Since its debut in 2002, the HMD has provided detailed mortality and population data to all those interested in the history of human longevity. With 50,000 registered users and over 5,000 publications citing the database as a main data source, the HMD is a leading resource for mortality research. The methods used in the HMD have become the gold standard to produce historical mortality indicators for countries with demographic data of good quality. One of the main advantages of the HMD is the standardized protocol used to construct life tables for all countries and time periods. Because the original data used to produce the HMD series are also published on the website (www.mortality.org), the whole process is transparent. Here, we applied the HMD process to evaluate the mortality data for Costa Rica. Though the HMD protocol cannot yield accurate mortality estimates for a country with defective data, the well-documented methodology can reveal questionable or implausible patterns. Indeed, the final goal of our study is not to produce a life table series that is as accurate as possible but to use it as an instrument to assess the reliability of national demographic statistics. 2. Data and methods The HMD methods protocol relies on classic demographic techniques (Wilmoth et al. 2017). The basic data used to construct mortality estimates are directly collected from national statistics offices. http://www.demographic-research.org 2.1 The Human Mortality Database protocol to construct life tables In principle, the raw data collected for the HMD (i.e., vital statistics and census counts or official population estimates) is not adjusted, though it might have been corrected for http://www.demographic-research.org 841 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 under- or overcount by the national statistics offices before publication. Following the HMD protocol, birth counts by sex are used mainly for estimating the relative size of individual cohorts. Death counts by sex and detailed age are used for the numerator of the age-specific mortality rates. Deaths, births, and population counts – also by sex and detailed age – are used to derive exposure counts below age 80 years. At age 80 and above, in part to account for lower data quality in most countries and for most historical periods, exposures-to-risk are estimated through a combination of the Extinct Cohort method and the Survival Ratio method that rely on the cohort-specific death counts. At very high ages (95+ years or below if there are fewer than 100 men or women surviving in any given country-year), the mortality rates are smoothed (to avoid the large random fluctuations associated with small numbers) using a technique initially proposed by Vaino Kannisto (Thatcher, Kannisto, and Andreev 2002). At these high ages, the underlying hazards are estimated by fitting a logistic curve with an asymptote at one to the age-specific mortality rates. The resulting age-specific death rates are combined to derive complete life tables by sex using conventional demographic formulas. The raw data for Costa Rica (see Supplementary archive, S1. Raw “Input Data” for Costa Rica) comprises official vital statistics, population estimates, and census counts available online (Centro Centroamericano de Población 2017a, 2017b; Palloni, Pinto- Aguirre, and Beltrán-Sánchez 2016). Since 1950, Costa Rica has conducted censuses on a regular basis (i.e., in 1950, 1963, 1973, 1984, 2000, and 2011). For the purpose of computing mortality rates and life tables, we use the official annual population estimates published by INEC-CCP rather than the original census counts. To produce these estimates, INEC-CCP adjusts for the underregistration of deaths, estimated net migration, age misreporting, and census underenumeration. 2.1 The Human Mortality Database protocol to construct life tables The INEC-CCP population estimates are constructed using backward projection, starting with the estimated population in 2011 – which has been adjusted for age misreporting and the undercount in the 2011 census – and using estimates of net migration and death counts that have been adjusted for underregistration (Rosero-Bixby, personal communication, November 2, 2016). We use the official census counts only for comparison. Our unadjusted life tables for Costa Rica (see Supplementary archive, S2. Unadjusted Life Tables for Costa Rica) were calculated following the HMD Methods Protocol, Version 5 (Wilmoth et al. 2007). http://www.demographic-research.org 2.2 Assessing data reliability through internal consistency checks We begin our data quality evaluation by graphing population and census counts by single year of age to visualize age-heaping patterns. Then, we review external evidence regarding problems of age exaggeration and use the intercensal cohort method (Elo and http://www.demographic-research.org http://www.demographic-research.org 842 Demographic Research: Volume 40, Article 29 Preston 1994) for diagnosing age exaggeration (see Supplementary archive, S3. Intercensal Cohort Method for details). Next, we evaluate relative completeness using Death Distribution Methods (DDM). Death Distribution Methods are designed to estimate the completeness of death registration relative to population counts. The two most well-established methods are the General Growth Balance method (GGB, Brass 1975; Hill 1987; Hill, You, and Choi 2009) and the Synthetic Extinct Generation method (SEG, Bennett and Horiuchi 1981; Bennett and Horiuchi 1984). Hill, You, and Choi (2009) have also developed a hybrid method (GGB-SEG), which addresses the sensitivity of the SEG method to changes in coverage between consecutive censuses. Palloni, Pinto-Aguirre, and Beltrán-Sánchez (2015) evaluate 12 different methods of estimating relative completeness and conclude that, in most cases, GGB-SEG performs best. Palloni, Beltrán-Sanchez, and Pinto- Aguirre (2017) also propose a procedure that uses all the plausible estimators from such methods, which they argue leads to more robust and conservative hypothesis tests than relying on a single estimate. Hill, You, and Choi (2009) also conclude that in populations not affected by migration, it is best to use GGB-SEG, fit to the age range 5+ to 65+ because age misreporting is more of a problem above age 65, independently from the issue of completeness per se. In populations substantially affected by migration, Hill, You, and Choi (2009) argue that the GGB and GGB-SEG methods underestimate coverage (thus, overestimating mortality), whereas the SEG method does the opposite. Thus, in the case of migration, they find the best strategy (i.e., smallest error) is to fit both GGB and SEG to the age range 30+ to 65+ and then average the results (Hill, You, and Choi 2009). Using this higher starting age reduces the effects of migration, which tends to be concentrated at younger ages. We used the DDM R package (Lima, Queiroz, and Riffe 2016) to compute the GGB and GGB-SEG estimates of relative completeness, fitted to three age ranges: 5+ to 65+; 30+ to 65+; and the ‘optimal’ age range as determined by the DDM package. http://www.demographic-research.org 2.2 Assessing data reliability through internal consistency checks We assess relative completeness based on the official population estimates rather than the original census counts because we rely on the former rather than the latter for the purpose of computing our life tables. These population estimates have already been adjusted by the national statistics office for census underenumeration, although the death counts have not been corrected for underregistration. Thus, our estimates may suggest lower relative completeness than they would if we had used the original census counts as inputs because the denominators have been inflated whereas the numerators have not, thus creating a numerator/denominator bias. Hill, You, and Choi (2009) conclude that these methods work very well when the only errors in the data are those for which the methods were developed. According to Hill, You, and Choi (2009), both the GGB and GGB-SEG methods do reasonably well in the presence of age misreporting or age variation in census coverage. Unfortunately, 843 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 the method is not robust when there is significant net migration or when the completeness of death registration varies by age, as these phenomena can create large distortions (double-digit percentage errors in adult mortality probabilities). While these methods can be adapted to account for migration, it is rare that estimates of net migration are accurate enough to be useful (IUSSP-UNFPA 2017a, 2017b). Net migration is often low enough to ignore when dealing with national populations, but caution should be exercised in interpreting the results if there is a substantial net migration (IUSSP-UNFPA 2017a, 2017b). Furthermore, the fundamental assumptions underlying these methods are often unrealistic, resulting in erroneous results (Li and Gerland 2016). 2.3 Assessing data reliability through external consistency checks We assess the external plausibility of the life table estimates by comparing our unadjusted estimates of e0 and e80 for Costa Rica with those produced by LAMBdA (Palloni, Pinto, and Beltrán-Sánchez 2016), UNPD (United Nations, Department of Economic and Social Affairs, Population Division 2015), and INEC-CCP (Centro Centroamericano de Población 2017c; Instituto Nacional de Estadística y Censos 2009, 2017a). In addition to comparing our results with those in LAMBdA, we compare the age structure of mortality resulting from our life tables with those for all countries and time periods in the HMD (University of California and Max Planck Institute for Demographic Research 2016). We evaluate how those comparisons differ when we use adjusted estimates from LAMBdA, which reports the highest mortality, rather than our own unadjusted estimates. 3.1.1 Age heaping Although there was clear evidence of age heaping in the 1950 census (Figure 3), age reporting improved considerably by the 1984 census (Figure 4) and age heaping had virtually disappeared by the most recent census in 2011 (Figure 5). In contrast, there is less age heaping in the official population estimates even in 1950, and by 1984, age heaping is no longer visible in the population estimates (Figure 4), as also confirmed by http://www.demographic-research.org http://www.demographic-research.org 844 Demographic Research: Volume 40, Article 29 the calculation of the classic Whipple’s and Myers’ indices of age attraction (Shryock, Siegel, and Larmon 1973). As noted in the Data section, the INEC-CCP population estimates are constructed using backward projection, starting with population estimates for 2011 that have been corrected for age misreporting. By relying on the official population estimates rather than using the original census counts, we avoid most of the problems associated with age heaping. Figure 3: Comparison of official population estimates with census counts, 1950, by sex e 3: Comparison of official population estimates with census counts, 1950, by sex http://www.demographic-research.org 845 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 4: Comparison of official population estimates with census counts, 1984, by sex Figure 4: Comparison of official population estimates with census counts, 1984, by sex Figure 4: Figure 4: Comparison of official population estimates with census counts, 1984, by sex Figure 5: Comparison of official population estimates with census counts, 2011, by sex 1984, by sex Figure 5: Comparison of official population estimates with census counts, 2011, by sex Figure 5: Comparison of official population estimates with census counts, 2011, by sex 846 http://www.demographic-research.org Figure 5: Comparison of official population estimates with census counts, 2011, by sex http://www.demographic-research.org 846 Demographic Research: Volume 40, Article 29 Age attraction, as measured by Whipple’s and Myers’ indices, is slightly more prominent in the death counts than in the population counts. However, they remain well within the range of good-quality data. For the period since 1980, Myers’ index (computed for the age range 30–99) hovers around 5 to 8 for the deaths (with an occasional value greater than 9) versus less than 2 (except for 1984, when values are slightly greater) for the population counts; Whipple’s index (computed for the age range 32–92) fluctuates between 96 and 110 for the death counts (with one value as high as 113 for females in 1981) and between 99 and 105 for the population counts (results not shown). There is little evidence of age heaping in the mortality rates after the mid-1970s. http://www.demographic-research.org 3.1.2 Age exaggeration Age exaggeration is a more insidious problem than age heaping because it is difficult to identify, quantify, and thus correct, but it can severely distort mortality estimates. Preston, Elo, and Stewart (1999) demonstrate that regardless of the nature of age misreporting (overstatement, understatement, or symmetric misreporting), mortality rates at the oldest ages are biased downward (i.e., old-age longevity is overestimated). External evaluation indicates that age exaggeration tends to bias census counts at older ages upward in Costa Rica. One study suggested that the population aged 80 years and older in the 1984 census was overcounted by as much as 50% (Ministerio de Planificación Nacional y Politica Económica, U.N. Centro Latinoamericano de Demografía, and Dirección General de Estadística y Censos 1988; cited in Rosero- Bixby 2008). Analysis of the 2000 census indicated that the population aged 90 years and older was still overestimated by 20% because of age exaggeration (Instituto Nacional de Estadistica y Censos and Centro Centroamericano de Población 2002; cited in Rosero-Bixby 2008). Another evaluation of the 2000 census found that age exaggeration inflated the population aged 95 and older by 22%, while the population aged 99 and older was overcounted by 42% (Rosero-Bixby, Brenes, and Collado 2004). However, official annual population estimates have been adjusted by the national statistics office for this problem, another reason for us to use these rather than the raw census counts to compute the denominator for the mortality rates. Unfortunately, in the absence of an external data source with correct dates of birth or age for comparison, it is difficult to diagnose age exaggeration. Most of the aforementioned evaluations were based on comparisons with the population voter registry. However, we do not have access to that population registry. Instead, we compared the population estimates built by the national statistics office using the cohort component method with the unadjusted census counts. Results indicate inconsistencies, 847 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 but it is unclear whether they stem from age exaggeration or from other data problems, such as large net-migration rates, underregistration of deaths, or incomplete enumeration in one or both censuses (see Supplementary archive, S3. Intercensal Cohort Method). 3.2 Relative completeness Table 1: Estimates of relative completeness of death registration, Costa Rica, 1950–2011 Intercensal period 1950–1963 1963–1973 1973–1984 1984–2000 2000–2011 Men GGB Ages 5+ to 65+ 96% 91% 90% 96% 99% Ages 30+ to 65+ 98% 93% 93% 97% 106% Optimal age range 98% 87% 97% 98% 101% GGB-SEG Ages 5+ to 65+ 94% 91% 89% 92% 96% Ages 30+ to 65+ 96% 92% 91% 93% 101% Optimal age range 96% 88% 95% 94% 98% Women GGB Ages 5+ to 65+ 95% 91% 91% 93% 88% Ages 30+ to 65+ 95% 92% 94% 95% 97% Optimal age range 98% 88% 99% 98% 99% GGB-SEG Ages 5+ to 65+ 94% 91% 91% 90% 88% Ages 30+ to 65+ 94% 92% 93% 92% 94% Optimal age range 96% 89% 97% 94% 96% Note: These estimates are calculated using the DDM R package developed by Tim Riffe based on deaths and the official population estimates (not the original census counts). Estimates of completeness between 95% and 105% are highlighted in green, values of 90–94.9% or 105.1–110% are highlighted in yellow, and values below 90% or above 110% are highlighted in red. Note: These estimates are calculated using the DDM R package developed by Tim Riffe based on deaths and the official population estimates (not the original census counts). Estimates of completeness between 95% and 105% are highlighted in green, values of 90–94.9% or 105.1–110% are highlighted in yellow, and values below 90% or above 110% are highlighted in red. Note: These estimates are calculated using the DDM R package developed by Tim Riffe based on deaths and the official population estimates (not the original census counts). Estimates of completeness between 95% and 105% are highlighted in green, values of 90–94.9% or 105.1–110% are highlighted in yellow, and values below 90% or above 110% are highlighted in red. Similar estimates (GGB and GGB-SEG) for existing HMD countries since the 1940s suggest that relative completeness is close to 100% in many, but not all, cases (Lima 2016, personal communication). Even for the period after 2000, some estimates are surprisingly low (e.g., 91% for men in Canada, 92% for US women, and 91% for US men). There are also many cases where estimated completeness is far above 100%, in some instances more than 115% (e.g., Icelandic men in the 2000s; Portuguese women in the 1960s). http://www.demographic-research.org 3.2 Relative completeness Demographers at Centro Centroamericano de Población (CCP) acknowledge that Costa Rica has had a problem of undocumented migration since the 1980s, and thus, the accuracy of migration estimates remains uncertain (Centro Centroamericano de Población 2013; Rosero-Bixby, Brenes Camacho, and Chen-Mok http://www.demographic-research.org http://www.demographic-research.org 848 Demographic Research: Volume 40, Article 29 2002), but it seems safe to assume that net migration has been positive and fluctuating over the past three to four decades. 2002), but it seems safe to assume that net migration has been positive and fluctuating over the past three to four decades. Table 1: Estimates of relative completeness of death registration, Costa Rica, 1950–2011 Intercensal period 1950–1963 1963–1973 1973–1984 1984–2000 2000–2011 Men GGB Ages 5+ to 65+ 96% 91% 90% 96% 99% Ages 30+ to 65+ 98% 93% 93% 97% 106% Optimal age range 98% 87% 97% 98% 101% GGB-SEG Ages 5+ to 65+ 94% 91% 89% 92% 96% Ages 30+ to 65+ 96% 92% 91% 93% 101% Optimal age range 96% 88% 95% 94% 98% Women GGB Ages 5+ to 65+ 95% 91% 91% 93% 88% Ages 30+ to 65+ 95% 92% 94% 95% 97% Optimal age range 98% 88% 99% 98% 99% GGB-SEG Ages 5+ to 65+ 94% 91% 91% 90% 88% Ages 30+ to 65+ 94% 92% 93% 92% 94% Optimal age range 96% 89% 97% 94% 96% Note: These estimates are calculated using the DDM R package developed by Tim Riffe based on deaths and the official population estimates (not the original census counts). Estimates of completeness between 95% and 105% are highlighted in green, values of 90–94.9% or 105.1–110% are highlighted in yellow, and values below 90% or above 110% are highlighted in red. 3.2 Relative completeness The estimates for GGB and GGB-SEG suggest that coverage of death registration was above 90% in most years, with a few exceptions (Table 1). Estimates for the 1950–1963 period appear surprisingly good. From 1963 to 1984, the results suggest lower levels of relative completeness (less than 90% by some estimates). Although relative completeness looks better in 1984–2000, the results indicate some problems even for the most recent period (2000–2011), particularly among women. The sensitivity of the results to the choice of age range for fitting is especially apparent for women in the most recent period: Estimates based on the optimal age range suggest that relative completeness is good (>95%), whereas estimates based on the age range 5+ to 65+ imply poor (88%) relative completeness. We are suspicious of these estimates because death registration is believed to be virtually complete since 2000. One report suggests that death records were already 92% complete in 1970 and that continuing progress has increased this figure to 99% as early as 1990 (Solano 2009). Also, if deaths were underregistered, we would have expected the underregistration to be concentrated among men rather than women because estimates of mortality for older Costa Rican men are lower than anticipated. Starting around ages 65–70 years, the sex ratio (male/female) in the Costa Rica mortality rates is lower than in other populations, even in those with a smaller sex difference in the expectation of life at birth (e.g., Sweden); data not shown. The GGB and GGB-SEG estimates should be interpreted with caution because it is likely that some important assumptions of the method have been violated, in particular the assumption of a closed population (i.e., no net migration), which is particularly problematic for Costa Rica, a country with fluctuating migration flows. Costa Rica has experienced positive and irregular net migration over the 1990s and 2000s with estimated net migration around 1,000 per year during 1990–1992, increasing sharply to more than 27,000 in 1993, remaining above 25,000 per year through 1998, and then falling to less than 10,000 per year after 2000 (Instituto Nacional de Estadística y Censos 2013). 3.2 Relative completeness Again, the results appear to be sensitive to the age range used for 849 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 fitting because the degree of violation of the method’s basic assumptions (with regard to migration in particular) varies across the age range. For example, when we estimated relative completeness for Swedish women in 2000–2011 (where we expect data to be virtually perfect), the GGB estimates ranged from 85% to 98% and the GGB-SEG estimates ranged from 83% to 97% depending on the ages used for fitting. It is thus difficult to determine whether the GGB and GGB-SEG estimates of completeness are in fact meaningful in Costa Rica given the international migration trends. The GGB and GGB-SEB estimates for Costa Rican men during 2000–2011 imply that relative completeness is as good as, or even better than, some HMD countries (i.e., Australia, Canada, Luxembourg, the Netherlands, and the United States) in the most recent time period. For Costa Rican women, it depends on the age range used for fitting: For ages 5+ to 65+, estimated completeness for 2000–2011 appears to have been worse than any HMD country, but for the optimal age range (35+ to 75+), estimated completeness appears to have been as good as many HMD countries. 3.3 Comparing Costa Rica mortality estimates across all sources Prior to 1980, our estimates of the probability of dying in the first year of life (q0) are lower than those provided by INEC-CCP and LAMBdA (Figure 6). After 1980, our estimates are similar to INEC-CCP, but the estimates for men are still lower than in LAMBdA (Figure 6). Surprisingly, the LAMBdA estimate for women in 2005 is notably lower than both our estimate or the one from INEC-CCP, although the absolute difference between the LAMBdA estimate of q0 and our own estimate is very small (i.e., less than 1.5 per thousand). The rapid decline in the infant mortality rate in Costa Rica – from a high of around 90 per thousand in 1950 down to less than 8 per thousand in 2015 (Instituto Nacional de Estadística y Censos 2017b) – appears to be real. With respect to life expectancy at birth (e0), our estimates prior to 1970 are notably higher (i.e., implying lower mortality) than estimates from INEC-CCP, LAMBdA, and UNPD (Figure 7). The difference between our estimates and those from the UNPD ranges from 3.1 to 7.8 years for the 1950s and 1960s; LAMBdA’s estimates are also more than 5 years lower than ours in 1956 and more than 4 years lower in 1968. 3.3 Comparing Costa Rica mortality estimates across all sources http://www.demographic-research.org 850 http://www.demographic-research.org Demographic Research: Volume 40, Article 29 Figure 6: Comparison of the probability of dying in the first year of life (q0, log scale)with external estimates, 1950–2014, by sex Figure 7: Comparison of life expectancy at birth (e0) with external estimates, 1950–2014, by sex .01 .025 .05 .1 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA (adjusted) INEC-CCP q0 (log scale) Year 55 65 75 85 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA INEC-CCP UNPD e0 Year Comparison of the probability of dying in the first year of life (q0, log scale)with external estimates, 1950–2014, by sex Figure 6: Comparison of the probability of dying in the first year of life (q0, log scale)with external estimates, 1950–2014, by sex .01 .025 .05 .1 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA (adjusted) INEC-CCP q0 (log scale) Year Figure 7: Comparison of life expectancy at birth (e0) with external estimates, 1950–2014, by sex 65 75 85 a) Males b) Females e0 Comparison of life expectancy at birth (e0) with external estimates, 1950–2014, by sex Figure 7: Comparison of life expectancy at birth (e0) with external estimates, 1950–2014, by sex 55 65 75 85 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA INEC-CCP UNPD e0 Year 55 65 75 85 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA INEC-CCP UNPD e0 Year http://www.demographic-research.org 851 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 Our estimates of e0 are closer to the INEC-CCP estimates in the 1970s and 1980s, but they are still substantially higher than those from LAMBdA and UNPD by as much as 4.6 years. Since 2000, our estimates are similar to the INEC-CCP and UNPD estimates, but the LAMBdA estimates suggest much lower e0 even as recently as 2005. The LAMBdA estimates of life expectancy in 2005 (73.7 years for men and 78.7 years for women) are more than 2.5 years lower than ours, which are the same as INEC- CCP’s (76.4 for men and 81.2 for women). 3.3 Comparing Costa Rica mortality estimates across all sources Our estimated e0 for women in 2005 would put Costa Rica on par with UK women, whereas the LAMDdA-adjusted estimate implies a level of life expectancy closer to that of Estonian women in that same year. A comparison of HMD estimates of e0 for Chile (the only Latin American country in the HMD) in 2006 with the corresponding LAMBdA estimates indicates a difference of similar magnitude (with the LAMBdA estimate 2.5 years lower than the HMD for men and 2.7 years lower for women), even though demographic statistics are believed to have been of high quality in Chile (Núñez and Icaza 2006). As mortality declines, old-age life expectancy becomes increasingly important in determining overall life expectancy. When we compare our estimates of e80 with those produced by others, we find that they are somewhat higher than those from INEC-CCP prior to 1980 but much higher than in LAMBdA throughout the period (Figure 8). Compared with LAMBdA, our estimate of e80 in 1956 is 64% higher for women (9.2 vs. 5.6 years) and 43% higher for men (7.7 vs. 5.4 years). Even in 2005, our estimates of e80 are still more than 20% higher than in LAMBdA. Our estimates are also substantially higher than the UNPD estimates for men prior to 1995 and for women prior to 1980. The patterns in our estimates are consistent with a progressive improvement in age reporting, with a decline in old-age life expectancy during the 1950s and 1960s and stagnation in the 1980s, continuing into the 1990s for men (Figure 8). Trends are more plausible since 2000. 852 http://www.demographic-research.org Demographic Research: Volume 40, Article 29 Figure 8: Comparison of life expectancy at age 80 (e80) with exte estimates, 1950–2014, by sex 5 6 7 8 9 10 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA INEC-CCP UNPD e80 Year Figure 8: Comparison of life expectancy at age 80 (e80) with external estimates, 1950–2014, by sex 5 6 7 8 9 10 1950 1960 1970 1980 1990 2000 2010 1950 1960 1970 1980 1990 2000 2010 a) Males b) Females Ours (unadjusted) LAMBdA INEC-CCP UNPD e80 Year Figure 8: Comparison of life expectancy at age 80 (e80) with external estimates, 1950–2014, by sex http://www.demographic-research.org 4.1 Life expectancy at birth (e0) At the beginning of the data series (1950), life expectancy at birth in Costa Rica fell near to the bottom of the range defined by HMD countries (25 with data for 1950) but increased rapidly, between 1950 and 1980 in particular (Figure 9). By 2013, Costa Rican men (with an unadjusted e0 of 77.8 years) would rank 25th among the 38 HMD countries (listed from the lowest to the highest level of life expectancy at birth), between Slovenia and Taiwan. For women, Costa Rica (at 82.3 years) would rank 26th, between Denmark and the Czech Republic. 853 http://www.demographic-research.org Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 Figure 9: Life expectancy at birth (e0), Costa Rica (unadjusted and adjusted estimates, shown in red) compared with HMD populations (shown in other colors), 1950–2015, by sex Our current (unadjusted) estimate of e0 for Costa Rica (79.9 years in 2013, both sexes combined) is higher than the United States (78.9 in 2015) but lower than the United Kingdom (80.9 in 2013) and well below Japan, the leader in life expectancy (83.8 in 2014). Life expectancy at birth (e0), Costa Rica (unadjusted and adjusted estimates, shown in red) compared with HMD populations (shown in other colors), 1950–2015, by sex Figure 9: Life expectancy at birth (e0), Costa Rica (unadjusted and adjusted estimates, shown in red) compared with HMD populations (shown in other colors), 1950–2015, by sex Figure 9: Life expectancy at birth (e0 estimates, shown in red) co other colors), 1950–2015, b Our current (unadjusted) estimate of e0 for Costa Rica (79.9 years in 2013, both sexes combined) is higher than the United States (78.9 in 2015) but lower than the United Kingdom (80.9 in 2013) and well below Japan, the leader in life expectancy (83.8 in 2014). Our current (unadjusted) estimate of e0 for Costa Rica (79.9 years in 2013, both sexes combined) is higher than the United States (78.9 in 2015) but lower than the United Kingdom (80.9 in 2013) and well below Japan, the leader in life expectancy (83.8 in 2014). 4.2 Life expectancy at age 80 (e80) In contrast with e0, e80 looks surprisingly high for Costa Rica (Figure 10). In 1950, our unadjusted estimates imply that old-age life expectancy was notably higher in Costa Rica than any HMD country. For women, the remaining length of life at age 80 was estimated at 8.5 years compared with 7.2 for the HMD leader, Iceland. Among men, it was estimated at 7.2 years in Costa Rica compared with 6.8 for Bulgaria and 6.4 for http://www.demographic-research.org http://www.demographic-research.org 854 Demographic Research: Volume 40, Article 29 Iceland. Between 1950 and 1970, e80 in Costa Rica appeared to have declined, which likely results from improved data quality as previously mentioned: In the early part of the data series e80 was probably overestimated in Costa Rica because of age exaggeration and/or incomplete death registration. Figure 10: Life expectancy at age 80 (e80), Costa Rica (unadjusted and adjusted estimates, shown in red) compared with HMD populations (shown in other colors), 1950–2015, by sex Figure 10: Life expectancy at age 80 (e80), Costa Rica (unadjusted and adjusted estimates, shown in red) compared with HMD populations (shown in other colors), 1950–2015, by sex In 2013, at 10.0 years, e80 in Costa Rican women would tie with the United States and New Zealand for the 8th position among the 38 HMD countries, and, at 8.9 years, Costa Rican men would tie with Iceland for 2nd place just below the world leader, France (9.0). In 2013, at 10.0 years, e80 in Costa Rican women would tie with the United States and New Zealand for the 8th position among the 38 HMD countries, and, at 8.9 years, Costa Rican men would tie with Iceland for 2nd place just below the world leader, France (9.0). 855 http://www.demographic-research.org Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 4.3 Relationship between child and older-age mortality In Figure 11, we plot the relationship between child mortality (as captured by the probability of dying in the first five years of life, 5q0) and older-age mortality (measured by the probability of dying between ages 60 and 80, 20q60) for Costa Rica alongside all HMD populations for all years available (i.e., back to the mid-1800s for Sweden). Clearly there is a direct relationship between child and early old-age mortality as the HMD estimates are clustered within an elongated point cloud. Over time, the estimates for any given country are likely to proceed from the upper right corner of the graph (high child and high older-age mortality) toward the lower left corner (lower levels of both child and older-age mortality). Child mortality (5q0, log scale) by old-age mortality (20q60, log scale), Costa Rica (unadjusted and adjusted) compared with HMD populations, by sex Figure 11: Child mortality (5q0, log scale) by old age mortality (20q60, log scale), Costa Rica (unadjusted and adjusted) compared with HMD populations, by sex For both women and men, Costa Rica is a clear outlier: At any given level of child mortality, mortality between ages 60 and 80 (based on our unadjusted estimates) falls well outside the range of HMD estimates even for the most recent years. This unusual pattern could reflect a problem of data quality. It could stem from overestimation of 1956 2005 .2 .4 .6 .8 1 20q60 (log scale) .001 .01 .1 .2 .4 .6 5q0 (log scale) a) Males 1956 2005 .2 .4 .6 .8 1 20q60 (log scale) .001 .01 .1 .2 .4 .6 5q0 (log scale) b) Females HMD populations CRI (unadjusted) CRI (LAMBdA-adjusted) populations, by sex For both women and men, Costa Rica is a clear outlier: At any given level of child 1956 2005 .2 .4 .6 .8 1 20q60 (log scale) .001 .01 .1 .2 .4 .6 5q0 (log scale) a) Males 1956 2005 .2 .4 .6 .8 1 20q60 (log scale) .001 .01 .1 .2 .4 .6 5q0 (log scale) b) Females HMD populations CRI (unadjusted) CRI (LAMBdA-adjusted) For both women and men, Costa Rica is a clear outlier: At any given level of child mortality, mortality between ages 60 and 80 (based on our unadjusted estimates) falls well outside the range of HMD estimates even for the most recent years. This unusual pattern could reflect a problem of data quality. http://www.demographic-research.org 4.4 Pattern of mortality at higher ages in comparison with Sweden Vaino Kannisto, renowned for his analysis of old age mortality, suggested looking at the ratios of mortality indicators at higher ages to assess their validity. Following this strategy, we selected Sweden for comparison as the excellent quality of demographic data in this country has been demonstrated (Statistiska Centralbyrån 1969; Wilmoth and Lundström 1996). We compared the relationship between life expectancy at birth and three sets of life table ratios: the ratio of the number of person-years lived at ages 100 and above (T100) to the number of person-years lived at ages 80 and above (T80); the ratio of the number of person-years lived at ages 85 and above (T85) to the number of person-years lived at ages 70 and above (T70); and the ratio of the probability of dying between the ages of 65 and 80 (15q65) to the expectation of life at age 80 (e80; see Supplementary archive, S4. Figures S1–S3). The first two ratios are much higher in Costa Rica compared to Sweden at the same level of life expectancy at birth, while the third ratio is much lower in Costa Rica than in Sweden. Thus, mortality at the oldest ages is lower than we would expect based on the age pattern of mortality observed in Sweden. 4.3 Relationship between child and older-age mortality It could stem from overestimation of For both women and men, Costa Rica is a clear outlier: At any given level of child mortality, mortality between ages 60 and 80 (based on our unadjusted estimates) falls well outside the range of HMD estimates even for the most recent years. This unusual pattern could reflect a problem of data quality. It could stem from overestimation of http://www.demographic-research.org 856 Demographic Research: Volume 40, Article 29 mortality below age 5 (5q0), although it is rare for child mortality to be overestimated; typically, the problem is underestimation because of incomplete registration of infants who died shortly after birth, but it is possible that the adjustment of census population counts carried out by the national statistics office below age 5 was insufficient, which would produce this pattern. Alternatively, it could result from the underestimation of mortality between the ages of 60 and 80 years (20q60) owing to uncomplete coverage of death registration or to age exaggeration, which has been shown to be widespread in Latin America (Palloni, Pinto-Aguirre, and Beltrán-Sánchez 2015). http://www.demographic-research.org 5. Intercountry comparisons with LAMBdA life tables In Figures 9 and 10, we also show the adjusted estimates for e0 and e80 from LAMBdA alongside our own unadjusted estimates for Costa Rica and HMD countries. The LAMBdA-adjusted estimates suggest that in 1950 e0 in Costa Rica was as low as or lower than in any HMD country and that e80 ranked near the bottom. By 2005, Costa Rica ranked near the middle of the pack in terms of both e0 and e80. As for the relationship between child and early old-age mortality (Figure 11), the LAMBdA estimates for Costa Rica are much more consistent with the mortality experience of HMD countries than the unadjusted estimates. Among women, the 857 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 LAMBdA estimates fall well within the range defined by the HMD estimates, though for men they are still at the bottom of the range in the most recent years. However, plausibility does not necessarily mean accuracy, and the documentation available for LAMBdA is too general to determine if the methods implemented to produce mortality indicators in this database were selected because they yielded reasonable estimates or if these are indeed closer to the reality than our unadjusted estimates. 6. Conclusion and discussion Using the HMD methods, which were developed for countries with full demographic coverage and reliable vital registration systems, we find that life expectancy at birth in Costa Rica has risen from 59.3 years in 1950 to 79.9 years in 2013, a level higher than in several high-income countries. Although our estimates indicate that Costa Ricans are long-lived, analysis of mortality trends and patterns as well as comparisons with other estimates suggest that longevity in Costa Rica may be overestimated. Compared with estimates constructed by others who have adjusted for relative completeness and age misreporting, our life expectancy estimates are substantially higher, especially prior to 1980. Mortality at older ages is even more unusual when compared with other estimates for Costa Rica and with other high-income countries. In particular, Costa Rica exhibits surprisingly low old-age mortality relative to child mortality, falling well outside the range defined by 38 high-income countries in the HMD. Our analysis suggests that mortality among the oldest Costa Ricans is almost certainly underestimated prior to 1984, probably because of some combination of incomplete census coverage, incomplete death registration, and age exaggeration. For the more recent period, we cannot draw a definitive conclusion regarding the reliability of the estimates because the evidence is circumstantial and because other studies suggest that Costa Ricans might in fact enjoy unusually low mortality at older ages (Payne 2015; Rosero-Bixby 2008, 2018; Rosero-Bixby and Dow 2009). For example, a prior study that carefully selected birth, death, and population data based on accuracy to compute mortality at ages 90 and over using the Extinct Cohort method found old-age mortality that was higher (i.e., life expectancy was lower) than what official estimates would suggest (Rosero-Bixby 2008). Nonetheless, life expectancy at age 90 among men was still about half a year higher in this study than for any other country, while corresponding estimates for women would put Costa Rica on par with two countries at the top of the distribution (i.e., Japan and the United States). One important source of uncertainty is the size of the immigrant population in Costa Rica, which is the most undercounted subpopulation. 6. Conclusion and discussion Although INEC-CCP has 858 http://www.demographic-research.org Demographic Research: Volume 40, Article 29 published estimates of net migration (Instituto Nacional de Estadística y Censos 2013), demographers admit that there is a problem of undocumented migration in Costa Rica since the 1980s, and thus, they do not know whether the net-migration figures are accurate (Brenes 2016, personal communication). Unaccounted migration may jeopardize the accuracy of adjustment methods, resulting in biased estimates. Migration assumptions are crucial in the implementation of evaluation and adjustment methods. In general, methods such as DDM may be useful for detecting gross errors, but it may be risky to rely on them for fine-tuning estimates, particularly in populations where the assumptions underlying the methods are violated. Although measures of relative completeness suggest some inconsistencies, it is difficult to determine if they truly represent data deficiencies or are simply the result of violations in the method’s assumptions. Part of the apparent longevity advantage of Costa Ricans may be an artifact of data deficiencies. The pattern of declining levels of e80 during 1950–1970, when e0 was rapidly increasing, is particularly suspicious. The most likely explanation for this pattern is improvement in age reporting throughout this period, which is supported by prior external evaluation of the census counts (Ministerio de Planificación Nacional y Politica Económica, U.N. Centro Latinoamericano de Demografía, and Dirección General de Estadística y Censos 1988; cited in Rosero-Bixby 2008). In any event, variability in the level of mortality estimated by diverse institutions together with unusual mortality patterns suggest that only in a context of improved reliability of the basic national statistics will we be able to determine with certainty the level of life expectancy in Costa Rica. http://www.demographic-research.org 7. Acknowledgements This work was supported by awards from the US National Institute on Aging [grant numbers R01-AG011552, R01-AG040245, P30-AG012839] as well as from contributions to the Human Mortality Database project from the Berkeley Center on the Economics and Demography of Aging; the Society of Actuaries in the United States; the United Kingdom Institute and Faculty of Actuaries; the AXA Research Fund in France; Hannover-Re; SCOR; and Reinsurance Group of America. However, any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors alone and do not necessarily represent the official views of the National Institute on Aging and other funders. We are grateful to Luis Rosero-Bixby, Gilbert Brenes, Dmitri Jdanov, William Dow, and Patrick Gerland for their comments on this manuscript. We also thank other HMD staff who assisted with the construction of this data series: Carl Boe, Lisa Yang, Celeste Winant, and Gabriel Borges. 859 Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 References Barbieri, M., Wilmoth, J.R., Shkolnikov, V.M., Glei, D.A., Jasilionis, D., Jdanov, D., Boe, C., Riffe, T., Grigoriev, P., and Winant, C. (2015). Data resource profile: The Human Mortality Database (HMD). International Journal of Epidemiology 44(5): 1549–1556. doi:10.1093/ije/dyv105. Beltrán-Sánchez, H., Palloni, A., Pinto-Aguirre, G., and Verhulst, A. (2018). The Latin American Mortality Database (LAMBdA): Methodological document, preliminary version [electronic resource]. Madison: University of Wisconsin. https://www.ssc.wisc.edu/cdha/latinmortality/wp-content/uploads/LAMBdA_ documentation.pdf. Bennett, N.G. and Horiuchi, S. (1981). Estimating the completeness of death registration in a closed population. Population Index 47(2): 207–221. doi:10.2307/2736447. Bennett, N.G. and Horiuchi, S. (1984). Mortality estimation from registered deaths in less developed countries. Demography 21(2): 217–233. doi:10.2307/2061041. Borges, G. and Sacco, N. (2016). Has census coverage in Latin America improved with time? Paper presented at the International Seminar on Registering and Counting the Population: The production and exploration of census information from early modern times to the end of 20th century, IUSSP and NEPO-UNICAMP, Campinas, Brazil, May 19–21, 2016. Brass, W. (1975). Methods for estimating fertility and mortality from limited and defective data: Based on seminars held 16–24 September 1971 at the Centro Latinoamericano de Demografía (CELADE), San José, Costa Rica. Chapel Hill: International Program of Laboratories for Population Statistics, Department of Biostatistics, University of North Carolina at Chapel Hill. Centro Centroamericano de Población (2013). Evaluación demográfica del X Censo Nacional de Población de Costa Rica 2011 y de otras fuentes de información. San José: Centro Centroamericano de Población, Universidad de Costa Rica. Centro Centroamericano de Población (2017a). Biblioteca virtual en población y salud [electronic resource]. San José: Centro Centroamericano de Población, Universidad de Costa Rica. http://biblioteca.ccp.ucr.ac.cr. Centro Centroamericano de Población (2017b). Sistema de consulta a bases de datos estadísticas [electronic resource]. San José: Centro Centroamericano de Población, Universidad de Costa Rica. http://censos.ccp.ucr.ac.cr. 860 http://www.demographic-research.org http://www.demographic-research.org Demographic Research: Volume 40, Article 29 Centro Centroamericano de Población (2017c). Tabla completa de mortalidad: Costa Rica 1950–55, 1955–60, …2005–10 [electronic resource]. San José: Centro Centroamericano de Población, Universidad de Costa Rica. http://ccp.ucr.ac.cr/ observa/CRindicadores/TVcompletas.html. Elo, I.T. and Preston, S.H. (1994). Estimating African-American mortality from inaccurate data. Demography 31(3): 427–458. doi:10.2307/2061751. Hill, K. (1987). Estimating census and death registration completeness. Asian Pacific Census Forum 1(3): 8–24. Hill, K., You, D., and Choi, Y. (2009). Death distribution methods for estimating adult mortality: Sensitivity analysis with simulated data error. Demographic Research 21(9): 235–254. doi:10.4054/DemRes.2009.21.9. Institute for Health Metrics and Evaluation (2016). References Global Burden of Disease Study 2015 (GBD 2015) results [electronic resource]. Seattle: Institute for Health Metrics and Evaluation. http://ghdx.healthdata.org/gbd-results-tool. Instituto Nacional de Estadística y Censos (2009). Esperanza de vida al nacer, a los 20 y a los 65 años de edad, Costa Rica 1930‒2008 [electronic resource]. San José: INEC. http://ccp.ucr.ac.cr/observa/CRindicadores/evida.html (accessed 13 November 2009). Instituto Nacional de Estadística y Censos (2013). Estimaciones y proyecciones de población por sexo y edad 1950–2050. San José: INEC, Centro Centro- americano de Población. Instituto Nacional de Estadística y Censos (2017a). Tablas completas de mortalidad 1995, 1996, …2015 [electronic resource]. San José: INEC. http://www.inec. go.cr/sites/default/files/documentos/poblacion/mortalidad/estadisticas/resultados /repoblacevYYYY-01.xls (where YYYY=Year). Instituto Nacional de Estadística y Censos (2017b). Cuadro 1: Principales indicadores demográficos, 1950–2015, Tasa global de fecundidad [electronic resource]. San José: INEC. http://www.inec.go.cr/sites/default/files/documetos-biblioteca- virtual/sepoblacev1950-2015-01.xls. Instituto Nacional de Estadística y Censos and Centro Centroamericano de Población (2002). Costa Rica: Estimaciones y proyecciones de población 1970–2050 actualizados al año 2000 y evaluación del censo del 2000 y otras fuentes de información. San José: INEC. 861 http://www.demographic-research.org Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 IUSSP-UNFPA (2017a). The generalized growth balance method: Tools for demographic estimation [electronic resource]. Paris: IUSSP. http://demo graphicestimation.iussp.org/content/generalized-growth-balance-method. IUSSP-UNFPA (2017b). Synthetic extinct generations methods: Tools for demographic estimation [electronic resource]. Paris: IUSSP. http://demographicestimation. iussp.org/content/synthetic-extinct-generations-methods. Latin America and the Caribbean Demographic Observatory (2007). Mortality: Methodology of mortality estimates and projections by age and sex. Santiago: CEPAL/ECLAC, United Nations. Li, N. and Gerland, P. (2016). Evaluating the completeness of death registration for developing countries at old age. Paper presented at the United Nations Expert Group Meeting on ‘Methodology and lessons learned to evaluate the completeness and quality of vital statistics data from civil registration,’ New York, USA, November 3–4, 2016. Lima, E., Queiroz, B., and Riffe, T.L. (2016). R implementations of three growth balance methods for estimating adult mortality coverage [electronic resource]. San Francisco: GitHub. https://github.com/timriffe/AdultCoverage. Ministerio de Planificación Nacional y Politica Económica, UN Centro Latinoamericano de Demografía, and Dirección General de Estadística y Censos (1988). Costa Rica: Estimaciones y proyecciones de población 1950–2025. San José: Imprenta Nacional. Núñez, M.L. and Icaza, M.G. (2006). Quality of mortality statistics in Chile, 1997– 2003. Revista Médica Chile 134(9): 1191–1196. Palloni, A., Beltrán-Sánchez, H., and Pinto Aguirre, G. (2017). Incertidumbre de los estimadores de mortalidad y pruebas de hipótesis: El caso de América Latina y el Caribe, 1850–2010. Notas de Población 44(104): 13–32. doi:10.18356/ e68ff37c-es. References Palloni, A., Pinto, G., and Beltrán-Sánchez, H. (2015). Chapter 2: Estimation of life tables 1850–2010: Adjustments for relative completeness and age misreporting. In: Two centuries of mortality decline in Latin America: From hunger to longevity. Madison: University of Wisconsin: 3–75. Palloni, A., Pinto, G., and Beltrán-Sánchez, H. (2016). Life tables by year, sex, and 5- year age groups, Latin American Mortality Database (LAMBdA) [electronic resource]. Madison: University of Wisconsin. http://www.ssc.wisc. edu/cdha/latinmortality/?page_id=28. 862 http://www.demographic-research.org Demographic Research: Volume 40, Article 29 Payne, C.F. (2015). Aging in the Americas: Disability-free life expectancy among adults aged 65 and older in the United States, Costa Rica, Mexico, and Puerto Rico. The Journals of Gerontology Series B: Psychological Sciences and Social Science 73(2): 337–348. doi:10.1093/geronb/gbv076. Preston, S.H., Elo, I.T., and Stewart, Q. (1999). Effects of age misreporting on mortality estimates at older ages. Population Studies 53(2): 165–177. doi:10.10 80/00324720308075. Rosero-Bixby, L. (2008). The exceptionally high life expectancy of Costa Rican nonagenarians. Demography 45(3): 673–691. doi:10.1353/dem.0.0011. Rosero-Bixby, L. (2018). High life expectancy and reversed socioeconomic gradients of elderly people in Mexico and Costa Rica. Demographic Research 38(3): 95– 108. doi:10.4054/DemRes.2018.38.3. Rosero-Bixby, L., Brenes Camacho, G., and Chen-Mok, M. (2002). Fecundidad diferencial e inmigrantes Nicaragüeneses en Costa Rica. Notas de población 74: 27–51. Rosero-Bixby, L., Brenes, G., and Collado, A. (2004). Tablas de vida para cálculo actuarial de rentas vitalicias y retiro programado: Costa Rica circa 2000. Población y Salud en Mesoamérica 1(2): 173–205. Rosero-Bixby, L. and Dow, W.H. (2009). Surprising SES gradients in mortality, health, and biomarkers in a Latin American population of adults. Journals of Gerontology Series B: Psychological Sciences and Social Sciences 64(1): 105– 117. doi:10.1093/geronb/gbn004. Shryock, H.S., Siegel, J.S., and Larmon, E.A. (1973). The methods and materials of demography. Suitland: US Bureau of the Census. Solano, E. (2009). Evaluacíon censal con métodos indirectos: Experiencia de Costa Rica. Paper presented at Conferencía Estadistica de las Américas – CEA CEPAL, Santiago, Chile, June 3–5, 2009. Statistiska Centralbyrån (1969). Brief historical survey of the organization of the Swedish population statistics. In: Historisk statistisk för Sverige del 1: Befolkning, andra upplagan 1720–1967. Stockholm: Statistiska Centralbyrån: 10–11. Thatcher, R., Kannisto, V., and Andreev, K. (2002). The survivor ratio method for estimating numbers at high ages. Demographic Research 6(1): 1–18. doi:10.40 54/DemRes.2002.6.1. 863 http://www.demographic-research.org Glei, Barbieri & Santamaría-Ulloa: Costa Rican mortality 1950–2013 United Nations Statistics Division (2017). Coverage of civil registration system, births and deaths [electronic resource]. References New York: United Nations. https://unstats. un.org/unsd/demographic-social/crvs. United Nations Statistics Division (2017). Coverage of civil registration system, births and deaths [electronic resource]. New York: United Nations. https://unstats. un.org/unsd/demographic-social/crvs. United Nations, Department of Economic and Social Affairs, Population Division (2015). World population prospects: The 2015 revision, DVD edition [electronic resource]. New York: United Nations. https://esa.un.org/unpd/wpp/ Download/Standard/Mortality. United Nations, Department of Economic and Social Affairs, Population Division (2017). World population prospects 2017 [electronic resource]. New York: United Nations. https://esa.un.org/unpd/wpp/Download/Standard/Mortality. University of California, Berkeley and Max Planck Institute for Demographic Research (2016). Human mortality database [electronic resource]. Berkeley: University of California, Berkeley. https://www.mortality.org. Wang, H., Naghavi, M., Allen, C., Barber, R.M., Carter, A., Casey, D.C., Charlson, F.J., Chen, A.Z., Coates, M.M., Coggeshall, M., and Dandona, L. (2016). Global, regional, and national life expectancy, all-cause mortality, and cause- specific mortality for 249 causes of death, 1980–2015: A systematic analysis for the Global Burden of Disease Study 2015. The Lancet 388(10053): 1459–1544. doi:10.1016/S0140-6736(16)31012-1. Wilmoth, J.R., Andreev, K., Jdanov, D., Glei, D.A., Boe, C., Bubenheim, M., Philipov, D., Shkolnikov, S., and Vachon, P. (2007). Methods protocol for the human mortality database, version 5 [electronic resource]. Berkeley: University of California, Berkeley. https://v5.mortality.org/Public/Docs/MethodsProtocol.pdf. Wilmoth, J.R., Andreev, K., Jdanov, D., Glei, D.A., Riffe, T., Boe, C., Bubenheim, M., Philipov, D., Shkolnikov, S., Vachon, P., Winant, C., and Barbieri, M. (2017). Methods protocol for the human mortality database, version 6 [electronic resource]. Berkeley: University of California, Berkeley. https://www.mortality. org/Public/Docs/MethodsProtocol.pdf. Wilmoth, J.R. and Lundström, H. (1996). Extreme longevity in five countries: Presentation of trends with special attention to issues of data quality. European Journal of Population 12(1): 63–93. doi:10.1007/BF01797166. World Health Organization (2014). Life expectancy at birth, WHO Data; downloaded from UNdata [electronic resource]. Geneva: World Health Organization. http://data.un.org/Data.aspx?d=WHO&f=MEASURE_CODE%3aWHOSIS_000 001. 864 http://www.demographic-research.org http://www.demographic-research.org
https://openalex.org/W3090526474	https://bmcpsychiatry.biomedcentral.com/track/pdf/10.1186/s12888-020-02888-3	English	null	Prevalence and associated factors of psychological distress among a national sample of in-school adolescents in Morocco	BMC psychiatry	2,020	cc-by	7,952	© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access Prevalence and associated factors of psychological distress among a national sample of in-school adolescents in Morocco Supa Pengpid1,2 and Karl Peltzer3* Abstract Background: The goal of the study was to estimate the prevalence and correlates of psychological distress among adolescent school children in Morocco. Methods: Nationally representative cross-sectional data were analysed from 6745 adolescents (15 years median age) that responded to questions on a two-item measure of psychological distress from “2016 Morocco Global School-Based Student Health Survey (GSHS).” Results: The prevalence of psychological distress was 23.3, 18.0% among males and 29.2% among females. In adjusted logistic regression analysis, female sex, older age, bullying victimization, infrequently physically attacked, frequent participation in physical fights, having no close friends, frequent experience of hunger, parental emotional neglect, parental disrespect of privacy, school truancy, sedentary behaviour and having sustained a single or multiple serious injuries (past year) were associated with psychological distress. In addition, in unadjusted analysis, low peer support, parents never check homework, exposure to passive smoking, substance use (current tobacco use, current cannabis use and ever used amphetamine), frequent soft drink and frequent fast food consumption were positively and fruit and vegetable intake was negatively associated with psychological distress. Conclusion: Almost one in four students reported psychological distress and several associated factors were identified which can aid prevention and control strategies. Keywords: Risk behaviour, Social factors, Psychological distress, Adolescents, Morocco characterised by vulnerability to psychological distress, and is therefore an important time for promotion of psy- chological well-being and early mental health interven- tion, in order to safeguard against the development of mental health issues” [4]. According to the American Psychological Association (APA) [5], psychological dis- tress is “a set of painful mental and physical symptoms that are associated with normal fluctuations of mood in most people. It is thought to be what is assessed by many putative self-report measures of depression and anxiety.” For example, the Kessler Psychological Distress Scale includes “symptoms of depression, anxiety, stress, and somatic complaints” [6]. Psychological distress has Background “Mental health conditions, including depression and anxiety, account for 16% of the global burden of disease and injury in people aged 10–19 years.” [1]. In children and adolescents, the “worldwide-pooled prevalence of mental disorders was 13.4%, including any anxiety dis- order 6.5% and any depressive disorder 2.6%” [2]. “First onset of mental disorders usually occurs in childhood or adolescence” [3]. “Adolescence is a critical period * Correspondence: kfpeltzer@gmail.com 3Department of Psychology, University of the Free State, Bloemfontein, South Africa Full list of author information is available at the end of the article Africa Full list of author information is available at the end of the article Pengpid and Peltzer BMC Psychiatry (2020) 20:475 https://doi.org/10.1186/s12888-020-02888-3 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 https://doi.org/10.1186/s12888-020-02888-3 Sources of data Nationally representative cross-sectional data from the “2016 Morocco GSHS” were analyzed [23]. More de- tailed information on the survey methodology and the data can be accessed [23]; the overall response rate was 91% [23]. The sampling approach included a two stage sampling design, including schools and classes. All school students present in a selected class room were eligible to participate by filling in a self-administered an- onymous questionnaire [23]. The prevalence of psychological distress among adoles- cents in Eastern Mediterranean countries was 27.7% (≥2 items of no close friends, loneliness, anxiety, suicidal idea- tion, and suicide attempt) in Afghanistan [11], 16.8% (≥14 scores on GHQ-28) in Egypt [12], 17.7% (≥3 items of worthless, anxious, angriness, confusion, and insomnia) in Iran [13], and 17.2% probable depression and 21.4% prob- able anxiety (≥10 scores and ≥12 scores on the Hospital Anxiety and Depression Scale, respectively) in Pakistan [14]. The proportion of moderate to severe psychological distress (measured with the Kessler K-10) was 23.0% in a community-based study among adolescents (N = 1000) in Tanzania [15] and 10.5% among school-going adolescents in India [16], and the prevalence of psychological distress measured with the GHQ was 35.1% among adolescents in Ontario, Canada [17], and 24.2% among urban out of school adolescents (N = 480) in Nigeria [18]. In Zambia, the prevalence of psychological distress among adoles- cents was 15.7% (scoring 3 of 4 items, anxiety, loneliness, sadness, and suicide plan [9], in Tanzania among adoles- cents 20.6% had single psychological distress and 10.3% had multiple psychological distress (based on five items: anxiety, loneliness, no close friends, suicidal ideation and suicide attempt) [10], and in four Asian countries psycho- logical distress was 32.9% (presence of any item: suicidal ideation, plan and suicide attempt, loneliness, sadness and anxiety) [19]. In a local survey among secondary school students in an urban area in Morocco (Tetouan), the most common mental problems “were memory problems, con- centration difficulties, restlessness, fear, nervosity and feel- ings of inadequacy during interpersonal interactions.” [20]. There is a lack of national data on the prevalence and correlates of psychological distress among adolescents in Morocco. In order to prevent and control psychological distress in adolescent populations, it is vital to assess its prevalence and risk factors [21]. Outcome variable Psychological distress was assessed with two items, 1) “During the past 12 months, how often have you been so worried about something that you could not sleep at night?” and 2) “During the past 12 months, how often have you felt lonely?” Response options included and were coded as “Never=0, Rarely=1, Sometimes=1, Most of the time=2, Always=3” Scores of the two items were summed and scores three or more were defined as psy- chological distress, following the scoring of other 2-item mental health screeners, the “Patient Health Questionnaire-2 (PHQ-2)” [24], and the “Generalized Anxiety Disorder-2 (GAD-2)” [25, 26]. Cronbach alpha for the 2-item “psychological distress” measure was 0.66 in this sample. The inter-item correlation was 0.44, and the item-total correlations ranged from .82 to .90. Measures The questionnaire (the questions, response options and coding) used is shown in supplementary file 1 [23]. Page 2 of 11 Page 2 of 11 Page 2 of 11 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 been assessed with different scales, such as the “Psycho- logical Distress Scale”, K-6 or K-10 [6, 7], the “General Health Questionnaire (GHQ-12)” [8] and several differ- ent psychological distress items, such as anxiety, loneli- ness, sadness and suicide plan [9], or no close friends, anxiety, loneliness, suicidal ideation and attempt [10]. Data analysis Statistical analyses were done with “STATA software version 15.0 (Stata Corporation, College Station, Texas, USA),” taking into account the complex study design. The data were described using frequencies, means, and standard deviations. Pearson Chi-square tests were used for testing differences in proportions. In addition, Cron- bach alpha coefficients were calculated and Principal Component Analysis with the 2-item psychological dis- tress measure. Unadjusted and adjusted (with all co- variates) logistic regression analyses were used to assess predictors of psychological distress. Co-variates included age group, social distress (no close friends, being bullied, physically attacked, and participation in physical fight), social-environmental factors (experience of hunger, low peer support, lack of parental support, passive smoking, and school truancy), and health risk behaviours (tobacco use, cannabis use, amphetamine use, sedentary behav- iour, injury, soft drink intake, fast food consumption, and fruit and vegetable intake). They were selected based on literature review [10]. Missing values (< 3.7% on any variable) were not included in the analysis. frequently truancy, 45.4% had low peer support, 24.1% experienced parental emotional neglect, 29.5% had par- ents who never checked on their home work, and 13.1% had parents who mostly or always disrespected their privacy. Almost one in four students (23.3%) reported psychological distress, 29.2% among females and 18.0% among males (see Table 2). Associations with psychological distress among both sexes Table 3 shows the unadjusted and adjusted associations between sociodemographic factors, social distress items, social-environmental factors and health risk behaviours with psychological distress. Compared to students aged 13 years or younger, older adolescents aged 16 years and older reported 129% (AOR = 2.29; 95% CI = 1.73–3.03) higher prevalence of psychological distress. Male adoles- cents were 70% (AOR = 0.30; 95% CI = 0.23–0.40) less likely having psychological distress than female adoles- cents do. Social distress factors associated with psycho- logical distress were frequent bullying victimization (AOR = 2.92; 95% CI = 2.16–3.94), having no close friends (AOR = 1.98; 95% CI = 1.44–2.73), frequent in- volvement on physical fighting (AOR = 1.89; 95% CI = 1.34–2.67), and having been attacked once (AOR = 1.47; 95% CI = 1.08–2.00). Social-environmental factors asso- ciated with psychological distress were frequent truancy (AOR = 1.87; 95% CI = 1.23–2.83), frequent experience of hunger (AOR = 1.49; 95% CI = 1.07–2.08), parental emotional neglect (AOR = 1.37; 95% CI = 1.07–1.75), and parental disrespect of privacy (AOR = 1.33; 95% CI = 1.01–1.77). Regarding health risk behaviours, compared to students who had not sustained a serious injury in the past year, students who had multiple injuries were 122% (AOR = 2.22; 95% CI = 1.69–2.93) more likely to have psychological distress. Compared to students who were less than 3 h a day engaged in leisure-time sedentary be- haviour, students who engaged eight or more hours a day in leisure-time sedentary behaviour had 71% (AOR = 1.71; 95% CI = 1.24–2.36) more likely psychological distress. In Co-variates Sociodemographic variables included sex and age group. Sociodemographic variables included sex and age group. Psychosocial distress items included the number of close friends, the number of days having been bullied in the past 30 days, the number of times having been phys- ically attacked in the past 12 months, and the number of times having been involved in a physical fight in the past 12 months. Social-environmental variables included, the frequency of feeling hungry in the past 30 days, the extend of peer support, parental disrespect of privacy, number of days exposed secondary smoke in the past 7 days, number of days truancy in the past 30 days, and parental emotional neglect was defined as never “parental or guardian un- derstanding of your problems and worries? AND never parents or guardians really know what you were doing with your free time when you were not at school or work?” [27]. Factors associated with psychological distress among adolescents can be divided into social distresses, socio- environmental factors and health risk behaviours [22], and may include, as reviewed in Pengpid and Peltzer [10], sociodemographic factors (older age, female sex), social distresses (interpersonal violence), socio- environmental factors (experience of hunger, low peer and low parental support and school truancy) and health risk behaviours (substance use, sedentary behaviour, sex- ual behaviour and injury). This investigation aimed at es- timating the prevalence and correlates of psychological distress among adolescents in Morocco. Health risk behaviour items included, current tobacco use, current cannabis use, ever used amphetamine, num- ber of hours spent sedentary during leisure time, Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Page 3 of 11 Table 1 Factor loadings of the 2-item psychological distress measure among adolescents in Morocco Psychological distress: 2 items Component 1 1. During the past 12 months, how often have you been so worried about something that you could not sleep at night? .85 2. During the past 12 months, how often have you felt lonely? .85 Eigenvalue 1.44 Percent of variance 72.21 Factor (items: 1 and 2): Psychological distress Table 1 Factor loadings of the 2-item psychological distress measure among adolescents in Morocco number of times seriously injured in the past 12 months, number of soft drinks consumed in a day, number of days having had fast food in the past week, and fruit and vegetable intake per day. Factor analysis of the 2-item psychological distress measure The Kaiser-Meyer-Olkin was .500, showing the adequacy of the sample, and the Bartlett’s Test of Sphericity (549, 195.047, df = 1, p < 0.001) found that the factor analysis was justified. One component with eigenvalues larger than one (1.44) was extracted and named “psychological distress” explaining 72.21% of the variance (see Table 1). Sample and psychological distress characteristics Sample and psychological distress characteristics The sample comprised 6745 school adolescents (15 years median age, 3 years interquartile range), 46.2% were fe- male and 9.2% were mostly or always hungry. One in ten of the participants (10.2%) had no close friends, 19.8% were lonely, 18.2% had frequently been in a phys- ical fight, 11.7% had frequently been attacked, and 14.7% had frequently been bullied. More than one in ten stu- dents (13.5%) reported current tobacco use, 6.3% current cannabis use, 7.8% had ever used amphetamine, 9.1% were daily exposed to secondary smoke, 17.2% had daily two or more soft drinks, 12.1% had multiple injuries (past year), 32.5% engaged in sedentary behaviour, 26.9% had on three or more days fast food in the past week and 33.0% had five or more servings of fruit and vegeta- bles per day. Discussion addition, in unadjusted analyses, low peer support, parents never check home work, exposure to passive smoking, substance use (current tobacco use, current cannabis use and ever used amphetamine), frequent soft drink and fre- quent fast food consumption were positively and fruit and vegetable intake was negatively associated with psycho- logical distress (see Table 3). addition, in unadjusted analyses, low peer support, parents never check home work, exposure to passive smoking, substance use (current tobacco use, current cannabis use and ever used amphetamine), frequent soft drink and fre- quent fast food consumption were positively and fruit and vegetable intake was negatively associated with psycho- logical distress (see Table 3). The current study aimed at estimating the prevalence and correlates of psychological distress in school adoles- cents in Morocco. The prevalence of past 12-month psy- chological distress (23.3%) in this study, which is similar to some previous studies among adolescents, e.g., in Tanzania (23.0%) [15], Nigeria (24.2%) [18], lower than in Afghanistan (27.7%) [11], in United Arab Emirates (28% anxiety disorders) [28], in Lebanon (Beirut) (26.1% mental disorders [29], in Canada (35.1%) [17], and higher than in Egypt (16.8%) [12], India (10.5%) [16], and Iran (17.7%) [13]. Other previous studies among ad- olescents in the Morocco seem to confirm that psycho- logical distress is common [20], calling for strategies and programmes to prevent and control psychological dis- tress in this adolescent population in Morocco. Sample and psychological distress characteristics One in ten of the students (10.1%) reported Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Page 4 of 11 Table 2 Sample and psychological distress characteristics among adolescents in Morocco Variable Sample Psychological distress Both sexes Male Female N (%) % % % Socio-demographics All 6745 23.3 18.0 29.2 Age in years 13 or less 1863 (26.8) 16.4 16.5 14.9 14–15 2212 (31.9) 21.9 14.4 28.6 16 or more 2558 (41.3) 30.3 21.2 41.0 Psychosocial distress No close friends 698 (10.2) 37.6 32.3 48.3 Bullied in past month 0 days 3869 (61.5) 15.9 11.1 19.7 1 or 2 days 1511 (23.8) 29.3 20.0 40.5 3–30 days 955 (14.7) 45.5 36.7 59.6 Physically attacked in past year 0 times 5080 (76.7) 20.2 14.1 25.6 time 795 (11.5) 32.5 26.6 40.1 2 or more times 793 (11.7) 38.8 31.6 53.1 In physical fight in past year 0 times 64.2 (63.4) 21.7 14.1 26.7 1 time 18.4 (18.4) 23.2 15.1 35.2 2 or more times 17.4 (18.2) 31.8 27.4 46.5 Social-environmental factors Mostly/always feeling hungry 680 (9.2) 38.5 36.6 42.7 Low peer support 3085 (45.4) 25.6 18.9 34.0 Parental emotional neglect 1655 (24.1) 25.1 20.2 34.2 Parents never check home work 2029 (29.5) 25.7 20.2 33.5 Parents disrespect privacy 818 (13.1) 30.7 18.0 36.0 Passive smoking in past week 0 days 3908 (59.0) 20.8 14.8 26.2 1–6 days 2104 (31.9) 24.7 18.4 32.2 All 7 days 580 (9.1) 38.2 30.7 43.4 School truancy (past month) 0 days 4584 (69.3) 20.6 14.5 25.7 1–2 days 1317 (20.6) 28.4 20.0 37.5 3 or more days 644 (10.1) 36.1 31.6 48.7 Health risk behaviours Current tobacco use 913 (13.5) 31.9 29.8 40.4 Current cannabis use 453 (6.3) 34.1 37.1 37.6 Ever used amphetamine 464 (7.8) 37.2 36.6 47.9 Leisure time sedentary behaviour/day < 3 h 4574 (67.5) 21.2 16.3 24.9 3–4 h 1100 (18.7) 26.2 15.6 36.3 Table 2 Sample and psychological distress characteristics among adolescents in Morocco 15.1 35.2 20.2 34.2 20.2 33.5 18.4 32.2 20.0 37.5 29.8 40.4 37.1 37.6 15.6 36.3 24.6 41.4 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Page 5 of 11 Table 2 Sample and psychological distress characteristics among adolescents in Morocco (Continued) Variable Sample Psychological distress Both sexes Male Female N (%) % % % ≥8 h 306 (5.4) 39.8 36.9 43.7 Injury in past 12 months 0 times 4251 (68.4) 18.1 12.1 22.2 1 time 1206 (19.5) 29.7 23.2 39.0 2 or more times 735 (12.1) 39.5 32.2 58.6 Soft drink intake/day 0 4488 (67.4) 23.5 16.6 29.4 1 1033 (15.3) 22.0 17.7 25.8 2 423 (6.6) 22.9 21.2 31.2 3 or more 706 (10.6) 27.1 23.5 31.3 Fast food consumption/week 0 days 2486 (35.5) 22.3 15.9 26.7 1 1579 (23.7) 22.4 16.7 26.8 2 899 (13.8) 23.5 18.3 28.8 3–7 days 1718 (26.9) 27.0 21.5 35.6 Fruit and vegetable intake < 1 serving/day 560 (8.2) 32.3 24.2 43.0 1 or 2 2222 (34.1) 25.0 18.8 31.3 3 or 4 1591 (24.7) 20.5 14.1 26.7 5 or more 2158 (33.0) 22.0 17.9 25.5 and psychological distress characteristics among adolescents in Morocco (Continued) Associations with psychological distress among boys and girls g Compared to 13 or less year-old girls, 16 or more year-old girls were 3.5 times more likely to have psy- chological distress. Among both boys and girls, fre- quent bullying victimization, frequently having been in a physical fight, having no close friends, frequent truancy and frequent injury increased the odds for psychological distress. Boys who frequently experi- enced hunger were 114% (AOR = 2.14; 95% CI = 1.31– 3.48) more likely to have psychological distress, and girls who had low peer support were 23% (AOR = 1.23; 95% CI = 1.05–1.44) and engaged 8 or more hours in leisure-time sedentary behaviour were 91% (AOR = 1.91; 95% CI = 1.14–3.20) more likely to have psychological distress (see Table 4). The study showed that being female increased the odds for psychological distress, which was also found in some previous investigations [11]. Generally, “girls are more likely than boys to report internalising problems such as psychological distress, depression, and anxiety.” [4, 30, 31]. The study showed that older age, in particu- lar among girls, increased the odds for psychological dis- tress. Similar results were found in a study among adolescents in India [16]. Associations with psychological distress among boys and girls Possible reasons for higher Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Page 6 of 11 Table 3 Associations with psychological distress in both sexes Variable Unadjusted Odds Ratio (95% CI) P-value Adjusted Odds Ratio (95% CI) P-value Socio-demographics Age in years 13 or less 1 (Reference) 1 (Reference) 14–15 1.46 (1.19, 1.79) < 0.001 1.42 (1.07, 1.89) 0.016 16 or more 2.26 (1.89, 2.71) < 0.001 2.29 (1.73, 3.03) < 0.001 Gender Female 1 (Reference) 1 (Reference) Male 0.53 (0.45, 0.63) < 0.001 0.30 (0.23, 0.40) < 0.001 Social distress No close friends 2.53 (0.25, 3.24) < 0.001 1.98 (1.44, 2.73) < 0.001 Bullied in past month 0 days 1 (Reference) 1 (Reference) 1 or 2 days 2.22 (1.87, 2.62) < 0.001 1.95 (1.58, 2.40) < 0.001 3–30 days 4.70 (3.70, 5.98) < 0.001 2.92 (2.16, 3.94) < 0.001 Physically attacked in past year 0 times 1 (Reference) 1 (Reference) 1 time 1.89 (1.51, 2.37) < 0.001 1.47 (1.08, 2.00) 0.015 2 or more times 2.63 (2.13, 3.23) < 0.001 1.02 (0.72, 1.46) 0.987 In physical fight in past year 0 times 1 (Reference) 1 (Reference) 1 time 1.00 (0.83, 1.21) 0.989 1.20 (0.89, 1.60) 0.232 2 or more times 1.66 (1.34, 2.04) < 0.001 1.89 (1.34, 2.67) < 0.001 Social-environmental factors Mostly/always feeling hungry 2.30 (1.78, 2.98) < 0.001 1.49 (1.07, 2.08) 0.019 Low peer support 1.27 (1.11, 1.45) < 0.001 1.15 (0.97, 1.35) 0.098 Parental emotional neglect 1.29 (1.09, 1.52) 0.003 1.37 (1.07, 1.75) 0.014 Parents never check home work 1.27 (1.02, 1.58) 0.031 1.11 (0.90, 1.36) 0.326 Parents disrespect privacy 1.27 (1.07, 1.50) 0.006 1.33 (1.01, 1.77) 0.045 Passive smoking in past week 0 days 1 (Reference) 1 (Reference) 1–6 days 1.18 (1.04, 1.33) 0.010 0.89 (0.67, 1.17) 0.388 All 7 days 2.17 (1.78, 2.64) < 0.001 1.19 (0.82, 1.71) 0.362 School truancy (past month) 0 days 1 (Reference) 1 (Reference) 1–2 days 1.49 (1.28, 1.74) < 0.001 1.37 (1.08, 1.74) 0.009 3 or more days 2.35 (1.81, 3.04) < 0.001 1.87 (1.23, 2.83) 0.004 Health risk behaviours Current tobacco use 1.67 (1.34, 2.07) < 0.001 0.68 (0.48, 1.01) 0.098 Current cannabis use 2.07 (1.56, 2.74) < 0.001 0.96 (0.57, 1.61) 0.877 Ever used amphetamine 2.34 (1.86, 2.94) < 0.001 1.58 (0.91, 2.75) 0.100 Leisure time sedentary behaviour/day < 3 h 1 (Reference) 1 (Reference) 3–4 h 1.29 (1.09, 1.53) 0.003 1.04 (0.73, 1.48) 0.820 5–7 h 1.74 (1.42, 2.14) < 0.001 1.68 (1.23, 2.30) < 0.001 Unadjusted Odds Ratio (95% CI) P-value Adjusted Odds Ratio (95% CI) P-value 1 (Reference) < 0.001 1.42 (1.07, 1.89) 0.016 < 0.001 2.29 (1.73, 3.03) < 0.001 1 (Reference) < 0.001 0.30 (0.23, 0.40) < 0.001 < 0.001 1.98 (1.44, 2.73) < 0.001 1 (Reference) < 0.001 1.95 (1.58, 2.40) < 0.001 < 0.001 2.92 (2.16, 3.94) < 0.001 1 (Reference) < 0.001 1.47 (1.08, 2.00) 0.015 < 0.001 1.02 (0.72, 1.46) 0.987 1 (Reference) 0.989 1.20 (0.89, 1.60) 0.232 < 0.001 1.89 (1.34, 2.67) < 0.001 < 0.001 1.49 (1.07, 2.08) 0.019 < 0.001 1.15 (0.97, 1.35) 0.098 0.003 1.37 (1.07, 1.75) 0.014 0.031 1.11 (0.90, 1.36) 0.326 0.006 1.33 (1.01, 1.77) 0.045 1 (Reference) 0.010 0.89 (0.67, 1.17) 0.388 < 0.001 1.19 (0.82, 1.71) 0.362 1 (Reference) < 0.001 1.37 (1.08, 1.74) 0.009 < 0.001 1.87 (1.23, 2.83) 0.004 < 0.001 0.68 (0.48, 1.01) 0.098 < 0.001 0.96 (0.57, 1.61) 0.877 < 0.001 1.58 (0.91, 2.75) 0.100 1 (Reference) 0.003 1.04 (0.73, 1.48) 0.820 < 0.001 1.68 (1.23, 2.30) < 0.001 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Page 7 of 11 Pengpid and Peltzer BMC Psychiatry Table 3 Associations with psychological distress in both sexes (Continued) Table 3 Associations with psychological distress in both sexes (Continued) Variable Unadjusted Odds Ratio (95% CI) P-value Adjusted Odds Ratio (95% CI) P-value ≥8 h 2.70 (2.10, 3.46) < 0.001 1.71 (1.24, 2.36) < 0.001 Injury in past 12 months 0 times 1 (Reference) 1 (Reference) 1 time 2.00 (1.74, 2.29) < 0.001 1.50 (1.20, 1.87) < 0.001 2 or more times 3.23 (2.73, 3.82) < 0.001 2.22 (1.69, 2.93) < 0.001 Soft drink intake/day 0 1 (Reference) 1 (Reference) 1 0.95 (0.74, 1.22) 0692 0.85 (0.61, 1.19) 0.335 2 1.21 (0.91, 1.63) 0.194 0.94 (0.65, 1.36) 0.756 3 or more 1.28 (1.06, 1.54) 0.011 1.00 (0.60, 1.64) 0.986 Fast food consumption/week 0 days 1 (Reference) 1 (Reference) 1 0.96 (0.80, 1.14) 0.618 0.92 (0.67, 1.26) 0.600 2 1.04 (0.90, 1.20) 0.593 0.86, 0.65, 1.13) 0.282 3–7 days 1.52 (1.19, 1.94) < 0.001 1.04 (0.75, 1.44) 0.828 Fruit and vegetable intake < 1 serving/day 1 (Reference) 1 (Reference) 1 or 2 0.66 (0.51, 0.86) 0.003 0.99 (0.74, 1.33) 0.965 3 or 4 0.51 (0.39, 0.67) < 0.001 0.86 (0.61, 1.20) 0.374 5 or more 0.55 (0.42, 0.72) < 0.001 0.88 (0.61, 1.28) 0.499 CI Confidence Interval CI Confidence Interval world to reduce the harmful effects of bullying are lack- ing.” [36] However, Morocco has “implemented coordi- nated national action plans to address violence against children, enforced legislation to protect victims, and pro- moted programmes aimed at changing societal beliefs and attitudes around violence.” [37] psychological distress among older than younger adoles- cents include increasing demands, physical and psycho- social changes [4, 32, 33]. Associations with psychological distress among boys and girls In addition, older adolescents in this study were more likely to be injured (p = 0.009), engage in substance use (tobacco use: p < 0.001, cannabis use: p = 0.008, amphetamine use: p = 0.003), truancy (p < 0.001), sedentary behaviour (p < 0.001), passive smoking (p < 0.001), had no close friends (p < 0.001), fre- quent fast food intake (p = 0.003) and ate less fruit and vegetables (p < 0.001) than younger adolescents, which may have contributed to increased psychological distress among older adolescents. Several social-environmental factors (experience of hun- ger, in particular among boys, parental emotional neglect, parental disrespect of privacy, infrequent truancy and in unadjusted analysis passive smoking, low peer support, in particular among girls, and parents never check home- work) were found associated with psychological distress. These results are consistent with various previous investi- gations [9, 10, 19, 38, 39] and call for programmes im- proving parental and peer support and food security. A previous review provides evidence that “parental training and school-based interventions can reduce symptoms of common mental disorders in adolescents” [40]. g In line with former research findings [9, 10, 19, 34], this survey showed that having social distress, such as having no close friends, bullying victimization, infre- quently physically attacked and frequently involved in physical fighting increased the odds for psychological distress. Students exposed to interpersonal violence victimization may worry about further or future victimization increasing psychological distress. In addition, our study findings demonstrate that students who had been frequently bullied had the highest odds for psychological distress, which concurs with former re- search [35]. This finding may highlight the relevance of anti-bullying programme activities in order to ameliorate psychological distress. “The design and implementation of school-based anti-bullying programmes in the Arab In terms of health risk behaviours, high sedentary be- haviour (in particular among girls) and having experi- enced single and multiple serious injuries, and in unadjusted analysis frequent fast food consumption, in- frequent fruit and vegetable intake, frequent soft drink, having no close friends, and substance use increased the odds for having psychological distress. These findings concur with previous studies [10, 17, 41– 45]. Associations with psychological distress among boys and girls Since this study did not assess the type of Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Pengpid and Peltzer BMC Psychiatry Page 8 of 11 Table 4 Associations with psychological distress among males and females Variable Male Female Adjusted Odds Ratio (95% CI) p-value Adjusted Odds Ratio (95% CI) p-value Socio-demographics Age in years 13 or less 1 (Reference) 1 (Reference) 14–15 0.77 (0.46, 1.29) 0.321 2.12 (1.48, 3.03) < 0.001 16 or more 1.23 (0.75, 2.02) 0.414 3.52 (2.50, 4.95) < 0.001 Social distress No close friends 2.88 (1.73, 4.81) < 0.001 1.61 (1.14, 2.28) 0.007 Bullied in past month 0 days 1 (Reference) 1 (Reference) 1 or 2 days 1.61 (1.08, 2.39) 0.020 2.24 (1.60, 3.15) < 0.001 3–30 days 2.50 (1.63, 3.82) < 0.001 3.41 (2.24, 5.18) < 0.001 Physically attacked in past year 0 times 1 (Reference) 1 (Reference) 1 time 1.64 (1.00, 2.70) 0.051 1.17 (0.73, 1.88) 0.516 2 or more times 1.02 (0.67, 1.57) 0.921 1.19 (0.62, 2.31) 0.594 In physical fight in past year 0 times 1 (Reference) 1 (Reference) 1 time 1.12 (0.77, 1.64) 0.549 1.38 (0.94, 2.03) 0.099 2 or more times 1.98 (1.33, 2.96) < 0.001 1.86 (1.21, 2.87) 0.005 Social-environmental factors Mostly/always feeling hungry 2.14 (1.31, 3.48) 0.002 1.05 (0.69, 1.61) 0.805 Low peer support 0.99 (0.77, 1.27) 0.915 1.23 (1.05, 1.44) 0.013 Parental emotional neglect 1.28 (0.91, 1.81) 0.404 1.43 (0.98, 2.08) 0.063 Parents never check home work 1.36 (0.99, 1.86) 0.057 0.92 (0.68, 1.26) 0.610 Parents disrespect privacy 1.20 (0.78, 1.85) 0.404 1.32 (0.88, 2.00) 0.182 Passive smoking in past week 0 days 1 (Reference) 1 (Reference) 1–6 days 0.88 (0.67, 1.14) 0.329 0.90 (0.65, 1.25) 0.519 All 7 days 1.15 (0.77, 1.71) 0.494 1.27 (0.76, 2.12) 0.353 School truancy (past month) 0 days 1 (Reference) 1 (Reference) 1–2 days 1.58 (1.15, 2.17) 0.006 1.28 (0.92, 1.79) 0.146 3 or more days 2.12 (1.28, 3.51) 0.004 2.01 (1.04, 3.89) 0.039 Health risk behaviours Current tobacco use 0.81 (0.53, 1.24) 0.326 0.50 (0.19, 1.28) 0.147 Current cannabis use 0.94 (0.55, 1.58) 0.804 1.09 (0.30, 3.90) 0.896 Ever used amphetamine 1.36 (0.75, 2.47) 0.303 1.96 (0.57, 6.76) 0.285 Leisure time sedentary behaviour/day < 3 h 1 (Reference) 1 (Reference) 3–4 h 0.75 (0.47, 1.19) 0.214 1.26 (0.83, 1.90) 0.273 Table 4 Associations with psychological distress among males and females p-value Adjusted Odds Ratio (95% CI) p-value 1 (Reference) 0.321 2.12 (1.48, 3.03) < 0.001 0.414 3.52 (2.50, 4.95) < 0.001 < 0.001 1.61 (1.14, 2.28) 0.007 1 (Reference) 0.020 2.24 (1.60, 3.15) < 0.001 < 0.001 3.41 (2.24, 5.18) < 0.001 1 (Reference) 0.051 1.17 (0.73, 1.88) 0.516 0.921 1.19 (0.62, 2.31) 0.594 1 (Reference) 0.549 1.38 (0.94, 2.03) 0.099 < 0.001 1.86 (1.21, 2.87) 0.005 0.002 1.05 (0.69, 1.61) 0.805 0.915 1.23 (1.05, 1.44) 0.013 0.404 1.43 (0.98, 2.08) 0.063 0.057 0.92 (0.68, 1.26) 0.610 0.404 1.32 (0.88, 2.00) 0.182 1 (Reference) 0.329 0.90 (0.65, 1.25) 0.519 0.494 1.27 (0.76, 2.12) 0.353 1 (Reference) 0.006 1.28 (0.92, 1.79) 0.146 0.004 2.01 (1.04, 3.89) 0.039 0.326 0.50 (0.19, 1.28) 0.147 0.804 1.09 (0.30, 3.90) 0.896 0.303 1.96 (0.57, 6.76) 0.285 1 (Reference) 0.214 1.26 (0.83, 1.90) 0.273 0.193 2.06 (1.25, 3.41) 0.005 0.250 1.91 (1.14, 3.20) 0.014 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Page 9 of 11 Pengpid and Peltzer BMC Psychiatry Table 4 Associations with psychological distress among males and females (Continued) Variable Male Female Adjusted Odds Ratio (95% CI) p-value Adjusted Odds Ratio (95% CI) p-value 0 times 1 (Reference) 1 (Reference) 1 time 1.55 (1.18, 2.04) 0.002 1.37 (0.95, 1.97) 0.090 2 or more times 1.75 (1.27, 2.42) < 0.001 3.09 (1.73, 5.48) < 0.001 Soft drink intake/day 0 1 (Reference) 1 (Reference) 1 1.18 (0.76, 1.84) 0.459 0.70 (0.50, 0.99) 0.043 2 1.56 (1.00, 2.45) 0.050 0.63 (0.33, 1.20) 0.160 3 or more 1.13 (0.56, 2.29) 0.729 0.89 (0.52, 1.52) 0.664 Fast food consumption/week 0 days 1 (Reference) 1 (Reference) 1 1.08 (0.72, 1.65) 0.689 0.80 (0.52, 1.22) 0.293 2 0.98 (0.67, 1.43) 0.905 0.77 (0.47, 1.27) 0.302 3–7 days 0.89 (0.62, 1.27) 0.515 1.13 (0.75, 1.71) 0.549 Fruit and vegetable intake < 1 serving/day 1 (Reference) 1 (Reference) 1 or 2 1.11 (0.65, 1.91) 0.705 0.99 (0.62, 1.59) 0.573 3 or 4 0.95 (0.58, 1.57) 0.852 0.86 (0.56, 1.32) 0.494 5 or more 1.16 (0.67, 2.00) 0.595 0.79 (0.51, 1.23) 0.294 CI Confidence Interval Table 4 Associations with psychological distress among males and females (Continued) CI Confidence Interval the data. Conclusion The study found among school-going nationally representative adolescents in Morocco that almost one in four students reported psychological distress. Several correlates for psychological distress were identified, including female sex, having no close friends, older age, bullying victimization, infre- quently physically attacked, frequent participation in physical fights, frequent experience of hunger, parental emotional neglect, parental disrespect of privacy, school truancy, sedentary behaviour and having sustained a single or multiple serious injur- ies (past year), which can potentially guide inter- ventions to prevent psychological distress in this adolescent school population. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12888-020-02888-3. Additional file 1. Variable description. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12888-020-02888-3. Associations with psychological distress among boys and girls An additional limitation was that the GSHS in Morocco only assessed psychological distress with two items which may not reflect a standardized scale nor a diagnostic interview, and did not assess help seeking be- haviours for psychological distress. sedentary behaviour, for example social media use, we are not able to show the potentially negative ef- fects of social media use on psychological distress [46]. In a systematic review among adolescents [47] found that sedentary behaviour was associated with poor mental health and psychological distress, which may be explained by “the beneficial pathophysio- logical, social and general health effects of being ac- tive may be omitted when sedentary, which may have a negative impact on mental health.” Another possible mechanism by which sedentary behaviour may increase psychological distress is via inflammatory pro- cesses [48]. For example, in a randomized controlled intervention, “a one-week sedentary behaviour-inducing intervention had deleterious effects on anxiety in an active, young adult population” [49]. The association between in- jury occurrence in the past 12 months and psychological distress in the past 12 months may be explained by “the injury occurrence being considered a particularly impact- ful stressful life event, and experiences of stressful life events have been strongly associated with prospective anx- iety symptom development” [50]. sedentary behaviour, for example social media use, we are not able to show the potentially negative ef- fects of social media use on psychological distress [46]. In a systematic review among adolescents [47] found that sedentary behaviour was associated with poor mental health and psychological distress, which may be explained by “the beneficial pathophysio- logical, social and general health effects of being ac- tive may be omitted when sedentary, which may have a negative impact on mental health.” Another possible mechanism by which sedentary behaviour may increase psychological distress is via inflammatory pro- cesses [48]. For example, in a randomized controlled intervention, “a one-week sedentary behaviour-inducing intervention had deleterious effects on anxiety in an active, young adult population” [49]. The association between in- jury occurrence in the past 12 months and psychological distress in the past 12 months may be explained by “the injury occurrence being considered a particularly impact- ful stressful life event, and experiences of stressful life events have been strongly associated with prospective anx- iety symptom development” [50]. Author details 1 Author details 1ASEAN Institute for Health Development, Mahidol University, Salaya, Phutthamonthon, Nakhon Pathom, Thailand. 2Department of Research Administration and Development, University of Limpopo, Polokwane, South Africa. 3Department of Psychology, University of the Free State, Bloemfontein, South Africa. 19. Lee H, Lee EY, Greene B, Shin YJ. Psychological distress among adolescents in Laos, Mongolia, Nepal, and Sri Lanka. Asian Nurs Res (Korean Soc Nurs Sci). 2019;13(2):147–53. https://doi.org/10.1016/j.anr.2019.04.001. 20. Zouini B, Sfendla A, Hedman Ahlström B, Senhaji M, Kerekes N. Mental health profile and its relation with parental alcohol use problems and/or the experience of abuse in a sample of Moroccan high school students: an explorative study. Ann General Psychiatry. 2019;18:27. https://doi.org/10. 1186/s12991-019-0251-5. Received: 7 May 2020 Accepted: 21 September 2020 Received: 7 May 2020 Accepted: 21 September 2020 Received: 7 May 2020 Accepted: 21 September 2020 21. Kieling C, Baker-Henningham H, Belfer M, et al. Child and adolescent mental health worldwide: evidence for action. Lancet. 2011;378(9801):1515–25. https://doi.org/10.1016/S0140-6736(11)60827-1. 21. Kieling C, Baker-Henningham H, Belfer M, et al. Child and adolescent mental health worldwide: evidence for action. Lancet. 2011;378(9801):1515–25. https://doi.org/10.1016/S0140-6736(11)60827-1. Funding 12. Ismail A, Abdelgaber A, Hegazi H, Lotfi M, Kamel A, Ramdan M. The prevalence and risk factors of anxiety disorders in an Egyptian sample of school and students at the age of 12-18 years. J Psychiatry. 2015;18:316 10. 4172/2378- 5756.1000316. 12. Ismail A, Abdelgaber A, Hegazi H, Lotfi M, Kamel A, Ramdan M. The prevalence and risk factors of anxiety disorders in an Egyptian sample of school and students at the age of 12-18 years. J Psychiatry. 2015;18:316 10. 4172/2378- 5756.1000316. Not applicable. Acknowledgements 8. Goldberg DP, Blackwell B. Psychiatric illness in general practice. A detailed study using a new method of case identification. Br Med J. 1970;1:439–43. 8. Goldberg DP, Blackwell B. Psychiatric illness in general practice. A detailed study using a new method of case identification. Br Med J. 1970;1:439–43. The data source, the World Health Organization NCD Microdata Repository (URL: https://extranet.who.int/ncdsmicrodata/index.php/catalog), is hereby acknowledged. 9. Siziya S, Mazaba ML. Prevalence and correlates for psychosocial distress among in-school adolescents in Zambia. Front Public Health. 2015;3:180. https://doi.org/10.3389/fpubh.2015.00180. Availability of data and materials The data for the current study are publicly available at the World Health Organization NCD Microdata Repository (URL: https://extranet.who.int/ ncdsmicrodata/index.php/catalog). 13. Ahadi Z, Qorbani M, Kelishadi R, Ardalan G, Taslimi M, Mahmoudarabi M, et al. Regional disparities in psychiatric distress, violent behavior, and life satisfaction in Iranian adolescents: the CASPIAN-III study. J Dev Behav Pediatr. 2014;35(9):582–90. https://doi.org/10.1097/DBP.0000000000000103. 13. Ahadi Z, Qorbani M, Kelishadi R, Ardalan G, Taslimi M, Mahmoudarabi M, et al. Regional disparities in psychiatric distress, violent behavior, and life satisfaction in Iranian adolescents: the CASPIAN-III study. J Dev Behav Pediatr. 2014;35(9):582–90. https://doi.org/10.1097/DBP.0000000000000103. Abbreviations 7. Furukawa TA, Kessler RC, Slade T, Andrews G. The performance of the K6 and K10 screening scales for psychological distress in the Australian National Survey of mental health and well-being. Psychol Med. 2003;33(2): 357–62. https://doi.org/10.1017/s0033291702006700. GSHS: Global School-Based Student Health Survey; STATA: Statistics and data Consent for publication 17. Arbour-Nicitopoulos KP, Faulkner GE, Irving HM. Multiple health-risk behaviour and psychological distress in adolescence. J Can Acad Child Adolesc Psychiatry. 2012;21(3):171–8. 17. Arbour-Nicitopoulos KP, Faulkner GE, Irving HM. Multiple health-risk behaviour and psychological distress in adolescence. J Can Acad Child Adolesc Psychiatry. 2012;21(3):171–8. Competing interests 18. Okwaraji FE, Obiechina KI, Onyebueke GC, Udegbunam ON, Nnadum GS. Loneliness, life satisfaction and psychological distress among out-of-school adolescents in a Nigerian urban city. Psychol Health Med. 2018;23(9):1106– 12. https://doi.org/10.1080/13548506.2018.147672. The authors declare that they have no competing interests. Authors’ contributions 10. Pengpid S, Peltzer K. Psychological distress and its associated factors among school-going adolescents in Tanzania. Psychol Stud. 2020;65(2):174. https:// doi.org/10.1007/s12646-020-00550-2. 10. Pengpid S, Peltzer K. Psychological distress and its associated factors among school-going adolescents in Tanzania. Psychol Stud. 2020;65(2):174. https:// doi.org/10.1007/s12646-020-00550-2. All authors fulfil the criteria for authorship. SP and KP conceived and designed the research, performed statistical analysis, drafted the manuscript and made critical revision of the manuscript for key intellectual content. All authors read and approved the final version of the manuscript and have agreed to authorship and order of authorship for this manuscript. 11. Pengpid S, Peltzer K. High psychological distress among school-going adolescents in Afghanistan: prevalence and correlates from a national survey. Vulnerable Child Youth Stud. 2020;15(1):40–7. https://doi.org/10. 1080/17450128.2019.1679937. Study limitations Study limitations include that this investigation was lim- ited because of its cross-sectional design, the inclusion of only school adolescents as well as the self-report of Additional file 1. Variable description. Page 10 of 11 Page 10 of 11 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 Ethics approval and consent to participate 14. Khalid A, Qadir F, Chan SWY, Schwannauer M. Adolescents’ mental health and well-being in developing countries: a cross-sectional survey from Pakistan. J Ment Health. 2019;28(4):389–96. https://doi.org/10.1080/ 09638237.2018.1521919. The present study was based on an analysis of the Morocco 2016 GSHS survey dataset freely available online with all identifier information detached. The Morocco 2016 GSHS was approved by the School and University Health Division, Ministry of Health, Rabat, Morocco and the World Health Organization. Therefore, the permission and ethical approval for the present analysis was automatically deemed unnecessary. Moreover, during the GSHS survey, written assent attached to a questionnaire was obtained from all eligible participants before filling the questionnaire. 15. Mwakanyamale AA, Yizhen Y. Psychological maltreatment and its relationship with self-esteem and psychological stress among adolescents in Tanzania: a community based, cross-sectional study. BMC Psychiatry. 2019;19(1):176. https://doi.org/10.1186/s12888-019-2139-y. 15. Mwakanyamale AA, Yizhen Y. Psychological maltreatment and its relationship with self-esteem and psychological stress among adolescents in Tanzania: a community based, cross-sectional study. BMC Psychiatry. 2019;19(1):176. https://doi.org/10.1186/s12888-019-2139-y. 16. Jaisoorya TS, Geetha D, Beena KV, Beena M, Ellangovan K, Thennarasu K. Prevalence and correlates of psychological distress in adolescent students from India. East Asian Arch Psychiatr. 2017;27(2):56–62. 16. Jaisoorya TS, Geetha D, Beena KV, Beena M, Ellangovan K, Thennarasu K. Prevalence and correlates of psychological distress in adolescent students from India. East Asian Arch Psychiatr. 2017;27(2):56–62. References 1. World Health Organization (WHO). Adolescent mental health; 2019. URL: https://www.who.int/news-room/fact-sheets/detail/adolescent-mental- health (Accessed 2 Jan 2020). 1. World Health Organization (WHO). Adolescent mental health; 2019. URL: https://www.who.int/news-room/fact-sheets/detail/adolescent-mental- health (Accessed 2 Jan 2020). 1. World Health Organization (WHO). Adolescent mental health; 2019. URL: https://www.who.int/news-room/fact-sheets/detail/adolescent-mental- health (Accessed 2 Jan 2020). 22. Khan MMA, Rahman MM, Islam MR, Karim M, Hasan M, Jesmin SS. Suicidal behavior among school-going adolescents in Bangladesh: findings of the global school-based student health survey. Soc Psychiatry Psychiatr Epidemiol. 2020. https://doi.org/10.1007/s00127-020-01867-z. 2. Polanczyk GV, Salum GA, Sugaya LS, Caye A, Rohde LA. Annual research review: a meta-analysis of the worldwide prevalence of mental disorders in children and adolescents. J Child Psychol Psychiatry. 2015;56(3):345–65. https://doi.org/10.1111/jcpp.12381. 23. World Health Organization (WHO). Global school-based student health survey (GSHS); 2019. URL: https://www.who.int/ncds/surveillance/gshs/en/ (Accessed 10 April 2020). 3. Kessler RC, Amminger GP, Aguilar-Gaxiola S, Alonso J, Lee S, Ustün TB. Age of onset of mental disorders: a review of recent literature. Curr Opin Psychiatry. 2007;20(4):359–64. https://doi.org/10.1097/YCO. 0b013e32816ebc8c. 24. Kroenke K, Spitzer RL, Williams JB. The patient health Questionnaire-2: validity of a two-item depression screener. Med Care. 2003;41(11):1284–92. https://doi.org/10.1097/01.MLR.0000093487.78664.3C. 4. Marsh IC, Chan SWY, MacBeth A. Self-compassion and psychological distress in adolescents-a meta-analysis. Mindfulness (N Y). 2018;9(4):1011–27. https:// doi.org/10.1007/s12671-017-0850-7. 25. Kroenke K, Spitzer RL, Williams JB, Monahan PO, Löwe B. Anxiety disorders in primary care: prevalence, impairment, comorbidity, and detection. Ann Intern Med. 2007;146(5):317–25. https://doi.org/10.7326/0003-4819-146-5- 200703060-00004. 5. American Psychological Association (APA) Dictionary of Psychology: Psychological distress, 2020. URL https://dictionary.apa.org/psychological- distress. 26. Plummer F, Manea L, Trepel D, McMillan D. Screening for anxiety disorders with the GAD-7 and GAD-2: a systematic review and diagnostic metaanalysis. Gen Hosp Psychiatry. 2016;39:24–31. https://doi.org/10.1016/j. genhosppsych.2015.11.005. 26. Plummer F, Manea L, Trepel D, McMillan D. Screening for anxiety disorders with the GAD-7 and GAD-2: a systematic review and diagnostic metaanalysis. Gen Hosp Psychiatry. 2016;39:24–31. https://doi.org/10.1016/j. genhosppsych.2015.11.005. 6. Kessler RC, Barker PR, Colpe LJ, Epstein JF, Gfroerer JC, Hiripi E, et al. Screening for serious mental illness in the general population. Arch Gen Psychiatry. 2003;60(2):184–9. https://doi.org/10.1001/archpsyc.60.2.184. Page 11 of 11 Page 11 of 11 Pengpid and Peltzer BMC Psychiatry (2020) 20:475 27. World Health Organization (WHO). Adverse childhood experiences international questionnaire (ACE-IQ) guidance for Analysing ACE-IQ; 2016. URL: https://www.who.int/violence_injury_prevention/violence/activities/ adverse_childhood_experiences/guidance_for_analysing.pdf?ua=1 (Accessed 2 April 2020). 46. Keles B, McCrae N. & Grealish. A systematic review: the influence of social media on depression, anxiety and psychological distress in adolescents. Int J Adolesc Youth. 2020;25(1):79–93. References https://doi.org/10.1080/02673843.2019. 1590851. 47. Hoare E, Milton K, Foster C, Allender S. The associations between sedentary behaviour and mental health among adolescents: a systematic review. Int J Behav Nutr Phys Act. 2016;13(1):108. https://doi.org/10.1186/s12966-016- 0432-4. 28. Al-Yateem N, Bani Issa W, Rossiter RC, Al-Shujairi A, Radwan H, Awad M, et al. Anxiety related disorders in adolescents in the United Arab Emirates: a population based cross-sectional study. BMC Pediatr. 2020;20(1):245. https:// doi.org/10.1186/s12887-020-02155-0. 48. Vancampfort D, Van Damme T, Stubbs B, Smith L, Firth J, Hallgren M, et al. Sedentary behavior and anxiety-induced sleep disturbance among 181,093 adolescents from 67 countries: a global perspective. Sleep Med. 2019;58:19– 26. https://doi.org/10.1016/j.sleep.2019.01.048. 29. Maalouf FT, Ghandour LA, Halabi F, Zeinoun P, Shehab AA, Tavitian L. Psychiatric disorders among adolescents from Lebanon: prevalence, correlates, and treatment gap. Soc Psychiatry Psychiatr Epidemiol. 2016; 51(8):1105–16. https://doi.org/10.1007/s00127-016-1241-4. 49. Edwards MK, Loprinzi PD. Experimentally increasing sedentary behavior results in increased anxiety in an active young adult population. J Affect Disord. 2016;204:166–73. https://doi.org/10.1016/j.jad.2016.06.045. 30. Angold A, Erkanli A, Silberg J, Eaves L, Costello EJ. Depression scale scores in 8-17-year-olds: effects of age and gender. J Child Psychol Psychiatry. 2002; 43(8):1052–63. https://doi.org/10.1111/1469-7610.00232. p g j j 50. McLaughlin KA, Hatzenbuehler ML. Stressful life events, anxiety sensitivity, and internalizing symptoms in adolescents. J Abnorm Psychol. 2009;118(3): 659–69. https://doi.org/10.1037/a0016499. 31. Rescorla L, Achenbach TM, Ivanova MY, Dumenci L, Almqvist F, Bilenberg N, et al. Epidemiological comparisons of problems and positive qualities reported by adolescents in 24 countries. J Consult Clin Psychol. 2007;75(2): 351–8. https://doi.org/10.1037/0022-006X.75.2.351. Publisher’s Note 32. Byrne DG, Davenport SC, Mazanov J. Profiles of adolescent stress: the development of the adolescent stress questionnaire (ASQ). J Adolesc. 2007; 30(3):393–416. https://doi.org/10.1016/j.adolescence.2006.04.004. 32. Byrne DG, Davenport SC, Mazanov J. Profiles of adolescent stress: the development of the adolescent stress questionnaire (ASQ). J Adolesc. 2007; 30(3):393–416. https://doi.org/10.1016/j.adolescence.2006.04.004. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 33. Spear LP. The adolescent brain and age-related behavioral manifestations. Neurosci Biobehav Rev. 2000;24(4):417–63. https://doi.org/10.1016/s0149- 7634(00)00014-2. 34. Landstedt E, Gillander GK. Experiences of violence among adolescents: gender patterns in types, perpetrators and associated psychological distress. Int J Public Health. 2011;56(4):419–27. https://doi.org/10.1007/s00038-011- 0258-4. 35. Fleming LC, Jacobsen KH. Bullying among middle-school students in low and middle income countries. Health Promot Int. 2010;25(1):73–84. https:// doi.org/10.1093/heapro/dap046. 36. Kazarian SS, Ammar J. School bullying in the Arab world: a review. Arab J Psychiatry. 2013;24(1):37–45. https://doi.org/10.12816/0000097. 37. Save the Children (Middle East and Eastern Europe Regional Office) and UNICEF (MENA Regional Office) November 2017 Violence against adolescents and youth: new evidence and key policy issues for Mena. URL: https://www.menayouthhub.org/sites/menayouthhub.org/files/2019- 03/51%20Violence%20Evidence%20Paper%20ESAY%202017.pdf. 38. Beattie TS, Prakash R, Mazzuca A, Kelly L, Javalkar P, Raghavendra T, et al. Prevalence and correlates of psychological distress among 13-14 year old adolescent girls in North Karnataka, South India: a cross-sectional study. BMC Public Health. 2019;19(1):48. https://doi.org/10.1186/s12889-018-6355-z. 39. Tabak I, Jodkowska M, Oblacińska A. Społeczne uwarunkowania podwyzszonego poziomu stresu psychologicznego u młodziezy 18-letniej w Polsce [social determinants of psychological distress in adolescents aged 18 years in Poland]. Med Wieku Rozwoj. 2008;12(2 Pt 1):569–76. 40. Klasen H, Crombag AC. What works where? A systematic review of child and adolescent mental health interventions for low and middle income countries. Soc Psychiatry Psychiatr Epidemiol. 2013;48(4):595–611. https:// doi.org/10.1007/s00127-012-0566-x. 40. Klasen H, Crombag AC. What works where? A systematic review of child and adolescent mental health interventions for low and middle income countries. Soc Psychiatry Psychiatr Epidemiol. 2013;48(4):595–611. https:// doi.org/10.1007/s00127-012-0566-x. 41. Hong SA, Peltzer K. Dietary behaviour, psychological well-being and mental distress among adolescents in Korea. Child Adolesc Psychiatry Ment Health. 2017;11:56. https://doi.org/10.1186/s13034-017-0194-z. 41. Hong SA, Peltzer K. Dietary behaviour, psychological well-being and mental distress among adolescents in Korea. Child Adolesc Psychiatry Ment Health. 2017;11:56. https://doi.org/10.1186/s13034-017-0194-z. 42. Jacob L, Smith L, Haro JM, Stickley A, Koyanagi A. Publisher’s Note Serious physical injury and depressive symptoms among adolescents aged 12-15 years from 21 low- and middle-income countries. J Affect Disord. 2020;264:172–80. https:// doi.org/10.1016/j.jad.2019.12.026. 42. Jacob L, Smith L, Haro JM, Stickley A, Koyanagi A. Serious physical injury and depressive symptoms among adolescents aged 12-15 years from 21 low- and middle-income countries. J Affect Disord. 2020;264:172–80. https:// doi.org/10.1016/j.jad.2019.12.026. 43. Pengpid S, Peltzer K. Early substance use initiation and psychological distress among adolescents in five ASEAN countries: a cross-sectional study. Psychol Res Behav Manag. 2019;12:1003–8. https://doi.org/10.2147/PRBM. S223624. 43. Pengpid S, Peltzer K. Early substance use initiation and psychological distress among adolescents in five ASEAN countries: a cross-sectional study. Psychol Res Behav Manag. 2019;12:1003–8. https://doi.org/10.2147/PRBM. S223624. 44. Pengpid S, Peltzer K. Leisure-time sedentary behavior is associated with psychological distress and substance use among school-going adolescents in five southeast Asian countries: a cross-sectional study. Int J Environ Res Public Health. 2019;16(12):2091. https://doi.org/10.3390/ijerph16122091. 45. Williams JL, Rheingold AA, Knowlton AW, Saunders BE, Kilpatrick DG. Associations between motor vehicle crashes and mental health problems: data from the National Survey of adolescents-replication. J Trauma Stress. 2015;28(1):41–8. https://doi.org/10.1002/jts.21983.
https://openalex.org/W4372324951	https://bcpublication.org/index.php/BM/article/download/4903/4766	English	null	Opportunities and Challenges of Enterprise Digital Transformation	BCP business & management	2,023	cc-by	3,841	BCP Business & Management Volume 44 (2023) BCP Business & Management Volume 44 (2023) FIBA 2023 1.1 Background introduction Many new technologies, including machine-to-machine communication and the Internet of things (IoTs), are brought into the public's awareness by the "Industrial Revolution 4.0" environment. With a new generation of industrial advanced technology like smart manufacturing, which combine virtual and physical reality, digital transformation is changing every aspect of how an organization runs, including adjustments made in tandem with earlier transformations like environmental and social transformations [1]. As a result of the high pace and unpredictable nature of many changes, complexity and dynamics are created, making effective forecasting and planning difficult or impossible. Due to the current business environment and competitive dynamics, businesses (especially those engaged in e-business) and their operations must be flexible and responsive, which entails being constantly driven by the need to innovate. The divergence among the prevalent belief among digital economies entrepreneurs and the reality and requirements of the digital economies, however, is expanding as the demands of the new generation of digital technologies and the problems of digital transformation increase. These expectations include a state that is far more active in policy, risk-taking, operating the market, and managing the economy [2]. Digital transformation both fuels and is fueled by innovation. It is vital to realize that the legacy of prior organizational and management issues can be applied to the new ecosystems, instead of being an encumbrance, think of them as models that could be used to make distinctions [3]. The digital economy has similar challenges for new industries in terms of innovation and adaptability of new waves of technology, as well as absorption and dissemination of new digital technologies throughout the entire economy [2]. Opportunities and Challenges of Enterprise Digital Transformation Yunrong Yan* Yunrong Yan* Shenzhen College of International Education, Guangdong, China Corresponding author: s20094.yan@stu.scie.com.cn Abstract. "Change or die!" Is the title and opening concept of Alan Deutschman's article for Fast Company magazine. As the aphorism indicates, change is crucial if an enterprise wants to remain invincible in the market competition. Therefore, we investigate and study several academic sources relating to enterprise innovation. After analyzing different economic innovation models and the problems confronted by innovation, we believe that although enterprise innovation may bring a lot of setbacks, only through continuous innovation can enterprises keep the leading position in the competition. By perfecting the transformation of public data space, the successful application of digital service innovation (DSI) digital transformation technology has become an indispensable part of innovation. Enterprises will gradually be obsolete if they are not able to keep innovating. Innovation enables enterprises to adapt to new opportunities in the new era and have the adaptability to "adapt to all changes", so as to cope with the rapidly changing market. By studying various kinds of economic innovation literature and using charts and other methods, we summarized the advantages and methods of innovation in a more simple way, so that a more intuitive understanding of why enterprises should innovate today, how to innovate and the direction of innovation is provided. Keywords: Innovate; benefits; complex. 1.3 Motivation and framework of this article Considering that the existing approaches are rather specialized and restricted to their domains, we aim to provide guidance and several suggesting solutions on how the industry should fulfill its goal of persistent innovation by producing a review based on existing research. Section 2 presents the focus of establishing a core innovation strategy. Section 3 identifies the changes that digital transformation caused on digital platforms, and possible combinations of digital transformation techniques, and puts forward absorptive capacity as a factor leading to innovation. The main research findings are outlined in the Conclusions section, which also explores the potential for further research. Section 4 selects an example innovation model to present its effects on an industry. Section 5 presents the limitations and future outlook of the research method used in section 4. 1.2 An overview of the progress of existing research and literature The term "digital transformation" has gained popularity in our day and age, sparking the emergence of numerous academic disciplines and a huge number of studies. To understand the origins, 631 BCP Business & Management Volume 44 (2023) FIBA 2023 FIBA 2023 circumstances, and impacts of disruptive technologies, researchers examine how each technology is used or how digitalization changes that use [4]. circumstances, and impacts of disruptive technologies, researchers examine how each technology is used or how digitalization changes that use [4]. A growing number of studies have been published in journals and conference proceedings. Researchers have identified the main research strands in a variety of important domains, including marketing, finance, and innovation management, with the aid of co-citation network analysis. It was shown that the main areas of study and research focus for digital transformation are related [4]. The publications in earlier research trend analyses were manually sorted according to the theories and methods employed, or according to the subjectivity of the researchers. The current study conducts network text analysis on papers that have recently been presented at conferences and published in scholarly journals to examine the trends of the research on digital transformation in order to shed some light on the service sector's future prospects for digital transformation [5]. There are also studies that analyze the possible methods to guide innovation in the digital transformation from a government perspective [2]; studies that research the method of improving business process management maturity models (BPM MMs); and studies that analyze small and medium-sized businesses' ongoing digital transformation processes, which have been accelerated by the fourth industrial revolution, in order to examine how they went through and discovered during the second and third industrial revolutions represents essential components that have shaped their way of operation and continue to promote innovation and transformation [3]. 2.1 Definition and characteristics of enterprise innovation In the face of complex and changing market environment, enterprises must keep innovating to adapt to the new opportunities in the new era, produce sudden changes, and so to have the flexibility to cope with the rapid change of the market. To adapt to the emerging markets and business environment of the Internet age, it is arduous for companies to achieve transformation and development without innovation. Innovation is a process that involves a wide variety of sectors that work in the organization and it can be the improvement of the existing products and service performance or new kinds of one for new markets or developing an unprecedented market for current products [6]. Although the success of innovation brings huge benefits to enterprises, a small portion of them is able to do it because the complicated market environment leads to methods that are involved to implement. And the knowledge that distinguishes effective business innovation strategies from futile attempts is difficult to obtain in one team [7]. Therefore, innovation is very time-consuming and will result in the situation of rush, delay, and regression. The consequence of it is a low success rate of innovation, which is characteristic of enterprise innovation. Innovation should be initiated from the demand, there will be a lot of obstacles on the way to innovation, and perseverance and daring to struggle are indispensable for the success of the enterprise innovation. 632 BCP Business & Management Volume 44 (2023) FIBA 2023 FIBA 2023 3. The effect of digital transformation on enterprise innovation The new and developing form of digital economies are sure to be confronted with challenges, in order to form a solution, possible combinations of digital transformation techniques and the crucial factor, absorptive capacity, are put forward below to support innovation. 2.3 Enterprise innovation model There are many ways for enterprises to innovate, so there are different models corresponding to various forms of innovation, such as the combinatorial innovation model. This model first finds the smallest unit that needs to be studied in innovation by dismantling the elements and then re-examines the combination of the product, technology, market, customer, channel, marketing, and other aspects, so as to improve the innovation ability. For example, the field of aerial UAV, can be divided into UAV and camera for separate research to improve the endurance and stability of UAV, as well as the tracking mode and clarity of the camera, so as to realize innovation [11]. 2.2 The core strategy of enterprise innovation The most important part of enterprise innovation is to establish the core ‘innovation strategy’. The goal is to enhance the core innovation ability. Changing the strategic ideology is the first step. Modifying the extensive mode of economic growth, Improve the level of industrial technology, enhance the core innovation ability as the strategic basis for enterprise development. Amazon is a successful precedent for updating the core innovation ability as they updated their previous product to innovate: Amazon FBA program which incentivizes third-party sellers to sell online [8]. The implementation of core innovation strategy should grasp the general trend of economic development, take social and market demand as guidance, and take the scientific outlook on development as guidance. Integrating original innovation, and system integration innovation with an introduction, digestion, and absorption innovation so that to acquire more core intellectual property in key fields, and occupy a place in the scientific frontier and strategic high-tech fields. Making core innovation become the engine and motivator of the sustainable development of enterprise finance. Strengthening science and technology planning, construction of digital, applied science and technology research, promoting the research and application of new technology, new method, and new way of industry, completely transfer the economic growth mode to technological progress and staff quality improvement in order to promote the optimization of industrial strategy and realize the sustainable development of economy[9]; Through constructing innovation strategy, the enterprise can drive and promote its target, business, brand, and talent strategy, form a scientific strategic innovation system, eventually make the enterprise core innovation strategy more standardized, networked and systematic. Take Apple as an example. They create a WWDC congress which allows the practitioners to provide outside-of-the-box ideas so that innovative ideas are brought to the company from the platform which is a form of the innovation strategy of Apple [10]. 3.3 Considering absorptive capacity as a mediating role that contributes to innovation The study's findings demonstrate how absorptive capacity, and to a lesser extent of digital capacity, contribute significantly and directly to the performance of innovation [14]. Businesses must develop their digital capacity, which includes measures to integrate fully and actively employ digital technology, in order to realize their aim of digital transformation. While absorptive capacity is often based on the amount of information currently available and the amount of effort being made to enhance technical capabilities, digital capacity is typically related to two dimensions: a supply-side one and a demand-side one. Innovation success depends on a firm's own knowledge base as well as its original capacity to integrate the usage of digital technologies for the generation of new value as innovation is not always the outcome of the adoption of digital technology alone [14]. 3.2 Possible combinations of digital transformation techniques that produce successful digital service innovation (DSI) Digital technologies have been used to transition from product-centric to service-centric business models, in order to form a digital transformation strategy. Researchers employ full-set Qualitative Comparative Analysis on a set of 17 case studies of digital transformation strategies from legitimate companies with a range of industrial backgrounds [13]. Innovation in digital services is described as "the creation of new or developing current processes and resources, or through integrating practices and resources in novel ways to offer new value propositions." The necessary condition results indicate that centralizing judgment is the only requirement for a successful DSI. The designs suggest that a company's innovation activities are negatively impacted by the danger of digital disruption. Additionally, researchers find that while competitive organizations may rely on automatic methods, corporations that are immediately threatened by digital disruption can benefit from strategic alliances. According to the patterns of the research findings, failure can occur when strategic outsourcing is not used. One strategy for success includes strategic outsourcing. However, rather than relying on outsourcing, small and medium-sized firms should work to construct a robust ecosystem of association members that offer the necessary competencies [13]. 3.1 Transforming into public data spaces Information and communication technology have enabled data interchange more rapid than ever since the internet was created 25 years ago, opening up new opportunities for organizational barriers to be broken through corporate innovation. Public data spaces differ significantly from private digital platforms in that they involve more third parties, have wider network effects, and necessitate the enactment of data protection legislation. A single company finds it difficult to adapt to a dynamic platform environment, which makes it difficult to address complex societal issues, properly divide earnings, involve physically connected digital things, and develop governance structures [12]. Modern digital solutions from the ecosystem and the organization are likely to have an impact on the company. The capacity to control this dynamic relationship makes it harder to manage digital findings in an efficient manner. 633 BCP Business & Management Volume 44 (2023) FIBA 2023 4.1 Improvements based on conventional models The Cyclic Innovation Model, which was created in the 1990s as a tool for ongoing reform of research and industry, is suggested as a fourth-generation innovation model. In contrast to the conventional CIM approaches, this paradigm integrates engineering, commercialization, and hard and soft sciences into a unified system of creative processes. It also demonstrates the necessity of adding feedback routes to models in order to explicitly represent adaptive steering and learning processes. 4. Model of the innovation Fig.1. the graph of Circle of change, The Circle of Change is a system model that depicts the foundations of the innovation economy as a collection of dynamic processes that work together to strengthen one another. Figure 1. The graph of Circle of change [15] Figure 1. The graph of Circle of change [15] 634 BCP Business & Management Volume 44 (2023) FIBA 2023 4.3 Linked cycle system Fig. 1 first distinctive feature is that architecture is circular rather than a string: Value creation is a continuous process since ideas generate new advances, successes generate new problems, and failures provide fresh concepts. To keep the circle's momentum, new macroeconomic mechanisms are required. The faith in the innovation economy is being eroded by massive failures like the recent dotcom disaster, which is also making venture money scarce. The change processes with reference to CIM are separated. Businesses are focusing on doing more of the same at a slower pace as the economy enters a phase of stagnation. where businesses are focusing on doing more of the same things at lower costs until confidence and private equity become available again to drive innovation. The circle's momentum is increasing once more. where businesses are focusing on doing more of the same things at lower costs until confidence and private equity become available again to drive innovation. The circle's momentum is increasing once more. 4.2 A key characteristic of fourth-generation innovation models Innovation models are divided into generations in the literature on innovation. However, well- defined models have not yet made it into the open literature despite discussions about the requirements for next-generation concepts. The innovation regime is described by a "circle of change" in this model, which is a fourth-generation innovation model. It ties together changes in markets, technological advancements, product design, and manufacturing. The model substitutes a circle with four "nodes of change" connected by four interlocking "cycles of change" for the conventional chain concept. When taken as a whole, they can be viewed as an area of opportunity where traditional boundaries are crossed by processes. These procedures are typical of open innovation today and are cyclical in nature. Knowledge, information, capital, labor, goods, and services, as well as technological and socioeconomic values, are all constantly traded in the circle. The role of entrepreneurship is crucial: Innovation is nonexistent without entrepreneurship. 5.2 Future outlook Future innovations will frequently be the outcome of fusing customer demands from various industries with technical capabilities. Models of innovation from the fourth generation should be able to demonstrate that [15]. In terms of CIM: takes into consideration the advancements that will be possible in the healthcare sector as a result of recent developments in the IC sector. p p countermeasure—turning scientific knowledge into socioeconomic value The proposed model's key implication is that, in order to transform scientific knowledge into socioeconomic value, Innovation in all industries necessitates early interactions between new business ideas and scientific discoveries, as well as new technological inventions and market opportunities. These cyclical linkages will transcend not just traditional sectoral borders but also those separating various stages of innovation. It is intended to imply that all nodes of the proposed innovation model are dynamic at every stage of the innovation track by eliminating the pipeline idea from models of innovation. The sluggish and expensive internal pipeline model is slowly being taken place by a more flexible idea that calls for "playing chess at four levels at once”. The distant distance among technology and markets is greatly reduced, there are little connections between new information and prompt adopters, and new innovations construct on previous ones as a result of this utilization of knowledge from other industrial sectors. 5.1 Current situation Digital transformation is a fundamental change process that is made possible by making smart use of crucial capabilities and resources and innovative use of digital innovations. This is one of the most common problems that all businesses face today. despite the fact that this subject has been investigated in a variety of settings and that numerous papers offer in-depth viewpoints on it. There are still a lot of unanswered questions about how various industries implement digital transformation, what drives businesses to move from using basic information technology to digital transformation, and the connection between these issues. A significant limitation is that the networks connecting multi-partnerships that can start quickly, change quickly, and learn quickly are required for the "nodes of change. “This indicates that in the current innovation landscape, the issue will not be "who is accessible," but rather "who is needed”. This implies, among other things, that the current system of a labor organization has to be reviewed: innovation's social component In fact, the proposed model alerts decision-makers to the need to redesign institutional elements like governmental regulations regarding the movement of capital, labor, and knowledge around the innovation circle in order to better support the innovation processes. The current structures of government need to be rethought in light of this. 4.4 Dynamic processes in a system - Circle of Change The proposed model's description of a system of processes that change—the circle of change, comprising four processes: market transitions, product development, scientific inquiry, and technological exploration—is also shown in Figure 1. However, more crucially, there are "cycles of change" between these nodes that guarantee that dynamic processes in the nodes influence one another. As a result, connected cycles is created, which in influences other connected cycles (higher- order dependencies). This enables a more or less connected web between industry and technology, encouraging creative engagement. This results in a system of interconnected cycles that have an impact on one another (higher-order dependencies). The end result is a regime of interconnected dynamic processes that are more or less coordinated and encourage creative collaboration among advancements in science (on the left) and industry (on the right), as well as advancements in technology (on the top) and the market (bottom). Few barriers will exist between nodes and cycles in an innovation economy that is successful because institutions and organizational structures support the change processes [16]. There is a constant exchange of knowledge, information, capital, labor, goods, services, and socioeconomic as well as technical values throughout the circle. 635 BCP Business & Management Volume 44 (2023) FIBA 2023 FIBA 2023 6. Conclusion In conclusion, digital innovation transformation has formed an irresistible trend for enterprises in modern society. Perfecting the transformation to public data space and successfully using the digital transformation technology of Digital Service innovation (DSI) has become an indispensable part of innovation. Similarly, having a good absorptive capacity can directly promote digital innovation. On this basis, the fourth-generation innovation model and the company's own positioning to properly use the innovation cycle model can be conflated, giving play to the effect of the linkage cycle, updating the innovation model, and forming a good innovation cycle. Although the research on enterprise innovation in the western era is not in-depth, it basically summarizes the current forms of innovation, which has certain reference and foresight. 636 FIBA 2023 BCP Business & Management Volume 44 (2023) FIBA 2023 6] Volberda, H.W. (1998) Building the Flexible Firm, Oxford University Press. References [1] Marek Szelągowski, Justyna Berniak‐Woźny, How to improve the assessment of BPM maturity in the era of digital transformation. Information Systems and e-Business Management, 2022, 20: 171 – 198. [2] Nagy Hanna, A role for the state in the digital age. Hanna Journal of Innovation and Entrepreneurship, 2018, 7: 5. [3] Elisa Martina Martinelli, Maria Cristina Farioli, Annalisa Tunisini, New companies’ DNA: the heritage of the past industrial revolutions in digital transformation. Journal of Management and Governance, 2021, 25: 1079 – 1106. [4] J. Piet Hausberg, Kirsten Liere‐Netheler, Sven Packmohr, Stefanie Pakura, Kristin Vogelsang, Research streams on digital transformation from a holistic business perspective: a systematic literature review and citation network analysis. Journal of Business Economics, 2019, 89:931 – 963 [5] Jin Sung Rha, Hong‐Hee Lee, Research trends in digital transformation in the service sector: a review based on network text analysis. Service Business, 2022, 16: 77 – 98. [6] Rim Jallouli, Mohamed Anis Bach Tobji, Deny Bélisle, Sehl Mellouli, Farid Abdallah, Ibrahim Osman, Emerging Technologies and Business Innovation, 2019. [7] Deny Bélisle, Sehl Mellouli, Farid Abdallah, Ibrahim Osman, Rim Jallouli, Mohamed Anis Bach Tobji, The Effect of Digital Transformation on Innovation and Entrepreneurship in the Tourism Sector: The Case of Lebanese Tourism Services Providers, 2019. [8] Rui Li, Jing Rao, Liangyong Wan, The digital economy, enterprise digital transformation, and enterprise innovation, 2022; 43:2875 - 2886. [9] Lei Zhang, Bank Competition, Financing Constraints, and Enterprise Innovation Investment, 2022. [10] Minshu Zhao, Fangying Yuan, the top management team and enterprise innovation: An empirical study from growth enterprise market listed companies in China, 2016; 20. 1] Mengjun Yang, Shilin Zheng, Lin Zhou, Broadband internet and enterprise innovation, 2022. [12] Daniel Beverungen, Thomas Hess, Antonia Köster, Christiane Lehrer, From private digital platforms to public data spaces: implications for the digital transformation. Electronic Markets, 2022, 32: 493 – 501. [13] David Soto Setzke, Tobias Riasanow, Markus Böhm, Helmut Krcmar, Pathways to Digital Service Innovation: The Role of Digital Transformation Strategies in Established Organizations. Information Systems Frontiers, 2021. [14] Ioanna Kastelli, Petros Dimas, Dimitrios Stamopoulos, Aggelos Tsakanikas, Linking Digital Capacity to Innovation Performance: The Mediating Role of Absorptive Capacity. Journal of the Knowledge Economy, 2022. [15] A. J Berkhout, Dap Hartmann, Patrick van der Duin and Roland Ortt, innovating the innovation process Nos. 3/4, 2006. [16] Volberda, H.W. (1998) Building the Flexible Firm, Oxford University Press. 637
https://openalex.org/W4308803722	https://www.frontiersin.org/articles/10.3389/fmicb.2022.1045660/pdf	English	null	Case report: Acute Talaromyces marneffei mediastinitis in an HIV-negative patient	Frontiers in microbiology	2,022	cc-by	5,329	OPEN ACCESS OPEN ACCESS EDITED BY Esaki M. Shankar, Central University of Tamil Nadu, India REVIEWED BY Alex Andrianopoulos, The University of Melbourne, Australia Koichi Tanabe, Ryukoku University, Japan CORRESPONDENCE Meichun Zhang drzhangmc@sina.com SPECIALTY SECTION This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology RECEIVED 15 September 2022 ACCEPTED 24 October 2022 PUBLISHED 11 November 2022 CITATION Chen L, Zhang M, Guo W, Ding W, Tan J, Du H, Zhao Z and Zhong W (2022) Case report: Acute Talaromyces marnefei mediastinitis in an HIV-negative patient. Front. Microbiol. 13:1045660. doi: 10.3389/fmicb.2022.1045660 OPEN ACCESS EDITED BY Esaki M. Shankar, Central University of Tamil Nadu, India REVIEWED BY Alex Andrianopoulos, The University of Melbourne, Australia Koichi Tanabe, Ryukoku University, Japan CORRESPONDENCE Meichun Zhang drzhangmc@sina.com SPECIALTY SECTION This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology RECEIVED 15 September 2022 ACCEPTED 24 October 2022 PUBLISHED 11 November 2022 CITATION Chen L, Zhang M, Guo W, Ding W, Tan J, Du H, Zhao Z and Zhong W (2022) Case report: Acute Talaromyces marnefei mediastinitis in an HIV-negative patient. Front. Microbiol. 13:1045660. doi: 10.3389/fmicb.2022.1045660 OPEN ACCESS EDITED BY Esaki M. Shankar, Central University of Tamil Nadu, India REVIEWED BY Alex Andrianopoulos, The University of Melbourne, Australia Koichi Tanabe, Ryukoku University, Japan CORRESPONDENCE Meichun Zhang drzhangmc@sina.com SPECIALTY SECTION This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology RECEIVED 15 September 2022 ACCEPTED 24 October 2022 PUBLISHED 11 November 2022 CITATION Chen L, Zhang M, Guo W, Ding W, Tan J, Du H, Zhao Z and Zhong W (2022) Case report: Acute Talaromyces marnefei mediastinitis in an HIV-negative patient. Front. Microbiol. 13:1045660. doi: 10.3389/fmicb.2022.1045660 Liangyu Chen1,2,3, Meichun Zhang3,4, Weihong Guo3,4, Wenshuang Ding4,5, Jinwen Tan3,4, Hong Du4,5, Ziwen Zhao3,4 and Weinong Zhong3,4 Liangyu Chen1,2,3, Meichun Zhang3,4*, Weihong Guo3,4, Wenshuang Ding4,5, Jinwen Tan3,4, Hong Du4,5, Ziwen Zhao3,4 and Weinong Zhong3,4 1Department of Respiratory and Critical Care Medicine, Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine, Zhuhai, China, 2Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, China, 3Department of Respiratory and Critical Care Medicine, Guangzhou First People’s Hospital, Guangzhou, China, 4South China University of Technology, Guangzhou, China, 5Department of Pathology, Guangzhou First People’s Hospital, Guangzhou, China Talaromyces marnefei (T. marnefei) is one of the most important opportunistic human pathogens endemic in Southeast Asia. TYPE Case Report PUBLISHED 11 November 2022 DOI 10.3389/fmicb.2022.1045660 TYPE Case Report PUBLISHED 11 November 2022 DOI 10.3389/fmicb.2022.1045660 COPYRIGHT © 2022 Chen, Zhang, Guo, Ding, Tan, Du, Zhao and Zhong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. mediastinal mass, nebulized amphotericin B, Talaromyces marnefei, mediastinitis, EBUS-TBNA, HIV-negative, immunocompetent OPEN ACCESS Talaromycosis, which was once regarded as an opportunistic infectious disease in patients with acquired immunodeﬁciency syndrome, is being increasingly reported in HIV-negative populations. Since T. marnefei infection can be localized or disseminated, patients may present with a variety of symptoms. However, mediastinal infection attributed to T. marnefei is extremely rare. We report the case of a 32-year-old female who manifested a large mediastinal mass and was eventually diagnosed as acute T. marnefei mediastinitis. The patient was HIV- negative and had no direct contact with intermediate hosts. We successfully managed to treat the patient with inhaled amphotericin B deoxycholate and observed lesion absorption in subsequent CT examinations. To our knowledge, this is the ﬁrst published case of T. marnefei mediastinitis and ﬁrst use of inhaled antifungal monotherapy on patients with T. marnefei infection. Front. Microbiol. 13:1045660. doi: 10.3389/fmicb.2022.1045660 Abbreviations: BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage ﬂuid; TBLB, conventional transbronchial lung biopsy; EBUS-TBNA, endobronchial ultrasound-guided transbronchial needle aspiration; TBNA, transbronchial needle aspiration; PAS, periodic acid-Schif; GMS, grocott methenamine silver; mNGS, metagenomic next-generation sequencing; AmBd, amphotericin B deoxycholate; MIC, minimum inhibitory concentration. Case presentation A 32-year-old woman presented with acute-onset shortness of breath and chest pain for 4 days. She also complained of cough with some small amounts of phlegm and occasional low- grade fever, but no night sweat, rash, hemoptysis, loss of weight, joint swelling, or any other discomfort. Her medical history included hypotension, hypoglycemia and bile reﬂux gastritis. She was also a hepatitis B virus carrier and once arranged for an interventional operation for spontaneous intracerebral hemorrhage at the age of 20. Luckily, no sequela was found after that operation. Her home medications included itopride, sucralfate and famotidine. She was a lifelong non-smoker and had no history of recent travel, wild animal contacts or sick contacts. She lived in Guangdong province in China. She had no known allergies. To obtain an accurate diagnosis, bronchoscopy, bronchoalveolar lavage (BAL), conventional transbronchial lung biopsy (TBLB) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) were performed on the 4th day of hospitalization. Bronchoscopy revealed severe trachea compression stenosis caused by an extratracheal neoplasm anterior to the lower trachea (Figure 2A), along with bronchial inﬂammation and airway secretion. Mixed bacteria were found yet indistinguishable by BALF smear and BALF culture. mNGS of BALF detected T. marneﬀei with 15 sequence reads identiﬁed out of 84 total reads. TBLB showed acute inﬂammation, negative for Acid-fast, PAS and GMS stains, and no tumor cells were seen (Figure 3A). Whereas, with the technology of EBUS-TBNA, we were able to observe the ultrasonogram of the extratracheal neoplasm (Figure 2B) and obtain the needle aspirates of the mass. On the 9th day of admission, culturing on Sabouraud dextrose agar medium at 25◦C conﬁrmed T. marneﬀei in the biopsied mass with colonies showing characteristic red-pigmented fungal colonies from the needle aspirates (Figure 2D). Under the microscope, the mold form of T. marneﬀei was also observed in imprint smear obtained from cultured samples of needle aspirates at 25◦C on the background of lactophenol cotton blue staining (Figure 2C). On the same day, histopathologic picture of needle aspirates demonstrated acute exudative inﬂammation and focal necrosis, where yeast cells with characteristic transverse septa were identiﬁed through PAS and GMS staining (Figures 3C,D). Based on the evidence above, we ﬁnally proved the diagnosis was acute Talaromyces marneﬀei mediastinitis. Case presentation After admission to hospital, physical examination revealed the following: blood pressure was 92 mmHg systolic and 65 mmHg diastolic, respiratory rate was 21 breaths/min, heart rate was 122 beats/min, body temperature was 99.7◦F, arterial oxygen saturation on room air was 95%. She had coarse breath sounds on auscultation without any wheezing, rhonchi or crackles. The remainder of the physical examination produced normal results. Introduction In terms of immune-related examinations, blood level of immunoglobulin G turned out 20.8 g/L, slightly above the normal value (8.6–17.4 g/L), while levels of immunoglobulin A, immunoglobulin M, immunoglobulin E, complement 3 and complement 4 were within normal range. Blood levels of 6 types of cytokines, TNF-α, IFN-γ, IL-2, IL-4, IL-6, and IL- 10 were also normal. Moreover, no abnormal results were obtained on lymphocyte subset counts test, anti-extractable nuclear antigen antibodies test, antinuclear antibody test and vasculitis-associated autoantibodies test. Sputum culture and an electrocardiogram showed no obvious abnormalities. Initial chest radiograph indicated bilateral pneumonia. The contrasted chest CT demonstrated a 4.0 × 3.6 cm non-uniformly enhanced middle mediastinal mass with compression of lower trachea and superior vena cava (Figure 1A). Enlarged intramediastinal lymph nodes were appreciated, as well as right middle lobar inﬁltration (Figure 1D). to have a variety of other immunocompromising conditions (You et al., 2021; Liu et al., 2022). Clinically, talaromycosis is usually referred to as an easily misdiagnosed, intractable and high-mortality disease, for its uncertain pathogenesis, non-speciﬁc symptoms, various imaging manifestations and complicated treatment with serious side eﬀects. This case report aims to inform physicians about the clinical presentation, imaging study, pathological characteristics and treatment of T. marneﬀei mediastinitis. Introduction T. marneﬀei was previously named Penicillium marneﬀei and the disease has variably been described as penicillosis or penicillosis marneﬀei. Among all discovered Talaromyces species, T. marneﬀei is the only thermally dimorphic species known to be pathogenic to humans. At 25◦C, the colonies of T. marneﬀei are greenish-yellow, granular, and circular in shape, surrounded by characteristic red diﬀusible pigment. Little or no red pigment occur in the yeast phase at 37◦C (Cooper, 1997). Microscopically, the mold form is like other Talaromyces species, with hyaline, septate and branched hyphae. Conidiophores give rise to three to ﬁve phialides, where chains of conidia are formed (Vanittanakom et al., 2006). The bamboo rat is considered as main natural reservoir host of T. marneﬀei (Deng et al., 1986). T. marneﬀei infections in humans are overwhelming in immunocompromised individuals, however a number of cases in apparent immunocompetent individuals have been described (Duong, 1996; Qiu et al., 2015; Pruksaphon et al., 2022). Increasingly these HIV-negative patients are found Frontiers in Microbiology 01 frontiersin.org Chen et al. 10.3389/fmicb.2022.1045660 normal. T-Spot test suggested positive while other blood tests showed negative for blood culture, cryptococcal antigen latex agglutination test, candida galactomannan antigen test, (1-3)- β-D glucan antigen test, tumor-associated antigens and HIV antibodies. In terms of immune-related examinations, blood level of immunoglobulin G turned out 20.8 g/L, slightly above the normal value (8.6–17.4 g/L), while levels of immunoglobulin A, immunoglobulin M, immunoglobulin E, complement 3 and complement 4 were within normal range. Blood levels of 6 types of cytokines, TNF-α, IFN-γ, IL-2, IL-4, IL-6, and IL- 10 were also normal. Moreover, no abnormal results were obtained on lymphocyte subset counts test, anti-extractable nuclear antigen antibodies test, antinuclear antibody test and vasculitis-associated autoantibodies test. Sputum culture and an electrocardiogram showed no obvious abnormalities. Initial chest radiograph indicated bilateral pneumonia. The contrasted chest CT demonstrated a 4.0 × 3.6 cm non-uniformly enhanced middle mediastinal mass with compression of lower trachea and superior vena cava (Figure 1A). Enlarged intramediastinal lymph nodes were appreciated, as well as right middle lobar inﬁltration (Figure 1D). normal. T-Spot test suggested positive while other blood tests showed negative for blood culture, cryptococcal antigen latex agglutination test, candida galactomannan antigen test, (1-3)- β-D glucan antigen test, tumor-associated antigens and HIV antibodies. Diagnostic studies FIGURE 2 (A) Bronchoscopic view of the lower trachea, revealing severe compression stenosis caused by an extratracheal neoplasm (blue arrows). (B) Ultrasonogram of the extratracheal neoplasm reveals an enlarged para-aortic hypoechoic zone (blue arrows), with blood ﬂow signals inside (orange arrow). (C) Micrograph of imprint smear obtained from cultured samples of needle aspirates at 25◦C, suggesting conidiophores of T. marnefei bearing phialides and chains of conidia on the background of lactophenol cotton blue staining. Scale = 10 µm. (D) 5-day culture of needle aspirates of mediastinal mass at 25◦C, demonstrating red-pigmented fungus colonies on Sabouraud dextrose agar plate. FIGURE 2 (A) Bronchoscopic view of the lower trachea, revealing severe compression stenosis caused by an extratracheal neoplasm (blue arrows). (B) Ultrasonogram of the extratracheal neoplasm reveals an enlarged para-aortic hypoechoic zone (blue arrows), with blood ﬂow signals inside (orange arrow). (C) Micrograph of imprint smear obtained from cultured samples of needle aspirates at 25◦C, suggesting conidiophores of T. marnefei bearing phialides and chains of conidia on the background of lactophenol cotton blue staining. Scale = 10 µm. (D) 5-day culture of needle aspirates of mediastinal mass at 25◦C, demonstrating red-pigmented fungus colonies on Sabouraud dextrose agar plate. FIGURE 1 (A) Selected axial image of the soft-tissue window on contrast-enhanced chest CT before treatment, showing a mass in middle mediastinum (orange arrows) with compression of lower trachea (red arrow) and superior vena cava (blue arrow), it is heterogeneous in density. (B) Selected axial image of the soft-tissue window on contrast-enhanced chest CT after 2-week therapy of nebulized AmBd, suggesting the middle mediastinal mass shrinks (orange arrows) and the compression of lower trachea (red arrow) and superior vena cava (blue arrow) is partially relieved. (C) Selected axial image of the soft-tissue window on chest CT scan at 3-month follow-up, demonstrating the signiﬁcant mass shrinkage in the middle mediastinum (orange arrows). The compression of lower trachea (red arrow) and superior vena cava (blue arrow) is almost completely relieved. (D) Selected axial image of the pulmonary window on chest CT before treatment, showing patchy inﬁltrates in the right middle lobe (orange arrows). (E) Selected axial image of the pulmonary window on chest CT after 2-week therapy of nebulized AmBd, suggesting the inﬁltrates in the right middle lobe are partially absorbed (orange arrows). Diagnostic studies (F) Selected axial image of the pulmonary window on chest CT at 3-month follow-up, demonstrating further absorption in the right middle lobar lesion (orange arrows). therapy of oral itraconazole (dose: 400 mg/d) and CT scan uncovered signiﬁcant improvement (Figures 1C,F). Diagnostic studies Laboratory results suggested white blood cell count of 14,360 cells/mm (3) [normal value, 3,500–9,500 cells/mm(3)], neutrophil count of 10,820 cells/mm(3) [normal value, 1,800– 6,300 cells/mm(3)], lymphocyte count of 1,960 cells/mm(3) [normal value, 1,100–3,200 cells/mm(3)], monocyte count of 850 cells/mm(3) [normal value, 100–600 cells/mm(3)] and procalcitonin level of 0.594 ng/ml (normal value, <0.1 ng/ml). Blood gas analysis, NT-proBNP, D-dimer, cardiac enzymes were We initially treated the patient with intravenous moxiﬂoxacin (dose: 400 mg/d), but the broad-spectrum Frontiers in Microbiology 02 frontiersin.org Chen et al. 10.3389/fmicb.2022.1045660 FIGURE 2 (A) Bronchoscopic view of the lower trachea, revealing severe compression stenosis caused by an extratracheal neoplasm (blue arrows). (B) Ultrasonogram of the extratracheal neoplasm reveals an enlarged para-aortic hypoechoic zone (blue arrows), with blood ﬂow signals inside (orange arrow). (C) Micrograph of imprint smear obtained from cultured samples of needle aspirates at 25◦C, suggesting conidiophores of T. marnefei bearing phialides and chains of conidia on the background of lactophenol cotton blue staining. Scale = 10 µm. (D) 5-day culture of needle aspirates of mediastinal mass at 25◦C, demonstrating red-pigmented fungus colonies on Sabouraud dextrose agar plate. FIGURE 1 (A) Selected axial image of the soft-tissue window on contrast-enhanced chest CT before treatment, showing a mass in middle mediastinum (orange arrows) with compression of lower trachea (red arrow) and superior vena cava (blue arrow), it is heterogeneous in density. (B) Selected axial image of the soft-tissue window on contrast-enhanced chest CT after 2-week therapy of nebulized AmBd, suggesting the middle mediastinal mass shrinks (orange arrows) and the compression of lower trachea (red arrow) and superior vena cava (blue arrow) is partially relieved. (C) Selected axial image of the soft-tissue window on chest CT scan at 3-month follow-up, demonstrating the signiﬁcant mass shrinkage in the middle mediastinum (orange arrows). The compression of lower trachea (red arrow) and superior vena cava (blue arrow) is almost completely relieved. (D) Selected axial image of the pulmonary window on chest CT before treatment, showing patchy inﬁltrates in the right middle lobe (orange arrows). (E) Selected axial image of the pulmonary window on chest CT after 2-week therapy of nebulized AmBd, suggesting the inﬁltrates in the right middle lobe are partially absorbed (orange arrows). (F) Selected axial image of the pulmonary window on chest CT at 3-month follow-up, demonstrating further absorption in the right middle lobar lesion (orange arrows). Discussion Although the mechanisms in the pathogenesis of T. marneﬀei are not fully understood, inhalation of conidia seems to be the main route of transmission (Pruksaphon et al., 2022). In China, most cases are reported in the southern part of the country, particularly Guangdong and Guangxi province, which indicates that talaromycosis is regionally related (Narayanasamy et al., 2021; Pruksaphon et al., 2022). Plenty of reports suggest talaromycosis in immunocompetent patients (Duong, 1996; Ye et al., 2015; Wang et al., 2017) but do not provide categorical evidence for immunocompetency. Recent studies have shown that some infected individuals thought to be non-immunosuppressed carry neutralizing anti-IFN-γ autoantibodies and associated HLA alleles (Guo et al., 2020). Additionally some HIV-negative patients have been shown to carry immune-related genetic mutations such as CD40L, STAT1, STAT3 and CARD9 (You et al., 2021; Liu et al., 2022). For this reason, in spite of the fact that there was no evidence antibiotic seemed to make little eﬀect on her. Her shortness of breath and chest pain continued, while the cough and fever were even exacerbated. We changed the treatment into intravenous amphotericin B deoxycholate (AmBd) (initial dose: 0.1 mg/kg/d) immediately on the day when the diagnosis was conﬁrmed. However, the patient experienced hypotension (BP: 70/43 mmHg) and refused to continue the therapy after the ﬁrst infusion. Therefore, on the 10th day of hospitalization, she was treated with inhaled AmBd (dose: 0.7 mg/kg/d). As expected, the patient’s symptoms were gradually alleviated during the therapy. She was eventually discharged with imaging improvement (Figures 1B,E) after 2-week treatment of nebulized AmBd. At 3-month follow-up, the patient was still stable with subsequent Frontiers in Microbiology 03 frontiersin.org 10.3389/fmicb.2022.1045660 Chen et al. on, usually along with enlargement in hilar and mediastinal lymph nodes (Shi et al., 2020; Wei et al., 2021). In this case report, it’s also shown that T. marneﬀei chest infection can even manifest a large mediastinal mass on imaging when it involves the mediastinum. Based on the anatomical structure of mediastinum and radiologic manifestation of T. marneﬀei chest infection, we assume that the formation of the mediastinal mass may derive from the infection of mediastinal lymph nodes. For the similarity in radiography, physicians should diﬀerentiate T. marneﬀei mediastinitis from other mediastinal diseases, such as mediastinal lymphatic tuberculosis, lymphoma, thymoma and mediastinal teratoma. We eventually diagnosed the patient by performing EBUS- TBNA to obtain mediastinal lesion samples. g Murthi et al., 2020). Even though the patient’s mNGS result proves its high- sensitivity and rapidness, it isn’t considered as convincing evidence to make a deﬁnite diagnosis of T. marneﬀei infection as the result of its relatively low speciﬁcity (Zhang et al., 2022). The gold standard for diagnosis of T. marneﬀei is microbiological culture. It is typiﬁed by fungus colonies with massive production of red pigment at 25◦C (Figure 2D). Microscopically, hyaline, septate and branched hyphae with conidiophores and conidia can be appreciated after incubation at 25◦C (Figure 2C). Histologically, the transverse septum within the yeast cell is the most distinguishable feature of T. marneﬀei (Vanittanakom et al., 2006). However, with hematoxylin and eosin staining, yeast cells with the characteristic septated structure are usually diﬃcult to identify on the background of inﬁltrations of neutrophils, lymphocytes and histiocytes (Figure 3B), which makes it hard to distinguish T. marneﬀei from Histoplasma capsulatum (Widaty et al., 2020). In contrast, once stained with PAS or GMS, especially GMS, yeast cells with transverse septa can have been more easily appreciated in histological observation (Figures 3C,D). Our patient was deﬁnitely diagnosed with T. marneﬀei mediastinitis according to the typical microbiological and pathological characteristics. suggesting this patient suﬀered from immune diseases, her immunocompetency remains undetermined. Genetic testing and the detection of anti–IFN-γ autoantibodies may have revealed a potential immune impairment, but unfortunately none of these had been arranged for this patient. Since T. marneﬀei infection may have been localized or disseminated, clinical manifestations are varied according to diﬀerent infection sites. Patients with T. marneﬀei chest infection are likely to suﬀer from fever, cough with little phlegm, dyspnea and chest pain (Duong, 1996; Wei et al., 2021). These symptoms are similar to our patient’s and non-speciﬁc compared with other infectious mediastinitis and pneumonia. It indicates that the clinical presentation of T. marneﬀei mediastinitis plays a less important role in the diagnosis. Though guidelines for T. marneﬀei infection in HIV- infected patients have been published (Kaplan et al., 2009; Nelson et al., 2011), standardized treatments for HIV-negative patients have not been established. In guidelines for HIV- positive patients, experts recommend intravenous infusion of AmBd as preferred treatment, but the adverse eﬀects of intravenous AmBd make plenty of patients unable to continue the therapy. For example, the intravenous treatment may The images of T. marneﬀei chest infection also lack speciﬁcity. Resemble with pulmonary tuberculosis, T. Frontiers in Microbiology Discussion In contrast with conventional TBNA and surgery to access mediastinal lesions, the use of EBUS-TBNA in mediastinal diseases has become increasingly prevalent due to its simplicity of operator, minimal invasiveness and fewer adverse events. Researches show with consistency that EBUS-TBNA has high speciﬁcity and accuracy, while its sensitivity in diagnosis of mediastinal diseases is controversial. Despite that, it makes for the ideal ﬁrst step in the diagnosis of pathology in mediastinal lesions (Divisi et al., 2018; Murthi et al., 2020). FIGURE 3 (A) Photomicrograph of TBLB. Inﬁltrations of massive neutrophils and lymphocytes are demonstrated by hematoxylin-eosin staining, which indicates acute inﬂammation (HEx200). Scale = 40 µm. (B) Photomicrograph of needle-aspirated material obtained by EBUS-TBNA with hematoxylin and eosin staining. Yeast cells of T. marnefei with transverse septa are difcult to observe on the background of acute inﬂammatory inﬁltration (HEx800). Scale = 10 µm. (C) Photomicrograph of needle-aspirated material obtained by EBUS-TBNA with PAS staining. The characteristic transverse septum within the sausage-shaped yeast cell (orange arrow) can be identiﬁed (PASx800). Scale = 10 µm. (D) Photomicrograph of needle-aspirated material obtained by EBUS-TBNA with GMS staining. Ovoid, elliptic and sausage-shaped yeast cells with characteristic transverse septa (orange arrows) are more distinguishable compared with PAS staining (GMSx800). Scale = 10 µm. Author contributions LC contributed to the clinical design and concept. LC, MZ, and WG acquired the clinical data. JT and WZ performed clinical practices. WD and HD performed pathological analyses. LC, MZ, and ZZ interpreted the data and drafted and revised the manuscript. All authors discussed, read, approved the manuscript, and authorized its submission for publication. Acknowledgments The authors would like to thank Dr. Huiling Chen for her professional opinions on the article. And also authors are indebted to two reviewers for valuable comments and suggestions. g Murthi et al., 2020). marneﬀei chest infection can show a variety of lung abnormalities on chest CT imaging, including inﬁltration, nodules, cavity, ground- glass shadows, diﬀuse miliary shadows, pleural eﬀusion and so Frontiers in Microbiology 04 frontiersin.org Chen et al. 10.3389/fmicb.2022.1045660 Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conclusion To sum up, when patients manifest a mediastinal mass in radiologic images, clinicians should consider mycotic mediastinal infection besides other common mediastinal diseases. EBUS-TBNA is the ideal ﬁrst step in the diagnosis of mediastinal lesions. Aerosolized AmBd has the potential to become ﬁrst-line treatment in HIV-negative patients with localized T. marneﬀei mediastinitis and pneumonia. However, as it’s the ﬁrst published case of nebulized AmBd monotherapy for T. marneﬀei chest infection, more studies and clinical trials are required. Data availability statement Moreover, the use of nebulized AmBd is simple and convenient. Patients may prefer to accept aerosolized treatment of AmBd for its simplicity, as it reduces the frequency of liver and kidney function tests, serum potassium tests, routine blood tests and urine tests, which are necessarily required during the therapy of intravenous AmBd. Nevertheless, based on the fact that AmBd inhalation results in high alveolar concentrations and no or very low systemic absorption, the nebulized treatment may not be appropriate for disseminated talaromycosis nor extrathoracic T. marneﬀei infection. Physicians should also notice the side eﬀects of inhaled AmBd, including cough, shortness of breath, diﬃculty breathing, chest tightness and bronchospasm (Lowry et al., 2007). In addition, liposomal amphotericin B may have been a better candidate for nebulization than AmBd, for it has higher drug concentration in the lungs and lower risk of toxicity (Allen et al., 1994; Ruijgrok et al., 2001). The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. Brunet, K., Martellosio, J. P., Tewes, F., Marchand, S., and Rammaert, B. (2022). Inhaled antifungal agents for treatment and prophylaxis of bronchopulmonary invasive mold infections. Pharmaceutics 14, 641. doi: 10.3390/pharmaceutics140 30641 Ethics statement Written informed consent was obtained from the individual(s) for the publication of any potentially identiﬁable images or data included in this article. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Data availability statement cause hypokalemia, hypotension, hyperpyrexia, arrhythmia, neurological symptoms and hepatic dysfunction. Besides, renal impairment has been seen among most patients (Saliba, 2006). Our patient failed to tolerate intravenous AmBd because she felt dizzy and her blood pressure dropped to 70/43 mmHg after the ﬁrst infusion. As a result, we attempted to apply inhaled AmBd to the patient, for researches prove that nebulized AmBd can have been well-delivered in the bronchial and alveolar compartments with concentrations in BALF above most fungal MICs, and no or very weak systemic absorption is detected (Lowry et al., 2007; Brunet et al., 2022). These highlights of pharmacokinetics ensure nebulized AmBd to maintain its eﬀect in the chest infection and limit its side reactions at the same time. Moreover, the use of nebulized AmBd is simple and convenient. Patients may prefer to accept aerosolized treatment of AmBd for its simplicity, as it reduces the frequency of liver and kidney function tests, serum potassium tests, routine blood tests and urine tests, which are necessarily required during the therapy of intravenous AmBd. Nevertheless, based on the fact that AmBd inhalation results in high alveolar concentrations and no or very low systemic absorption, the nebulized treatment may not be appropriate for disseminated talaromycosis nor extrathoracic T. marneﬀei infection. Physicians should also notice the side eﬀects of inhaled AmBd, including cough, shortness of breath, diﬃculty breathing, chest tightness and bronchospasm (Lowry et al., 2007). In addition, liposomal amphotericin B may have been a better candidate for nebulization than AmBd, for it has higher drug concentration in the lungs and lower risk of toxicity (Allen et al., 1994; Ruijgrok et al., 2001). cause hypokalemia, hypotension, hyperpyrexia, arrhythmia, neurological symptoms and hepatic dysfunction. Besides, renal impairment has been seen among most patients (Saliba, 2006). Our patient failed to tolerate intravenous AmBd because she felt dizzy and her blood pressure dropped to 70/43 mmHg after the ﬁrst infusion. As a result, we attempted to apply inhaled AmBd to the patient, for researches prove that nebulized AmBd can have been well-delivered in the bronchial and alveolar compartments with concentrations in BALF above most fungal MICs, and no or very weak systemic absorption is detected (Lowry et al., 2007; Brunet et al., 2022). These highlights of pharmacokinetics ensure nebulized AmBd to maintain its eﬀect in the chest infection and limit its side reactions at the same time. Allen, S. D., Sorensen, K. N., Nejdl, M. J., Durrant, C., and Proﬃt, R. T. (1994). Prophylactic eﬃcacy of aerosolized liposomal (ambisome) and non-liposomal (fungizone) amphotericin B in murine pulmonary aspergillosis. J. Antimicrob. Chemother. 34, 1001–1013. doi: 10.1093/jac/34.6.1001 Frontiers in Microbiology References Frontiers in Microbiology frontiersin.org 05 Chen et al. Chen et al. 10.3389/fmicb.2022.1045660 and host defense mechanisms. J. Fungi 8, 200. doi: 10.3390/jof80 20200 Cooper, C. R. Jr, and McGinnis, M. R. (1997). Pathology of Penicillium marneﬀei. An emerging acquired immunodeﬁciency syndrome-related pathogen. Arch. Pathol. Lab. Med. 121, 798–804. Qiu, Y., Liao, H., Zhang, J., Zhong, X., Tan, C., Lu, D., et al. (2015). Diﬀerences in clinical characteristics and prognosis of Penicilliosis among HIV-negative patients with or without underlying disease in Southern China: a retrospective study. BMC Infect. Dis. 15, 525. doi: 10.1186/s12879-015-1243-y Deng, Z. L., Yun, M., and Ajello, L. (1986). Human penicilliosis marneﬀei and its relation to the bamboo rat (Rhizomys pruinosus). J. Med. Vet. Mycol. 24, 383–389. doi: 10.1080/02681218680000581 Divisi, D., Zaccagna, G., Barone, M., Gabriele, F., and Crisci, R. (2018). Endobronchial ultrasound-transbronchial needle aspiration (EBUS/TBNA): a diagnostic challenge for mediastinal lesions. Ann. Transl. Med. 6, 92. doi: 10.21037/atm.2017.12.19 Ruijgrok, E. J., Vulto, A. G., and Van Etten, E. W. (2001). Eﬃcacy of aerosolized amphotericin B desoxycholate and liposomal amphotericin B in the treatment of invasive pulmonary Aspergillosis in severely immunocompromised rats. J. Antimicrob. Chemother. 48, 89–95. doi: 10.1093/jac/48.1.89 Duong, T. A. (1996). Infection due to Penicillium marneﬀei, an emerging pathogen: review of 155 reported cases. Clin. Infect. Dis. 23, 125–130. doi: 10.1093/clinids/23.1.125 Saliba, F. (2006). Antifungals and renal safety–getting the balance right. Int. J. Antimicrob. Agents. 27(Suppl. 1), 21–24. doi: 10.1016/j.ijantimicag.2006.03.015 Shi, X., Yan, Q., Zhan, Y., Shi, C., Song, F., Wang, L., et al. (2020). Eﬀect of combination antiretroviral therapy on the clinical manifestations, radiological characteristics, and disease severity of HIV-associated Talaromyces marneﬀei infection. Int. J. STD AIDS 31, 747–752. doi: 10.1177/0956462420925248 Guo, J., Ning, X. Q., Ding, J. Y., Zheng, Y. Q., Shi, N. N., Wu, F. Y., et al. (2020). Anti-IFN-γ autoantibodies underlie disseminated Talaromyces marneﬀei infections. J. Exp. Med. 217, e20190502. doi: 10.1084/jem.20190502 Kaplan, J. E., Benson, C. A., Brooks, J. T., Holmes, K. K., Masur, H., Pau, A., et al. (2009). Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm. Rep. 58:1-CE4. Available online at: https:// www.cdc.gov/mmwr/preview/mmwrhtml/rr58e324a1.htm Vanittanakom, N., Cooper, C. R. Jr., Fisher, M. C., and Sirisanthana, T. (2006). Penicillium marneﬀei infection and recent advances in the epidemiology and molecular biology aspects. Clin. Microbiol. Rev. References 19, 95–110. doi: 10.1128/CMR.19.1.95-110.2006 Wang, P., Chen, Y., Xu, H., Ding, L., Wu, Z., Xu, Z., et al. (2017). Acute disseminated Talaromyces marneﬀei in an immunocompetent patient. Mycopathologia 182, 751–754. doi: 10.1007/s11046-017-0127-7 Liu, L., Sun, B., Ying, W., Liu, D., Wang, Y., Sun, J., et al. (2022). Rapid diagnosis of Talaromyces marneﬀei infection by metagenomic next-generation sequencing technology in a Chinese cohort of inborn errors of immunity. Front. Cell. Infect. Microbiol. 12, 987692. doi: 10.3389/fcimb.2022.987692 Wei, H. Y., Liang, W. J., Li, B., Wei, L. Y., Jiang, A. Q., Chen, W. D., et al. (2021). Clinical characteristics and risk factors of Talaromyces marneﬀei infection in human immunodeﬁciency virus-negative patients: a retrospective observational study. World J. Emerg. Med. 12, 281–286. doi: 10.5847/wjem.j.1920-8642.2021.04.005 Lowry, C. M., Marty, F. M., Vargas, S. O., Lee, J. T., Fiumara, K., Deykin, A., et al. (2007). Safety of aerosolized liposomal versus deoxycholate amphotericin B formulations for prevention of invasive fungal infections following lung transplantation: a retrospective study. Transpl. Infect. Dis. 9, 121–125. doi: 10.1111/j.1399-3062.2007.00209.x Widaty, S., Santoso, I. D., Ricky, D., Yunihastuti, E., Rihatmadja, R., Wahyuningsih, R., et al. (2020). Talaromycosis clinically and histopathologically mimicking histoplasmosis in an immunocompromised patient. Dermatol. Online J. 26, 13030./qt9r49g227. doi: 10.5070/D3269050160 Murthi, M., Donna, E., Arias, S., Villamizar, N. R., Nguyen, D. M., Holt, G., et al. (2020). Diagnostic accuracy of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in real life. Front. Med. 7, 118. doi: 10.3389/fmed.2020.00118 Ye, F., Luo, Q., Zhou, Y., Xie, J., Zeng, Q., Chen, G., et al. (2015). Disseminated penicilliosis marneﬀei in immunocompetent patients: a report of two cases. Indian J. Med. Microbiol. 33, 161–165. doi: 10.4103/0255-0857.148433 Narayanasamy, S., Dat, V. Q., Thanh, N. T., Ly, V. T., Chan, J. F. W., Yuen, K. Y., et al. (2021). A global call for talaromycosis to be recognised as a neglected tropical disease. Lancet Glob. Health 9, e1618–e1622. doi: 10.1016/S2214-109X(21)00350-8 You, C. Y., Hu, F., Lu, S. W., Pi, D. D., Xu, F., Liu, C. J., et al. (2021). Talaromyces marneﬀei infection in an HIV-negative child with a CARD9 mutation in China: a case report and review of the literature. Mycopathologia 186, 553–561. doi: 10.1007/s11046-021-00 576-8 Nelson, M., Dockrell, D., Edwards, S., Angus, B., Simson, B., and Nick, B. (2011). British HIV Association and British Infection Association guidelines for the treatment of opportunistic infection in HIV-seropositive individuals 2011. HIV Med. 12(Suppl. 2), 1–140. References doi: 10.1111/j.1468-1293.2011.00 944_1.x Zhang, D., Yang, X., Wang, J., Xu, J., and Wang, M. (2022). Application of metagenomic next-generation sequencing for bronchoalveolar lavage diagnostics in patients with lower respiratory tract infections. J. Int. Med. Res. 50, 3000605221089795. doi: 10.1177/030006052210 89795 Pruksaphon, K., Nosanchuk, J. D., Ratanabanangkoon, K., and Youngchim, S. (2022). Talaromyces marneﬀei infection: virulence, intracellular lifestyle 06 Frontiers in Microbiology frontiersin.org 06
https://openalex.org/W2884529889	http://pure-oai.bham.ac.uk/ws/files/53177673/Sharmila_Velapasamy_et_al_The_Dynamic_Roles_of_TGF_Cancers_2018.pdf	English	null	The Dynamic Roles of TGF-β Signalling in EBV-Associated Cancers	Cancers	2,018	cc-by	16,070	The Dynamic Roles of TGF-β Signalling in EBV- Associated Cancers Velapasamy, Sharmila; Dawson, Christopher W; Young, Lawrence S; Paterson, Ian C; Yap, Lee Fah DOI: 10.3390/cancers10080247 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Velapasamy, S, Dawson, CW, Young, LS, Paterson, IC & Yap, LF 2018, 'The Dynamic Roles of TGF-β Signalling in EBV-Associated Cancers', Cancers, vol. 10, no. 8, 247. https://doi.org/10.3390/cancers10080247 Link to publication on Research at Birmingham portal Publisher Rights Statement: Checked for eligibility: 06/08/2018 Publisher Rights Statement: Checked for eligibility: 06/08/2018 Publisher Rights Statement: Checked for eligibility: 06/08/2018 General rights U l li Download date: 24. Oct. 2024 Received: 9 July 2018; Accepted: 25 July 2018; Published: 27 July 2018 Abstract: The transforming growth factor-β (TGF-β) signalling pathway plays a critical role in carcinogenesis. It has a biphasic action by initially suppressing tumorigenesis but promoting tumour progression in the later stages of disease. Consequently, the functional outcome of TGF-β signalling is strongly context-dependent and is inﬂuenced by various factors including cell, tissue and cancer type. Disruption of this pathway can be caused by various means, including genetic and environmental factors. A number of human viruses have been shown to modulate TGF-β signalling during tumorigenesis. In this review, we describe how this pathway is perturbed in Epstein-Barr virus (EBV)-associated cancers and how EBV interferes with TGF-β signal transduction. The role of TGF-β in regulating the EBV life cycle in tumour cells is also discussed. Keywords: TGF-β signalling; Epstein-Barr virus; nasopharyngeal carcinoma; gastric cancer; B-cell lymphoma The Dynamic Roles of TGF-β Signalling in EBV-Associated Cancers Sharmila Velapasamy 1, Christopher W. Dawson 2, Lawrence S. Young 3 ID , Ian C. Paterson 1,4 and Lee Fah Yap 1,4,* ID Sharmila Velapasamy 1, Christopher W. Dawson 2, Lawrence S. Young 3 ID , Ian C. Paterson 1,4 and Lee Fah Yap 1,4,* ID 1 Department of Oral & Craniofacial Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia; vsharmila84@gmail.com (S.V.); ipaterson@um.edu.my (I.C.P) 1 Department of Oral & Craniofacial Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia; vsharmila84@gmail.com (S.V.); ipaterson@um.edu.my (I.C.P) p y g ( ) p y ( ) 2 Institute of Cancer and Genomic Medicine, University of Birmingham, Birmingham B15 2TT, UK; c.w.dawson@bham.ac.uk 3 Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK; L.S.Young@warwick.ac.uk 4 Oral Cancer Research and Coordinating Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia * Correspondence: yapleefah@um.edu.my; Tel.: +60-3-7967-6471; Fax: +60-3-7967-6456 3 Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK; L.S.Young@warwick.ac.uk 4 Oral Cancer Research and Coordinating Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia * Correspondence: yapleefah@um.edu.my; Tel.: +60-3-7967-6471; Fax: +60-3-7967-6456 Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK; L.S.Young@warwick.ac.uk 4 Oral Cancer Research and Coordinating Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia * Correspondence: yapleefah@um edu my; Tel : +60 3 7967 6471; Fax: +60 3 7967 6456 4 Oral Cancer Research and Coordinating Centre, University of Malaya, 50603 Kuala Lumpur, Malaysia d l f h d l Received: 9 July 2018; Accepted: 25 July 2018; Published: 27 July 2018 cancers cancers General rights l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. y p oad and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private mercial research y y y p sers may download and/or print one copy of the publication from the University of Birmingham research portal for the pu udy or non-commercial research. y •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and P •Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of th When citing, please reference the published version. y ty of Birmingham exercises care and attention in making items available there are rare occasions when an item has bee or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we the work immediately and investigate. Download date: 24. Oct. 2024 cancers 2. TGF-β Signalling [ ] , p plays in EBV-assoc In mammals, there are three TGF-β isoforms (TGF-β1, TGF-β2 and TGF-β3), each encoded by different genes [20–22]. These isoforms are highly similar and share approximately 70–80% sequence homology [22–24]. TGF-β1 was the ﬁrst isoform to be characterized and is the most studied to date [20,21]. TGF-β1 is synthesized in a latent form as a large precursor protein which binds to and is stored in the extracellular matrix (ECM) [25]. The precursor protein undergoes proteolytic digestion by the endopeptidase furin to produce two proteins, namely latency-associated peptide (LAP; 278 amino acids) and mature TGF-β1 (112 amino acids) [26,27]. Despite the cleavage of the precursor protein, the LAP remains bound to the mature TGF-β1 making the TGF-β1 biologically inactive [28]. The release of active TGF-β1 from the ECM can be triggered by several factors, such as changes in the cellular environment, tissue injury or inﬂammation [29–31]. Activated TGF ligands mediate signalling through the TGF-β type I and type II receptors (TGFR-1 and TGFR-2, respectively) that are endowed with serine/threonine kinase activity [32,33]. Upon binding of an active TGF-β ligand to TGFR-2, TGFR-1 is recruited and phosphorylated by TGFR-2. The activated heterotetramer TGFR-1/TGFR-2 complex triggers the canonical Smad-dependent, as well as non-canonical Smad-independent signalling pathways (Figure 1). 2. TGF-β Signalling In mammals, there are three TGF-β isoforms (TGF-β1, TGF-β2 and TGF-β3), each encoded by different genes [20–22]. These isoforms are highly similar and share approximately 70–80% sequence homology [22–24]. TGF-β1 was the first isoform to be characterized and is the most studied to date [20,21]. TGF-β1 is synthesized in a latent form as a large precursor protein which binds to and is stored in the extracellular matrix (ECM) [25]. The precursor protein undergoes proteolytic digestion by the endopeptidase furin to produce two proteins, namely latency-associated peptide (LAP; 278 amino acids) and mature TGF-β1 (112 amino acids) [26,27]. Despite the cleavage of the precursor protein, the LAP remains bound to the mature TGF-β1 making the TGF-β1 biologically inactive [28]. The release of active TGF-β1 from the ECM can be triggered by several factors, such as changes in the cellular environment, tissue injury or inflammation [29–31]. Activated TGF ligands mediate signalling through the TGF-β type I and type II receptors (TGFR-1 and TGFR-2, respectively) that are endowed with serine/threonine kinase activity [32,33]. Upon binding of an active TGF-β ligand to TGFR-2, TGFR-1 is recruited and phosphorylated by TGFR-2. 1. Introduction The transforming growth factor-beta (TGF-β) superfamily is a group of multifunctional proteins comprising more than 40 members that are clustered in several subfamilies, which include TGF-β, activins/inhibins, bone morphogenetic proteins (BMPs), nodal and growth differentiation factors (GDFs) [1,2]. The prototypic member, TGF-β1, is produced by a diverse range of cell types and modulates cell proliferation, migration, adhesion, differentiation and survival [2,3]. Consequently, a malfunctioning TGF-β pathway is central to many diseases including cancer. TGF-β functions as a tumour suppressor by inhibiting the growth of untransformed epithelial, endothelial and lymphoid cells [4–6] and resistance to TGF-β is regarded as one of the crucial steps in malignant progression [2,7]. In the early stages of cancer development, TGF-β signalling functions as a tumour suppressor by inhibiting cell cycle progression from G1 to S phase and inducing apoptosis, senescence and differentiation [2,5,8,9]. Conversely, in late stage disease, it acts as a tumour promoter by inducing epithelial-to-mesenchymal transition (EMT), migration, invasion, metastasis, angiogenesis and immune suppression [2,9–12]. Frequently, cancer cells become resistant to the tumour suppressive effects of TGF-β, however functional TGF-β signalling often persists in these cells enabling TGF-β-induced tumour promoting phenotypes [13–16]. Accumulating evidence has revealed that the TGF-β signalling pathway is targeted by many oncogenic viruses, including Epstein-Barr virus (EBV), during the course of tumorigenesis [17]. www.mdpi.com/journal/cancers Cancers 2018, 10, 247; doi:10.3390/cancers10080247 www.mdpi.com/journal/cancers Cancers 2018, 10, 247 2 of 23 EBV was the ﬁrst human cancer virus to be discovered [18] and the most common viral infection in humans. Following its discovery in Burkitt lymphoma (BL), EBV has been linked to the aetiology of multiple human cancers of both lymphoid and epithelial origin, including classical Hodgkin lymphoma (HL), diffuse large B cell lymphoma (DLBCL), post-transplant lymphoproliferative disorders (PTLD), nasopharyngeal carcinoma (NPC) and EBV-associated gastric cancer (EBVaGC) [19]. Here, we provide an overview of our current understanding of the dynamic roles that TGF-β plays in EBV-associated malignancies. Cancers 2018, 10, x 2 of 23 EBV was the first human cancer virus to be discovered [18] and the most common viral infection in humans. 1. Introduction Following its discovery in Burkitt lymphoma (BL), EBV has been linked to the aetiology of multiple human cancers of both lymphoid and epithelial origin, including classical Hodgkin lymphoma (HL), diffuse large B cell lymphoma (DLBCL), post-transplant lymphoproliferative disorders (PTLD), nasopharyngeal carcinoma (NPC) and EBV-associated gastric cancer (EBVaGC) [19] H id i f d di f h d i l h TGF β 2.1. Canonical Smad-Dependent Signalling Smad proteins were the ﬁrst identiﬁed downstream signalling transducers of TGF-β [34]. The proteins of the SMAD family are the vertebrate homologs of the Drosophila mothers against decapentaplegic (MAD) protein and the Caenorhabditis elegans small body size (SMA) protein [35,36]. These proteins are divided into three groups based on their functions: receptor-activated Smads (R-Smad; Smad2 and Smad3), common mediator Smad (Co-Smad; Smad4) and inhibitory Smads (I-Smads; Smad6 and Smad7) [37–39]. Following TGF-β binding to TGFR-2 and receptor activation, the Smad anchor for receptor activation (SARA) protein binds TGFR-1 and Smad2 and/or Smad3 simultaneously, resulting in the phosphorylation of the Smad2 and/or Smad3 by TGFR-1 [40–42]. Upon activation, Smad2 and/or Smad3 are released from the receptor complex and SARA, and oligomerize with Smad4 through their MH2 domains [40,42,43]. The Smad2/Smad4 and/or Smad3/Smad4 complexes then translocate to the nucleus to either stimulate or repress the transcription of their target genes, depending on interactions with various transcription factors [44]. A variety of transcription factor families have been identiﬁed that act in concert with Smad proteins, including p300/CBP, AP1, and Forkhead [45–47]. The two I-Smads, Smad6 and Smad7, tightly control the activation of TGF-β signalling. Compared to Smad6, Smad7 has been shown to inhibit TGF-β signalling more efﬁciently through a number of mechanisms [48,49]. These include inhibition of the phosphorylation of R-Smads by forming a complex with activated TGFR-1, degradation of the activated TGFR-1 by recruiting ubiquitin E3 ligases, such as Smurf1/2 or disruption of the formation of functional Smad-DNA complexes in the nucleus [50–53]. Recent evidence also demonstrated that Smad7 might directly oligomerize with R-Smads and inhibit their activities [54]. 2. TGF-β Signalling [ ] , p plays in EBV-assoc The activated heterotetramer TGFR- 1/TGFR-2 complex triggers the canonical Smad-dependent, as well as non-canonical Smad- independent signalling pathways (Figure 1). Figure 1. The TGF-β signalling pathway. Binding of an activated TGF-β ligand to TGFR-2 recruits and activates TGFR-1. This, in turn, phosphorylates Smad2 and/or Smad3 (R-Smads), which then form complexes with Smad4 (Co-Smad) and translocate into nucleus to regulate the transcription of various target genes. Smad7 (I-Smad) inhibits the pathway through various mechanisms, including mediating the degradation of TGFR-1, inhibiting phosphorylation of Smad2/Smad3 or inhibiting the formation of the Smad2/3-Samd4 complex. In addition to the canonical Smad-dependent signalling, activated TGF-β receptors can trigger other signalling pathways including ERK-MAPK, p38-MAPK, PI3K-Akt and JNK. Figure 1. The TGF-β signalling pathway. Binding of an activated TGF-β ligand to TGFR-2 recruits and activates TGFR-1. This, in turn, phosphorylates Smad2 and/or Smad3 (R-Smads), which then form complexes with Smad4 (Co-Smad) and translocate into nucleus to regulate the transcription of various target genes. Smad7 (I-Smad) inhibits the pathway through various mechanisms, including mediating the degradation of TGFR-1, inhibiting phosphorylation of Smad2/Smad3 or inhibiting the formation of the Smad2/3-Samd4 complex. In addition to the canonical Smad-dependent signalling, activated TGF-β receptors can trigger other signalling pathways including ERK-MAPK, p38-MAPK, PI3K-Akt and JNK. Figure 1. The TGF-β signalling pathway. Binding of an activated TGF-β ligand to TGFR-2 recruits and activates TGFR-1. This, in turn, phosphorylates Smad2 and/or Smad3 (R-Smads), which then form complexes with Smad4 (Co-Smad) and translocate into nucleus to regulate the transcription of various target genes. Smad7 (I-Smad) inhibits the pathway through various mechanisms, including mediating the degradation of TGFR-1, inhibiting phosphorylation of Smad2/Smad3 or inhibiting the formation of the Smad2/3-Samd4 complex. In addition to the canonical Smad-dependent signalling, activated TGF-β receptors can trigger other signalling pathways including ERK-MAPK, p38-MAPK, PI3K-Akt and JNK. Figure 1. The TGF-β signalling pathway. Binding of an activated TGF-β ligand to TGFR-2 recruits and activates TGFR-1. This, in turn, phosphorylates Smad2 and/or Smad3 (R-Smads), which then form complexes with Smad4 (Co-Smad) and translocate into nucleus to regulate the transcription of various target genes. Smad7 (I-Smad) inhibits the pathway through various mechanisms, including mediating the degradation of TGFR-1, inhibiting phosphorylation of Smad2/Smad3 or inhibiting the formation of the Smad2/3-Samd4 complex. In addition to the canonical Smad-dependent signalling, activated TGF-β receptors can trigger other signalling pathways including ERK-MAPK, p38-MAPK, PI3K-Akt and JNK. Cancers 2018, 10, 247 3 of 23 2.2. Non-Canonical Smad-Independent Signalling While the canonical Smad-dependent pathway has been regarded as the major signalling route of TGF-β, the ligand can also signal through non-canonical Smad-independent pathways, engaging the ERK-MAPK, p38-MAPK, PI3K-AKT and JNK pathways [1,2]; different mechanisms are utilised to trigger these signalling pathways. For example, activation of the ERK-MAPK pathway is mediated by the phosphorylation of TGFR-1, whereas the activation of both TGFR-1 and TGFR-2 are required for activation of the PI3K-AKT signalling pathway [55–57]. Notably, activation of the canonical Smad-dependent and the non-canonical Smad-independent signalling pathways is not mutually exclusive [58,59]. In breast cancer cells lines, for example, both pathways act together to mediate TGF-β-induced growth arrest [59]. 3. Resistance of EBV-Positive Cells to TGF-β-Mediated Cytostasis In normal epithelial and neuronal cells, TGF-β1 inhibits progression from G1 to S phase of the cell cycle by inducing the expression of CDK inhibitors, p15 and p21, thereby blocking the phosphorylation of the Rb protein [60–62]. In addition, the increase in p15 levels induces the release of p27 from CDK4 and/or CDK6 [60]. p21 and the free p27 bind to CDK2, inhibiting formation of cyclin A-CDK2 and/or cyclin E-CDK2, thereby blocking the progression to S phase [60,62]. Further, TGF-β1 suppresses the expression of the c-MYC protein, preventing c-MYC from inhibiting the expression of p15, p21 and p27 [63–65]. This safeguards the induction of the CDK inhibitors and thereby leads to G1 cell cycle arrest. Additionally, TGF-β1 has been shown to induce both the intrinsic and extrinsic apoptotic programs in a cell-type dependent manner [2]. In lymphoma cells, TGF-β1 induces the intrinsic apoptotic pathway by stimulating the expression of several pro-apoptotic Bcl-2 family members (such as Bmf, Bim and Bax), which in turns suppress the expression of anti-apoptotic proteins (Bcl-XL and Bcl-2) [66]. The ability of TGF-β1 to induce the extrinsic apoptotic program has been shown in liver and lung cancer cells, in which expression of death-associated protein kinase (DAPK) and Fas-mediated apoptosis was increased upon the exogenous addition of TGF-β1, respectively [67,68]. Cancers 2018, 10, 247 4 of 23 EBV-positive and -negative B cells exhibit differential responses to TGF-β. EBV-negative B cells are sensitive to TGF-β-mediated growth inhibition and apoptosis [69–71], whilst these responses are lost in EBV-positive B cells [72–74]. Similarly, gastric tissue-derived EBV-infected epithelial cell lines (GT38 and GT39) have been shown to be resistant to TGF-β1-mediated growth inhibition and apoptosis, as opposed to a TGF-β1-responsive EBV-negative gastric carcinoma cell line HSC-39 [75]; similar observations were also obtained in our laboratory with the EBV-positive NPC cell line, C666-1 (Yap L.F.; Dawson C.W. (University of Malaya, Kuala Lumpur, Malaysia) Personal observation, 2013). However, the growth of an EBV-negative NPC cell line CNE-2 was not suppressed by exogenous TGF-β1 [76]. CNE-2 cells were originally derived from a poorly differentiated NPC which was likely to be EBV-positive [77]. Although CNE-2 cells do not carry EBV genomes in long-term culture, it is possible that these cells developed resistance towards the cytostatic effect of TGF-β at the initial stage of EBV infection and retained this characteristic even after they lost the EBV genomes. 3. Resistance of EBV-Positive Cells to TGF-β-Mediated Cytostasis It is reasonable to hypothesize that EBV-infected cells can selectively outgrow the neighboring cells (EBV-negative) which are growth inhibited by TGF-β1 produced by EBV-infected cells through the expression of Zta (discussed below). Such EBV-infected cells can then clonally expand to drive the transformation process. Indeed, it was shown that LMP1-transfected BALB/c 3T3 cells exhibited non-transformed phenotypes in vitro but those that lost sensitivity to TGF-β-mediated growth inhibition formed tumours in severe combined immunodeﬁciency (SCID) mice [78], implying that loss of TGF-β responsiveness is a critical event for the tumorigenicity of EBV-infected cells. 3.1. Contribution of EBV Latent Genes It has been shown that the EBV oncoprotein LMP1 was responsible for mediating resistance to the cytostatic effects of TGF-β1 in BL cells (BL41) by elevating levels of cyclin D2 [73]. A modest sensitization to TGF-β was observed in EBV-positive lymphoblastoid cell lines (LCLs) following treatment with LMP1 antisense oligodeoxynucleotides [87], although studies of EBV-converted and stably transfected BL cell lines have shown that LMP1 was not sufﬁcient or necessary to block the TGF-β1 response [88]. Using epithelial cells as study models, LMP1 has been shown to abrogate TGF-β signalling through NF-κB-dependent depletion of transcriptional coactivators required for Smad-mediated transcription [89,90]. Additional evidence showed that LMP1 induction of Id1 through suppression of ATF3 (a SMAD-induced transcriptional repressor) attenuated TGF-β-Smad-mediated transcription and counteracted the cytostatic action of TGF-β1 in epithelial cells [91]. The ability of LMP1 to suppress Smad-dependent transcription was also demonstrated in SCC12F epithelial cells [92]. Further, it has been shown that LMP1 can down-regulate the expression of limb-bud and heart (LBH) resulting in the alleviation of TGF-β1-induced NF-kB signalling inhibition, rendering NPC cells refractory to TGF-β1-mediated cytostasis [93]. y β y [ ] Other EBV-encoded proteins have also been shown to repress TGF-β signalling and this could facilitate the evasion of TGF-β-mediated cytostatic effects during EBV infection. In BL Ramous cells and gastric carcinoma cells (HSC-39), LMP2A has been shown to inhibit TGF-β1-induced apoptosis through the PI3K/AKT pathway [94]. A negative feedback loop between EBNA1 and TGF-β was also postulated, in which expression of EBNA1 in a nasopharyngeal adenocarcinoma cell line, AdAH, has been shown to repress TGF-β1-induced transcription by increasing Smad2 protein turnover [95], an effect that may overcome the ability of TGF-β to repress the Q promoter (Qp), which is responsible for EBNA1 expression in NPC [96]. The strategic inhibition of B-cell apoptosis is central to EBV biology. In germinal centres, only those B-cells that express the highest-afﬁnity immunoglobulins are rescued from stringent pro-apoptotic pathways that signal through TGF-β, FAS and B-cell receptors [97–99]. The ability of EBNA1 to disable TGF-β signalling was also demonstrated in HL cells, where EBNA1 increased Smad2 protein degradation which subsequently inhibited transcription of the TGF-β target gene, PTPRK tumour suppressor, contributing to the growth and survival of HL cells [100]. In B-cells exhibiting a group III latency program, EBNA2 antagonized the apoptotic effects of TGF-β1, partly by repressing the pro-apoptotic “sensitizer” protein, BIK, resulting in B-cell survival [101,102]. 3.1. Contribution of EBV Latent Genes EBV displays two distinct lifecycles, namely the lytic and latent cycles. The lytic cycle is associated with viral replication in which new virions are produced while latent cycle is a state of persistent infection and the absence of productive viral replication [79]. During latent infection, a limited set of EBV genes is expressed and to date, three latency programmes have been identiﬁed. Different types of malignancies are associated with a distinct latency programme (Table 1). The EBV lytic cycle is initiated by the expression of the viral immediate-early gene BZLF1 (the gene product is commonly known as Zta or ZEBRA) [80]. While the lytic cycle can be triggered in vitro by diverse stimuli such as phorbol ester and sodium butyrate [81], this process is closely associated with the differentiation of both B cells and epithelial cells in vivo [82–84]. Table 1. Characteristics of Epstein-Barr virus (EBV)-associated cancers. Malignancy %EBV+ Cases Latency EBV Latent Genes Endemic Burkitt Lymphoma 100% I EBNA1, EBER1, EBER2, BARTs, miR-BARTs Sporadic Burkitt Lymphoma 10–85% HIV-associated Burkitt Lymphoma 30–40% T/NK cell lymphoma 100% II EBNA1, LMP1, LMP2A, EBER1, EBER2, BARTs, miR-BARTs Hodgkin’s Lymphoma 80–90% Hodgkin’s Lymphoma (Nodular sclerosing) 15–20% HIV-associated Hodgkin’s Lymphoma <90% Diffuse large B-cell lymphoma (Pythorax lymphoma) 100% II/III EBNA1, LMP1, LMP2A, EBER1, EBER2, BARTs, miR-BARTs and/or EBNA2, 3A, 3B, 3C, LP Diffuse large B-cell lymphoma (in Elderly patients) >50% Diffuse large B-cell lymphoma (late post-transplant) >50% HIV-associated diffuse large B-cell lymphoma 30% Table 1. Characteristics of Epstein-Barr virus (EBV)-associated cancers. 5 of 23 Cancers 2018, 10, 247 Table 1. Cont. Malignancy %EBV+ Cases Latency EBV Latent Genes Post-transplant B-lymphoproliferative disorder 100% III EBNA 1, 2, 3A, 3B, 3C, LP, LMP1, LMP2A, LMP2B, EBER1, EBER2, BARTs, miRNAs-BARTs, BHRF1 HIV-associated B-lymphoproliferative disease 100% Nasopharyngeal carcinoma 98% II EBNA1, LMP1, LMP2A, EBER1, EBER2, BARTs, miR-BARTs, BARF1 EBV-associated gastric cancer 10% Epstein-Barr nuclear antigens (EBNA), Latent membrane proteins (LMP), Epstein-Barr virus-encoded RNAs (EBERs), BamH1-A fragment transcripts (BARTs), BamH1 fragment H rightward open reading frame 1 (BHRF1), BamH1 fragment A rightward open reading frame 1 (BARF1). Table adapted from Rickinson [85] and Yap & Lo [86]. Epstein-Barr nuclear antigens (EBNA), Latent membrane proteins (LMP), Epstein-Barr virus-encoded RNAs (EBERs), BamH1-A fragment transcripts (BARTs), BamH1 fragment H rightward open reading frame 1 (BHRF1), BamH1 fragment A rightward open reading frame 1 (BARF1). Table adapted from Rickinson [85] and Yap & Lo [86]. 3.1. Contribution of EBV Latent Genes Further, BARF1 was found to promote gastric cancer cell proliferation through a mechanism involving the downregulation of Smad4 via an increase in NF-κB-dependent miR-146a [103]. Taken together, it is apparent that TGF-β signalling is disrupted by EBV-encoded latent genes by a variety of mechanisms leading to malignant transformation. 3.2. Dysregulation of TGF-β Receptors In order to evade the tumour suppressive effects of TGF-β1, cancer cells often develop genetic abnormalities within key molecules of the TGF-β signalling pathway, particularly the TGF-β receptors, TGFBR1 and TGFBR2. However, the contribution of alterations in TGF-β receptor expression to the loss of responsiveness towards TGF-β1-mediated growth inhibition in EBV-positive cells is inconclusive. While 6 of 23 Cancers 2018, 10, 247 some studies showed that the levels of TGFBR1 and TGFBR2 did not correlate with resistance [73,75,78], others reported that the lack of responsiveness appeared to correlate with a down-regulation of TGFBR2 expression [88,104]. These observations suggest that multiple mechanisms regulate the growth inhibitory response to TGF-β in EBV-positive cells. Nonetheless, alterations in the expression of TGF-β receptors have been shown in EBV-associated cancers in vivo. The down-regulation of TGF-β receptors in cancer cells can be caused by multiple mechanisms. For example, the expression of TGFBR2 can be reduced by mutation, promoter hypermethylation or miRNA regulation. It is noteworthy that TGFR-2 expression has been suggested as a positive prognostic marker in DLBCL patients [105]. Further, the mRNA and/or protein levels of TGFBR1 and TGFBR2 were found to be significantly reduced in primary NPC tissues compared with non-cancerous controls, and their decreased expression correlated with poor survival [106–109]. However, a recent report described contradictory results in which TGFR-1 was found to be up-regulated in primary NPC tissues [110]. We previously had reported the expression of TGFR-2 in oral cancer by immunohistochemical analysis [111] and accurate staining results could only be achieved by applying stringent methodologies and assessment. The discrepancy between studies could be due to differences in antibody specificities. It is worth noting that TGFBR2 is located at chromosome 3p, a region with the most frequent loss of heterozygosity in NPC [112,113], implying that TGFBR2 might be a tumour suppressor gene that is altered in the early stages of NPC pathogenesis. Using advanced next-generation sequencing technology, several studies have reported genetic abnormalities of key molecules within the TGF-β pathway, including the TGF-β receptors, in EBV-associated cancers (Table 2). Although the frequency of the genetic alterations appears to be low, further studies are warranted to confirm the results and investigate the functional significance of these alterations. It is important to recognize, however, that these results do not take into account possible transcriptional alterations of the receptors and/or signalling molecules. 3.2. Dysregulation of TGF-β Receptors The involvement of cellular miRNAs in the disruption of TGF-β signalling has also been reported. For example, miR-93 and miR-19a, paralogues of the oncogenic miR-17-92 cluster, were shown to promote NPC aggressiveness by down-regulating TGFR-2 [108,109]. Several studies on global miRNA proﬁling in NPC have identiﬁed a number of differentially expressed miRNAs that target the TGF-β pathway [114–116], but the exact targets within the pathway are yet to be identiﬁed. Notably, a susceptibility gene TNFRSF19 in NPC, was shown to render NPC cells resistant to TGF-β-mediated cell cycle arrest [117]. TNFRSF19 was highly expressed in NPC and binds speciﬁcally to the kinase domain of TGFR-1, thereby blocking Smad2/3 association with TGFR-1 and subsequent signal transduction. 7 of 23 Cancers 2018, 10, 247 Table 2. Genetic alterations of TGF-β pathway components in EBV-associated cancers identiﬁed by next-generation sequencing. 3.2. Dysregulation of TGF-β Receptors Cancer Total Number of Cases EBV Status Genes Alterations Number of Cases with Alterations References NPC 56 primary tumours Positive SMAD3 Missense mutation 1 (primary tumour) [118] NPC • 51 primary tumours • 8 recurrent tumours • 3 local metastatic tumours Positive TGF-β1 Missense mutation 1 (primary tumour) [119] TGF-β2 Missense mutation 1 (primary tumour) TGFBR2 Missense mutation 1 (primary tumour) NPC • 78 primary tumours • 11 local recurrent tumours • 22 distant metastatic tumours Positive TGF-β1 Missense mutation 1 (primary tumour) [120] TGF-β1 Nonsense mutation 1 (local recurrent tumour) TGF-β1 Silent mutation 1 (primary tumour) TGF-β2 Frame shift deletion 1 (local recurrent tumour) TGF-β2 Inversion 1 (primary tumour) TGFBR1 Missense mutation 1 (primary tumour) 1 (local recurrent tumour) TGFBR2 Inter chromosomal translocation 1 (primary tumour) SMAD3 Silent mutation 1 (local recurrent tumour) SMAD4 Missense mutation 1 (primary tumour) SMAD4 Nonsense mutation 1 (primary tumour) SMAD7 Missense mutation 1 (local recurrent tumour) EBVaGC 134 primary tumours • Positive: n = 34 • Negative: n = 100 TGFBR1 Nonsynonymous mutation • 9 (EBV-positive) • 8 (EBV-negative) [121] AGS cell line Before and after EBV infection Missense mutation EBV-infected AGS cells EBVaGC 22 primary tumours Positive SMAD4 Missense mutation 2 [122] HL 7 cell lines • Positive: L591 • Negative: SUPHD1, L540, L428, L1236, KMH2, DEV SMAD9 Missense mutation 1 (KMH2) [123] HL 5 cell lines Negative (HDML2, KMH2, UH01, L540, L428) TGF-β1 Ampliﬁcation 2 (L540, L428) [124] TGF-β2 Ampliﬁcation 3 (KMH2, L540, L428) Deletion 1 (UH01) TGFBR2 Ampliﬁcation 3 (KMH2, L540, L428) s of TGF-β pathway components in EBV-associated cancers identiﬁed by next-generation sequencing. 8 of 23 Cancers 2018, 10, 247 Table 2. Cont. Table 2. Cont. 4. Tumour Promoting Roles of TGF-β TGF-β1 exerts its tumour promoting effects by inducing EMT, migration, invasion, metastasis, angiogenesis and immune suppression [2,9]. High levels of TGF-β1 are commonly detected in many types of solid tumour and positively correlate with disease stage [130–133]. TGF-β1 can be produced by tumour cells or by stromal cells in the tumour microenvironment, including macrophages and platelets [134,135]. As tumours progress, many cancer cells develop genetic abnormalities within the pathway to escape the tumour suppressive effects of TGF-β signalling and, therefore, the excessive production of TGF-β1 drives tumour progression [2,7]. Although it is suggested that the tumour promoting effects of TGF-β1 are mainly mediated through the non-canonical Smad-independent signalling pathway [136–139], there is evidence to show that the canonical Smad-dependent signalling pathway can also be responsible for mediating some of these effects [1–3]. 3.2. Dysregulation of TGF-β Receptors Cancer Total Number of Cases EBV Status Genes Alterations Number of Cases with Alterations References TGFBR3 Ampliﬁcation 2 (KMH2, L428) SMAD1 Ampliﬁcation 3 (KMH2, L540, L428) Deletion 2 (HDML2, UH01) SMAD5 Ampliﬁcation 3 (KMH2, L540, L428) DLBCL • 73 primary tumours • 21 DLBCL cell lines Unreported TGF-β1 Missense mutation 1 (primary tumours) [125] TGF-β1 Intronic mutation 1 (primary tumours) TGFBR2 Intronic mutation 2 (primary tumours) TGFBR3 Intronic mutation 2 (primary tumours) SMAD9 Intronic mutation 1 (primary tumours) DLBCL 51 primary tumours & immunochemotherapy-treated tumours Unreported TGF-β1 CNA 3 (treated tumours) [126] DLBCL 6 refractory & 7 responsive tumours to R-Chop Negative TGFBR2 Missense mutation 1 (refractory tumour) [127] DLBCL • 47 relapsed/ refractory tumours • 65 primary tumours Unreported TGFBR2 Missense mutation 6 (relapsed/refractory tumours) [128] DLBCL • 295 activated B-cell like DLBCL (ABC) • 164 germinal-center B-cell like DLBCL (GCB) • 115 unclassiﬁed DLBCL Unreported TGF-β1 Missense mutation 5 (4 ABC, 1 GCB) [129] Truncated mutation 2 (ABC) TGF-β2 Truncated mutation 2 (1 ABC, 1 GCB) TGF-β3 Missense mutation 1 (ABC) TGFBR1 Missense mutation 1 (GCB) TGFBR2 Missense mutation 2 (1 ABC, 1 GCB) Truncated mutation 2 (1 GCB, 1 unclassiﬁed) TGFBR3 Missense mutation 2 (1 ABC, 1 unclassiﬁed) Truncated mutation 1 (GCB) SMAD1 Missense mutation 1 (ABC) Truncated mutation 1 (GCB) SMAD2 Missense mutation 3 (1 ABC, 1 GCB, 1 unclassiﬁed) SMAD4 Missense mutation 3 (2 ABC, 1 unclassiﬁed) Truncated mutation 1 (ABC) SMAD5 Missense mutation 5 (4 ABC, 1 GCB) SMAD6 Missense mutation 1 (GCB) SMAD7 Missense mutation 2 (1 ABC, 1 GCB) SMAD9 Truncated mutation 1 (ABC) 9 of 23 Cancers 2018, 10, 247 4.1. High Levels of TGF-β in EBV-Associated Cancers Several early studies showed that TGF-β1 and TGF-β2 were produced by Hodgkin’s Reed-Sternberg (H-RS) cells in vitro and in vivo [140–143]. It was subsequently shown that HL cells produced TGF-β, which contributes to the shift from a Th1-biased towards a Th2-biased T-cell inﬁltrate characteristics of HL [144]. EBV and its lytic gene product (Zta) have been shown to induce TGF-β1 production and secretion in BL and HeLa cells [145,146]. In patients with NPC, the levels of both the total and active TGF-β1 in serum samples have been reported to be elevated compared to those from healthy individuals with levels positively correlating with disease staging [147]. In support of these observations, our previous study has conﬁrmed that TGF-β1 was up-regulated in EBV-positive NPC tissues compared to non-malignant nasopharyngeal mucosa [148]. There is also evidence to show that EBNA1 and LMP1 induced the expression and secretion of TGF-β1 in epithelial cells in vitro [92,149]. Interestingly, a relatively high intracellular expression of TGF-β1 protein was detected following miR-93-mediated down-regulation of TGFBR2 in NPC cells [108]. In gastric cancer, high levels of TGF-β1 and TGF-β3 expression were detected in tissue samples of gastric carcinoma compared to gastric mucosa, although the status of EBV in these cancer samples examined was unreported [150]. Further, both gastric tissue-derived EBV-infected epithelial cell lines, GT38 and GT39, spontaneously produce biologically active TGF-β1 [75]. These data are consistent with the more recent report that TGF-β1 levels were elevated in EBVaGC [151]. Notably, several mutations on TGF-β1 and TGF-β2 have been detected in EBV-associated cancers (Table 2), pointing to a possible role of EBV in regulating the expression of TGF-β ligands. Further investigations are warranted to examine whether these are gain-of-function mutations that might result in increased levels of TGF-β ligands. 4.2. Contribution of TGF-β Signalling to the Aggressive Phenotypes of EBV-Associated Cancers 5. Induction of EBV Lytic Reactivation by TGF-β EBV is able to induce its lytic cycle by switching on the expression of BZLF1 gene which encodes protein Zta [80]. TGF-β was initially shown to induce the viral productive cycle in marmoset B lymphocytes immortalized with EBV [162]. It was subsequently shown that TGF-β induces latent EBV to enter into lytic cycle (as shown by EA expression) in two BL cell lines P3HR-1 and Akata [145,163]. These observations were later conﬁrmed in a series of BL cell lines (Mutu-I, Raji and B95-8) in which TGF-β1 induced BZLF1/Zta expression by an indirect mechanism which required the ERK 1/2 MAPK kinase pathway; Smad signalling alone was not sufﬁcient to mediate TGF-β1 induction of Zta [164]. It was further shown in additional BL cells (Mutu-I, Kem-I and Sav-I) that the PI3K/AKT pathway, acting downstream of ERK 1/2, enabled Smad3 to be acetylated by direct interaction with the co-activator CREB-binding protein to stimulate TGF-β1-induced Zta expression [165]. Different mechanisms of TGF-β1-mediated activation of BZLF1 gene have also been reported. In BL cell lines, Rael and P3HR-1, BZLF1 gene expression appeared to be activated by TGF-β through its mediator Smad proteins [166]. A Smad4-binding element (later termed SBE1) located within the BZLF1 Z promoter (Zp) was identiﬁed and both SBE1 and AP-1 motifs were required for TGF-β to activate the expression of BZLF1 through the complex of Smad3/Smad4 associated with the c-Jun/c-Fos proteins of the AP-1 complex [166]. However, this mechanism accounted for only 20–30% of the total TGF-β-mediated activation of transcription from Zp. Subsequently, Iempridee and colleagues identiﬁed an additional four SBEs (termed SBE2-5) and showed that TGF-β induced EBV lytic reactivation via the canonical Smad pathway by alleviating ZEB-mediated repression of Zp through multiple SBEs acting in concert [167]. In epithelial cells, TGF-β1 partially induced EBV reactivation in gastric cancer cells GT38 and GT39, as shown by the expression of BZLF1/Zta and early antigen-D, possibly primarily through junB pathway [75]. These studies have collectively demonstrated that TGF-β induces lytic reactivation in latently EBV-infected cells by stimulating the expression of BZLF1 gene/Zta protein through both canonical and non-canonical pathways. In addition to the BZLF1 Zp, the EBNA1 Qp is also a direct target of the TGF-β signaling pathway. EBNA1 is a DNA-binding protein that binds to the ori-P region of the EBV genome and allows the viral genome to be present as an episome in infected cells [168]. 4.2. Contribution of TGF-β Signalling to the Aggressive Phenotypes of EBV-Associated Cancers Flot1 was shown to stimulate the expression and secretion of TGF-β1, facilitate the activation of TGF-β/Smad3 signalling to effectuate EMT in NPC cells [155]. Whereas Zhao and colleagues showed that Flot2 was required for TGF-β1-induced EMT in NPC cells through activation of Src [156]. Further, the ability of high-mobility group AT-hook 2 (HMGA2) to induce EMT in NPC cells was attributed to the activation of TGF-β/Smad3 signalling pathway [157]. TGF-β1 was also shown to induce EMT in NPC cells by enhancing the expression of formin-like 3 (FMNL3) and Y-box binding protein-1 (YBX1) [153,154]. More recently, it was found that TGF-β1 induced NPC cell growth and migration by down-regulating miRNA-124 which inhibited TGF-β1-mediated responses by targeting the pro-oncogenic lncRNA MALAT1 primarily via the ERK/MAPK pathway [158]. In addition, LMP1-mediated activin/ TGF-β signalling through the JNK/SAPK pathway was also involved in the induction of the extracellular matrix protein, ﬁbronectin, a process that may contribute to tumour invasiveness in NPC [92]. In addition to promoting aggressive phenotypes of cancer cells, an emerging role for TGF-β signalling in cancer drug resistance has also been proposed [159,160]. Very recently, it was reported that overexpression of miR-449b in NPC down-regulated TGF-β-induced (TGFβI), a target gene of TGF-β pathway, leading to increased pro-TGF-β1 activation and cisplatin resistance [161]. The effect of TGF-β in inducing aggressive phenotypes in EBVaGC is currently unexplored and further studies are warranted. 4.2. Contribution of TGF-β Signalling to the Aggressive Phenotypes of EBV-Associated Cancers Several lines of evidence have shown that TGF-β signalling promotes aggressive phenotypes of EBV-associated epithelial cancers. TGF-β signalling is a major inducer of EMT in cancer cells [152]. EMT is morphologically characterized by changes from an epithelial cell phenotype to a spindle ﬁbroblast-like appearance and functionally characterized by decreased cell adhesion and increase cell migration. Accordingly, TGF-β signalling-associated induction of EMT is considered an important step in the progression of tumour metastasis. Alterations in EMT markers (increased Vimentin and decreased E-cadherin) were detected in clinical NPC samples [153,154], indicating that NPC cells undergo EMT in vivo. Although EBNA1 has been shown to suppress TGF-β-mediated transcription in AdAH and HL cells [95,100], in NPC cells, EBNA1 appeared to up-regulate the expression of TGF-β1 protein leading to a reduction in expression of miR-200a and miR-200b which in turn, up-regulated their target genes ZEB1 and ZEB2, well known mediators of EMT [149]. Recent reports have repeatedly described the underlying mechanisms of the EMT process induced by TGF-β signalling in NPC, and a number of effectors have been identiﬁed. There is evidence to demonstrate that components of lipid rafts, ﬂotillin-1 and -2 (Flot1 and Flot2), were highly expressed in primary 10 of 23 Cancers 2018, 10, 247 NPC tissues [155,156] and that this contributed to the TGF-β1 induction of EMT in NPC. Flot1 was shown to stimulate the expression and secretion of TGF-β1, facilitate the activation of TGF-β/Smad3 signalling to effectuate EMT in NPC cells [155]. Whereas Zhao and colleagues showed that Flot2 was required for TGF-β1-induced EMT in NPC cells through activation of Src [156]. Further, the ability of high-mobility group AT-hook 2 (HMGA2) to induce EMT in NPC cells was attributed to the activation of TGF-β/Smad3 signalling pathway [157]. TGF-β1 was also shown to induce EMT in NPC cells by enhancing the expression of formin-like 3 (FMNL3) and Y-box binding protein-1 (YBX1) [153,154]. More recently, it was found that TGF-β1 induced NPC cell growth and migration by down-regulating miRNA-124 which inhibited TGF-β1-mediated responses by targeting the pro-oncogenic lncRNA MALAT1 primarily via the ERK/MAPK pathway [158]. In addition, LMP1-mediated activin/ TGF-β signalling through the JNK/SAPK pathway was also involved in the induction of the extracellular matrix protein, ﬁbronectin, a process that may contribute to tumour invasiveness in NPC [92]. NPC tissues [155,156] and that this contributed to the TGF-β1 induction of EMT in NPC. 6. Concluding Remarks Since its discovery in 1964, EBV has been implicated in the aetiology of several tumours of both lymphoid and epithelial origin. Although the mechanisms of EBV infection in lymphoid and epithelial cells are different, it is well-recognised that the virus drives cancer development by de-regulating a diverse range of signalling pathways that regulate essential cellular processes [19]. It is perhaps not surprising that EBV acts as a modulator of the TGF-β signalling pathway, a key network that controls various vital processes, such as proliferation, differentiation, apoptosis and migration. Like many other tumours, EBV-associated cancers produce high levels of TGF-β and do not respond to the cytostatic effects of TGF-β, but yet often sustain a functional TGF-β core machinery to promote more aggressive malignant phenotypes. EBV utilises different mechanisms to manipulate the “double-edged sword” nature of TGF-β signalling to ﬁne-tune the TGF-β response at various levels (Figure 2). A balance between latent and lytic infection is crucial for EBV oncogenesis. In latently infected cells, EBV-encoded proteins (EBNA1, LMP1, LMP2A and BARF1) suppress TGF-β-mediated transcription, rendering cells refractory to TGF-β cytostatic effects. When lytic cycle is needed for the spreading of the virus, TGF-β disrupts the latency by stimulating the expression of BZLF1/Zta via both Smad-dependent and Smad-independent pathways which in turn, promotes the production of TGF-β and inhibits the transcription of EBNA1. Intriguingly, it has been shown that the EBV infection rate of epithelial cells can be enhanced by exogenous TGF-β1 and TGF-β1 derived from the epithelial cells facilitated viral transmission by inducing lytic cycle in the donor B-cells in co-culture systems [171,172]. These observations imply that TGF-β signalling might play a critical role in regulating persistent EBV infection, particularly in epithelial cells. Several lines of evidence have shown that the expression of TGFBR2 is down-regulated in NPC and TGF-β/Smad signalling is defective [76,106–108,173] and mutations in SMAD genes have been reported (Table 2). It is currently unclear whether these defects in the canonical pathway would be adequate to result in the loss of BZLF1/Zta expression, thereby facilitate the maintenance of EBV genomes in the nasopharyngeal epithelial cells. This is also relevant to the ability of TGF-β in the induction of differentiation of epithelial cells [174,175]. EBV infection is intimately associated with a number of undifferentiated carcinomas [86], implying that undifferentiated properties of epithelial cells are likely to be a prerequisite for stable EBV latent infection. 5. Induction of EBV Lytic Reactivation by TGF-β Transcription of the EBNA1 gene in BL and NPC cells is initiated from the Qp [169]. Qp expression is subject to regulation by a number of mechanisms and interestingly, in BL cells, it has been shown that TGF-β transcriptionally repressed EBNA1 Qp through cooperativity of a Smad3/Smad4 complex and the 11 of 23 Cancers 2018, 10, 247 transcriptional repressor TGIF at the SBE site within Qp [96]. It has also been shown that the Zta protein interfered with JAK/STAT activation of Qp [170] and induced TGF-β production in HeLa cells [146]. Thus, the autocrine/paracrine function of TGF-β is generated by up-regulation of Zta, which then activates the transcription of TGF-β, thereby forming a positive feedback loop to initiate the EBV lytic cycle. In parallel, both TGF-β and Zta repressed Qp to disrupt EBV latency. 6. Concluding Remarks Modulation of TGF-β signalling by EBV. EBV infection or EBV-encoded latent proteins (LMP1 and EBNA1) can stimulate the expression and secretion of TGF-β1 in epithelial cells. However, cancer cells often do not respond to the cytostatic effects of TGF-β, partly through the repression of signal transduction by the EBV-encoded proteins (EBNA1, LMP1, LMP2A and BARF1) through various mechanisms. The cancer cells often sustain a functional TGF-β core machinery and the excessive production of TGF-β drives aggressive malignant phenotypes. TGF-β signalling also appears to be crucial in regulating the balance between latent and lytic cycles in EBV-infected cells. TGF-β facilitates lytic reactivation in EBV-infected cells by stimulating the expression of BZLF1/Zta via both Smad-dependent and Smad-independent pathways. Zta induces the production of TGF-β1 which in turn, together with Zta, suppress the transcription of EBNA1 from Qp to disrupt EBV latency. Figure 2. Modulation of TGF-β signalling by EBV. EBV infection or EBV-encoded latent proteins (LMP1 and EBNA1) can stimulate the expression and secretion of TGF-β1 in epithelial cells. However, cancer cells often do not respond to the cytostatic effects of TGF-β, partly through the repression of signal transduction by the EBV-encoded proteins (EBNA1, LMP1, LMP2A and BARF1) through various mechanisms. The cancer cells often sustain a functional TGF-β core machinery and the excessive production of TGF-β drives aggressive malignant phenotypes. TGF-β signalling also appears to be crucial in regulating the balance between latent and lytic cycles in EBV-infected cells. TGF-β facilitates lytic reactivation in EBV-infected cells by stimulating the expression of BZLF1/Zta via both Smad-dependent and Smad-independent pathways. Zta induces the production of TGF-β1 which in turn, together with Zta, suppress the transcription of EBNA1 from Qp to disrupt EBV latency. β p y Smad2/3/4-dependent signalling, emerging evidence has revealed the contribution of non-canonical Smad1/5/9 signalling dysregulation to lymphomagenesis. In DLBCL, oncogenic miR-155 inhibited Smad5 expression and rendered cells resistant to the growth inhibitory effects of both TGF-β1 and BMPs, via a defective p21 induction and decreased formation of the RB/E2F1 complex [177,178]. More recently, Stelling and colleagues identified TGF-β/TGFR-2/Smad1 axis as the upstream regulator in suppressing the expression of sphingosine-1-phosphate (S1P) receptor 2 (S1PR2), a bona fide tumour suppressor in DLBCL, to provide a significant proliferative advantage to DLBCL cells in vitro and in vivo [179]. 6. Concluding Remarks Interestingly, we have previously reported that EBV infection contributed to aberrant S1P signalling in NPC [180] and also have data showing that BMP signalling is de-regulated in NPC (manuscript in preparation). It will be intriguing to unravel the contribution of EBV infection to the non-canonical Smad TGF-β signalling in the development of EBV-associated cancers. A better understanding of these mechanisms may also provide an explanation for the seemingly contradicting roles of EBV-encoded latent proteins in regulating the TGF-β pathway. EBNA1 and LMP1 have been shown to stimulate the production of TGF-β, and yet, they disrupted the signal transduction rendering the cells refractory to the TGF-β-mediated cytostasis. It is now clear that the high TGF-β production promotes aggressive phenotypes through the EMT induction arm; however, the contribution of EBV to this process is not well-explored. Additional mechanistic studies are warranted to elucidate how EBV fine-tunes the response to TGF-β and utilises this pathway to achieve malignant transformation. Further, it has been shown that TGF-β-induced EMT can drive tumour cells towards a more stem cell-like phenotype [181,182]. Significantly, a decrease in the While most of the studies on the TGF-β pathway to date have focused on the roles of canonical Smad2/3/4-dependent signalling, emerging evidence has revealed the contribution of non-canonical Smad1/5/9 signalling dysregulation to lymphomagenesis. In DLBCL, oncogenic miR-155 inhibited Smad5 expression and rendered cells resistant to the growth inhibitory effects of both TGF-β1 and BMPs, via a defective p21 induction and decreased formation of the RB/E2F1 complex [177,178]. More recently, Stelling and colleagues identiﬁed TGF-β/TGFR-2/Smad1 axis as the upstream regulator in suppressing the expression of sphingosine-1-phosphate (S1P) receptor 2 (S1PR2), a bona ﬁde tumour suppressor in DLBCL, to provide a signiﬁcant proliferative advantage to DLBCL cells in vitro and in vivo [179]. Interestingly, we have previously reported that EBV infection contributed to aberrant S1P signalling in NPC [180] and also have data showing that BMP signalling is de-regulated in NPC (manuscript in preparation). It will be intriguing to unravel the contribution of EBV infection to the non-canonical Smad TGF-β signalling in the development of EBV-associated cancers. A better understanding of these mechanisms may also provide an explanation for the seemingly contradicting roles of EBV-encoded latent proteins in regulating the TGF-β pathway. EBNA1 and LMP1 have been shown to stimulate the production of TGF-β, and yet, they disrupted the signal transduction rendering the cells refractory to the TGF-β-mediated cytostasis. 6. Concluding Remarks It has been shown that differentiation of epithelial and B cells triggered EBV lytic reactivation in the latently infected cells [176]. In line with this concept, defects in TGF-β signalling might impair cellular differentiation which results in the suppression of lytic cycle, thereby facilitating latent infection in epithelial cells. Interestingly, it has been shown that in patients with EBVaGC, TGF-β1 levels were signiﬁcantly associated with the expression of EBV lytic genes in the absence of Helicobacter pylori (H. pylori) infection [151]. These data imply that H. pylori infection prevents EBV lytic induction by suppressing TGF-β1 expression in EBVaGC patients, observations that warrant further investigation. 12 of 23 of 23 Cancers 2018, 10, 247 Cancers 2018 10 Figure 2. Modulation of TGF-β signalling by EBV. EBV infection or EBV-encoded latent proteins (LMP1 and EBNA1) can stimulate the expression and secretion of TGF-β1 in epithelial cells. However, cancer cells often do not respond to the cytostatic effects of TGF-β, partly through the repression of signal transduction by the EBV-encoded proteins (EBNA1, LMP1, LMP2A and BARF1) through various mechanisms. The cancer cells often sustain a functional TGF-β core machinery and the excessive production of TGF-β drives aggressive malignant phenotypes. TGF-β signalling also appears to be crucial in regulating the balance between latent and lytic cycles in EBV-infected cells. TGF-β facilitates lytic reactivation in EBV-infected cells by stimulating the expression of BZLF1/Zta via both Smad-dependent and Smad-independent pathways. Zta induces the production of TGF-β1 which in turn, together with Zta, suppress the transcription of EBNA1 from Qp to disrupt EBV latency. Figure 2. Modulation of TGF-β signalling by EBV. EBV infection or EBV-encoded latent proteins (LMP1 and EBNA1) can stimulate the expression and secretion of TGF-β1 in epithelial cells. However, cancer cells often do not respond to the cytostatic effects of TGF-β, partly through the repression of signal transduction by the EBV-encoded proteins (EBNA1, LMP1, LMP2A and BARF1) through various mechanisms. The cancer cells often sustain a functional TGF-β core machinery and the excessive production of TGF-β drives aggressive malignant phenotypes. TGF-β signalling also appears to be crucial in regulating the balance between latent and lytic cycles in EBV-infected cells. TGF-β facilitates lytic reactivation in EBV-infected cells by stimulating the expression of BZLF1/Zta via both Smad-dependent and Smad-independent pathways. Zta induces the production of TGF-β1 which in turn, together with Zta, suppress the transcription of EBNA1 from Qp to disrupt EBV latency. Figure 2. 6. Concluding Remarks It is now clear that the high TGF-β production promotes aggressive phenotypes through the EMT induction arm; however, the contribution of EBV to this process is not well-explored. Additional mechanistic studies are warranted to elucidate how EBV ﬁne-tunes the response to TGF-β and utilises this pathway to achieve malignant transformation. Further, it has been shown that TGF-β-induced EMT can drive tumour cells towards a more stem cell-like phenotype [181,182]. Signiﬁcantly, a decrease in the number of stem cells was observed after Cancers 2018, 10, 247 13 of 23 treatment with TGFBR1 inhibitors in gliobastoma [183]. This would be an important research area to be explored in EBV-associated cancers, particularly as EBV latent proteins (LMP1 and LMP2A) have been shown to contribute to the induction and maintenance of cancer stem-like cell (CSC) population in NPC [184,185]. [ ] Many drugs that target TGF-β signalling have been developed for the treatment of a number of diseases [186]. Some of which have reached clinical trials, including a phase I trial for EBV-positive lymphoma using recombinant cytotoxic-T-lymphocytes with a virus encoding a dominant negative TGFBR2 [187]; however, the results have not been posted for this trial. The main goal of utilising anti-TGF-β therapies in cancer is to reduce excessive levels of TGF-β ligands. However, there are clearly concerns and issues with this approach, such as that TGF-β inhibitors are not cytotoxic and might disrupt the stem cells niche resulting in releasing cancer stem cells from dormancy [186]. TGF-β action is highly context-dependent and inﬂuenced by multiple factors, such as interactions with other signalling pathways, disease stage and innate genetic background among individuals. EBV infection is likely to add another level of complexity to anti-TGF-β therapies in EBV-associated cancers. It is also noteworthy that the function of TGF-β signalling in the tumour immune microenvironment (TIME) is important in this regard. TGF-β signalling is a crucial mediator not only of changes to the tumour cell phenotype but also of changes in the stromal environment [7]. This is particularly relevant to the development of EBV-based immunotherapies. It has recently been shown that a transcriptional signature of TGF-β pathway activation was associated with low levels of stromal tumour-inﬁltrating lymphocytes (TILs) and poor prognosis in NPC patients [188]. 6. Concluding Remarks The TIME in both NPC and HL may inﬂuence the response to immunotherapeutic interventions and it might be important to determine the level of TGF-β expression in these tumours as a prognostic indicator of response to such therapies. Therefore, a more complete understanding of the multifaceted function of TGF-β signalling in EBV-associated cancers is required to determine if this pathway can be manipulated therapeutically for the management of patients with these diseases. Funding: This work was supported by Newton-Ungku Omar Fund MR/P013201/1 (IF016-2017) from the Academy of Sciences Malaysia and Medical Research Council UK, Fundamental Research Grant Scheme (FP013-2016) from the Ministry of Higher Education Malaysia and High Impact Research Grant UM.C/625/1/HIR/MOHE/DENT/23 from the University of Malaya. The APC was funded by Newton-Ungku Omar Fund MR/P013201/1 (IF016-2017) and Institute of Research Management & Service (IPPP), University of Malaya. Conﬂicts of Interest: The authors declare no conﬂict of interest. s of Interest: The authors declare no conﬂict of interest. References [CrossRef] [PubMed] 10. Oft, M.; Heider, K.H.; Beug, H. TGF-β signaling is necessary for carcinoma cell invasiveness and metastasis. Curr. Biol. 1998, 8, 1243–1252. [CrossRef] 11. Ueki, N.; Nakazato, M.; Ohkawa, T.; Ikeda, T.; Amuro, Y.; Hada, T.; Higashino, K. Excessive production of transforming growth-factor beta 1 can play an important role in the development of tumorigenesis by its action for angiogenesis: Validity of neutralizing antibodies to block tumor growth. Biochim. Biophys. Acta 1992, 1137, 189–196. [CrossRef] 12. Torre-Amione, G.; Beauchamp, R.D.; Koeppen, H.; Park, B.H.; Schreiber, H.; Moses, H.L.; Rowley, D.A. A highly immunogenic tumor transfected with a murine transforming growth factor type beta 1 cDNA escapes immune surveillance. Proc. Natl. Acad. Sci. USA 1990, 87, 1486–1490. [CrossRef] [PubMed] 12. Torre-Amione, G.; Beauchamp, R.D.; Koeppen, H.; Park, B.H.; Schreiber, H.; Moses, H.L.; Rowley, D.A. A highly immunogenic tumor transfected with a murine transforming growth factor type beta 1 cDNA 13. McEarchern, J.A.; Kobie, J.J.; Mack, V.; Wu, R.S.; Meade-Tollin, L.; Arteaga, C.L.; Dumont, N.; Besselsen, D.; Seftor, E.; Hendrix, M.J.; et al. Invasion and metastasis of a mammary tumor involves TGF-β signaling. Int. J. Cancer 2001, 91, 76–82. [CrossRef] 14. Lehmann, K.; Janda, E.; Pierreux, C.E.; Rytomaa, M.; Schulze, A.; McMahon, M.; Hill, C.S.; Beug, H.; Downward, J. Raf induces TGF-β production while blocking its apoptotic but not invasive responses: A mechanism leading to increased malignancy in epithelial cells. Genes Dev. 2000, 14, 2610–2622. [CrossRef] [PubMed] 15. Zhang, Y.Y.; Li, X.; Sui, L.H.; Wang, Q.; Li, P.; Fu, S.B. Functional study on TGF-β/Smads signaling pathway in human ovarian cancer cells. Yi Chuan Xue Bao 2004, 31, 759–765. [PubMed] 16. Chen, C.R.; Kang, Y.; Massague, J. Defective repression of c-myc in breast cancer cells: A loss at the core of the transforming growth factor beta growth arrest program. Proc. Natl. Acad. Sci. USA. 2001, 98, 992–999. [CrossRef] [PubMed] 17. Mirzaei, H.; Faghihloo, E. Viruses as key modulators of the TGF-β pathway; a double-edged sword involved in cancer. Rev. Med. Virol. 2018, 28. [CrossRef] [PubMed] 8. Epstein, M.A.; Achong, B.G.; Barr, Y.M. Virus Particles in Cultured Lymphoblasts from Burkitt's Lympho Lancet 1964, 1, 702–703. [CrossRef] 19. Young, L.S.; Yap, L.F.; Murray, P.G. Epstein-Barr virus: More than 50 years old and still providing surprises. Nat. Rev. Cancer 2016, 16, 789–802. [CrossRef] [PubMed] 20. Assoian, R.K.; Komoriya, A.; Meyers, C.A.; Miller, D.M.; Sporn, M.B. Transforming growth factor-beta in human platelets. References 1. Derynck, R.; Zhang, Y.E. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 2003, 425, 577–584. [CrossRef] [PubMed] 1. Derynck, R.; Zhang, Y.E. Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 2003, 425, 577–584. [CrossRef] [PubMed] 2. Siegel, P.M.; Massague, J. Cytostatic and apoptotic actions of TGF-β in homeostasis and cancer. Nat. Rev. Cancer 2003, 3, 807–821. [CrossRef] [PubMed] 2. Siegel, P.M.; Massague, J. Cytostatic and apoptotic actions of TGF-β in homeostasis and cancer. Nat. Rev. Cancer 2003, 3, 807–821. [CrossRef] [PubMed] 3. Derynck, R.; Akhurst, R.J.; Balmain, A. TGF-β signaling in tumor suppression and cancer progression. Nat. Genet. 2001, 29, 117–129. [CrossRef] [PubMed] 3. Derynck, R.; Akhurst, R.J.; Balmain, A. TGF-β signaling in tumor suppression and cancer progression. Nat. Genet. 2001, 29, 117–129. [CrossRef] [PubMed] 4. Takehara, K.; LeRoy, E.C.; Grotendorst, G.R. TGF-β inhibition of endothelial cell proliferation: Alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Cell 1987, 49, 415–422. [CrossRef] 5. Moses, H.L.; Coffey, R.J., Jr.; Leof, E.B.; Lyons, R.M.; Keski-Oja, J. Transforming growth factor beta regulation of cell proliferation. J. Cell. Physiol. Suppl. 1987, (Suppl. 5), 1–7. [CrossRef] 5. Moses, H.L.; Coffey, R.J., Jr.; Leof, E.B.; Lyons, R.M.; Keski-Oja, J. Transforming growth factor beta regulation of cell proliferation. J. Cell. Physiol. Suppl. 1987, (Suppl. 5), 1–7. [CrossRef] 6. Stoeck, M.; Howe, R.C.; Miescher, S.; von Fliedner, V.; MacDonald, H.R. Effect of transforming growth factor beta on the EL4 thymoma variant EL4/6.1: Dissociation of inhibition of proliferation from expression of IL-1 and IL-2 receptors. Immunobiology 1990, 181, 13–21. [CrossRef] 6. Stoeck, M.; Howe, R.C.; Miescher, S.; von Fliedner, V.; MacDonald, H.R. Effect of transforming growth factor beta on the EL4 thymoma variant EL4/6.1: Dissociation of inhibition of proliferation from expression of IL-1 and IL-2 receptors. Immunobiology 1990, 181, 13–21. [CrossRef] 7. Pickup, M.; Novitskiy, S.; Moses, H.L. The roles of TGF-β in the tumour microenvironment. Nat. Rev. Cancer 2013, 13, 788–799. [CrossRef] [PubMed] 7. Pickup, M.; Novitskiy, S.; Moses, H.L. The roles of TGF-β in the tumour microenvironment. Nat. Rev. Cancer 2013, 13, 788–799. [CrossRef] [PubMed] Cancers 2018, 10, 247 14 of 23 8. Yanagihara, K.; Tsumuraya, M. Transforming growth factor beta 1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 1992, 52, 4042–4045. [PubMed] 9. Lebrun, J.J. The Dual Role of TGF-β in Human Cancer: From Tumor Suppression to Cancer Metastasis. ISRN Mol. Biol. 2012, 2012, 381–428. References Cloning of a type I TGF-β receptor and its effect on TGF-β binding to the type II receptor. Science 1993, 260, 1344–1348. [CrossRef] [PubMed] 34. Zhang, Y.; Feng, X.; We, R.; Derynck, R. Receptor-associated Mad homologues synergize as effectors of the TGF-β response. Nature 1996, 383, 168–172. [CrossRef] [PubMed] 35. Sekelsky, J.J.; Newfeld, S.J.; Raftery, L.A.; Chartoff, E.H.; Gelbart, W.M. Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics 1995, 139, 1347–1358. [PubMed] 36. Raftery, L.A.; Twombly, V.; Wharton, K.; Gelbart, W.M. Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila. Genetics 1995, 139, 241–254. [PubMed] 37. Moustakas, A.; Souchelnytskyi, S.; Heldin, C.H. Smad regulation in TGF-β signal transduction. J. Cell Sci. 2001, 114, 4359–4369. [PubMed] 38. Savage, C.; Das, P.; Finelli, A.L.; Townsend, S.R.; Sun, C.Y.; Baird, S.E.; Padgett, R.W. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 deﬁne a conserved family of transforming growth factor beta pathway components. Proc. Natl. Acad. Sci. USA 1996, 93, 790–794. [CrossRef] [PubMed] 39. Heldin, C.H.; Miyazono, K.; ten Dijke, P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature 1997, 390, 465–471. [CrossRef] [PubMed] 40. Wu, G.; Chen, Y.G.; Ozdamar, B.; Gyuricza, C.A.; Chong, P.A.; Wrana, J.L.; Massague, J.; Shi, Y. Structural basis of Smad2 recognition by the Smad anchor for receptor activation. Science 2000, 287, 92–97. [CrossRef] [PubMed] 41. Tsukazaki, T.; Chiang, T.A.; Davison, A.F.; Attisano, L.; Wrana, J.L. SARA, a FYVE domain protein that recruits Smad2 to the TGF-β receptor. Cell 1998, 95, 779–791. [CrossRef] 42. Wu, J.W.; Hu, M.; Chai, J.; Seoane, J.; Huse, M.; Li, C.; Rigotti, D.J.; Kyin, S.; Muir, T.W.; Fairman, R.; et al. Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-β signaling. Mol. Cell 2001, 8, 1277–1289. [CrossRef] 43. Chacko, B.M.; Qin, B.Y.; Tiwari, A.; Shi, G.; Lam, S.; Hayward, L.J.; De Caestecker, M.; Lin, K. Structural basis of heteromeric smad protein assembly in TGF-β signaling. Mol. Cell 2004, 15, 813–823. [CrossRef] [PubMed] 44. Nakao, A.; Imamura, T.; Souchelnytskyi, S.; Kawabata, M.; Ishisaki, A.; Oeda, E.; Tamaki, K.; Hanai, J.; Heldin, C.H.; Miyazono, K.; et al. TGF-β receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J. 1997, 16, 5353–5362. [CrossRef] [PubMed] 45. Topper, J.N.; DiChiara, M.R.; Brown, J.D.; Williams, A.J.; Falb, D.; Collins, T.; Gimbrone, M.A., Jr. References Identiﬁcation of a major storage site, puriﬁcation, and characterization. J. Biol. Chem. 1983, 258, 7155–7160. [PubMed] 21. Childs, C.B.; Proper, J.A.; Tucker, R.F.; Moses, H.L. Serum contains a platelet-derived transforming growth factor. Proc. Natl. Acad. Sci. USA 1982, 79, 5312–5316. [CrossRef] [PubMed] 22. Derynck, R.; Lindquist, P.B.; Lee, A.; Wen, D.; Tamm, J.; Graycar, J.L.; Rhee, L.; Mason, A.J.; Miller, D.A.; Coffey, R.J.; et al. A new type of transforming growth factor-beta, TGF-β 3. EMBO J. 1988, 7, 3737–3743. [PubMed] [ ] 23. Segarini, P.R.; Roberts, A.B.; Rosen, D.M.; Seyedin, S.M. Membrane binding characteristics of two forms of transforming growth factor-beta. J. Biol. Chem. 1987, 262, 14655–14662. [PubMed] 24. Sporn, M.B.; Roberts, A.B.; Wakeﬁeld, L.M.; Assoian, R.K. Transforming growth factor-beta: Biological function and chemical structure. Science 1986, 233, 532–534. [CrossRef] [PubMed] 25. Derynck, R.; Jarrett, J.A.; Chen, E.Y.; Eaton, D.H.; Bell, J.R.; Assoian, R.K.; Roberts, A.B.; Sporn, M.B.; Goeddel, D.V. Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature 1985, 316, 701–705. [CrossRef] [PubMed] 26. Gentry, L.E.; Lioubin, M.N.; Purchio, A.F.; Marquardt, H. Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide. Mol. Cell. Biol. 1988, 8, 4162–4168. [CrossRef] [PubMed] 27. Dubois, C.M.; Laprise, M.H.; Blanchette, F.; Gentry, L.E.; Leduc, R. Processing of transforming growth factor beta 1 precursor by human furin convertase. J. Biol. Chem. 1995, 270, 10618–10624. [CrossRef] [PubMed] 15 of 23 15 of 23 Cancers 2018, 10, 247 28. Gentry, L.E.; Nash, B.W. The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor. Biochemistry 1990, 29, 6851–6857. [CrossRef] [PubMed] 9. Barcellos-Hoff, M.H.; Derynck, R.; Tsang, M.L.; Weatherbee, J.A. Transforming growth factor-beta activa in irradiated murine mammary gland. J. Clin. Investig. 1994, 93, 892–899. [CrossRef] [PubMed] 30. Jullien, P.; Berg, T.M.; Lawrence, D.A. Acidic cellular environments: Activation of latent TGF-β and sensitization of cellular responses to TGF-beta and EGF. Int. J. Cancer 1989, 43, 886–891. [CrossRef] [PubMed] 31. Khalil, N. TGF-β: From latent to active. Microbes Infect 1999, 1, 1255–1263. [CrossRef] 32. Lin, H.Y.; Wang, X.F.; Ng-Eaton, E.; Weinberg, R.A.; Lodish, H.F. Expression cloning of the TGF-β type II receptor, a functional transmembrane serine/threonine kinase. Cell 1992, 68, 775–785. [CrossRef] 33. Ebner, R.; Chen, R.H.; Shum, L.; Lawler, S.; Zioncheck, T.F.; Lee, A.; Lopez, A.R.; Derynck, R. References Yan, X.; Liao, H.; Cheng, M.; Shi, X.; Lin, X.; Feng, X.H.; Chen, Y.G. Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-beta (TGF-β)/Smad Signaling. J. Biol. Chem. 2016, 291, 382–392. [CrossRef] [PubMed] 55. Galliher, A.J.; Schiemann, W.P. Src phosphorylates Tyr284 in TGF-β type II receptor and regulates TGF-β stimulation of p38 MAPK during breast cancer cell proliferation and invasion. Cancer Res. 2007, 67, 3752–3758. [CrossRef] [PubMed] 56. Lee, M.K.; Pardoux, C.; Hall, M.C.; Lee, P.S.; Warburton, D.; Qing, J.; Smith, S.M.; Derynck, R. TGF-β activates Erk MAP kinase signalling through direct phosphorylation of ShcA. EMBO J. 2007, 26, 3957–3967. [CrossRef] [PubMed] 57. Yi, J.Y.; Shin, I.; Arteaga, C.L. Type I transforming growth factor beta receptor binds to and activates phosphatidylinositol 3-kinase. J. Biol. Chem. 2005, 280, 10870–10876. [CrossRef] [PubMed] 58. Ohshima, T.; Shimotohno, K. Transforming growth factor-beta-mediated signaling via the p38 MAP kinase pathway activates Smad-dependent transcription through SUMO-1 modiﬁcation of Smad4. J. Biol. Chem. 2003, 278, 50833–50842. [CrossRef] [PubMed] 59. Kamaraju, A.K.; Roberts, A.B. Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo. J. Biol. Chem. 2005, 280, 1024–1036. [CrossRef] [PubMed] 60. Reynisdottir, I.; Polyak, K.; Iavarone, A.; Massague, J. Kip/Cip and Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response to TGF-β. Genes Dev. 1995, 9, 1831–1845. [CrossRef] [PubMed] 61. Laiho, M.; DeCaprio, J.A.; Ludlow, J.W.; Livingston, D.M.; Massague, J. Growth inhibition by TGF-β linked to suppression of retinoblastoma protein phosphorylation. Cell 1990, 62, 175–185. [CrossRef] 62. Harper, J.W.; Adami, G.R.; Wei, N.; Keyomarsi, K.; Elledge, S.J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993, 75, 805–816. [CrossRef] 63. Yang, W.; Shen, J.; Wu, M.; Arsura, M.; FitzGerald, M.; Suldan, Z.; Kim, D.W.; Hofmann, C.S.; Pianetti, S.; Romieu-Mourez, R.; et al. Repression of transcription of the p27(Kip1) cyclin-dependent kinase inhibitor gene by c-Myc. Oncogene 2001, 20, 1688–1702. [CrossRef] [PubMed] 64. Claassen, G.F.; Hann, S.R. A role for transcriptional repression of p21CIP1 by c-Myc in overcoming transforming growth factor beta -induced cell-cycle arrest. Proc. Natl. Acad. Sci. USA 2000, 97, 9498–9503. [CrossRef] [PubMed] 65. Warner, B.J.; Blain, S.W.; Seoane, J.; Massague, J. Myc downregulation by transforming growth factor beta required for activation of the p15(Ink4b) G(1) arrest pathway. Mol. Cell. Biol. 1999, 19, 5913–5922. [CrossRef] [PubMed] 66. References CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor beta transcriptional responses in endothelial cells. Proc. Natl. Acad. Sci. USA 1998, 95, 9506–9511. [CrossRef] [PubMed] 46. Seoane, J.; Le, H.V.; Shen, L.; Anderson, S.A.; Massague, J. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004, 117, 211–223. [CrossRef] 46. Seoane, J.; Le, H.V.; Shen, L.; Anderson, S.A.; Massague, J. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 2004, 117, 211–223. [CrossRef] 47. Yamamura, Y.; Hua, X.; Bergelson, S.; Lodish, H.F. Critical role of Smads and AP-1 complex in transforming growth factor-beta -dependent apoptosis. J. Biol. Chem. 2000, 275, 36295–36302. [CrossRef] [PubMed] 47. Yamamura, Y.; Hua, X.; Bergelson, S.; Lodish, H.F. Critical role of Smads and AP-1 complex in transforming growth factor-beta -dependent apoptosis. J. Biol. Chem. 2000, 275, 36295–36302. [CrossRef] [PubMed] 48. Hanyu, A.; Ishidou, Y.; Ebisawa, T.; Shimanuki, T.; Imamura, T.; Miyazono, K. The N domain of Smad7 is essential for speciﬁc inhibition of transforming growth factor-beta signaling. J. Cell Biol. 2001, 155, 1017–1027. [CrossRef] [PubMed] 49. Yan, X.; Liu, Z.; Chen, Y. Regulation of TGF-β signaling by Smad7. Acta Biochim. Biophys. Sin. 2009, 41, 263–272. [CrossRef] [PubMed] 16 of 23 16 of 23 Cancers 2018, 10, 247 50. Kavsak, P.; Rasmussen, R.K.; Causing, C.G.; Bonni, S.; Zhu, H.; Thomsen, G.H.; Wrana, J.L. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF -β receptor for degradation. Mol. Cell 2000, 6, 1365–1375. [CrossRef] 51. Ebisawa, T.; Fukuchi, M.; Murakami, G.; Chiba, T.; Tanaka, K.; Imamura, T.; Miyazono, K. Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J. Biol. Chem. 2001, 276, 12477–12480. [CrossRef] [PubMed] 52. Hayashi, H.; Abdollah, S.; Qiu, Y.; Cai, J.; Xu, Y.Y.; Grinnell, B.W.; Richardson, M.A.; Topper, J.N.; Gimbrone, M.A., Jr.; Wrana, J.L.; et al. The MAD-related protein Smad7 associates with the TGF-β receptor and functions as an antagonist of TGF-β signaling. Cell 1997, 89, 1165–1173. [CrossRef] 53. Zhang, S.; Fei, T.; Zhang, L.; Zhang, R.; Chen, F.; Ning, Y.; Han, Y.; Feng, X.H.; Meng, A.; Chen, Y.G. Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation. Mol. Cell. Biol. 2007, 27, 4488–4499. [CrossRef] [PubMed] 54. References Bakhshayesh, M.; Zaker, F.; Hashemi, M.; Katebi, M.; Solaimani, M. TGF- β1-mediated apoptosis associated with SMAD-dependent mitochondrial Bcl-2 expression. Clin. Lymphoma Myeloma Leuk. 2012, 12, 138–143. [CrossRef] [PubMed] 67. Jang, C.W.; Chen, C.H.; Chen, C.C.; Chen, J.Y.; Su, Y.H.; Chen, R.H. TGF-β induces apoptosis through Smad-mediated expression of DAP-kinase. Nat. Cell Biol. 2002, 4, 51–58. [CrossRef] [PubMed] 68. Hagimoto, N.; Kuwano, K.; Inoshima, I.; Yoshimi, M.; Nakamura, N.; Fujita, M.; Maeyama, T.; Hara, N. TGF-β 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells. J. Immunol. 2002, 168, 6470–6478. [CrossRef] [PubMed] 17 of 23 Cancers 2018, 10, 247 69. MacDonald, I.; Wang, H.; Grand, R.; Armitage, R.J.; Fanslow, W.C.; Gregory, C.D.; Gordon, J. Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines. Blood 1996, 87, 1147–1154. [PubMed] 70. Saltzman, A.; Munro, R.; Searfoss, G.; Franks, C.; Jaye, M.; Ivashchenko, Y. Transforming growth factor-beta-mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL downregulation. Exp. Cell Res. 1998, 242, 244–254. [CrossRef] [PubMed] 71. Inman, G.J.; Allday, M.J. Apoptosis induced by TGF-β 1 in Burkitt's lymphoma cells is caspase 8 dependent but is death receptor independent. J. Immunol. 2000, 165, 2500–2510. [CrossRef] [PubMed] 72. Blomhoff, H.K.; Smeland, E.; Mustafa, A.S.; Godal, T.; Ohlsson, R. Epstein-Barr virus mediates a switch in responsiveness to transforming growth factor, type beta, in cells of the B cell lineage. Eur. J. Immunol. 1987, 17, 299–301. [CrossRef] [PubMed] 73. Arvanitakis, L.; Yaseen, N.; Sharma, S. Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-β1-mediated growth inhibition in EBV-positive B cells. J. Immunol. 1995, 155, 1047–1056. [PubMed] 74. Kumar, A.; Rogers, T.; Maizel, A.; Sharma, S. Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells. J. Immunol. 1991, 147, 998–1006. [PubMed] 75. Fukuda, M.; Ikuta, K.; Yanagihara, K.; Tajima, M.; Kuratsune, H.; Kurata, T.; Sairenji, T. Effect of transforming growth factor-β1 on the cell growth and Epstein-Barr virus reactivation in EBV-infected epithelial cell lines. Virology 2001, 288, 109–118. [CrossRef] [PubMed] 76. Xiao, J.; Xiang, Q.; Xiao, Y.C.; Su, Z.J.; Huang, Z.F.; Zhang, Q.H.; Tan, Y.; Li, X.K.; Huang, Y.D. The effect of transforming growth factor-β1 on nasopharyngeal carcinoma cells: Insensitive to cell growth but functional to TGF-β/Smad pathway. J. Exp. Clin. Cancer Res. 2010, 29, 35. References J. Gen. Virol. 2000, 81, 1567–1578. [CrossRef] [PubMed] 89. Prokova, V.; Mosialos, G.; Kardassis, D. Inhibition of transforming growth factor beta signaling and Smad-dependent activation of transcription by the Latent Membrane Protein 1 of Epstein-Barr virus. J. Biol. Chem. 2002, 277, 9342–9350. [CrossRef] [PubMed] 90. Mori, N.; Morishita, M.; Tsukazaki, T.; Yamamoto, N. Repression of Smad-dependent transforming growth factor-beta signaling by Epstein-Barr virus latent membrane protein 1 through nuclear factor-κB. Int. J. Cancer 2003, 105, 661–668. [CrossRef] [PubMed] 91. Lo, A.K.; Dawson, C.W.; Lo, K.W.; Yu, Y.; Young, L.S. Upregulation of Id1 by Epstein-Barr Virus-encoded LMP1 confers resistance to TGFβ-mediated growth inhibition. Mol. Cancer 2010, 9, 155. [CrossRef] [PubMed] 92. Morris, M.A.; Dawson, C.W.; Laverick, L.; Davis, A.M.; Dudman, J.P.; Raveenthiraraj, S.; Ahmad, Z.; Yap, L.-F.; Young, L.S. The Epstein-Barr virus encoded LMP1 oncoprotein modulates cell adhesion via regulation of activin A/TGFβ and β1 integrin signalling. Sci. Rep. 2016, 6, 19533. [CrossRef] [PubMed] 93. Liu, Q.; Guan, X.; Lv, J.; Li, X.; Wang, Y.; Li, L. Limb-bud and Heart (LBH) functions as a tumor suppressor of nasopharyngeal carcinoma by inducing G1/S cell cycle arrest. Sci. Rep. 2015, 5, 7626. [CrossRef] [PubMed] 94. Fukuda, M.; Longnecker, R. Latent membrane protein 2A inhibits transforming growth factor-β1-induced 93. Liu, Q.; Guan, X.; Lv, J.; Li, X.; Wang, Y.; Li, L. Limb-bud and Heart (LBH) functions as a tumor suppr nasopharyngeal carcinoma by inducing G1/S cell cycle arrest. Sci. Rep. 2015, 5, 7626. [CrossRef] [P 93. Liu, Q.; Guan, X.; Lv, J.; Li, X.; Wang, Y.; Li, L. Limb bud and Heart (LBH) functions as a tumor suppressor of nasopharyngeal carcinoma by inducing G1/S cell cycle arrest. Sci. Rep. 2015, 5, 7626. [CrossRef] [PubMed] 94. Fukuda, M.; Longnecker, R. Latent membrane protein 2A inhibits transforming growth factor-β1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. J. Virol. 2004, 78, 1697–1705. [CrossRef] [PubMed] p y g y g y p 94. Fukuda, M.; Longnecker, R. Latent membrane protein 2A inhibits transforming growth factor-β1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. J. Virol. 2004, 78, 1697–1705. [CrossRef] [PubMed] 95. Wood, V.H.; O’Neil, J.D.; Wei, W.; Stewart, S.E.; Dawson, C.W.; Young, L.S. Epstein-Barr virus-encoded EBNA1 regulates cellular gene transcription and modulates the STAT1 and TGF-β signaling pathways. Oncogene 2007, 26, 4135–4147. [CrossRef] [PubMed] 96. Liang, C.L.; Tsai, C.N.; Chung, P.J.; Chen, J.L.; Sun, C.M.; Chen, R.H.; Hong, J.H.; Chang, Y.S. References [CrossRef] [PubMed] 77. Gu, S.; Tann, B.; Zeng, Y.; Zhou, W.; Li, K.; Zhao, M. Establishment of an epithelial cell line (CNE-2) from an NPC patient with poorly differentiated squamous cell carcinoma. Chin. J. Cancer 1983, 2, 70. 78. Takanashi, M.; Li, J.; Shirakata, M.; Mori, S.; Hirai, K. Tumorigenicity of mouse BALB/c 3T3 ﬁbroblast cells which express Epstein-Barr virus-encoded LMP1 and show normal growth phenotypes in vitro is correlated with loss of transforming growth factor-β1-mediated growth inhibition. Arch. Virol. 1999, 144, 241–257. [CrossRef] [PubMed] 79. Tsurumi, T.; Fujita, M.; Kudoh, A. Latent and lytic Epstein-Barr virus replication strategies. Rev. Med. Virol. 2005, 15, 3–15. [CrossRef] [PubMed] 80. Countryman, J.; Miller, G. Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA. Proc. Natl. Acad. Sci. USA 1985, 82, 4085–4089. [CrossRef] [PubMed] 81. Countryman, J.; Gradoville, L.; Bhaduri-McIntosh, S.; Ye, J.; Heston, L.; Himmelfarb, S.; Shedd, D.; Miller, G. Stimulus duration and response time independently inﬂuence the kinetics of lytic cycle reactivation of Epstein-Barr virus. J. Virol. 2009, 83, 10694–10709. [CrossRef] [PubMed] 82. Bhende, P.M.; Dickerson, S.J.; Sun, X.; Feng, W.H.; Kenney, S.C. X-box-binding protein 1 activates lytic Epstein-Barr virus gene expression in combination with protein kinase D. J. Virol. 2007, 81, 7363–7370. [CrossRef] [PubMed] 83. Sun, C.C.; Thorley-Lawson, D.A. Plasma cell-speciﬁc transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter. J. Virol. 2007, 81, 13566–13577. [CrossRef] [PubMed] 84. Temple, R.M.; Zhu, J.; Budgeon, L.; Christensen, N.D.; Meyers, C.; Sample, C.E. Efﬁcient replication of Epstein-Barr virus in stratiﬁed epithelium in vitro. Proc. Natl. Acad. Sci. USA 2014, 111, 16544–16549. [CrossRef] [PubMed] 85. Rickinson, A.B. Co-infections, inﬂammation and oncogenesis: future directions for EBV research. Semin. Cancer Biol. 2014, 26, 99–115. [CrossRef] [PubMed] 86. Yap, L.F.; Lo, K.W. Epstein-Barr virus and epithelial carcinogenesis. In DNA Tumour Viruses: Virology, Pathogenesis and Vaccines; Roberts, I.S., Ed.; Caister Academic Press: Poole, UK, 2018; pp. 139–161. ISBN 978-1-910190-80-7. 87. Kenney, J.L.; Guinness, M.E.; Reiss, M.; Lacy, J. Antisense to the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) sensitizes EBV-immortalized B cells to transforming growth factor-beta and chemotherapeutic agents. Int. J. Cancer 2001, 91, 89–98. [CrossRef] 18 of 23 18 of 23 Cancers 2018, 10, 247 88. Inman, G.J.; Allday, M.J. Resistance to TGF-β1 correlates with a reduction of TGF-β type II receptor expression in Burkitt’s lymphoma and Epstein-Barr virus-transformed B lymphoblastoid cell lines. References Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells. Virology 2000, 277, 184–192. [CrossRef] [PubMed] 97. Foote, L.C.; Marshak-Rothstein, A.; Rothstein, T.L. Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with speciﬁc antigen unless a surface immunoglobulin threshold is exceeded. J. Exp. Med. 1998, 187, 847–853. [CrossRef] [PubMed] 98. Holder, M.J.; Knox, K.; Gordon, J. Factors modifying survival pathways of germinal center B cells. Glucocorticoids and transforming growth factor-beta, but not cyclosporin A or anti-CD19, block surface immunoglobulin-mediated rescue from apoptosis. Eur. J. Immunol. 1992, 22, 2725–2728. [CrossRef] [PubMed] 99. Sater, R.A.; Sandel, P.C.; Monroe, J.G. B cell receptor-induced apoptosis in primary transitional murine B cells: signaling requirements and modulation by T cell help. Int. Immunol. 1998, 10, 1673–1682. [CrossRef] [PubMed] 100. Flavell, J.R.; Baumforth, K.R.; Wood, V.H.; Davies, G.L.; Wei, W.; Reynolds, G.M.; Morgan, S.; Boyce, A.; Kelly, G.L.; Young, L.S.; et al. Down-regulation of the TGF-beta target gene, PTPRK, by the Epstein-Barr virus encoded EBNA1 contributes to the growth and survival of Hodgkin lymphoma cells. Blood 2008, 111, 292–301. [CrossRef] [PubMed] 101. Horndasch, M.; Raschke, E.E.; Bommer, G.; Schuhmacher, M.; Dumont, E.; Kuklik-Roos, C.; Eick, D.; Kempkes, B. Epstein-Barr virus antagonizes the antiproliferative activity of transforming growth factor-beta but does not abolish its signaling. Int. J. Cancer 2002, 101, 442–447. [CrossRef] [PubMed] 102. Campion, E.M.; Hakimjavadi, R.; Loughran, S.T.; Phelan, S.; Smith, S.M.; D'Souza, B.N.; Tierney, R.J.; Bell, A.I.; Cahill, P.A.; Walls, D. Repression of the proapoptotic cellular BIK/NBK gene by Epstein-Barr virus antagonizes transforming growth factor beta1-induced B-cell apoptosis. J. Virol. 2014, 88, 5001–5013. [CrossRef] [PubMed] 103. Kim, D.H.; Chang, M.S.; Yoon, C.J.; Middeldorp, J.M.; Martinez, O.M.; Byeon, S.J.; Rha, S.Y.; Kim, S.H.; Kim, Y.S.; Woo, J.H. Epstein-Barr virus BARF1-induced NFkappaB/miR-146a/SMAD4 alterations in stomach cancer cells. Oncotarget 2016, 7, 82213–82227. [CrossRef] [PubMed] 104. Fukuda, M.; Kurosaki, H.; Sairenji, T. Loss of functional transforming growth factor (TGF)-beta type II receptor results in insensitivity to TGF-beta1-mediated apoptosis and Epstein-Barr virus reactivation. J. Med. Virol. 2006, 78, 1456–1464. [CrossRef] [PubMed] 105. Mao, S.; Yang, W.; Ai, L.; Li, Z.; Jin, J. Transforming growth factor beta type II receptor as a marker in diffuse large B cell lymphoma. Tumour Biol. 2015, 36, 9903–9908. [CrossRef] [PubMed] 19 of 23 19 of 23 Cancers 2018, 10, 247 106. References Fang, W.; Li, X.; Jiang, Q.; Liu, Z.; Yang, H.; Wang, S.; Xie, S.; Liu, Q.; Liu, T.; Huang, J.; et al. Transcriptional patterns, biomarkers and pathways characterizing nasopharyngeal carcinoma of Southern China. J. Transl. Med. 2008, 6, 32. [CrossRef] [PubMed] 107. Zhang, W.; Zeng, Z.; Fan, S.; Wang, J.; Yang, J.; Zhou, Y.; Li, X.; Huang, D.; Liang, F.; Wu, M.; et al. Evaluation of the prognostic value of TGF-beta superfamily type I receptor and TGF-beta type II receptor expression in nasopharyngeal carcinoma using high-throughput tissue microarrays. J. Mol. Histol. 2012, 43, 297–306. [CrossRef] [PubMed] 108. Lyu, X.; Fang, W.; Cai, L.; Zheng, H.; Ye, Y.; Zhang, L.; Li, J.; Peng, H.; Cho, W.C.; Wang, E.; et al. TGFbetaR2 is a major target of miR-93 in nasopharyngeal carcinoma aggressiveness. Mol. Cancer 2014, 13, 51. [CrossRef] [PubMed] 109. Ma, F.; Wang, Z.; Wang, J.; Liu, X.; Hu, C. MicroRNA-19a promotes nasopharyngeal carcinoma by targeting transforming growth factor beta receptor 2. Exp. Ther. Med. 2017, 14, 1419–1426. [CrossRef] [PubMed] 110. Ma, J.; Xuan, S.H.; Li, Y.; Zhang, Z.P.; Li, X.H. Role of the TGFbeta/PDCD4/AP-1 Signaling Pathway in Nasopharyngeal Carcinoma and Its Relationship to Prognosis. Cell. Physiol. Biochem. 2017, 43, 1392–1401. [CrossRef] [PubMed] 111. Paterson, I.C.; Matthews, J.B.; Huntley, S.; Robinson, C.M.; Fahey, M.; Parkinson, E.K.; Prime, S.S. Decreased expression of TGF-beta cell surface receptors during progression of human oral squamous cell carcinoma. J. Pathol. 2001, 193, 458–467. [CrossRef] 112. Chan, A.S.; To, K.F.; Lo, K.W.; Mak, K.F.; Pak, W.; Chiu, B.; Tse, G.M.; Ding, M.; Li, X.; Lee, J.C.; et al. High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese. Cancer Res. 2000, 60, 5365–5370. [PubMed] 113. Lo, K.W.; Teo, P.M.; Hui, A.B.; To, K.F.; Tsang, Y.S.; Chan, S.Y.; Mak, K.F.; Lee, J.C.; Huang, D.P. High resolution allelotype of microdissected primary nasopharyngeal carcinoma. Cancer Res. 2000, 60, 3348–3353. [PubMed] 114. Chen, H.C.; Chen, G.H.; Chen, Y.H.; Liao, W.L.; Liu, C.Y.; Chang, K.P.; Chang, Y.S.; Chen, S.J. MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br. J. Cancer 2009, 100, 1002–1011. [CrossRef] [PubMed] 115. Wan, X.X.; Yi, H.; Qu, J.Q.; He, Q.Y.; Xiao, Z.Q. Integrated analysis of the differential cellular and EBV miRNA expression proﬁles in microdissected nasopharyngeal carcinoma and non-cancerous nasopharyngeal tissues. Oncol. Rep. 2015, 34, 2585–2601. [CrossRef] [PubMed] 116. References Xu, Y.F.; Li, Y.Q.; Guo, R.; He, Q.M.; Ren, X.Y.; Tang, X.R.; Jia, W.H.; Kang, T.B.; Zeng, M.S.; Sun, Y.; et al. Identiﬁcation of miR-143 as a tumour suppressor in nasopharyngeal carcinoma based on microRNA expression proﬁling. Int. J. Biochem. Cell Biol. 2015, 61, 120–128. [CrossRef] [PubMed] 117. Deng, C.; Lin, Y.X.; Qi, X.K.; He, G.P.; Zhang, Y.; Zhang, H.J.; Xu, M.; Feng, Q.S.; Bei, J.X.; Zeng, Y.X.; et al. TNFRSF19 inhibits TGFbeta signaling through interaction with TGFbeta receptor type I to promote tumorigenesis. Cancer Res. 2018. [CrossRef] [PubMed] 118. Lin, D.C.; Meng, X.; Hazawa, M.; Nagata, Y.; Varela, A.M.; Xu, L.; Sato, Y.; Liu, L.Z.; Ding, L.W.; Sharma, A.; et al. The genomic landscape of nasopharyngeal carcinoma. Nat. Genet. 2014, 46, 866–871. [CrossRef] [PubMed] 119. Zheng, H.; Dai, W.; Cheung, A.K.; Ko, J.M.; Kan, R.; Wong, B.W.; Leong, M.M.; Deng, M.; Kwok, T.C.; Chan, J.Y.; et al. Whole-exome sequencing identiﬁes multiple loss-of-function mutations of NF-kappaB pathway regulators in nasopharyngeal carcinoma. Proc. Natl. Acad. Sci. USA 2016, 113, 11283–11288. [CrossRef] [PubMed] 120. Li, Y.Y.; Chung, G.T.; Lui, V.W.; To, K.F.; Ma, B.B.; Chow, C.; Woo, J.K.; Yip, K.Y.; Seo, J.; Hui, E.P.; et al. Exome and genome sequencing of nasopharynx cancer identiﬁes NF-kappaB pathway activating mutations. Nat. Commun. 2017, 8, 14121. [CrossRef] [PubMed] 121. Liang, Q.; Yao, X.; Tang, S.; Zhang, J.; Yau, T.O.; Li, X.; Tang, C.M.; Kang, W.; Lung, R.W.; Li, J.W.; et al. Integrative identiﬁcation of Epstein-Barr virus-associated mutations and epigenetic alterations in gastric cancer. Gastroenterology 2014, 147, 1350–1362. [CrossRef] [PubMed] 122. Boger, C.; Kruger, S.; Behrens, H.M.; Bock, S.; Haag, J.; Kalthoff, H.; Rocken, C. Epstein-Barr virus-associated gastric cancer reveals intratumoral heterogeneity of PIK3CA mutations. Ann. Oncol. 2017, 28, 1005–1014. [CrossRef] [PubMed] 20 of 23 Cancers 2018, 10, 247 20 of 23 123. Liu, Y.; Abdul Razak, F.R.; Terpstra, M.; Chan, F.C.; Saber, A.; Nijland, M.; van Imhoff, G.; Visser, L.; Gascoyne, R.; Steidl, C.; et al. The mutational landscape of Hodgkin lymphoma cell lines determined by whole-exome sequencing. Leukemia 2014, 28, 2248–2251. [CrossRef] [PubMed] 24. Hudnall, S.D.; Meng, H.; Lozovatsky, L.; Li, P.; Strout, M.; Kleinstein, S.H. Recurrent genetic defect classical Hodgkin lymphoma cell lines. Leuk. Lymphoma 2016, 57, 2890–2900. [CrossRef] [PubMed] 125. Zhang, J.; Grubor, V.; Love, C.L.; Banerjee, A.; Richards, K.L.; Mieczkowski, P.A.; Dunphy, C.; Choi, W.; Au, W.Y.; Srivastava, G.; et al. Genetic heterogeneity of diffuse large B-cell lymphoma. Proc. Natl. Acad. References Sci. USA 2013, 110, 1398–1403. [CrossRef] [PubMed] 126. Novak, A.J.; Asmann, Y.W.; Maurer, M.J.; Wang, C.; Slager, S.L.; Hodge, L.S.; Manske, M.; Price-Troska, T.; Yang, Z.Z.; Zimmermann, M.T.; et al. Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma. Blood Cancer J. 2015, 5, e346. [CrossRef] [PubMed] 127. Park, H.Y.; Lee, S.B.; Yoo, H.Y.; Kim, S.J.; Kim, W.S.; Kim, J.I.; Ko, Y.H. Whole-exome and transcriptome sequencing of refractory diffuse large B-cell lymphoma. Oncotarget 2016, 7, 86433–86445. [CrossRef] [PubMed] 128. Greenawalt, D.M.; Liang, W.S.; Saif, S.; Johnson, J.; Todorov, P.; Dulak, A.; Enriquez, D.; Halperin, R.; Ahmed, A.; Saveliev, V.; et al. Comparative analysis of primary versus relapse/refractory DLBCL identiﬁes shifts in mutation spectrum. Oncotarget 2017, 8, 99237–99244. [CrossRef] [PubMed] 129. Schmitz, R.; Wright, G.W.; Huang, D.W.; Johnson, C.A.; Phelan, J.D.; Wang, J.Q.; Roulland, S.; Kasbekar, M.; Young, R.M.; Shaffer, A.L.; et al. Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma. N. Engl. J. Med. 2018, 378, 1396–1407. [CrossRef] [PubMed] 130. Dalal, B.I.; Keown, P.A.; Greenberg, A.H. Immunocytochemical localization of secreted transforming growth factor-beta 1 to the advancing edges of primary tumors and to lymph node metastases of human mammary carcinoma. Am. J. Pathol. 1993, 143, 381–389. [PubMed] 131. Friedman, E.; Gold, L.I.; Klimstra, D.; Zeng, Z.S.; Winawer, S.; Cohen, A. High levels of transforming growth factor beta 1 correlate with disease progression in human colon cancer. Cancer Epidemiol. Biomark. Prev. 1995, 4, 549–554. 132. Gorsch, S.M.; Memoli, V.A.; Stukel, T.A.; Gold, L.I.; Arrick, B.A. Immunohistochemical staining for transforming growth factor beta 1 associates with disease progression in human breast cancer. Cancer Res. 1992, 52, 6949–6952. [PubMed] 133. Shim, K.S.; Kim, K.H.; Han, W.S.; Park, E.B. Elevated serum levels of transforming growth factor-beta1 in patients with colorectal carcinoma: Its association with tumor progression and its signiﬁcant decrease after curative surgical resection. Cancer 1999, 85, 554–561. [CrossRef] 134. Calon, A.; Espinet, E.; Palomo-Ponce, S.; Tauriello, D.V.; Iglesias, M.; Cespedes, M.V.; Sevillano, M.; Nadal, C.; Jung, P.; Zhang, X.H.; et al. Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation. Cancer Cell 2012, 22, 571–584. [CrossRef] [PubMed] 135. Hawinkels, L.J.; Verspaget, H.W.; van Duijn, W.; van der Zon, J.M.; Zuidwijk, K.; Kubben, F.J.; Verheijen, J.H.; Hommes, D.W.; Lamers, C.B.; Sier, C.F. Tissue level, activation and cellular localisation of TGF-beta1 and association with survival in gastric cancer patients. Br. J. References Cancer 2007, 97, 398–404. [CrossRef] [PubMed] 136. Bakin, A.V.; Tomlinson, A.K.; Bhowmick, N.A.; Moses, H.L.; Arteaga, C.L. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration. J. Biol. Chem. 2000, 275, 36803–36810. [CrossRef] [PubMed] g 137. Dumont, N.; Bakin, A.V.; Arteaga, C.L. Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells. J. Biol. Chem. 2003, 278, 3275–3285. [CrossRef] [PubMed] 138. Han, G.; Lu, S.L.; Li, A.G.; He, W.; Corless, C.L.; Kulesz-Martin, M.; Wang, X.J. Distinct mechanisms of TGF-beta1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis. J. Clin. Investig. 2005, 115, 1714–1723. [CrossRef] [PubMed] 139. Vinals, F.; Pouyssegur, J. Transforming growth factor beta1 (TGF-beta1) promotes endothelial cell survival during in vitro angiogenesis via an autocrine mechanism implicating TGF-α signaling. Mol. Cell. Biol. 2001, 21, 7218–7230. [CrossRef] [PubMed] 140. Newcom, S.R.; Kadin, M.E.; Ansari, A.A. Production of transforming growth factor-beta activity by Ki-1 positive lymphoma cells and analysis of its role in the regulation of Ki-1 positive lymphoma growth. Am. J. Pathol. 1988, 131, 569–577. [PubMed] 21 of 23 21 of 23 Cancers 2018, 10, 247 141. Newcom, S.R.; Kadin, M.E.; Ansari, A.A.; Diehl, V. L-428 nodular sclerosing Hodgkin’s cell secretes a unique transforming growth factor-beta active at physiologic pH. J. Clin. Investig. 1988, 82, 1915–1921. [CrossRef] [PubMed] 142. Kadin, M.E.; Agnarsson, B.A.; Ellingsworth, L.R.; Newcom, S.R. Immunohistochemical evidence of a role for transforming growth factor beta in the pathogenesis of nodular sclerosing Hodgkin’s disease. Am. J. Pathol. 1990, 136, 1209–1214. [PubMed] 143. Hsu, S.M.; Lin, J.; Xie, S.S.; Hsu, P.L.; Rich, S. Abundant expression of transforming growth factor-beta 1 and -beta 2 by Hodgkin’s Reed-Sternberg cells and by reactive T lymphocytes in Hodgkin’s disease. Hum. Pathol. 1993, 24, 249–255. [CrossRef] 144. Maeda, H.; Shiraishi, A. TGF-beta contributes to the shift toward Th2-type responses through direct and IL-10-mediated pathways in tumor-bearing mice. J. Immunol. 1996, 156, 73–78. [PubMed] 145. Di Renzo, L.; Altiok, A.; Klein, G.; Klein, E. Endogenous TGF-beta contributes to the induction of the EBV l ti l i t B kitt l h ll li I t J C 1994 57 914 919 [C R f] [P bM d] 145. Di Renzo, L.; Altiok, A.; Klein, G.; Klein, E. Endogenous TGF-beta contributes to the induction of the EBV lytic cycle in two Burkitt lymphoma cell lines. Int. J. Cancer 1994, 57, 914–919. [CrossRef] [PubMed] 146. References Cayrol, C.; Flemington, E.K. Identiﬁcation of cellular target genes of the Epstein-Barr virus transactivator Zta: Activation of transforming growth factor beta igh3 (TGF-beta igh3) and TGF-beta 1. J. Virol. 1995, 69, 4206–4212. [PubMed] 47. Xu, J.; Menezes, J.; Prasad, U.; Ahmad, A. Elevated serum levels of transforming growth factor beta Epstein-Barr virus-associated nasopharyngeal carcinoma patients. Int. J. Cancer 1999, 84, 396–399. [Cross 148. Hu, C.; Wei, W.; Chen, X.; Woodman, C.B.; Yao, Y.; Nicholls, J.M.; Joab, I.; Sihota, S.K.; Shao, J.Y.; Derkaoui, K.D.; et al. A global view of the oncogenic landscape in nasopharyngeal carcinoma: An integrated analysis at the genetic and expression levels. PLoS ONE 2012, 7, e41055. [CrossRef] [PubMed] 149. Wang, L.; Tian, W.D.; Xu, X.; Nie, B.; Lu, J.; Liu, X.; Zhang, B.; Dong, Q.; Sunwoo, J.B.; Li, G.; et al. Epstein-Barr virus nuclear antigen 1 (EBNA1) protein induction of epithelial-mesenchymal transition in nasopharyngeal carcinoma cells. Cancer 2014, 120, 363–372. [CrossRef] [PubMed] 150. Naef, M.; Ishiwata, T.; Friess, H.; Buchler, M.W.; Gold, L.I.; Korc, M. Differential localization of transforming growth factor-beta isoforms in human gastric mucosa and overexpression in gastric carcinoma. Int. J. Cancer 1997, 71, 131–137. [CrossRef] 151. Shukla, S.K.; Khatoon, J.; Prasad, K.N.; Rai, R.P.; Singh, A.K.; Kumar, S.; Ghoshal, U.C.; Krishnani, N. Transforming growth factor beta 1 (TGF-β1) modulates Epstein-Barr virus reactivation in absence of Helicobacter pylori infection in patients with gastric cancer. Cytokine 2016, 77, 176–179. [CrossRef] [PubMed] py p g y 152. Xu, J.; Lamouille, S.; Derynck, R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 2009, 19, 156–172. [CrossRef] [PubMed] 153. Wu, Y.; Shen, Z.; Wang, K.; Ha, Y.; Lei, H.; Jia, Y.; Ding, R.; Wu, D.; Gan, S.; Li, R.; et al. High FMNL3 expression promotes nasopharyngeal carcinoma cell metastasis: Role in TGF-beta1-induced epithelia-to-mesenchymal transition. Sci. Rep. 2017, 7, 42507. [CrossRef] [PubMed] 154. Zhou, L.L.; Ni, J.; Feng, W.T.; Yao, R.; Yue, S.; Zhu, Y.N.; Tang, H.Y.; Lv, L.Y.; Feng, J.F.; Zhu, W.G. High YBX1 expression indicates poor prognosis and promotes cell migration and invasion in nasopharyngeal carcinoma. Exp. Cell Res. 2017, 361, 126–134. [CrossRef] [PubMed] 155. Cao, S.; Cui, Y.; Xiao, H.; Mai, M.; Wang, C.; Xie, S.; Yang, J.; Wu, S.; Li, J.; Song, L.; et al. Upregulation of ﬂotillin-1 promotes invasion and metastasis by activating TGF-beta signaling in nasopharyngeal carcinoma. Oncotarget 2016, 7, 4252–4264. [CrossRef] [PubMed] 156. References Zhao, L.; Lin, L.; Pan, C.; Shi, M.; Liao, Y.; Bin, J.; Liao, W. Flotillin-2 promotes nasopharyngeal carcinoma metastasis and is necessary for the epithelial-mesenchymal transition induced by transforming growth factor-β. Oncotarget 2015, 6, 9781–9793. [CrossRef] [PubMed] 157. Xia, Y.Y.; Yin, L.; Jiang, N.; Guo, W.J.; Tian, H.; Jiang, X.S.; Wu, J.; Chen, M.; Wu, J.Z.; He, X. Downregulating HMGA2 attenuates epithelial-mesenchymal transition-induced invasion and migration in nasopharyngeal cancer cells. Biochem. Biophys. Res. Commun. 2015, 463, 357–363. [CrossRef] [PubMed] 158. Du, M.; Chen, W.; Zhang, W.; Tian, X.K.; Wang, T.; Wu, J.; Gu, J.; Zhang, N.; Lu, Z.W.; Qian, L.X.; et al. TGF-β regulates the ERK/MAPK pathway independent of the SMAD pathway by repressing miRNA-124 to increase MALAT1 expression in nasopharyngeal carcinoma. Biomed. Pharmacother. 2018, 99, 688–696. [CrossRef] [PubMed] 159. Oshimori, N.; Oristian, D.; Fuchs, E. TGF-β promotes heterogeneity and drug resistance in squamous cell carcinoma. Cell 2015, 160, 963–976. [CrossRef] [PubMed] 22 of 23 Cancers 2018, 10, 247 160. Brunen, D.; Willems, S.M.; Kellner, U.; Midgley, R.; Simon, I.; Bernards, R. TGF-β: An emerging player in drug resistance. Cell cycle 2013, 12, 2960–2968. [CrossRef] [PubMed] 161. Bissey, P.A.; Law, J.H.; Bruce, J.P.; Shi, W.; Renoult, A.; Chua, M.L.K.; Yip, K.W.; Liu, F.F. Dysregulation of the MiR-449b target TGFBI alters the TGFbeta pathway to induce cisplatin resistance in nasopharyngeal carcinoma. Oncogenesis 2018, 7, 40. [CrossRef] [PubMed] 162. Wittmann, P.; Hoﬂer, P.; Bauer, G. Epstein-barr virus induction by a serum factor: IV. Ubiquitous occurrence of the factor within vertebrates and its interaction with deﬁned lymphoid cell lines. Int. J. Cancer 1982, 30, 503–510. [CrossRef] [PubMed] 163. Chasserot-Golaz, S.; Schuster, C.; Dietrich, J.B.; Beck, G.; Lawrence, D.A. Antagonistic action of RU38486 on the activity of transforming growth factor-β in ﬁbroblasts and lymphoma cells. J. Steroid Biochem. 1988, 30, 381–385. [CrossRef] 164. Fahmi, H.; Cochet, C.; Hmama, Z.; Opolon, P.; Joab, I. Transforming growth factor beta 1 stimulates expression of the Epstein-Barr virus BZLF1 immediate-early gene product ZEBRA by an indirect mechanism which requires the MAPK kinase pathway. J. Virol. 2000, 74, 5810–5818. [CrossRef] [PubMed] 165. Oussaief, L.; Hippocrate, A.; Ramirez, V.; Rampanou, A.; Zhang, W.; Meyers, D.; Cole, P.; Khelifa, R.; Joab, I. Phosphatidylinositol 3-kinase/Akt pathway targets acetylation of Smad3 through Smad3/CREB-binding protein interaction: Contribution to transforming growth factor beta1-induced Epstein-Barr virus reactivation. J. Biol. Chem. 2009, 284, 23912–23924. [CrossRef] [PubMed] 166. Liang, C.L.; Chen, J.L.; Hsu, Y.P.; Ou, J.T.; Chang, Y.S. References Epstein-Barr virus BZLF1 gene is activated by transforming growth factor-beta through cooperativity of Smads and c-Jun/c-Fos proteins. J. Biol. Chem. 2002, 277, 23345–23357. [CrossRef] [PubMed] 167. Iempridee, T.; Das, S.; Xu, I.; Mertz, J.E. Transforming growth factor beta-induced reactivation of Epstein-Barr virus involves multiple Smad-binding elements cooperatively activating expression of the latent-lytic switch BZLF1 gene. J. Virol. 2011, 85, 7836–7848. [CrossRef] [PubMed] 168. Yates, J.L.; Warren, N.; Sugden, B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature 1985, 313, 812–815. [CrossRef] [PubMed] 169. Tsai, C.-N.; Liu, S.-T.; Chang, Y.-S. Identiﬁcation of a novel promoter located within the Bam HI Q region of the Epstein-Barr virus genome for the EBNA 1 gene. DNA Cell Biol. 1995, 14, 767–776. [CrossRef] [PubMed] 170. Chen, H.; Lee, J.M.; Wang, Y.; Huang, D.P.; Ambinder, R.F.; Hayward, S.D. The Epstein-Barr virus latency BamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss of BamHI-Q promoter activity. Proc. Natl. Acad. Sci. USA 1999, 96, 9339–9344. [CrossRef] [PubMed] 171. Tsang, C.M.; Zhang, G.; Seto, E.; Takada, K.; Deng, W.; Yip, Y.L.; Man, C.; Hau, P.M.; Chen, H.; Cao, Y.; et al. Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: Regulation of infection and phenotypic characterization. Int. J. Cancer 2010, 127, 1570–1583. [CrossRef] [PubMed] 172. Nanbo, A.; Ohashi, M.; Yoshiyama, H.; Ohba, Y. The Role of Transforming Growth Factor beta in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission. Front. Microbiol. 2018, 9, 984. [CrossRef] [PubMed] 172. Nanbo, A.; Ohashi, M.; Yoshiyama, H.; Ohba, Y. The Role of Transforming Growth Factor beta in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission. Front. Microbiol. 2018, 9, 984. [CrossRef] [PubMed] 173 Poh YW ; Gan S Y; Tan E L Effects of IL-6 IL-10 and TGF-beta on the expression of survivin and apoptosis 172. Nanbo, A.; Ohashi, M.; Yoshiyama, H.; Ohba, Y. The Role of Transforming Growth Factor beta in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission. Front. Microbiol. 2018, 9, 984. [CrossRef] [PubMed] 173. Poh, Y.W.; Gan, S.Y.; Tan, E.L. Effects of IL-6, IL-10 and TGF-beta on the expression of survivin and apoptosis h l ll l [ b d] 173. Poh, Y.W.; Gan, S.Y.; Tan, E.L. Effects of IL-6, IL-10 and TGF-beta on the expression of survivin and apoptos in nasopharyngeal carcinoma TW01 cells. Exp. Oncol. 2012, 34, 85–89. [PubMed] 174. Iglesias, M.; Frontelo, P.; Gamallo, C.; Quintanilla, M. References Blockade of Smad4 in transformed keratinocytes containing a Ras oncogene leads to hyperactivation of the Ras-dependent Erk signalling pathway associated with progression to undifferentiated carcinomas. Oncogene 2000, 19, 4134–4145. [CrossRef] [PubMed] 175. Huntley, S.P.; Davies, M.; Matthews, J.B.; Thomas, G.; Marshall, J.; Robinson, C.M.; Eveson, J.W.; Paterson, I.C.; Prime, S.S. Attenuated type II TGF-β receptor signalling in human malignant oral keratinocytes induces a less differentiated and more aggressive phenotype that is associated with metastatic dissemination. Int. J. Cancer 2004, 110, 170–176. [CrossRef] [PubMed] 176. Reusch, J.A.; Nawandar, D.M.; Wright, K.L.; Kenney, S.C.; Mertz, J.E. Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters. J. Virol. 2015, 89, 1731–1743. [CrossRef] [PubMed] 177. Rai, D.; Kim, S.W.; McKeller, M.R.; Dahia, P.L.; Aguiar, R.C. Targeting of SMAD5 links microRNA-155 to the TGF-beta pathway and lymphomagenesis. Proc. Natl. Acad. Sci. USA 2010, 107, 3111–31116. [CrossRef] [PubMed] 178. Jiang, D.; Aguiar, R.C. MicroRNA-155 controls RB phosphorylation in normal and malignant B lymphocytes via the noncanonical TGF-β1/SMAD5 signaling module. Blood 2014, 123, 86–93. [CrossRef] [PubMed] 23 of 23 Cancers 2018, 10, 247 179. Stelling, A.; Hashwah, H.; Bertram, K.; Manz, M.G.; Tzankov, A.; Muller, A. The tumor suppressive TGF-beta/SMAD1/S1PR2 signaling axis is recurrently inactivated in diffuse large B-cell lymphoma. Blood 2018, 131, 2235–2246. [CrossRef] [PubMed] 180. Lee, H.M.; Lo, K.W.; Wei, W.; Tsao, S.W.; Chung, G.T.Y.; Ibrahim, M.H.; Dawson, C.W.; Murray, P.G.; Paterson, I.C.; Yap, L.F. Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3. J. Pathol. 2017, 242, 62–72. [CrossRef] [PubMed] Lee, H.M.; Lo, K.W.; Wei, W.; Tsao, S.W.; Chung, G.T.Y.; Ibrahim, M.H.; Dawson, C.W.; Murray, P.G Paterson, I.C.; Yap, L.F. Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates Paterson, I.C.; Yap, L.F. Oncogenic S1P signalling in EBV associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3. J. Pathol. 2017, 242, 62–72. [CrossRef] [PubMed] AKT and promotes cell migration through S1P receptor 3. J. Pathol. 2017, 242, 62–72. [CrossRef] [PubMed] AKT and promotes cell migration through S1P receptor 3. J. Pathol. 2017, 242, 62–72. [CrossRef] [PubMed] 181. Mani, S.A.; Guo, W.; Liao, M.J.; Eaton, E.N.; Ayyanan, A.; Zhou, A.Y.; Brooks, M.; Reinhard, F.; Zhang, C.C.; Shipitsin, M.; et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008, 133, 704–715. [CrossRef] [PubMed] 182. References Shipitsin, M.; Campbell, L.L.; Argani, P.; Weremowicz, S.; Bloushtain-Qimron, N.; Yao, J.; Nikolskaya, T.; Serebryiskaya, T.; Beroukhim, R.; Hu, M.; et al. Molecular deﬁnition of breast tumor heterogeneity. Cancer Cell 2007, 11, 259–273. [CrossRef] [PubMed] 183. Anido, J.; Saez-Borderias, A.; Gonzalez-Junca, A.; Rodon, L.; Folch, G.; Carmona, M.A.; Prieto-Sanchez, R.M.; Barba, I.; Martinez-Saez, E.; Prudkin, L.; et al. TGF-beta Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma. Cancer Cell 2010, 18, 655–668. [CrossRef] [PubMed] 184. Kondo, S.; Wakisaka, N.; Muramatsu, M.; Zen, Y.; Endo, K.; Murono, S.; Sugimoto, H.; Yamaoka, S.; Pagano, J.S.; Yoshizaki, T. Epstein-Barr virus latent membrane protein 1 induces cancer stem/progenitor-like cells in nasopharyngeal epithelial cell lines. J. Virol. 2011, 85, 11255–11264. [CrossRef] [PubMed] 185. Kong, Q.L.; Hu, L.J.; Cao, J.Y.; Huang, Y.J.; Xu, L.H.; Liang, Y.; Xiong, D.; Guan, S.; Guo, B.H.; Mai, H.Q.; et al. Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma. PLoS Pathog. 2010, 6, e1000940. [CrossRef] [PubMed] 186. Akhurst, R.J.; Hata, A. Targeting the TGFβ signalling pathway in disease. Nat. Rev. Drug Discov. 2012, 11, 790–811. [CrossRef] [PubMed] 187. Foster, A.E.; Dotti, G.; Lu, A.; Khalil, M.; Brenner, M.K.; Heslop, H.E.; Rooney, C.M.; Bollard, C.M. Antitumor activity of EBV-speciﬁc T lymphocytes transduced with a dominant negative TGF-beta receptor. J. Immunother. 2008, 31, 500–505. [CrossRef] [PubMed] 188. Zhang, L.; MacIsaac, K.D.; Zhou, T.; Huang, P.Y.; Xin, C.; Dobson, J.R.; Yu, K.; Chiang, D.Y.; Fan, Y.; Pelletier, M.; et al. Genomic Analysis of Nasopharyngeal Carcinoma Reveals TME-Based Subtypes. Mol. Cancer Res. 2017, 15, 1722–1732. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2998927592	http://old.scielo.br/pdf/jbchs/v31n6/0103-5053-jbchs-31-06-1161.pdf	English	null	Matrix Effects on the Degradation of Gemfibrozil, Hydrochlorothiazide, and Naproxen by Heterogeneous Photocatalysis	Journal of the Brazilian Chemical Society	2,020	cc-by	6,606	aInstituto de Química, Universidade Federal de Uberlândia, 38400-902 Uberlândia-MG, Brazil bDepartamento de Ciências Naturais e Matemática, Universidade Save, 1200, Chongoene, Gaza, Mozambique The degradation of a pharmaceutical mixture (gemfibrozil, hydrochlorothiazide and naproxen) by TiO2/UV-A in different aqueous matrices was evaluated. The matrix components affected the operational conditions, rate constants, and removal efficiency of the pharmaceuticals, as well as toxicity. For sewage treatment plant effluent, a TiO2 concentration three-fold higher (450 mg L−1) and an irradiation time 1.5-3 times longer were required to reach the same efficiency of degradation as the pharmaceuticals in deionized water and surface water matrices. An improvement in the rate constants and the removal efficiency of the pharmaceuticals was achieved by adding 6 mg L−1 of H2O2 and 150 mg L−1 of TiO2 in deionized water and surface water, but not in the sewage treatment plant effluent, due to the coupling of 450 mg L−1 of TiO2 and 50-200 mg L−1 of H2O2. Overall, TiO2/UV-A efficiently degraded all compounds, but an additional step is needed for the removal of residual toxicity. Keywords: advanced oxidation process, contaminants of emerging concern, environmental aqueous matrices, operational conditions, titanium dioxide e-mail: alamtrovo@ufu.br http://dx.doi.org/10.21577/0103-5053.20200002 http://dx.doi.org/10.21577/0103-5053.20200002 Article J. Braz. Chem. Soc., Vol. 31, No. 6, 1161-1169, 2020 Printed in Brazil - ©2020 Sociedade Brasileira de Química Cleiseano E. S. Paniagua,a Eduardo O. Marson,a Ivo A. Ricardo,a,b Vinícius A. B. Paiva,a Bárbara R. Gonçalvesa and Alam G. Trovó ,a Cleiseano E. S. Paniagua,a Eduardo O. Marson,a Ivo A. Ricardo,a,b Vinícius A. B. Paiva,a Bárbara R. Gonçalvesa and Alam G. Trovó ,a Cleiseano E. S. Paniagua,a Eduardo O. Marson,a Ivo A. Ricardo,a,b Vinícius A. B. Paiva, Bárbara R. Gonçalvesa and Alam G. Trovó ,a Cleiseano E. S. Paniagua,a Eduardo O. Marson,a Ivo A. Ricardo,a,b Vinícius A. B. Paiva, Bárbara R. Gonçalvesa and Alam G. Trovó *,a Introduction A previous study of our research group8 showed that the degradation of these pharmaceuticals by UV-C and UV-C/H2O2 processes was strongly affected by the matrix composition. These results have motivated the evaluation of the degradation of these target compounds by heterogeneous photocatalysis, since this process has the advantage that sunlight can be used as a source of radiation, and the process has applications over a wide pH range.9 Pharmaceuticals are commonly detected and quantified in environmental aqueous matrices at trace levels (ng L−1 to μg L−1) due to their low removal efficiency in municipal wastewater treatment plants.1,2 Among them, gemfibrozil (GEM), hydrochlorothiazide (HCTZ) and naproxen (NAP) (Figure S1, Supplementary Information (SI) section) have been detected at levels of 8.91-15.12,3 33.13,4 and 0.0038‑0.0133 ng L−1,5 respectively. In addition, these compounds were detected in influents and sewage effluents at levels higher than 1000 ng L−1 in most samples of the five sewage treatment plants (STP) monitored in Spain over a period of two years.6 Thus, these compounds were chosen as representative compounds for different classes of contaminants of emerging concern (CEC) because of their frequent occurrence in different aqueous matrices and their environmental relevance. Although present at low concentrations, they cause deleterious effects to aquatic biota.7 Alvarez-Corena et al.10 evaluated the influence of pH (3-9) and TiO2 dosage (0.1-2.5 g L−1) by TiO2/UV-C during the simultaneous degradation of five contaminants including gemfibrozil in deionized water (DW) (initially with 2 mg L−1 for each compound). Optimal results were obtained with 1.5 g L−1 of the photocatalyst and at pH 5.0. Márquez et al.11 evaluated the coupling of O3 and TiO2 under solar radiation, aiming to degrade a mixture of pharmaceuticals (atenolol, hydrochlorothiazide, ofloxacin and trimethoprim; 10 mg L−1 of each compound) in DW, with a UV-A radiation dose of 0.94 kJ per mg pharmaceutical, obtaining 100% degradation efficiency for hydrochlorothiazide and ofloxacin and a maximum efficiency of 80% for the remaining pharmaceuticals. In 1162 Matrix Effects on the Degradation of Gemfibrozil, Hydrochlorothiazide, and Naproxen J. Braz. Chem. Soc. assessing the degradation of 60 and 440 mg L−1 of NAP in DW by TiO2 under UV-C and solar radiation using 0.4 g L−1 of the photocatalyst at a pH of 6.5, Jallouli et al.12 obtained higher NAP (98%) removals with the heterogeneous photocatalysis process compared to 83% removal efficiency obtained with photolysis. Sampling of the environmental aqueous matrices The sampling was carried out in municipal wastewater treatment plants and the main physical-chemical parameters of the samples were determined (Table S1, SI section). The collection took place in the autumn and spring of 2017. The sampling of SW was performed from a river used to supply water to the city of Uberlândia, Brazil (18°55’08”S, 48°16’37”W). The sampling of STP effluent was carried out after complete treatment in the municipal wastewater, which consists of steps of: (i) removal of coarse solids and sand; (ii) use of upflow anaerobic reactors; and (iii) use of a FlotFlux® channel based on sequential application of coagulation- flocculation-flotation. The SW and STP effluent samples were kept at 4 °C for two to three weeks while conducting the photodegradation experiments. As it can been observed, most of the scientific papers10-13 concerning degradation of these target compounds were performed in DW, and by using high concentrations of the target compounds (2-440 mg L−1), values well above those detected in real matrices were obtained. Since this technology is affected by the operational conditions (temperature, concentrations of the target compounds, TiO2 and H2O2, pH, inorganic anions, natural organic matter (NOM), etc.) and since, in the present work, low concentrations (413-536 µg L−1) of these pharmaceuticals were evaluated, the best experimental conditions were specific to each matrix and they had to be determined separately. In addition, monitoring the toxicity profile for each treated matrix is important once the mineralization monitoring is no longer feasible for real matrices. This happens because it is not possible to distinguish between the mineralization of the pharmaceuticals and the NOM. Besides, according to our research in literature, no comparative study of the simultaneous degradation of these target compounds at low concentrations has been performed at a natural pH value for each matrix, with the main operational conditions (concentration of TiO2 and H2O2) being evaluated and optimized, the kinetic parameters determined, and the toxicity monitored. Introduction constants and removal efficiency of the target compounds, and on toxicity, using the results in DW (a free matrix of interfering species) as basis for comparison. Chemicals In parallel, the influence of chloride and phosphate anions (isolated and mixture) was also evaluated on the removal of 30 mg L−1 of NAP using heterogeneous photocatalysis.13 The influence of the matrix components (DW and STP effluent) on the operational conditions of heterogeneous photocatalysis was also evaluated in an experiment involving the degradation of 0.05 g L−1 of metoprolol.14 A similar study15 was carried out with ibuprofen, with concentrations of 0.006 to 213 mg L−1 in DW, surface water (SW), and effluent from the pharmaceutical industry. Generally, it was found that (i) the relative natural abundance of optically active substances limits the penetration of light; (ii) the possible elimination of hydroxyl radicals and generation of less reactive radical species may be due to the chloride, sulfate and bicarbonate anions and, organic matter of the matrix; and (iii) inorganic ions and organic matter may also adsorb on the catalyst surface, inhibiting the production of HO•.13,16 The solutions were made in DW and with analytical- grade reagents. Standards of the target pharmaceuticals (99 wt.%) were acquired from Sigma-Aldrich (St. Louis, USA). High-performance liquid chromatography (HPLC)‑grade methanol was purchased from J.T. Baker (Xalostoc, Mexico). NH4VO3 from Vetec (Rio de Janeiro, Brazil) was used as received. The remaining reagents employed in this work, which were titanium dioxide P25 (Degussa/Evonick, Essen, Germany), C4K2O9Ti·2H2O, H2O2 (30 wt.%), Na2SO3, and 2-propanol, all from Synth (São Paulo, Brazil), were used without further purification. Photolytic and photocatalytic experiments The simultaneous degradation of the pharmaceuticals (451 µg L−1 of GEM, 536 µg L−1 of HCTZ and 413 µg L−1 of NAP) was performed by irradiating a 500-mL aqueous solution. An amber glass flask of 4.3 cm of depth and 15.5 cm of diameter was used as reactor (Figure S2, SI section). The experiments were performed under constant magnetic stirring at 25-28 °C (room temperature). The initial concentrations of the pharmaceuticals were settled to allow for monitoring their degradation by direct injection of the sample into the HPLC system, without the pre-concentration step, with efficiencies above 95%. Therefore, in order to clarify this issue, this work aimed to evaluate the impact of each matrix on the operational conditions of heterogeneous photocatalysis, on the rate Vol. 31, No. 6, 2020 Paniagua et al. 1163 of 150 μL of 2.0 g L−1 bovine catalase was added to the samples followed by stirring for 30 s. Two 10 W, low pressure, black-light lamps (λmax = 365 nm) (Figure S2, SI section) were placed side by side at a distance of 3.5 cm between them and at 1 cm of height from the top of the flask and used as a radiation source. A radiometer PMA 2100 Solar Light Co. equipped with a UV-A (320-400 nm) sensor was used to monitor de irradiance and an average of 32 W m−2 was obtained. Prior to analyses, the removal of the suspended TiO2 particles was carried out through filtration by membranes of 0.45 µm mean pore size. Adsorption-desorption equilibrium experiments Since in heterogeneous photocatalysis the degradation of the compounds occurs on the surface of the photocatalyst, the adsorption-desorption equilibrium has important consequences for the treatment.21 To determine the equilibrium time between the catalyst and the target compounds, an experiment was carried out in DW at pH of 6.0 (the natural pH of this matrix) using 80 mg L−1 of TiO2 (Figure S3, SI section). As it can be observed in the results presented in Figure S3, extending the time to 30 min, a maximum adsorption of 26-34% was obtained for all target compounds, followed by complete desorption after 120 min. Once degradation of the target compounds by heterogeneous photocatalysis occurs on the surface of the catalyst, either by a direct (holes) or indirect (hydroxyl radicals) mechanism,21,22 and based on the results of the Figure S3 after 120 min, it is expected to find no difference in degradation of the target compounds irradiating the solutions without and with equilibrium establishment. To confirm this hypothesis, two sets of experiments were carried out: one in which activation of the black-light lamp occurred after 120 min (Figure S4, SI section, open symbols) and the other in which the solution was irradiated just after the addition of TiO2 (Figure S4, solid symbols). No difference in the degradation of the pharmaceuticals was found. Thus, additional experiments were carried out by activating the black-light lamp soon after the addition of TiO2. Using the ideal conditions (150 mg L−1 of TiO2 and 6 mg L−1 of H2O2 for DW and SW and only 450 mg L−1 of TiO2 for STP1 effluent), the irradiation time was doubled from 120 to 240 min for the DW and SW, and from 240 to 360 min for the STP effluent while monitoring the evolution of toxicity as well as mineralization. Results and Discussion First, at an initial pH of 5.8-6.0 (the natural pH of these matrices), the influence of TiO2 concentration (50‑200 mg L−1) on the degradation of the target compounds in DW and SW was evaluated. The same study was performed with STP1 effluent at a pH of 7.7 (the natural pH for this matrix); however, higher concentrations of TiO2 (150-600 mg L−1) were used due to its higher complexity. Then, by using the optimal concentrations of TiO2 for each matrix (150 mg L−1 for DW and SW and 450 mg L−1 for STP1 effluent), the combination with H2O2 was studied. Different concentrations of this reagent (1-9 mg L−1 for DW and SW and, 50-200 mg L−1 for STP1 effluent) were evaluated taking into account the highest dissolved organic carbon (DOC) concentration (Table S1, SI section). The concentrations of H2O2 for each matrix were chosen based on a previous work of our group.8 Chemical analyses and toxicity bioassays The concentrations of the target compounds were determined following the methodology described by Paiva et al.17 H2O2 was spectrophotometrically quantified by the titanium oxalate method when H2O2 concentrations below 50 mg L−1 were used,18 and the metavanadate method was used when monitoring H2O2 concentrations that were greater than 50 mg L−1.19 Mineralization was monitored by carbon analyzer equipment (Shimadzu TOC VCPN model). In addition, to verify the main mechanism responsible (direct or indirect) for the degradation of pharmaceuticals during heterogeneous photocatalysis, experiments were performed using 80 mg L−1 of TiO2 at pH of 6.0, with the 2-propanol varied from 5 to 50 mmol L−1 (Figure S5, SI section). The addition of 2-propanol inhibited the degradation of the target compounds, but no complete inhibition was obtained by ranging the 2-propanol concentration from 5 to 50 mmol L−1 (Figure S5). In addition, a distinct profile during the degradation of HCTZ (Figure S5b, SI section) can be observed when compared to GEM and NAP (Figures S5a and S5c, SI section). High inhibition of the degradation of HCTZ occurred using Acute toxicity toward V. fischeri bacteria followed the Brazilian norm.20 The bioluminescence emitted by the V. fischeri bacteria to the non-treated and treated solutions obtained during the heterogeneous photocatalysis process (after adjustment of the salinity to 2%) was monitored. The values obtained were compared with a solution of 2% NaCl, used as a control. A solution of 13.4 mg L−1 of Cr6+ was employed as a positive toxicant control. A thermoblock was used to maintain the temperature at 15 °C. In order to remove the residual H2O2 present in the samples, a volume Matrix Effects on the Degradation of Gemfibrozil, Hydrochlorothiazide, and Naproxen 1164 J. Braz. Chem. Soc. 25 mmol L−1 of 2-propanol (Figure S5b), compared to the inhibition of GEM and NAP in absence of 2-propanol (Figures S5a and S5c). These results indicate that major degradation of HCTZ was through an indirect mechanism via the HO•, since 2-propanol is a good trapping agent for HO• (k = 1.9 × 109 L mol−1 s−1).23,24 In contrast, the majority degradation of GEM and NAP primarily occurred via direct mechanism by holes (h+), since a lower inhibition of the degradation of GEM and NAP (Figures S5a and S5c) occurred using 25 mmol L−1 of 2-propanol in comparison to HCTZ (Figure S5b). the pharmaceuticals (Figure 1). Chemical analyses and toxicity bioassays The kinetic parameters (Table S2, SI section) were determined by plotting the results of (C is the target compound concentration at a determined time X, C0 is the initial target compound concentration) versus time, which is characteristic of a pseudo-first-order kinetics model. An improvement in the degradation rates of the pharmaceuticals was obtained by increasing the TiO2 dosage (compared to experiments in which no TiO2 was used) (Figure 1). A quantitative comparison of the results in terms of the kinetic parameters (rate constant, k; and half‑life, t1/2) was also made (Table S2, SI section). For DW and SW matrices, an increase in k and, consequently, a decrease in the t1/2 was observed using 150 mg L−1 TiO2 (Table S2 and Figure 1). In addition, a negative effect occurred when using 200 mg L−1 of TiO2 due to a raise in the turbidity and aggregation of the catalyst particles, which limits light penetration and reduces the surface area of the active sites, respectively.25,27 Similar behavior was observed Influence of TiO2 concentration The determination of the optimal TiO2 concentration is crucial to ensure the maximum degradation of the pharmaceuticals involved in this study.25,26 Therefore, different dosages of TiO2 were tested in the aqueous matrices, aiming to verify how the matrix components affect this operational parameter in the degradation of Figure 1. Influence of the TiO2 concentration on the degradation of (a-c) gemfibrozil (GEM); (d-f) hydrochlorothiazide (HCTZ); and (g-i) naproxen (NAP) in different aqueous matrices. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, pH = 5.8-6.0 in DW and SW, and 7.7 in STP1 effluent. Figure 1. Influence of the TiO2 concentration on the degradation of (a-c) gemfibrozil (GEM); (d-f) hydrochlorothiazide (HCTZ); and (g-i) naproxen (NAP) in different aqueous matrices. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, pH = 5.8-6.0 in DW and SW, and 7.7 in STP1 effluent. Paniagua et al. Vol. 31, No. 6, 2020 1165 for the STP effluent matrix; there was an improvement in the value of k up to 450 mg L−1 of TiO2, followed by a negative effect when using 600 mg L−1 of TiO2 (Table S2 and Figure 1). degradation efficiency of the pharmaceuticals when compared to the DW and SW, which is a consequence of the high concentration of organic matter and inorganic ions (Table S1). The same profile was obtained during the degradation of ibuprofen; 1000 mg L−1 of TiO2 was necessary with DW and 2500 mg L−1 of TiO2 was required with the effluent from the pharmaceutical industry.15 In addition, the composition of the aqueous matrices significantly affected the k values; the higher their complexity (STP effluent > SW > DW), the lower the k values and the higher the t1/2 of the target compounds (Table S2, SI section). Although the same TiO2 concentration (150 mg L−1) resulted in the best results for k and t1/2 for the DW and SW, respectively, lower values of k and higher values of t1/2 were obtained in SW when compared to DW (Table S2). Influence of TiO2 concentration This is justified by the matrix components (Table S1, SI section), which was 7.3 mg C L−1 of dissolved inorganic carbon (DIC), equivalent to 37 mg L−1 HCO3 −, 1.4 mg L−1 Cl−, 0.8 mg L−1 SO4 2−, 0.2 mg L−1 NH4 + and less than 0.05 mg L−1 HPO4 2−, and by the NOM (6.7 mg C L−1 of DOC). The NOM can absorb the target compounds as they compete for TiO2 adsorption sites. This is in accordance with a previous research28 that studied the influencing power of inorganic salts on the photocatalytic efficiency of TiO2; the amount of methylene blue (84.2 µmol L−1) adsorbed on the TiO2 surface (0.5 g L−1) at pH of 6-7 decreased in the following sequence: HCO3 − > HPO4 2− > SO4 2− > Cl− > NO3 −. At the present working pH of 5.8 (the natural pH of the SW matrix), the semiconductor surface is positive, with strong competition expected between the target compounds, with neutral or negative charges (Figure S6, SI section), and the major inorganic anions (HCO3 −, SO4 2−, and Cl−; Table S1, SI section), justifying the reduction in k values (Table S2, SI section). In addition, there is the strong contribution of 6.7 mg L−1 of DOC (Table S1), which mostly inhibits the photocatalytic removal of oestrogenic activity in secondary effluent when compared to inorganic ions.29 In parallel, these inorganic ions and DOC are also capable of competing with the target compounds through hydroxyl radicals (equations 1-5),30,31 decreasing their availability and, consequently, affecting the degradation efficiency or reacting with the holes (equation 6), generating Cl• radicals, which are then converted into Cl2 •−radicals (equation 7).16,29,32 Influence of H2O2 concentration Influence of H2O2 concentration The coupling of H2O2 and TiO2 can improve the degradation of the target compounds since this minimizes recombination of the electron/hole pair and raises the production of HO• (equation 8):33 H2O2 + eBC − → HO• + OH− (8) (8) The action of the hydrogen peroxide dosage on the degradation efficiency of the target compounds for the different aqueous matrices (Figure 2) was evaluated, and the kinetic parameters were determined (Table S3, SI section). The efficiency of the degradation of the pharmaceuticals increased by adding 6 mg L−1 of H2O2 in the DW and SW matrices (Figure 2), contributing to the high k and low t1/2 values, respectively (Table S2, SI section). Using 9 mg L−1 of H2O2, there was a drop in k values and an increase in t1/2 values (Table S3, SI section). This is attributed to the excess H2O2 (Figures S7a and S7b, SI section), which can adsorb onto the surface of the TiO2, causing changes to its surface and its catalytic activity,34 and it can compete with the target compounds for hydroxyl radicals (equation 9) or reacting with the holes (equation 10): H2O2 + HO• → H2O + HO2 • k = 2.7 × 107 L mol−1 s−1 (9) H2O2 + 2hBV + → O2 + 2H+ (10) 1 (9) (10) ( ) (10) No synergistic effect on the degradation of the pharmaceuticals in the STP effluent was obtained when compared to the results in DW and SW matrices by adding H2O2 in the range evaluated (50-200 mg L−1) (Figure 2 and Table S3, SI section). Thus, the consumption of H2O2 (Figure S7c, SI section) can be attributed to the inefficient parallel reactions presented by equations 1-5, radical recombination (equations 11-15), or reactions between H2O2 and radical species, such as HO•, HO2 • (equations 9 and 16), SO4 •− (equation 17), or radicals derived from chlorine (equations 18 and 19), which generate less reactive hydroperoxyl radicals (HO2 •).35 Thus, in the STP effluent matrix, the addition of H2O2 is unnecessary. Influence of H2O2 concentration HO2 • + HO• → H2O + O2 k = 7.1 × 109 L mol−1 s−1 (11) HO2 • + HO2 • → H2O2 + O2 k = 8.3 × 105 L mol−1 s−1 (12) O2 •− + HO• → HO− + O2 k = 1.01 × 1010 L mol−1 s−1 (13) HO2 • + O2 •− → HO2 − + O2 k = 9.7 × 107 L mol−1 s−1 (14) HO• + HO• → H2O2 k = 5.2 × 109 L mol−1 s−1 (15) HO2 • + H2O2 → HO• + H2O + O2 k = 0.5 L mol−1 s−1 (16) SO4 •− + H2O2 → SO4 2− + HO2 • + H+ k = 1.2 × 107 L mol−1 s−1 (17) Cl• + H2O2 → Cl− + HO2 • + H+ k = 1.0 × 109 L mol−1 s−1 (18) Cl2 •− + H2O2 → 2Cl− + HO2 • + H+ k = 4.1 × 104 L mol−1 s−1 (19) HO2 • + HO• → H2O + O2 k = 7.1 × 109 L mol−1 s−1 (11) HO2 • + HO2 • → H2O2 + O2 k = 8.3 × 105 L mol−1 s−1 (12) O2 •− + HO• → HO− + O2 k = 1.01 × 1010 L mol−1 s−1 (13) HO2 • + O2 •− → HO2 − + O2 k = 9.7 × 107 L mol−1 s−1 (14) HO• + HO• → H2O2 k = 5.2 × 109 L mol−1 s−1 (15) HO2 • + H2O2 → HO• + H2O + O2 k = 0.5 L mol−1 s−1 (16) SO4 •− + H2O2 → SO4 2− + HO2 • + H+ k = 1.2 × 107 L mol−1 s−1 (17) Cl• + H2O2 → Cl− + HO2 • + H+ k = 1.0 × 109 L mol−1 s−1 (18) Cl2 •− + H2O2 → 2Cl− + HO2 • + H+ k = 4.1 × 104 L mol−1 s−1 (19) conditions and considering the limit of detection (LOD) of the equipment (24.8, 3.3, and 0.76 µg L−1 for GEM, HCTZ, and NAP, respectively), degradation efficiencies of 95.0, 99.3 and 99.8% for GEM, HCTZ and NAP, respectively, were obtained in all matrices, with the exception of GEM in the STP effluent (Figure 3). Control experiments (Figures S8 and S9, SI section) using the optimal experimental conditions for each matrix were performed. Influence of H2O2 concentration Cl− + HO• → ClHO•− k = 4.3 × 109 L mol−1 s−1 (1) ClHO•− → Cl− + HO• k = 6.0 × 109 s−1 (2) ClHO•− + H+ → Cl• + H2O k = 2.4 × 1010 L mol−1 s−1 (3) HCO3 − + HO• → H2O + CO3 •− k = 8.5 × 106 L mol−1 s−1 (4) SO4 2− + HO• → OH− + SO4 •− k = 3.5 × 105 L mol−1 s−1 (5) h+ BV + Cl− → Cl• (6) Cl− + Cl• → Cl2 •− k = 8.5 × 109 L mol−1 s−1 (7) Cl− + HO• → ClHO•− k = 4.3 × 109 L mol−1 s−1 (1) ClHO•− → Cl− + HO• k = 6.0 × 109 s−1 (2) ClHO•− + H+ → Cl• + H2O k = 2.4 × 1010 L mol−1 s−1 (3) HCO3 − + HO• → H2O + CO3 •− k = 8.5 × 106 L mol−1 s−1 (4) SO4 2− + HO• → OH− + SO4 •− k = 3.5 × 105 L mol−1 s−1 (5) h+ BV + Cl− → Cl• (6) Cl− + Cl• → Cl2 •− k = 8.5 × 109 L mol−1 s−1 (7) For the STP matrix, a TiO2 concentration three times higher (450 mg L−1) was required to achieve the same Matrix Effects on the Degradation of Gemfibrozil, Hydrochlorothiazide, and Naproxen 1166 J. Braz. Chem. Soc. Figure 2. Influence of the H2O2 dosage on the degradation efficiency of (a-c) gemfibrozil (GEM); (d-f) hydrochlorothiazide (HCTZ); and (g-i) naproxen (NAP) in different aqueous matrices during heterogeneous photocatalysis. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, [TiO2] = 150 mg L−1 at pH = 5.8-6.0 (natural pH of the DW and SW solutions); [TiO2] = 450 mg L−1 at pH = 7.7 (natural pH of the STP1 effluent). Figure 2. Influence of the H2O2 dosage on the degradation efficiency of (a-c) gemfibrozil (GEM); (d-f) hydrochlorothiazide (HCTZ); and (g-i) naproxen (NAP) in different aqueous matrices during heterogeneous photocatalysis. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, [TiO2] = 150 mg L−1 at pH = 5.8-6.0 (natural pH of the DW and SW solutions); [TiO2] = 450 mg L−1 at pH = 7.7 (natural pH of the STP1 effluent). Influence of H2O2 concentration No significant adsorption (Figure S8) and/or degradation through the oxidizing action of H2O2 (Figure S9, SI section, open symbols) was obtained, demonstrating that the degradation of the pharmaceuticals occurs by photocatalytic treatment. On the other hand, a significant contribution of H2O2/UV-A was obtained (Figure S9, solid symbols). However, comparing these results with the TiO2/UV-A results (Figures 1a-1f), an improvement in the degradation of the pharmaceuticals was obtained by the coupling of TiO2, H2O2, and UV-A (Figures 2a-2f). Furthermore, using the optimal experimental conditions for the different aqueous matrices, new experiments were carried out by extending the irradiation time to 240 min for the DW and SW and 360 min for the STP effluent, aiming for higher degradation efficiencies (Figure 3). Under these 1167 Vol. 31, No. 6, 2020 Paniagua et al. Figure 3. Degradation of the target compounds: (a) gemfibrozil (GEM); (b) hydrochlorothiazide (HCTZ); and (c) naproxen (NAP) by heterogeneous photocatalysis using the best experimental conditions. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, [TiO2] = 150 mg L−1, and [H2O2] = 6.0 mg L−1 in DW and SW at an initial pH of 5.8-6.0; [TiO2] = 450 mg L−1 at an initial pH of 7.4 in the STP2 effluent. Figure 3. Degradation of the target compounds: (a) gemfibrozil (GEM); (b) hydrochlorothiazide (HCTZ); and (c) naproxen (NAP) by heterogeneous photocatalysis using the best experimental conditions. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, [TiO2] = 150 mg L−1, and [H2O2] = 6.0 mg L−1 in DW and SW at an initial pH of 5.8-6.0; [TiO2] = 450 mg L−1 at an initial pH of 7.4 in the STP2 effluent. It is important to emphasize that a new sample of STP effluent was used, named STP2 (Table S1, SI section). For this new sample, lower concentrations for most of the analyzed parameters were obtained (Table S1) since the sample was collected during a rainy period. Influence of H2O2 concentration For better comparison with the previous results (Table S3, SI section), using the results present in Figure 3, and considering a pseudo-first-order kinetics model, the values of k and t1/2 were calculated, with the following results: k = 0.065 min−1 for GEM (t1/2 = 1.0 min), 0.012 min−1 for HCTZ (t1/2 = 1.0 min), and 0.020 min−1 for NAP (t1/2 = 0.9 min). A difference in the k values of only 10‑13% was obtained for GEM and NAP, compared to a difference of 39% for HCTZ (Table S3). Similar behavior was obtained by Soriano-Molina et al.,36 using the photo- Fenton process as an alternative to degrade CEC in effluents of five municipal wastewater treatment plants. These same researchers noticed that the nature of the NOM strongly affected the degradation, but not the origin or composition of the effluents. In that work,36 the interference of HCO3 − was eliminated by adding H2SO4 prior to experimentation. solutions toward the bacteria V. fischeri (Figure 4b) were evaluated. Complete mineralization was not obtained for any matrix (Figure 4a). According to the results in the DW matrix (free of NOM), by-products were formed with the degradation of the pharmaceuticals since their concentrations were below the LOD of the equipment (Figure 3), and 65% mineralization was obtained (Figure 4a). Although similar behavior in terms of degradation (Figure 3) and mineralization (Figure 4a) was obtained in the SW and STP effluent matrices, a high residual organic load was present in the SW and STP effluent as a consequence of the greater initial DOC values in these matrices (6.7 mg L−1 in the SW and 25 mg L−1 in the STP2 effluent). Thus, the toxicity of the treated solution was also evaluated to analyze the behavior of this technology as another method for the treatment of wastewater containing the studied compounds (Figure 4b). No inhibition in bioluminescence towards V. fischeri was obtained for the initial concentration of the pharmaceuticals in the DW (Figure 4b). This corroborated the results obtained for the initial solutions in SW and STP effluent in the absence and presence of the pharmaceuticals (Figure 4b) In addition, the mineralization (Figure 4a) and the changes in the acute toxicity of the non-treated and treated Figure 4. (a) Mineralization and (b) bioluminescence inhibition of the bacteria V. References 1. Richardon, S. D.; Kimura, S. Y.; Environ. Technol. Innovation 2017, 8, 40. 1. Richardon, S. D.; Kimura, S. Y.; Environ. Technol. Innovation 2017, 8, 40. Conclusions 2. K’oreje, K. O.; Okoth, M.; Langenhove, H. V.; Demeestere, K.; J. Environ. Manage. 2020, 254, 109752. This work demonstrated that the matrix components strongly affects the operational parameters evaluated, which demonstrates the need to evaluate and determine the best operating conditions for each aqueous matrix so as to obtain the maximum degradation efficiency of the target compounds. A TiO2 concentration that was three times higher and treatment time that was 1.5-3 times longer were required to obtain the same degradation efficiency in the matrix of higher complexity (STP effluent) when compared to the DW and SW. Overall, heterogeneous photocatalysis efficiently degraded the target compounds GEM, HCTZ, and NAP in the matrices studied, being an option for the degradation of this type of pollutant. Besides affecting the operational conditions, rate constants, and removal efficiency, the components of the matrix also affected the toxicity of the solutions after the photocatalytic treatment, requiring a complementary step to remove the residual toxicity. 3. Fang, T.-H.; Lin, C.-W.; Kao, C.-H.; Mar. Pollut. Bull. 2019, 146, 509. 4. Liu, M.; Yin, H.; Wu, Q.; Ecotoxicol. Environ. Saf. 2019, 183, 109497. 5. Brumovský, M.; Bečanová, J.; Kohoutek, J.; Borghini, M.; Nizzetto, L.; Environ. Pollut. 2017, 229, 976. 6. Bueno, M. J. M.; Gomez, M. J.; Herrera, G. S.; Hernando, M. D.; Aguera, A.; Fernández-Alba, A. R.; Environ. Pollut. 2012, 164, 267. 7. Grenni, P.; Patrolecco, L.; Ademollo, N.; Di Lenola, M.; Caracciolo, A. B.; Microchem. J. 2018, 136, 49. Caracciolo, A. B.; Microchem. J. 2018, 136, 49. 8. Paniagua, C. E. S.; Ricardo, I. A.; Marson, E. O.; Gonçalves, B. R.; Trovó, A. G.; J. Environ. Chem. Eng. 2019, 7, 103164. 9. Cabrera-Reina, A.; Martínez-Piernas, A. B.; Bertakis, Y.; Nikolaos, P.; Xekoukoulotakis, N. P.; Agüera, A.; Pérez, J. A. S.; Water Res. 2019, 166, 115037. S.; Water Res. 2019, 166, 115037. 10. Alvarez-Corena, J. R.; Bergendahl, J. A.; Hart, F. L.; J. Environ. Manage. 2016, 181, 544. Author Contributions Cleiseano E. S. Paniagua, Eduardo O. Marson, Ivo A. Ricardo, Vinícius A. B. Paiva and Bárbara R. Gonçalves were responsible for conceptualization, formal analysis, data curation, methodology, investigation, software, resources, validation, visualization and writing original draft; Alam G. Trovó for the conceptualization, formal analysis, data curation, methodology, funding acquisition, visualization, project administration, writing original draft, review and editing. Acknowledgments since no difference in toxicity was observed. In addition, the higher initial value of inhibition (50%) obtained in the STP2 effluent was a consequence of the natural components present in this matrix (Figure 4b). The authors thank FAPEMIG (PPM-00509-18), CNPq (Projects 405043/2018-0 and 305215/2018-3), and Laboratory Multiuser of the Institute of Chemistry (Federal University of Uberlândia) for use of the HPLC- DAD equipment. By applying heterogeneous photocatalysis to the DW, an increase in the bioluminescence inhibition from 0 to 28% was obtained in the DW after 240 min (Figure 4b), indicating that by-products of higher toxicity were generated. Similar behavior was observed in SW and STP effluent matrices (Figure 4b). However, for the real matrices under study, the majority toxicity was due to the by-products formed from the NOM (blank experiments) present in these matrices (Figure 4b) since similar degrees of mineralization were obtained (Figure 4a). Similar behavior was obtained by Michael et al.,37 during the degradation of a mixture of antibiotics in urban wastewater by the photo-Fenton process under sunlight radiation as a consequence of the oxidation of the dissolved organic matter.37 In parallel, these same authors37 applied an additional step with granular activated carbon, and almost complete removal of toxicity was obtained. Thus, this procedure can be applied as an alternative method in the removal of the residual toxicity obtained in our work. Influence of H2O2 concentration fischeri obtained during the degradation of the different environmental aqueous matrices in the absence (open symbols) and presence (solid symbols) of the pharmaceuticals. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, [TiO2] = 150 mg L−1, and [H2O2] = 6.0 mg L−1 in the DW and SW at an initial pH of 5.8‑6.0; [TiO2] = 450 mg L−1 at an initial pH of 7.4 in the STP2 effluent. [DOC] = 0.77, 6.7, and 25 mg L−1 in DW, SW, and STP2, respectively. Figure 4. (a) Mineralization and (b) bioluminescence inhibition of the bacteria V. fischeri obtained during the degradation of the different environmental aqueous matrices in the absence (open symbols) and presence (solid symbols) of the pharmaceuticals. The initial conditions were the following: [GEM] = 451 µg L−1, [HCTZ] = 536 µg L−1, [NAP] = 413 µg L−1, [TiO2] = 150 mg L−1, and [H2O2] = 6.0 mg L−1 in the DW and SW at an initial pH of 5.8‑6.0; [TiO2] = 450 mg L−1 at an initial pH of 7.4 in the STP2 effluent. [DOC] = 0.77, 6.7, and 25 mg L−1 in DW, SW, and STP2, respectively. 1168 Matrix Effects on the Degradation of Gemfibrozil, Hydrochlorothiazide, and Naproxen J. Braz. Chem. Soc. Supplementary Information 11. Márquez, G.; Rodríguez, E. M.; Maldonado, M. I.; Álvarez, P. M.; Sep. Purif. Technol. 2014, 136, 18. 11. Márquez, G.; Rodríguez, E. M.; Maldonado, M. I.; Álvarez, P. Supplementary information (chemical structures of the target compounds, control experiments, influence of TiO2 and H2O2 concentration, and H2O2 consumption) is available free of charge at http://jbcs.sbq.org.br as PDF file. M.; Sep. Purif. Technol. 2014, 136, 18. 12. Jallouli, N.; Elghniji, K.; Hentati, O.; Ribeiro, A. R.; Silva, A. M.; Ksibi, A. M.; J. Hazard. Mater. 2016, 304, 329. 13. Kanakaraju, D.; Motti, C. A.; Glass, B. D.; Oelgemoller, M.; Chemosphere 2015, 139, 579. Vol. 31, No. 6, 2020 1169 Paniagua et al. 14. Cavalcante, R. P.; Dantas, R. F.; Wender, H.; Bayarri, B.; González, O.; Giménez, J.; Esplugas, S.; Júnior, A. M.; Appl. Catal., B 2015, 176-177, 173. 26. Abdel-Wahab, A.-M.; Al-Shirbin, A.-S.; Mohamed, O.; Nasr, O.; J. Photochem. Photobiol., A 2017, 347, 186. 27. Roushenas, P.; Ong, Z. C.; Ismail, Z.; Majidnia, Z.; Ang, B. C.; Asadsangabifard, M.; Onn, C. C.; Tam, J. H.; Desalin. Water Treat. 2018, 120, 109. 15. Jallouli, N.; Pastrana-Martínez, L. M.; Ribeiro, A. R.; Moreira, N. F. F.; Faria, J. L.; Adrián, O. H.; Silva, A. M. T.; Ksibi, M.; Chem. Eng. J. 2018, 334, 976. 28. Guillard, C.; Puzenat, E.; Lachheb, H.; Houas, A.; Herrmann, J.-M.; Int. J. Photoenergy 2005, 7, DOI 10.1155/ S1110662X05000012. 16. Ismail, L.; Ferronato, C.; Fine, L.; Jaber, F.; Chovelon, J.-M.; Environ. Sci. Pollut. Res. 2018, 25, 2651. 17. Paiva, V. A. B.; Paniagua, C. E. S.; Ricardo, I. A.; Gonçalves, B. R.; Martins, S. P.; Daniel, D.; Trovó, A. G.; J. Environ. Chem. Eng. 2018, 6, 1086. 29. Zhang, W.; Li, Y.; Su, Y.; Mao, K.; Wang, Q.; J. Hazard. Mater. 2012, 215-216, 252. 30. Dimitroula, H.; Daskalaki, V. M.; Frontistis, Z.; Kondarides, D. I.; Panagiotopoulou, P.; Xekoukoulotakis, N. P.; Mantzavinos, D.; Appl. Catal., B 2012, 117-118, 283. 18. http://www.h2o2.com/technical-library/analytical- methods/default.aspx?pid=71&name=Titanium-Oxalate- Spectrophotometric, accessed in December 2019. D.; Appl. Catal., B 2012, 117-118, 283. 31. Plantard, G.; Azais, A.; Mendret, J.; Brosillon, S.; Goetz, V.; Chem. Eng. Process. 2018, 134, 115. 19. Nogueira, R. F. P.; Oliveira, M. C.; Paterlini, W. C.; Talanta 2005, 66, 86. 32. Hassan, M.; Zhao, Y. X.; Xie, B.; Chem. Eng. J. 2016, 285, 264. 20. This is an open-access article distributed under the terms of the Creative Commons Attribution License. Supplementary Information ABNT NBR 15411-3: Aquatic Ecotoxicology - Determination of Inhibitory Effect of Aqueous Samples on the Bioluminescence of Vibrio fischeri (Luminescent Bacteria Essay). Part 3: Method Using Freeze-Dried Bacteria; ABNT: Rio de Janeiro, 2012. 33. Reza, K. M.; Kurny, A. S. W.; Gulshan, F.; Appl. Water Sci. 2017, 7, 1569. 34. Nosaka, Y.; Nosaka, A. Y.; Chem. Rev. 2017, 117, 11302. 35. Marson, E. O.; Paiva, V. A. B.; Gonçalves, B. R.; Gomes Jr., O.; Borges Neto, W.; Machado, A. E. H.; Trovó, A. G.; Environ. Sci. Pollut. Res. 2017, 24, 6176. 21. Hu, X.; Hu, X.; Peng, Q.; Zhou, L.; Tan, X.; Jiang, L.; Tang, C.; Wang, H.; Liu, S.; Wang, Y.; Ning, Z.; Chem. Eng. J. 2020, 380, 122366. 36. Soriano-Molina, P.; Plaza-Bolaños, P.; Lorenzo, A.; Agüera, A.; García-Sánchez, J. L.; Malato, S.; Sánchez-Pérez, J. A.; Chem. Eng. J. 2019, 366, 141. 22. Legrini, O.; Oliveros, E.; Braun, A. M.; Chem. Rev. 1993, 93, 671. 23. Buxton, G. F.; Greenstock, C. L.; Helman, W. P.; Ross, A. B.; J. Phys. Chem. Ref. Data 1988, 17, 513. 37. Michael, S. G.; Michael-Kordatou, I.; Beretsou, V. G.; Jäger, T.; Michael, C.; Schwartz, T.; Fatta-Kassinos, D.; Appl. Catal., B 2019, 244, 871. 24. Huang, W.; Luo, M.; Wei, C.; Wang, Y.; Hanna, K.; Mailhot, G.; Environ. Sci. Pollut. Res. 2017, 24, 10421. G.; Environ. Sci. Pollut. Res. 2017, 24, 10421. Submitted: October 21, 2019 Published online: January 9, 2020 Submitted: October 21, 2019 Published online: January 9, 2020 25. Gomes Jr., O.; Borges Neto, W.; Machado, A. E. H.; Daniel, D.; Trovó, A. G.; Water Res. 2017, 110, 133. Published online: January 9, 2020 This is an open-access article distributed under the terms of the Creative Commons Attribution Licen
https://openalex.org/W3048028213	https://www.frontiersin.org/articles/10.3389/fimmu.2020.01965/pdf	English	null	Use of Cell and Genome Modification Technologies to Generate Improved “Off-the-Shelf” CAR T and CAR NK Cells	Frontiers in immunology	2,020	cc-by	13,999	Edited by: John Maher, King’s College London, United Kingdom Reviewed by: Marcus Konrad Odendahl, German Red Cross Blood Donor Services, Germany Robin Parihar, Baylor College of Medicine, United States Reviewed by: Marcus Konrad Odendahl, German Red Cross Blood Donor Services, Germany Robin Parihar, Baylor College of Medicine, United States Correspondence: Michael A. Morgan morgan.michael@mh-hannover.de Axel Schambach schambach.axel@mh-hannover.de; Axel.Schambach@ childrens.harvard.edu Correspondence: Michael A. Morgan morgan.michael@mh-hannover.de Axel Schambach schambach.axel@mh-hannover.de; Axel.Schambach@ childrens.harvard.edu Specialty section: This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology Keywords: chimeric antigen receptor, T cell, immunotherapy, genome editing, CRISPR-Cas9 Received: 30 April 2020 Accepted: 21 July 2020 Published: 07 August 2020 Use of Cell and Genome Modiﬁcation Technologies to Generate Improved “Off-the-Shelf” CAR T and CAR NK Cells Michael A. Morgan1,2, Hildegard Büning1,2, Martin Sauer3 and Axel Schambach1,2,4 1 Institute of Experimental Hematology, Hannover Medical School, Hanover, Germany, 2 REBIRTH Research Center for Translational Regenerative Medicine, Hannover Medical School, Hanover, Germany, 3 Department of Pediatric Hematology, Oncology, and Blood Stem Cell Transplantation, Hannover Medical School, Hanover, Germany, 4 Division of Hematology/Oncology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, United States The broad success of adoptive immunotherapy to treat human cancer has resulted in a paradigm shift in modern medicine. Modiﬁcation of autologous and allogenic immune cells with chimeric antigen receptors (CAR) designed to target speciﬁc antigens on tumor cells has led to production of CAR T and CAR NK cell therapies, which are ever more commonly introduced into cancer patient treatment protocols. While allogenic T cells may offer advantages such as improved anti-tumor activity, they also carry the risk of adverse reactions like graft-versus-host disease. This risk can be mitigated by use of autologous immune cells, however, the time needed for T and/or NK cell isolation, modiﬁcation and expansion may be too long for some patients. Thus, there is an urgent need for strategies to robustly produce “off-the-shelf” CAR T and CAR NK cells, which could be used as a bridging therapy between cancer diagnosis or relapse and allogeneic transplantation. Advances in genome modiﬁcation technologies have accelerated the generation of designer cell therapy products, including development of “off-the-shelf” CAR T cells for cancer immunotherapy. The feasibility and safety of such approaches is currently tested in clinical trials. This review will describe cell sources for CAR-based therapies, provide background of current genome editing techniques and the applicability of these approaches for generation of universal “off-the-shelf” CAR T and NK cell therapeutics. REVIEW published: 07 August 2020 doi: 10.3389/ﬁmmu.2020.01965 REVIEW published: 07 August 2020 doi: 10.3389/ﬁmmu.2020.01965 published: 07 August 2020 doi: 10.3389/ﬁmmu.2020.01965 Keywords: chimeric antigen receptor, T cell, immunotherapy, genome editing, CRISPR-Cas9 Citation: The severity of on-target-oﬀ-tumor activity may be modulated by the dose of CAR T cells applied, as another study that tested HER2-CAR T cells in sarcoma patients showed this to be safe if administered up to 1 × 108 CAR T/m2 compared to 1010 (or 6.25 × 1010 based on average female body surface area of 1.6 m2) CAR T cells in the former study (16). Disease relapse due to lack of CAR T cell persistence has also been reported. Loss of anti-CD19 CAR T cells was found to result from CD8+ immunity against the CAR T cells in some patients, which may have been due to the use of a murine scFv in the clinical CAR construct (17). To decrease potential immunogenic eﬀects of CAR scFv sequences derived from mouse monoclonal antibodies, and, thus improve CAR T cell persistence, these should be humanized (18) (NCT02659943). To increase safety of CAR T cell therapies, vectors designed to deliver the CAR can be engineered to co-express suicide genes to allow removal of CAR T cells in case of uncontrollable severe adverse events. Examples of clinically available suicide gene strategies include the HSV-tk suicide gene (19), which makes the cells sensitive to ganciclovir- induced cytotoxicity or the inducible caspase 9 (iCasp9) gene cassette, which leads to rapid caspase-mediated apoptosis of expressing cells (e.g., CAR T cells) upon application of a synthetic inducer of dimerization, such as AP1903 or AP20187 (20, 21). T and NK cells engineered to express CARs still eliminate target cells via the same cytotoxic mechanisms as unmodiﬁed T and NK cells, i.e., by release of perforins and granzymes, the cytotoxic T cells transferred in the graft also provided improved anti-cancer (leukemia) activity (1). These seminal discoveries led to investigation of cellular therapies in clinical modalities, including infusion of tumor inﬁltrating lymphocytes (TIL) for greater disease control [reviewed in (2)]. The cytotoxic activities of immune cells, like T and NK cells, can be exploited to generate more eﬀective anti-cancer cell therapies. In the case of T cells, the T cell receptor (TCR) is activated upon recognition of and binding to “foreign” peptides presented by the major histocompatibility complex (MHC) class I on antigen presenting cells. A cascade of signaling events then ensues, including co-receptor binding that leads to activation of the SRC tyrosine kinase LCK, which phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3ζ complex. Citation: ZAP-70 is recruited to the phosphorylated CD3ζ and orchestrates downstream signaling events that lead to NFAT and AP-1 activation, resulting in T cell expansion, cytokine production (e.g., IL2, IFNγ) and stimulation of cytotoxic activity (3, 4). To avoid detection and subsequent elimination by T cells, transformed cells often exhibit repressed levels of MHC expression (5, 6). In contrast, NK cells become activated depending on the balance of activating and inhibitory signals that are generated by NK cell receptors during surveillance of cells that they contact (7). As loss of MHC on a tumor cell results in decreased inhibitory signaling in the NK cell, cancer cells must use other mechanisms to inhibit the cytotoxic function of NK cells. Alternative tumor immune escape mechanisms include upregulation of HLA-E on the tumor cell surface and release of soluble NKG2D ligands, such as MICA and MICB (8–10). The idea of combining the anti-cancer activity of immune cells, such as T and NK cells, with the concept of antibody- speciﬁcity to redirect the cytotoxic activity of these cells to target tumor cells that express a particular antigen led to the development of chimeric antigen receptor (CAR) T and NK cells (11). Speciﬁcally, CARs are synthetic receptors that contain an extracellular antibody-like region designed to target a speciﬁc antigen called the single chain variable fragment (scFv), a hinge region that can be of diﬀerent lengths, the choice of which may be guided by the proximity of the recognized epitope to the target cell surface, a transmembrane domain, one or more co-stimulatory domains and a signaling domain to induce cytotoxicity upon antigen binding (Figure 1). The choice of the co-stimulatory and signaling domains have been largely based upon components of the T cell receptor (TCR), i.e., containing CD28 and/or 4-1BB costimulatory domains and a CD3ζ signaling domain. Clinically approved second generation CARs contain a CD3ζ signaling domain in combination with either a CD28 (Yescarta R⃝) or 4-1BB (Kymriah R⃝) co-stimulatory domain. While the CD28-CD3ζ-containing CAR T cells were shown to exhibit more rapid and stronger signaling and to favor development of eﬀector cell phenotypes, 4-1BB-CD3ζ-containing CAR T cells had a memory cell phenotype with greater persistence (12). Citation: The clinical usefulness of cellular therapies was increasingly demonstrated through decades of successful hematopoietic stem cell transplantations (HSCT) in both autologous and allogeneic settings. In the case of allogeneic HSCT, some patients develop a complication called graft- versus-host disease (GVHD) due to cytotoxic alloreactivity of donor T cells that were transferred from the donor graft and which destroy tissues in the recipient. GVHD occurs due to immuno- incompatibility, e.g., human leukocyte antigen (HLA) mismatches between the donor and recipient. Although GVHD can result in increased transplant-related mortality, it was observed that Morgan MA, Büning H, Sauer M and Schambach A (2020) Use of Cell and Genome Modiﬁcation Technologies to Generate Improved “Off-the-Shelf” CAR T and CAR NK Cells. Front. Immunol. 11:1965. doi: 10.3389/ﬁmmu.2020.01965 August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 1 Off-the-Shelf CAR T Cells Morgan et al. in B cell non-Hodgkin’s lymphoma (B-NHL) patients (13). The eﬃcacy of CAR T cells, and recently CAR NK cells, has been shown for liquid tumors, most prominently in CD19+ lymphoid- derived cancers and several clinical studies currently explore the translation of these promising results to solid tumors (link to these studies on clinicaltrials.gov). However, there are important clinical challenges that must be addressed to further improve CAR T cell approaches. For example, one major adverse event that commonly occurs during CAR T cell therapy is cytokine release syndrome (CRS), in which greatly elevated levels of inﬂammatory cytokines such as interleukin (IL)-6 are observed. Severity of CRS was correlated with patient IL-6 levels and the anti-IL-6 receptor antibody tocilizumab can be used to reverse CRS symptoms without interfering with CAR T cell anti-tumor activity (14). CRS was more severe in B-NHL patients treated with CD28-CD3ζ CAR T cells as compared to 4-1BB-CD3ζ CAR T cells, possibly due to the high immune response induced by CD28 stimulation (13). As it is not always possible to identify a neoantigen that is only expressed on the tumor cell to be targeted by a CAR-modiﬁed cell, healthy cells may also be eliminated by on-target-oﬀ-tumor activity. While this may be clinically manageable in some cases, e.g., loss of healthy B cells with CARs directed against CD19, the adverse events due to on-target-oﬀ- tumor activity may be more severe with other targets, such as unwanted destruction of lung tissue after administration of anti- ERBB2(HER2)-CAR T cells designed to treat metastatic ERBB2+ cancer (15). Frontiers in Immunology \| www.frontiersin.org Citation: are introduced into regulatory T cells (Tregs), which have anti- inﬂammatory activities [(24) and reviewed in (25)]. receptor that fused NKG2D to CD3ζ (NKG2D.ζ) eliminated myeloid-derived suppressor cells (MDSC) and the anti-cancer activity of these modiﬁed NK cells was not suppressed by the tumor microenvironment (TME) (31). Of note, CAR T cells that were administered following NKG2D.ζ-NK cells had improved tumor inﬁltration and anti-cancer activity. While most studies to date collect and modify autologous T cells to produce CAR T cell therapies, use of allogeneic CAR NK cells derived from primary NK cells (e.g., cord or peripheral blood) or from NK cell lines derived from lymphoma patients (e.g., NK-92) is increasing and other “oﬀ-the-shelf” cell sources are also being tested. Development of cell-based immunotherapeutic treatment strategies is, at least partially, directed by the characteristics of the disease to be treated and the available technology or feasibility to generate the necessary technologies. In the case of generating new CAR therapeutics to treat cancer, one of the key decisions to be made is which tumor-associated antigens to target with the scFv design. This will largely determine the speciﬁcity of tumor targeting and the extent of on-target, but also oﬀ-tumor side eﬀects. Another important consideration is the design of the remaining domains of the CAR, for example which transmembrane, co-stimulatory and signaling domains should be incorporated. This decision may also be inﬂuenced by the cell type (e.g., T cell, NK cell, other immune cells) to be used as the “living” drug as well as the temporal window in which these cell therapies should be active. Interestingly, CAR designs based on the T cell receptor also function in NK cells (26– 28). However, this does not rule out the possibility to engineer immune cell type-speciﬁc CARs for optimal use in the chosen cell type (Figure 1). For example, modiﬁcation of NK cells with a chimeric receptor consisting of the NK cell activating receptor NKG2D, DNAX-activation protein 10 (DAP10) and CD3ζ led to increased cytotoxic activity against cancer cell lines and improved activity in an osteosarcoma mouse model (29, 30). Citation: CAR NK cells designed to target the prostate stem cell antigen (PSCA) on prostate cancer cells were modiﬁed with a CAR vector in which the CD28 transmembrane and costimulatory domains as well as the CD3ζ signaling domain were exchanged for DNAX-activation protein 12 (DAP12) transmembrane and intracellular signaling domains, which resulted in speciﬁc cytotoxicity against PSCA- positive tumor cell lines as compared to PSCA-negative tumor cells in vitro and in vivo. NK cells modiﬁed with a chimeric In the following sections, important concepts of how to generate “oﬀ-the-shelf” CAR cell therapies, such as the source of immune cells to be modiﬁed, strategies to overcome tumor immune escape mechanisms and genome engineering approaches that can be applied to improve CAR T and CAR NK cell function will be considered. Citation: Direct comparison of CD28 and 4-1BB co-stimulatory domains in anti-CD19-CARs showed that 4-1BB contributes to greater CAR T cell persistence and a more favorable toxicity proﬁle T and NK cells engineered to express CARs still eliminate target cells via the same cytotoxic mechanisms as unmodiﬁed T and NK cells, i.e., by release of perforins and granzymes, as well as death receptor interactions (22, 23). However, the cytotoxic activity is speciﬁcally ampliﬁed through binding of the scFv to the respective tumor-associated antigen. In addition, the concept of CAR T cell therapy is also applicable to other disease indications, incl. autoimmune diseases, in which CARs August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 2 Off-the-Shelf CAR T Cells Morgan et al. FIGURE 1 \| Modiﬁcation of T or NK cells with CAR-encoding retro- and lentiviral vectors. On the left, a lentiviral vector is shown that transfers the genetic cargo into the T or NK cells leading to the expression of a chimeric antigen receptor (CAR) on the cell surface. On the right, the structure of a 3rd generation CAR is depicted with single chain variable fragment (scFv, including VH and VL chains), hinge, transmembrane and signaling domains shown. CARs can be engineered with cell-type speciﬁc modules to enhance CAR T or CAR NK cell activity. Examples of cytoplasmic signaling domains that can be engineered into CARs for T and NK cells are shown. Combining such strategies with additional genome modiﬁcation approaches described in later sections of this review will lead to improved “off-the-shelf” cell products. FIGURE 1 \| Modiﬁcation of T or NK cells with CAR-encoding retro- and lentiviral vectors. On the left, a lentiviral vector is shown that transfers the genetic cargo into the T or NK cells leading to the expression of a chimeric antigen receptor (CAR) on the cell surface. On the right, the structure of a 3rd generation CAR is depicted with single chain variable fragment (scFv, including VH and VL chains), hinge, transmembrane and signaling domains shown. CARs can be engineered with cell-type speciﬁc modules to enhance CAR T or CAR NK cell activity. Examples of cytoplasmic signaling domains that can be engineered into CARs for T and NK cells are shown. Combining such strategies with additional genome modiﬁcation approaches described in later sections of this review will lead to improved “off-the-shelf” cell products. Frontiers in Immunology \| www.frontiersin.org T CELL SOURCES: AUTOLOGOUS, ALLOGENEIC, INDUCED PLURIPOTENT STEM CELL-DERIVED AND EXPANDED PROGENITOR-DERIVED The most common source of CAR T cells currently applied clinically is patient-derived autologous T cells, which are then genetically modiﬁed to express the CAR of choice, expanded and re-infused into the patient. Lentiviral or gammaretroviral vectors are often used in clinical trials to deliver the CAR into the T cell genome (Figure 1) (32, 33), but also non-viral integrating technologies such as Sleeping Beauty transposons were shown to eﬃciently generate CAR T cells (34). While use August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 3 Off-the-Shelf CAR T Cells Morgan et al. of autologous cells is enticing as this avoids challenges with immuno-incompatibilities, such as complications like GVHD, there are also disadvantages with autologous cell sources. For example, the immune cell populations may be adversely aﬀected in heavily pre-treated patients so that the quality and number of cells for ex vivo modiﬁcation and expansion may be suboptimal. Additionally, patients who have infections or rapidly advancing cancers might not survive the several weeks needed to produce autologous CAR T cells, as the cells have to be collected by apheresis, shipped to the facility site for genetic modiﬁcation, expansion and formulation, before being shipped back to the hospital where the patient will be infused with the CAR T cells. Advantages of allogeneic CAR T cells include a lower risk of genetically modifying and re-infusing leukemic cells (35), and allogeneic cells can be prepared and stored for future use so that there is a shorter waiting period for infusion into the patient. Thus, “oﬀ-the-shelf” allogeneic cell sources could provide greater ﬂexibility for treatment protocols, potentially lower overall costs if multiple patients can be treated from a single CAR T cell product and could be expected to allow broader access to these clinical procedures (36). cells/kg) peripheral blood stem cells from matched sibling donors (1 of 5) or matched unrelated donors (4 of 4) (44). The authors propose that allogeneic NK-DLI may have contributed to the observed GVHD by aggravating an existing subclinical T cell- mediated GVHD. This is supported by the assessment of donor chimerism based upon CD3, which showed signiﬁcantly higher donor chimerism in GVHD patients, and that allogeneic NK-DLI was accomplished shortly prior to detection of the high donor chimerism in three of the ﬁve patients who developed GVHD. T CELL SOURCES: AUTOLOGOUS, ALLOGENEIC, INDUCED PLURIPOTENT STEM CELL-DERIVED AND EXPANDED PROGENITOR-DERIVED The relative risk of GVHD following NK cell application will become clearer as more data accumulates with CAR NK cells, which are increasingly incorporated into clinical trials (Table 1). which are increasingly incorporated into clinical trials (Table 1). Diﬀerent NK sources have been used to generate pre-clinically and clinically tested CAR NK cells, including cell lines such as NK-92 cells (45), cord blood-derived NK cells (43, 46) and peripheral blood-derived NK cells (28). Of note, a recent landmark phase 1 and 2 study showed the feasibility of cord- blood-derived CAR NK cells to treat relapsed or refractory CD19+ B-cell cancers (43). Eight of eleven (73%) patients responded rapidly (within 30 days after CAR NK cell infusion), including seven complete remissions. Of particular interest, the only major adverse events were related to the lymphodepletion strategy (i.e., neutropenia, lymphopenia) and no cytokine release syndrome, neurologic events or GVHD were observed, even with 2-5 HLA allelic mismatches (43). CAR NK cells persisted for at least 12 months after infusion, which may have been at least partially due to inclusion of an IL-15 expression cassette in the CAR construct, a cytokine known to enhance NK cell survival and proliferation (43). The same group previously showed that one cord-blood unit could be used to produce over 100 CAR NK doses, further highlighting allogeneic CAR NK cells as potential “oﬀ-the-shelf” drugs (47). While regulatory guidelines may vary depending on the country in which the study is performed, cell therapeutics should be viable (e.g., ≥70%) and demonstrated to be negative for endotoxin, mycoplasma or bacterial contaminations. For CAR NK cells, the cell product should contain mostly CD56+ cells (≥90%), and be free of CD3+ cells (e.g., ≤0.2%) and CD14+ cells (e.g., ≤5%). In the case that CAR NK cells are expanded via co-culture with irradiated feeder cells, for example, membrane bound IL-15 and 4-1BB ligand expressing K562 cells or membrane bound IL-21 expressing OCI-AML3 cells, the ﬁnal CAR NK cell product should be demonstrated to be free from contamination of co-cultured cells (e.g., ≤1%) (48, 49). Contamination of primary NK cell therapeutics with feeder cells may be mitigated by alternative expansion methods, such as use of coated beads or cytokine combinations to expand NK cells. Primary NK cells can be activated and expanded with cytokines such as IL-2, IL-12, IL-15, IL18, and IL-21 (50–53). NK CELL SOURCES An alternative approach is to exploit the natural cytotoxic activity of NK cells to generate allogeneic “oﬀ-the-shelf” CAR NK cells to target cancer cells. One advantage of NK cells is that they were shown to not induce GVHD even in mismatched settings (43). However, an earlier study observed acute GVHD in ﬁve of nine patients who received donor-derived allogeneic NK-donor lymphocyte infusions (NK-DLI) after HLA-matched transplantation of T cell-depleted (for delivery of ≤2 × 104 T T CELL SOURCES: AUTOLOGOUS, ALLOGENEIC, INDUCED PLURIPOTENT STEM CELL-DERIVED AND EXPANDED PROGENITOR-DERIVED Similarly to expansion of primary T cells with CD3/CD28 beads, primary NK cells can also be expanded with CD335 (NKp46)/CD2 beads. Therefore, methods for eﬃcient and reliable production of “oﬀ-the-shelf” T cells remain highly sought goals in the ﬁeld of cellular immunotherapy. Important conditions that these cells must meet include avoidance of rejection due to recognition by host T cells via HLA class I molecules or host NK cells by HLA class II receptors. “Oﬀ-the-shelf” immune cells should also lack alloreactivity to limit unwanted toxicities due to recognition and destruction of healthy host tissues. Several strategies to avoid host cell recognition have been explored, such as knock-down or knock-out of MHC molecules to block recognition by host T cells. However, this can result in elimination of the modiﬁed cells due to NK cell activity against cells lacking MHC expression (37– 39). Expression of ligands that inhibit NK cell cytotoxicity, like HLA E or HLA G, can ameliorate elimination of the engineered cells, but since receptors for these ligands are not expressed on all NK cells, veritable “oﬀ-the-shelf” cell therapeutics will likely require genetic engineering strategies that address multiple layers of immune cell recognition patterns and cytotoxic mechanisms (40–42). Importantly, these modiﬁcations would ideally not negatively impact immune cell expansion that is necessary for clinical application or cell persistence and function, which are important for immune cell anti-cancer eﬃcacy. Additionally, potential risks of genetically modiﬁed immune cells must be evaluated, although such risks are generally low in diﬀerentiated somatic cells like T cells. Frontiers in Immunology \| www.frontiersin.org iPSC AND OTHER CELL SOURCES Additional cell sources to produce “oﬀ-the-shelf” CAR cells include stem cell and progenitor cell populations such as induced pluripotent stem cells (iPSC) and precursor T cells. Frontiers in Immunology \| www.frontiersin.org August 2020 \| Volume 11 \| Article 1965 4 Off-the-Shelf CAR T Cells Morgan et al. TABLE 1 \| Selected clinical trials testing potential “off-the-shelf” CAR cell therapies. iPSC AND OTHER CELL SOURCES Cells CAR Diseases/Patients 1◦/2◦outcomes References No genome modiﬁcation Allogeneic T cells CD19-CAR Relapsed or refractory CD19+ B cell malignancies 1◦: DLT, CR 2◦: ORR, DOR, safety, tolerability, TRM NCT04384393 ThisCART19 Allogeneic T cells CD19-CAR Elderly relapsed or refractory B-ALL 1◦: occurrence of adverse events 2◦: overall response rate, DFS, OS NCT02799550 Allogeneic T cells alloCART-19 Pediatric relapsed or refractory ALL 1◦: DLT 2◦: AE, ORR, BOR NCT04173988 Allogeneic T cells NKG2D-based CAR-T plus inhibitory peptide T cell receptor (TCR) inhibiting molecule (TIM) to reduce signaling of the TCR complex through a non-gene edited approach Metastatic colorectal cancer (mCRC) 1◦: DLT, ORR 2◦: AE, safety, ORR, BOR, kinetics, clinical activity, PFS, EFS, OS NCT03692429 alloSHRINK trial Genome modiﬁed Allogeneic T cells (TCRα/β disruption) Anti-CS1 CAR (UCARTCS1A) Relapsed or refractory MM 1◦: safety NCT04142619 MELANI-01 Allogeneic T cells (TCRα/β disruption) Anti-CD123 (UCART123) Relapsed or refractory AML 1◦: safety, tolerability NCT03190278 AMELI-01 Allogeneic T cells (TCRα/β disruption) CD19-UCART Relapsed or refractory B cell malignancies 1◦: DLT 2◦: ORR, CART persistence NCT03229876 Allogeneic T cells (TCRα/β disruption) BCMA-UCART Relapsed or refractory MM 1◦: ORR 2◦: safety, tolerability, CART persistence NCT03752541 Allogeneic T cells (TCRα/β disruption) CD22-CAR (UCART22) Relapsed or refractory CD22+ B-cell B-ALL 1◦: safety, tolerability NCT04150497 BALLI-01 Allogeneic T cells (TCRα/β disruption) CD19-UCART Relapsed or refractory B-ALL 1◦: DLT 2◦: safety, tolerability, objective remission rate and duration, PFS, OS NCT02746952 CALM Allogeneic T cells (TCRα/β and B2M disruption) UCART019 Relapsed or refractory CD19+ leukemia and lymphoma 1◦: safety, feasibility, persistence 2◦: tumor response, test for humoral immunity against murine CD19 scFv NCT03166878 Allogeneic T cells (TCRα/β and B2M disruption) CTX110 (CD19-CAR) Relapsed or refractory B cell malignancies 1◦: DLT, ORR 2◦: DOR, PFS, OS NCT04035434 Allogeneic T cells (TCRα/β and B2M disruption) CTX120 (BCMA-CAR) Relapsed or refractory MM 1◦: AE, DLT, ORR 2◦: PFS, OS NCT04244656 Allogeneic T cells (TCRα/β and B2M disruption) CTX130 (CD70-CAR) Relapsed or refractory renal cell carcinoma 1◦: AE, DLT, ORR 2◦: PFS, OS NCT04438083 Allogeneic T cells (TCRα/β and B2M disruption) Universal Dual CD19 + CD20-CAR or CD19 + CD22-CAR Relapsed or refractory B-cell malignancies 1◦: safety, feasibility, persistence 2◦: anti-tumor response, test for humoral immunity against murine CD19 scFv NCT03398967 Donor T cells (CMV- or EBV-speciﬁc T cells derived from donor CD62L + TCM cells) CD19-CAR B cell malignancies after allogeneic transplant 1◦: safety, feasibility 2◦: persistence, trafﬁcking to bone marrow, function, CMV/EBV reactivation, elimination of CD19+ tumor NCT01475058 TABLE 1 \| Selected clinical trials testing potential “off-the-shelf” CAR cell therapies. iPSC AND OTHER CELL SOURCES cells demonstrated superior anti-tumor activity when directly compared to T-CAR-iPSC-NK cells in an ovarian cancer xenograft model and had similar activity as observed for CAR T cells that expressed a typical CAR designed for T cells (CD28- CD3ζ) (56). Advantages of iPSC-derived CAR T/CAR NK cells include their enormous proliferative and expansion capacities as well as the relative ease of genomic modiﬁcation, which provides the possibility to create cell banks with diﬀerent CAR constructs as standardized “oﬀ-the-shelf” immunotherapies. iPSC possess a nearly unlimited proliferative potential and can be diﬀerentiated into various cell types, including T and NK cells. Thus, iPSC oﬀer a renewable source of potentially standardized cells for immunotherapies and can be easily genetically modiﬁed to generate immune cells with improved characteristics (54). The feasibility of producing CAR T cells from iPSC was demonstrated by transduction of peripheral blood lymphocyte-derived iPSC with a lentiviral vector encoding for a second-generation anti-CD19-CAR (55). After hematopoietic speciﬁcation and expansion, the authors used a T-lymphoid commitment co-culture protocol to generate anti-CD19-CAR-T- iPSC-T. The authors directly compared the iPSC-derived CAR T cells with TCR-αβ and TCR-γδ peripheral blood lymphocytes from the same donor and transduced with the same CAR and demonstrated that the iPSC-derived CAR T cells showed a similar anti-cancer activity as the CAR TCR-γδ cells in an immunodeﬁcient mouse xenograft tumor model using the CD19+ Raji human Burkitt lymphoma cell line (55). p A recently described inducible transcription factor-mediated forward programming approach to eﬃciently produce large numbers of hemato-endothelial progenitor cells and hematopoietic progenitor cells may also become useful for generating “oﬀ-the-shelf” cell therapies, such as CAR NK cells (57). While this strategy led to sustained production of myeloid lineages, diﬀerentiation into the lymphoid lineages was less robust. However, RNAseq interrogation of gene expression patterns revealed several transcription factor targets whose expression could potentially be modulated to overcome this. Similarly, methods to produce conditionally immortalized murine lymphoid progenitors might be exploited to eﬃciently generate CAR T cells, although this remains to be tested and applied to human lymphoid progenitors (58, 59). Expression of an anti-CD19-CAR in lymphoid progenitors was shown to suppress T cell development with the generation of cells with NK cell-like characteristics that had strong cytotoxic activity Adaptation of CAR designs to exploit the signal transduction pathways naturally used for cell activity may lead to improved CAR NK or other CAR-cell type activities. iPSC AND OTHER CELL SOURCES Allogeneic T cells (TCRα/β and B2M disruption) UCART019 Allogeneic T cells (TCRα/β and B2M disruption) CTX110 (CD19-CAR) Allogeneic T cells (TCRα/β and B2M disruption) CTX120 (BCMA-CAR) Allogeneic T cells (TCRα/β and B2M disruption) CTX130 (CD70-CAR) Allogeneic T cells (TCRα/β and B2M disruption) Universal Dual CD19 + CD20-CAR or CD19 + CD22-CAR August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 5 Off-the-Shelf CAR T Cells Morgan et al. TABLE 1 \| Continued Cells CAR Diseases/Patients 1◦/2◦outcomes References proliferation, long-term status of treated patients Allogeneic EBV speciﬁc T cells Anti-CD30 CAR Relapsed or refractory CD30+ lymphoma 1◦: DLT 2◦: ORR, DOR, SD, PFS NCT04288726 Other “off-the-shelf” CAR cells NK-92 cell line CD33-CAR (CD28-CD137 (4-1BB)-CD3ζ) Relapsed or refractory CD33+ AML 1◦: safety, feasibility 2◦: anti-leukemia response, in vitro anti-AML cytotoxicity, test for development of humoral immunity against the murine anti-CD33 scFv NCT02944162 NK-92 cell line CD7-CAR (CD28-4-1BB-CD3ζ) CD7 + leukemia and lymphoma 1◦: AE, toxicity proﬁle 2◦: clinical response, persistence NCT02742727 NK-92 cell line CD19-CAR CD19+ leukemia and lymphoma 1◦: AE 2◦: ORR NCT02892695 Allogeneic NKT cells CD19-CAR + IL-15 Relapsed or refractory B cell malignancies 1◦: DLT 2◦: persistence of modiﬁed cells, overall response NCT03774654 ANCHOR Haploidentical/Allogeneic Gamma Delta (γδ) T cells NKG2DL-targeting CAR Relapsed or refractory solid tumors 1◦: DLT 2◦: AE, efﬁcacy, PFS, DOR NCT04107142 Allogeneic Gamma Delta (γδ) T cells CD19-CAR High risk, relapsed CD19+ B cell malignancies 1◦: safety (adverse events) 2◦: CAR γδ persistence, antitumor activity, MTD NCT02656147 AE, adverse events; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; BOR, best overall response; CR, complete remission; DFS, disease-free survival; DLT, dose limiting toxicities; DOR, duration of response; EBV, Epstein Barr virus; MM, multiple myeloma; ORR, objective response rate; PFS, progression-free survival; OS, overall survival; scFv, single chain variable fragment; SD, stable disease; TRM, treatment related mortality. AE, adverse events; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; BOR, best overall response; CR, complete remission; DFS, disease-free survival; DLT, dose limiting toxicities; DOR, duration of response; EBV, Epstein Barr virus; MM, multiple myeloma; ORR, objective response rate; PFS, progression-free survival; OS, overall survival; scFv, single chain variable fragment; SD, stable disease; TRM, treatment related mortality. Frontiers in Immunology \| www.frontiersin.org TALEN “Off-the-Shelf”Cell Products TALEN Off the Shelf Cell Products Universal CAR19T (UCART19) cells were generated using TALENs to target the constant region of the TCRα chain (TRAC) and the CD52 gene to make UCART19 cells resistant to Alemtuzumab (Campath R⃝), which is an antibody used to eliminate CD52+ lymphocytes in B-cell chronic lymphocytic leukemia (66). UCART cells led to rapid molecular remissions (28 days) in two infants with refractory high-risk B-ALL. One patient had grade 2 skin GVHD and the second patient had a possible mild skin GVHD that was quickly resolved with topical steroids (66). TALENs were also used to disrupt the TCRαβ locus to generate universal allogeneic CAR T cells directed against the tumor-associated antigen CS1 (UCARTCS1A), which are currently tested in relapsed and refractory multiple myeloma patients (NCT04142619) (Table 1). A similar approach was used to generate universally applicable anti-CD22 CAR T cells (UCART22) to treat patients with relapsed and refractory CD22+ B-cell B-ALL (NCT04150497). ZFN “Off-the-Shelf" Cell Products ZFN “Off-the-Shelf" Cell Products In order to produce “oﬀ-the-shelf” allogeneic CAR T cell products, it is necessary to disrupt adverse reactions such as GVHD due to endogenous TCR activation that occurs with HLA-mismatched donors and recipients. Possible gene editing strategies to circumvent this include elimination of endogenous TCR expression to generate universal donor T cells (66) and targeted insertion of CAR into the TCR alpha constant (TRAC) locus (67). Eﬃcient genome editing of CD8 and CD4 T cells by HDR was accomplished using Adeno-associated virus (AAV) serotype 6 vectors (AAV6) to deliver the homologous donor template and electroporation of ZFN mRNA (68). Thus, much progress has been made in identiﬁcation of alternative “oﬀ-the-shelf” therapeutic CAR cells. As any manipulation of the genome, such as insertion of therapeutic CAR vectors (Figure 1), carries an inherent risk, these must be carefully evaluated. While possible genotoxic risks such as transformation of a healthy cell to a cancer cell are low in terminally diﬀerentiated somatic cells like T and NK cells, such modiﬁcations in stem cells (e.g., HSC, iPSC) or progenitor populations that can be diﬀerentiated into T or NK cells may carry higher risks, which should be assessed and mitigated as necessary. There are also diﬀerences in regulatory requirements for clinical use of primary lymphocytes and cell lines. For example, cell lines must be irradiated to minimize the risk of secondary lymphoma (e.g., due to uncontrolled proliferation of immortalized cell lines), and it has to be documented that culture conditions did not include animal based supplements (e.g., fetal bovine serum) or antibiotics (e.g., penicillin, streptomycin). Electrotransfer was also used to deliver designer ZFN to delete TCR α or β chains in CD19-CAR T cells, and the TCR−CAR+ population maintained CD19 speciﬁcity without responding to TCR stimulation (69). GENOME MODIFICATION TECHNIQUES AND APPLICATION TO CAR T CELLS In addition to gene transfer technologies to improve CAR T cell function, genomic modiﬁcation strategies have been used to advance “oﬀ-the-shelf” cell therapeutics. Zinc-ﬁnger nucleases (ZFN), transcription activator-like nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 (CRISPR-associated protein 9) systems are currently the most commonly employed genome editing technologies. ZFN and TALEN technologies target speciﬁc genomic loci via protein-DNA interactions, and require protein engineering expertise that is not available in every laboratory. In contrast, the CRISPR-Cas9 system uses RNA-guided DNA recognition to deﬁne the genomic modiﬁcation locus, which makes tailored design of CRISPR-Cas9 to target speciﬁc genes relatively easy and has led to wide-spread use of this technique in the scientiﬁc community. Genome editing occurs after double strand break (DSB) induction through two main DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), with NHEJ active throughout all phases of the cell cycle and the less eﬃcient HDR mainly conﬁned to the S phase. The eﬃciency of targeted insertion by HDR can be improved by TP53 inactivation to block the TP53 damage response and interruption of the cell-cycle induced by DNA double-stranded breaks caused by CRISPR-Cas9 (62–64). Interestingly, NHEJ was further improved by a new method called CRISPR-HOT, which stands for CRISPR–Cas9-mediated homology-independent organoid transgenesis (65). This method CRISPR-Cas9 “Off-the-Shelf”Cell Products and Developing Technologies Frontiers in Immunology \| www.frontiersin.org iPSC AND OTHER CELL SOURCES For example, an “NK-CAR” engineered to contain the NKG2D transmembrane domain, the 2B4 co-stimulatory domain and the CD3ζ signaling domain was used to modify iPSC cells, which were subsequently diﬀerentiated into NK-CAR-iPSC-NK cells (iPSC-derived NK cells equipped with an NK-CAR). The NK-CAR-iPSC-NK August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 6 Off-the-Shelf CAR T Cells Morgan et al. against CD19+ leukemia cells across MHC barriers and without causing GVHD. Importantly, this shift in diﬀerentiation was dependent upon ongoing signaling activity of the respective CAR early during hematopoietic development (60). Most recently, CAR-macrophages (CAR-Ms) were shown to phagocytose tumor cells in an antigen-speciﬁc manner, decrease tumor burden in two solid tumor xenograft mouse models and to promote anti-cancer T cell activity by inducing a pro-inﬂammatory tumor microenvironment (61). enables the eﬃcient generation of knock-in human organoids in diﬀerent tissues and achieves precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53. CRISPR-Cas9 “Off-the-Shelf”Cell Products and Developing Technologies p g g CRISPR-Cas9 RNPs and AAV6 were used to speciﬁcally deliver an engineered 2.3-kb-long TCR construct TCR25D6, which recognizes a peptide derived from myeloperoxidase as a tumor- associated antigen in myeloid neoplasia patients when presented on HLA-B7, into the TRAC locus (70). CRISPR-Cas9-mediated knockout of TCRα/β and B2M in combination with lentiviral delivery of an anti-CD19 CAR into allogeneic T cells resulted in universal CAR T cells (UCART019) that are clinically tested in relapsed or refractory CD19+ leukemia and lymphoma patients (NCT03166878). CRISPR-Cas9-mediated TCRα/β and B2M knockout to generate “oﬀ-the-shelf” allogeneic CAR T cells is also evaluated in other clinical trials for CD19+ leukemia and lymphoma patients (NCT04035434), multiple myeloma August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 7 Off-the-Shelf CAR T Cells Morgan et al. patients (NCT04244656) and renal cell carcinoma patients (NCT04438083) (Table 1). coupled with the possibility to target speciﬁc cell populations (76). High-ﬁdelity CRISPR-Cas9 nuclease variants designed to have fewer interactions with non-speciﬁc DNA sequences, but maintaining on-target DNA activity were also developed (77). As mentioned above, novel Cas9 fusion proteins were engineered to create base editors, i.e., cytosine base editors (CBE) and adenosine base editors (ABE), capable of editing single bases (78, 79). CBE were generated by fusing a cytidine deaminase to a catalytically impaired Cas9 protein (i.e., that is unable to induce double- strand DNA breaks) and uracil glycosylase inhibitor. Since Cas9- independent oﬀ-target DNA editing was observed with CBEs largely due to cytidine deaminase activity, additional Cas9 fusion variants were generated and shown to have up to 100-fold less Cas9-independent oﬀ-target DNA editing, but retained 50–90% of on-target DNA editing (80). Approaches like these will make it easier to safely modify allogeneic T cells into universal CAR T cells via disruption of TCRα/β and B2M without the need to introduce double strand DNA breaks. Multiplex CRISPR-Cas9 allows simultaneous editing of several genomic loci. The feasibility and safety of using multiplex CRISPR-Cas9 to engineer autologous T cells with enhanced anticancer activity was recently demonstrated in a phase I trial (NCT03399448) (71). CRISPR guide RNA was electroporated into T cells to delete endogenous TCRα and TCRβ chains as well as the PDCD1 gene that encodes the programmed cell death protein 1 (PD-1). Endogenous TCR disruption was done to allow enhanced expression of the cancer-speciﬁc TCR NY-ESO- 1, which was introduced by lentiviral transduction. Technology Speciﬁcity and Safety In addition to enhancing immune cell recognition of tumor cells via CAR expression, additional modiﬁcations of CAR cells may be necessary to eﬀectively overcome tumor cell resistance mechanisms. One mechanism tumor cells use to evade immune cell-mediated cytotoxicity is exploitation of immune checkpoint signaling, which is used to inhibit immunologic damage of “self” cells in the healthy state. Immune checkpoints are critical components of autoimmune tolerance to avoid autoimmune diseases like rheumatoid arthritis (81, 82), type I diabetes (83) and multiple sclerosis (84). Checkpoint receptors on immune cells recognize ligands expressed on cells being surveilled and activation of these immune checkpoint receptors by the ligands leads to inactivation of the immune cells. This mechanism is exploited by tumor cells, which may overexpress these ligands or induce other cells [e.g., tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs)] within the tumor microenvironment (TME) to express checkpoint ligands to create an immunosuppressive shield throughout the TME and thus help tumor cells evade immunosurveillance (85–87). Secretion of immunosuppressive factors like transforming growth factor-β (TGF-β) by cells in the TME can directly inhibit CAR T cell cytotoxic activity and even direct diﬀerentiation of eﬀector T cells to regulatory T cells (88–90). gy p y y While RNA-guided (sgRNA) programmable nucleases based on CRISPR-Cas9 are very versatile and useful tools and mostly generate accurate and precise DNA DSBs, potentially also oﬀ-target eﬀects can occur. Furthermore, chromosomal translocations are rare unwanted side eﬀects, especially in case of multiplexing (72). To decrease the risk of these unwanted events, further engineering, e.g., CRISPR-Cas9 systems with less oﬀ-target eﬀects, and newer gene editing approaches are being developed as discussed below. Such advances will lead to more eﬃcient and safer generation of genome modiﬁed “oﬀ-the-shelf” CAR T and CAR NK cell products (Figure 2). CRISPR-Cas9 has two nuclease domains and introduction of inactivating mutations into one of these domains results in so- called nickases, which cleave only one strand of the targeted DNA (73). As a further improvement, “dead” Cas9 variants with inactivating mutations in both nuclease domains were created that could be fused to DNA modifying enzymes, such as Apobec- like nucleobase deaminase enzymes. These “base editors” lead to deﬁned base alterations without the need to cut the DNA and thus further reduce the likelihood of side eﬀects (74). CRISPR-Cas9 “Off-the-Shelf”Cell Products and Developing Technologies In addition, PD-1 knockout was accomplished to improve activity of the engineered T cells by avoiding checkpoint inhibition through tumor-associated cells. This may be an important strategy as disruption of PD-1 on T cells may help avoid immune-related side eﬀects observed upon systemic administration of anti-PD-1 monoclonal antibodies, while still improving CAR T cell anti- tumor activity. Frontiers in Immunology \| www.frontiersin.org Technology Speciﬁcity and Safety More recently, a catalytically impaired Cas9 was fused to an engineered (Murine Leukemia Virus-derived) reverse transcriptase to enable “prime editing” as a new technology to write new genetic information into a speciﬁed DNA site (75). Accordingly, prime editing further expands the capabilities of gene editing to create new options for immunotherapeutics. Immune checkpoint molecules include cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), PD-1 (PDCD1, CD279), lymphocyte activation gene 3 (LAG-3), and T cell membrane protein 3 (TIM3, HAVCR2) (91–94). Interaction of immune checkpoints with their cognate ligands results in suppression of immune cell function. Thus, tumor cells may express CD80/86 to suppress T cell activity via binding to CTLA4, or express PD-1 ligands PD-L1 (CD274) or PD-L2 (PDCD1LG2, PD-2 ligand). Accordingly, binding of LAG-3 to MHC class Additional approaches have been developed with the intent to minimize possibly deleterious activity of genome editing described above. For example, as long-term CRISPR-Cas9 expression is not required for eﬃcient genome modiﬁcation, transient RNA-protein (RNP) complexes can be delivered into the target cell population in place of viral vectors or DNA constructs. Alternatively, non-integrating lentiviral vectors can be engineered for transient delivery of CRISPR-Cas9 editing August 2020 \| Volume 11 \| Article 1965 8 Off-the-Shelf CAR T Cells Morgan et al. FIGURE 2 \| CRISPR-Cas9-mediated gene editing of CAR T cells. The TCR can be knocked out to lessen the likelihood of graft versus host disease (GVHD). The HLAs can be knocked out to increase persistence of gene-modiﬁed cells. Knockout of receptors that can be targeted by other medications, such as antibodies, can be accomplished to allow selective survival of gene-modiﬁed cells, e.g., CD52. FIGURE 2 \| CRISPR-Cas9-mediated gene editing of CAR T cells. The TCR can be knocked out to lessen the likelihood of graft versus host disease (GVHD). The HLAs can be knocked out to increase persistence of gene-modiﬁed cells. Knockout of receptors that can be targeted by other medications, such as antibodies, can be accomplished to allow selective survival of gene-modiﬁed cells, e.g., CD52. II or ﬁbrinogen-like protein 1 (FGL1), or ligation of TIM3 to galectin 9, carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), high-mobility group box protein 1 (HMGB1) or the non-protein ligand phosphatidylserine was shown to negatively regulate immune cell cytotoxic activity (95–97). Technology Speciﬁcity and Safety In addition to application of immune checkpoint inhibitors, anti-tumor activity was demonstrated by CAR-mediated cytokine secretion at the tumor site using T cells redirected for universal cell killing (TRUCKs) (102–105). This strategy involves modiﬁcation of T cells with a constitutively expressed CAR and a cytokine expression cassette that is controlled by an inducible promoter. The TRUCK concept uses the NFAT signaling pathway to produce pro-inﬂammatory cytokines upon activation of the CAR CD3ζ signaling domain after tumor antigen recognition. This results in modiﬁcation of the TME via cytokine secretion and recruitment of additional anti-tumor immune cells to increase anti-cancer activity (see Figure 3). In the original design, TRUCKs were generated by retroviral vector-mediated transfer of two separate vectors – one for the CAR and the second for the inducible cytokine expression cassette. Recent work showed the feasibility to deliver the necessary genetic cargo on a single lentiviral vector (106), thus advancing the potential use of this technology for “oﬀ-the-shelf” immunotherapy. g y g y y Several antibodies were developed to inhibit the activity of immune checkpoint molecules and clinical anti-cancer activity was demonstrated for some of these checkpoint inhibitors. Currently, most studies have investigated checkpoint inhibition of CTLA4 and PD-1 activities (98, 99). However, strategies to inhibit LAG-3 may be even more promising as antibodies that target LAG-3 were shown to enhance cytotoxic T cell activation and may inhibit Treg-induced immunosuppressive activity as elevated levels of a LAG-3+ subpopulation of Tregs was found at tumor sites and in peripheral blood mononuclear cells of patients with melanoma or colorectal cancer (100). Combination of the anti-LAG-3 antibody IMP321 with paclitaxel led to improved immune responses and greater antitumor activity in metastatic breast cancer patients (101). Currently, more than 240 clinical studies are evaluating the eﬃcacy of checkpoint inhibitors in several diﬀerent treatment modalities in cancer patients (link to respective studies on clinicaltrials.gov). As CAR- modiﬁed immune cells can become functionally inactivated or depleted due to tumor escape mechanisms such as immune checkpoints, checkpoint inhibition can help promote CAR T and perhaps CAR NK cell persistence and anti-tumor activity. The eﬃcacy of CAR T cells directed against mesothelin with concomitant CRISPR-Cas9-mediated knockout of TCRαβ and PD-1 is currently tested in a clinical trial (NCT03545815) of patients with mesothelin positive solid tumors. Such studies will help elucidate the feasibility of combining checkpoint inhibition in an “oﬀ-the-shelf” CAR T cell setting. Frontiers in Immunology \| www.frontiersin.org DIRECT IN VIVO APPLICATION OF CAR-BASED PRINCIPLES USING TARGETED VIRAL VECTOR NANO-PARTICLES As the whole genesis of CAR-harboring cellular products is highly demanding, future-oriented approaches for “oﬀ-the- shelf” applications are also considering the direct application of GMP-grade viral vector preparations to deliver the CAR-based principles directly into the target immune cells of choice, e.g., T and NK cells. This method entirely circumvents immunological August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 9 Off-the-Shelf CAR T Cells Morgan et al. FIGURE 3 \| Reshaping of the tumor microenvironment using T cells redirected for universal cytokine killing (TRUCK). Upon antigen binding, the CAR activates CD3z(eta) signaling, which leads to activation of an NFAT-driven promoter that controls expression of antitumor-cytokine cassette, e.g., IL12 or IL18. The cytokines are then secreted from the CAR T or CAR NK cells into the tumor microenvironment, where they recruit additional immune cells to enhance the antitumor activity. FIGURE 3 \| Reshaping of the tumor microenvironment using T cells redirected for universal cytokine killing (TRUCK). Upon antigen binding, the CAR activates CD3z(eta) signaling, which leads to activation of an NFAT-driven promoter that controls expression of antitumor-cytokine cassette, e.g., IL12 or IL18. The cytokines are then secreted from the CAR T or CAR NK cells into the tumor microenvironment, where they recruit additional immune cells to enhance the antitumor activity. assigned target speciﬁcity. Proof-of-concept for targeted delivery was demonstrated for a variety of target cells, including various hematopoietic and endothelial cells. Of particular importance for CAR technology, surface-engineered lentiviral vectors were successfully applied to mediate selective gene transfer into various subtypes of lymphocytes, including T cells (110, 113) that, impressively, led to the in vivo generation of human CD19- CAR T cells with B-cell depletion and signs of cytokine release syndrome in a humanized mouse model (114). rejection barriers, avoids time consuming ex vivo manipulation and cultivation of cells, and would directly reach the target eﬀector cells of the individual. Of note, receptor-targeted vector particles can be as selective for their targeted cell type as antibodies for their antigen when applied systemically or locally in preclinical studies. In this regard, receptor targeting using viral vector nano-particles opens up the possibility for novel concepts in immunotherapy and cell type-speciﬁc delivery of CARs in in vivo settings (107). Similar delivery principles were shown for non-viral delivery of mRNA in lipid nanoparticles (108). AAV Vectors for “Off-the-Shelf” CAR Delivery In the case of AAV vectors, which are derived from non- enveloped viruses, the capsid is the target of engineering. Prominent capsid structures are protrusions, which host the natural receptor binding motifs and pores used for loading of vector DNA. Genetic targeting approaches are the currently preferred strategy to modify vector tropism, and were used to insert receptor binding peptides [reviewed in (115)], immunoglobulin binding domains (116) or nanobodies (117) at the tip of the protrusions. Alternatively, the N-terminus of the non-essential capsid protein VP2 can be used as an insertion site. This is especially useful (I) to incorporate large peptides, (II) to target moieties that depend upon their 3D structure for DIRECT IN VIVO APPLICATION OF CAR-BASED PRINCIPLES USING TARGETED VIRAL VECTOR NANO-PARTICLES As a prerequisite for this approach, the natural tropism of the used viral vectors, e.g., gammaretro-/lentiviral and AAV vectors, needs to be blinded, so that the viral vectors no longer bind to their natural target receptors. In a second step, a deﬁned and speciﬁed target selectivity has to be added by incorporation of a new selective target cell-binding principle, e.g., scFv antibody, peptide or DARPin (designed ankyrin repeat protein) (107, 109, 110). Retroviral Vectors for “Off-the-Shelf” CAR Delivery In the case of enveloped gammaretro- and lentiviral vectors, the envelope for pseudotyping is substituted for a “targeted” Measles (111) or Nipah virus-derived envelope (112) with a newly August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 10 Off-the-Shelf CAR T Cells Morgan et al. function or (III) to incorporate entire proteins (118–122). The respective fusion proteins become exposed on the capsid surface through the pore structures. Tropism can either be expanded or re-directed, depending on the speciﬁcity of the targeting moiety that is inserted, and whether or not the natural tropism has been ablated, for example by site-directed mutagenesis. The feasibility that oﬀ-target free, on-target delivery following intravenous administration of viral vector particles is possible was demonstrated by incorporating DARPins with antibody- like speciﬁcity via fusion to VP2 into AAV2 capsids blinded for binding to their primary receptor heparan sulfate proteoglycan (109). These AAV particles can eﬃciently discriminate between target and non-target cells ex vivo in mixed cell cultures as well as in vivo, e.g., as demonstrated by delivery of a suicide gene precisely into tumor tissue and speciﬁc targeting of CD4+ lymphocytes in vivo (109, 122). will become even more challenging with advances like epigenome editing technologies. Combinations of cell therapies may also be useful in this context, especially considering the complex interactions between diﬀerent cell types during immune responses. For example, CAR T cell activity against colorectal cancer cells was recently shown to be improved by co-application of mesenchymal stem cells (MSC) genetically modiﬁed to release IL7 and IL12 (123). The authors exploited the natural capacity of MSC to home to tumor sites and thus support CAR T cells. Cross-talk between CAR T and MSC led to a greater persistence of CAR T, less activation- induced cell death and better anti-tumor activity as shown in in vitro and in vivo models. Therefore, generation of master cell banks of diﬀerent types of universal allogeneic cells available as “oﬀ-the-shelf” living drugs may help increase the eﬃcacy of immune cell therapies. These systems will further enrich the portfolio of “oﬀ-the- shelf” applications for cancer immunotherapy. Therapeutic eﬃcacy can be limited by loss of CAR T/CAR NK cell persistence due to rejection. As discussed above, humoral responses raised against murine-derived scFv may lead to loss of CAR-modiﬁed cells. In such cases, strategies to humanize the scFv can result in greater CAR cell persistence. DISCUSSION/OUTLOOK Several factors impact the potency and successful translation of adoptive cell therapies like CAR T and CAR NK cells to treat cancer. As discussed above, selection of the cell source is a critical decision. The majority of CAR-based therapies use autologous T cells, which have been successfully administered in several clinical studies, with broader success in hematologic malignancies (especially of the lymphoid compartment) than in solid tumors thus far. Autologous CAR T cells have advantages such as no risk of GVHD and lower risk of rejection than allogeneic CAR T cells. However, autologous CAR T cells may have some immunologic defects and the patient must wait several weeks before the autologous CAR T cells are ready for application. As we seek to extend the clinical usefulness of CAR cell strategies, one obvious path forward is to commit more resources toward development of “oﬀ-the-shelf” CAR cell therapies, such as genetically modiﬁed “universal” allogeneic CAR T cells, NK cells, iPSC and progenitor-derived cells. Universal allogeneic CAR cell therapies are derived from healthy donors, so the immune cells should function properly, and the TCR and MHC are disrupted to avoid induction of GVHD or elimination by the host T cells. While such genome editing strategies to generate “oﬀ-the-shelf” CAR T cells are already in clinical practice (66, 71), these may have higher regulatory burden to demonstrate lack of oﬀ-target hits and translocations. Another advantage of allogeneic CAR cells is that they can be prepared in advance and stored until needed, thus reducing the time a patient must wait for treatment. As CRISPR-Cas9 genome modiﬁcation procedures continue to become more eﬃcient and precise, potential risks of genome modiﬁed cell therapies will decrease. For example, advances such as base editing make it possible to speciﬁcally edit the genome without the necessity to induce double strand DNA breaks, thus potentially increasing the safety of genome editing by reducing the risk of unwanted complications like chromosomal translocations in cell therapies. However, detection of oﬀ-target hits is even more diﬃcult and Development of strategies to overcome tumor-induced immune suppression has been widely studied and use of immune checkpoint inhibitors or genetically engineering CAR T and CAR NK cells to be less responsive to checkpoint signaling are two main approaches to address this challenge. Retroviral Vectors for “Off-the-Shelf” CAR Delivery Genetic ablation of MHC may also help to increase CAR cell persistence by evading the host T cell responses, but may also lead to increased detection by host NK cells. Frontiers in Immunology \| www.frontiersin.org DISCUSSION/OUTLOOK For example, CRISPR-Cas9-mediated elimination of the checkpoint receptor PD-1 from CAR T cells led to improved activity against the solid tumor glioblastoma in preclinical models (124). The possibility to simultaneously and eﬃciently modify multiple genes with CRISPR-Cas9 seems to be an advantage over ZFN and TALEN genome editing technologies. Emergence of or selection for tumor cells that do not express the target antigen, a concept called “antigen loss,” can also negatively impact CAR T and CAR NK cell antitumor activity. For example, relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) patients who were previously administered blinatumomab, a bispeciﬁc antibody that targets CD3 on T cells and CD19 on B cells, were less likely to achieve minimal residual disease deep remission and were more likely to experience relapse due to antigen loss after treatment with anti-CD19 CAR T cells (125). Use of dual CAR concepts to target two tumor-associated antigens can lead to improved tumor control. However, the increased risk of on-target-oﬀ-tumor activity has to be taken into account. In addition to antigen loss to avoid CAR T cell activity, decreased death receptor activity (FADD, BID, CASP8 and TNFRSF10B) was shown to be a mechanism of resistance to anti- CD19 CAR T cells (126). Importantly, pre-treatment leukemia- inﬁltrated bone marrow samples from patients who were treated with anti-CD19 CAR T cell therapy showed that lower death receptor gene expression associated with worse overall survival (126). Methods to restore or elevate death receptor expression and signal transduction activity in tumor cell target populations August 2020 \| Volume 11 \| Article 1965 11 Off-the-Shelf CAR T Cells Morgan et al. could lead to improved tumor control. Identiﬁcation of target molecules that are more speciﬁcally expressed on tumor cells and absent on healthy tissues will also increase the therapeutic eﬃcacy of CAR T and CAR NK cell therapies. The recently described “Sequentially Tumor-selected Antibody and antigen Retrieval (STAR)” method led to isolation of nanobodies that preferentially bound acute myeloid leukemia (AML) cells, and identiﬁed CD13 as an AML-speciﬁc target (127). Generation of bi-speciﬁc CAR T cells that targeted CD13 and TIM3, a checkpoint inhibitor that was found to be upregulated in leukemic stem cells, led to improved elimination of AML. Furthermore, determination of general cancer-speciﬁc targets would alleviate the current need to target diﬀerent antigens for diﬀerent types of cancer. REFERENCES 9. Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature. (2002) 419:734–8. 1. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, et al. and et al.: graft-versus-leukemia reactions after bone marrow transplantation. Blood. (1990) 75:555–62. 10. Raﬀaghello L, Prigione I, Airoldi I, Camoriano M, Levreri I, Gambini C, et al. Downregulation and/or release of NKG2D ligands as immune evasion strategy of human neuroblastoma. Neoplasia. (2004) 6:558–68. 11. Gross G, Gorochov G, Waks T, Eshhar Z. Generation of eﬀector T cells expressing chimeric T cell receptor with antibody type-speciﬁcity. Transplant Proc. (1989) 21(1 Pt 1):127–30. 2. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME. Adoptive cell transfer: a clinical path to eﬀective cancer immunotherapy. Nat Rev Cancer. (2008) 8:299–308. doi: 10.1038/nrc2355 3. Samelson LE, Donovan JA, Isakov N, Ota Y, Wange RL. Signal transduction mediated by the T-cell antigen receptor. Ann N Y Acad Sci. (1995) 766:157– 72. 12. Salter AI, Ivey RG, Kennedy JJ, Voillet V, Rajan A, Alderman EJ, et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative diﬀerences that aﬀect cell function. Sci Signal. (2018) 11:eaat6753. doi: 10.1126/scisignal.aat6753 4. Wange RL, Samelson LE. Complex complexes: signaling at the TCR. Immunity. (1996) 5:197–205. doi: 10.1016/s1074-7613(00)80315-5 13. Ying Z, He T, Wang X, Zheng W, Lin N, Tu M, et al. Parallel comparison of 4-1BB or CD28 Co-stimulated CD19-Targeted CAR-T Cells for B Cell Non-Hodgkin’s Lymphoma. Mol Ther Oncolytics. (2019) 15:60–8. 5. Andrews PW, Bronson DL, Wiles MV, Goodfellow PN. The expression of MHC antigens by human teratocarcinoma derived cell lines. Tissue Antigens. (1981) 17:493–500. doi: 10.1111/j.1399-0039.1981.tb00736.x 14. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in children and young adults with b-cell lymphoblastic leukemia. N Engl J Med. (2018) 378:439–48. 6. Garrido F, Ruiz-Cabello F, Aptsiauri N. Rejection versus escape: the tumor MHC dilemma. Cancer Immunol Immunother. (2016) 66:259–71. 6. Garrido F, Ruiz-Cabello F, Aptsiauri N. Rejection versus escape: the tumor MHC dilemma. Cancer Immunol Immunother. (2016) 66:259–71. 7. Lanier LL. NK cell recognition. Annu Rev Immunol. (2005) 23:225–74. 7. Lanier LL. NK cell recognition. Annu Rev Immunol. (2005) 23:225–74. 15. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. FUNDING This work was supported by grants from the Deutsche Forschungsgemeinschaft [SFB738, Cluster of Excellence REBIRTH (EXC 62/2)], the REBIRTH Center for Translational Regenerative Medicine funded through the State of Lower Saxony (MWK: ZN3440), the Bundesministerium für Bildung und Forschung (BMBF, Joint Research Project IFB-Tx), the Wihelm Sander-Stiftung (2018.046.1), the Deutsche Krebshilfe (70113658), the CARs to TRUCKs (Krebshilfe-Priority Program in Translational Oncology), and the Comprehensive Cancer Center (CCC) Hannover. This project received funding from the European Union’s Horizon 2020 Research and Innovation Program under grant agreement Nos. 755170, 666908, and from the European Research Council (ERC) under grant agreement No. 819531. In summary, several possibilities to generate “oﬀ-the-shelf” anti-cancer immunotherapeutics are currently being explored. For example, control of TCR expression by genomic knockout or down-regulation via RNAi demonstrated the feasibility of generating “oﬀ-the-shelf” allogeneic CAR T cell products. However, also other allogeneic cell sources, such as NK cells AUTHOR’S NOTE Figures were created with BioRender.com. 9. Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature. (2002) 419:734–8. AUTHOR CONTRIBUTIONS All authors contributed to conception and writing of the manuscript. All authors contributed to conception and writing of the manuscript. DISCUSSION/OUTLOOK For example, designing CARs to target cancer-speciﬁc post- translational modiﬁcations such as Tn-glycosylated podoplanin (Tn-PDPN) (128) would be expected to result in fewer oﬀ-cancer eﬀects as the 237Ab-derived 237 CAR T cells target Tn-PDPN, which is not present on normal tissue. As Tn glycosylation was present on all cancer cells evaluated, tumor escape is also less likely, which makes this a novel approach to improve CAR T cell eﬃcacy (128). Along these lines, a genome-wide CRISPR-Cas9 screening method was recently used to discover a TCR that recognized the monomorphic MHC class I-related protein MR1 and T cells engineered to express this TCR killed several diﬀerent types of human cancers without damaging healthy cells (129). Once veriﬁed, development of strategies such as these in allogeneic “oﬀ-the-shelf” cell sources could have great potential to exhibit anti-cancer activity against a broad spectrum of malignancies. and macrophages, appear to be suitable as “oﬀ-the-shelf” anti- cancer CAR cells. In addition to delivery of cell therapies, the possibility to apply viral vectors engineered for targeted in vivo modiﬁcation of immune cells with CARs is another potent “oﬀ- the-shelf” strategy to generate CAR T and CAR NK cells. As research in these areas is rapidly progressing, we look forward to development of eﬃcient “oﬀ-the-shelf” therapies that will be made broadly available to the many cancer patients world-wide. REFERENCES Clinical and immunological signiﬁcance of HLA-E in stem cell transplantation and cancer. Tissue Antigens. (2014) 84:523–35. 21. Hubner J, Hoseini SS, Suerth JD, Hoﬀmann D, Maluski M, Herbst J, et al. generation of genetically engineered precursor T-cells from human umbilical cord blood using an optimized alpharetroviral vector platform. Mol Ther. (2016) 24:1216–26. 42. Celik AA, Simper GS, Huyton T, Blasczyk R, Bade-Doding C. HLA-G mediated immune regulation is impaired by a single amino acid exchange in the alpha 2 domain. Hum Immunol. (2018) 79:453–62. 43. Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N Engl J Med. (2020) 382:545–53. 22. Prager I, Liesche C, van Ooijen H, Urlaub D, Verron Q, Sandstrom N, et al. NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing. J Exp Med. (2019) 216:2113–27. 23. Prager I, Watzl C. Mechanisms of natural killer cell-mediated cellular cytotoxicity. J Leukoc Biol. (2019) 105:1319–29. 44. Shah NN, Baird K, Delbrook CP, Fleisher TA, Kohler ME, Rampertaap S, et al. Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation. Blood. (2015) 125:784–92. 24. Noyan F, Zimmermann K, Hardtke-Wolenski M, Knoefel A, Schulde E, Geﬀers R, et al. Prevention of allograft rejection by use of regulatory T cells with an MHC-speciﬁc chimeric antigen receptor. Am J Transplant. (2017) 17:917–30. doi: 10.1111/ajt.14175 45. Nowakowska P, Romanski A, Miller N, Odendahl M, Bonig H, Zhang C, et al. Clinical grade manufacturing of genetically modiﬁed, CAR-expressing NK- 92 cells for the treatment of ErbB2-positive malignancies. Cancer Immunol Immunother. (2018) 67:25–38. doi: 10.1007/s00262-017-2055-2 j 25. Romano M, Fanelli G, Albany CJ, Giganti G, Lombardi G. Past, Present, and future of regulatory T cell therapy in transplantation and autoimmunity. Front Immunol. (2019) 10:43. 46. Knorr DA, Ni Z, Hermanson D, Hexum MK, Bendzick L, Cooper LJ, et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl Med. (2013) 2:274–83. 26. Suerth JD, Morgan MA, Kloess S, Heckl D, NeudorﬂC, Falk CS, et al. Eﬃcient generation of gene-modiﬁed human natural killer cells via alpharetroviral vectors. J Mol Med (Berl). (2015) 94:83–93. doi: 10.1007/s00109-015-1327-6 47. Liu E, Tong Y, Dotti G, Shaim H, Savoldo B, Mukherjee M, et al. REFERENCES Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity. Leukemia. (2018) 32:520–31. 27. Klapdor R, Wang S, Hacker U, Buning H, Morgan M, Dork T, et al. Improved killing of ovarian cancer stem cells by combining a novel chimeric antigen receptor-based immunotherapy and chemotherapy. Hum Gene Ther. (2017) 28:886–96. doi: 10.1089/hum.2017.168 48. Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res. (2009) 69:4010–7. 28. Kloess S, Oberschmidt O, Dahlke J, Vu XK, NeudoerﬂC, Kloos A, et al. Preclinical assessment of suitable natural killer cell sources for chimeric antigen receptor natural killer-based “oﬀ-the-shelf” acute myeloid leukemia immunotherapies. Hum Gene Ther (2019) 30:381–401. doi: 10.1089/hum. 2018.247 49. Ojo EO, Sharma AA, Liu R, Moreton S, Checkley-Luttge MA, Gupta K, et al. Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells. Sci Rep. (2019) 9:14916. 50. Brehm C, Huenecke S, Quaiser A, Esser R, Bremm M, Kloess S, et al. IL-2 stimulated but not unstimulated NK cells induce selective disappearance of peripheral blood cells: concomitant results to a phase I/II study. PLoS One. (2011) 6:e27351. doi: 10.1371/journal.pone.0027351 29. Topfer K, Cartellieri M, Michen S, Wiedemuth R, Muller N, Lindemann D, et al. DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy. J Immunol. (2015) 194:3201–12. 30. Chang YH, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A chimeric receptor with NKG2D speciﬁcity enhances natural killer cell activation and killing of tumor cells. Cancer Res. (2013) 73:1777–86. 51. Suck G, Oei VY, Linn YC, Ho SH, Chu S, Choong A, et al. Interleukin-15 supports generation of highly potent clinical-grade natural killer cells in long- term cultures for targeting hematological malignancies. Exp Hematol. (2011) 39:904–14. 31. Parihar R, Rivas C, Huynh M, Omer B, Lapteva N, Metelitsa LS, et al. NK cells expressing a chimeric activating receptor eliminate MDSCs and rescue impaired CAR-T cell activity against solid tumors. Cancer Immunol Res. (2019) 7:363–75. 52. Sakamoto N, Ishikawa T, Kokura S, Okayama T, Oka K, Ideno FS, et al. Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer. J Transl Med. (2015) 13:277. 32. REFERENCES Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. (2010) 18:843–51. 8. Marin R, Ruiz-Cabello F, Pedrinaci S, Mendez R, Jimenez P, Geraghty DE, et al. Analysis of HLA-E expression in human tumors. Immunogenetics. (2003) 54:767–75. August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 12 Off-the-Shelf CAR T Cells Morgan et al. 16. Ahmed N, Brawley VS, Hegde M, Robertson C, Ghazi A, Gerken C, et al. Human epidermal growth factor receptor 2 (HER2) -speciﬁc chimeric antigen receptor-modiﬁed T cells for the immunotherapy of HER2-positive sarcoma. J Clin Oncol. (2015) 33:1688–96. 36. The quest for oﬀ-the-shelf CAR T cells. Cancer Discov. (2018) 8:787–8. 37. Piontek GE, Taniguchi K, Ljunggren HG, Gronberg A, Kiessling R, Klein G, et al. YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage. J Immunol. (1985) 135:4281–8. 17. Turtle CJ, HanaﬁLA, Berger C, Gooley TA, Cherian S, Hudecek M, et al. CD19 CAR-T cells of deﬁned CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest. (2016) 126:2123–38. 38. Ljunggren HG, Karre K. Host resistance directed selectively against H-2- deﬁcient lymphoma variants. Analysis of the mechanism. J Exp Med. (1985) 162:1745–59. 18. Harris WJ. Production of humanized monoclonal antibodies for in vivo imaging and therapy. Biochem Soc Trans. (1995) 23:1035–8. 39. Diefenbach A, Raulet DH. The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol Rev. (2002) 188:9–21. doi: 10.1034/j.1600-065x.2002.18802.x 19. Bordignon C, Bonini C, Verzeletti S, Nobili N, Maggioni D, Traversari C, et al. and et al.: Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Hum Gene Ther. (1995) 6:813–9. 40. Pazmany L, Mandelboim O, Vales-Gomez M, Davis DM, Reyburn HT, Strominger JL. Protection from natural killer cell-mediated lysis by HLA-G expression on target cells. Science. (1996) 274:792–5. logeneic bone marrow transplantation. Hum Gene Ther. (1995) 6 20. Straathof KC, Pule MA, Yotnda P, Dotti G, Vanin EF, Brenner MK, et al. An inducible caspase 9 safety switch for T-cell therapy. Blood. (2005) 105:4247– 54. 41. Wieten L, Mahaweni NM, Voorter CE, Bos GM, Tilanus MG. REFERENCES doi: 10.1158/1078-0432.CCR-10-1966 65. Artegiani B, Hendriks D, Beumer J, Kok R, Zheng X, Joore I, et al. Fast and eﬃcient generation of knock-in human organoids using homology- independent CRISPR-Cas9 precision genome editing. Nat Cell Biol. (2020) 22:321–31. doi: 10.1038/s41556-020-0472-5 86. Norde WJ, Maas F, Hobo W, Korman A, Quigley M, Kester MG, et al. PD- 1/PD-L1 interactions contribute to functional T-cell impairment in patients who relapse with cancer after allogeneic stem cell transplantation. Cancer Res. (2011) 71:5111–22. doi: 10.1158/0008-5472.CAN-11-0108 66. Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Staﬀord S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med. (2017) 9:eaaj2013. doi: 10.1126/ scitranslmed.aaj2013 87. Gordon SR, Maute RL, Dulken BW, Hutter G, George BM, McCracken MN, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature. (2017) 545:495–9. doi: 10.1038/ nature22396 67. MacLeod DT, Antony J, Martin AJ, Moser RJ, Hekele A, Wetzel KJ, et al. Integration of a CD19 CAR into the TCR Alpha chain locus streamlines production of allogeneic gene-edited CAR T cells. Mol Ther. (2017) 25:949– 61. doi: 10.1016/j.ymthe.2017.02.005 88. Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, von Boehmer H, et al. Regulatory T cells suppress tumor-speciﬁc CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc Natl Acad Sci USA. (2005) 102:419– 24. 68. Wang J, DeClercq JJ, Hayward SB, Li PW, Shivak DA, Gregory PD, et al. Highly eﬃcient homology-driven genome editing in human T cells by combining zinc-ﬁnger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res. (2016) 44:e30. doi: 10.1093/nar/gkv1121 89. Pickup M, Novitskiy S, Moses HL. The roles of TGFbeta in the tumour microenvironment. Nat Rev Cancer. (2013) 13:788–99. 69. Torikai H, Reik A, Liu PQ, Zhou Y, Zhang L, Maiti S, et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19- speciﬁc chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood. (2012) 119:5697–705. 90. Liu VC, Wong LY, Jang T, Shah AH, Park I, Yang X, et al. Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol. (2007) 178:2883–92. 91. Simmons AD, Moskalenko M, Creson J, Fang J, Yi S, VanRoey MJ, et al. Local secretion of anti-CTLA-4 enhances the therapeutic eﬃcacy of a cancer immunotherapy with reduced evidence of systemic autoimmunity. REFERENCES Stable lines and clones of long-term proliferating normal, genetically unmodiﬁed murine common lymphoid progenitors. Blood. (2018) 131:2026–35. 79. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of A∗T to G∗C in genomic DNA without DNA cleavage. Nature. (2017) 551:464–71. doi: 10.1038/nature24644 59. Koniaeva E, Stahlhut M, Lange L, Sauer MG, Kustikova OS, Schambach A. Conditional immortalization of lymphoid progenitors via tetracycline- regulated LMO2 expression. Hum Gene Ther. (2020) 31:183–98. 80. Doman JL, Raguram A, Newby GA, Liu DR. Evaluation and minimization of Cas9-independent oﬀ-target DNA editing by cytosine base editors. Nat Biotechnol. (2020) 38:620–28. doi: 10.1038/s41587-020-0414-6 60. Maluski M, Ghosh A, Herbst J, Scholl V, Baumann R, Huehn J, et al. Chimeric antigen receptor-induced BCL11B suppression propagates NK-like cell development. J Clin Invest. (2019) 129:5108–22. 81. Samuels J, Ng YS, Coupillaud C, Paget D, Meﬀre E. Impaired early B cell tolerance in patients with rheumatoid arthritis. J Exp Med. (2005) 201:1659– 67. doi: 10.1084/jem.20042321 61. Klichinsky M, Ruella M, Shestova O, Lu XM, Best A, Zeeman M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat. Biotechnol. (2020) [Epub ahead of print]. doi: 10.1038/s41587-020-0462-y 82. Chamberlain N, Massad C, Oe T, Cantaert T, Herold KC, Meﬀre E. Rituximab does not reset defective early B cell tolerance checkpoints. J Clin Invest. (2016) 126:282–7. doi: 10.1172/JCI83840 62. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. (2018) 24:927–30. doi: 10.1038/s41591-018-0049-z 83. Henry RA, Kendall PL, Thomas JW. Autoantigen-speciﬁc B-cell depletion overcomes failed immune tolerance in type 1 diabetes. Diabetes. (2012) 61:2037–44. doi: 10.2337/db11-1746 63. Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med. (2018) 24:939–46. doi: 10.1038/s41591-018-0050-6 84. Joller N, Peters A, Anderson AC, Kuchroo VK. Immune checkpoints in central nervous system autoimmunity. Immunol Rev. (2012) 248:122–39. doi: 10.1111/j.1600-065X.2012.01136.x 64. Schiroli G, Conti A, Ferrari S, Della Volpe L, Jacob A, Albano L, et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell. (2019) 24:551-565.e8. doi: 10.1016/j.stem.2019.02.019 85. Yu P, Steel JC, Zhang M, Morris JC, Waldmann TA. Simultaneous blockade of multiple immune system inhibitory checkpoints enhances antitumor activity mediated by interleukin-15 in a murine metastatic colon carcinoma model. Clin Cancer Res. (2010) 16:6019–28. REFERENCES Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokine- associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor- transduced T cells. Blood. (2012) 119:2709–20. 53. Becker PS, Suck G, Nowakowska P, Ullrich E, Seifried E, Bader P, et al. Selection and expansion of natural killer cells for NK cell-based immunotherapy. Cancer Immunol Immunother. (2016) 65:477–84. 33. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modiﬁed T cells for acute lymphoid leukemia. N Engl J Med. (2013) 368:1509–18. 54. Nianias A, Themeli M. Induced pluripotent stem cell (iPSC)-Derived lymphocytes for adoptive cell immunotherapy: recent advances and challenges. Curr Hematol Malig Rep. (2019) 14:261–8. 34. Kebriaei P, Singh H, Huls MH, Figliola MJ, Bassett R, Olivares S, et al. Phase I trialls using Sleeping Beauty to generate CD19-speciﬁc CAR T cells. J Clin Invest. (2016) 126:3363–76. 55. Themeli M, Kloss CC, Ciriello G, Fedorov VD, Perna F, Gonen M, et al. Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat Biotechnol. (2013) 31:928–33. 35. Ruella M, Xu J, Barrett DM, Fraietta JA, Reich TJ, Ambrose DE, et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat Med. (2018) 24:1499–503. doi: 10.1038/s41591-018-0201-9 56. Li Y, Hermanson DL, Moriarity BS, Kaufman DS. Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti- tumor activity. Cell Stem Cell. (2018) 18:e5. August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 13 Off-the-Shelf CAR T Cells Morgan et al. 57. Lange L, Hoﬀmann D, Schwarzer A, Ha TC, Philipp F, Lenz D, et al. Inducible forward programming of human pluripotent stem cells to hemato- endothelial progenitor cells with hematopoietic progenitor potential. Stem Cell Rep. (2020) 14:122–37. 77. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-ﬁdelity CRISPR-Cas9 nucleases with no detectable genome-wide oﬀ-target eﬀects. Nature. (2016) 529:490–5. doi: 10.1038/nature16526 78. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. (2016) 533:420–4. doi: 10.1038/nature17946 58. Kawano Y, Petkau G, Stehle C, Durek P, Heinz GA, Tanimoto K, et al. REFERENCES (2008) 15:1618–22. doi: 10.1038/gt.2008.127 103. Chmielewski M, Abken H. CAR T Cells Releasing IL-18 Convert to T-Bet(high) FoxO1(low) eﬀectors that exhibit augmented activity against advanced solid tumors. Cell Rep. (2017) 21:3205–19. 121. Rybniker J, Nowag A, Janicki H, Demant K, Hartmann P, Buning H. Incorporation of antigens into viral capsids augments immunogenicity of adeno-associated virus vector-based vaccines. J Virol. (2012) 86:13800–4. doi: 10.1128/JVI.01708-12 104. Chmielewski M, Kopecky C, Hombach AA, Abken H. IL-12 release by engineered T cells expressing chimeric antigen receptors can eﬀectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Res. (2011) 71:5697–706. 122. Munch RC, Janicki H, Volker I, Rasbach A, Hallek M, Buning H, et al. Displaying high-aﬃnity ligands on adeno-associated viral vectors enables tumor cell-speciﬁc and safe gene transfer. Mol Ther. (2013) 21:109–18. doi: 10.1038/mt.2012.186 105. Chmielewski M, Hombach AA, Abken H. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma. Immunol Rev. (2014) 257:83–90. doi: 10.1111/ imr.12125 123. Hombach AA, Geumann U, Gunther C, Hermann FG, Abken H. IL7-IL12 Engineered mesenchymal stem cells (MSCs) improve A CAR T cell attack against colorectal cancer cells. Cells. (2020) 9:873. doi: 10.3390/cells9040873 106. Zimmermann K, Kuehle J, Dragon AC, Galla M, Kloth C, Rudek LS, et al. Design and characterization of an “All-in-One” lentiviral vector system combining constitutive anti-GD2 CAR expression and inducible cytokines. Cancers (Basel). (2020) 12:375. doi: 10.3390/cancers12020375 124. Choi BD, Yu X, Castano AP, Darr H, Henderson DB, Bouﬀard AA, et al. CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. J Immunother Cancer. (2019) 7:304. 107. Buchholz CJ, Friedel T, Buning H. Surface-engineered viral vectors for selective and cell type-speciﬁc gene delivery. Trends Biotechnol. (2015) 33:777–90. doi: 10.1016/j.tibtech.2015.09.008 125. Pillai V, Muralidharan K, Meng W, Bagashev A, Oldridge DA, Rosenthal J, et al. CAR T-cell therapy is eﬀective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy. Blood Adv. (2019) 3:3539–49. 108. Parhiz H, Shuvaev VV, Pardi N, Khoshnejad M, Kiseleva RY, Brenner JS, et al. PECAM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake. J Control Release. (2018) 291:106–15. doi: 10.1016/j.jconrel.2018.10.015 126. Singh N, Lee YG, Shestova O, Ravikumar P, Hayer KE, Hong SJ, et al. REFERENCES Cancer Immunol Immunother. (2008) 57:1263–70. doi: 10.1007/s00262-008-0451-3 70. Albers JJ, Ammon T, Gosmann D, Audehm S, Thoene S, Winter C, et al. Gene editing enables T-cell engineering to redirect antigen speciﬁcity for potent tumor rejection. Life Sci Alliance. (2019) 2:e201900367. doi: 10.26508/lsa. 201900367 92. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. (2012) 12:252–64. doi: 10.1038/nrc3239 71. Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, et al. CRISPR-engineered T cells in patients with refractory cancer. Science. (2020) 367:6481. 93. Stremitzer S, Sunakawa Y, Zhang W, Yang D, Ning Y, Stintzing S, et al. Variations in genes involved in immune response checkpoints and association with outcomes in patients with resected colorectal liver metastases. Pharmacogenomics J. (2015) 15:521–9. doi: 10.1038/tpj.2015.14 72. Wu X, Kriz AJ, Sharp PA. Target speciﬁcity of the CRISPR-Cas9 system. Quant Biol. (2014) 2:59–70. doi: 10.1007/s40484-014-0030-x 73. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing speciﬁcity. Cell. (2013) 154:1380–9. doi: 10.1016/j.cell.2013.08.021 94. Gorris MAJ, Halilovic A, Rabold K, van Duﬀelen A, Wickramasinghe IN, Verweij D, et al. Eight-color multiplex immunohistochemistry for simultaneous detection of multiple immune checkpoint molecules within the tumor microenvironment. J Immunol. (2018) 200:347–54. doi: 10.4049/ jimmunol.1701262 74. Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet. (2018) 19:770–88. doi: 10.1038/ s41576-018-0059-1 75. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. (2019) 576:149–57. doi: 10.1038/s41586-019-1711-4 95. Wang J, Sanmamed MF, Datar I, Su TT, Ji L, Sun J, et al. Fibrinogen- like protein 1 is a major immune inhibitory ligand of LAG-3. Cell. (2019) 176:334–47.e12. doi: 10.1016/j.cell.2018.11.010 96. Meggyes M, Miko E, Polgar B, Bogar B, Farkas B, Illes Z, et al. Peripheral blood TIM-3 positive NK and CD8+ T cells throughout pregnancy: TIM- 3/galectin-9 interaction and its possible role during pregnancy. PLoS One. (2014) 9:e92371. doi: 10.1371/journal.pone.0092371 76. Knopp Y, Geis FK, Heckl D, Horn S, Neumann T, Kuehle J, et al. Transient retrovirus-based CRISPR/Cas9 all-in-one particles for eﬃcient, targeted gene knockout. Mol Ther Nucleic Acids. (2018) 13:256–74. doi: 10.1016/j.omtn. 2018.09.006 August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 14 Morgan et al. Off-the-Shelf CAR T Cells 97. REFERENCES Impaired Death receptor signaling in leukemia causes antigen-independent resistance by inducing CAR T-cell dysfunction. Cancer Discov. (2020) 10:552–67. 109. Munch RC, Muth A, Muik A, Friedel T, Schmatz J, Dreier B, et al. Oﬀ- target-free gene delivery by aﬃnity-puriﬁed receptor-targeted viral vectors. Nat Commun. (2015) 6:6246. doi: 10.1038/ncomms7246 127. He X, Feng Z, Ma J, Ling S, Cao Y, Gurung B, et al. Bispeciﬁc and split CAR T cells targeting CD13 and TIM3 eradicate acute myeloid leukemia. Blood. (2020) 135:713–23. 110. Frank AM, Buchholz CJ. Surface-engineered lentiviral vectors for selective gene transfer into subtypes of lymphocytes. Mol Ther Methods Clin Dev. (2019) 12:19–31. doi: 10.1016/j.omtm.2018.10.006 128. He Y, Schreiber K, Wolf SP, Wen F, Steentoft C, Zerweck J, et al. Multiple cancer-speciﬁc antigens are targeted by a chimeric antigen receptor on a single cancer cell. JCI Insight. (2019) 4:e135306. 111. Kneissl S, Abel T, Rasbach A, Brynza J, Schneider-Schaulies J, Buchholz CJ. Measles virus glycoprotein-based lentiviral targeting vectors that avoid neutralizing antibodies. PLoS One. (2012) 7:e46667. doi: 10.1371/journal. pone.0046667 129. Crowther MD, Dolton G, Legut M, Caillaud ME, Lloyd A, Attaf M, et al. Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nat Immunol. (2020) 21:178–85. 112. Bender RR, Muth A, Schneider IC, Friedel T, Hartmann J, Pluckthun A, et al. Receptor-targeted nipah virus glycoproteins improve cell-type selective gene delivery and reveal a preference for membrane-proximal cell attachment. PLoS Pathog. (2016) 12:e1005641. doi: 10.1371/journal.ppat.1005641 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 113. Frank AM, Weidner T, Brynza J, Uckert W, Buchholz CJ, Hartmann J. CD8- Speciﬁc designed ankyrin repeat proteins improve selective gene delivery into human and primate T lymphocytes. Hum Gene Ther. (2020) 31:679–91. doi: 10.1089/hum.2019.248 Copyright © 2020 Morgan, Büning, Sauer and Schambach. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 114. Pfeiﬀer A, Thalheimer FB, Hartmann S, Frank AM, Bender RR, Danisch S, et al. REFERENCES Sabatos-Peyton CA, Nevin J, Brock A, Venable JD, Tan DJ, Kassam N, et al. Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional eﬃcacy. Oncoimmunology. (2018) 7:e1385690. doi: 10.1080/(2162402X): 2017.1385690 115. Buning H, Srivastava A. Capsid modiﬁcations for targeting and improving the eﬃcacy of AAV vectors. Mol Ther Methods Clin Dev. (2019) 12:248–65. doi: 10.1016/j.omtm.2019.01.008 116. Ried MU, Girod A, Leike K, Buning H, Hallek M. Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody- mediated vector retargeting to speciﬁc cell surface receptors. J Virol. (2002) 76:4559–66. doi: 10.1128/jvi.76.9.4559-4566.2002 98. Yamazaki N, Uhara H, Fukushima S, Uchi H, Shibagaki N, Kiyohara Y, et al. Phase II study of the immune-checkpoint inhibitor ipilimumab plus dacarbazine in Japanese patients with previously untreated, unresectable or metastatic melanoma. Cancer Chemother Pharmacol. (2015) 76:969–75. doi: 10.1007/s00280-015-2870-0 117. EichhoﬀAM, Borner K, Albrecht B, Schafer W, Baum N, Haag F, et al. Nanobody-enhanced targeting of AAV gene therapy vectors. Mol Ther Methods Clin Dev. (2019) 15:211–20. doi: 10.1016/j.omtm.2019. 09.003 99. Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med. (2015) 373:123–35. doi: 10.1056/NEJMoa1504627 118. Warrington KH Jr., Gorbatyuk OS, Harrison JK, Opie SR, Zolotukhin S, Muzyczka N. Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol. (2004) 78:6595–609. doi: 10.1128/JVI.78.12.6595-66 09.2004 100. Camisaschi C, Casati C, Rini F, Perego M, De Filippo A, Triebel F, et al. LAG- 3 expression deﬁnes a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites. J Immunol. (2010) 184:6545–51. 119. Lux K, Goerlitz N, Schlemminger S, Perabo L, Goldnau D, Endell J, et al. Green ﬂuorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear traﬃcking. J Virol. (2005) 79:11776–87. doi: 10.1128/JVI.79.18.11776-11787.2005 101. Brignone C, Gutierrez M, Mefti F, Brain E, Jarcau R, Cvitkovic F, et al. First- line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity. J Transl Med. (2010) 8:71. doi: 10.1186/1479-5876-8-71 y 102. Chmielewski M, Abken H. TRUCKs: the fourth generation of CARs. Expert Opin Biol Ther. (2015) 15:1145–54. 120. Asokan A, Johnson JS, Li C, Samulski RJ. Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals. Gene Ther. REFERENCES In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome. EMBO Mol Med. (2018) 10:e915. doi: 10.15252/emmm.201809158 August 2020 \| Volume 11 \| Article 1965 Frontiers in Immunology \| www.frontiersin.org 15
https://openalex.org/W1941683264	https://europepmc.org/articles/pmc4572358?pdf=render	English	null	The Independent Probabilistic Firing of Transcription Factors: A Paradigm for Clonal Variability in the Zebrafish Retina	Developmental cell	2,015	cc-by	12,427	The Independent Probabilistic Firing of Transcription Factors: A Paradigm for Clonal Variability in the Zebraﬁsh Retina The Independent Probabilistic Firing of Transcription Factors: A Paradigm for Clonal Variability in the Zebraﬁsh Retina Graphical Abstract Article Article Boije et al., 2015, Developmental Cell 34, 532–543 September 14, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.devcel.2015.08.011 Authors Henrik Boije, Steffen Rulands, Stefanie Dudczig, Benjamin D. Simons, William A. Harris Correspondence wah20@cam.ac.uk SUMMARY clone size distributions and lineage patterns (Gomes et al., 2011; He et al., 2012). Early retinal progenitor cells (RPCs) in vertebrates produce lineages that vary greatly both in terms of cell number and fate composition, yet how this vari- ability is achieved remains unknown. One possibility is that these RPCs are individually distinct and that each gives rise to a unique lineage. Another is that stochastic mechanisms play upon the determinative machinery of equipotent early RPCs to drive clonal variability. Here we show that a simple model, based on the independent ﬁring of key fate-inﬂuencing tran- scription factors, can quantitatively account for the intrinsic clonal variance in the zebraﬁsh retina and predict the distributions of neuronal cell types in clones where one or more of these fates are made unavailable. ) Work from many laboratories has uncovered a gene regulatory network (GRN) of key transcription factors (TFs) that control some of the earliest cell fate decisions among the ﬁve main neuronal cell types of the vertebrate retina (Figure 1A; reviewed in Boije et al., 2014; Xiang, 2013). This GRN is activated when the repressive TF, Vsx2, is downregulated in RPCs, thus releasing these cells to express various fate-specifying TFs (Bur- meister et al., 1996; Levine and Green, 2004; Vitorino et al., 2009). The ﬁrst of these is the bHLH TF, Atoh7, which is neces- sary and sufﬁcient for the generation of ganglion cells (GCs) (Hernandez et al., 2007; Kanekar et al., 1997; Liu et al., 2001; Pra- sov et al., 2012; Vetter and Brown, 2001; Yang et al., 2003). Vsx2 downregulation also de-represses FoxN4, which turns on Ptf1a, a TF that is necessary and sufﬁcient for the generation of ama- crine cells (ACs) and horizontal cells (HCs), and is capable of overriding Atoh7’s GC-promoting activity (Dullin et al., 2007; Fu- jitani et al., 2006; Jusuf et al., 2011; Lelie` vre et al., 2011; Vitorino et al., 2009). Some Ptf1a expressing cells co-express Lhx1, and these adopt HC fates (Boije et al., 2013; Lelie` vre et al., 2011). Other cells, released from Vsx2 repression, express Vsx1 and give rise to the majority of bipolar cells (BCs) in the zebraﬁsh retina (Chow et al., 2001; Ohtoshi et al., 2001; Vitorino et al., 2009). The Independent Probabilistic Firing of Transcription Factors: A Paradigm for Clonal Variability in the Zebraﬁsh Retina http://dx.doi.org/10.1016/j.devcel.2015.08.011 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). SUMMARY Finally, a small proportion of RPCs in the zebraﬁsh retina re-express Vsx2 and give rise to Muller cells (MCs) and a single subclass of BCs, distinct from the subclasses that express Vsx1 (Burmeister et al., 1996; Livne-Bar et al., 2006; Vitorino et al., 2009). Within this GRN, photoreceptors (PRs) can be considered as a default fate (Dorval et al., 2006; Le et al., 2006; Toy et al., 2002). While the exploration of this GRN has revealed several of the earliest TFs involved in cell fate diversiﬁcation in the retina, little light has been shed on how Atoh7, Ptf1a, Lhx1, and Vsx1 come to be expressed in a way that ensures that all of the main retinal cell types are generated in consistent proportions. In Brief Boije et al. show that a simple model, based on independent and probabilistic ﬁring of key fate-inﬂuencing transcription factors, can quantitatively account for intrinsic clonal variance in zebraﬁsh retina. Robust distributions generated through stochastic processes can explain how one retina is almost invariant from the next despite high clonal variability. d A simple quantitative model can explain clonal variability in the retina d This model is based on the ﬁring probabilities of key transcription factors d These probabilities are shown to be largely independent of each other d The environment has only a minor effect on these probabilities Developmental Cell Article Developmental Cell Article 532 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors Generation of Clones In order to generate retinal clones in which all cell types could be identiﬁed based on nuclear position and reporter gene expres- sion, blastomeres were transplanted from H2B-GFP; Ptf1a- dsRed double-transgenic zebraﬁsh embryos into WT embryos at 3.5 hr post fertilization (hpf) (Figure 1B). H2B-GFP labels all nuclei while Ptf1a-dsRed is expressed in cells destined to become HCs or ACs (Jusuf and Harris 2009). The cell-cycle of RPCs during optic cup formation is very slow but speeds up by at least a factor of four at about 24 hpf (Li et al., 2000), making this an ideal time point to screen host embryos for single, isolated, GFP-labeled cells in the optic cup (Figure 1C). We also found pairs of labeled cells that appeared to be derived from a single progenitor that had recently divided (see Supple- mental Experimental Procedures). However, as we did not actu- ally witness the divisions generating these pairs, we classiﬁed single-cell origin and two-cell origin clones separately. By 72 hpf, central retinal development is complete with radial clones generated by the transplanted cells, allowing quantiﬁcation of their size and fate composition (Figures 1D and 1E; Tables S1 and S2). The cell fate distribution in these clones agrees well with previously published fate distributions in the zebraﬁsh retina (He et al., 2012). The model (shown in Figure 2) initiates at the time when RPCs in the retina begin to cycle rapidly at around 24 hpf. After three rounds of symmetric PP divisions, the eight descendants of a RPC reach the ﬁrst neurogenic phase where they may upregu- late the two TFs, Ptf1a and Atoh7, with ﬁxed probabilities (i.e., pAtoh7, pPtf1a) (Figure 2B). This creates four classes of RPCs (Fig- ure 2A): those that express Atoh7 but not Ptf1a and as a result generate GCs through PD divisions; those that express of Ptf1a but not Atoh7 and generate ACs and HCs through terminal DD divisions; those that express both Atoh7 and Ptf1a leading to the production of ACs and HCs through PD divisions; and ﬁnally, those that express neither Atoh7 nor Ptf1a. Cells in this last class produce BCs and PRs through terminal DD divisions or remain proliferative (PP). INTRODUCTION It is estimated that the human brain contains over 100 billion cells of more than 10,000 different types (Azevedo et al., 2009). Understanding how all of these cells are generated in the correct proportions is one of the great challenges of devel- opmental neuroscience. To address this question, it is critical to investigate how individual CNS progenitors generate clones of mature neurons. In the vertebrate CNS, it is known that retinal progenitor cells (RPCs) at the optic cup stage are multipotent and give rise to clones that are highly variable both in size and neuronal fate composition (Holt et al., 1988; Turner and Cepko, 1987; Wetts and Fraser, 1988). The ﬁnding that clones derived from isolated individual rat RPCs grown in vitro are just as var- iable as those in vivo, suggests that this variability is an intrinsic property of RPCs (Cayouette et al., 2003). One possibility is that these RPCs are individually programmed to go through unique and determined lineage trees. An alternative possibility, how- ever, is that early RPCs are essentially equivalent but that probabilistic mechanisms drive differences in clonal sizes and compositions. The latter hypothesis is supported by recent studies on rat and zebraﬁsh RPCs, where it has been shown that simple stochastic models can accurately account for the It seemed possible that the probabilistic ﬁring of the genes en- coding the TFs released from Vsx2 repression in this network could explain the variability of fate distributions within clones. To test this idea, we developed a simple model based on the assumption that these TFs ﬁre probabilistically and indepen- dently of each other within a set of equipotent RPCs. We then tested this model against a large array of clonal datasets from RPCs in which we deliberately perturbed the probabilities of 532 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors expressing each of these factors individually. In all cases, this model was capable of making good predictions about the distri- butions of cellular compositions and sizes of clones arising from these RPCs. We thus conclude that the independent and prob- abilistic expression of these TFs is capable of explaining most of the variance in cell type composition seen in zebraﬁsh retinal clones. (A) A core network of four key TFs can explain much of the cellular diversity in the retina. expressing each of these factors individually. INTRODUCTION In all cases, this model was capable of making good predictions about the distri- butions of cellular compositions and sizes of clones arising from these RPCs. We thus conclude that the independent and prob- abilistic expression of these TFs is capable of explaining most of the variance in cell type composition seen in zebraﬁsh retinal clones. 2003). The model is simply based on the idea that the genes en- coding these key TFs in this GRN ﬁre probabilistically and inde- pendently of each other. RPCs progress through three distinct phases starting with a proliferative phase in which cells divide symmetrically, termed PP type. The proliferative phase is followed by the ﬁrst neuro- genic phase in which RPCs choose between all three modes of division, PP, PD and DD, according to deﬁned probabilities, and a late neurogenic phase in which the predominant mode of division is terminal, DD (He et al., 2012; Livesey and Cepko, 2001). Atoh7 is upregulated prior to mitosis in PD divisions lead- ing to one differentiating and one proliferative cell (He et al., 2012), while Ptf1a is expressed immediately following mitosis (He et al., 2012; Jusuf and Harris, 2009; Poggi et al., 2005). Based on this behavior and additional studies that suggest that the TFs Atoh7, Ptf1a, and Vsx are expressed during restricted time windows, we sought to deﬁne the simplest model of fate choice that is compatible with this observed progression (Boije et al., 2008; Brzezinski et al., 2012; Decembrini et al., 2009; Vitor- ino et al., 2009). Generation of Clones As there are an almost equal numbers of PRs and BCs in the zebraﬁsh retina, we suggest that the decision between these two fates can be effectively described as a ‘‘coin ﬂip.’’ Half of these differentiating cells will express Vsx and become BCs, while the other half will become PRs. We deﬁne one more parameter, which reﬂects the probability of differenti- ating (the neurogenic probability, png). After three cell cycles in the ﬁrst phase of neurogenesis, any remaining RPCs enter a sec- ond neurogenic phase, where pAtoh7 and pPtf1a both drop to zero Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 533 Figure 2. Clone Size and Cell Fate Distribu- tions of Retinal Clones Can Be Recapitu- lated by a Minimal Model (A) Combinatorial expression of Ptf1a and Atoh7 gives rise to four distinct groups that adopt fates differently, and where cells either continue to proliferate (P) or differentiate (D). (B) Temporal progression of the probabilities of expressing Atoh7, Ptf1a and undergoing a neurogenic division (png). (C) Averages of the fate distributions generated in the experimental clones compared with a set of virtual RPCs allowed to ﬂow through the model. Error bars depict SDs. (D) Clone size distribution of binned experimental data (black boxes) compared with the model. Note that in this and in similar ﬁgures the shaded re- gions denote the expected variation (95% conﬁ- dence intervals) around the theoretical curves due to the limited experimental sample size. (E) Cell fate distributions in experimental clones compared with the model. For visualization rea- sons PRs and BCs are merged but individual dis- tributions are available in Figure S2. P, proliferative progenitor; D, differentiated cell. See also Table S4. Ptf1a, or Vsx1 into WT hosts, thus keeping the environment constant. while png remains unchanged (see Supplemental Experimental Procedures). Our next challenge was to assign ﬁxed values to pAtoh7, pPtf1a, and png. Previous studies of clone size distributions in zebraﬁsh found that, in late-stage retinal development, roughly 80% of divisions are of DD type, which translates to png = 0.8 (He et al., 2012). With png constrained, we were then left with just two parameters to ﬁt from analysis of the experimental data. We calculated these by minimizing the sum of the squared errors between the mean cell numbers predicted by the model and the corresponding mean values obtained in the above clonal dataset, from WT RPCs in WT hosts. Thus, pAtoh7 = 0.32 ± (0.04, 0.03), and pPtf1a = 0.30 ± (0.04, 0.05) (Figure 2B). With all parameters ﬁxed by calibration to the WT mean values, we then asked whether a theoretical set of RPCs following the model dynamics could produce a set of clones that match not only the means but also higher moments of the experimental distributions. Signiﬁcantly, as well as capturing the average abundances of the various cell types (Figure 2C), this simple model also provides an excellent ﬁt to the detailed distributions for both cell numbers and cell fates (Figures 2D, 2E, and S2; Table S4). Figure 2. Clone Size and Cell Fate Distribu- tions of Retinal Clones Can Be Recapitu- lated by a Minimal Model Clones derived from RPCs of Atoh7 morphant embryos re- sulted in a major (93%) reduction in the number of GCs (Figures 3A and 3C; Table S3). Concomitantly, there was an increase in the average number of ACs/HCs, PRs, and BCs as well as a signiﬁcantly larger average clone size (Figures 3A, 3B, and S3A). To test the model against the experimental data, we simply reduced the probability pAtoh7 by the same 93%, while the other parameters remained unchanged. We accounted for the fact that RPCs that would have undergone asymmetrical PD divisions, due to Atoh7 expression, now undergo symmetric PP divisions (He et al., 2012). The model then faithfully recapitulates the data (Figures 4A and 4B; Table S4). Tellingly, while the number of ACs/HCs was increased, the proportion of ACs/HCs within these clones was not signiﬁcantly different (Figures S3A and S3B). This suggests that the additional cells, generated by the in- crease in PP-divisions, have the same probability of expressing Ptf1a as WT RPCs. Also, the 50/50 split between PRs and BCs observed in WT clones was preserved following the increase in both populations due to the loss of GCs, strongly suggesting that the reduction in pAtoh7 did not affect the probability of ex- pressing Vsx (Figure 4B, inset). 534 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors Modeling Cell Fate Distributions To derive a model for the generation of clonal cell type variability, we began by suggesting that the downregulation of Vsx2 allows RPCs to express the key TFs in this fate specifying GRN with certain ﬁxed probabilities. The three TFs we consider here are Atoh7, Ptf1a, and Vsx (which includes Vsx1 and Vsx2) (Figure 1A) (Chow et al., 2001; Fujitani et al., 2006; He et al., 2012; Jusuf et al., 2011; Le et al., 2006; Vitorino et al., 2009; Yang et al., Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 533 Figure 2. Clone Size and Cell Fate Distribu- tions of Retinal Clones Can Be Recapitu- lated by a Minimal Model (A) Combinatorial expression of Ptf1a and Atoh7 gives rise to four distinct groups that adopt fates differently, and where cells either continue to proliferate (P) or differentiate (D). (B) Temporal progression of the probabilities of expressing Atoh7, Ptf1a and undergoing a neurogenic division (png). (C) Averages of the fate distributions generated in the experimental clones compared with a set of virtual RPCs allowed to ﬂow through the model. Error bars depict SDs. (D) Clone size distribution of binned experimental data (black boxes) compared with the model. Note that in this and in similar ﬁgures the shaded re- gions denote the expected variation (95% conﬁ- dence intervals) around the theoretical curves due to the limited experimental sample size. (E) Cell fate distributions in experimental clones compared with the model. For visualization rea- sons PRs and BCs are merged but individual dis- tributions are available in Figure S2. P, proliferative progenitor; D, differentiated cell. See also Table S4. Figure 2. Clone Size and Cell Fate Distribu- tions of Retinal Clones Can Be Recapitu- lated by a Minimal Model Figure 2. Clone Size and Cell Fate Distribu- tions of Retinal Clones Can Be Recapitu- lated by a Minimal Model Figure 3. Intrinsic Impact on Cell Fate While Vsx2-positive BCs are all of the same S4 subtype in WT embryos with a single terminal button stratifying in the IPL (Connaughton and Nelson, 2000; Vitorino et al., 2009), the Vsx2-positive BCs in Vsx1 morphant clones stratify in multiple layers with varying complexity, as is seen within the Vsx1 lineage (Figures S4A and S4B). Thus, the probability of ex- pressing either Vsx1 or Vsx2 appears to make no difference to the probability of expressing Atoh7 or Ptf1a. Moreover, the ratio of BCs and PRs, which is linked to the expression of Vsx, remains constant at one-half in all these datasets, which strongly suggests that the expression of Vsx is independent of Ptf1a and Atoh7. To further challenge the basis of the independent probabilistic model, we then compared its behavior to two alternative models based on a component of interdependent TF expression (see Supplemental Information). In the ﬁrst alternative model, Ptf1a and Atoh7 are positively interdependent; i.e., the knockdown of Ptf1a leads to a knockdown in Atoh7 and vice versa. In the second alternative model, Ptf1a and Atoh7 are negatively inter- dependent; i.e., the knockdown of Ptf1a leads to overexpression of Atoh7 and vice versa. As the probabilities of TF factor expres- sion in all these models were derived from the means of the WT to WT dataset, it is not surprising that all three models ﬁt the WT to WT dataset (Figure S7). However, both interdependent models fail when they are asked to predict clonal distributions derived from Atoh7 and the Ptf1a morphant RPCs (Figure S7). This analysis shows that a simple model of independent TF expression does a good job at explaining the experimental out- comes in the different treatments, whereas introducing an inter- dependent component makes the predictions worse (Figure S7). Taken together, these results strongly support the view that the independent probabilistic expression of TFs is the simplest To challenge the model even further, we asked whether it could predict size and fate distributions of clones generated by Atoh7, Ptf1a double morphant RPCs, i.e., RPCs in which GC, AC, and HC fates are compromised. Our data show that such clones are signiﬁcantly larger than WT clones and contain, as ex- pected, primarily PRs and BCs (Figures 3A and 3B). We modeled the double knockdown by reducing the probabilities of express- ing Atoh7 and Ptf1a by the same amounts as estimated for single morphants individually. Testing the Independent Firing of TFs Moreover, the ratio of BCs and PRs, which is linked to the expression of Vsx, remains constant at one-half in all these datasets, which strongly suggests that the expression of Vsx is independent of Ptf1a and Atoh7. To challenge the model even further, we asked whether it could predict size and fate distributions of clones generated by Atoh7, Ptf1a double morphant RPCs, i.e., RPCs in which GC, AC, and HC fates are compromised. Our data show that such clones are signiﬁcantly larger than WT clones and contain, as ex- pected, primarily PRs and BCs (Figures 3A and 3B). We modeled the double knockdown by reducing the probabilities of express- ing Atoh7 and Ptf1a by the same amounts as estimated for single morphants individually. The model again does a good job of pre- dicting clone size an Table S4). Thus, in a mental data strongly clones in which a pa selves among the re with the stochastic r pAtoh7 and pPtf1a wer that all these distribu we ﬁnd it striking tha theoretical prediction To further challeng model, we then com based on a compon Supplemental Inform and Atoh7 are posit of Ptf1a leads to a k second alternative m dependent; i.e., the k of Atoh7 and vice ve sion in all these mod to WT dataset, it is n to WT dataset (Fig models fail when the derived from Atoh7 This analysis shows expression does a g comes in the differen dependent compone Taken together, thes independent probab the distributions of cell fates and cell numbers (Figures 4C and 4D; Table S4). dicting clone size an Table S4). Thus, in a Figure 3. Intrinsic Impact on Cell Fate (A) Fate distribution of clones generated by Ptf1a, Atoh7, or Vsx1 morphant cells transplanted into WT hosts. * indicates signiﬁcance for the merged p values of one- and two-cell compared with WT, while # denotes signiﬁcant difference between one- and two-cell clones within a particular morphant. /# p < 0.05, /## p < 0.01, */### p < 0.001. Error bars depict SEM. The ﬁgure legend in (A) is also valid for (B). For the number of clones for the different treatments, see Table S1, and for statistical calculations, see Table S3. (B) Average clone sizes generated from single RPCs scored at 24 hpf for the various morphants in WT environment. (B) Average clone sizes generated from single RPCs scored at 24 hpf for the various morphants in WT environment. (C–F) Representative micrographs of clones generated by Atoh7, Ptf1a, or Vsx1 morphant cells in a WT environment, respectively. The red chan- nel is shown individually for the Ptf1a morphant clone in (E) to reveal the fate switch performed by the Ptf1a lineage to PRs, BCs (marked by arrow head) and GCs (marked by hollow arrow head). Arrowhead in (F) indicates a Ptf1a-positive BC. The scale bar in (C) represents 10 mm and is also valid for (D)–(F). See also Figure S4 and Table S2. the distributions of cell fates and cell numbers (Figures 4C and 4D; Table S4). dicting clone size and fate redistributions (Figures 4G and 4H; Table S4). Thus, in all the cases that we examined, the experi- mental data strongly support the suggestion that the cells of clones in which a particular fate is unavailable distribute them- selves among the remaining fates in a manner that is consistent with the stochastic rules of the model. Considering the fact that pAtoh7 and pPtf1a were calibrated only against the WT means and that all these distributions were deduced without further ﬁtting, we ﬁnd it striking that the experimental distributions match the theoretical predictions so well. We also looked at clones generated from Vsx1 morphant RPCs in a WT environment. However, as Vsx1 and Vsx2 are reciprocally repressive, the reduction of Vsx1-positive BCs leads to a compensating increase in Vsx2-positive BCs (Figures S4A and 4B). Thus, the model, which treats Vsx1 and Vsx2 as equivalent TFs, and the data agree well with each other and with the results from WT RPCs (Figures 3A, 3B, 3F, 4E, and 4F). Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 535 Testing the Independent Firing of TFs Ptf1a morphants displayed a 79% decrease in ACs and HCs, allowing us to estimate pPtf1a = 0.06 for these RPCs (Figures 3A, 3D, and 3E). The reduced probability of making ACs and HCs in morphant clones, according to our model, should trans- late into an increased probability of making other cell types, and indeed, these clones showed such increases (Figure 3A). As expected, Ptf1a morphant clones showed no signiﬁcant change in clone size compared with WT clones, suggesting that Ptf1a knockdown does not signiﬁcantly affect the distribu- tion of division modes as is reﬂected in the model (Figure 3B). Assuming that Ptf1a knockdown does not produce additional PP divisions, the model again does a good job at predicting A basic prediction of our model is that when the choice of a particular cell type becomes unavailable due to the knockdown of a single TF, cells must choose among all the other available cell types in accordance with set probabilities. Speciﬁcally, the model posits that pAtoh7 and pPtf1a are speciﬁed independently of each other, and if this assumption is right, the independent probabilistic model should be able to predict the distributions of clone sizes and compositions from RPCs in which these factors are individually knocked down. To test the model exper- imentally, we transplanted blastomeres from H2B-GFP; Ptf1a- dsRed embryos injected with morpholinos targeting Atoh7, 534 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors the distributions of cell fates and cell numbers (Figures 4C and 4D; Table S4). We also looked at clones generated from Vsx1 morphant RPCs in a WT environment. However, as Vsx1 and Vsx2 are reciprocally repressive, the reduction of Vsx1-positive BCs leads to a compensating increase in Vsx2-positive BCs (Figures S4A and 4B). Thus, the model, which treats Vsx1 and Vsx2 as equivalent TFs, and the data agree well with each other and with the results from WT RPCs (Figures 3A, 3B, 3F, 4E, and 4F). While Vsx2-positive BCs are all of the same S4 subtype in WT embryos with a single terminal button stratifying in the IPL (Connaughton and Nelson, 2000; Vitorino et al., 2009), the Vsx2-positive BCs in Vsx1 morphant clones stratify in multiple layers with varying complexity, as is seen within the Vsx1 lineage (Figures S4A and S4B). Thus, the probability of ex- pressing either Vsx1 or Vsx2 appears to make no difference to the probability of expressing Atoh7 or Ptf1a. A Minor Inﬂuence of Extrinsic Feedback Several studies suggest that the retinal environment ﬁne tunes fate assignments (Yang, 2004). To look for extrinsic inﬂuences, we simply reversed the experimental situation and transplanted WT RPCs, marked by H2B-GFP and Ptf1a-dsRed expression, into different morphant or mutant environments (Figures 6 and S6). Previous experiments in which WT cells were transplanted into lak mutant zebraﬁsh, which have a mutation in the Atoh7 gene, indicated an increase in GCs in such clones (Poggi et al., 2005). Unexpectedly, in the present study, using Atoh7 mor- phants as hosts, we did not observe this homeostatic compen- sation of GCs, but found instead simply a larger average clone size (Figures 6A–6C). To see whether the discrepancy was due to the use of morphant rather than mutant hosts, we also analyzed clones generated by WT RPCs transplanted into lak mutant retinas, but the results were essentially identical (Figures S6A and S6B). Interestingly, we also observed HCs displaced in the GC layer in morphant hosts, suggesting that the abnormal plexiform layer formation in these embryos may be responsible for trapping both ACs and HCs (Figures S5E–S5G; Table S5). As currently deﬁned, our simple model does not deal with ACs and HCs as separate populations. To assess whether indepen- dent probabilistic expression of TFs could be a feature of the HC fate decision, we began by investigating the quantitative rela- tionship between Lhx1 and Ptf1a expression. It has previously been noted that Lhx1 is expressed in a subpopulation of HCs (Edqvist et al., 2006; Lelie` vre et al., 2011; Suga et al., 2009). We found, however, that all HCs are generated from the Lhx1 lineage within the larger Ptf1a-positive population (Figure 5A). We also found that there are numerous Lhx1-positive cells, which are not Ptf1a positive, that become PRs (Figures 5B and 5D). If the probabilities of expressing Ptf1a and Lhx1 are inde- pendent of each other, then the proportion of cells that are HCs (i.e., the population of cells that express both TFs) should simply translate to the product of these two probabilities. To ﬁnd these probabilities, we used quadruple transgenic em- bryos (Atoh7-gapGFP; Ptf1a-dsRed; Lhx1-GFP; Crx-gapCFP), which allowed us to accurately quantitate the expression of Ptf1a and Lhx1 in dissociated cells at 72 hpf (Figures 5C and 5D). Figure 3. Intrinsic Impact on Cell Fate See also Figure S7 and Table S4 (C and D) Modeling of clone size and fate distribution of Ptf1a morphant clones in WT hosts. (E and F) Modeling of clone size and fate distribution of Vsx1 morphant clones in WT hosts. (C and D) Modeling of clone size and fate distribution of Ptf1a morphant clones in WT hosts. (E and F) Modeling of clone size and fate distribution of Vsx1 morphant clones in WT hosts. (G and H) Modeling of clone size and fate distribution of Atoh7, Ptf1a double morphant clones See also Figure S7 and Table S4 (C and D) Modeling of clone size and fate distribution of Ptf1a morphant clones in WT hosts. (E and F) Modeling of clone size and fate distribution of Vsx1 morphant clones in WT hosts. (G and H) Modeling of clone size and fate distribution of Atoh7, Ptf1a double morphant clones in WT hosts. See also Figure S7 and Table S4. ( ) g p (G and H) Modeling of clone size and fate distribution of Atoh7, Ptf1a double morphant clones in WT hosts. See also Figure S7 and Table S4 modeling paradigm capable of predicting these experimental distributions. ﬁnding is therefore consistent with the model paradigm for cell fate speciﬁcation conditioned by the independent probabilistic expression of corresponding key TFs. Figure 3. Intrinsic Impact on Cell Fate The model again does a good job of pre- Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 535 Figure 4. Modeling of Intrinsic Factors (A and B) Modeling of clone size (A) and fate distribution (B) of Atoh7 morphant clones in WT hosts. Inset in (A) depicts the part of the network that has been primarily affected in the donor RPCs. Inset in (B) depicts averages of the experimental values and the values from the modeling with corresponding SDs. Dotted red line represents the WT value. (C and D) Modeling of clone size and fate distribution of Ptf1a morphant clones in WT hosts. (E and F) Modeling of clone size and fate distribution of Vsx1 morphant clones in WT hosts. (G and H) Modeling of clone size and fate distribution of Atoh7, Ptf1a double morphant clones in WT hosts. See also Figure S7 and Table S4. Figure 4. Modeling of Intrinsic Factors Figure 4. Modeling of Intrinsic Factors (A and B) Modeling of clone size (A) and fate distribution (B) of Atoh7 morphant clones in WT hosts. Inset in (A) depicts the part of the network that has been primarily affected in the donor RPCs. Inset in (B) depicts averages of the experimental values and the values from the modeling with corresponding SDs. Dotted red line represents the WT value. (C and D) Modeling of clone size and fate distribution of Ptf1a morphant clones in WT hosts. (E and F) Modeling of clone size and fate distribution of Vsx1 morphant clones in WT hosts. (G and H) Modeling of clone size and fate distribution of Atoh7, Ptf1a double morphant clones in WT hosts. See also Figure S7 and Table S4. g g (A and B) Modeling of clone size (A) and fate distribution (B) of Atoh7 morphant clones in WT hosts. Inset in (A) depicts the part of the network that has been primarily affected in the donor RPCs. Inset in (B) depicts averages of the experimental values and the values from the modeling with corresponding SDs. Dotted red line represents the WT value. (C and D) Modeling of clone size and fate distribution of Ptf1a morphant clones in WT hosts. (E and F) Modeling of clone size and fate distribution of Vsx1 morphant clones in WT hosts. (G and H) Modeling of clone size and fate distribution of Atoh7, Ptf1a double morphant clones in WT hosts. 536 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors A Minor Inﬂuence of Extrinsic Feedback E) P t f ll th t Lh 1 Ptf1 b th Lh 1 d Ptf1 Figure 5. HCs Are Generated as the Intersection between the Lhx1 and Ptf1a Lineages (A) The retina of a quadruple transgenic zebraﬁsh (Atoh7-gapGFP; Ptf1a-dsRed; Lhx1-GFP; Crx-gapCFP) at 96 hpf. Individual channels with arrowheads illustrating the overlap between GFP and dsRed. (B) Quadruple transgenic retina at 48 hpf. Arrows indicate Lhx1-GFP, Ptf1a-dsRed double positive cells, i.e., HCs. Individual channels reveal overlap between Crx-CFP and Lhx1-GFP, as indicated by arrowheads. pPR, putative PR. (C) Combinatorial expression in the quadruple transgenic allows identiﬁcation of the different cell types in a dissociated sample. Channel designation below is valid for both (C) and (D). (D) Lhx1-positive PR cell. (E) Percentages of cells that express Lhx1, Ptf1a, or both Lhx1 and Ptf1a. The scale bar in (A) represents 20 mm and is also valid for (B), while the scale bar in (C) represents 5 mm and is also valid for (D). Figure 5. HCs Are Generated as the Intersection between the Lhx1 and Ptf1a Lineages Figure 5. HCs Are Generated as the Intersection between the Lhx1 and Ptf1a Lineages (A) The retina of a quadruple transgenic zebraﬁsh (Atoh7-gapGFP; Ptf1a-dsRed; Lhx1-GFP; Crx-gapCFP) at 96 hpf. Individual channels with arrowheads illustrating the overlap between GFP and dsRed. (B) Quadruple transgenic retina at 48 hpf. Arrows indicate Lhx1-GFP, Ptf1a-dsRed double positive cells, i.e., HCs. Individual channels reveal overlap between Crx-CFP and Lhx1-GFP, as indicated by arrowheads. pPR, putative PR. (C) Combinatorial expression in the quadruple transgenic allows identiﬁcation of the different cell types in a dissociated sample. Channel designation below is valid for both (C) and (D). (D) Lhx1-positive PR cell. (E) Percentages of cells that express Lhx1, Ptf1a, or both Lhx1 and Ptf1a. The scale bar in (A) represents 20 mm and is also valid for (B), while the scale bar in (C) represents 5 mm and is also valid for (D). many HCs in these clones reside in the AC layer (Figures S5A– S5C; Table S5). Since all HCs initially migrate to the AC layer before migrating apically toward the OPL (Edqvist and Hallbo¨ o¨ k, 2004), the fact that intrinsically WT HCs do not make this migration in the morphant hosts suggests the existence of an extrinsic signal. Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 537 A Minor Inﬂuence of Extrinsic Feedback Our counts from four separate experiments revealed that 20.9% ± 2.3% of WT retinal cells expressed Ptf1a and 10.8% ± 1.1% expressed Lhx1, with a predicted intersectional population, assuming independent expression, of 2.3% ± 0.47%. We found that 3.1% ± 0.55% of the dissociated cells ex- pressed both Ptf1a and Lhx1 (i.e., are HCs), which is not statis- tically different from the predicted percentage (Figure 5E). This Similar experiments with WT RPCs in Ptf1a morphant retinas revealed similar extrinsic effects. Compared with WT clones in a WT environment, WT clones in Ptf1a morphant retinas were larger (Figures 6A, 6B, and 6D). However, in this case, there was a clear underproduction of GCs. As extrinsic signaling from GCs is thought to inhibit further production of GCs, the increased numbers of GCs in the Ptf1a MO hosts seemed like 536 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors Figure 5. HCs Are Generated as the Intersection between the Lhx1 and Ptf1a Lineages (A) The retina of a quadruple transgenic zebraﬁsh (Atoh7-gapGFP; Ptf1a-dsRed; Lhx1-GFP; Crx-gapCFP) at 96 hpf. Individual channels with arrowheads illustrating the overlap between GFP and dsRed. (B) Quadruple transgenic retina at 48 hpf. Arrows indicate Lhx1-GFP, Ptf1a-dsRed double positive cells, i.e., HCs. Individual channels reveal overlap between Crx-CFP and Lhx1-GFP, as indicated by arrowheads. pPR, putative PR. (C) Combinatorial expression in the quadruple transgenic allows identiﬁcation of the different cell types in a dissociated sample. Channel designation below is valid for both (C) and (D). (D) Lhx1-positive PR cell. (E) Percentages of cells that express Lhx1, Ptf1a, or both Lhx1 and Ptf1a. The scale bar in (A) represents 20 mm and is also valid for (B), while the scale bar in (C) represents 5 mm and is also valid for (D). igure 5. HCs Are Generated as the Intersection between the Lhx1 and Ptf1a Lineages A) The retina of a quadruple transgenic zebraﬁsh (Atoh7-gapGFP; Ptf1a-dsRed; Lhx1-GFP; Crx-gapCFP) at 96 hpf. Individual channels with arro ustrating the overlap between GFP and dsRed. B) Quadruple transgenic retina at 48 hpf. Arrows indicate Lhx1-GFP, Ptf1a-dsRed double positive cells, i.e., HCs. Individual channels reveal overlap b Crx-CFP and Lhx1-GFP, as indicated by arrowheads. pPR, putative PR. C) Combinatorial expression in the quadruple transgenic allows identiﬁcation of the different cell types in a dissociated sample. Channel designation b alid for both (C) and (D). D) Lhx1-positive PR cell. A Minor Inﬂuence of Extrinsic Feedback This prompted us to see whether a single minor adjustment of the model could account for this effect. Indeed, we were able to describe most of the changesthatoccurinbothmorphantenvironmentsbypostponing the onset of neurogenesis by approximately half a cell cycle, i.e., by assuming that 60% of the RPCs enter neurogenesis one divi on retinal lineages. the extrinsic effect fate distributions in cells from H2B-GFP beled morphant hos 6I) it is clear that mo Figure 6. Extent of Extrinsic Feedback dur- ing Retina Development (A) Fate distribution of clones generated by WT cells transplanted into Ptf1a, Atoh7, Vsx1 or Atoh7, Ptf1a double morphant hosts. indicates signiﬁcance compared with WT, while # denotes signiﬁcant difference between one- and two-cell clones within a particular morphant. Figure legend is also valid for (B). Error bars depict SEM. (B) Average clone size generated from single RPCs scored at 24 hpf for WT cells and the various environments previously described. (C–E) Representative micrographs of WT clones generated in Atoh7, Ptf1a, or Vsx1 morphant en- vironments, respectively. (F) Checker-plot visualizing the extent of intrinsic regulation and extrinsic feedback in the different Atoh7 morphant scenarios. Color denotes devia- tion from WT to WT, such that a brighter color corresponds to an increase while a darker corre- sponds to a decrease of cell numbers of a given cell type. (G and H) The intrinsic model is modiﬁed by the introduction of a delay postponing the onset of neurogenesis in 60% of the RPCs by one division. The resulting clone size (G) and fate (H) distribu- tions match the experimental clones. Inset depicts averages of the experimental values and the values from the modeling with corresponding SDs. (I–K) As in (F)–(H) but for the Ptf1a morphant environment with the same delay introduced to the intrinsic model. The scale bar in (E) represents 10 mm and is also valid for (C) and (D). See also Figures S5 and S6 and Table S5. Lineages in Whole Knockdown Retinas: Combining Intrinsic Potential with Extrinsic Inﬂuence In many knockout studies, both the pro- genitor cells and the environment they develop in are mutant, making it difﬁcult to quantitatively account for intrinsic versus extrinsic effects. A Minor Inﬂuence of Extrinsic Feedback We suggest that ACs might be the origin of this external signal as the failure of HC migration is inversely pro- portional to the number of ACs in the transplanted clones, the only source of ACs in these otherwise AC-less retinas (Figures S5D and S5G). a possible explanation for this underproduction of GCs in the transplanted WT RPCs. However, transplantation of WT RPCs into Atoh7, Ptf1a double morphant hosts in which there are no GCs in the host, revealed the same reduction in GCs generated from the transplanted WT RPCs ruling out this hypothesis (Fig- ures 6A and 6B). As the Ptf1a morpholino only prevents roughly 70% of the ACs from being formed, we also transplanted WT cells into retinas that were further deprived of ACs and HCs, generated by injection of a mixture of two different Ptf1a transla- tion blocking morpholinos. This Ptf1a morpholino mixture elimi- nates 95% of all ACs and HCs, which should further reduce the amount of feedback from generated HCs and ACs (Randlett et al., 2013). WT clones in such retinas are, however, similar to those in which the single morpholino was used (Figures S6A, S6B, and S6G). We also transplanted WT H2B-GFP; Ptf1a-dsRed cells into Vsx1 morphant hosts, but here we found no signiﬁcant extrinsic effects on fate composition or clone size when comparing to WT clones in WT hosts (Figures 6A, 6B, and 6E). This is not surprising considering that the reciprocal repression of Vsx1 and Vsx2 means that the loss of Vsx1 is largely compensated by the upre- gulation of Vsx2 resulting in little change in the number BCs or indeed of any of the main cell types in the morphant retinas. Interestingly, we also noted an apparent reduction in HCs, but no general difference in the number of Ptf1a-positive cells in WT clones that developed in Ptf1a morphant hosts (Figures 6A and S3A). To see whether some of Ptf1a-positive cells in the AC layer were HCs, we transplanted cells from Lhx1-GFP; Ptf1a-dsRed expressing donors into Ptf1a morphant hosts and found that The similar increase in clone sizes and change in fate distribu- tion observedbothin theAtoh7 and Ptf1a morphantenvironments suggests that there might be a uniﬁed explanation for these Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 537 extrinsic inﬂuences (Figures 6F and 6I). 538 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors A Minor Inﬂuence of Extrinsic Feedback To understand what happens in a retina when the expression of particular fate inﬂuencing gene is lost or knocked down in a whole animal, we have to consider mutant or morphant RPCs developing within a mutant or morphant environment, i.e., both the intrinsic and extrinsic inﬂuences etinal lineages. To test whether the model that incorporates extrinsic effect (viz. the delay in neurogenesis) can predict distributions in such scenarios, we transplanted morphant from H2B-GFP; Ptf1a-dsRed transgenic embryos into unla- d morphant hosts. By comparing deviations (Figures 6F and is clear that morphant clones in morphant hosts are roughly ar to morphant clones in WT hosts, showing that intrinsic in- ces dominate in these experiments. extrinsic inﬂuences (Figures 6F and 6I). This prompted us to see whether a single minor adjustment of the model could account for this effect. Indeed, we were able to describe most of the changesthatoccurinbothmorphantenvironmentsbypostponing the onset of neurogenesis by approximately half a cell cycle, i.e., by assuming that 60% of the RPCs enter neurogenesis one divi- sion later than they do in a WT environment (Figures 6G, 6H, 6J, and 6K). The ability of the model to largely predict the complex clonal distribution and compositional data following such a major change in the environmental input provides conﬁdence that the basic model captures the key regulatory machinery. Experimentally, Atoh7 morphant clones in an Atoh7 morphant environment show a large decrease in the frequency of GCs and an increased frequency of all other cell types, accompanied by 538 538 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors Figure 7. Combining the Intrinsic and Extrinsic Effects (A) Fate distribution of clones generated by morphant cells transplanted into morphant hosts. * indicates signiﬁcance compared with WT, while # denotes signiﬁcant difference between 1- and 2-cell clones within a particular morphant. Figure legend is also valid for (B). Error bars depict SEM. (B) Average clone size generated from single RPCs scored at 24 hpf for morphant cells in morphant environments. (C–G) Predicted distributions of clone sizes and cell types obtained by combining the intrinsic and extrinsic theory for various morphant conditions. For individual distributions, see Figure S2. See also Figure S6 and Tables S4 and S6. Figure 7. A Minor Inﬂuence of Extrinsic Feedback Combining the Intrinsic and Extrinsic Effects gu e Co b g e s c a d s c ec s (A) Fate distribution of clones generated by morphant cells transplanted into morphant hosts. * indicates signiﬁcance compared with WT, while # denotes signiﬁcant difference between 1- and 2-cell clones within a particular morphant. Figure legend is also valid for (B). Error bars depict SEM. (B) Average clone size generated from single RPCs scored at 24 hpf for morphant cells in morphant environments. (C–G) Predicted distributions of clone sizes and cell types obtained by combining the intrinsic and extrinsic theory for various morphant conditions. For individual distributions, see Figure S2. See also Figure S6 and Tables S4 and S6. g g (A) Fate distribution of clones generated by morphant cells transplanted into morphant hosts. * indicates signiﬁcance compared with WT, while # denotes signiﬁcant difference between 1- and 2-cell clones within a particular morphant. Figure legend is also valid for (B). Error bars depict SEM. (B) Average clone size generated from single RPCs scored at 24 hpf for morphant cells in morphant environments. (C–G) Predicted distributions of clone sizes and cell types obtained by combining the intrinsic and extrinsic theory for various morphant conditions. For individual distributions, see Figure S2. See also Figure S6 and Tables S4 and S6. an increase in total clone size (Figures 7A and 7B). These results are consistent with clonal data generated by an alternative assay in which WT and Atoh7 morphant embryos had single cells in Maze-Kaede transgenic retinas photoconverted (see Supple- mental Experimental Procedures), allowing us to quantify clonal expansion from single RPCs (Figures S6C–S6F; Table S6). ronment, both the intrinsic and combined model worked equally well, while in the case of Ptf1a morphant clones in an Atoh7 mor- phant environment, the model incorporating the delay due to the extrinsic effect does a better job at predicting clonal distributions than the purely intrinsic model (Table S4). Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 539 DISCUSSION We found that, while the purely intrinsic model generated dis- tributions similar to the experimental clones, we could favorably increase the quality of the ﬁt in the Atoh7 morphant by adding the delay in neurogenesis due to the extrinsic effect of the morphant environment (Figure 7C; Table S4). In the case of Ptf1a morphant clones within a Ptf1a morphant host, the full model also ﬁts the data well, but not signiﬁcantly better than the intrinsic model (Fig- ure 7D). For Vsx1 morphant clones in Vsx1 morphant retinas, as there are no discernable extrinsic effects, the purely intrinsic model continues to ﬁt the data well (Figures 7A, 7B, and 7E). Interestingly, we also challenged the model to predict clonal dis- tributions in cross-morphant scenarios, i.e., clones derived from Atoh7 morphant RPCs in Ptf1a morphant environments or vice versa. Again, the description for the cross morphant clones fol- lowed straightforwardly by combining the extrinsic effects found for the WT to morphant transplantation with the intrinsic model; in the case of Atoh7 morphant clones in a Ptf1a morphant envi- The ﬁndings above show that the variability in the clonal compo- sition seen in the zebraﬁsh retina can be quantitatively explained by the probabilistic and independent ﬁring of fate inﬂuencing TFs. The independent nature of TF expression here means that when one of these TFs is reduced in RPCs, new clonal distribu- tions can be predicted by the unchanged probabilities of the other TFs ﬁring, which we show is also the case. Indeed, the changes in the fraction of total cells expressing Atoh7 and Ptf1a in WT and morphant Spectrum of Fate lines of zebraﬁsh (Almeida et al., 2014 and unpublished data) are in good agree- ment with the results presented here. Our results also show how the independent ﬁring of fate inﬂuencing TFs can robustly generate the regular proportions of all the different neuronal types within the retina from a pool of equipotent progenitors even though there is great clone-to-clone variability. Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 539 539 Despite its success, it is important to note that the model presented here has many limitations. First, it is a minimal model, meant only to cover the major classes of retinal neu- rons. There are, however, many neuronal subtypes of each major class that this model makes no attempt to account for. DISCUSSION Second, to formulate this minimal model, we employed a reduced level of description, capturing only the core transcrip- tional network in the zebraﬁsh retina. This reduction was essen- tial in order to deﬁne a well-constrained and testable model that included biological mechanisms and just two free parame- ters that could be ﬁtted to the means of a WT to WT dataset. It should therefore not be too surprising that such a minimal model does not match all the datasets perfectly. Interestingly, the few signiﬁcant deviations between the predicted and the experimental distributions might be explainable by reasonable biological possibilities (see below). However, the capability of such a simple model to predict clonal statistics (clone compo- sition, average clone sizes, and detailed size distributions) in so many contexts suggests that it might have essential validity, despite its limitations. et al., 2006; Shkumatava et al., 2004; Wall et al., 2009) and quicken the onset of neurogenesis (Shkumatava et al., 2004). It may therefore be that the reduced levels of Shh in both morphant environments cause a delay in the onset of neurogenesis, increasing clone sizes and biasing these lineages toward later fates. Surprisingly, we did not observe the expected homeostatic compensations by WT RPCs in environments lacking GCs and ACs, as has been suggested in some other studies (Jusuf et al., 2011; Poggi et al., 2005; Yang, 2004). Instead, in our hands, WT RPCs in morphant environments did not overpro- duce cell types that were missing from that environment. What may account for these differences? In the previous studies, donor RPCs expressed only the cell-type speciﬁc transgenes, while in the present study, we use H2B-GFP to la- bel all the cells in a clone and Ptf1a-DsRed to label all the ACs and HCs. Thus, for example, in the study of Poggi et al. (2005), it is possible that some of the Atoh7:GFP cells in the GC layer may have been misclassiﬁed as GCs when they were actually displaced ACs. In Atoh7 mutants and morphants, due to the absence of GCs, almost all the cells in the RGC layer are dis- placed amacrine cells (dACs). If these are misclassiﬁed as GCs, it would look like a signiﬁcant increase in GCs. The current techniques would not allow such misclassiﬁcations. DISCUSSION Conﬁdence in the present ﬁndings comes from our ability here to count and identify all the cells, including displaced ACs, in large sets of individual clones, combined with the consistency of the sta- tistical effects in morphants and mutants. The conclusion of minimal homeostatic compensation is independently supported by the fact that rat RPCs in clonal cultures give rise to clonal distributions that are similar to those in vivo (Cayouette et al., 2003); i.e., there is not an overproduction of GCs or ACs in these clones even though they are grown in the absence of any feed- back cues. It is also important to note that this model does not address the ﬁne scale structure of clonal distributions, especially the ‘‘tips’’ of lineages. Indeed, there are a number of cases, highlighted in a recent review, where terminal and penultimate divisions are biased toward particular outcomes (Cepko, 2014). Among these are the symmetric PR-PR, BC-BC, and HC-HC terminal divisions seen in the zebraﬁsh retina (He et al., 2012). Some of these biases in late RPCs are clearly species speciﬁc, as BC-BC pairs are not common in mammals (Gomes et al., 2011). In the case of zebraﬁsh, we suggest that it is the early RPCs in which the choice is apparently stochastic, i.e., at the beginning of neurogenesis, when RPCs sort themselves into one of four intermediary pro- genitor types (Atoh7+Ptf1a, Atoh7Ptf1a+, Atoh7+Ptf1a+, and Atoh7Ptf1a) via the independent probabilistic expression of these factors. Once this sorting period is over, each of these progenitor classes is endowed with a different potential. The model outlined here has three phases. In the ﬁrst of these phases, all cells are proliferative. The retina then enters two suc- cessive phases of neurogenesis. In the early neurogenic phase of mammalian embryos, mainly GCs, ACs/HCs and cone PRs are generated, while BCs and rod PRs largely appear in the late neurogenic phase. Previous studies, in a variety of verte- brates, have also suggested that there are two phases of retinal neurogenesis, with early cell types generated in the ﬁrst and late cell types in the second (Elliott et al., 2008; Georgi and Reh, 2010; Morrow et al., 2008). Our model ﬁts these ﬁndings well, but it posits that both the early and late neurogenic phases are stochastic in the sense that, within each phase, cells have ﬁxed probabilities of expressing particular TFs and of leaving the cell cycle. 540 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors DISCUSSION An interesting question is what drives the cells through these phases. Recent work in mice has shown that Ikaros and Casz1, the vertebrate homologs of Hunchback and Castor, which control temporal identity in Drosophila CNS neu- roblasts, may regulate the early and mid/late phases of retinal neurogenesis (Elliott et al., 2008; Mattar et al., 2015). It will be interesting to understand whether these factors drive the RPCs from one phase to the next and are themselves stochas- tically expressed as has recently been suggested (Barton and Fendrik, 2015). In our previous work, eliminating particular cell types from the zebraﬁsh retina, we have been struck by the small effect on the overall size of the retina (Almeida et al., 2014; Randlett et al., 2013) and that RPCs have a strong intrinsic potential to produce clones of a given mean size (He et al., 2012). In one extreme example, for instance, we used a combination of morpholinos, mutants, and pharmacological agents to generate a retina con- taining only two cell types, namely BCs and PRs (Randlett et al., 2013). Yet these retinas were only slightly smaller than WT retinas. The present model shows how clone size may be speciﬁed relatively independently of fate. In our model it is only the expression of Atoh7 that has an effect on clone size, as Atoh7, besides assigning GC fate, also inﬂuences the mode of RPC division (He et al., 2012). Thus, the model predicts changes in clone size distributions only when Atoh7 is knocked down, but these changes are relatively small, as Atoh7 is only expressed in a minority of RPCs during a brief temporal window. Interestingly, by transplanting WT cells into either Atoh7 or Ptf1a morphants, we also found an extrinsic effect on clone size, as WT RPCs developing in these environments tend to produce larger clones than they do in a WT environment. There is a potential biological explanation for this as both ACs and GCs are sources of Shh, a factor known to affect proliferation in the zebraﬁsh retina (Locker The idea that TFs are probabilistically and independently ex- pressed in retinal precursors means that there should be Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors EXPERIMENTAL PROCEDURES predictable populations of precursors that express certain com- binations of TFs. This is similar to the idea that, in a large popu- lation of dice rolls, there will be a predictable number of snake eyes. Previous work has shown that speciﬁc cell fates in the retina may be greatly inﬂuenced by combinatorial coding mech- anisms (Ohsawa and Kageyama, 2008; Wang and Harris, 2005). If a retinal precursor expressing particular combination of TFs is the product of the probability of expressing each TF individually, then the proportions of certain cell types should simply reﬂect this product of probabilities. Here, we show that this could explain why only about 3% of the cells in the retina are HCs, as these cells represent the intersection of independently ex- pressed Ptf1a and Lhx1. Theoretically, this concept can explain how relatively few TFs could, from a pool of equipotent precur- sors, create a large and well-proportioned array of cell types and subtypes with high ﬁdelity corresponding to the intersection sets of probabilistically expressed TFs. Animals and Transgenic Lines Zebraﬁsh lines were maintained and bred at 26.5C. Embryos were raised at 28.5C or 32C and staged as described previously in hpf (Kimmel et al., 1995). Embryos were treated with 0.003% phenylthiourea (PTU) (Sigma) from 10 hpf to prevent pigmentation. All procedures were performed under the project license PL80/2198 approved by the UK Home Ofﬁce and by the Local Ethical Review Panel at the University of Cambridge. The transgenic lines used have all been described previously and are listed in Supplemental Information. Blastomere Transplantation Embryos from H2B-GFP, Ptf1a-dsRed double transgenic zebraﬁsh were dechorionated by pronase digestion (0.6 mg/ml; Sigma) and placed in agarose molds (Adaptive Science Tools), and one to ﬁve blastomeres were transplanted into an unlabeled embryo at 3.5 hpf using a ﬂame-pulled glass capillary (Sutter instruments, #b100-50-10) connected to a 2 ml syringe. The host embryos were allowed to recover at 32C overnight in agarose- coated dishes in order to catch up developmentally. At 24 hpf, embryos were anaesthetized by 0.04% MS-222 (Sigma) and screened on an upright ﬂuorescent microscope where isolated GFP-positive RPCs could be identi- ﬁed. Position and number of cells were logged before the ﬁsh were placed in individual wells at 28.5C. At 72 hpf, embryos were ﬁxed for 1 hr in 4% PFA, the eye dissected out, and mounted in 1% low melting agarose (Sigma) for imaging. Morpholino Injections Antisense translation blocking morpholinos were obtained from Gene Tools, reconstituted as 1 or 3 mM stock solutions in water, and injected into the yolk at the one-cell stage. Morpholinos targeting Ptf1a, Atoh7, and Vsx1 have all been described previously and sequences are listed in Supplemental Information. Control embryos were injected with 2 ng of standard control mor- pholino from Gene Tools. There are other systems where stochastic phenomena regu- late neural cell fates. For example, in the mouse olfactory epithe- lium, the choice of which receptor a sensory neuron expresses is partially stochastic. However, once one odorant receptor gene is expressed in a sensory cell, all other odorant receptor genes are repressed (Lomvardas et al., 2006). In the case of Dscam and clustered protocadherins, stochasticity is generated at the level of mRNA splicing rather than gene expression (Hattori et al., 2009; Lefebvre et al., 2012). In contrast to these systems, here we quantitatively account for the variability in clonal fates in the retina by a model in which the probabilities of expressing all, none, or any combination of these key fate determining genes is governed by the independent probability of expressing each of them individually. Recent studies into noisy gene expression systems show that stochastic mechanisms can indeed explain such probabilistic ﬁring of genes in multicellular and microbial systems (Boettiger, 2013; Frank, 2013; Rister and Desplan, 2011). It has also been shown that fate determining bHLH TFs in mouse neural progenitor cells oscillate at rates much faster than the cell cycle (Imayoshi et al., 2013). Such oscillations in Atoh7 and Ptf1a, if asynchronous, might explain their indepen- dent probabilities of expression, but there are also several other reasonable possibilities such as gene position in the nucleus or epigenetic variability. Cell Dissociation C Cell suspensions were prepared from freshly dissected retinal tissue from quadruple transgenic embryos (Atoh7-gapGFP; Ptf1a-dsRed; Lhx1-GFP; Crx-gapCFP). Quadruple positive embryos were, at 72 hpf, transferred to cold (4C) Ca2+-free medium (116.6 mM NaCl, 0.67 mM KCl, 4.62 mM Tris; 0.4 mM EDTA [pH 7.8]) (Harris and Messersmith, 1992) supplemented with 100 mg/ml of heparin and 0.04% MS-222. Fifteen to 20 retinas were dissected and transferred to glass Petri dishes. Without disturbing the retinas, the Ca2+- free medium was removed, and 0.25% Trypsin-EDTA was added. After a 10-min incubation at 37C, the trypsin was removed, and the retinas were mechanically dissociated by pipetting using ﬂame-pulled glass Pasteur pipette. For confocal imaging, single-cell suspensions were plated into 35-mm imaging dishes, seeded for 1 hr at 28.5C, followed by imaging. Whatever the molecular mechanisms may be in the case of the zebraﬁsh retina, we show here that the high degree of vari- ability in the lineages of RPC cells can be explained using a sim- ple stochastic model based on these ﬁxed probabilities of TF expression. It is important to note that, in this regard, whether or not a process is stochastic or follows some complicated deterministic rules is a matter of the level of description. Com- plex systems in which many variables interact often produce data that can best be described in terms of probabilities even though at the level of individual elements each of the interac- tions may be determinative. Statistically, however, stochastic processes produce robust and well-behaved distributions, as does the nervous system. This, we propose, is therefore a possible basis for understanding how it is that even though there is a high variability in the size and composition of individual clones, the total number of differentiated cells and the relative proportions of each cell type are almost invariant from one ze- braﬁsh retina to the next. Confocal Image Acquisition and Analysis Retinal clones or entire retinas were imaged under 60 3 (NA = 1.30) or 30 3 (NA = 1.05) silicon oil objectives on an inverted laser-scanning confocal micro- scope (Olympus FV1000) ﬁtted with GaAsP detectors. Image analysis was per- formed using Volocity Software (Perkin Elmer). Based on nuclear position and absence or presence of the Ptf1a reporter gene, the different cell types could be scored. The identity of MCs was difﬁcult to discern, and in many cases, these would be counted as BCs. However, when located among the ACs, MCs were easily spotted, but were still quantiﬁed as unknown. Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 541 REFERENCES Almeida, A.D., Boije, H., Chow, R.W., He, J., Tham, J., Suzuki, S.C., and Harris, W.A. (2014). Spectrum of Fates: a new approach to the study of the developing zebraﬁsh retina. Development 141, 1971–1980. Georgi, S.A., and Reh, T.A. (2010). Dicer is required for the transition from early to late progenitor state in the developing mouse retina. J. Neurosci. 30, 4048– 4061. Azevedo, F.A., Carvalho, L.R., Grinberg, L.T., Farfel, J.M., Ferretti, R.E., Leite, R.E., Jacob Filho, W., Lent, R., and Herculano-Houzel, S. (2009). Equal Azevedo, F.A., Carvalho, L.R., Grinberg, L.T., Farfel, J.M., Ferretti, R.E., Leite, R.E., Jacob Filho, W., Lent, R., and Herculano-Houzel, S. (2009). Equal numbers of neuronal and nonneuronal cells make the human brain an isomet- rically scaled-up primate brain. J. Comp. Neurol. 513, 532–541. Gomes, F.L., Zhang, G., Carbonell, F., Correa, J.A., Harris, W.A., Simons, B.D., and Cayouette, M. (2011). Reconstruction of rat retinal progenitor cell lineages in vitro reveals a surprising degree of stochasticity in cell fate decisions. Development 138, 227–235. numbers of neuronal and nonneuronal cells make the human brain an isomet- rically scaled-up primate brain. J. Comp. Neurol. 513, 532–541. Barton, A., and Fendrik, A.J. (2015). Retinogenesis: stochasticity and the com- petency model. J. Theor. Biol. 373, 73–81. Harris, W.A., and Messersmith, S.L. (1992). Two cellular inductions involved in photoreceptor determination in the Xenopus retina. Neuron 9, 357–372. Boettiger, A.N. (2013). Analytic approaches to stochastic gene expression in multicellular systems. Biophys. J. 105, 2629–2640. Hattori, D., Chen, Y., Matthews, B.J., Salwinski, L., Sabatti, C., Grueber, W.B., and Zipursky, S.L. (2009). Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms. Nature 461, 644–648. Boije, H., Edqvist, P.H., and Hallbo¨ o¨ k, F. (2008). Temporal and spatial expres- sion of transcription factors FoxN4, Ptf1a, Prox1, Isl1 and Lim1 mRNA in the developing chick retina. Gene Expr. Patterns 8, 117–123. He, J., Zhang, G., Almeida, A.D., Cayouette, M., Simons, B.D., and Harris, W.A. (2012). How variable clones build an invariant retina. Neuron 75, 786–798. Boije, H., Shirazi Fard, S., Ring, H., and Hallbook, F. (2013). Forkheadbox N4 (FoxN4) triggers context-dependent differentiation in the developing chick retina and neural tube. Differentiation 85, 11–19. Hernandez, J., Matter-Sadzinski, L., Skowronska-Krawczyk, D., Chiodini, F., Alliod, C., Ballivet, M., and Matter, J.M. (2007). Highly conserved sequences mediate the dynamic interplay of basic helix-loop-helix proteins regulating ret- inogenesis. J. Biol. Chem. 282, 37894–37905. Boije, H., MacDonald, R.B., and Harris, W.A. (2014). Modeling and Statistics The essence of the model is explained in Results and detailed in Supplemental Experimental Procedures. Statistical methods, also detailed in Supplemental Experimental Procedures, are used to compare the experimental clonal distri- butions to distributions generated by computer according to the rules of the model (explained in the Results). Similarly the statistical analyses are straight- forward except for the Goodness of Fit, which is described in Supplemental Experimental Procedures. Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 541 ACKNOWLEDGMENTS Edqvist, P.H., Myers, S.M., and Hallbo¨ o¨ k, F. (2006). Early identiﬁcation of retinal subtypes in the developing, pre-laminated chick retina using the tran- Edqvist, P.H., Myers, S.M., and Hallbo¨ o¨ k, F. (2006). Early identiﬁcation of retinal subtypes in the developing, pre-laminated chick retina using the tran- scription factors Prox1, Lim1, Ap2alpha, Pax6, Isl1, Isl2, Lim3 and Chx10. Eur. J. Histochem. 50, 147–154. We thank Neil Hukriede for supplying the Lhx1-GFP ﬁsh line. B.D.S. and S.R. acknowledge the support of the Wellcome Trust (098357/Z/12/Z). W.A.H. and We thank Neil Hukriede for supplying the Lhx1-GFP ﬁsh line. B.D.S. and S.R. acknowledge the support of the Wellcome Trust (098357/Z/12/Z). W.A.H. and H.B. also acknowledge the support of the Wellcome Trust (100329/Z/12/Z). H.B. was also supported by the Swedish Research Council (2011-7054). scription factors Prox1, Lim1, Ap2alpha, Pax6, Isl1, Isl2, Lim3 and Chx10. Eur. J. Histochem. 50, 147–154. g pp ( ) H.B. also acknowledge the support of the Wellcome Trust (100329/Z/12/Z). H.B. was also supported by the Swedish Research Council (2011-7054). Elliott, J., Jolicoeur, C., Ramamurthy, V., and Cayouette, M. (2008). Ikaros con- fers early temporal competence to mouse retinal progenitor cells. Neuron 60, 26–39. Received: December 16, 2014 Revised: June 29, 2015 Accepted: August 13, 2015 Published: September 3, 2015 Frank, S.A. (2013). Evolution of robustness and cellular stochasticity of gene expression. PLoS Biol. 11, e1001578. Revised: June 29, 2015 Accepted: August 13, 2015 Fujitani, Y., Fujitani, S., Luo, H., Qiu, F., Burlison, J., Long, Q., Kawaguchi, Y., Edlund, H., MacDonald, R.J., Furukawa, T., et al. (2006). Ptf1a determines horizontal and amacrine cell fates during mouse retinal development. Development 133, 4439–4450. Published: September 3, 2015 AUTHOR CONTRIBUTIONS Dullin, J.P., Locker, M., Robach, M., Henningfeld, K.A., Parain, K., Afelik, S., Pieler, T., and Perron, M. (2007). Ptf1a triggers GABAergic neuronal cell fates in the retina. BMC Dev. Biol. 7, 110. H.B. carried out all the experimental work with some help from S.D. S.R. car- ried out the bulk of the modeling and most of the statistical analyses. S.R., H.B., B.S., and W.A.H. worked to frame the essence of the model, and B.S. and W.A.H. supervised the study. H.B., W.A.H., S.R., and B.S. all contributed to writing the manuscript. Edqvist, P.H., and Hallbo¨ o¨ k, F. (2004). Newborn horizontal cells migrate bi-di- rectionally across the neuroepithelium during retinal development. Development 131, 1343–1351. SUPPLEMENTAL INFORMATION Decembrini, S., Bressan, D., Vignali, R., Pitto, L., Mariotti, S., Rainaldi, G., Wang, X., Evangelista, M., Barsacchi, G., and Cremisi, F. (2009). MicroRNAs couple cell fate and developmental timing in retina. Proc. Natl. Acad. Sci. USA 106, 21179–21184. Supplemental Information includes Supplemental Experimental Procedures, seven ﬁgures, and six tables and can be found with this article online at http://dx.doi.org/10.1016/j.devcel.2015.08.011. Dorval, K.M., Bobechko, B.P., Fujieda, H., Chen, S., Zack, D.J., and Bremner, R. (2006). CHX10 targets a subset of photoreceptor genes. J. Biol. Chem. 281, 744–751. 542 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors REFERENCES Reconciling competence and transcriptional hierarchies with stochasticity in retinal lineages. Curr. Opin. Neurobiol. 27, 68–74. Holt, C.E., Bertsch, T.W., Ellis, H.M., and Harris, W.A. (1988). Cellular determi- nation in the Xenopus retina is independent of lineage and birth date. Neuron 1, 15–26. Brzezinski, J.A., 4th, Prasov, L., and Glaser, T. (2012). Math5 deﬁnes the gan- glion cell competence state in a subpopulation of retinal progenitor cells exit- ing the cell cycle. Dev. Biol. 365, 395–413. Imayoshi, I., Isomura, A., Harima, Y., Kawaguchi, K., Kori, H., Miyachi, H., Fujiwara, T., Ishidate, F., and Kageyama, R. (2013). Oscillatory control of fac- tors determining multipotency and fate in mouse neural progenitors. Science 342, 1203–1208. Burmeister, M., Novak, J., Liang, M.Y., Basu, S., Ploder, L., Hawes, N.L., Vidgen, D., Hoover, F., Goldman, D., Kalnins, V.I., et al. (1996). Ocular retarda- tion mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nat. Genet. 12, 376–384. Jusuf, P.R., and Harris, W.A. (2009). Ptf1a is expressed transiently in all types of amacrine cells in the embryonic zebraﬁsh retina. Neural Dev. 4, 34. Cayouette, M., Barres, B.A., and Raff, M. (2003). Importance of intrinsic mech- anisms in cell fate decisions in the developing rat retina. Neuron 40, 897–904. Jusuf, P.R., Almeida, A.D., Randlett, O., Joubin, K., Poggi, L., and Harris, W.A. (2011). Origin and determination of inhibitory cell lineages in the vertebrate retina. J. Neurosci. 31, 2549–2562. Cepko, C. (2014). Intrinsically different retinal progenitor cells produce speciﬁc types of progeny. Nat. Rev. Neurosci. 15, 615–627. Kanekar, S., Perron, M., Dorsky, R., Harris, W.A., Jan, L.Y., Jan, Y.N., and Vetter, M.L. (1997). Xath5 participates in a network of bHLH genes in the devel- oping Xenopus retina. Neuron 19, 981–994. Chow, R.L., Snow, B., Novak, J., Looser, J., Freund, C., Vidgen, D., Ploder, L., and McInnes, R.R. (2001). Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells. Mech. Dev. 109, 315–322. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T.F. (1995). Stages of embryonic development of the zebraﬁsh. Dev. Dyn 203, 253–310. Connaughton, V.P., and Nelson, R. (2000). Axonal stratiﬁcation patterns and glutamate-gated conductance mechanisms in zebraﬁsh retinal bipolar cells. J. Physiol. 524, 135–146. Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 542 Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors Poggi, L., Vitorino, M., Masai, I., and Harris, W.A. (2005). REFERENCES Inﬂuences on neural lineage and mode of division in the zebraﬁsh retina in vivo. J. Cell Biol. 171, 991–999. Le, T.T., Wroblewski, E., Patel, S., Riesenberg, A.N., and Brown, N.L. (2006). Math5 is required for both early retinal neuron differentiation and cell cycle pro- gression. Dev. Biol. 295, 764–778. Prasov, L., Nagy, M., Rudolph, D.D., and Glaser, T. (2012). Math5 (Atoh7) gene dosage limits retinal ganglion cell genesis. Neuroreport 23, 631–634. Lefebvre, J.L., Kostadinov, D., Chen, W.V., Maniatis, T., and Sanes, J.R. (2012). Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 488, 517–521. Randlett, O., MacDonald, R.B., Yoshimatsu, T., Almeida, A.D., Suzuki, S.C., Wong, R.O., and Harris, W.A. (2013). Cellular requirements for building a retinal neuropil. Cell Rep. 3, 282–290. Lelie` vre, E.C., Lek, M., Boije, H., Houille-Vernes, L., Brajeul, V., Slembrouck, A., Roger, J.E., Sahel, J.A., Matter, J.M., Sennlaub, F., et al. (2011). Ptf1a/ Rbpj complex inhibits ganglion cell fate and drives the speciﬁcation of all hor- izontal cell subtypes in the chick retina. Dev. Biol. 358, 296–308. Rister, J., and Desplan, C. (2011). The retinal mosaics of opsin expression in invertebrates and vertebrates. Dev. Neurobiol. 71, 1212–1226. Levine, E.M., and Green, E.S. (2004). Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors. Semin. Cell Dev. Biol. 15, 63–74. Shkumatava, A., Fischer, S., Mu¨ ller, F., Strahle, U., and Neumann, C.J. (2004). Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebraﬁsh retina. Development 131, 3849–3858. Li, Z., Hu, M., Ochocinska, M.J., Joseph, N.M., and Easter, S.S., Jr. (2000). Modulation of cell proliferation in the embryonic retina of zebraﬁsh (Danio re- rio). Dev. Dyn 219, 391–401. Suga, A., Taira, M., and Nakagawa, S. (2009). LIM family transcription factors regulate the subtype-speciﬁc morphogenesis of retinal horizontal cells at post- migratory stages. Dev. Biol. 330, 318–328. Liu, W., Mo, Z., and Xiang, M. (2001). The Ath5 proneural genes function up- stream of Brn3 POU domain transcription factor genes to promote retinal gan- glion cell development. Proc. Natl. Acad. Sci. USA 98, 1649–1654. Toy, J., Norton, J.S., Jibodh, S.R., and Adler, R. (2002). Effects of homeobox genes on the differentiation of photoreceptor and nonphotoreceptor neurons. Invest. Ophthalmol. Vis. Sci. 43, 3522–3529. Livesey, F.J., and Cepko, C.L. (2001). Vertebrate neural cell-fate determina- tion: lessons from the retina. Nat. Rev. Neurosci. 2, 109–118. Turner, D.L., and Cepko, C.L. (1987). REFERENCES A common progenitor for neurons and glia persists in rat retina late in development. Nature 328, 131–136. Livne-Bar, I., Pacal, M., Cheung, M.C., Hankin, M., Trogadis, J., Chen, D., Dorval, K.M., and Bremner, R. (2006). Chx10 is required to block photore- ceptor differentiation but is dispensable for progenitor proliferation in the post- natal retina. Proc. Natl. Acad. Sci. USA 103, 4988–4993. Vetter, M.L., and Brown, N.L. (2001). The role of basic helix-loop-helix genes in vertebrate retinogenesis. Semin. Cell Dev. Biol. 12, 491–498. Vitorino, M., Jusuf, P.R., Maurus, D., Kimura, Y., Higashijima, S., and Harris, W.A. (2009). Vsx2 in the zebraﬁsh retina: restricted lineages through derepres- sion. Neural Dev. 4, 14. Locker, M., Agathocleous, M., Amato, M.A., Parain, K., Harris, W.A., and Perron, M. (2006). Hedgehog signaling and the retina: insights into the mech- anisms controlling the proliferative properties of neural precursors. Genes Dev. 20, 3036–3048. Wall, D.S., Mears, A.J., McNeill, B., Mazerolle, C., Thurig, S., Wang, Y., Kageyama, R., and Wallace, V.A. (2009). Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity. J. Cell Biol. 184, 101–112. Lomvardas, S., Barnea, G., Pisapia, D.J., Mendelsohn, M., Kirkland, J., and Axel, R. (2006). Interchromosomal interactions and olfactory receptor choice. Cell 126, 403–413. Wang, J.C., and Harris, W.A. (2005). The role of combinational coding by ho- meodomain and bHLH transcription factors in retinal cell fate speciﬁcation. Dev. Biol. 285, 101–115. Mattar, P., Ericson, J., Blackshaw, S., and Cayouette, M. (2015). A conserved regulatory logic controls temporal identity in mouse neural progenitors. Neuron 85, 497–504. Wetts, R., and Fraser, S.E. (1988). Multipotent precursors can give rise to all major cell types of the frog retina. Science 239, 1142–1145. Morrow, E.M., Chen, C.M., and Cepko, C.L. (2008). Temporal order of bipolar cell genesis in the neural retina. Neural Dev. 3, 2. Xiang, M. (2013). Intrinsic control of mammalian retinogenesis. Cell. Mol. Life Sci. 70, 2519–2532. Ohsawa, R., and Kageyama, R. (2008). Regulation of retinal cell fate speciﬁca- tion by multiple transcription factors. Brain Res. 1192, 90–98. Yang, X.J. (2004). Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis. Semin. Cell Dev. Biol. 15, 91–103. Ohtoshi, A., Justice, M.J., and Behringer, R.R. (2001). Isolation and character- ization of Vsx1, a novel mouse CVC paired-like homeobox gene expressed during embryogenesis and in the retina. Biochem. Biophys. Res. Commun. 286, 133–140. Developmental Cell 34, 532–543, September 14, 2015 ª2015 The Authors 543 REFERENCES Yang, Z., Ding, K., Pan, L., Deng, M., and Gan, L. (2003). Math5 determines the competence state of retinal ganglion cell progenitors. Dev. Biol. 264, 240–254.
https://openalex.org/W2109420298	https://academicjournals.org/journal/AJMR/article-full-text-pdf/0CD8FF916036.pdf	English	null	Levels of Sclerotium rolfsii inoculum influence identification of resistant genotypes in Jerusalem artichoke	African journal of microbiology research	2,012	cc-by	4,710	Corresponding author. E-mail: sanun@kku.ac.th. Tel: +66 043 364637. Fax: +66 043 364637. Full Length Research Paper Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen Khon Kaen 40002, Thailand. Accepted 24 September, 2012 Improvement of Jerusalem artichoke genotypes with resistance to stem rot caused by the soil borne fungus Sclerotium rolfsii is a sustainable means for controlling the disease. However, this crop is rather new to breeders. A consensus screening procedure for resistance to the disease is not yet available. The aim of this study was to determine the level of inoculum that provides the reliable and effective results for screening trials. In the experiment in Khon Kaen, Thailand, four levels of sorghum seed infested with S. rolfsii (1, 2, 3 or 4 seeds/plant) were tested with 10 Jerusalem artichoke genotypes. Plants inoculated with one sorghum seed had the lowest disease incidence, whereas plants inoculated with four sorghum seeds had the highest disease incidence. Most pairings of inoculum levels were statistically different for disease incidence except for two vs. three seeds. In addition, permanent wilting and area under disease progress curve occurred more rapidly with four seeds compared to the other inoculum levels. The highest variation among Jerusalem artichoke genotypes was observed in the plants inoculated with three sorghum seeds. Therefore, three sorghum seeds inoculum was suitable to identify Jerusalem artichoke genotypes resistant to S. rolfsii. Based on days to permanent wilting, resistant and susceptible genotypes were identified. Key words: Helianthus tuberosus L., sunchoke, stem rot, disease incidence, genotypes, sorghum seed, area under disease progress curve. African Journal of Microbiology Research Vol. 6(38), pp. 6755-6760, 4 October, 2012 Available online at http://www.academicjournals.org/ajmr DOI: 10.5897/AJMR12.1449 ISSN 1996-0808 ©2011 Academic Journals African Journal of Microbiology Research Vol. 6(38), pp. 6755-6760, 4 October, 2012 Available online at http://www.academicjournals.org/ajmr DOI: 10.5897/AJMR12.1449 ISSN 1996-0808 ©2011 Academic Journals Key words: Helianthus tuberosus L., sunchoke, stem rot, disease incidence, genotypes, sorghum seed, area under disease progress curve. ull Length Research Paper Levels of Sclerotium rolfsii inoculum influence identification of resistant genotypes in Jerusalem artichoke R. Sennoi, S. Jogloy , W. Saksirirat, T. Kesmala, N. Singkham and A. Patanothai Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand. Full Length Research Paper Statistical analysis Error variances between two experiments were tested for homogeneity. For analysis of variances, data with homogeneity of variance were pooled for the two experiments and assessed for factor main effects and interactions (Hoshmand, 2006). The interaction of genotype × level of inoculum was significant for all traits; therefore, each inoculum level was analyzed separately for each parameter. Least significant difference (LSD) was used to compare mean differences. All calculations were done using STATISTIX8 software program. Simple linear regression was used to determine the relationship between inoculum level and disease incidence, lesion length, days to permanent wilting and area under progress curve. Data collection ) In a recent study at Khon Kaen University in Thailand as well as in earlier work on peanut, inoculum grown on agar and on sorghum seed was compared, and sorghum seed was shown to be more effective (Shokes et al., 1996). However, the question of how many S. rolfsii- infested seeds should be used per inoculated plant remains unresolved. The objective of this study was to determine levels of sorghum seed inoculum that provide the most reliable and effective results for evaluation S. rolfsii resistance of Jerusalem artichoke. Number of infected plants and lesion length (cm) were recorded every two days after inoculation. Days to permanent wilting was observed daily after inoculation. Number of symptomatic plants was later converted to disease incidence (% plants exhibiting symptoms). Area under disease progress curve (AUDPC) was calculated from disease incidence according to the formula suggested by Marcel et al. (2008). RESULTS Base on high F-ratio values for genotypes and low coefficient of variation, data collected for disease incidence and lesion length were reported 3 and 5 days, respectively, after inoculation. Significant differences (P<0.01) between the two runs of the experiment were found for disease incidence, lesion length, days to permanent wilting and area under disease progress curve (AUDPC) (Table 1). The first experiment had higher disease incidence (88%) than the second experiment (67%) (data not shown). Genotypes were also significantly different (P<0.01) for disease incidence, lesion length, days to permanent wilting and AUDPC at all inoculum levels. Differences in the levels of inoculum were significant (P<0.01) for disease incidence, lesion length, days to permanent wilting and AUDPC. Genotype × level of inoculum interactions were also significant (P<0.01) for all four dependent variables. INTRODUCTION The isolate was transferred to potato dextrose agar (PDA) medium in petri dishes and incubated at room temperature (25 ± 2°C) for 3 days. After incubation, mycelium plugs were transferred to steamed sorghum seeds and incubated at room temperature for 10 days; the inoculum was then ready to use. At the 6- to 8-leaf stage, plants were inoculated by placing infested sorghum seeds at the crowns of stems at determined levels (1, 2, 3 and 4 sorghum seeds/plant). Cotton wool was used to cover infested sorghum seeds in order to maintain moisture. Preparation of plant materials Tubers of Jerusalem artichoke were cut into small pieces with 2 to 3 active buds, and incubated for one week in charred rice husks to facilitate germination under open-side greenhouse. Water was regularly applied in order to avoid drying out of the medium used. The germinated tuber pieces were then transferred to plug trays for one week until each seedling had two leaves. They were later transferred to experimental pots (8 × 8 × 9 cm) containing steamed soil and charred rice husks (1:1). The soil used in the experiment belonged to the Roi-et series (Re; fine-loamy, mixed, subactive, isohyperthermic Aeric Kandiaquults). From the regression analysis, the plants inoculated with one sorghum seed had the lowest disease incidence (62.5%), whereas the plants inoculated with four sorghum seeds had the highest disease incidence (92.5%) (Figure 1a). Most pairings of seed treatments were statistically different for disease incidence except for the plants INTRODUCTION animal feed (Zaky, 2009), and bio-ethanol (Yildiz et al., 2006). Jerusalem artichoke (Helianthus tuberosus L.) is currently an important crop for production of healthy food (Danilčenko et al., 2008), as the crop produces substantial amounts of the carbohydrate inulin rather than starch in its tubers. Because inulin is absorbed by the human body at lower rates than starches it can prevent obesity, enhance immunity, and reduce blood cholesterol and the risk of insulin-dependent diabetes mellitus (type 2) and heart disease (Orafti, 2005). Jerusalem artichoke is also used to produce a variety of products such as Jerusalem artichoke (Helianthus tuberosus L.) is currently an important crop for production of healthy food (Danilčenko et al., 2008), as the crop produces substantial amounts of the carbohydrate inulin rather than starch in its tubers. Because inulin is absorbed by the human body at lower rates than starches it can prevent obesity, enhance immunity, and reduce blood cholesterol and the risk of insulin-dependent diabetes mellitus (type 2) and heart disease (Orafti, 2005). Jerusalem artichoke is also used to produce a variety of products such as Jerusalem artichoke originated in North America (Kays and Nottingham, 2008) but can also be grown commercially in the tropics (Pimsaen et al., 2010; Puangbut et al., 2011). In tropical regions, stem rot disease caused by S. rolfsii can be a severe disease of the crop (Sennoi et al., 2010). The first occurrence of S. rolfsii on Jerusalem artichoke was reported in the United States (Koike, 2004), but diseases incited by S. rolfsii are more prevalent in warm climates, especially under high temperature and high humidity (Kwon et al., 2008), and the pathogen infects a wide variety of host plants, including most vegetables, flowers, legumes, cereals, and forage plants as well as many weeds (Agrios, 2005). Afr. J. Microbiol. Res. Afr. J. Microbiol. Res. 6756 Selection and improvement of Jerusalem artichoke varieties with resistance to S. rolfsii may provide a sustainable means of disease management. Many attempts have been made to find resistant genotypes against S. rolfsii (Gorbet et al., 2004; Infantino et al., 2006; Akram et al., 2008). As Jerusalem artichoke is rather new to breeders and plant pathologists, an optimal screening procedure for resistance is not available. Previous work on disease resistance in Jerusalem artichoke studied only Sclerotinia sclerotiorum (Cassells and Walsh, 1995). was used in the trials. Experimental setup Five Jerusalem artichoke genotypes (HEL 278, HEL 246, HEL 280, JA 1 and HEL 65) that exhibited a high level of resistance to S. rolfsii in our previous work and five genotypes (CN 52867, HEL 62, JA 102, JA 37 and JA 122) that were most susceptible were used in this study. Four levels of inoculum density (1, 2, 3 or 4 S. rolfsii- infested sorghum seeds/plant) were tested with the 10 Jerusalem artichoke genotypes in a factorial design in randomized complete block (RCBD) with four replications. There were four plants in each treatment unit. The experiments were carried out in an open-sided greenhouse at Khon Kaen University (KKU) Agronomy Farm, Khon Kaen, Thailand. The first run of the experiment was carried out in September 2010 and the second run was done in October 2010. Temperatures in the first and the second runs ranged from 23 to 29°C and 21 to 31°C, respectively. Average of all day’s relative humidity was 91% for the first experiment and 95% for the second experiment. Preparation of S. rolfsii inoculum and inoculation method One seed treatment gave the lowest AUDPC, whereas the highest AUDPC was observed in four seed treatment (Figure 1d). The correlation coefficient between seed treatment and AUDPC was positive and significant (R2 = 0.98). The highest variation among Jerusalem artichoke genotypes was observed for days to permanent wilting in the plants inoculated with three sorghum seeds as indicated by high F-ratio (21.4) (Table 2). Disease incidence and AUDPC in the plants inoculated with three sorghum seeds also had the highest variation (data not shown). Inoculation with three sorghum seeds resulted in the greatest range of variation in disease response among Jerusalem artichoke genotypes. Using three sorghum seeds per plant, disease incidence ranged from 50 to 100% among genotypes (Figure 2a). Genotypes HEL 278, JA 1, HEL 65, CN 52867 and JA 37 showed the lower of disease incidence (50 to 68.8%), whereas genotypes HEL 246, HEL 280, HEL 62, JA 102 and JA 122 had the higher disease incidence (90.6 to 100%). inoculated with two seeds (76.5%) vs. those inoculated with three seeds (79.1%). Lesion length ranged from 1.1 to 1.5 cm (Figure 1b). Plants inoculated with one sorghum seed had the shortest lesions (1.1 cm), but lesion length did not differ significantly from the plants inoculated with three sorghum seeds (1.2 cm) nor did two seeds differ significantly from four seeds (1.4 and 1.5 cm, respectively). Days to permanent wilting ranged from 2.2 to 3.2 (Figure 1c). Treatment with one sorghum seed required 3.2 days to permanent wilting. The other inoculum levels with two, three and four sorghum seeds required 2.8, 2.6 and 2.2 days to permanent wilting, respectively. One seed treatment gave the lowest AUDPC, whereas the highest AUDPC was observed in four seed treatment (Figure 1d). The correlation coefficient between seed treatment and AUDPC was positive and significant (R2 = 0.98). inoculated with two seeds (76.5%) vs. those inoculated with three seeds (79.1%). Lesion length ranged from 1.1 to 1.5 cm (Figure 1b). Plants inoculated with one sorghum seed had the shortest lesions (1.1 cm), but lesion length did not differ significantly from the plants inoculated with three sorghum seeds (1.2 cm) nor did two seeds differ significantly from four seeds (1.4 and 1.5 cm, respectively). Days to permanent wilting ranged from 2.2 to 3.2 (Figure 1c). Treatment with one sorghum seed required 3.2 days to permanent wilting. Preparation of S. rolfsii inoculum and inoculation method The other inoculum levels with two, three and four sorghum seeds required 2.8, 2.6 and 2.2 days to permanent wilting, respectively. One seed treatment gave the lowest AUDPC, whereas the highest AUDPC was observed in four seed treatment (Figure 1d). The correlation coefficient between seed treatment and AUDPC was positive and significant (R2 = 0.98). g ( ) The genotypes that required the longer time (2.8 to 3.3 days) to reach permanent wilting were HEL 278, HEL 246, HEL 280, JA 1, HEL 65, CN 52867 and JA 37. In contrast, genotypes HEL 62, JA 102 and JA 122 required only 1.4 to 2.3 days to reach permanent wilting (Figure 2b). Higher AUDPC were observed for HEL 62, JA 102 and JA 122 (540.6 to 565.6), and lower AUDPC were observed for HEL 278, HEL 246, HEL 280, JA 1, HEL 65, CN 52867 and JA 37 (403.1 to 487.5) (Figure 2c). The highest variation among Jerusalem artichoke genotypes was observed for days to permanent wilting in the plants inoculated with three sorghum seeds as indicated by high F-ratio (21.4) (Table 2). Disease incidence and AUDPC in the plants inoculated with three sorghum seeds also had the highest variation (data not shown). Preparation of S. rolfsii inoculum and inoculation method Isolate 1 of S. rolfsii, which was obtained from a KKU field in Khon Kaen, Thailand, and was very aggressive on Jerusalem artichoke as determined by previous screening assays (Sennoi et al., 2010), Sennoi et al. 6757 6757 Sennoi et al. Table 1. Mean squares from combined ANOVA for disease incidence (at 3 days after inoculation), lesion length (at 5 days after inoculation), days to permanent wilting and area under disease progress curve (AUDPC). Table 1. Mean squares from combined ANOVA for disease incidence (at 3 days after inoculation), lesion length (at 5 days after inoculation), days to permanent wilting and area under disease progress curve (AUDPC). SOV df Disease incidence Lesion length Days to permanent wilting AUDPC Experiment (E) 1 34031.3 2.3 37.8** 303195* Rep/experiment 6 134.1 0.1 2.1 9945 Genotype (G) 9 4397.6 4.2 12.7 79010 G × E 9 5094.6 0.8 2.4 18699 Inoculum level (I) 3 12085.9 2.0 12.3 129607 I × E 3 2536.5 0.1 0.4 22617 G × I 27 1366.9 1.1 1.2 7810 G × I × E 27 1979.5 0.7 0.9 5841 Pooled error 234 110.1 0.1 0.4 2135 C.V. (%) 13.5 22.8 20.3 9.9 Significant at P<0.01. Table 2. Mean squares from combined ANOVA for days to permanent wilting at different level of Sclerotium rolfsii inoculum in Jerusalem artichoke. SOV df 1 seed 2 seeds 3 seeds 4 seeds Experiment (E) 1 16.2** 7.2* 9.1* 6.6 Rep/experiment 6 0.9 0.7 0.8 0.4 Genotype (G) 9 4.5 4.4 4.5 3.0 G × E 9 2.0 1.5 1.4 0.2 Pooled error 54 0.4 0.4 0.2 0.3 F-ratio for genotypes 11.6 12.3 21.4 11.1 C.V. (%) 19.7 21.1 17.7 23.2 ,* Significant at P<0.05 and P<0.01, respectively. ,* Significant at P<0.05 and P<0.01, respectively. inoculated with two seeds (76.5%) vs. those inoculated with three seeds (79.1%). Lesion length ranged from 1.1 to 1.5 cm (Figure 1b). Plants inoculated with one sorghum seed had the shortest lesions (1.1 cm), but lesion length did not differ significantly from the plants inoculated with three sorghum seeds (1.2 cm) nor did two seeds differ significantly from four seeds (1.4 and 1.5 cm, respectively). Days to permanent wilting ranged from 2.2 to 3.2 (Figure 1c). Treatment with one sorghum seed required 3.2 days to permanent wilting. The other inoculum levels with two, three and four sorghum seeds required 2.8, 2.6 and 2.2 days to permanent wilting, respectively. DISCUSSION In our trials, higher levels of inoculum resulted in severer disease. In other investigations, chickpea seedling Afr. J. Microbiol. Res. 6758 y = 9.25x* + 54.55 R² = 0.94 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 Disease incidence (%) Rates of inoculum (sorghum seed) y = 0.1x + 1.05 R² = 0.50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 1 2 3 4 Lesion length (cm) Rates of inoculum (sorghum seed) y = -0.32x + 3.5 R² = 0.98 0 1 2 3 4 0 1 2 3 4 Days to permanent wilting (days) Rates of inoculum (sorghum seed) y = 30.873x + 388.29 R² = 0.98 0 100 200 300 400 500 600 0 1 2 3 4 AUDPC Rates of inoculum (sorghum seed) a b c d Figure 1. Disease incidence (a), lesion length (b), days to permanent wilting (c) and area under disease progress curve (AUDPC) (d) of Jerusalem artichoke at four different levels of S. rolfsii-infested seeds per plant; , * Significant at P < 0.05 and P < 0.01, respectively. y = 9.25x* + 54.55 R² = 0.94 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 Disease incidence (%) Rates of inoculum (sorghum seed) y = 0.1x + 1.05 R² = 0.50 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 1 2 3 4 Lesion length (cm) Rates of inoculum (sorghum seed) a b Rates of inoculum (sorghum seed) Rates of inoculum (sorghum seed) y = 30.873x + 388.29 R² = 0.98 0 100 200 300 400 500 600 0 1 2 3 4 AUDPC Rates of inoculum (sorghum seed) d g y = -0.32x + 3.5 R² = 0.98 0 1 2 3 4 0 1 2 3 4 Days to permanent wilting (days) Rates of inoculum (sorghum seed) c Figure 1. Disease incidence (a), lesion length (b), days to permanent wilting (c) and area under disease progress curve (AUDPC) (d) of Jerusalem artichoke at four different levels of S. rolfsii-infested seeds per plant; , * Significant at P < 0.05 and P < 0.01, respectively. mortality caused by S. DISCUSSION 0 20 40 60 80 100 Genotype Disease incidence (%) HEL 278 HEL 246 HEL 280 JA 1 HEL 65 CN 52867 HEL 62 JA 102 JA 37 JA 122 a LSD = 4.86 Days to permanent wilting (days) HEL 278 HEL 246 HEL 280 JA 1 HEL 65 CN 52867 HEL 62 JA 102 JA 37 JA 122 Genotype 0 1 2 3 4 5 LSD = 0.23 b 0 100 200 300 400 500 600 Genotype HEL 278 HEL 246 HEL 280 JA 1 HEL 65 CN 52867 HEL 62 JA 102 JA 37 JA 122 C LSD = 18.26 AUDPC Figure 2. Disease incidence (a), days to permanent wilting (b) and area under disease progress curve (c) of Jerusalem artichoke genotypes after inoculation with three Sclerotium rolfsii-infested seeds per plant. 0 20 40 60 80 100 Disease incidence (%) HEL 278 HEL 246 HEL 280 JA 1 HEL 65 CN 52867 HEL 62 JA 102 JA 37 JA 122 a LSD = 4.86 Days to permanent wilting (days) HEL 278 HEL 246 HEL 280 JA 1 HEL 65 CN 52867 HEL 62 JA 102 JA 37 JA 122 0 1 2 3 4 5 LSD = 0.23 b Genotype Genotype 0 100 200 300 400 500 600 Genotype HEL 278 HEL 246 HEL 280 JA 1 HEL 65 CN 52867 HEL 62 JA 102 JA 37 JA 122 C LSD = 18.26 AUDPC Figure 2. Disease incidence (a), days to permanent wilting (b) and area under disease progress curve (c) of Jerusalem artichoke genotypes after inoculation with three Sclerotium rolfsii-infested seeds per plant. Figure 2. Disease incidence (a), days to permanent wilting (b) and area under disease progress curve (c) of Jerusalem artichoke genotypes after inoculation with three Sclerotium rolfsii-infested seeds per plant. other traits (Sennoi et al., 2010). Therefore, this trait was used to identify resistance and susceptible genotypes. For the present study, the resistance ratings of genotypes were similar to those of the previous study based on days to permanent wilting except for CN 52867 and JA 37, showing good repeatability of the time to permanent wilting method, although, the inoculation methods of studies were different. A single S. rolfsii-infested sorghum seed was placed contacting a wound at the base of the stem. In contrast, the present study did not wound the stems. Stem wounding may destroy some structural defenses. DISCUSSION rolfsii increased with an increase in inoculum load (Hussain et al., 2006), but differences in the concentrations of sclerotia by weight between 0.1 and 0.5 g, by number of infested wheat between 1 grain and 5 grains and mycelium by weight between 3 and 20 g were not statistically significant. Similarly, increase in disease incidence of a lettuce crop was proportional to density of Sclerotinia sclerotiorum sclerotia used as inoculum (Chitrampalam et al., 2010). High inoculum concentration led to increased disease severity (Sugha et al., 1991). In this study, the effect of inoculation level is the main focus followed by genotypic differences. The questions underlying the experiment are that what inoculation level is more suitable for screening of Jerusalem artichoke genotypes and what traits are more appropriate for identification of resistant genotypes. Higher inoculation level gave higher disease incidence, lesion length and AUDPC but it gave lower days to permanent wilting. However, more suitable inoculation level was observed for three sorghum seeds because this seed rate had high variations among Jerusalem artichoke genotypes for disease incidence, days to permanent wilting and AUDPC as indicated by high F-ratios. Three main effects including environment, genotype and inoculation level were important for the variations in disease incidence, lesion length, permanent wilting point and AUDPC. Environment was more important for disease incidence, permanent wilting and AUDPC. Genotype was more important for lesion length, whereas inoculation level also contributed to high variations for disease incidence, days to permanent wilting and AUDPC although the contributions were somewhat lower than environment for disease incidence, days to permanent wilting and AUDPC and lower than genotype for lesion length and days to permanent wilting. Sorghum seed inoculum is efficient for mass production of inoculum and can be used for screening a large number of plants. S. rolfsii-infested cereal grains were generally used in greenhouse (Pande et al., 1994) and field screening in peanut (Gorbet et al., 2004). Infested millet and oat inoculum were also used in screening of S. sclerotiorum resistance in sunflower (Gulya, 2004). In our previous study, Jerusalem artichoke genotypes showed higher variation in days to permanent wilting than 6759 Sennoi et al. ACKNOWLEDGEMENTS This research was funded by a grant from the program Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program Thai Doctoral Degree from the Office of the Higher Education Commission, Thailand. Grateful acknowledgement are made to the Thailand Research Fund (TRF), the Commission for Higher Education (CHE) and Khon Kaen University (KKU) for providing financial supports to this research through the Distinguish Research Professor Grant of Professor Dr Aran Patanothai and the Peanut and Jerusalem Artichoke Improvement Research Group, KKU and Plant Breeding Research Center for Sustainable Agricultural, Khon Kaen, Thailand. The Plant Gene Resource of Canada and Leibniz Institute of Plant Genetics and Crop Plant Research, Germany, are acknowledged for their donation of Jerusalem artichoke germplasm. Finally, thanks are extended to Dr. Mark L. Gleason, Dept. of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa, USA for critical review of the manuscript. Infantino A, Kharrat M, Riccioni L, Coyne CJ, McPhee KE, Grünwald NJ (2006). Screening techniques and sources of resistance to root diseases in cool season food legumes. Euphytica. 147: 201-221. Kays SJ, Nottingham SF (2008). Introduction: An underutilized resource. In: Biology and chemistry of Jerusalem artichoke (Helianthus tuberosus L.) CRC press, Florida, pp. 1-5. Koike ST (2004). Southern blight of Jerusalem artichoke caused by Sclerotium rolfsii in California. Plant Dis. 88:769. Kwon JH, Park CS (2002). Stem rot of tomato caused by Sclerotium rolfsii in Korea. Mycobiol. 30(4):244–246. y ( ) Kwon JH, Jee HJ, Park CS (2008). Occurrence of bulb rot of Allium victorialis var. platyphyllum caused by Sclerotium rolfsii in Korea. Plant Pathol. J. 24(1):90–92. ( ) Marcel TC, Gorguet B, Ta MT, Kohutova Z, Vels A, Niks RE (2008). Isolate specificity of quantitative trait loci for partial resistance of barley to Puccinia hordei confirmed in mapping populations and near- isogenic lines. New Phytol. 177(3):743-755. Orafti L (2005). Active food scientific monitor. An Orafti Newsletter, Nr 12- spring 2005. Pande S, Rao JN, Reddy MV, McDonald D (1994). Development of a greenhouse screening technique for stem rot resistance in groundnut. International Arachis Newsletter. 14:23-24. Pimsaen W, Jogloy S, Suriharn B, Kesmala T, Pensuk V, Patanothai A (2010). Genotype by environment (G × E) interactions for yield components of Jerusalem artichoke (Helianthus tuberosus L.). Asian J. Plant Sci. 9(1):11-19. Puangbut D, Jogloy S, Srijaranai S, Vorasoot N, Kesmala T, Patanothai A (2011). Rapid assessment of inulin content in Helianthus tuberosus L. tubers. DISCUSSION For example, a thick and/or tough cuticle can increase resistance to a pathogen that enters the plant by direct penetration. The difference in methods of inoculation might be the main cause of the difference of the results between two studies. Since wounding does not normally accompany infection of Jerusalem artichoke in the field, inoculation without wounding should more accurately represent genotype resistance levels. Higher disease incidence during the first experiment may have been due to the fact that temperature at the time of inoculation was 30.2°C compared to 34.5°C for the second experiment. Punja (1985) reported that the optimum temperature for S. rolfsii mycelium growth ranged from 27 to 30°C. In another trial, S. rolfsii mycelium did not grow at temperatures < 15°C or > 40°C and the optimum was 30°C (Kwon and Park, 2002). Inoculation with three sorghum seeds obtained the highest variations in Jerusalem artichoke genotypes and provided replicable results for most genotypes. This method will be further used to evaluate Jerusalem artichoke genotypes for resistance to stem rot disease Afr. J. Microbiol. Res. 6760 caused by S. rolfsii. caused by S. rolfsii. caused by S. rolfsii. caused by S. rolfsii. Hoshmand AR (2006). Key assumptions of experimental designs. In: Design of experiments for agriculture and the natural sciences 2 nd ed. Chapman & Hall/CRC, Boca Raton, Florida, pp. 19-27. p pp Hussain A, Iqbal SM, Ayub N, Zahid MA (2006). Factors affecting development of collar rot disease in chickpea. Pak. J. Bot. 38(1):211– 216. ACKNOWLEDGEMENTS Sabrao J. Breed. Genet. 43(2):188–200. REFERENCES Agrios GN (2005). Sclerotium disease. In: Plant Pathology (5 th Ed). Academic Press, London, pp. 599-600. Punja ZK (1985). The biology, ecology, and control of Sclerotium rolfsii. Annu. Rev. Phytopathol. 23: 97-127. Akram A, Iqbal SM, Rauf CA, Aleem R (2008). Detection of resistant sources for collar rot disease in chickpea germplasm. Pak. J. Bot. 40(5):2211-2215. Sennoi R, Jogloy S, Saksirirat W, Patanothai A (2010). Pathogenicity test of Sclerotium rolfsii, a causal agent of Jerusalem artichoke (Helianthus tuberosus L.) stem rot. Asian J. Plant Sci. 95:281–284. Cassells AC, Walsh M (1995). Screening for Sclerotinia resistance in Helianthus tuberosus L. (Jerusalem artichoke) varieties, lines and somaclones, in the field and in vitro. Plant Pathol. 44:428–437. Shokes FM, Rozalski K, Gorbet DW, Brenneman TB, Berger DA (1996). Techniques for inoculation of peanut with Sclerotium rolfsii in the greenhouse and field. Peanut Sci. 23:124-128. Chitrampalam P, Turini TA, Matheron ME, Pryor BM (2010). Effect of sclerotium density and irrigation on disease incidence and on efficacy of Coniothyrium minitans in suppressing lettuce drop caused by Sclerotinia sclerotiorum. Plant Dis. 94:1118-1124. Sugha SK, Sharma KB, Tyagi PD (1991). A modified technique for screening chickpea (Cicer arietinum L.) varieties against collar rot caused by Sclerotium rolfsii. Indian J. Agric. Sci. 61(4):289–290. Yildiz G, Sacakli P, Gungorhe T (2006). The effect of dietary Jerusalem artichoke (Helianthus tuberosus L.) on performance, egg quality characteristics and egg cholesterol content in laying hens. Czech J. Anim. Sci. 51:349-354. Danilčenko H, Jarienė E, Aleknavičienė P, Gajewski M (2008). Quality of Jerusalem artichoke (Helianthus tuberosus L.) tubers in relation to storage conditions. Not. Bot. Hort. Agrobot. Cluj. 36:23-27. storage conditions. Not. Bot. Hort. Agrobot. Cluj. 36:23-2 Gorbet DW, Kucharek TA, Shokes EM, Brenneman TB (2004). Field evaluations of peanut germplasm for resistance to stem rot. Peanut Sci. 31:91-95. Zaky EA (2009). Physiological response to diets fortiﬁed with Jerusalem artichoke tubers (Helianthus tuberosus L.) powder by diabetic rats. American-Eurasian J. Agric. Environ. Sci. 5:682-68. Gulya TJ (2004). An inoculation method for Sclerotinia stalk rot. Sunflower Research Workshop Proceedings. 26th Sunflower Research Workshop, January 14-15, 2004, Fargo, ND. Available: http://www.sunflowernsa.com/research/research- workshop/documents/Gulya_StalkRot_04.PDF
https://openalex.org/W4381623849	http://jurnal.umb.ac.id/index.php/JIMAKUKERTA/article/download/4454/3045	Indonesian	null	PENGABDIAN MASYARAKAT DI DESA TALANG JAWA KABUPATEN EMPAT LAWANG	Jurnal Ilmiah Mahasiswa Kuliah Kerja Nyata	2,022	cc-by-sa	2,943	E ISSN : 2808-5566 P:ISSN : 2808-7569 E ISSN : 2808-5566 P:ISSN : 2808-7569 E ISSN : 2808-5566 ABSTRAK Program Kuliah Kerja Nyata (KKN) Universitas Muhammadiyah Bengkulu menerjunkan mahasiswa baik jurusan pendidikan maupun non pendidikan untuk mengabdikan diri dalam masyarakat. Di salah satu daerah yang dipilih oleh mahasiswa untuk menjadi tempat KKN Individu adalah yaitu desa Talang Jawa Kec. Pendopo Kab. Empat Lawang Prov. Sumatra Selatan. Desa Talang Jawa merupakan salah satu desa yang ada di kecamatan Pendopo Kabupaten Empat Lawang Provinsi Sumatra Selatan. Desa Talang Jawa memiliki jumlah penduduk sekitar 1300 jiwa. Metode dalam kegiatan ini menggunakan metode Menurut jenisnya, dalam pelaksanaan program KKN ini menggunakan metode lapangan (field research). Penelitian tersebut termasuk ke dalam penelitian deskriptif Kualitatif (Moelong, 2011). Metode pengumpulan data yang digunakan adalah wawancara, observasi, dan dokumentasi. Metode wawancara ini bertujuan untuk menggali informasi mengenai kompetensi sosial mahasiswa dan kompetensi kepribadian mahasiswa yang dirasakan oleh masyarakat selama KKN berlangsung. Penelitian kualitatif ini menggunakan analisis data model Miles dan Huberman. Miles dan Huberman mengemukakan terdapat dua aktivitas dalam penelitian kualitatif yaitu data reduction, data display, dan conclusion drawing/ verification (Sugiyono, 2012). Kegiatan yang dilakukan meliputi yaitu: 1. Pengembangan sastra dan pemanfaatan limbah organik padat pada warga Desa Talang Jawa Kabupaten Empat Lawang. Received [25-10-2022] Revised [06-11-2022] Accepted [26-12-2022] Kata Kunci: kegiatan, pengabdian, masyarakat Dengan demikian, kegiatan pengabdian masyarakat boleh dikatakan mengalami degradasi atau tidak efektif. Oleh sebab itu, pengabdian masyarakat diarahkan untuk menjamin keisnambunagn antara dunia akademik-teoritik dan dunia empirik- praktis. Dengan demikian akan terjadi interaksi yang bersinergi, saling menerima dan memberi, saling asah, asih dan asuh antara mahasiswa dan masyarakat. Pengabdian masyarakat juga merupakan wahana pengambdian serta pengembangan ilmu dan teknologi, dilaksanakan di luar kampus dalam waktu, mekanisme kerja, dan persyaratan tertentu (Syardiansah 2017). PENGABDIAN MASYARAKAT DI DESA TALANG JAWA KABUPATEN EMPAT LAWANG Legista Sari1, Tio Dwi Cahyo2, Diah Khairiyah3 , Yanti Paulia4, Fiana Podesta5 Universitas Muhammadiyah Bengkulu, Bengkulu, Indonesia Email: Legistasari12@gmail.com http://jurnal.umb.ac.id/index.php/JIMAKUKERTA I. PENDAHULUAN Pengabdian Masyarakat merupakan suatu bentuk pengamalan Tri Dharma Perguruan Tinggi. Namun dalam prakteknya, tidak mustahil sasaran pengabdian masyarakat dapat melenceng dari harapan semula, sehingga setelah pengabdian masyarakat berakhir, justru para mahasiswa pengabdian masyarakat tetap saja tidak memperoleh pembelajaran diri yang berarti. Begitu pula, kualitas kehidupan masyarakat di lokasi pengabdian masyarakat tidak menunjukkan perubahan yang signifikan. Bahkan, di mata masyarakat bisa saja citra perguruan tinggi malah semakin merosot. http://jurnal.umb.ac.id/index.php/JIMAKUKERTA 604 http://jurnal.umb.ac.id/index.php/JIMAKUKERTA dengan realita pembangunan dalam masyarakat. Berbagai kegiatan yang bisa dilakukan mahasiswa dalam mengabdikan diri pada masyarakat, diantaranya mengembangkan pembelajaran sastra dan pemanfaatan limbah kotoran. Pengembangan sastra merupakan salah- satu program yang dilaksanakan pada pengabdian masyarakat yang di lakukan di Desa Talang Jawa. Sesuai dengan bidag keilmuan yang di ambil pada perguruan tinggi di Universitas Muhammadiyah Bengkulu maka kegiatan ini merupakan salah satu kegiatan yang di kembangkan pada SDN 21 dan SDN 14 Kabuapaten Empat Lawang. Pengabdian masyarakat Muhammadiyah untuk Negeri adalah kegiatan yang dilaksanakan dalam rangka pembelajaran dan pemberdayaan melalui kegiatan pendidikan dan pengajaran, penelitian, pengabdian kepada masyarakat, dan dakwah amar makruf nahi munkaroleh sebuah atau beberapa Perguruan Tinggi Muhammadiyah. Pengabdian masyarakat Muhammadiyah untuk Negeri dilaksanakan dengan prinsip co-creation (gagasan dari berbagai pihak terkait), co- financing (dukungan dari berbagai pihak terkait), flexibility (menyesuaikan dengan situasi kondisi lingkungan dan kebutuhan pemerintah, mitra kerja, dan masyarakat dalam proses pembangunan di daerah), sustainability (kegiatan harus melahirkan pembangunan/ pengembangan berkelanjutan di tengah masyarakat sasaran), dan research based community services (setiap kegiatan didasarkan/ dilaksanakan berdasarkan hasil kajian ilmiah). Selain itu kegiatan pengembangan pembelajaran sastra kami juga melakukan pengabdian pada masyarakat dengan program pemanfaatan limbah kotoran pada desa Talang Jawa. Pemanfatan limbah kotoran ini sangan berguna bagi masyarakat Desa Talang Jawa dalam bertani, selain Limbah kotoran bisa dimanfaatkan sebagai pupuk bagi tumbuhan, juga bisa diperjual belikan untuk menambah nilai ekonomis bagi masyarakat yang beternak dalam pemanfaatan limbah kotoran. Pengabdian masyarakat adalah suatu bentuk pendidikan dengan cara memberikan pengalaman belajar kepada mahasiswa untuk hidup di tengah-tengah masyarakat di luar kampus dan secara langsung mengidentifikasi serta menangani masalah-masalah pembangunan yang dihadapi. Pengabdian masyarakat dilaksanakan oleh perguruan tinggi dalam upaya meningkatkan isi dan bobot pendidikan bagi mahasiswa dan untuk mendapatkan nilai tambah yang lebih besar pada perguruan tinggi (Al’afghani 2021). Maka dari itu dalam kegiatan pengabdian masyarakat yang kami lakukan ini berjudul Pengembangan Pembelajaran Sastra Dan Pemanfaatan Limbah Kotoran Di Desa Talang Jawa Kabupaten Empat Lawang” yang bertujuan supaya dengan pengabdian ini bisa bermanfaat bagi masyarakat Desa Talang Jawa Kabupaten Empat Lawang. Kegiatan pengabdian masyarakat dilaksanakan di luar kampus dengan maksud meningkatkan relevansi pendidikan tinggi dengan perkembangan dan kebutuhan masyarakat akan ilmu pengetahuan, teknologi, serta seni untuk melaksanakan pembangunan yang semakin meningkat, serta meningkatkan persepsi mahasiswa tentang relevansi antara kurikulum yang dipelajari di kampus adalah yaitu desa Talang Jawa Kec. Pendopo Kab. Empat Lawang Prov. Sumatra Selatan. Secara umum keadaan Desa Talang Jawa dapat saya ketahui dengan cara melakukan observasi di lapangan yang merupakan langkah awal untuk memperoleh data tentang kondisi masyarakat dan kondisi lingkungan setempat serta kekurangan apa saja yang menjadi titik lemah masyarakat didesa tersebut, hal ini bermaksud agar program yang dibuat sesuai dengan kebutuhan masyarakat setempat. Observasi dilakukan dengan cara datang langsung ke lapangan (lokasi) dan melalui sumber yang lain seperti perangkat desa dan warga setempat. Desa Talang Jawa merupakan salah satu desa yang ada di kecamatan Pendopo Kabupaten Empat Lawang Provinsi Sumatra Selatan. Desa Talang Jawa memiliki jumlah penduduk sekitar 1300 jiwa. Desa Talang Jawa terbagi menjadi 4 dusun yaitu: menumbuhkan jiwa dan raga yang sehat. Selain itu, masih minimnya kegiatan- kegiatan yang berhubungan dengan menumbuhkan budaya hidup sehat seperti senam, menjaga kebersihan, bergotong royong bersama salaing membantu untuk melestarikan desa. Kesehatan merupakan hal yang penting guna mencegah datangnya suatu penyakit. Melihat permasalahan tersebut, sangat perlu diadakan beberapa program yang menunjang perbaikan kesehatan bagi warga di Desa Talang Jawa. Kurangnya pengetahuan siswa terhadap perilaku hidup bersih dan sehat dapat menjadi salah satu penyebab lingkungan menjadi kumuh. Karena masih adanya anak-anak yang jajan dan membuang sampahnya sembarangan. Desa Talang Jawa merupakan salah satu desa yang ada di kecamatan Pendopo Kabupaten Empat Lawang Provinsi Sumatra Selatan. Desa Talang Jawa memiliki jumlah penduduk sekitar 1300 jiwa. Desa Talang Jawa terbagi menjadi 4 dusun yaitu: Dalam bidang kesehatan terutama di untuk infrastruktur yang ada di Desa Talang Jawa belum tersedia klinik kesehatan atau puskesmas yang dapat di gunakan oleh masyarakat. Oleh karena itu masyarakat disana biasanya datang berobat kedesa lain. Melihat pendidikan di Desa Talang Jawa seperti halnya di daerah lain dimana meskipun sudah tersedianya lembaga pendidikan, tetapi masih tetap dibutuhkan pengembangan kualitas pendidikan. Selain sekolah formal seperti SD juga terdapat Taman Pendidikan Al- qur’an (TPA). TPA yang ada di desa Talang Jawa sudah difungsikan dengan baik oleh anak-anak, namun tenaga pendidik yang mengajar di TPA masih kurang. Oleh karena itu, saya akan mengadakan program belajar bersama untuk menghimpun siswa-siswi agar mereka memiliki semangat yang tinggi untuk belajar di TPA. a. Dusun 1 berjumlah : 320 KK b. Dusun 2 berjumlah : 330 KK c. Dusun 3 berjumlah : 315 KK d. Dusun 4 berjumlah : 335 KK Analisis Situasi Lokasi Melalui program pengabdian masyarakat Universitas Muhammadiyah Bengkulu menerjunkan mahasiswa baik jurusan pendidikan maupun non pendidikan untuk mengabdikan diri dalam masyarakat. Di salah satu daerah yang dipilih oleh mahasiswa untuk menjadi tempat pengabdian masyarakat Individu http://jurnal.umb.ac.id/index.php/JIMAKUKERTA 605 http://jurnal.umb.ac.id/index.php/JIMAKUKERTA Masih terdapat beberapa infrastruktur lingkungan di Desa Talang Jawa yang belum merata seperti halnya tempat sampah yang tidak disediakan di jalan, pembuangan sampah yang merusak lingkungan seperti yang saya lihat masih banyak warga desa yang membuang sampah ke air sungai. Agar membiasakan masyarakatnya untuk selalu membuang sampah pada tempatnya,seharusnya diperlukan pasang poster di setiap penjuru tempat tentang pentingnya membuang sampah pada tempatnya.Hal tersebut dapat menjadi langkah bagi Kelurahan Bruge ilir untuk lebih menjaga lingkungan desa dan infrastruktur. Secara keseluruhan dari kondisi wilayah yang ada di Desa Talang Jawa memiliki potensi, antara lain : memanfaatkan alam yang begitu indah, berpotensi untuk mengembangkan usaha, serta lahan kosong yang dimiliki oleh beberapa warga yang dimanfaatkan untuk bercocok tanam yang dapat menghasilkan. Didesa ini juga minimnya kegiatan keagamaan, seperti majelis taklim tidak berjalan dengan apa yang diharapkan. Kondisi sosial yang ada di Desa Talang Jawa berpotensi untuk di adakannya program yang dapat meningkatkan kualitas sumberdaya manusia (penyuluhan dan pelatihan keterampilan). III. HASIL DAN PEMBAHASAN 1. Bimbingan belajar dan menulis cerpen Berdasarkan analisis situasi lokasi pengabdian masyarakat terhadap Desa Talang Jawa yang telah dipaparkan sebelumnya, perlu dikembangkan potensi- potensi yang ada dan cara mengatasi permasalahan yang ada di Desa Talang Jawa khususnya terkait pengelolaan sampah yang perlu diperhatian lagi 1. Bimbingan belajar dan menulis cerpen II. METODE KEGIATAN Pelaksanaan Program Pengabdian Mayarakat dilakukan di SDN 21 Talang Jawa Kabupaten Lintang Epat Lawang, dan di Desa Bruge Hilir Kecamatan Pendopo Kabupaten Empat Lawang Kegiatan ini dilaksanakan pada pada tanggal 10 Agustus 2022 sampai pada Tanggal 25 September 2022 dan dilaksanakan pada tanggal 21 September secara langsung (Offline) kepada masyarakat dengan durasi waktu 120 -160 menit. d. Dusun 4 berjumlah : 335 KK Dalam 4 dusun tersebut tidak hanya warga Desa Talang Jawa tetapi ada juga warga lainnya dari luar dusun atau bahkan luar daerah. Desa Talang Jawa dapat lebih dikembangkan lagi dari berbagai aspek seperti pendidikan, ekonomi, kesehatan, agama, sosial dan budaya, infrastruktur serta lingkungannya. Sebagian besar warga di desa Talang Jawa bekerja sebagai petani, pedagang, Pegawai Swasta, buruh/tukang, dan ada beberapa bagian yang menjadi pns. Sehingga dapat dikatakan bahwa tingkat perekonomian masyarakat Desa Talang Jawa tergolong dalam kelas menengah dengan penghasilan rata-rata yang telah mampu mencukupi kebutuhan hidup dan kebutuhan keluarga. Kondisi kesehatan masyarakatnya merupakan hal yang sangat perlu untuk diperhatikan. Namun, masih belum aktif dalam upaya Untuk sarana infrastruktur seperti jalan yang ada di Desa Talang Jawa sudah dikatakan baik pada umumnya jalannya sudah aspal semua dan tidak ditemukan lagi jalan yang buruk. Seperti yang saya lihat saat observasi di desa Talang Jawa kurangnya kegiatan warga desa terutama http://jurnal.umb.ac.id/index.php/JIMAKUKERTA 606 sehingga terwujudnya lingkungan yang bersih dan asri serta peningkatan ekonomi warga sehingga tercipta kemandirian ekonomi warga. Hal-hal yang telah dijelaskan dalam analisis situasi lokasi pengabdian masyarakat merupakan serangkaian kegiatan yang akan dilaksanakan oleh mahasiswa pengabdian masyarakat untuk pengabdian kepada masyarakat. Mahasiswa yang tergabung dalam pengabdian masyarakat diharapkan dapat menangkap dan menghayati kehidupan masyarakat di Desa Talang Jawa dengan berbagai permasalahan yang ada sehingga persoalan persoalan sumber daya yang telah ada dan belum dimiliki, dan solusi-solusi yang perlu dilakukan sesuai dapat diwujudkan. Disisi lain, partisipasi dan dukungan warga sangat diperlukan dalam mewujudkan program- program pengabdian masyarakat. Sehingga dapat mewujudakan Desa Talang Jawa sebagai desa yang baik di bidang ekonomi, sosial, kesehatan, dan lingkungan. untuk ibu-ibu dalam melakukan kegiatan wirausahaan/ home industry seperti membuat produk yang bisa dihasilan untuk menambah dan mengangkat perekonomian masyarakat, seharusnya disini ibu-ibu bisa lebih kreatif dan berinovasi dalam membuat produk apa saja misalnya seperti makanan ringan, kue, mengolah limbah sampah menjadi barang yang mempunyai nilai jual tinggi. Masih terdapat beberapa infrastruktur lingkungan di Desa Talang Jawa yang belum merata seperti halnya tempat sampah yang tidak disediakan di jalan, pembuangan sampah yang merusak lingkungan seperti yang saya lihat masih banyak warga desa yang membuang sampah ke air sungai. Agar membiasakan masyarakatnya untuk selalu membuang sampah pada tempatnya,seharusnya diperlukan pasang poster di setiap penjuru tempat tentang pentingnya membuang sampah pada tempatnya.Hal tersebut dapat menjadi langkah bagi Kelurahan Bruge ilir untuk lebih menjaga lingkungan desa dan infrastruktur. Secara keseluruhan dari kondisi wilayah yang ada di Desa Talang Jawa memiliki potensi, antara lain : memanfaatkan alam yang begitu indah, berpotensi untuk mengembangkan usaha, serta lahan kosong yang dimiliki oleh beberapa warga yang dimanfaatkan untuk bercocok tanam yang dapat menghasilkan. Didesa ini juga minimnya kegiatan keagamaan, seperti majelis taklim tidak berjalan dengan apa yang diharapkan. Kondisi sosial yang ada di Desa Talang Jawa berpotensi untuk di adakannya program yang dapat meningkatkan kualitas sumberdaya manusia (penyuluhan dan pelatihan keterampilan). Berdasarkan analisis situasi lokasi pengabdian masyarakat terhadap Desa untuk ibu-ibu dalam melakukan kegiatan wirausahaan/ home industry seperti membuat produk yang bisa dihasilan untuk menambah dan mengangkat perekonomian masyarakat, seharusnya disini ibu-ibu bisa lebih kreatif dan berinovasi dalam membuat produk apa saja misalnya seperti makanan ringan, kue, mengolah limbah sampah menjadi barang yang mempunyai nilai jual tinggi. http://jurnal.umb.ac.id/index.php/JIMAKUKERTA E ISSN : 2808-5566 P:ISSN : 2808-7569 E ISSN : 2808-5566 P:ISSN : 2808-7569 2. Bimbingan menulis indah dan rapi Pada kesempatan ini saya membantu mengajari anak-anak kelas 1 untuk menulis rapi dan indah sehingga bisa dibaca dan dipahami. Adapun hasil dari kegiatan ini dapat dilihat pada gambar berikut : 3. Pelatihan membaca Banyak anak-anak umumnya disekolah dasar masih belum lancar dalam membaca pada foto dibawa saya sedang mengajari anak kelas enam membaca, kasus ini disebabkan kurang pedulinya orang tua terhadap pendidikan anak padahal sekolah pertamanya adalah rumah. Gambar 4. Persiapan Bahan baku pembuatan pupuk limbah organik padat Kegiatan ini dapat kita lihat pada gambar berikut : cerpen dan bagaimana cara pembuatannya serta saya juga mengajak mereka membaca cerpen agar mereka paham dan mengerti ketika dinanti disuruh membuat karangan cerita pendek. Dapat kita lihat pada gambar berikut : Gambar 3. mengajar membaca Gambar 1. Membimbing Pembuatan Cerpen Gambar 3. mengajar membaca Gambar 3. mengajar membaca Dalam kegitan ini kami melakukan kegiatan mengajar anak-anak kelas enam SDN 21 Kabupaten Empat Lawang membaca sebagian anak-anak sudah lancar membaca namun ada beberapa siswa yang masih kesulitan membaca, kegiatan ini merupakan kegiatan yang harus inten dilakukan mengingat siswa kelas enam SDN 21 Kabupaten Empat lawang masih ada yang belum lancar membaca. Gambar 1. Membimbing Pembuatan Cerpen 4. Pelatihan dalan pemanfatan limbah organik padat Pada kegiatan ini saya melakukan pembuatan limbah organik padat bersama warga, yang mana limbah ini di ambil dari kotoran sapi yang kemudian di caampur dengan sekam padi, lalu taburkan dekomposer secara merata, selanjutnya molases di encerkan dan disiram secara merata diatasan adukan secara merata. kegitan ini dapat kita lihat pada gambara berikut : Gambar 2. Mengawasi anak –anak yang sedang belajar menulis yang indah dan rapi Gambar 4. Persiapan Bahan baku pembuatan pupuk limbah organik padat Gambar 2. Mengawasi anak –anak yang sedang belajar menulis yang indah dan rapi 1. Bimbingan belajar dan menulis cerpen Pada kegiatan ini saya melalukan Edukasi di SD N 21 dan SDN 14 tanjung baru kecamatan pendopo kabupaten empat Lawang Provinsi Sumatra Selatan. disini saya memupuk minat anak-anak tingkat SD untuk belajar mengetahui apa itu http://jurnal.umb.ac.id/index.php/JIMAKUKERTA 607 http://jurnal.umb.ac.id/index.php/JIMAKUKERTA http://jurnal.umb.ac.id/index.php/JIMAKUKERTA 608 E ISSN : 2808-5566 P:ISSN : 2808-7569 E ISSN : 2808-5566 UCAPAN TERIMA KASIH Persiapan bahan baku ini dilaksanakan sebelum pembuatan Pupuk organik dan bahan baku berasal dari limbah tumbuhan dan kotoran hewan. Setelah persiapan bahan baku selesai maka selanjutnya menyiapkan tempat fermentasi dari limpah organik untuk dijadikan pupuk. Alhamdullilahirobbil’alamin, puji syukur kehadirat Allah SAW yang maha pengasih lagi maha penyayang telah memberikan kita kesempatan dan kemudahan kepada kita semua dalam menjalankan amanah yang menjadi tanggung jawab kita. Sholawat beserta salam tercurahkan kepada baginda kita baginda Nabi Muhammad SAW yang telah membawa kita dari jaman Jahiliyah menuju zaman yang penuh dengan teknologi yang dapat kita rasakan seperti sekarang ini. Atas rahmat dan Karunia- NYA, program Kuliah Kerja Nyata (KKN) di Desa Talang Jawa kecamatan Pendopo dimulai sejak tanggal 10 Agustus – 24 September 2022 dapat berjalan dengan lancar sehingga dapat terselesaikannya laporan akhir ini. Pada hasil dalam kegiatan ini bahwah warga bisa dan paham dalam pembuatan pupuk dari limbah organik dan dari hasil pembuatan pupuk dari limbah organik warga bisa memanfaatkan limbah tadi untuk menyuburkan tanah dan kegunaan dalam kesuburan tanaman. Selain itu limbah yang telah di produksi bisa di perjual belikan guna menambah penghasilan warga. Laporan ini disusun sebagai salah satu penilaian dari seluruh program Kuliah Kerja Nyata (KKN) serta untuk mengetahui sejauh mana program kegiatan mahasiswa dalam melaksanakan kegiatan KKN dapat terelisasi dengan baik.dengan tujuan untuk melakukan pengabdian kepada masyarakat, kami berharap semoga seluruh program yang telah berjalan dapat bermanfaat bagi kedua belah pihak. Kami menyadari bahwa pelaksanaan Kuliah kerja Nyata dan penyusunan laporan akhir ini tidak lepas dari bimbingan,bantuan dan dorongan dari berbagai pihak. Oleh karena itu saya ucapkan terima kasih kepada : P:ISSN : 2808-7569 Covid-19 Dengan Mengoptimalkan Aktifitas Masyarakat Sebagai Upaya Menghadapi Adaptasi Kebiasaan Baru, 137. 3. Ketua Karng Taruna yang ikut serta berpartisipasi dalam melaksakan kegiatan pada saat 17 Agustus. g 4. Mayarakat setempat yang ikut berpartisipasi dalam kegiatan KKN, dan Syardiansah, S. (2019). Peranan Kuliah Kerja Nyata Sebagai Bagian dari Pengembangan Kompetensi Mahasiswa: Studi Kasus Mahasiswa Universitas Samudra KKN Tahun 2017. JIM UPB (Jurnal Ilmiah Manajemen Universitas Putera Batam), 7(1), 57-68. 5. Kawan-kawan kelompok yang ikut serta dalam pelaskanan KKN dan memberikan semangat serta kerja sama yang baik selama kegiatan KKN. IV. KESIMPULAN DAN SARAN Setelah melakukan kegiatan Kuliah Kerja Nyata Universitas Muhammadiyah Bengkulu di SDN 21 Kabupaten Empat Lawang dan di Desa Talang Jawa. Saya dapat membuat kesimpulan bahwa pelaksanaan berjalan sesuai dengan program yang telah direncanakan, meskipun masih ada program yang harus dirubah karena harus menyesuaikan dengan situasi dan kondisi di lokasi KKN. Semua program dapat terlaksana dengan baik dan lancar meskipun ada beberapa kendala, namun hal tersebut dapat teratasi. Kegiatan program kerja dilaksanakan pada pagi hari, siang dan sore. Dengan terlaksananya kegiatan tersebut semoga dapat bermanfaat bagi SDN 21 dan SDN 14 Kabupaten Empat Lawang serta masyarakat yang tinggal di Desa Talang Jawa. Terselesaikannya program kerja ini tidak terlepas dari adanya kerja sama dengan pihak sekolah SDN 21 dan SDN 14 Kabupaten Empat Lawang, Lurah, karang Taruna dan kerja sama dengan team-teman kelompok lainnya. 1. Selaku Dosen pembingan lapangan yang telah memberikan bimbingan dan sarannya selama kegiatan. 2. Ibu Izan Ulpa selaku Ibu Lurah yang telah memperbolehkan saya dan kawan-kawan kelompok lainnya melaksanakan kegiatan KKN di Desa Talang Jawa, kecamatan Empat Lawang Ibu PKK yang slalu membantu dalam kesulitan apapun http://jurnal.umb.ac.id/index.php/JIMAKUKERTA 609 E ISSN : 2808-5566 P:ISSN : 2808-7569 Covid-19 Dengan Mengoptimalkan Aktifitas Masyarakat Sebagai Upaya Menghadapi Adaptasi Kebiasaan Baru, 137. DAFTAR PUSTAKA Al’afghani, M. R., & Djati, L. U. S. G. (2021). KKN-Dr Di Rw 18 Nangkasuni Kelurahan Tamansari Kecamatan Bandung Wetan. Penguatan Atas Kesadaran Dan Kepedulian Terhadap Wabah 610 http://jurnal.umb.ac.id/index.php/JIMAKUKERTA
https://openalex.org/W4280615473	https://www.research-collection.ethz.ch/bitstream/20.500.11850/549169/2/s12915-022-01304-4.pdf	English	null	Optimization of the antimicrobial peptide Bac7 by deep mutational scanning	BMC biology	2,022	cc-by	19,847	ETH Library Journal Article Author(s): Koch, Philipp; Schmitt, Steven; Heynisch, Alexander; Gumpinger, Anja; Wüthrich, Irene; Gysin, Marina; Shcherbakov, Dimitri; Hobbie, Sven N.; Panke, Sven; Held, Martin Author(s): Koch, Philipp; Schmitt, Steven; Heynisch, Alexander; Gumpinger, Anja; Wüthrich, Irene; Gysin, Marina; Shcherbakov, Dimitri; Hobbie, Sven N.; Panke, Sven; Held, Martin © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Intracellularly active antimicrobial peptides are promising candidates for the development of antibiot- ics for human applications. However, drug development using peptides is challenging as, owing to their large size, an enormous sequence space is spanned. We built a high-throughput platform that incorporates rapid investigation of the sequence-activity relationship of peptides and enables rational optimization of their antimicrobial activity. The platform is based on deep mutational scanning of DNA-encoded peptides and employs highly parallelized bacte- rial self-screening coupled to next-generation sequencing as a readout for their antimicrobial activity. As a target, we used Bac71-23, a 23 amino acid residues long variant of bactenecin-7, a potent translational inhibitor and one of the best researched proline-rich antimicrobial peptides. Results: Using the platform, we simultaneously determined the antimicrobial activity of >600,000 Bac71-23 variants and explored their sequence-activity relationship. This dataset guided the design of a focused library of ~160,000 variants and the identification of a lead candidate Bac7PS. Bac7PS showed high activity against multidrug-resistant clinical isolates of E. coli, and its activity was less dependent on SbmA, a transporter commonly used by proline- rich antimicrobial peptides to reach the cytosol and then inhibit translation. Furthermore, Bac7PS displayed strong ribosomal inhibition and low toxicity against eukaryotic cells and demonstrated good efficacy in a murine septicemia model induced by E. coli. Conclusion: We demonstrated that the presented platform can be used to establish the sequence-activity relation- ship of antimicrobial peptides, and showed its usefulness for hit-to-lead identification and optimization of antimicro- bial drug candidates. Keywords: Antibiotics, Antimicrobial peptides, Drug discovery, High-throughput screening, Antimicrobials, Deep mutational scanning, Proline-rich antimicrobial peptides, Protein synthesis inhibitor, Antimicrobial resistance, Sequence-activity relationship Optimization of the antimicrobial peptide Bac7 by deep mutational scanning Philipp Koch1†, Steven Schmitt1†, Alexander Heynisch1, Anja Gumpinger2, Irene Wüthrich1, Marina Gysin3, Dimitri Shcherbakov3, Sven N. Hobbie3, Sven Panke1 and Martin Held1* Backgroundh The drug development field is in urgent need of novel compounds to deliver the next generation of antibiotics to combat multidrug-resistant (MDR) bacteria [1]. Sev- eral promising leads have been identified in the group of antimicrobial peptides (AMPs), some of which are cur- rently in drug development pipelines [2]. However, fail- ure rates in clinical trials [3] are high, often because a majority of these molecules form pores in bacterial mem- branes or disintegrate them completly, a mode of action © The Author(s) 2022. Open Access This art permits use, sharing, adaptation, distributio original author(s) and the source, provide a other third party material in this article are in to the material. If material is not included in regulation or exceeds the permitted use, yo licence, visit http://creativecommons.org/lic mmons.org/publicdomain/zero/1.0/) applie †Philipp Koch and Steven Schmitt contributed equally to this work. Correspondence: martin.held@bsse.ethz.ch 1 Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Full list of author information is available at the end of the article †Philipp Koch and Steven Schmitt contributed equally to this work. Correspondence: martin.held@bsse.ethz.ch 1 Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland Full list of author information is available at the end of the article Originally published in: This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Koch et al. BMC Biology (2022) 20:114 https://doi.org/10.1186/s12915-022-01304-4 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 2 of 21 (MoA) that is prone to cause toxicity against human cells [4]. (MoA) that is prone to cause toxicity against human cells [4]. abundance of peptide-encoding DNA fragments [19]. Recently, DMS studies investigated the effect of amino acid residue substitutions of the proline-rich AMP api- daecin and oncocin on critical interactions with the ribo- some [20, 21]. However, DMS has never been exploited as a platform for hit-to-lead optimization of antimicro- bials. If applied successfully, the coverage of the AMP sequence-activity relationships could be increased con- siderably and deliver valuable clues for the design of AMP drug candidates. [ ] Proline-rich AMPs usually do not lyse but are interfer- ing with the activity of intracellular targets essential for survival such as ribosomes [5]. One of the most inten- sively researched proline-rich AMPs is bactenecin-7 (Bac7). It is a 60 amino acid long linear peptide that was first isolated from bovine neutrophils [6]. In vitro studies on Bac7 truncates indicate that the two N-terminal argi- nine residues are needed for efficient uptake [7] and its C-terminus can be truncated resulting in peptides with a length of 35, 23, and 16 amino acids at only a minor loss of antimicrobial activity [8]. Moreover, the antimi- crobial activity can be increased via modulation of the amino acid sequence [7, 9, 10]. To interact with the ribo- some and inhibit protein translation, Bac7 crosses the outer membrane of Gram-negative bacteria via not yet fully elucidated mechanisms and then traverses the inner membrane through the SbmA transporter [11]. Bac7 displays high activity against many species of the Gram- negative Enterobacteriaceae [8, 12], a family of bacteria in which sbmA is expressed, and which are currently listed as “critical priority pathogens” by the WHO [13]. Research on Bac7 may thus offer a path towards develop- ing a treatment against these threatening pathogens.h In this study, we optimized the 23 amino acid truncate of Bac7 (Bac71-23) in two DMS rounds. In the first round, we screened a Bac71-23 library consisting of 601,551 ran- domly mutagenized variants and assessed their growth inhibitory effects when expressed intracellularly in E. coli. This enabled us to determine the contribution of each amino acid residue substitution to antimicrobial activ- ity. Guided by these results, we performed a second DMS round with a focused, semi-rationally designed library of Bac71-23 covering 156,779 variants. After assessing the effect on growth inhibition of each variant, we were able to build a peptide bearing the most activity-enhancing amino acid residue combination. This new-to-nature peptide called Bac7PS has a higher activity towards a broad panel of bacterial pathogens than Bac71-23, low toxicity against eukaryotic cells, and good efficacy in vivo studies. The standard method to study the sequence-activity relationship of AMPs relies on the chemical synthesis of mildly modulated peptides followed by activity tests using antimicrobial susceptibility assays. Due to limited throughput and costs of peptide synthesis, these studies can typically deliver only very few data points [9, 14]. Deep mutational scanning of Bac71‑23 using random mutagenesis z is empirically divided into four groups, corresponding to very positive (yellow; z ≥ 40), positive (green; z ≥ 4), negative (blue; z ≤ -4), or very negative (purple; z ≤ −40) effects on the antimicrobial activity. No effect on growth inhibition is detectable if the z is close to 0 (white; −4 < z < 4). Black dots are used for indication of Bac71-23 wild-type amino acid residues. The underlined positions are chosen as targets for the subsequent site-saturation mutagenesis. Fig. 1 Sequence-activity relationship of Bac71-23. a DMS workflow. epPCR: the Bac71-23 gene is amplified at a high error rate using an error-prone DNA polymerase in the presence of Mn2+. Cloning: the mutagenized DNA sequences are inserted into plasmids downstream of inducible promoters. Transformation: E. coli TOP10 is transformed with the generated peptide-encoding DNA library. Growth: the pooled transformants are grown in a single shaking flask (n = 3), peptide synthesis is induced and plasmids are isolated after 4 h. NGS: the abundance of each peptide-encoding DNA sequence is determined by NGS at the time of induction and 4 h later. Analysis: for each peptide-encoding DNA sequence, the log2-fold change is determined (log2 ratio of abundances at the two time points). Histogram showing the log2-fold changes of the abundance of the peptide-encoding DNA of all 601,551 variants. Ranking: peptide sequences are ranked by the degree of the observed antimicrobial effect. The more negative a log2-fold change, the higher the observed antimicrobial effect and vice versa. b Bac71-23 sequence-activity relationship displaying the magnitude of the observed antimicrobial effect. For each amino acid residue substitution (and stop codon), the enrichment in higher or lower antimicrobial peptides is determined and a z-score (z) is calculated (see the “Methods” section). z corresponds to the number of standard deviations by which the calculated enrichment lies above (positive values) or below (negative values) the mean a null distribution indicating no enrichment. z is empirically divided into four groups, corresponding to very positive (yellow; z ≥ 40), positive (green; z ≥ 4), negative (blue; z ≤ -4), or very negative (purple; z ≤ −40) effects on the antimicrobial activity. No effect on growth inhibition is detectable if the z is close to 0 (white; −4 < z < 4). Black dots are used for indication of Bac71-23 wild-type amino acid residues. The underlined positions are chosen as targets for the subsequent site-saturation mutagenesis. Deep mutational scanning of Bac71‑23 using random mutagenesis First, we performed DMS of Bac71-23 to identify all amino acid residues essential for antimicrobial activity and those amendable to further activity optimization. To do so, the Bac71-23 coding gene was randomly mutagenized by epPCR, and the modified DNA fragments were ligated into plasmids allowing their expression from the tightly regulated PBAD promotor (Fig. 1a). To determine the anti- microbial activity of the synthesized peptide variants, we transformed E. coli TOP10 and induced their intra- cellular synthesis. Growth curves recorded for 94 ran- domly selected strains in microtiter plates indicated that about half of the Bac71-23 variants efficiently suppressed the growth of the respective host (Additional file 1: Fig. S1). To assess the antimicrobial effect of a much larger proportion of the randomly mutated library at once, we grew around 500 million transformed E. coli TOP10 cells expressing the entire peptide-encoding library in a shake flask (n = 3; Additional file 1: Fig. S2), induced peptide synthesis, and counted the abundance of each peptide- encoding DNA sequence at the time of induction and 4 h later by NGS (Additional file 2). As the synthesis of active peptides limits the growth of their hosts and the To expand the coverage of protein or peptide sequence- activity relationships in general, deep mutational scan- ning (DMS) methods are widely used as they grant access to millions of variants and data points in single experi- ments [15]. Most frequently, these methods are used to study the effects of single amino acid residue substitu- tions [16], since introducing multiple substitutions leads to a combinatorial explosion of possible variants. In DMS, first, large libraries are produced by systematically varying the coding DNA sequence by chemical DNA syn- thesis methods or error-prone polymerase chain reaction (epPCR) [17]. The resulting peptide or protein variants are then expressed recombinantly in cells and subjected to screening or selection protocols, allowing the cor- relation of phenotype of the variants with a measurable output, e.g., cell survival or the level to which genetically encoded fluorophores are synthesized. To also ascertain the genotype, next-generation sequencing (NGS) is used to identify and quantify the encoding DNA fragments [18]. In the case of AMPs, DMS can be based on self- screening where the degree to which growth is affected by AMPs is estimated via quantification of the relative Page 3 of 21 Koch et al. BMC Biology (2022) 20:114 Fig. 1 Sequence-activity relationship of Bac71-23. Deep mutational scanning of Bac71‑23 using random mutagenesis b Bac71-23 sequence-activity relationship displaying the magnitude of the observed antimicrobial effect. For each amino acid residue substitution (and stop codon), the enrichment in higher or lower antimicrobial peptides is determined and a z-score (z) is calculated (see the “Methods” section). z corresponds to the number of standard deviations by which the calculated enrichment lies above (positive values) or below (negative values) the mean a null distribution indicating no enrichment. z is empirically divided into four groups, corresponding to very positive (yellow; z ≥ 40), positive (green; z ≥ 4), negative (blue; z ≤ -4), or very negative (purple; z ≤ −40) effects on the antimicrobial activity. No effect on growth inhibition is detectable if the z is close to 0 (white; −4 < z < 4). Black dots are used for indication of Bac71-23 wild-type amino acid residues. The underlined positions are chosen as targets for the subsequent site-saturation mutagenesis. Fig. 1 Sequence-activity relationship of Bac71-23. a DMS workflow. epPCR: the Bac71-23 gene is amplified at a high error rate using an error-prone DNA polymerase in the presence of Mn2+. Cloning: the mutagenized DNA sequences are inserted into plasmids downstream of inducible promoters. Transformation: E. coli TOP10 is transformed with the generated peptide-encoding DNA library. Growth: the pooled transformants are grown in a single shaking flask (n = 3), peptide synthesis is induced and plasmids are isolated after 4 h. NGS: the abundance of each peptide-encoding DNA sequence is determined by NGS at the time of induction and 4 h later. Analysis: for each peptide-encoding DNA sequence, the log2-fold change is determined (log2 ratio of abundances at the two time points). Histogram showing the log2-fold changes of the abundance of the peptide-encoding DNA of all 601,551 variants. Ranking: peptide sequences are ranked by the degree of the observed antimicrobial effect. The more negative a log2-fold change, the higher the observed antimicrobial effect and vice versa. b Bac71-23 sequence-activity relationship displaying the magnitude of the observed antimicrobial effect. For each amino acid residue substitution (and stop codon), the enrichment in higher or lower antimicrobial peptides is determined and a z-score (z) is calculated (see the “Methods” section). z corresponds to the number of standard deviations by which the calculated enrichment lies above (positive values) or below (negative values) the mean a null distribution indicating no enrichment. Deep mutational scanning of Bac71‑23 using random mutagenesis a DMS workflow. epPCR: the Bac71-23 gene is amplified at a high error rate using an error-prone DNA polymerase in the presence of Mn2+. Cloning: the mutagenized DNA sequences are inserted into plasmids downstream of inducible promoters. Transformation: E. coli TOP10 is transformed with the generated peptide-encoding DNA library. Growth: the pooled transformants are grown in a single shaking flask (n = 3), peptide synthesis is induced and plasmids are isolated after 4 h. NGS: the abundance of each peptide-encoding DNA sequence is determined by NGS at the time of induction and 4 h later. Analysis: for each peptide-encoding DNA sequence, the log2-fold change is determined (log2 ratio of abundances at the two time points). Histogram showing the log2-fold changes of the abundance of the peptide-encoding DNA of all 601,551 variants. Ranking: peptide sequences are ranked by the degree of the observed antimicrobial effect. The more negative a log2-fold change, the higher the observed antimicrobial effect and vice versa. b Bac71-23 sequence-activity relationship displaying the magnitude of the observed antimicrobial effect. For each amino acid residue substitution (and stop codon), the enrichment in higher or lower antimicrobial peptides is determined and a z-score (z) is calculated (see the “Methods” section). z corresponds to the number of standard deviations by which the calculated enrichment lies above (positive values) or below (negative values) the mean a null distribution indicating no Fig. 1 Sequence-activity relationship of Bac71-23. a DMS workflow. epPCR: the Bac71-23 gene is amplified at a high error rate using an error-prone DNA polymerase in the presence of Mn2+. Cloning: the mutagenized DNA sequences are inserted into plasmids downstream of inducible promoters. Transformation: E. coli TOP10 is transformed with the generated peptide-encoding DNA library. Growth: the pooled transformants are grown in a single shaking flask (n = 3), peptide synthesis is induced and plasmids are isolated after 4 h. NGS: the abundance of each peptide-encoding DNA sequence is determined by NGS at the time of induction and 4 h later. Analysis: for each peptide-encoding DNA sequence, the log2-fold change is determined (log2 ratio of abundances at the two time points). Histogram showing the log2-fold changes of the abundance of the peptide-encoding DNA of all 601,551 variants. Ranking: peptide sequences are ranked by the degree of the observed antimicrobial effect. The more negative a log2-fold change, the higher the observed antimicrobial effect and vice versa. Sequence‑activity relationship of Bac71‑23f To determine the effect of each amino acid residue on antimicrobial activity, we investigated the ranking of 601,551 Bac71-23 variants (Additional file 3). As NGS- based abundancy rankings can be error-prone, especially at low DNA fragment read counts [22], here ranking should be considered a qualitative instead of a quantita- tive measure. This means that interpreting the effect of an amino acid residue should not be inferred from a single peptide (one data point in the ranking) but rather using groups of peptides all having the same amino acid residue at a specific position (multiple data points in the ranking). Fig. 2 DMS of Bac71-23 site-saturation mutagenesis library. a Enrichment curves. Peptides are first ranked according to their antimicrobial activity (x-axis; from left to right starting from the most growth inhibitory) and then a running enrichment score for each amino acid residue at each of the four substitution sites is calculated (y-axis). Increasing y-values indicate the presence of that particular amino acid residue in the ranking segment while decreasing y-values indicate the absence. In all cases, the AUC is calculated, whereby positive AUC values represent an enrichment among more active peptides (left side of the x-axis) and negative AUC values represent an enrichment among less active peptides (right side of the x-axis). An example is shown for the glutamate (E) at position 5 (AUCE5). b AUC values for each amino acid residue substitution. Effects on antimicrobial activity are binned empirically: very positive (yellow; AUC ≥ 0.2), positive (green; AUC ≥ 0.07), no effect (white; −0.07 < AUC < 0.07 = interquartile range of all values), negative (blue; AUC ≤ −0.07), or very negative (purple; AUC ≤ −0.2). Black dots correspond to the Bac7 parental amino acid residue at each position Fig. 2 DMS of Bac71-23 site-saturation mutagenesis library. a Enrichment curves. Peptides are first ranked according to their antimicrobial activity (x-axis; from left to right starting from the most growth inhibitory) and then a running enrichment score for each amino acid residue at each of the four substitution sites is calculated (y-axis). Increasing y-values indicate the presence of that particular amino acid residue in the ranking segment while decreasing y-values indicate the absence. Deep mutational scanning of Bac71‑23 using random mutagenesis propagation of the peptide-encoding DNA [19], a reduc- tion of the relative abundance of the peptide-encoding DNA of each variant, expressed as log2-fold change, is representative of antimicrobial activity (Fig. 1a). In total, the library consisted of 601,551 different Bac71-23 variants, ranked from the most active (=lowest negative log2-fold change) to the least active peptide (=highest positive log2-fold change) (Additional file 3). Among more heavily substituted variants (Additional file 1: Fig. S3), we found 398 peptides with one amino acid substi- tutions to Bac71-23 (87% out of 460 possible variants), 21,567 double substitutions (21% out of 101,200 possible Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 4 of 21 Page 4 of 21 Page 4 of 21 variants), 185,993 triple substitutions (1.3% out of ~14 million possible variants), and 228,433 quadruple substi- tutions (0.01% out of ~1.4 billion possible variants). However, amino acid residue substitutions were unevenly introduced in the library; for example, only one peptide incorporated a tryptophan at position 17 or methionine at position 11, while 2685 different peptides incorpo- rated the substitution to glycine at position 1 (Additional file 1: Fig. S4). Thus, to robustly measure the effect of a particular amino acid residue on antimicrobial activ- ity, we investigated whether specific amino acid residues were significantly enriched in peptides with a higher or lower antimicrobial activity using a permutation scheme (Fig. 1b; enrichment calculation is explained in more depth for Fig. 2a, or fully in the “Methods” section). Based on these results, we could not assign a contribu- tion to antimicrobial activity for 250 out of 483 possible single amino acid residues (23 positions x 20 amino acids plus the stop codon leading to truncated peptides; white boxes in Fig. 1b). Out of these 250, we could not draw any Sequence‑activity relationship of Bac71‑23f Deep mutational scanning of Bac71‑23 using site‑directed mutagenesisi Sequence‑activity relationship of Bac71‑23f In all cases, the AUC is calculated, whereby positive AUC values represent an enrichment among more active peptides (left side of the x-axis) and negative AUC values represent an enrichment among less active peptides (right side of the x-axis). An example is shown for the glutamate (E) at position 5 (AUCE5). b AUC values for each amino acid residue substitution. Effects on antimicrobial activity are binned empirically: very positive (yellow; AUC ≥ 0.2), positive (green; AUC ≥ 0.07), no effect (white; −0.07 < AUC < 0.07 = interquartile range of all values), negative (blue; AUC ≤ −0.07), or very negative (purple; AUC ≤ −0.2). Black dots correspond to the Bac7 parental amino acid residue at each position Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 5 of 21 statistical conclusion for 100 amino acid residues because they were too underrepresented in the library (Addi- tional file 1: Fig. S4, and Additional file 1: Fig. S5; p-value > 0.1, Benjamini-Hochberg adjusted). The remaining 233 amino acid residues showed an influence on antimicro- bial activity. Peptides bearing the amino acid residues of the wild-type Bac7 ‘RLPRPR’ sequence at position 9-14 and an arginine residue at position 6 were considerably enriched in the fraction of the highly antimicrobial vari- ants thereby indicating the crucial importance of these amino acids for the activity of Bac71-23 (yellow boxes in Fig. 1b). Additionally, we found that the random incor- poration of stop codons at any of the first 16 positions had mostly negative effects on antimicrobial activity, while insertions downstream of position 16 had no nega- tive effect (Fig. 1b). These results point towards a mini- mal requirement for Bac7-truncates of 16 amino acids by length, previously also reported by others [7]. Interest- ingly, substituting the wild-type amino acid residues at positions 3, 5, 7, and 15 and at positions 17-23 allowed to considerably increase the antimicrobial activity of the respective peptide variant. Hence, those 11 positions are potential targets for further activity optimization in Bac71-23 (Fig. 1b). However, proposing a Bac71-23 lead compound is hardly possible based on the sequence- activity relationship data obtained from the epPCR library mainly for two reasons: firstly, many amino acid substitutions were too underrepresented in the library to infer statistical significance concerning their effect on antimicrobial activity (Additional file 1: Fig. S4 and Additional file 1: Fig. S5). Sequence‑activity relationship of Bac71‑23f As only a small portion of the sequence space was cov- ered for simultaneous substitution (combinations), we decided to create in-depth knowledge on amino acid resi- due combinations in the course of a second DMS round using a focused Bac71-23 library. The focused library was generated by site-saturation mutagenesis, using one NNK codon for position 18 and a mixture of codons NDT, VMA, ATG, and TGG as described by Tang et al. [25] for the remaining positions. This reduced the need for oversampling drastically, as it limits the bias in amino acid distribution compared to less restrictive schemes (e.g., NNK) [26]. We again grew E. coli TOP10 cells expressing the entire library in a sin- gle flask (n=3; Additional file 1: Fig. S6), sequenced the peptide-encoding DNA at the time of induction and 4.5 h later (Additional file 4), and used its relative abundance as a proxy for growth inhibition (comparable to the DMS workflow display in Fig. 1a; Additional file 5). In total, 156,779 Bac71-23 derivatives were traced back thereby indicating 98% coverage.i Analogous to the epPCR library, we then quantified the effect of each amino acid residue substitution on anti- microbial activity. As each amino acid substitution (e.g., alanine in position 5) appeared in roughly 8000 different peptides (1 × 203=8000; one residue in a specific position fixed and combined with the entire set of possible sub- stitutions at the remaining three positions), no permu- tation scheme had to be used to calculate the respective effect (z) on antimicrobial activity. Instead, we directly inferred the effect of single amino acid residue substitu- tions by a method inspired by gene set enrichment analy- sis (GSEA) [27]. First, we ranked all peptides according to their antimicrobial activity (log2-fold change; similarly to Fig. 1a) and then drew enrichment curves for each amino acid residue (Fig. 2a). From those curves, the area under the curve (AUC) was computed giving values between −1 and 1. Positive AUC values indicate enrichment of a specific amino acid residue in peptides with higher antimicrobial activity (left side of the x-axis in Fig. 2a), while negative AUC values indicate enrichment in pep- tides with lower antimicrobial activity (right side of the x-axis in Fig. 2a). Amino acid residues with AUC values between −0.07 and 0.07 (covering the interquartile range (IQR), or middle 50%, of all AUC values) did not affect the enrichment and thus on antimicrobial activity. Sequence‑activity relationship of Bac71‑23f Secondly, Bac71-23 is already very active, and single, or even double substitution, may not suffice to considerably boost antimicrobial activity in minimal inhibitory concentration (MIC) assays [9]. As only a small portion of the sequence space was cov- ered for simultaneous substitution (combinations), we decided to create in-depth knowledge on amino acid resi- due combinations in the course of a second DMS round using a focused Bac71-23 library. residues downstream of position 16 with the ribosome [23, 24], and we sought to increase knowledge about the effect those C-terminal amino acid residue substitutions, positions 18-20 were also selected. statistical conclusion for 100 amino acid residues because they were too underrepresented in the library (Addi- tional file 1: Fig. S4, and Additional file 1: Fig. S5; p-value > 0.1, Benjamini-Hochberg adjusted). The remaining 233 amino acid residues showed an influence on antimicro- bial activity. Peptides bearing the amino acid residues of the wild-type Bac7 ‘RLPRPR’ sequence at position 9-14 and an arginine residue at position 6 were considerably enriched in the fraction of the highly antimicrobial vari- ants thereby indicating the crucial importance of these amino acids for the activity of Bac71-23 (yellow boxes in Fig. 1b). Additionally, we found that the random incor- poration of stop codons at any of the first 16 positions had mostly negative effects on antimicrobial activity, while insertions downstream of position 16 had no nega- tive effect (Fig. 1b). These results point towards a mini- mal requirement for Bac7-truncates of 16 amino acids by length, previously also reported by others [7]. Interest- ingly, substituting the wild-type amino acid residues at positions 3, 5, 7, and 15 and at positions 17-23 allowed to considerably increase the antimicrobial activity of the respective peptide variant. Hence, those 11 positions are potential targets for further activity optimization in Bac71-23 (Fig. 1b). However, proposing a Bac71-23 lead compound is hardly possible based on the sequence- activity relationship data obtained from the epPCR library mainly for two reasons: firstly, many amino acid substitutions were too underrepresented in the library to infer statistical significance concerning their effect on antimicrobial activity (Additional file 1: Fig. S4 and Additional file 1: Fig. S5). Secondly, Bac71-23 is already very active, and single, or even double substitution, may not suffice to considerably boost antimicrobial activity in minimal inhibitory concentration (MIC) assays [9]. Design of the optimized variant Bac7PS Cooperativity alone (∆AUC values>0.09) cannot be used for the design of highly optimized peptide vari- ants because these effects also appeared among peptide variants with low activity. For example, even though the strongest cooperativity resulted from the combination of proline at position 19 with cysteine at position 18 (∆AUC P19&C18 = + 0.18; asterisk in Fig. 3b), proline at position 19 occurred mostly among less active peptides (AUCP19 = −0.149; Fig. 2a, b). To investigate if such phenomena occurred, we meas- ured if the effect on antimicrobial activity of one amino acid residue substitution (AUCAA1; as shown in Fig. 2a, b) changed upon conditioning the same calculation on a second substitution at another position (AUCAA1 \| AA2). This change can be described as ∆AUCAA1&AA2=AUCAA1 \| AA2–AUCAA1. For example, we compared the effect on antimicrobial activity for the single substitution histidine at position 5 (AUCH5 = 0.089; Fig. 3a) among all peptides (~160,000) to the effect on antimicrobial activity of his- tidine at position 5 among peptides (~8000) that have a cysteine residue at position 18 (AUCH5\| C18 = 0.072; Fig. 3a). Note that the latter set of peptides is a subset of the former. In this case, ∆AUCH5&C18 is small (−0.016), suggesting the effect of histidine in a peptide does not change if cysteine is positioned at position 18, i.e., the combination behaved additively (Fig. 3a). Comparing the ∆AUC of all double combinations (4800) indicated that 96.1% (4613) behaved additively, (−0.09<∆AUC<+0.09 = within IQR ± 1.5 × IQR; Fig. 3b; all data in Addi- tional file 6). The remaining 187 combinations displayed non-additivity, of which 83 combinations showed coop- erativity (∆AUC>+0.09), and 104 combinations showed antagonism (∆AUC<−0.09). As exemplarily illustrated (Fig. 3a), we discovered cooperativity for phenylala- nine on position 19 and tyrosine at position 18 (∆AUC F19&Y18 =+0.144) and antagonism for two prolines at position 18 and 19 (∆AUCP19 &P18 =−0.207). In fact, two prolines at any of the positions 18, 19, and 20 behaved antagonistically (Additional file 6). Single substitu- tions to tyrosine had a positive or very positive effect on antimicrobial activity (Fig. 2a), but combining two tyrosines, e.g., at positions 18 and 19 (∆AUCY18&Y19 = −0.099), 5 and 18 (∆AUC Y5&Y18 =−0.152), or 18 and 20 (∆AUC Y18&Y20=−0.133), was antagonistic. Deep mutational scanning of Bac71‑23 using site‑directed mutagenesisi To study the focused library, we first performed com- binatorial saturation mutagenesis at four positions of Bac71-23 (204 = 160,000 possible variants). From the 11 positions that seemed to offer room for the accommo- dation of potentially activity-enhancing amino acid resi- dues (Fig. 1b), we chose to saturate positions 5 and 18-20. Position 5 was selected as it is not part of the crucial N-terminal ‘RRIR’ motif important for cellular uptake and ribosomal binding, nor of a conserved core region among proline-rich AMPs [23]. As little is known from crystallization studies about potential interactions of Our results indicated that peptides that had alanine, glycine, or arginine residues at any of the four positions experienced negative or very negative effects on anti- microbial activity, while isoleucine and tyrosine had positive or very positive effects (Fig. 2b). However, even though these analyses revealed the effect of each sin- gle amino acid residue substitution at all four positions in higher detail, they did not reveal the effect of residue Koch et al. BMC Biology (2022) 20:114 Page 6 of 21 combinations, likely to be substantial for generation if an antimicrobial lead compound. effects among serine (0 times), valine (1 time), and glu- tamate (2 times) (Additional file 1: Fig. S7). Moreover, non-additivity occurred more frequently for neighbor- ing amino acids, e.g., at positions 19 and 20, or 18 and 19 (see boxplots in Fig. 3b). Interestingly, we also discov- ered cooperativity between the proline and phenylalanine residues (∆AUC P19&F20=0.140) at positions 19 and 20 both being part of the Bac71-23 wild type sequence. We hence showed that for design of optimized peptide vari- ants, single substitutions cannot always be combined as antagonistic effects could strongly limit the antimicrobial activity of the peptide. Effects of amino acid residue combinations When substituting multiple amino acid residues in pro- teins or peptides, the effect on function usually equals the sum of the effect of the single substitutions (=addi- tive effects) [28]. In this case, we could select the most activity-enhancing amino acid residues of each position and combine them in one peptide. However, it has been shown that the effect of such combinations can be non- additive [28, 29], that is, it can become larger (coopera- tive) or smaller (antagonistic) than the sum of the single substitution. Naturally, especially antagonistic combina- tions have to be avoided. Design of the optimized variant Bac7PS Right: the influence of the second amino acid on the effect on antimicrobial activity of the first amino acid is calculated by subtracting the two previous calculations, resulting in ∆AUC. Indicators ~ and ^ are used to link the values to Fig. 3b. b ∆AUC values for all 4800 amino acid residue combinations. For each of the 20 amino acid residues at each of the four positions, there are 60 (20 amino acid residues at the remaining three positions) combinations with a second amino acid residue. Non-additive effects are estimated if a second amino acid residue changes the effect that a first amino acid residue has on antimicrobial activity, resulting in either larger positive or negative ∆AUC values. Cooperativity is measured when ∆AUC is larger than 0.09. Antagonism is measured when ∆AUC is smaller than −0 09 (=outliers of a boxplot containing all results; IQR ± 1 5 IQR) Exemplary ∆AUC values: Fig. 3 Effect of amino acid residue double combinations. a Examples of double amino acid residue combinations resulting in small (top), large positive (middle), and large negative (bottom) ∆AUC values. Left: to calculate the AUC of a single amino acid residue substitution (AUCAA1), all peptides are ranked on the horizontal axis according to their antimicrobial activity (as shown in Fig. 2a). Middle: the AUC of the same residue substitution is recalculated for the subset of peptides with a fixed second AA residue at another position (AUCAA1 \| AA2). Right: the influence of the second amino acid on the effect on antimicrobial activity of the first amino acid is calculated by subtracting the two previous calculations, resulting in ∆AUC. Indicators ~ and ^ are used to link the values to Fig. 3b. b ∆AUC values for all 4800 amino acid residue combinations. For each of the 20 amino acid residues at each of the four positions, there are 60 (20 amino acid residues at the remaining three positions) combinations with a second amino acid residue. Non-additive effects are estimated if a second amino acid residue changes the effect that a first amino acid residue has on antimicrobial activity, resulting in either larger positive or negative ∆AUC values. Cooperativity is measured when ∆AUC is larger than 0.09. Antagonism is measured when ∆AUC is smaller than −0.09 (=outliers of a boxplot containing all results; IQR ± 1.5 IQR). Exemplary ∆AUC values: P19 and C18, ~F19 and Y18, ^P19 and P18 Fig. Design of the optimized variant Bac7PS In gen- eral, we observed most non-additive effects among aro- matic amino acid residues (57 times), proline (24 times), and arginine (18 times), and very few non-additive We thus aimed to extract a design for the most opti- mized peptide variant using significant pattern mining [30]. This method looks for significantly enriched combi- nations of three amino acid residues among the 10% and 25% (arbitrary threshold set by us; Table 1; full dataset in Additional file 7) most and least antimicrobial peptides. A specific combination of four amino acid residues can- not be enriched in a fraction, as it appears only in a single peptide. In addition, as also previously stated, single data points in an NGS-based ranking can be error-prone. We thus investigated enrichment among all triple amino acid residue combinations (in total 32,000 = 203 × 4 3 ) shown in Additional file 7), each appearing in 20 different peptides (20 possible combinations for the fourth amino acid residue). Peptides with the triple combination of ala- nine-proline-proline residues at positions 5, 18, and 19 were the least active peptides in the entire library (Table 1). Indeed, our previous analysis showed that com- bining two prolines at positions 18 and 19 was strongly antagonistic for activity (∆AUCP19 &P18 =−0.207; Fig. 3a, b). Additionally, we found that, even though single tyros- ine residues were the most positive substitutions for anti- microbial activity (Fig. 2a, b), peptides with combinations of three tyrosine residues were not among the most active peptides (two examples shown in Table 1). This can be explained by the measured antagonism in combi- nations of two tyrosine residues shown before. We recorded the highest antimicrobial activity among pep- tides bearing tyrosine-phenylalanine-methionine at posi- tions 18–20, including cooperativity (∆AUCF19&Y18 Koch et al. BMC Biology (2022) 20:114 Page 7 of 21 Fig. 3 Effect of amino acid residue double combinations. a Examples of double amino acid residue combinations resulting in small (top), large positive (middle), and large negative (bottom) ∆AUC values. Left: to calculate the AUC of a single amino acid residue substitution (AUCAA1), all peptides are ranked on the horizontal axis according to their antimicrobial activity (as shown in Fig. 2a). Middle: the AUC of the same residue substitution is recalculated for the subset of peptides with a fixed second AA residue at another position (AUCAA1 \| AA2). Design of the optimized variant Bac7PS 3 Effect of amino acid residue double combinations. a Examples of double amino acid residue combinations resulting in small (top), large positive (middle), and large negative (bottom) ∆AUC values. Left: to calculate the AUC of a single amino acid residue substitution (AUCAA1), all peptides are ranked on the horizontal axis according to their antimicrobial activity (as shown in Fig. 2a). Middle: the AUC of the same residue substitution is recalculated for the subset of peptides with a fixed second AA residue at another position (AUCAA1 \| AA2). Right: the influence of the second amino acid on the effect on antimicrobial activity of the first amino acid is calculated by subtracting the two previous calculations, resulting in ∆AUC. Indicators ~ and ^ are used to link the values to Fig. 3b. b ∆AUC values for all 4800 amino acid residue combinations. For each of the 20 amino acid residues at each of the four positions, there are 60 (20 amino acid residues at the remaining three positions) combinations with a second amino acid residue. Non-additive effects are estimated if a second amino acid residue changes the effect that a first amino acid residue has on antimicrobial activity, resulting in either larger positive or negative ∆AUC values. Cooperativity is measured when ∆AUC is larger than 0.09. Antagonism is measured when ∆AUC is smaller than −0.09 (=outliers of a boxplot containing all results; IQR ± 1.5 IQR). Exemplary ∆AUC values: *P19 and C18, ~F19 and Y18, ^P19 and P18 Page 8 of 21 Koch et al. BMC Biology (2022) 20:114 Table 1 Significant pattern mining results A subset of triple amino acid combinations was obtained by significant pattern mining (see Additional file 7 for all combinations). The first column indicates the rank of each combination (from most to least significantly enriched in 10% most inhibitory peptides by p-values). Columns 2–5 indicate the amino acid residue at each position in the triple combination. “–” indicates the open fourth position of the peptide. Design of the optimized variant Bac7PS Columns 6–9 indicate the number of peptides containing the respective combination among the top 25% and top 10% most and least active peptides Rank Position 5 Position 18 Position 19 Position 20 # in 25% most actives # in 25% least actives # in 10% most actives # in 10% least actives 1 - Y F M 20 0 20 0 2 - N H N 20 0 20 0 3 C N N - 20 0 19 0 1602 Y Y - Y 11 0 7 0 4815 Y Y Y - 10 0 3 0 32,000 A P P - 0 20 0 20 A subset of triple amino acid combinations was obtained by significant pattern mining (see Additional file 7 for all combinations). The first column indicates the rank of each combination (from most to least significantly enriched in 10% most inhibitory peptides by p-values). Columns 2–5 indicate the amino acid residue at each position in the triple combination. “–” indicates the open fourth position of the peptide. Columns 6–9 indicate the number of peptides containing the respective combination among the top 25% and top 10% most and least active peptides A subset of triple amino acid combinations was obtained by significant pattern mining (see Additional file 7 for all combinations). The first column indicates the rank of each combination (from most to least significantly enriched in 10% most inhibitory peptides by p-values). Columns 2–5 indicate the amino acid residue at each position in the triple combination. “–” indicates the open fourth position of the peptide. Columns 6–9 indicate the number of peptides containing the respective combination among the top 25% and top 10% most and least active peptides activity of the peptides against the microbial pathogen model used for DMS (E. coli TOP10) in MIC assays. The set was completed by E. coli ATCC 25922, a qual- ity control strain often used in clinical microbiology, the transporter-loss mutant E. coli BW25113 ΔsbmA [32], which is less susceptible to Bac71-23 [33], and its paren- tal strain E. coli BW25113 [32]. Remarkably, we found an approximately twofold reduction of the MIC with Bac7PS for the DMS strain E. coli TOP10 (MIC of 2.1 μM for Bac7PS, 4.6 μM for Bac71-23). Design of the optimized variant Bac7PS Furthermore, Bac7PS showed a MIC of 14.4 μM against the transporter-loss mutant BW25113 ΔsbmA, while Bac71-23 was only active at the highest tested concentration of 52.1 μM (Table 2). Interestingly, the MIC for the quality control strain was similar for both peptides (2.6 μM for Bac7PS, 2.8 μM for Bac71-23). Next, we recorded the MICs of both peptides against a panel of 45 E. coli clinical isolates also contain- ing 23 MDR strains expressing extended-spectrum beta- lactamases (ESBL) or carbapenemase (CRE) collected from Swiss hospitals. The results indicated that the anti- microbial activity of Bac7PS exceeded that of Bac71-23 with an MIC50 for Bac7PS of 2.9 μM as compared to 7.5 μM for Bac71-23 (Fig. 4a), hence proofing the superiority of the DMS optimized variant Bac7PS. =+0.144; Fig. 3a, b), or asparagine-histidine-asparagine at positions 18–20, including cooperativity (∆AUC H19&N18 =+0.108) (Table 1). To design an optimized peptide variant as our lead compound, we build a peptide containing asparagine-his- tidine-asparagine at positions 18–20. This was the most significantly enriched triple combination among highly activity peptides in the library and incorporated one of the largest cooperative effects. For the remaining posi- tion 5, we avoided a second tyrosine and chose isoleucine because it showed the second most positive effects on growth inhibition at position 5 (Fig. 2a, b). In addition, among all 20 variants containing the chosen triple com- bination in the library, the peptide containing isoleucine at position 5 was the most active (Additional file 5). This Bac71-23 P5I R18Y L19F P20M variant is from here on referred to as Bac7PS. Characterization of Bac7PS and Bac71‑23 To investigate if our optimization strategy resulted in a potential antimicrobial drug lead compound with improved activity over Bac71-23, we chemically synthe- sized Bac7PS (92% purity) and Bac71-23 (95% purity) and characterized them further. While strictly adhering to CLSI standards [31], we first evaluated the antimicrobial Table 2 Summary of susceptibility assays MIC values are averaged (n>3) and performed under CSLI standards in the MHB II medium. Hemolysis assays and toxicity measurements were performed in triplicates. The therapeutic index (TI) is calculated by dividing the IC50 values measured with HeLa cells by the MIC50 obtained from clinical isolates (see Fig. 4a) MIC against E. coli strains [μM] Hemolysis of mouse red blood cells [%] Toxicity IC50 [μM] TI Peptide TOP10 ATCC 25922 BW 25113 BW 25113 ∆sbmA Clinical isolates (MIC50) 1xMIC 4xMIC HeLa HEK 293 Toxicity/MIC Bac71-23 4.6 2.8 7.4 52.1 7.5 2.1% 6.4% 1460 1970 195 Bac7PS 2.1 2.6 3.6 14.4 2.9 3.1% 3.8% 521 755 180 MIC values are averaged (n>3) and performed under CSLI standards in the MHB II medium. Hemolysis assays and toxicity measurements were performed in triplicates. The therapeutic index (TI) is calculated by dividing the IC50 values measured with HeLa cells by the MIC50 obtained from clinical isolates (see Fig. 4a) Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 9 of 21 We next investigated whether an increased propen- sity to damage membranes could be the reason for the improved antimicrobial activity of Bac7PS. Mem- brane damage is the most frequent way AMPs kill bac- teria and is often considered the reason for high toxicity against eukaryotic cells [4]. However, Bac7 is known to not damage bacterial membranes at its MIC [12]. Con- sequently, we investigated if Bac7PS had acquired a tendency to damage membranes, which could imply a greater risk for systemic human applications. Cell dam- age was assessed by measuring leakage of green fluores- cent protein (GFP), expressed in E. coli TOP10 cells, and uptake of (membrane-impermeable) propidium iodide (PI) [34]. For both peptides, we confirmed that the integ- rity of the membrane is neither affected at the MIC nor 8-fold higher concentrations (~1% PI-positive cells for Bac71-23 and Bac7PS, ~99% of cells retained GFP levels; Fig. 4b). In contrast, the known membrane-active peptide melittin rapidly induced PI uptake and loss of GFP at its MIC (Fig. 4b). Characterization of Bac7PS and Bac71‑23 However, at approximately 16-fold above its MIC (~35 μM), we noticed minor membrane dam- age of cells only treated with Bac7PS, indicated by loss of GFP (26% of cells lost GFP) and minor uptake of PI (~3% PI-positive cells). This effect was more pronounced when comparing these peptides in MHB I medium, which is not cation-adjusted and therefore less ionic, an effect that is often taken advantage of to increase membrane interaction of cationic AMPs [35, 36] (Additional file 1: Fig. S8). In conclusion, the growth inhibitory activity of Bac7PS was not due to membrane damage. To ensure that mammalian membranes were also not affected, we performed a hemolysis assay using red blood cells from mice. The results indicated that only a low frac- tion of membranes lysed (<4%) when applying Bac7PS and Bac71-23 at the MIC measured for E. coli ATCC 25922, and only very minor lysis (<7%) when exceeding Fig. 4 Characterization of Bac7PS and Bac71-23. a MICs of a panel of clinical isolates of E. coli (n=45) including MDR bacteria (ESBL, CRE, n = 25). MIC50 of Bac71-23 = 7.5 μM, MIC50 of Bac7PS = 2.9 μM. b Membrane damage assays measuring GFP loss (% of cells that lost GFP fluorescence) and PI uptake (% cells that gained PI fluorescence) when incubating E. coli TOP10 in MHB II for 30 min in the presence of increasing concentrations of Bac7PS and Bac71-23 (n = 3; error bars = 1SD). MIC of melittin against E. coli TOP10 is 5.0 μM (data not shown). c In vitro translation inhibition assays against E. coli ATCC 29522 (left) and HEK 293 ribosomes (right). IC50 values are extracted from a luminescence assay translating firefly luciferase mRNA peptide concentrations between 800 and 0.08 μM (n = 9). p-values (p) are calculated by performing a Wilcoxon rank-sum test, testing for differences in mean IC50 values of Bac71-23 and Bac7PS Fig. 4 Characterization of Bac7PS and Bac71-23. a MICs of a panel of clinical isolates of E. coli (n=45) including MDR bacteria (ESBL, CRE, n = 25). MIC50 of Bac71-23 = 7.5 μM, MIC50 of Bac7PS = 2.9 μM. b Membrane damage assays measuring GFP loss (% of cells that lost GFP fluorescence) and PI uptake (% cells that gained PI fluorescence) when incubating E. Characterization of Bac7PS and Bac71‑23 coli TOP10 in MHB II for 30 min in the presence of increasing concentrations of Bac7PS and Bac71-23 (n = 3; error bars = 1SD). MIC of melittin against E. coli TOP10 is 5.0 μM (data not shown). c In vitro translation inhibition assays against E. coli ATCC 29522 (left) and HEK 293 ribosomes (right). IC50 values are extracted from a luminescence assay translating firefly luciferase mRNA peptide concentrations between 800 and 0.08 μM (n = 9). p-values (p) are calculated by performing a Wilcoxon rank-sum test, testing for differences in mean IC50 values of Bac71-23 and Bac7PS Fig. 4 Characterization of Bac7PS and Bac71-23. a MICs of a panel of clinical isolates of E. coli (n=45) including MDR bacteria (ESBL, CRE, n = 25). MIC50 of Bac71-23 = 7.5 μM, MIC50 of Bac7PS = 2.9 μM. b Membrane damage assays measuring GFP loss (% of cells that lost GFP fluorescence) and PI uptake (% cells that gained PI fluorescence) when incubating E. coli TOP10 in MHB II for 30 min in the presence of increasing concentrations of Bac7PS and Bac71-23 (n = 3; error bars = 1SD). MIC of melittin against E. coli TOP10 is 5.0 μM (data not shown). c In vitro translation inhibition assays against E. coli ATCC 29522 (left) and HEK 293 ribosomes (right). IC50 values are extracted from a luminescence assay translating firefly luciferase mRNA peptide concentrations between 800 and 0.08 μM (n = 9). p-values (p) are calculated by performing a Wilcoxon rank-sum test, testing for differences in mean IC50 values of Bac71-23 and Bac7PS Koch et al. BMC Biology (2022) 20:114 Page 10 of 21 Page 10 of 21 the MIC fourfold (Table 2). The reduced dependency on the inner membrane transporter SbmA (Table 2) and the slight increase of membrane damage at very high concen- trations (Fig. 4b and Additional file 1: Fig. S8) indicated a higher degree of membrane interaction of Bac7PS com- pared to Bac71-23. This could potentially lead to facili- tated uptake of Bac7PS and thus a decreased MIC of the peptide. and extracted the IC50 value, the concentration at which half-maximal protein translation inhibition was achieved. Bac7PS triggered a mean 10% increase of the activity of E. coli ribosomes (Fig. 4c) compared to Bac71-23, which, however, did not reach statistical significance (p-value = 0.15, Wilcoxon rank-sum test). Characterization of Bac7PS and Bac71‑23 For HEK 293 ribosomes, Bac7PS showed a mean 38% increase in ribosomal bind- ing (Fig. 4c) compared to Bac71-23 (p<10−5, Wilcoxon rank-sum test), but at much higher concentrations com- pared to the bacterial ribosome. However, in a HEK 293 whole-cell assay, increased translational inhibition of Bac7PS relative to Bac71-23 only appeared at concentra- tions above 200 μM (Additional file 1: Fig. S9). At this concentration, we also observed general toxicity against HEK 293 cells (IC50 values in Table 2), which suggests that cell viability was rather affected by other mecha- nisms than translational inhibition (e.g., membrane damage). Thus, Bac7PS remained a very strong, but still selective ribosomal inhibitor for bacteria. While low MIC values are desirable for an antimicro- bial compound, low toxicity to eukaryotic cells is impera- tive. To measure the general toxicity of both peptides, we added them to human epithelioid cervix carcinoma (Henrietta Lacks = HeLa) and human embryonic kid- ney 293 (HEK 293) cells and quantified the degree of reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide (MTT), only catalyzed by meta- bolically active and viable cells [37]. Bac7PS showed an increase in toxicity against HeLa with an IC50 of 521 μM compared to an IC50 of 1460 μM for Bac71-23 (Table 2). Toxicity values against HEK 293 cells were similar when compared to HeLa cells, with an IC50 of 755 μM for Bac7PS compared to an IC50 of 1970 μM for Bac71-23 (Table 2). However, the TI (Table 2) remained very high (>180) for both peptides, especially when compared to most other AMPs, suggesting low toxicity at therapeuti- cally valuable concentrations [38]. Discussion DMS methods have been widely applied to study the sequence-activity relationship of proteins [39], optimize enzymes [40], or design therapeutics [41]. One of the major potential advantages of implementing DMS for the development of AMPs to antimicrobial drug candidates is that a large number of peptide variants can be func- tionally assessed without the need for their production and purification using chemical or biological methods. This advantage can be exploited to largely increase the sequence space assessed for studying sequence-activity relationships eventually allowing hit-to-lead optimization of AMPs. About the applied intracellular screening method, we note that intracellular bacterial self-screenings might lead to identifying better binders to intracellular targets, variants with increased membrane damaging properties, or variants that reach higher intracellular concentration because of increased solubility, mRNA stability, or pro- teolytic resistance. However, uptake across the outer and inner membrane, for example via the SbmA transporter, cannot be selected for. As for the proline-rich peptides used in this study, the N-terminal “RRIR” motif seems to be most critical for cellular uptake [8, 42], we decided not to modify this part and accepted the risk for contracting unknown effects regarding uptake when modifying other regions of Bac71-23. Still, even if intracellularly produced peptides would later fail to pass over the bacterial mem- brane, uptake could perhaps be recovered post-screen, for example by fusing them to motifs of cell-penetrating peptides [43]. Our quest towards the identification of non-additive effects in intracellular activity screenings allowed the design of Bac7PS bearing two amino acids that coopera- tively contributed to antimicrobial activity. While not researched well in the peptide field, results from directed evolution of proteins indicate that non-additive (i.e., epistatic) effects, must be considered to fully optimize a given scaffold for a specific function [46]. However, other than proteins, which usually adopt complex struc- tural confirmation, linear peptides display less structural flexibility, potentially leading to a reduced propensity for interactions between amino acid residues. Still, our results prove that non-additivity occurs (Fig. 3a/b). We reckon that it might predominately result from phenom- ena such as the modulation of the affinity for molecular We present an in-depth characterization of the impor- tance of the antimicrobial activity of each amino acid residue substitution in Bac71-23. Bac7PS activity in a murine modelfi The study was performed for each condition as described in a Koch et al. BMC Biology (2022) 20:114 Page 11 of 21 Page 11 of 21 to incorporate additional mutations in the same pep- tide. Much of the information acquired upon parallel- ized intracellular expression of Bac71-23 variants is in high agreement with previously reported data (Fig. 1b). Except position seven, we retrieved the core motif of Bac71-23 between positions 9 and 14, known to be largely evolu- tionarily conserved among proline-rich AMPs [5]. By the random insertion of stop codons, we found that peptides shorter than 16 amino acids are mostly inactive. As the peptides in our assay are synthesized in the cytosol, we attributed the loss of activity to weaker inhibition of pro- tein translation, which is supported by earlier research [42], but contested in recently made claims attributing the loss of antimicrobial activity of Bac71-15 to impaired uptake into the cytosol [5, 7]. Strikingly, we found that the C-terminus of Bac71-23 offered a large potential for optimization even though most research currently focuses on the substitution of the first 16 residues [9, 10]. Unlike the core motifs of proline-rich AMPs, which well overlapped in the crystal structure when bound to the ribosome, the residues of the C-termini of proline-rich AMPs are less conserved [23]. The higher spatial flexibil- ity of the upper ribosome chamber is illustrated by the fact that also Gram-positive targeting macrolide antibi- otics bind in that region [44] and that longer Bac7 vari- ants (60 or 35 amino acids by length) showed a broader (against different Gram-negative bacteria) antimicrobial activity than their shorter counterparts [8]. Surprisingly, we found that the insertion of a tyrosine residue is one of the most activity-enhancing single amino acid resi- due substitutions in the site-specific library, especially at positions 18 and 19 (Fig. 2b). Interestingly, even though a tyrosine residue is not present in full-length Bac7 com- prised of 60 amino acids, it is common among other proline-rich AMPs [5, 45], potentially because, similarly to arginine residues, an aromatic residue can well fill the space and interact with the amino acid residues or ribo- nucleotides of the surrounding ribosomal exit tunnel [9].i at administrated concentrations of 30 and 10 mg kg−1, respectively. Bac7PS activity in a murine modelfi Bac7PS activity in a murine model Finally, we investigated the efficacy of Bac7PS in a murine septicemia infection model (Fig. 5a). The maximal dose at which all CD-1 mice survived intraperitoneal Bac7PS treatment for 3 days was 50 mg kg−1 (Additional file 1: Fig. S10). Additionally, we saw that a second adminis- tration of 40 mg kg−1 Bac7PS was also tolerated by the animals (data not shown). For the efficacy study (Fig. 5a), we used two concentrations of Bac7PS and applied them twice 4 h apart: 30 mg kg−1, a concentration close to the maximum tolerated dose, and 10 mg kg−1. IP infection of CD-1 mice using E. coli ATCC 25922 resulted in the death of all 10 mice after 72 h if treated using the vehi- cle control and survival of all mice when treated with 30 mg kg−1 ciprofloxacin (CIP) (Fig. 5b). Bac7PS showed a dose-dependent efficacy with 80% and 60% survival Furthermore, we investigated the effect of the amino acid substitutions in Bac7PS on the expected main activ- ity, bacterial protein synthesis inhibition. For this, Bac7PS and Bac71-23 were incubated with bacterial S30 extracts (E. coli ATCC 25922) together with a luciferase encoding mRNA. To determine target selectivity for the bacterial ribosome, we also performed the same experiment using S30 extracts from HEK 293 cells. We measured the result- ing luminescence of each sample (peptide concentration range: 800 to 0.08 μM in 2.5-fold dilutions steps; n = 9) Fig. 5 Efficacy of Bac7PS in a murine model. a Efficacy study scheme. Drugs are applied to mice infected with E. coli ATCC 25922. Bacteria and drugs are administered intraperitoneally (IP). b Survival rates after infection. Mice are infected with E. coli ATCC 25922 and then treated with CIP as positive control and without a drug (vehicle) as the negative control. The study was performed for each condition as described in a e. Drugs are applied to mice infected with E. coli ATCC 25922. Bacteria and drugs Fig. 5 Efficacy of Bac7PS in a murine model. a Efficacy study scheme. Drugs are applied to mice infected with E. coli ATCC 25922. Bacteria and drugs are administered intraperitoneally (IP). b Survival rates after infection. Mice are infected with E. coli ATCC 25922 and then treated with CIP as positive control and without a drug (vehicle) as the negative control. Bac7PS activity in a murine modelfi Taken together, our optimization strategy guided us in the design of an antimicrobial lead compound with activ- ity improvements over Bac71-23, which has to be further evaluated when entering additional drug development stages. Discussion Compared to generating each amino acid residue substitution in a peptide indi- vidually, we used an epPCR for library generation, which also resulted in a large number of highly mutated (e.g., 38% were quadrupole mutants; Additional file 1: Fig. S3) Bac71-23 variants. Measuring the antimicrobial activity of heavily mutated variants was valuable to determine the sequence-activity relationship of Bac71-23 because (i) the activity of specific amino acid residue substitution can be robustly determined by averaging the activity of thousands of variants sharing the same specific exchange, and (ii) it allows to obtain information on the possibility Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 12 of 21 report examining the efficacy of Bac71-35 (35 amino acid long) in vivo, which, however, does not allow for direct comparison to our results [48]. The authors treated mice, infected with Gram-negative Salmonella typh- imurium intraperitoneally, with 30 mg kg−1 of Bac71-35 and showed an efficacy of 36%, and increased the mean survival from 10 days (untreated control) to 24.5 days (treated). In our study, all untreated mice already died within 30 h after an intraperitoneal injection of E. coli. However, we demonstrated a treatment efficacy of 60% when applying 10 mg kg−1 and 80% when applying 30 mg kg−1 of Bac7PS. More studies, especially on the pharmacokinetics and using different infection models are needed to evaluate the suitability of Bac7 variants and Bac7PS for further antimicrobial drug development stages. targets (e.g., macromolecules or membranes), compo- nents of the cellular defense machinery (e.g., drug efflux pumps or proteases), or exceedance of crucial physico- chemical parameters (e.g., solubility). We hence hypothe- size that also in the peptide field, non-additive effects are of considerable importance. Bac7PS excelled over Bac71-23 in terms of antimicrobial activity against clinical isolates including MDR bacteria and a decreased dependency on the SbmA transporter (Fig. 4a; Table 2). As Bac71-23 was largely inactive against the SbmA transporter knockout strain, this improvement is a very favorable new property, potentially making bac- terial resistance development harder. We mainly attrib- ute the activity gain to the increased ability to penetrate membranes, which was accompanied by a slight increase in membrane damage at higher concentrations compared to Bac71-23 (Fig. 4b; Additional file 1: Fig. S8; Table 2). This effect likely correlates with the observed cooperativ- ity between the phenylalanine and tyrosine residues. Conclusions In summary, we provided an example of a successful AMP hit-to-lead optimization by DMS, resulting in an antimicrobial lead compound. Bac7PS is a strong ribo- somal inhibitor that is non-toxic to human cells and displays a far higher TI than most other AMPs. Com- pared to Bac71-23, which already displays high activity against pathogenic bacteria, Bac7PS has increased activ- ity against multidrug-resistant clinical isolates. The gen- erated sequence-activity relationship landscape might inspire further engineering approaches on Bac71-23, and the principal strategy could be easily expanded for opti- mization of other intracellularly active, or even mem- brane damaging AMPs if displaying the peptides on the surface [19]. We envision coupling this platform to tar- get-based discovery, to directed evolution approaches, or to further increase the activity spectrum of AMPs when directly expressing the peptides in drug-resistant strains. Discussion Our hypothesis could be supported by the fact that aromatic amino acids, such as the now introduced tyrosine residue at position 18 or the phenylalanine residue at position 19, are known to enhance membrane penetration [47]. Simi- lar beneficial effects were observed in the study of Mard- irossian et al., relying on the activity assessment of 133 chemically synthesized Bac71-16 (16 amino acids long) derivatives [9]. The most promising candidate mentioned in that study, a peptide called B7-005 incorporating mul- tiple tryptophan residues, was also more active against a broader panel of bacteria including a SbmA knock- out. However, B7-005 was not more active than Bac71-16 against the initial screening strain and seemed to show slightly decreased inhibition of protein translation. On the contrary, Bac7PS showed increased activity against the screening strain E. coli TOP10 and an at least equally strong inhibition of protein translation than the wild- type peptide Bac71-23. Bac7PS displayed a small (non- significant) shift in binding the E. coli ribosome (+10%; p = 0.15) while binding to the human ribosome was more clearly improved (+38%; p < 10−5) (Fig. 4c). However, as toxicity and translational inhibition remained low for human cells (Table 2; Additional file 1: Fig. S9), there appears to be a large window at which bacteria can be efficiently eradicated with no detectable impairment of human cell viability. We assume that the absence of the SbmA transporter in human cells, as well as structural differences of the bacterial and mammalian membrane and cell surface, is responsible for the observed insen- sitivity of human cells, corroborating the potential for Bac7PS as an antimicrobial candidate. Generation of the site‑saturation Bac71‑23 library Generation of the site‑saturation Bac71‑23 library A focused strategy was pursued to generate diversity at positions 5, 18, 19, and 20 in the Bac71-23 peptide. These sites were simultaneously randomized on the genetic level by site-saturation mutagenesis using a single NNK codon for position 18 and a mixture of codons as sug- gested in the small intelligent approach [25] for the remaining three positions (see Additional file 1: Table S1 for sequences and mixing ratios). Degeneration was introduced by using the QuikChange technique [50], amplifying the pBAD plasmid containing the Bac71-23 gene using a mixture of 20 oligonucleotides (Additional file 1: Table S1) at a final concentration of 0.3 μM and Phusion® High-Fidelity PCR Master Mix in HF buffer. The amplification was performed using 20 cycles of 98°C for 10 s, 60°C for 15 s, and 72°C for 10 s. The PCR prod- uct was treated with the enzyme Dpn1 to remove the template plasmid and subsequently purified. The purified product was used to transform 20 μl of CloneCatcher™ Gold DH5G Electrocompetent E. coli (Genlantis, Burl- ington, USA) cells using electroporation. Transforma- tion, recovery, and storage were performed as explained for the random mutagenesis protocol, but this time we recovered approximately 2.3 million (E. coli Clone- catcher) and approximately 1.9 million (E. coli TOP10) colonies. Generation of the randomly mutagenized Bac71‑23 library To mutate the Bac71-23 gene randomly, we used epPCR. We amplified the Bac71-23 gene using primer 1 and primer 2 (Additional file 1: Table S1), which bind upstream of the first codon, including the start codon, and down- stream of the last codon, not including the stop codon. For amplification, we used the Pfu DNA Polymerase exo− mutant (D141A/E143A), Pfu reaction buffer includ- ing dNTPs, and 0.3 mM final concentration of MnCl2 to increase the error rate. The amplification was performed using 30 cycles of 98°C for 10 s, 60°C for 15 s, and 72°C for 10 s. The PCR product (118 bp in length) was purified and used for a restriction digest using enzymes HindIII- HF and PstI-HF. The digested product was again puri- fied using a DNA purification kit and ligated to plasmid pBAD [49] (Thermo Fisher Scientific) previously digested with the same enzymes using T4 ligase (800 units). The ligation product was used to transform 20 μl of Clone- Catcher™ Gold DH5G Electrocompetent E. coli (Gen- lantis, Burlington, USA) cells using electroporation. Chemicals and reagents Unless otherwise stated, all chemicals, reagents, and primers were obtained from Sigma Aldrich (Buchs, CH). Restriction enzymes and their buffers were obtained from New England Biolabs (Ipswich, USA). Kits for the isola- tion of plasmid isolation and DNA purification kits were obtained from Zymo Research (Irvine, USA). Peptides in either purified (>90%) or crude form were obtained from Pepscan (Lelystad, NL) or Genescript (Piscataway Town- ship, USA). Sanger sequencing was done at Microsynth (Balgach, CH). Lastly, it is hard to judge if the activity increase of Bac7PS compared to Bac71-23 measured in vitro can be translated to in vivo experiments. There has been one Koch et al. BMC Biology (2022) 20:114 Page 13 of 21 Generation of the site‑saturation Bac71‑23 library Recovered cells were plated and incubated overnight on LB agar plates supplemented with carbenicillin. Approxi- mately 5.6 million colonies were washed off several plates using LB medium and the plasmids containing the Bacterial strains and cultivations peptide-encoding DNA sequences were extracted from 2.5 × 109 cells. An aliquot of 5 ng of these plasmids was used to transform E. coli TOP10 cells. Approximately 10 million colonies were recovered from the plates after overnight incubation by washing with LB medium. The suspension was diluted to OD = 1 with LB medium, glyc- erol was added to a final concentration of 20% (v/v), and aliquots of 500 million cells were stored at −80°C. Unless otherwise stated, all experiments were performed using E. coli TOP10 (F– mcrA Δ (mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK λ– rpsL(StrR) endA1 nupG; Thermo Fisher Scientific, Waltham, USA). In this study, overnight cul- tivations were performed either in 14-ml polypropylene tubes (Greiner, Kremsmuenster, AT), filled with 5 ml of lysogeny broth (LB) medium (Difco, Becton Dickinson, Franklin Lakes, USA) or in 96-deep-well polypropylene plates (Greiner, Kremsmuenster, AT) filled with 500 μl of LB medium. All samples were incubated at 37°C with agitation on a shaker (Kuhner, Birsfelden, CH) operated at 200 r.p.m. and 25 mm amplitude. All media were sup- plemented with the appropriate antibiotic for plasmid maintenance (50 μg ml−1 kanamycin; 100 μg ml−1 car- benicillin) and 1% (w/v) d-glucose for repression of gene expression from catabolite-repression sensitive promoter PBAD. In the case of peptide expression experiments, cul- tures were incubated without d-glucose and 0.3% (w/v) of the inducer l-arabinose was used for induction. For all cultivations on solid medium, 15 mg ml−1 agar (Difco) was added to the broth, and incubation was performed in an incubator (Kuhner) at 37°C. If not indicated dif- ferently, the optical densities (OD) of bacterial cultures were determined by measuring light scattering at 600 nm using a UV/VIS spectrophotometer (Eppendorf, Ham- burg, DE). Next‑generation sequencing Peptide-encoding DNA sequences on plasmids, collected from both experiments (three replicates across the dif- ferent time points) were sequenced using NGS. To first amplify the peptide-encoding DNA, we added primer 1 and primer 2 (Additional file 1: Table S1), 100 ng of plas- mid, and thermocycled (10 cycles of 98°C for 10 s, 60°C for 15 s, and 72°C for 10 s). The amplification product was purified using an agarose gel. Single Index PentA- dapters from Pentabase were used to prepare PCR-free libraries with the KAPA HyperPrep Kit (Roche, Basel, CH) according to the manufacturer’s specifications. Libraries were quantified using the qPCR KAPA Library Quantification Kit. Libraries were pooled and sequenced single read with 101 cycles using an Illumina NovaSeq 6000 SP flow cell. Roughly 10% genomic PhiX library was used as a spike-in to increase sequence diversity. Base- calling was done with bcl2fastq v2.20.0.422. The resulting fastq files were processed using the software Geneious Prime 2020 (Biomatters, Auckland, NZ) and an in-house software written in R. For the randomly mutagenized library, we first discarded all sequences that missed the combination of a start codon and 69 bases downstream stop codon. Next, we discarded all sequences that appeared less than five times in at least two replicates. Next, all DNA sequences were translated and the result- ing peptide sequences were counted for each replicate and time point. All NGS counts can be seen in Additional file 2. For the site-saturation library, we first discarded all sequences that did not have a start codon and 69 bases downstream stop codon, translated the DNA sequences into peptide sequences, aligned them to our reference table of 160,000 possible peptide variants, and counted them for each replicate and time point. All NGS counts can be seen in Additional file 4. Single‑strain growth experiments To assess the antimicrobial activity of single peptides when expressed intracellularly in E. coli TOP10, a mon- oclonal strain carrying the pBAD plasmid containing a single peptide gene was picked from solid media, incu- bated overnight, and inoculated into a fresh LB medium containing 0.3% (w/v) l-arabinose to a final OD of 0.01 into 96-well microtiter plate (Greiner). Growth of strains was recorded by measuring OD in a Tecan Infinite 200 PRO (Tecan, Männedorf, CH) for at least 4 h (37°C, 1.5 mm orbital shaking). Parallel growth experiment To assess the antimicrobial activity of multiple pep- tides in parallel when expressed intracellularly in E. coli TOP10, previously prepared aliquots containing E. coli plasmid libraries created by either random mutagen- esis or site-saturation mutagenesis were used. For both approaches, three aliquots containing approximately 500 million cells each of E. coli TOP10 harboring peptide- encoding DNA sequences on the pBAD plasmid were thawed and added to three 1-l baffled shake flasks con- taining 100 ml of LB medium + 100 μg ml−1 carbeni- cillin. The cultures were grown for roughly 7 h at 37°C. When the OD reached approximately 0.2, the cultures were supplemented with l-arabinose to a final concen- tration of 0.3% (w/v) to induce peptide expression. When analyzing the randomly mutagenized library, cell samples were taken from each biological replicate at the point of Koch et al. BMC Biology (2022) 20:114 Page 14 of 21 induction and 4 h post-induction. When analyzing the library created by site-saturation mutagenesis, cell sam- ples were taken from each biological replicate at the point of induction and 4.5 h post-induction. The plasmids were extracted from all samples using a plasmid isolation kit. Ranking of peptides based on antimicrobial activity induction and 4 h post-induction. When analyzing the library created by site-saturation mutagenesis, cell sam- ples were taken from each biological replicate at the point of induction and 4.5 h post-induction. The plasmids were extracted from all samples using a plasmid isolation kit. Ranking of peptides based on antimicrobial activity To analyze the NGS read counts of both libraries, we used the standard workflow of DESeq2 [51] (NGS read count normalization, dispersion estimates, and Wald’s test). We calculated the log2-fold changes of the NGS read counts (listed for each peptide in Additional files 2 and 4) between the time of induction and 4.0 h (random mutagenesis) as well as 4.5 h (site-saturation mutagen- esis) post-induction. A Bayesian shrinkage estimator was employed to shrink the log2-fold change for each sequence using the R/Bioconductor package ‘apeglm’ [52]. Finally, the shrunken log2-fold change was used as a proxy for the antimicrobial activity of each peptide, as the propagation rate of the peptide-encoding DNA would follow the growth rate of the respective host. The more negative a log2-fold change, the higher the observed antimicrobial effect. The ranked peptide list from the randomly mutagenized library and the focused library are in Additional files 3 and 5, respectively. Enrichment curve‑derived AUCs to quantify the degreed of growth inhibitionf To determine the effect of amino acid residue substi- tutions on antimicrobial activity, we applied a varia- tion of the GSEA [27]. This adjusted method was based on drawing what we refer to here as enrichment curves (Fig. 2a). In those plots, each value on the x-axis repre- sented a peptide ranked by their shrunken log2-fold changes, giving rise to the ranked peptide set L. More active peptides are assigned to the left spectrum of the x-axis (lowest log2-fold change), while less active pep- tides are assigned to the right spectrum of the x-axis. For each single amino acid residue substitution, e.g., alanine at position 1, we defined S to be a set of all peptides that exhibit this substitution. Each y-value indicated whether the corresponding peptide pertains to the peptide-set or not. Formally, if a peptide pi in the ranked list L pertains to the peptide set S, its value is defined as (1) Phit(S, i) = pi∈S;j≤i 1 \| S \| (1) If peptide pi is not present in the set S, its value will correspond to (2) Pmiss(S, i) = pi /∈S;j≤i 1 N−\| S \| (2) where N corresponds to the total number of peptides in the ranked list. Subramanian et al. developed a so-called enrichment score (ES) that is defined as the maximum deviation of Page 15 of 21 ch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 15 of 21 measurements under the null hypothesis. It is defined as Phit − Pmiss from zero. We proposed a slightly different approach, which we referred to as the AUC. Compared to the ES, the AUC describes the complete dynamic of enrichment curves. We computed the AUCs as follows: Phit − Pmiss from zero. We proposed a slightly different approach, which we referred to as the AUC. Compared to the ES, the AUC describes the complete dynamic of enrichment curves. We computed the AUCs as follows: as as (5) pSM = #abs auc(SM) ≥ ˆ auc(SM) nperm (5) (3) AUC(S) = 1 \| L \| i=1,...,\|L\| [Phit(S, i) + Pmiss(S, i)] (3) To account for multiple testing, p-values were adjusted using the Benjamini-Hochberg procedure with a false discovery rate of α = 0.1. Determination of non‑additivityhf The AUCAA1 describes the effect of a single amino acid residue substitutions (AA1) on antimicrobial activity and can be calculated as described in Eqs. (1-3). We defined the conditional AUCAA1 \| AA2 as the effect of a single amino acid residue substitutions (AA1) on antimicro- bial activity using a ranked peptide set L′ containing only peptides that have a fixed amino acid residue (AA2) at a specific position. To determine if two amino acid residues behave non-additively, we calculated the difference of the AUC calculated for the conditional ranked peptide set L′ (with a size of approximately 8000 peptides) and the AUC calculated for the entire ranked peptide set L (with a size of approximately 160,000 peptides). This difference can be described as ∆AUCAA1&AA2 = AUCAA1 \| AA2 − AUC AA1 (see Fig. 3a for examples). ∆AUC values were calcu- lated for all possible 4800 (considering both directions of the combinations) combinations of two amino acid residues (see Additional file 6). By creating a boxplot of all resulting ∆AUC values, we defined that combinations resulting in ∆AUC values that lie between both whisk- ers of the boxplot (1.5 times the lower limit of the IQR to 1.5 times the upper limit of the IQR) behave additively. Non-additivity in the form of antagonism, i.e., one amino acid residue decreased the effect of the other amino acid residue on antimicrobial activity, was defined for combi- nations with ∆AUC values below −0.09. Non-additivity in the form of cooperativity, i.e., one amino acid residue increased the effect of the other amino acid residue on antimicrobial activity was defined for combinations with ∆AUC values above +0.9. In cases where the peptide-set S is small, i.e., if there are only a few observations for an amino acid residue substitution, the computation of AUCs can be mislead- ing. To present a more robust measurement for the randomly mutagenized Bac71-23 library, we resorted to a permutation scheme that allowed us to derive better estimates of the effect of each amino acid residue sub- stitution on activity. These permutation schemes relied on drawing from the null distribution, i.e., assum- ing that a single substitution, in the following denoted as SM, is not overrepresented in peptide with higher or lower antimicrobial activity. Determination of non‑additivityhf The following scheme is executed for each permutation: • Randomly permute the peptides in list L, giving rise to permuted list ˆL , which destroys the activity- based ranking ˆ • Compute enrichment curves for permuted list ˆL and single substitution SM ˆ • Compute the permuted ˆ AUC(SM) This scheme is repeated nperm times, where commonly nperm = 103, or 104, giving rise to the null distribution of the AUC values. z-scores were derived as: (4) zSM = auc(S) −mean ˆ auc(SM) std ˆ auc(SM) (4) Enrichment curve‑derived AUCs to quantify the degreed of growth inhibitionf Positive AUC values indicated that the corresponding set S is overrepresented in peptides with higher anti- microbial activity (top of the list), while negative AUC values indicate that the corresponding peptide set is overrepresented in peptides with lower antimicrobial activity (bottom of the list). AUC values close to zero indicated that the peptide set was randomly distributed across the list, or exhibited bimodal behaviors. Significant pattern mining to rank amino acid residue combinationsi z-scores represented how many standard deviations the observed AUC value differed from the mean of all AUC values derived from the null distribution. A large positive z-score indicated an enrichment of SM among more active peptides, and a large negative z-score denotes enrichment of SM among less active peptides. We furthermore computed a two-sided p-value ( pSM ) to assess the statistical significance of the observed z-scores represented how many standard deviations the observed AUC value differed from the mean of all AUC values derived from the null distribution. A large positive z-score indicated an enrichment of SM among more active peptides, and a large negative z-score denotes enrichment of SM among less active peptides. We furthermore computed a two-sided p-value ( pSM ) to assess the statistical significance of the observed Significant pattern mining emerged recently within the field of machine learning [53] and is devoted to find patterns that occur significantly more often in one ver- sus another group of observations. In our specific case, we defined a pattern to be any combination of three amino acid residues present in our data set. To find significantly enriched patterns in the data set, we first had to generate two classes. We achieved this by using the activity-based ranking according to the shrunken Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 16 of 21 concentration profile previously used for purification. The concentration was measured using the integrated peak area at 205 nm and then calculated using peptide- specific absorption properties [54, 55]. log2-fold change and focused our analysis on the most and least antimicrobial 10% and 25% of all peptides. To identify patterns that occur significantly more often in either the most and least antimicrobial fraction, we applied a tool named fast automatic conditional search [30]. It is based on the creation of a 2-by-2 contingency table for each pattern, and a subsequent two-sided Fish- er’s exact test (enrichment in either 10% or 25% most or least growth inhibitory peptides). Results from all triple combinations can be seen in Additional file 7. Purification of chemically synthesized peptides Purification of chemically synthesized peptides Peptide Bac71-23 (H-RRIRPRPPRLPRPRPRPLPFPRP- OH) and Bac7PS (H-RRIRIRPPRLPRPRPRPYFMPRP- OH) were obtained from Pepscan (Lelystad, NL) or Genscript (Piscatawa, USA) in >90% purity or in crude format and subsequently purified to >90% in house. For the latter, crude peptides were dissolved in 5 ml DMSO and 15 ml 0.1% aqueous trifluoroacetic acid, TFA. RP- HPLC-purification of the dissolved crude peptides was performed on an ӒKTAexplorer chromatography sys- tem (GE Healthcare, SE). The entire peptide sample was loaded onto a C18 column (PRONTOSIL 120 C18 AQ 10 μm, 250 × 20 mm, 50 × 20 mm precolumn, Bischoff, Leonberg, DE), heated to 30°C and operated at a flow rate of 10 ml min−1 using 0.1% aqueous TFA as solvent A and acetonitrile supplemented with 0.1% TFA as sol- vent B. The ratios of A to B were adapted for each pep- tide and typical values are given below. The column was equilibrated with the peptide-specific mixture of solvent A and solvent B (0–20%) prior to injection. After injec- tion and an initial wash step of 6 min, a gradient was imposed with the same eluent mixture, and then a gra- dient was applied, in the course of which the amount of solvent B was increased to 50–90% in 40 min. The col- umn was washed with 95% solvent B for 8 min and equil- ibrated with the specific solvent A/solvent B mixture for the next run for 13 min. Peptide elution was monitored spectrophotometrically at 205 nm and generally, the main peptide peak was collected. The sample was frozen at −80°C for >2 h and lyophilized (approx. 18 h) using a freeze-dryer (Alpha 2-4 LDplus, Christ, DE), connected to a vacuum pump (RC6, Vacuubrand, DE). The lyophi- lized peptides were dissolved in 1 ml DMSO and stored at −20°C. The concentration of the peptide stocks was determined via HPLC using an Agilent 1200 series RP- HPLC system. Each peptide stock was analyzed as a 1:100 dilution in water. An aliquot of 10 μl of the peptide stock was injected onto a C18 column (ReproSil-Pur Basic C18, 50 × 3 mm, Dr. Maisch, DE) operated with water supple- mented with 0.1% TFA as solvent A and acetonitrile sup- plemented with 0.1% TFA as solvent B. Separation was performed at a flow rate of 0.6 ml min−1 using the same Measurement of the MIC Bacterial cells were grown in cation-adjusted Mueller Hinton Broth (MHB II) overnight to stationary phase. The cultures were then supplemented with 200 g l−1 glycerol, aliquoted, and frozen at −80°C. For MIC meas- urements, an aliquot of the cells was thawed and resus- pended in MHB II to a final volume of 750 μl and cell concentration of 1 × 106 CFU ml−1. The purified pep- tides were thawed and the concentration was determined by RP-HPLC as described before. The peptides were diluted with sterile water to 4-fold the desired assays starting concentration and to a final volume of 50 μl. Pipetting of the MIC dilution series was done by a Ham- ilton Microlab STAR Liquid Handling System (Hamil- ton, Bonaduz, CH) and in 384-well plates (PP, F-bottom, 781201, Greiner, Kremsmünster, AT) with a final assay volume of 40 μl. The first well of each MIC dilution series was filled with 20 μl of 2-fold concentrated MHB II, wells 2–11 with 20 μl of MHB II, and well 12 with 40 μl MHB II (sterility control). Next, 20 μl of the peptide dilution was added to the first well, mixed, and a log2-serial dilution was performed from wells 1 to 10 (20 μl transfer volume). Well 11 served as growth control (i.e., no peptide added). In the last step, 20 μl of the bacterial cell suspension was added to wells 1–11 either using the pipetting robot (E. coli TOP10, BW25113, BW25113 ΔsbmA, and ATCC 25922) or by manual pipetting (E. coli clinical isolates). The plates were sealed airtight and incubated for 18 h without shaking at 37°C before reading the OD using an Infinite 200 PRO plate reader (Tecan, Männedorf, CH). The MIC value corresponded to the concentration at which no growth of the bacterial strain was observed (< 5% of the OD value of the growth control) and was evalu- ated using a custom-written script in the programming language R. MIC values of E. coli TOP10, BW25113, BW25113 ΔsbmA, and ATCC 25922 were determined at least in biological triplicates. All MIC experiments were determined in technical replicate. Membrane damage assay For membrane damage assays, the bacterial strain E. coli ATCC 25922 [pSEVA271-sfGFP] and the peptide dilu- tions were prepared as described for the MIC measure- ments but by scaling all volumes 5-fold and using 96-well plates (polypropylene, U-bottom, 650201, Greiner, Kremsmünster, AT) with a final assay volume of 200 μl. The bacterial strain suspension was furthermore supple- mented with a final concentration of 1 μg ml−1 propidium Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 17 of 21 Page 17 of 21 CO2. After incubation, 10 μl of (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (from a 5 mg ml−1 stock in DPBS) was added to each well. The plate was then incubated for 2 h. After incuba- tion, the cell culture medium containing residual MTT was removed from each well. The formed formazan crys- tals were dissolved by adding 100 μl of DMSO to each well. The formazan content in each well was determined by measuring the absorbance at 575 nm using an Infinite M1000 PRO plate reader (Tecan) and corrected for light scattering by subtracting the absorbance at 690 nm used as the reference wavelength. For each dilution series, the IC50 value was determined by computing a weighted n-parameters logistic regression using the “nplr” package in R (https://CRAN.R-project.org/package=nplr). The in vitro toxicity assays were performed at least in biologi- cal triplicates. iodide (PI, from a 1 mg ml−1 stock in water) just before pipetting the assay. After 1 h incubation at room temper- ature, the cell membrane integrity was assessed by flow cytometry using a Fortessa Analyzer (BD Biosciences) and appropriate filters for GFP and PI (488-nm laser with 530/30-nm bandpass filter and 579-nm laser with 610/20-nm bandpass filter). The fractions of PI-positive and PI-negative cells, as well as GFP-positive and GFP- negative cells, were determined with the same gate for all populations using the FlowJo V10 software (BD Bio- sciences). The membrane integrity assay was performed in biological triplicates. In vitro toxicity assay HeLa epithelioid cervix carcinoma cells (originally pur- chased: 93021013, Sigma Aldrich) and HEK 293 human embryonic kidney (originally purchased: 85120602, Sigma Aldrich) were routinely cultivated in Dulbecco’s MEM high glucose (DMEM, with l-glutamine, without phenol red, 1-26P32, Bioconcept, Allschwil, CH), supplemented with 10% fetal bovine serum (heat inactivated, sterile filtered, F9665, Sigma Aldrich) and 100 IU ml−1/100 μg ml−1 Penicillin/streptomycin (4-01F00, Bioconcept) at 37°C with 5% CO2. Cells were split at a confluency of ≤ 90% (every 3 to 4 days) and maintained for max. 10 pas- sages before a fresh aliquot of cells were seeded. For the tox assay, cells were cultivated for at least two passages after thawing, detached from the cultivation flask using Trypsin-EDTA (25300054, Gibco, Thermo Fisher Scien- tific), centrifuged at 200 × g for 4 min, and washed once by resuspending the pellet in an equal volume of Dul- becco’s phosphate-buffered saline (DPBS, D8537, Sigma Aldrich). The DPBS was removed by another centrifuga- tion step and the cell pellets were resuspended in fresh, prewarmed DMEM. The cell concentration was deter- mined using a Countess 2 device (Thermo Fisher Scien- tific) and approx. 5000 (HeLa) or 25,000 (HEK 293) cells were seeded into wells of a 96-well plate (F-bottom, PS, 655180, Greiner) together with 100 μl DMEM. The last row of each plate was filled with DMEM only. After cell seeding, the plate was incubated for 24 h at 37°C with 5% CO2. The following day, a log2-dilution series of the peptides were prepared as described for the MIC assays but using a 96-well plate (V-bottom, PP, 651201, Greiner) with a final volume of 50 μl. For the first well, 2-fold con- centrated DMEM medium was used; in wells 2–9 and 11, DMEM medium was used. Well 10 served as killing control (100% DMSO) and well 11 as non-treated con- trol (no peptide added). From the cell culture plate, 50 μl of the medium in each well (except the last row) was removed, discarded, and replaced with the 50 μl of liq- uid from the equivalent well on the peptide dilution plate. The plate was incubated again for 24 h at 37°C with 5% Hemolysis assay bl d Mouse blood was obtained from the ETH Phenomics Center. The erythrocytes were isolated by centrifugation at 500 × g for 10 min and removal of the blood plasma. The cells were washed three times by gently resuspending them in an equal volume of DPBS followed by centrifu- gation. After the last resuspension, the cells were diluted 1:50 in DPBS. For the hemolysis assay, a log2-serial dilu- tion of each peptide was prepared as described for the MIC but using DPBS and 96-well plates (U-bottom, PP, 650201, Greiner) with a final volume of 100 μl. As lysis control, 2.5% Triton-X100 in DPBS was used in well 10, well 11 served as non-treated control (no peptide added), and well 12 as blank. To each well of the dilution plate, 100 μl of the red blood cells suspension was added. The plate was incubated for 1 h at 37°C. After incubation, the plate was centrifuged at 500 × g for 10 min and 100 μl of the supernatant was transferred to a clean 96-well plate (F-bottom, PS, 655101, Greiner). The absorbance was measured at 540 nm using an Infinite M1000 PRO plate reader (Tecan) and corrected by the measurements from the untreated wells. The lysis of each peptide con- centration was expressed relative to the lysis control (set as 100% lysis). The hemolysis assay was performed in bio- logical triplicates. Production of S30 extracts To measure inhibition of the E. coli ATCC 29522 ribo- some, an S30 extract from E. coli cells was purified. E. coli ATCC 25922 was grown in 2 l of liquid LB medium at 37°C and 160 rpm. Cells were harvested in the logarith- mic phase by centrifugation for 30 min at 3500 × g at 4°C. The cell pellet was washed with ice-cold S30 buffer (20 mM Tris-acetate pH 8.2, 60 mM potassium acetate, 14 mM magnesium acetate, and 3 mM β-mercaptoethanol) Koch et al. BMC Biology (2022) 20:114 Page 18 of 21 Page 18 of 21 μl. The HEK293 translation master mix contained 7 μl HEK S30 extract, 20 mM HEPES-KOH buffer pH 7.4, 95 mM potassium acetate, 10 U RNAse inhibitor (Ribolock, Thermo Fisher), 0.125 mM amino acid mix, 12.5 mM cre- atine phosphate, 0.25 U creatine phosphokinase, 1.25 mM ATP, 0.25 mM GTP, 0.5 μg hFluc mRNA, 1.875 mM DTT, and 2.9 mM magnesium acetate to result in a final reac- tion volume of 20 μl. Sealed plates were incubated for 1 h at 37°C and stopped on ice before adding 75 μl of lucif- erase assay reagent (Promega) to each reaction. (BIO-TEK FLx80, Witec AG, Littau, CH). Regression analysis for IC50 calculation was performed using GraphPad Prism version 8.3.0 by using the built-in equation: [log(inhibitor) vs. response – Variable slope (four parameters)] with the built-in fitting method: “least squares (ordinary) fit. Y=Bottom + (Top-Bottom)/(1+10^((X-LogIC50)))”. and pelleted again for 30 min at 3500 × g at 4°C. The resulting cell pellet was shock-frozen in liquid nitrogen and stored at −80°C until further processing. Frozen E. coli cells were thawed on ice and resuspended in four times their volume ice-cold S30 buffer. The resulting cell suspension was lysed by passing it twice through a microfluidizer processor (Microfluidics, Westwood, MA, USA) at 25,000 lb/in2 and the cell lysate was immediately amended with DTT to a final concentration of 1 mM. The lysate was then centrifuged twice for 30 min at 30,000 × g at 4°C. After each centrifugation cycle, the clear superna- tant was carefully decanted and the pellet discarded. Ali- quots of S30 extract were shock-frozen in liquid nitrogen and stored at −80°C. To measure inhibition of the HEK 293 ribosome, an S30 extract was prepared from HEK 293-F cells. Whole cell translation inhibition assay HEK293 cells were cultivated in DMEM medium supple- mented with 10% fetal bovine serum and transfected with vector pRM-hFluc carrying a Fluc gene under constitu- tive CMV promotor control using Xfect™ Transfection Reagent (Takara, Japan) according to the manufacturer’s protocol [56]. Transfected cells were seeded into a 24-well plate 6 h post-transfection and pre-incubated overnight prior to addition of peptides in the next morning. Follow- ing 24 h of incubation in the presence of the peptides, cells were lysed using Lysis Buffer (Promega) and the lumines- cence was measured in a white microtiter plate using the FLx800 luminometer (Bio-Tek Instruments, USA). Animals CD-1 mice (7 weeks old, 27–28 g female) were used (Charles River, France). These animals were housed for a week of acclimation period before the experiment in a protected area in the ‘Centre de Zootechnie de l’Université de Bourgogne’ (Biosafety level 2 facility) and fed ad libitum according to the current recommendations by the European Institute of Health. Housing took place in rooms where a 12-h/12 h light/dark cycle is applied, the temperature ranges from 18 to 21°C and the relative humidity from 45 to 65%. The animal facility is author- ized by the French authorities (Agreement N° C 21 464 04 EA). Animal housing and experimental procedures were performed according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals. All procedures using animals were submitted to the Animal Care and Use Committee C2EA agreed by French authorities. Any animal showing poor conditions (20% body weight loss, signs of pain or dis- tress, lack of activity) was humanely euthanized. Production of S30 extracts Suspension cultures of HEK 293-F cells (Gibco, Thermo Fisher) were maintained in 293 SFM II medium (Thermo Fisher) supple- mented with 4 mM of L-glutamine according to the manu- facturer’s instructions. Cells in the logarithmic phase were harvested by centrifugation for 18 min at 200 × g at 4°C. The cell pellet was washed twice with ice-cold PBS and pel- leted again for 5 min at 200 × g at 4°C. Subsequently, cells were washed twice with ice-cold washing buffer (35 mM HEPES-KOH buffer pH 7.4, 146 mM NaCl, 11 mM D-(+)- glucose) and centrifuged for 5 min at 200 × g and 4°C. Cells were then resuspended in twice the volume of hypotonic buffer (20 mM HEPES-KOH buffer pH7.4, 10 mM KCl, 1.5 mM magnesium acetate, 1 mM DTT) and incubated for 10 min on ice. Cells were disrupted using a glass Dounce tissue homogenizer (Kimble, DWK Life Sciences, Mainz, Germany). After disruption, 0.1 volume of 10fold buffer (200 mM HEPES-KOH buffer pH 7.4, 1.2 M potassium acetate, 40 mM magnesium acetate, 50 mM DTT) of the total amount of hypotonic buffer was added. Cell debris was pelleted by centrifuging the lysate for 10 min at 700 × g at 4°C. The supernatant was then centrifuged for 15 min at 30,000 × g at 4°C. Aliquots of S30 extract were shock- frozen in liquid nitrogen and stored at −80°C. Acknowledgements We kindly thank all members of the Genomics Facility Basel for NGS library preparation and sequencing. We want to thank Dr. Gregor Schmidt for his sup- port using lab automation. We want to thank Dr. Bastian Rieck for useful dis- cussions on computational analyses. We thank Susanne Freedrich (Phenomics Center; ETH Zürich) for providing the mouse blood, and Rosmarie Sütterlin (University Basel) for providing human cell lines. We thank Dr. Tania Roberts for her helpful comments on the manuscript. In vitro translation inhibition assay y Bac71-23 and Bac7PS were dissolved in water and 0.3% Tween20 at a concentration of 3 mM. The two peptides and 0.3% Tween20 blank matrix were co-dispensed into white 96-well plates (Eppendorf) with a TECAN D300e digital dispenser to result in a concentration range of 0.08 to 800 μM in the final reaction mixture. The E. coli trans- lation master mix contained 4 μl E. coli S30 extract, 0.2 mM amino acid mix, 6 μg tRNA (Sigma), 0.4 μg hFluc mRNA, 0.3 μl protease inhibitor (cOmplete, EDTA-free, Roche), 12 U RNAse inhibitor (Ribolock, Thermo Sci- entific), 1.3 μl H2O, and 6 μL S30 premix without amino acids (Promega) to result in a final reaction volume of 15 Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 Page 19 of 21 Funding Open access funding provided by Swiss Federal Institute of Technology Zurich. S.S. was supported by a BRIDGE Proof of Concept grant (20B1-1_186286). Authors’ contributions P.K., S.S., and M.H. conceived the project. M.H., S.P., and S.N.H. supervised exper- imental work. P.K., S.S., and A.H. performed and analyzed experimental work. S.S. and I.W. conceived mouse experiments. M.G. and D.S. performed in vitro translation inhibition assays. A.G. and P.K. developed and performed computa- tional methods. P.K. and A.H. analyzed NGS data. P.K., A.G., and A.H. performed statistical analyses. P.K., S.S., M.H., and S.P. wrote the manuscript with input from all authors. All authors read and approved the final manuscript Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12915-022-01304-4. Additional file 1: Fig. S1. Intracellular expression of randomly mutated Bac71-23 variants. Fig. S2. Growth of E. coli TOP10 expressing the Bac71-23 error-prone library. Fig. S3. Histogram of Bac71-23 variants derived from the epPCR. Fig. S4. Amino acid residue counts per position. Fig. S5. Sta- tistical significance of the observed growth inhibitory measurements for each amino acid substitution. Fig. S6. Growth of E. coli TOP10 expressing the Bac71-23 focused library. Fig. S7. Interactions observed per amino acid residue. Fig. S8. Membrane damage assay. Fig. S9. Whole-cell translation inhibition (n = 1). Fig. S10. In vivo toxicity. Table S1. Primers and genes used. Additional file 1: Fig. S1. Intracellular expression of randomly mutated Bac71-23 variants. Fig. S2. Growth of E. coli TOP10 expressing the Bac71-23 error-prone library. Fig. S3. Histogram of Bac71-23 variants derived from the epPCR. Fig. S4. Amino acid residue counts per position. Fig. S5. Sta- tistical significance of the observed growth inhibitory measurements for each amino acid substitution. Fig. S6. Growth of E. coli TOP10 expressing the Bac71-23 focused library. Fig. S7. Interactions observed per amino acid residue. Fig. S8. Membrane damage assay. Fig. S9. Whole-cell translation inhibition (n = 1). Fig. S10. In vivo toxicity. Table S1. Primers and genes used. Additional file 2. NGS read counts for each peptide of the epPCR library. Additional file 2. NGS read counts for each peptide of the epPCR library. Additional file 3. Ranking of each peptide of the epPCR library according to antimicrobial activity (log2-fold change). Additional file 4. NGS read counts for each peptide of the site-saturation library. Additional file 5. Ranking of each peptide of the site-saturation library according to antimicrobial activity (log2-fold change). Additional file 6. ∆AUC-values of all double combinations of the site- saturation library. Additional file 7. Ranking of significant pattern mining of triple amino acid residue combinations. Availability of data and materials The computational workflow to reproduce the NGS count data analysis and ranking of peptides is available on GitHub (https://github.com/derpkoch/ Bac7) [57]. Additionally, the computational workflow to reproduce rankings, AUC calculations, permutation test, and calculation of ∆AUC to determine non-additivity is also available on GitHub using the same link. NGS data are available at the NCBI Sequence Read Archive (SRA) under accession number PRJNA730488 [58]. Additional files supporting the conclusions of this article are included within the article. Materials used in this study are available from the corresponding author upon reasonable request. Tools General data analysis and plotting were performed using R (version 4.1.2) and Python (version 3.6). MDR: Multidrug-resistant; AMP: Antimicrobial peptide; MoA: Mode of action; Bac7: Bactenecin-7; DMS: Deep mutational scanning; epPCR: Error-prone polymerase chain reaction; NGS: Next-generation sequencing; z: z-Score; MIC: Minimal inhibitory concentration; GSEA: Gene set enrichment analysis; AUC: Area under the curve; IQR: Interquartile range; GFP: Green fluorescent protein; PI: Propidium iodide; HeLa: cervical cancer cells from Henrietta Lacks; HEK 293: Human embryonic kidney 293; MTT: 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; TI: Therapeutic index; CIP: Ciprofloxacin; IP: Intraperitoneally; LB: Lysogeny broth; OD: Optical density. Supplementary Information For animal experiments, Bac7PS was synthesized at Genscript as acetate salt and with a purity of 92%. The peptide was reconstituted in Dulbecco’s phosphate- buffered saline (DPBS) at a concentration of 200 mg ml−1 and sterile filtered. All animal experiments were performed at Vivexia (Dijon, France) according to a pro- tocol submitted and approved by the local ethic com- mittee and authorities (Ethics Committee of Burgundy and the Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation). First, the maximum tol- erated dose (MTD) for the peptide was determined by testing different peptide doses in 8 groups with 5 ani- mals per group. The peptide solution was administered once by intraperitoneal (IP) injection with a volume of between 94 and 240 μl (depending on the weight of the animal and the dosage) and the animals were moni- tored for 2 h following the injection, then 6 to 8 h later, and then once or twice a day, depending on the clini- cal status, up to 5 days post-injection. For each group, a different, predefined peptide dose was tested starting with 500 and proceeding in the following sequence 50, 100, 75, 15, 25, 30, and 40 mg kg−1). The 40 mg kg−1 con- centration was administered twice with the 4-h time dif- ference. The MTD was defined as a dose where no dead animals were observed 2 days after injection. Second, the in vivo efficacy of the peptide was tested in a murine septicemia model induced by E. coli ATCC 25922. For this, a total of 4 groups with 10 animals per group were infected by IP injection of the bacterial inoculum (1 × 106 CFU per animal, +5% mucin) and each group was treated differently: A first group received ciprofloxacin (as positive control) administered IP once (1 h post- infection) at a dose of 30 mg kg−1. A second group received DPBS (as vehicle control) administered IP once (1 h post-infection). The two other groups received the peptide administered IP twice (1 and 4 h post-infection) either at a dose of 10 mg kg−1 or of 30 mg kg−1. As end- point, the wellbeing and survival rate, on a twice daily based evaluation for up to 3 days, was monitored. The online version contains supplem org/10.1186/s12915-022-01304-4. The online version contains supplementary material available at https://doi. org/10.1186/s12915-022-01304-4. The online version contains supplementary material available at https://doi. org/10.1186/s12915-022-01304-4. References 25. Tang L, Gao H, Zhu X, Wang X, Zhou M, Jiang R. Construction of “small- intelligent” focused mutagenesis libraries using well-designed combina- torial degenerate primers. Biotechniques. 2012;52:149–58. 1. Lewis K. The science of antibiotic discovery. Cell. 2020;181:29–45. 1. Lewis K. The science of antibiotic discovery. Cell. 2020;181:29–45. 2. Mookherjee N, Anderson MA, Haagsman HP, Davidson DJ. Antimicrobial host defence peptides: functions and clinical potential. Nat Rev Drug Discov. 2020;19:311–32. 2. Mookherjee N, Anderson MA, Haagsman HP, Davidson DJ. Antimicrobial host defence peptides: functions and clinical potential. Nat Rev Drug Discov. 2020;19:311–32. 26. Reetz MT, Kahakeaw D, Lohmer R. Addressing the numbers problem in directed evolution. Chem Bio Chem. 2008;9:1797–804. 3. Haney EF, Straus SK, Hancock REW. Reassessing the host defense peptide landscape. Front Chem. 2019;7 February:1–22. 3. Haney EF, Straus SK, Hancock REW. Reassessing the host defense peptide landscape. Front Chem. 2019;7 February:1–22. 27. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50. 4. Magana M, Pushpanathan M, Santos AL, Leanse L, Fernandez M, Ioannidis A, et al. The value of antimicrobial peptides in the age of resistance. Lancet Infect Dis. 2020;20:e216–30. 4. Magana M, Pushpanathan M, Santos AL, Leanse L, Fernandez M, Ioannidis A, et al. The value of antimicrobial peptides in the age of resistance. Lancet Infect Dis. 2020;20:e216–30. 28. Reetz MT. The importance of additive and non-additive mutational effects in protein engineering. Angew Chemie - Int Ed. 2013;52:2658–66. 5. Graf M, Mardirossian M, Nguyen F, Seefeldt AC, Guichard G, Scocchi M, et al. Proline-rich antimicrobial peptides targeting protein synthesis. Nat Prod Rep. 2017;34:702–11. 5. Graf M, Mardirossian M, Nguyen F, Seefeldt AC, Guichard G, Scocchi M, et al. Proline-rich antimicrobial peptides targeting protein synthesis. Nat Prod Rep. 2017;34:702–11. 29. Fjell CD, Hiss JA, Hancock REW, Schneider G. Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov. 2012;11:37–51. 6. Frank RW, Gennarot R, Schneiderq K, Przybylskin M, Romeo D. Amino acid sequences of two proline-rich bactenecins. J Biol Chem. 1990;265:18974. 6. Frank RW, Gennarot R, Schneiderq K, Przybylskin M, Romeo D. Amino acid sequences of two proline-rich bactenecins. J Biol Chem. 1990;265:18974. 30. Papaxanthos L, Llinares-López F, Bodenham D, Borgwardt K. Finding significant combinations of features in the presence of categorical covari- ates. Proc 30th Int Conf Neural Inf Process Syst. References 2016;30;2279–87. 7. Guida F, Benincasa M, Zahariev S, Scocchi M, Berti F, Gennaro R, et al. Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7. J Med Chem. 2015;58:1195–204. 7. Guida F, Benincasa M, Zahariev S, Scocchi M, Berti F, Gennaro R, et al. Effect of size and N-terminal residue characteristics on bacterial cell penetration and antibacterial activity of the proline-rich peptide Bac7. J Med Chem. 2015;58:1195–204. 31. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc. 2008;3:163–75. 8. Benincasa M, Scocchi M, Podda E, Skerlavaj B, Dolzani L, Gennaro R. Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates. Peptides. 2004;25:2055–61. 32. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006;2:2006.0008 9. Mardirossian M, Sola R, Beckert B, Valencic E, Collis DWP, Borišek J, et al. Peptide inhibitors of bacterial protein synthesis with broad spectrum and SbmA-independent bactericidal activity against clinical pathogens. J Med Chem. 2020;63:9590–602.i 33. Runti G, Lopez Ruiz M d. C, Stoilova T, Hussain R, Jennions M, Choud- hury HG, et al. Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35). J Bacteriol. 2013;195:5343–51. 10. Lai P, Tresnak DT, Hackel BJ. Identification and elucidation of proline-rich antimicrobial peptides with enhanced potency and delivery. Biotechnol Bioeng. 2019;116:2439–50. 34. Lehtinen J, Nuutila J, Lilius EM. Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability. Cytom Part A. 2004;60:165–72. 11. Mattiuzzo M, Bandiera A, Gennaro R, Benincasa M, Pacor S, Antcheva N, et al. Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides. Mol Microbiol. 2007;66:151–63. 35. Holfeld L, Knappe D, Hoffmann R. Proline-rich antimicrobial peptides show a long-lasting post-antibiotic effect on Enterobacteriaceae and Pseudomonas aeruginosa. J Antimicrob Chemother. 2018;73:933–41. proline-rich peptides. Mol Microbiol. 2007;66:151–63. 12. Podda E, Benincasa M, Pacor S, Micali F, Mattiuzzo M, Gennaro R, et al. Dual mode of action of Bac7, a proline-rich antibacterial peptide. Biochim Biophys Acta - Gen Subj. 1760;2006:1732–40. 36. Hancock REW, Sahl H-G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol. 2006;24:1551–7. 37. Mosmann T. Consent for publication 19. Tucker AT, Leonard SP, DuBois CD, Knauf GA, Cunningham AL, Wilke CO, et al. Discovery of next-generation antimicrobials through bacterial self- screening of surface-displayed peptide libraries. Cell. 2018;172:1–11. Competing interests 20. Baliga C, Brown TJ, Florin T, Colon S, Shah V, Skowron KJ, et al. Charting the sequence-activity landscape of peptide inhibitors of translation termination. Proc Natl Acad Sci. 2021;118:e2026465118. The authors declare that they have no competing interests. termination. Proc Natl Acad Sci. 2021;118:e2026465118. Author details 1 21. DeJong MP, Ritter SC, Fransen KA, Tresnak DT, Golinski AW, Hackel BJ. A platform for deep sequence–activity mapping and engineering antimi- crobial peptides. ACS Synth Biol. 2021;10:2689–704. 1 Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland. 2 Machine Learning and Computational Biol- ogy, Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland. 3 Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland. y 22. Rubin AF, Gelman H, Lucas N, Bajjalieh SM, Papenfuss AT, Speed TP, et al. A statistical framework for analyzing deep mutational scanning data. Genome Biol. 2017;18:1–15. 23. Gagnon MG, Roy RN, Lomakin IB, Florin T, Mankin AS, Steitz TA. Structures of proline-rich peptides bound to the ribosome reveal a common mech- anism of protein synthesis inhibition. Nucleic Acids Res. 2016;44:2439–50. Received: 10 September 2021 Accepted: 30 March 2022 24. Seefeldt AC, Graf M, Pérébaskine N, Nguyen F, Arenz S, Mardirossian M, et al. Structure of the mammalian antimicrobial peptide Bac7(1–16) bound within the exit tunnel of a bacterial ribosome. Nucleic Acids Res. 2016;44:2429–38. Ethics approval and consent to participate All animal experiments were performed at Vivexia (Dijon, France) according to a protocol submitted and approved by the local ethics committee and author- ities (Ethics Committee of Burgundy and the Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation). The animal facility is authorized Page 20 of 21 Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 by the French authorities (Agreement N° C 21 464 04 EA). Animal housing and experimental procedures were performed according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals. All procedures using animals were submitted to the Animal Care and Use Committee C2EA agreed upon by French authorities. Any animal showing poor conditions (20% body weight loss, signs of pain or distress, lack of activ- ity) was humanely euthanized. 16. Gray VE, Hause RJ, Fowler DM. Analysis of large-scale mutagenesis data to assess the impact of single amino acid substitutions. Genetics. 16. Gray VE, Hause RJ, Fowler DM. Analysis of large-scale mutagenesis data to assess the impact of single amino acid substitutions. Genetics. 2017;207:53–61. 16. Gray VE, Hause RJ, Fowler DM. Analysis of large-scale mutage y y g g data to assess the impact of single amino acid substitutions. Genetics. 2017;207:53–61. 17. Davis AM, Plowright AT, Valeur E. Directing evolution: the next revolution in drug discovery? Nat Rev Drug Discov. 2017;16:681–98. 18. Fowler DM, Araya CL, Fleishman SJ, Kellogg EH, Stephany JJ, Baker D, et al. High-resolution mapping of protein sequence-function relationships. Nat Methods. 2010;7:741–6. References Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. 13. Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018;18:318–27. 38. Bacalum M, Radu M. Cationic antimicrobial peptides cytotoxicity on mammalian cells: an analysis using therapeutic index integrative con- cept. Int J Pept Res Ther. 2015;21:47–55. 14. Torres MDT, Sothiselvam S, Lu TK, de la Fuente-Nunez C. Pep- tide design principles for antimicrobial applications. J Mol Biol. 2019;431:3547–67. 39. Sarkisyan KS, Bolotin DA, Meer MV, Usmanova DR, Mishin AS, Sharonov GV, et al. Local fitness landscape of the green fluorescent protein. Nature. 2016;533:397–401. 15. Fowler DM, Fields S. Deep mutational scanning : a new style of protein science. 2014;11:801–807. Page 21 of 21 Koch et al. BMC Biology (2022) 20:114 Koch et al. BMC Biology (2022) 20:114 40. Melnikov A, Rogov P, Wang L, Gnirke A, Mikkelsen TS. Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes. Nucleic Acids Res. 2014;42:1–8. 41. Linsky TW, Vergara R, Codina N, Nelson JW, Walker MJ, Su W, et al. De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2. Science. 2020;370:1208–14. 42. Sadler K, Eom KD, Yang JL, Dimitrova Y, Tam JP. Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7. Biochemistry. 2002;41:14150–7. 43. Milletti F. Cell-penetrating peptides: classes, origin, and current land- scape. Drug Discov Today. 2012;17:850–60. 44. Kannan K, Vázquez-Laslop N, Mankin AS. Selective protein synthesis by ribosomes with a drug-obstructed exit tunnel. Cell. 2012;151:508–20. 45. Mardirossian M, Pérébaskine N, Benincasa M, Gambato S, Hofmann S, Huter P, et al. The dolphin proline-rich antimicrobial peptide Tur1A inhib- its protein synthesis by targeting the bacterial ribosome. Cell Chem Biol. 2018;25:530–9. 46. Romero PA, Arnold FH. Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol. 2009;10:866–76. 47. Zhang W, Sato T, Smith SO. NMR spectroscopy of basic/aromatic amino acid clusters in membrane proteins. Prog Nucl Magn Reson Spectrosc. 2006;48:183–99. 48. Benincasa M, Pelillo C, Zorzet S, Garrovo C, Biffi S, Gennaro R, et al. The proline-rich peptide Bac7(1-35) reduces mortality from Salmonella typh- imurium in a mouse model of infection. BMC Microbiol. 2010;10. 49. Guzman LM, Belin D, Carson MJ, Beckwith J. References Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177:4121–30. 50. Liu H, Naismith JH. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008;8:91. 51. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21. 52. Zhu A, Ibrahim JG, Love MI. Heavy-Tailed prior distributions for sequence count data: removing the noise and preserving large differences. Bioin- formatics. 2019;35:2084–92. 53. Llinares-Lopez,Felipe; Borgwardt K. Machine learning for biomarker dis- covery: significant pattern mining. In: Analyzing network data in biology and medicine. Cambridge University Press; 2019. p. 313–68. 54. Anthis NJ, Clore GM. Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci. 2013;22:851–8. 55. Pelillo C, Benincasa M, Scocchi M, Gennaro R, Tossi A, Pacor S. Cellular internalization and cytotoxicity of the antimicrobial proline-rich peptide Bac7(1-35) in monocytes/macrophages, and its activity against phagocy- tosed Salmonella typhimurium. Protein Pept Lett. 2014;21:382–90. 56. Shcherbakov D, Teo Y, Boukari H, Cortes-Sanchon A, Mantovani M, Osin- nii I, et al. Ribosomal mistranslation leads to silencing of the unfolded protein response and increased mitochondrial biogenesis. Commun Biol. 2019;2:381. 57. Anja Gumpinger, Philipp. Zenodo. 2022. https://doi.org/10.5281/zenodo. 6377314. Accessed 22 Mar 2022. 58. Optimization of the antimicrobial peptide Bac7 by deep mutational scan- ning. NCBI BioProject. 2021. https://www.ncbi.nlm.nih.gov/bioproject? term=PRJNA730488. Accessed 17 May 2021. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note S i N i • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W2029287469	https://www.frontiersin.org/articles/10.3389/fnagi.2013.00032/pdf	English	null	Abnormal iron homeostasis and neurodegeneration	Frontiers in aging neuroscience	2,013	cc-by	11,350	INTRODUCTION ferritin aggregation, and oxidative damage. These processes are linked to a structural defect in the FTL C-terminus leading to cel- lular dysfunction that can be broadly classiﬁed as a loss of normal function and gain of toxic function as discussed below. Hereditary ferritinopathy (HF) or neuroferritinopathy is an auto- somal dominant, adult onset neurodegenerative disease caused by mutations in the ferritin light chain (FTL) gene (Curtis et al., 2001; Vidal et al., 2004a; Mancuso et al., 2005; Ohta et al., 2008; Devos et al., 2009; Kubota et al., 2009). The disease was ﬁrst reported in members of two families from England and France and was named neuroferritinopathy (Curtis et al., 2001). Sequence analysis of the FTL gene in members of the English family disclosed an adenine duplication, which predicts alteration of the C-terminal FTL polypeptide sequence and length (Curtis et al., 2001). Thus, far, six different mutations in exon four of the FTL gene have been reported, all affecting the FTL C-terminus. The clinical phenotype of HF is characterized by a movement dis- order, behavioral abnormalities, and cognitive impairment. The brain shows cerebral and cerebellar atrophy and cavitation of the putamen. The main neuropathologic ﬁndings are the presence of intranuclear and intracytoplasmic ferritin inclusion bodies (IBs) in glial cells and in some subsets of neurons, and abnormal iron deposition. Molecular level investigations of ferritin containing the mutant subunit reveal functional defects of iron mishandling, Barry B. Muhoberac 1* and Ruben Vidal 2 Barry B. Muhoberac 1* and Ruben Vidal 2 1 Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA 2 Division Neuropathology, Indiana Alzheimer Disease Center and Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 1 Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA 2 Division Neuropathology, Indiana Alzheimer Disease Center and Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA Abnormal iron metabolism is observed in many neurodegenerative diseases, however, only two have shown dysregulation of brain iron homeostasis as the primary cause of neurodegeneration. Herein, we review one of these - hereditary ferritinopathy (HF) or neuroferritinopathy, which is an autosomal dominant, adult onset degenerative disease caused by mutations in the ferritin light chain (FTL) gene. HF has a clinical phenotype characterized by a progressive movement disorder, behavioral disturbances, and cognitive impairment. The main pathologic ﬁndings are cystic cavitation of the basal ganglia, the presence of ferritin inclusion bodies (IBs), and substantial iron deposition. Mutant FTL subunits have altered sequence and length but assemble into soluble 24-mers that are ultrastructurally indistinguishable from those of the wild type. Crystallography shows substantial localized disruption of the normally tiny 4-fold pores between the ferritin subunits because of unraveling of the C-termini into multiple polypeptide conformations. This structural alteration causes attenuated net iron incorporation leading to cellular iron mishandling, ferritin aggregation, and oxidative damage at physiological concentrations of iron and ascorbate. A transgenic murine model parallels several features of HF, including a progressive neurological phenotype, ferritin IB formation, and misregulation of iron metabolism. These studies provide a working hypothesis for the pathogenesis of HF by implicating (1) a loss of normal ferritin function that triggers iron accumulation and overproduction of ferritin polypeptides, and (2) a gain of toxic function through radical production, ferritin aggregation, and oxidative stress. Importantly, the ﬁnding that ferritin aggregation can be reversed by iron chelators and oxidative damage can be inhibited by radical trapping may be used for clinical investigation. This work provides new insights into the role of abnormal iron metabolism in neurodegeneration. Correspondence: Correspondence: Barry B. Muhoberac, Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N Blackford Street LD 326, Indianapolis, IN 46202, USA e-mail: bmuhober@iupui.edu Keywords: neurodegeneration, neuroferritinopathy, ferritin, inclusion bodies, iron, oxidative stress Correspondence: Barry B. Muhoberac, Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, 402 N Blackford Street LD 326, Indianapolis, IN 46202, USA e-mail: bmuhober@iupui.edu Reviewed by: Reviewed by: Catarina Oliveira, University of Coimbra, Portugal Luis F. Gonzalez-Cuyar, University of Washington School of Medicine, USA REVIEW ARTICLE published: 30 July 2013 doi: 10.3389/fnagi.2013.00032 REVIEW ARTICLE published: 30 July 2013 doi: 10.3389/fnagi.2013.00032 AGING NEUROSCIENCE REVIEW ARTICLE published: 30 July 2013 doi: 10.3389/fnagi.2013.00032 Frontiers in Aging Neuroscience Edited by: Edited by: Katja Kanninen, University of Eastern Finland, Finland Reviewed by: Catarina Oliveira, University of Coimbra, Portugal Luis F. Gonzalez-Cuyar, University of Washington School of Medicine, USA Edited by: Katja Kanninen, University of Eastern Finland, Finland Reviewed by: Catarina Oliveira, University of Coimbra, Portugal Luis F. Gonzalez-Cuyar, University of Washington School of Medicine, USA FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION Along these lines, during iron excess the likelihood of inappropriate iron coordination by normally available small molecule cellular constituents, which are not part of proteins and usually not problematic, is increased potentially causing the generation of ROS beyond basal levels and protein oxidation. Proper disposal or reconstitution of damaged or misfolded proteins to avoid aggregation is an ongoing necessary cellu FIGURE 1 \| Structure of ferritin, one of its 24 subunits, and its pores. Ferritin assembles spontaneously from 24 conformationally equivalent subunits into a hollow spherical shell (A) with 2-, 3-, and 4-fold symmetry axes. The subunits (B) are each composed of ﬁve α-helices with four of them parallel and tightly associated and the ﬁfth (the E-helix) at an angle and pointed inwards. The junctions of the subunits form hydrophilic pores at the 3-fold axes (C), which are implicated as the entry path for iron ions, and hydrophobic pores at the 4-fold axes (D), which are smaller and considered closed, especially to ions. Pores are viewed from the ferritin shell interior with the inwardly pointing E-helices from the 4 subunits clearly visible in (D). Structures were taken from RCSB (code 2FG8). FIGURE 1 \| Structure of ferritin, one of its 24 subunits, and its pores. Ferritin assembles spontaneously from 24 conformationally equivalent subunits into a hollow spherical shell (A) with 2-, 3-, and 4-fold symmetry axes. The subunits (B) are each composed of ﬁve α-helices with four of them parallel and tightly associated and the ﬁfth (the E-helix) at an angle and pointed inwards. The junctions of the subunits form hydrophilic pores at the 3-fold axes (C), which are implicated as the entry path for iron ions, and hydrophobic pores at the 4-fold axes (D), which are smaller and considered closed, especially to ions. Pores are viewed from the ferritin shell interior with the inwardly pointing E-helices from the 4 subunits clearly visible in (D). Structures were taken from RCSB (code 2FG8). the 24-mer formed from two conformationally equivalent sub- units with slightly different masses and 54% sequence identity. The two subunits are folded from the ferritin heavy chain (FTH1) and light chain (FTL) polypeptides with the former longer by 8 amino acids. FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION Ferritin is uniquely suited for its crucial iron sequestration and storage function. Ferritin consists of 24 subunits that can self-assemble into a 480 kDa hollow sphere of ∼110 Å outer and ∼80 Å inner diameter (Figures 1A, B), which can store up to 4500 atoms of iron as a ferrihydrite biomineral (Vidal et al., 2004b; Crichton and Declercq, 2010). The exterior and interior of the ferritin shell are connected via channels (pores) along 3- fold and 4-fold symmetry axes at subunit junctions. There are eight 3-fold pores that are larger diameter and shorter relative to the six 4-fold pores. The 3-fold pores (Figure 1C), which are hydrophylic, have been implicated as the iron entry pathway in a number of studies, whereas the 4-fold pores (Figure 1D), which are hydrophobic, are smaller and considered essentially closed, especially to ions. Human ferritin is usually heteropolymeric with July 2013 \| Volume 5 \| Article 32 \| 1 www.frontiersin.org www.frontiersin.org www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal (Zecca et al., 2004; Berg and Youdim, 2006). The normal cellular response to abundant iron is to decrease the synthesis of the transferrin receptor, which transports iron into the cells, and to increase ferritin synthesis for appropriate iron sequestration and storage. During iron deﬁciency the synthesis of ferritin is inhibited in part by an increase in Iron Responsive Protein (IRP) binding to the Iron Responsive element (IRE) on target mRNAs. Clearly, iron must be made available to form the essential catalytic centers of neuroenzymes, e.g., tryptophan hydroxylase, which is required for serotonin synthesis, and tyrosine hydroxylase leading to dopamine. However, local iron concentration and its ligation (coordination) and oxidation state must all be carefully reg- ulated to prevent cellular damage. Indeed, the chemistry that occurs at the iron can vary substantially depending on iron lig- ation, which could be provided by protein donors (e.g., certain amino acid side chains) or small molecules normally available in cells. Much cellular chemistry is driven by the process of redox change, and cells have available the redox drivers of reductants such as NADPH, glutathione, and ascorbate, as well as oxygen. FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION Hydrogen peroxide and superoxide are generated in cells as part of several routine metabolic redox processes as well as by non- enzymatic reduced iron, and although they can deactivate a few enzymes, they are not particularly damaging at appropriate lev- els (Zecca et al., 2004). In fact, hydrogen peroxide has recently been characterized as a neuromodulator in striatal dopamine release (Rice, 2011). However, improperly coordinated iron has the potential to convert hydrogen peroxide and superoxide into the highly toxic hydroxyl radical, which is extraordinarily reac- tive. This radical indiscriminately attacks proteins, lipids, and DNA causing protein oxidation, fragmentation, and covalent crosslinking leading to their loss of function. Hydroxyl radi- cal production occurs through redox change in metal-centered Fenton- and Haber-Weiss- type reactions, and when iron is coor- dinated by less than 6 strongly-bound ligands this conversion is facilitated (Graf et al., 1984). Hydrogen peroxide, superoxide, and the hydroxyl radical are generally termed reactive oxygen species (ROS), and cells produce enzymes (e.g., glutathione peroxidase, catalase, and superoxide dismutase) and small molecules (glu- tathione) to help control their levels (Murphy et al., 2011). The ability of ferritin to convert Fe2+ to Fe3+ and store it internally as a non-reactive biomineral can be considered a “detoxiﬁcation” function by removing it from potentially inappropriate ligation and reactions. Although usually considered as antioxidants, glu- tathione and ascorbate have concentrations in the brain that are sufﬁciently high enough to provide reducing equivalents that can generate ROS and thus lead to cellular damage in the presence of improperly coordinated iron. Along these lines, during iron excess the likelihood of inappropriate iron coordination by normally available small molecule cellular constituents, which are not part of proteins and usually not problematic, is increased potentially causing the generation of ROS beyond basal levels and protein oxidation. Proper disposal or reconstitution of damaged or misfolded (Zecca et al., 2004; Berg and Youdim, 2006). The normal cellular response to abundant iron is to decrease the synthesis of the transferrin receptor, which transports iron into the cells, and to increase ferritin synthesis for appropriate iron sequestration and storage. During iron deﬁciency the synthesis of ferritin is inhibited in part by an increase in Iron Responsive Protein (IRP) binding to the Iron Responsive element (IRE) on target mRNAs. FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION Clearly, iron must be made available to form the essential catalytic centers of neuroenzymes, e.g., tryptophan hydroxylase, which is required for serotonin synthesis, and tyrosine hydroxylase leading to dopamine. However, local iron concentration and its ligation (coordination) and oxidation state must all be carefully reg- ulated to prevent cellular damage. Indeed, the chemistry that occurs at the iron can vary substantially depending on iron lig- ation, which could be provided by protein donors (e.g., certain amino acid side chains) or small molecules normally available in cells. Much cellular chemistry is driven by the process of redox change, and cells have available the redox drivers of reductants such as NADPH, glutathione, and ascorbate, as well as oxygen. Hydrogen peroxide and superoxide are generated in cells as part of several routine metabolic redox processes as well as by non- enzymatic reduced iron, and although they can deactivate a few enzymes, they are not particularly damaging at appropriate lev- els (Zecca et al., 2004). In fact, hydrogen peroxide has recently been characterized as a neuromodulator in striatal dopamine release (Rice, 2011). However, improperly coordinated iron has the potential to convert hydrogen peroxide and superoxide into the highly toxic hydroxyl radical, which is extraordinarily reac- tive. This radical indiscriminately attacks proteins, lipids, and DNA causing protein oxidation, fragmentation, and covalent crosslinking leading to their loss of function. Hydroxyl radi- cal production occurs through redox change in metal-centered Fenton- and Haber-Weiss- type reactions, and when iron is coor- dinated by less than 6 strongly-bound ligands this conversion is facilitated (Graf et al., 1984). Hydrogen peroxide, superoxide, and the hydroxyl radical are generally termed reactive oxygen species (ROS), and cells produce enzymes (e.g., glutathione peroxidase, catalase, and superoxide dismutase) and small molecules (glu- tathione) to help control their levels (Murphy et al., 2011). The ability of ferritin to convert Fe2+ to Fe3+ and store it internally as a non-reactive biomineral can be considered a “detoxiﬁcation” function by removing it from potentially inappropriate ligation and reactions. Although usually considered as antioxidants, glu- tathione and ascorbate have concentrations in the brain that are sufﬁciently high enough to provide reducing equivalents that can generate ROS and thus lead to cellular damage in the presence of improperly coordinated iron. Frontiers in Aging Neuroscience CLINICAL PRESENTATION, GENETICS, AND PATHOLOGY OF HF The neurodegenerative disease HF has been reported in members of Caucasian and Japanese families, being inherited in an autoso- mal dominant pattern. Linkage analysis, performed on members of an English family, linked the disease to a locus on chromosome 19q13.3, which contains the FTL gene. Affected individuals from this family were found to have an adenine insertion at position 460–461 of the FTL gene (c.460dupA) that was predicted to alter the carboxyl-terminal region of the protein (Curtis et al., 2001). Five additional mutations have been reported, all in exon 4 of the FTL gene, which consists of 4 exons and 3 introns. In all cases, the mutations (Table 1) affect the E-helix of the FTL polypeptide by altering both the C-terminal sequence and extending its length. In addition to these, two more cases of HF have been described. The ﬁrst case was diagnosed pathologically and no genetic data is available (Schröder, 2005), while the second case was described as an individual with a missense mutation (A96T) in FTL (Maciel et al., 2005). In the later, it remains to be seen whether this case reﬂects a bigger spectrum of the disease or a different condi- tion since the mother of the proband (also a carrier of the A96T mutation who displayed similar MRI ﬁndings) was asymptomatic (lack of autosomal dominant transmission), and the patient did not have signiﬁcant involvement of the putamen, thalamus, and substantia nigra. Transition metal ions such as iron, copper and zinc are often found at elevated levels in neurodegenerative diseases and are intimately connected with protein aggregation, misfolding and cellular dysfunction (Ayton et al., 2012). For example, the addi- tion of iron to solutions of α-synuclein or amyloid β enhances the rate of aggregation and/or ﬁbril formation even to the extent of inﬂuencing the structure of the aggregates formed, and ele- vated iron is associated with the pathology of these disorders in both animal models and humans. Metal ion binding can be causal to aggregation and misfolding, or be advantageous after they occur. However, metal ion-induced aggregated and misfolded proteins, while problematic for cellular processes and elimination, could foster an additional cellular dysfunction through provid- ing binding sites that improperly ligate (coordinate) iron and thus generate ROS. The resulting oxidative damage itself can enhance protein aggregation. FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION Both subunits consist of four parallel α-helices (A–D) of ∼45 Å length and a shorter, carboxy terminal α-helix (E) at a 60◦angle to the parallel helix bundle pointing into the inte- rior of the shell (Figure 1B) (Crichton and Declercq, 2010). The 4-fold pores are formed from four hydrophobically-associated E-helices donated from four different subunits (Figure 1D), while 3-fold pores are formed from C- and D-helices from three sub- units (Figure 1C). The FTH1 subunit contains the ferroxidase site in the interior of the parallel helix bundle, which converts Fe2+ to Fe3+ in the presence of O2 on the pathway to biomineraliza- tion. Iron then migrates to the ferrihydrite nucleation site on the interior surface of the FTL subunit to complete biomineral forma- tion. Interestingly, each organ ﬁne tunes the ratio of FTH1 to FTL subunits, which can vary substantially, for optimal physiologi- cal function. No homopolymers of the FTH1 subunit have been reported, but homopolymeric ferritin formed from 24 FTL sub- units is capable of a slow rate of iron sequestration and storage. The FTL subunit is more stable than the FTH1 subunit toward increasing temperature and denaturants, and when incorporated into the heteropolymer stabilizes it and hinders iron-induced aggregation (Santambrogio et al., 1992, 1993). Proper disposal or reconstitution of damaged or misfolded proteins to avoid aggregation is an ongoing necessary cellu- lar function, and protein aggregation is strongly linked with neurodegenerative diseases (Lansbury and Lashuel, 2006). Cells devote signiﬁcant resources energetically through specialized Properly functioning ferritin of the appropriate cellular con- centration is essential for iron homeostasis, and a number of neurodegenerative diseases have links with misregulation of iron July 2013 \| Volume 5 \| Article 32 \| 2 www.frontiersin.org www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal protein synthesis and transport toward the disposal or reconstitu- tion of oxidized or misfolded proteins, which are generally prone to aggregate formation. The aggregation process is dependent upon a variety of factors besides the identity and extent of abnor- mality of the primary protein involved such as its concentration, cellular crowding by other proteins, and the presence of small molecules and metal ions. However, the aggregation process is generally thought to follow one of two paths. The misfolded pro- tein ﬁrst forms dimers, trimers, and oligomers with a structural rearrangement, and then forms highly ordered and symmet- ric structures, e.g., ﬁbrils in Alzheimer disease. CLINICAL PRESENTATION, GENETICS, AND PATHOLOGY OF HF In Huntington disease IBs form iron-dependent centers of oxidative damage causing alterations in the cellular morphology of their surroundings (Firdaus et al., 2006). Cells devote resources to respond to damaged or aggre- gated proteins by synthesis of enzymes to repair or degrade Clinically, HF presents as a movement disorder syndrome sim- ilar to Huntington disease or Parkinson disease. The disease may present with tremor, cerebellar signs, Parkinsonism, psychiatric problems, abnormal involuntary movements (dystonia, chorea), pyramidal syndrome, pseudo-bulbar symptoms, and cognitive deﬁcit (Caparros-Lefebvre et al., 1997; Curtis et al., 2001; Vidal et al., 2004a; Mancuso et al., 2005; Chinnery et al., 2007; McNeill et al., 2008; Ohta et al., 2008; Devos et al., 2009; Kubota et al., 2009; Ory-Magne et al., 2009). The clinical presentation of HF has Table 1 \| Sequence alignment of WT- and MT-FTL polypeptides starting at residue 142. L I K K M G D H L T N L H R L G G P E A G L G E Y L F E R L T L K H D L I K K M G D H L T N L H K A G W P G G W A G R V S L R K A H S Q A R L R A F ………………………. 1 L I K K M G D H L T N L Q Q A G W P G G W A G R V S L R K A H S Q A R L R A F ………………………. 2 L I K K M G D R P P D Q P P Q A G W P G G W A G R V S L R K A H S Q A R L R A F …………………….. 3 L I K K M G D P P D Q P P Q A G W P G G W A G R V S L R K A H S Q A R L R A F ……………………….. FERRITIN STRUCTURE, IRON CHEMISTRY, AND PROTEIN AGGREGATION Alternately the aggregation process may involve less ordered clumping or pack- ing together of proteins that leads to various tangles or IBs. Often the most toxic forms are the smaller oligomers and poorly ordered aggregates, and there is substantial evidence for this with the larger structures considered neuroprotective. However, the larger, microscopically visible IBs may not be inert cel- lular end products, but inﬂuence metabolism, transport and structure, perhaps even through causing detrimental mechanical crowding. them. As will be discussed below, mutant ferritin is intimately associated with iron mishandling, aggregation, and radical dam- age of proteins, which can be traced directly to a protein structural abnormality resulting from its disordered C-terminal helices and unstructured 4-fold pores (Baraibar et al., 2010). CLINICAL PRESENTATION, GENETICS, AND PATHOLOGY OF HF July 2013 \| Volume 5 \| Article 32 \| 3 CLINICAL PRESENTATION, GENETICS, AND PATHOLOGY OF HF These areas show severe nerve cell loss, extracellular ferritin deposits, and loss of neuropil. In the cerebral cortex, IBs are seen in perineuronal cells and in perivascular glia. The presence of IBs in neurons is clearly observed in the putamen, globus pallidus and thala- mus, and in cerebellar granule cells and in Purkinje cells (Vidal et al., 2004a). Intranuclear inclusions are large enough to almost completely occupy the nucleus mechanically forcing chromatin against the nuclear membrane. IBs can be seen as homogenous, eosinophilic bodies, which can be labeled by antibodies against been reported to differ both within and between families (Ory- Magne et al., 2009), usually becoming evident in the third to ﬁfth decade of life (depending on the speciﬁc mutation) and progress- ing unalleviated thereafter. Neuroimaging studies show abnormal signals in the globus pallidus and putamen, and cavitation of the putamen, while serum ferritin levels were reported to be decreased in some patients (Curtis et al., 2001; Ory-Magne et al., 2009). Neuropathological data is available for individuals with the c.442dupC, c.460dupA, and c.497_498dupTC mutations (Curtis et al., 2001; Vidal et al., 2004a; Mancuso et al., 2005). Examination of the brain shows mild cerebral and cerebellar atrophy as well as cavitations of the putamen. The main neuropathologic ﬁndings (Figure 2) are the presence of intranuclear and intracytoplasmic ferritin IBs in glial cells and in some subsets of neurons, substan- tial iron deposition, and mild to moderate nerve cell loss and gliosis. Glial cells containing IBs are mostly found in the cau- date nucleus, putamen, and globus pallidus. These areas show severe nerve cell loss, extracellular ferritin deposits, and loss of neuropil. In the cerebral cortex, IBs are seen in perineuronal cells and in perivascular glia. The presence of IBs in neurons is clearly observed in the putamen, globus pallidus and thala- mus, and in cerebellar granule cells and in Purkinje cells (Vidal et al., 2004a). Intranuclear inclusions are large enough to almost completely occupy the nucleus mechanically forcing chromatin against the nuclear membrane. IBs can be seen as homogenous, eosinophilic bodies, which can be labeled by antibodies against FTL and FTH1 polypeptides of ferritin and by antibodies speciﬁc for the mutant FTL polypeptide. Inclusions also contain Fe2+ and Fe3+, as determined by Turnbull blue and by Perls’ or Prussian blue, respectively. FUNCTIONAL ABNORMALITIES IN MUTANT-CONTAINING FERRITIN The isolation and biochemical analysis of IBs from individu- als with HF identiﬁed wild-type FTL, FTH1 and mutant FTL polypeptides as the main components of IBs (Vidal et al., 2004a). Although ferritin is generally isolated as a heteropolymer of FTH1 and FTL subunits, initial structural and functional stud- ies focused on the biological signiﬁcance of the mutant as a 24-mer in its homopolymeric form. Thus, recombinant wild type (WT)- and mutant (MT)-FTL (p.Phe167SerfsX26) polypep- tides were separately expressed in E. coli, reconstituted with all iron removed, and analyzed. Both polypeptides were soluble and assembled as 24-mer homopolymers by size exclusion chro- matography (SEC). TEM analysis showed that the ferritin particle had a spherical shape and size (outer diameter ∼110 Å) similar to that of human ferritin (Baraibar et al., 2008). When WT-FTL apoferritin homopolymers (1 μM) were iron-loaded following a routine procedure by aerobic incubation with up to 4500 iron atoms per 24-mer of ferrous ammonium sulfate for 2 h, no precipitation was found. However, precipitation of the Mt-FTL homopolymer was observed to begin when the number of iron atoms was higher than ∼1500 iron atoms per ferritin 24-mer. By monitoring direct iron incorporation by native PAGE followed by Prussian blue stain, it was observed that at moderate iron loading of up to ∼1000 iron atoms per ferritin 24-mer and 2 h incuba- tion, WT- and MT-apoferritin homopolymers incorporated very similar amounts of iron, which implies a degree of functional- ity for both. However, at higher iron to ferritin ratios, WT-FTL homopolymers continued incorporating iron, whereas incorpo- ration by MT-FTL homopolymers dropped precipitously. The change in absorbance at 310 nm measured during the ﬁrst 500 s after mixing iron and ferritin in a 1000 to 1 ratio was substan- tially larger for WT-FTL vs. MT-FTL homopolymers, uncovering a clear difference in iron handling between mutant and wild type ferritin at early times after mixing (Baraibar et al., 2010). When in separate solutions, both WT- and MT-FTL homopolymers showed signiﬁcant ability to incorporate iron, but when in direct competition (in the same solution), there was complete absence of iron incorporation by MT-FTL. No precipitation was noted, which highlights the importance of direct iron mishandling by mutant ferritin without the effects of the iron-induced precip- itation. The direct role of iron in the precipitation of MT-FTL homopolymers was further emphasized by using the iron chela- tor deferoxamine (DFX). CLINICAL PRESENTATION, GENETICS, AND PATHOLOGY OF HF By transmission electron microscopy (TEM), nuclear IBs are seen as composed of small (∼100 Å) granular electron-dense particles that resemble ferritin and occupy a large portion of the nucleoplasm. IBs have been reported in the skin, kidney, liver, and muscle in affected individuals from French and American families (Vidal et al., 2004a; Mancuso et al., 2005). The presence of ferritin IBs in skin or muscle biopsies may help in the diagnosis of the disease. CLINICAL PRESENTATION, GENETICS, AND PATHOLOGY OF HF 4 L I K K M G D H L T N L H R L G G P E A G R P G G W A G R V S L R K A H S Q A R L R A F …………… 5 L I K K M G D H L T N L H R L G G P E A G L G E Y L S S K G S L S S T T K S L L S P A T S E G P L A K 6 Wild-type p.Arg154LysfsX27 p.His153GlufsX28 p.His148ArgfsX34 p.His148ProfsX33 p.Leu162ArgfsX24 p.Phe167SerfsX26 142 150 160 175 D E Part of the helical domain D and the complete helical domain E of the WT-FTL subunit are shown above their respective sequences (Protein Data Bank code 2fg4). MT-FTL polypeptides have a C-terminus that is altered in sequence and length. References are: (1) Curtis et al., 2001; (2) Devos et al., 2009; (3) Kubota et al., 2009; (4) Mancuso et al., 2005; (5) Ohta et al., 2008; (6) Vidal et al., 2004a. Frontiers in Aging Neuroscience www.frontiersin.org July 2013 \| Volume 5 \| Article 32 \| 3 Table 1 \| Sequence alignment of WT- and MT-FTL polypeptides starting at residue 142. E D www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal been reported to differ both within and between families (Ory- Magne et al., 2009), usually becoming evident in the third to ﬁfth decade of life (depending on the speciﬁc mutation) and progress- ing unalleviated thereafter. Neuroimaging studies show abnormal signals in the globus pallidus and putamen, and cavitation of the putamen, while serum ferritin levels were reported to be decreased in some patients (Curtis et al., 2001; Ory-Magne et al., 2009). Neuropathological data is available for individuals with the c.442dupC, c.460dupA, and c.497_498dupTC mutations (Curtis et al., 2001; Vidal et al., 2004a; Mancuso et al., 2005). Examination of the brain shows mild cerebral and cerebellar atrophy as well as cavitations of the putamen. The main neuropathologic ﬁndings (Figure 2) are the presence of intranuclear and intracytoplasmic ferritin IBs in glial cells and in some subsets of neurons, substan- tial iron deposition, and mild to moderate nerve cell loss and gliosis. Glial cells containing IBs are mostly found in the cau- date nucleus, putamen, and globus pallidus. FUNCTIONAL ABNORMALITIES IN MUTANT-CONTAINING FERRITIN It was found that both forms of heterepolymers con- taining the MT-FTL subunit (MT-FTL/FTH1, MT-FTL/WT-FTL) and again MT-FTL homopolymers themselves were signiﬁ- cantly more susceptible to iron-loading induced precipitation than either the WT-FTL/FTH1 heterepolymer or the WT-FTL homopolymer, when analyzed by the same above-mentioned routine procedure. Thus, reconstitution of mutant with either wild type does not rescue ferritin from the iron-induced aggregative behavior (Muhoberac et al., 2011). Furthermore, a direct measure of iron incorporation using native PAGE and Prussian blue stain showed that WT-FTL/FTH1 was twice as capable of incorporating iron than MT-FTL/FTH1 heteropolymers. Importantly, some iron was still incorpo- rated with mutant-containing ferritin suggesting some level of functionality. FIGURE 3 \| Structural disruption of the 4-fold pores of ferritin caused by the C-terminal mutation. Each wild type 4-fold pore is formed from four tightly associated E-helices, one donated from each subunit, as seen in Figure 1D. With the mutant, the E-helices are unraveled causing signiﬁcant disruption of the 4-fold pores and providing extended and disordered C-termini, which are not visible by X-ray crystallography. This MT-FTL 4-fold pore is an example of several, which vary somewhat in disruption details, and is viewed from the ferritin shell interior. The structure was taken from RCSB (code 3HX5). Recently, oxidation of MT-FTL was observed to occur both in vitro and in vivo in individuals with HF (Baraibar et al., 2012). Incubation of MT-FTL homopolymers with physiological concentrations of iron and ascorbate resulted in shell structural disruption and polypeptide cleavage not found under the same conditions with WT-FTL. Along with the ∼21 kDa FTL polypep- tide were found fragments of ∼6 and ∼14 K Da, as well as one of ∼27 kDa suggesting covalent crosslinking. Mutant ferritin also underwent a 2.5-fold increase in carbonyl group formation over wild type. Polypeptide cleavage and shell disruption was com- pletely inhibited by addition of the radical trap 5,5-dimethyl-1- pyrroline N-oxide, indicating an enhanced propensity of MT-FTL 24-mers toward free radical-induced, oxidative damage in vitro. Importantly, IBs from a patient with HF exhibited extensive car- bonylation together with an isolatable C-terminal MT-FTL frag- ment of ∼14 kDa, which are both indicative of in vivo oxidative ferritin damage. These data point toward a connection between oxidative damage, mutant ferritin, and HF, and suggest that radical scavengers (i.e., more generally antioxidants) and iron chelators have the potential to be therapeutic agents for treatment of HF. itself. FUNCTIONAL ABNORMALITIES IN MUTANT-CONTAINING FERRITIN Importantly, it was observed that greater than 50% of mutant homopolymers that were precipitated by the FIGURE 2 \| Inclusion bodies, iron deposits, and immunohistochemistry from a patient with hereditary ferritinopathy. Sections of putamen show numerous ferritin IBs of various sizes (A–E), which are ubiquitinated (E). Ferritin IBs were also present in neurons and glial cells of the cerebellum (F). Hematoxylin and eosin (A); Perls’ Prusian blue method for iron (B); and immunohistochemistry using antibodies against mutant FTL (C), wild-type FTL (D), ubiquitin (E), and FTH1 (F). Scale bars: (A–F), 50 μm. FIGURE 2 \| Inclusion bodies, iron deposits, and immunohistochemistry from a patient with hereditary ferritinopathy. Sections of putamen show numerous ferritin IBs of various sizes (A–E), which are ubiquitinated (E). Ferritin IBs were also present in neurons and glial cells of the cerebellum (F). Hematoxylin and eosin (A); Perls’ Prusian blue method for iron (B); and immunohistochemistry using antibodies against mutant FTL (C), wild-type FTL (D), ubiquitin (E), and FTH1 (F). Scale bars: (A–F), 50 μm. July 2013 \| Volume 5 \| Article 32 \| 4 Frontiers in Aging Neuroscience www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal addition of iron (3500 iron atoms per 24-mer) were re-solubilized by incubation with DFX (Baraibar et al., 2008), highlighting chelation as a major in vitro modulator of MT-FTL aggregation, which is an important marker for HF in vivo. FIGURE 3 \| Structural disruption of the 4-fold pores of ferritin caused by the C-terminal mutation. Each wild type 4-fold pore is formed from four tightly associated E-helices, one donated from each subunit, as seen in Figure 1D. With the mutant, the E-helices are unraveled causing signiﬁcant disruption of the 4-fold pores and providing extended and disordered C-termini, which are not visible by X-ray crystallography. This MT-FTL 4-fold pore is an example of several, which vary somewhat in disruption details, and is viewed from the ferritin shell interior. The structure was taken from RCSB (code 3HX5). These homopolymeric studies were followed by examina- tion of ferritin heteropolymers reconstituted with an equal number of mutant and wild type subunits in each 24-mer (Muhoberac et al., 2011). The intermediate mobility on SDS- PAGE and overlap in SEC proﬁles were indicative of appropriate co-assembly. FUNCTIONAL ABNORMALITIES IN MUTANT-CONTAINING FERRITIN The C-terminal sequence of the mutant contains a number of groups known to bind iron, e.g., the C-terminal carboxylate, glutamate, tyrosinate and the hydroxyl groups of several serines and threonines. Considering the sensitivity of ferritin-containing MT-FTL to in vitro and in vivo (see below) iron-induced aggrega- tion, a model was proposed in which iron binds to the unraveled and extended mutant C-termini on two different ferritin shells bridging them and initiating a gradual aggregation of ferritin and iron into a precipitate (Figure 4). Bridging is not necessar- ily restricted to C-termini and may become more general, e.g., between a C-terminal group and a surface amino acid which both have afﬁnity for iron (Baraibar et al., 2008, 2010). This model has potential additional complexities because of (1) the very strong dependence of the strength of iron binding to certain groups on its redox state and (2) the existence of small iron hydroxide nucle- ation centers that may form in solution without the presence of protein. The role of the mutant C-terminus and its protrusion above the protein shell in the iron-induced aggregation process was characterized further by comparing the iron loading of apo- ferritin homopolymers composed of WT-FTL, MT-FTL and a C-terminally truncated FTL polypeptide (p.S167X). The assem- bly status of the truncated FTL polypeptide as a homopolymer (24-mer) was veriﬁed by SEC and gel electrophoresis. In con- trast to MT-FTL homopolymers, which began to precipitate at ∼1500 iron atoms per 24-mer, both the WT-FTL and truncated FTL homopolymers remained in solution up to a ratio of 4000 to 1. Thus, removal of the mutant portion of the C-terminus pre- vented iron-induced precipitation reinforcing the importance of the interaction between iron and the disordered C-terminus in the aggregation process (Baraibar et al., 2010). Frontiers in Aging Neuroscience E-HELIX DISRUPTION AND ENHANCED AGGREGATION IN MUTANT-CONTAINING FERRITIN Unraveled MT-FTL C-terminal E-helices (brown lines) can extend a substantial distance from the ferritin shell surface into the solvent providing a number of groups that can coordinate iron. Addition of iron to a solution of MT-FTL-containing ferritin initiates bridging of C-termini by iron (or iron nucleation complexes) reducing their translational motion. Cross-linking may occur between two separate ferritin 24-mers through iron bridges between C-termini (A), between a C-terminus and surface iron-binding amino acid side chain (B), and/or eventually through surface amino acids that bind iron on both 24-mers (C) forming ferritin aggregates [adapted from Baraibar et al. (2008)]. FIGURE 4 \| Model of iron-induced aggregation of ferritin containing the C-terminal mutation. Unraveled MT-FTL C-terminal E-helices (brown lines) can extend a substantial distance from the ferritin shell surface into the solvent providing a number of groups that can coordinate iron. Addition of iron to a solution of MT-FTL-containing ferritin initiates bridging of C-termini by iron (or iron nucleation complexes) reducing their translational motion. Cross-linking may occur between two separate ferritin 24-mers through iron bridges between C-termini (A), between a C-terminus and surface iron-binding amino acid side chain (B), and/or eventually through surface amino acids that bind iron on both 24-mers (C) forming ferritin aggregates [adapted from Baraibar et al. (2008)]. FIGURE 5 \| Inclusion bodies and immunohistochemistry from a transgenic mouse model of hereditary ferritinopathy. Sections of cerebral cortex (A), globus pallidus (B), and cerebellum (C) of FTL-Tg mice show the presence of numerous ferritin IBs. Sections were from a 9 month old homozygous male (A,B) and an 11 month old heterozygous male (C). Immunohistochemistry was performed using antibodies against the N-terminus of wild-type and mutant FTL (A–C). Scale bars: (A–C), 50 μm. GURE 5 \| Inclusion bodies and immunohistochemistry from a ansgenic mouse model of hereditary ferritinopathy. Sections of rebral cortex (A), globus pallidus (B), and cerebellum (C) of FTL-Tg mice ow the presence of numerous ferritin IBs. Sections were from a 9 month d homozygous male (A,B) and an 11 month old heterozygous male (C). munohistochemistry was performed using antibodies against the FIGURE 4 \| Model of iron-induced aggregation of ferritin containing the FIGURE 4 \| Model of iron-induced aggregation of ferritin containing the C-terminal mutation. Unraveled MT-FTL C-terminal E-helices (brown lines) FIGURE 4 \| Model of iron-induced aggregation of ferritin containing the C-terminal mutation. E-HELIX DISRUPTION AND ENHANCED AGGREGATION IN MUTANT-CONTAINING FERRITIN The structure of the spherical protein shell is maintained in mutant ferritin as was seen in the crystallographic structures of the MT-FTL (p.Phe167SerfsX26) homopolymers (Baraibar et al., 2010). However, a close up examination of the 4-fold pores showed remarkable disruption of the MT-FTL C-terminal helices making the pores unstable and leaky (Figure 3 vs. Figure 1D). Because as many as the last 26 amino acids of MT-FTL remained unaccounted for crystallographically, mutant C-termini may extended and reach as far as the diameter of the ferritin shell July 2013 \| Volume 5 \| Article 32 \| 5 Frontiers in Aging Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal FIGURE 4 \| Model of iron-induced aggregation of ferritin containing the C-terminal mutation. Unraveled MT-FTL C-terminal E-helices (brown lines) can extend a substantial distance from the ferritin shell surface into the solvent providing a number of groups that can coordinate iron. Addition of iron to a solution of MT-FTL-containing ferritin initiates bridging of C-termini by iron (or iron nucleation complexes) reducing their translational motion. Cross-linking may occur between two separate ferritin 24-mers through iron bridges between C-termini (A), between a C-terminus and surface iron-binding amino acid side chain (B), and/or eventually through surface amino acids that bind iron on both 24-mers (C) forming ferritin aggregates [adapted from Baraibar et al. (2008)]. IRON CHELATION AND RADICAL TRAPPING IN ANIMAL AND CELLULAR MODELS OF HF A transgenic animal model of HF (FTL-Tg) was generated in order to increase our understanding of the effects of MT-FTL on brain iron metabolism and ferritin expression and disposition. A human FTL cDNA carrying a thymidine and cytidine inser- tion at position 498 (c.497_498dupTC) was expressed in mouse, l di t i i h dli f iti l ti d id FIGURE 5 \| Inclusion bodies and immunohistochemistry from a transgenic mouse model of hereditary ferritinopathy. Sections of cerebral cortex (A), globus pallidus (B), and cerebellum (C) of FTL-Tg mice show the presence of numerous ferritin IBs. Sections were from a 9 month old homozygous male (A,B) and an 11 month old heterozygous male (C). Immunohistochemistry was performed using antibodies against the N-terminus of wild-type and mutant FTL (A–C). Scale bars: (A–C), 50 μm. FIGURE 4 \| Model of iron-induced aggregation of ferritin containing the C-terminal mutation. E-HELIX DISRUPTION AND ENHANCED AGGREGATION IN MUTANT-CONTAINING FERRITIN Unraveled MT-FTL C-terminal E-helices (brown lines) can extend a substantial distance from the ferritin shell surface into the solvent providing a number of groups that can coordinate iron. Addition of iron to a solution of MT-FTL-containing ferritin initiates bridging of C-termini by iron (or iron nucleation complexes) reducing their translational motion. Cross-linking may occur between two separate ferritin 24-mers through iron bridges between C-termini (A), between a C-terminus and surface iron-binding amino acid side chain (B), and/or eventually through surface amino acids that bind iron on both 24-mers (C) forming ferritin aggregates [adapted from Baraibar et al. (2008)]. FIGURE 5 \| Inclusion bodies and immunohistochemistry from a transgenic mouse model of hereditary ferritinopathy. Sections of cerebral cortex (A), globus pallidus (B), and cerebellum (C) of FTL-Tg mice show the presence of numerous ferritin IBs. Sections were from a 9 month old homozygous male (A,B) and an 11 month old heterozygous male (C). Immunohistochemistry was performed using antibodies against the N-terminus of wild-type and mutant FTL (A–C). Scale bars: (A–C), 50 μm. Frontiers in Aging Neuroscience IRON CHELATION AND RADICAL TRAPPING IN ANIMAL AND CELLULAR MODELS OF HF A transgenic animal model of HF (FTL-Tg) was generated in order to increase our understanding of the effects of MT-FTL on brain iron metabolism and ferritin expression and disposition. A human FTL cDNA carrying a thymidine and cytidine inser- tion at position 498 (c.497_498dupTC) was expressed in mouse, leading to iron mishandling, ferritin accumulation, and oxida- tive stress (Vidal et al., 2008). Expression of the transgene caused the formation of nuclear and cytoplasmic ferritin IBs in glia and neurons throughout the CNS (Figure 5), as well as in cells of other organ systems. The size and number of nuclear inclusions increased with age becoming large enough to cause mechanical crowding and displacement of chromatin, as was found in HF patients (Vidal et al., 2004a). FTL-Tg mice had a progressive neu- rological phenotype, a signiﬁcant decrease in motor performance, and a shorter lifespan. These mutant mice showed an increase in brain iron and altered levels of associate proteins. Cytoplasmic FTL and FTH1 polypeptides increased and the transferrin recep- tor level decreased, as would be the expected in response to excess iron. Ubiquinated proteins and portions of the proteosome (20S, 11S, and 19S) accumulated in the IBs, implying cellular recogni- tion of the presence of abnormal protein aggregates. FTL-Tg mice showed accumulation of oxidative DNA damage in brain mito- chondria, but no signiﬁcant damage to nuclear DNA (Deng et al., 2010). Furthermore, markers for oxidative stress such as protein carbonyl formation, nitrone-protein adducts, and lipid peroxida- tion, were found in the brain indicative of cellular damage by ROS (Barbeito et al., 2009). Ferritin levels in primary cultures of astrocytes from the cerebral cortex of FTL-Tg mice respond dramatically to expo- sure to iron and chelators. Cell treatment with 50 uM ferric ammonium citrate (FAC) caused a switch of MT-FTL ferritin from the detergent-soluble to the detergent-insoluble fraction, strongly supporting a role for iron in the formation of IBs. ABNORMAL FERRITIN FUNCTION CAUSED BY THE MUTANT POLYPEPTIDE Currently there is no effective treatment for HF. Therapies aimed at decreasing iron levels or inhibiting ferritin synthesis would appear to be indicated in view of the pivotal roles played by ferritin and iron in cellular metabolism. Decreasing iron levels toward normal or eliminating mutant FTL polypeptide synthe- sis in HF patients by the more direct approach of gene therapy using viral transfer and expression that could inﬂuence iron or ferritin levels would be time consuming, costly to develop, and with some uncertainty of efﬁcacy. Decreasing iron levels toward normal with appropriately designed chelators would reduce ROS production, pathological iron-induced aggregation, and IB for- mation. However, use of the iron chelators desferrioxamine and deferiprone (as well as venesection) was reported to cause pro- found and refractory iron depletion without clinical beneﬁts (Chinnery et al., 2007). Although this initially may sound dis- couraging, the relatively limited number and variety of chelators examined until now as treatment for HF patients should be care- fully considered. Chelators are characterized by a large number of molecular properties that need to be optimized to match both the complexities of the cellular system and the disease being treated. The choice of a chelator (and in the longer term its discovery and design) is difﬁcult because optimization of one par- ticularly molecular property to the cellular system and disease may adversely affect the optimization of another. For example, adding a particular group to the skeleton of a chelator to opti- mize lipid- vs. aqueous-solubility may adversely affect its redox properties or binding strength. Indeed, there are a number of considerations to chelator efﬁcacy beyond facile blood-brain bar- rier penetration and increasing the binding strength as follows: Hereditary ferritinopathy provides a direct link between abnor- mal iron homeostasis and neurodegeneration. The pronounced cellular dysfunction in the pathogenesis of HF centers on three observable and well-characterized cellular abnormalities - iron accumulation, IB formation, and protein oxidation - as described in this review. All of these abnormalities involve a speciﬁc molecular-level defect of the MT-FTL C-terminal sequence caus- ing (1) 4-fold pore disruption with reduced ability to store and sequester iron, and (2) unraveling and extension of the C-terminus causing iron-induced aggregation of ferritin. IRON CHELATION AND RADICAL TRAPPING IN ANIMAL AND CELLULAR MODELS OF HF Although this couraging, the relatively limited number examined until now as treatment for HF fully considered. Chelators are characte of molecular properties that need to be o the complexities of the cellular system treated. The choice of a chelator (and discovery and design) is difﬁcult because ticularly molecular property to the cell may adversely affect the optimization o adding a particular group to the skeleto mize lipid- vs. aqueous-solubility may a properties or binding strength. Indeed, considerations to chelator efﬁcacy beyon i t ti d i i th bi d ar iron accumulation and ove des, and (2) gain of toxic functio erritin aggregation, and oxidat normal function and gain of to ity to the understanding of o when considering interactions available cellular reductants. M or conformational change tha ion may also allow the ligatio mal protein binding sites with f ROS generation and protein ain of toxic function. It shou not redox active, can induce su d may thus play an important r of normal function could lead ompensating cellular protein syn protein metal binding sites, and f the protein in question norm transport or removal. It is som n storage protein ferritin acqu inability to properly handle e sensitivity toward the metal th stasis. K FOR TREATMENT there is no effective treatment f sing iron levels or inhibiting f be indicated in view of the nd iron in cellular metabolism. ormal or eliminating mutant FT peptides, and (2) gain of toxic f on, ferritin aggregation, and ss of normal function and gain cability to the understanding cially when considering intera and available cellular reductan tion or conformational chang function may also allow the bnormal protein binding sites ces of ROS generation and pr a gain of toxic function. It ugh not redox active, can indu n, and may thus play an impor loss of normal function could in compensating cellular prote rmal protein metal binding site ted if the protein in question l ion transport or removal. It r iron storage protein ferritin the inability to properly ha egative sensitivity toward the m meostasis. IRON CHELATION AND RADICAL TRAPPING IN ANIMAL AND CELLULAR MODELS OF HF LOOK FOR TREATMENT ently there is no effective treatm ecreasing iron levels or inhibi ar to be indicated in view of in and iron in cellular metab rd normal or eliminating mut n HF patients by the more dire g viral transfer and expression Primary cultures of human skin ﬁbroblasts from patients with HF were also used to characterize the effects of MT-FTL on cellular iron metabolism (Barbeito et al., 2010). These cells exhib- ited iron mishandling, ferritin accumulation, and evidence of oxidative stress, paralleling the dysfunction seen in both patients with HF and the mouse model. Mutant ﬁbroblasts showed a sig- niﬁcant increase in the level of total iron content under basal metabolic conditions when compared to wild type ﬁbroblasts, but interestingly, without a signiﬁcant difference in the level of the liable iron pool, which is the iron more readily available for metabolism. Cellular levels of MT-FTL, WT-FTL, and FTH1 polypeptides were all substantially increased in HF vs. wild type ﬁbroblasts. IRE-IRP binding was reduced in HF ﬁbroblasts con- sistent with the observed enhanced ferritin and decreased trans- ferrin receptor-1 synthesis, and broadly consistent with higher total iron levels in HF ﬁbroblasts. Signiﬁcant higher levels of ROS were found in HF vs. non-HF ﬁbroblasts (Barbeito et al., 2010), which supports antioxidant therapy as a potential treatment for HF. Frontiers in Aging Neuroscience IRON CHELATION AND RADICAL TRAPPING IN ANIMAL AND CELLULAR MODELS OF HF After removal of FAC solution, addition of 50 uM of the lipophilic iron chelator 1,10-phenanthroline to the FAC-treated cells led to a large reduction in detergent-insoluble ferritin and Frontiers in Aging Neuroscience July 2013 \| Volume 5 \| Article 32 \| 6 www.frontiersin.org www.frontiersin.org www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal for the role of MT-FTL in the pathogenesis of HF consistent with the results presented here implies both (1) a loss of nor- mal ferritin function (decreased iron incorporation) that triggers intracellular iron accumulation and overproduction of ferritin polypeptides, and (2) gain of toxic function through radical pro- duction, ferritin aggregation, and oxidative stress. The concept of loss of normal function and gain of toxic function may have applicability to the understanding of other disease processes, especially when considering interactions with transition metal ions and available cellular reductants. More generally, a protein mutation or conformational change that leads to loss of nor- mal function may also allow the ligation of transition metals to abnormal protein binding sites with the two major conse- quences of ROS generation and protein aggregation, which are both a gain of toxic function. It should be noted that zinc, although not redox active, can induce substantial protein aggre- gation, and may thus play an important role (Ayton et al., 2012). The loss of normal function could lead to a positive feedback loop in compensating cellular protein synthesis producing more abnormal protein metal binding sites, and could be further com- plicated if the protein in question normally handles transition metal ion transport or removal. It is somewhat ironic that the major iron storage protein ferritin acquires through mutation both the inability to properly handle iron and an enhanced aggregative sensitivity toward the metal that it is designed to keep in homeostasis. the reappearance of ferritin in the detergent-soluble fraction. Phenanthroline is a freely cell-permeable chelator and was chosen for the study over the weakly cell-permeable chelator deferroxam- ine (Baraibar et al., 2008). These studies show that IB formation is strongly dependent on iron levels and can be reversed by using iron chelators in vivo, which supports chelation therapy as a potential treatment to inhibit aggregation and reduce IB formation in HF. IRON CHELATION AND RADICAL TRAPPING IN ANIMAL AND CELLULAR MODELS OF HF presented here imp tion (decreased iron n accumulation and d (2) gain of toxic fu aggregation, and ox al function and gain the understanding considering interact le cellular reductant nformational change ay also allow the li otein binding sites w generation and pro toxic function. It s dox active, can induc thus play an importa mal function could sating cellular protein n metal binding sites rotein in question n port or removal. It i age protein ferritin ity to properly han tivity toward the met TREATMENT s no effective treatme on levels or inhibiti dicated in view of n in cellular metabol or eliminating mutan t b th di - n y y B h n - f s - al s, f e 1 e - - r S , t - d e n d c - e e ) - r p g s n n o s - e t - t e - ) mal ferritin function (decreased iron inc intracellular iron accumulation and ov polypeptides, and (2) gain of toxic funct duction, ferritin aggregation, and oxida of loss of normal function and gain of applicability to the understanding of especially when considering interaction ions and available cellular reductants. M mutation or conformational change th mal function may also allow the ligati to abnormal protein binding sites with quences of ROS generation and protein both a gain of toxic function. It shou although not redox active, can induce su gation, and may thus play an important The loss of normal function could lead loop in compensating cellular protein sy abnormal protein metal binding sites, an plicated if the protein in question norm metal ion transport or removal. It is so major iron storage protein ferritin acq both the inability to properly handle aggregative sensitivity toward the metal t in homeostasis. OUTLOOK FOR TREATMENT Currently there is no effective treatment at decreasing iron levels or inhibiting appear to be indicated in view of the ferritin and iron in cellular metabolism toward normal or eliminating mutant F sis in HF patients by the more direct ap using viral transfer and expression that ferritin levels would be time consuming with some uncertainty of efﬁcacy. Decre normal with appropriately designed chel production, pathological iron-induced a mation. However, use of the iron chelat deferiprone (as well as venesection) was found and refractory iron depletion w (Chinnery et al., 2007). ABNORMAL FERRITIN FUNCTION CAUSED BY THE MUTANT POLYPEPTIDE Both (1) and (2) contribute to increasing iron levels leading to the misdi- rected cellular response of synthesis of more ferritin to sequester iron, which in turn, creates a destructive positive feedback loop that accumulates ferritin and taxes cellular resources. Handling increasing levels of improperly sequestered and stored iron is problematic. Improperly ligated iron, whether this occurs on smaller ferritin aggregates, in IBs, or even through coordination by small molecule cellular constituents, produces ROS leading to protein oxidation, as is evident in the in vitro and in vivo studies reviewed here. Increasing levels of iron also enhance the aggre- gation of MT-FTL-containing ferritin, further exacerbating the situation. IBs contain both Fe2+ and Fe3+, which suggests that ROS could be generated within them. Although HF is an auto- somal dominant disease, the age at onset of the disease is not in early childhood, suggesting that the cells are apparently able to handle the many insults initially, but later succumb to cumu- lative damage. Taken together, a working hypothesis (Figure 6) July 2013 \| Volume 5 \| Article 32 \| 7 www.frontiersin.org www.frontiersin.org www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal FIGURE 6 \| Interrelationship between iron accumulation, ROS generation, inclusion body formation, and neurodegeneration in hereditary ferritinopathy. The pathogenesis of HF is consistent with (1) a loss of normal ferritin function through a decrease in iron incorporation into ferritin that triggers intracellular iron accumulation and overproduction of ferritin polypeptides (a positive feedback loop) and (2) a gain of toxic function through radical production, ferritin aggregation, and oxidative stress. ROS can form at improperly ligate iron bound to small ferritin aggregates and IBs, as well as to other cellular constituents (e.g., small molecules) because of elevated iron as represented by the lowest curved arrow in ﬁgure. The most toxic ROS is the hydroxyl radical that indiscriminately attack proteins, lipids and DNA causing extensive cellular damage. through radical production, ferritin aggregation, and oxidative stress. ROS can form at improperly ligate iron bound to small ferritin aggregates and IBs, as well as to other cellular constituents (e.g., small molecules) because of elevated iron as represented by the lowest curved arrow in ﬁgure. The most toxic ROS is the hydroxyl radical that indiscriminately attack proteins, lipids and DNA causing extensive cellular damage. FIGURE 6 \| Interrelationship between iron accumulation, ROS generation, inclusion body formation, and neurodegeneration in hereditary ferritinopathy. ABNORMAL FERRITIN FUNCTION CAUSED BY THE MUTANT POLYPEPTIDE More generally this list is in contrast to the few chelators currently approved by the FDA for iron-overload therapy implying that chelation therapy for HF remains mainly unexplored. Although the preceding discussion targets the ability of chela- tors to remove and redistribute iron and to resolubilize or prevent ferritin aggregation, as was demonstrated to be oper- ational both in vitro and in vivo by examples in this review, inhibition of oxidative damage by a free radical scavenger was also described. Damage by ROS could be reduced by removal of excess iron, but could also be reduced by the presence of radical scavengers. This points to the investigation of com- bined drug therapy for HF patients in the form of adminis- tering an optimal iron chelator simultaneously with a power- ful antioxidant known to easily cross the blood brain barrier. The combined therapy as outlined here, especially if initiated early, may be more successful, not only for HF, but also for other neurodegenerative diseases characterized by brain iron deposition. Although additional in vitro characterization of mutant ferritin in combination with computational approaches may eventually prove useful in deﬁning compounds with opti- mal molecular properties for drug candidates for HF therapy, it is important to consider application of currently available in vivo approaches employing existing animal and cellular mod- els. One approach would be to develop a screening proce- dure using ﬁbroblasts or astrocytes from HF patients against a small molecule library akin to the NIH Molecular Libraries Frontiers in Aging Neuroscience ABNORMAL FERRITIN FUNCTION CAUSED BY THE MUTANT POLYPEPTIDE The pathogenesis of HF is consistent with (1) a loss of normal ferritin function through a decrease in iron incorporation into ferritin that triggers intracellular iron accumulation and overproduction of ferritin polypeptides (a positive feedback loop) and (2) a gain of toxic function (1) The chelator must selectively bind and release iron and not interfere signiﬁcantly with the concentration, transport, and dis- tribution of other metals. (2) Iron binding and removal must occur in a manner as not to sequester iron from function- ing enzymes that require it for activity. (3) The chelator and iron-chelator complex must be hydrophobic enough to perme- ate multiple membrane barriers to redistribute iron for eventual excretion. (4) The iron-chelator complex should have redox and coordination properties such that it does not itself serve as a source of ROS using available oxygen and reducing equivalents in the brain. This list of considerations and the ﬁne tuning of chela- tor molecular properties that it implies stand in contrast to the two chelators already explored to treat HF both in number and molecular composition. More generally this list is in contrast to the few chelators currently approved by the FDA for iron-overload therapy implying that chelation therapy for HF remains mainly unexplored. Program or other similar programs. Whole animal studies using the mouse model would follow. In the best case scenario, the drug candidates would be the same or similar to an approved FDA drug. A more focused approach would perhaps be to screen approved drugs known to have iron deﬁciency as a side effect. (1) The chelator must selectively bind and release iron and not interfere signiﬁcantly with the concentration, transport, and dis- tribution of other metals. (2) Iron binding and removal must occur in a manner as not to sequester iron from function- ing enzymes that require it for activity. (3) The chelator and iron-chelator complex must be hydrophobic enough to perme- ate multiple membrane barriers to redistribute iron for eventual excretion. (4) The iron-chelator complex should have redox and coordination properties such that it does not itself serve as a source of ROS using available oxygen and reducing equivalents in the brain. This list of considerations and the ﬁne tuning of chela- tor molecular properties that it implies stand in contrast to the two chelators already explored to treat HF both in number and molecular composition. REFERENCES Schröder, J. M. (2005). Ferritinopathy: diagnosis by muscle or nerve biopsy, with a note on other nuclear inclusion body diseases. Acta Neuropathol. 109, 109–114. doi: 10.1007/s00401-004-0949-5 history of neuroferritinopathy caused by the FTL1 460InsA muta- tion. Brain 130, 110–119. doi: 10.1093/brain/awl319 Mancuso, M., Davidzon, G., Kurlan, R. M., Tawil, R., Bonilla, E., Di Mauro, S., et al. (2005). Hereditary ferritinopathy: a novel mutation, its cellular pathology, and pathogenetic insights. J. Neuropathol. Exp. Neurol. 64, 280–294. Ayton, S., Lei, P., and Bush, A. I. (2012). Metallostasis in Alzheimer’s disease. Free Radic. Biol. Med. doi: 10.1016/ j.freeradbiomed.2012.10.558. [Epub ahead of print]. Crichton, R. R., and Declercq, J. P. (2010). X-ray structures of fer- ritins and related proteins. Biochim. Biophys. Acta 1800, 706–718. doi: 10.1016/j.bbagen.2010.03.019 Vidal, R., Ghetti, B., Takao, M., Brefel- Courbon, C., Uro-Coste, E., Glazier, B. S., et al. (2004a). Intracellular ferritin accumulation in neural and extraneural tissue character- izes a neurodegenerative disease associated with a mutation in the ferritin light polypeptide gene. J. Neuropathol. Exp. Neurol. 63, 363–380. Baraibar, M. A., Barbeito, A. G., Muhoberac, B. B., and Vidal, R. (2008). Iron-mediated aggrega- tion and a localized structural change characterize ferritin from a mutant light chain polypeptide that causes neurodegeneration. J. Biol. Chem. 283, 31679–31689. doi: 10.1074/jbc.M805532200 McNeill, A., Birchall, D., Hayﬂick, S. J., Gregory, A., Schenk, J. F., Zimmerman, E. A., et al. (2008). T2 and FSE MRI distinguishes four subtypes of neurodegeneration with brain iron accumulation. Neurology 70, 1614–1619. doi: 10.1212/01.wnl. 0000310985.40011.d6 Curtis, A. R., Fey, C., Morris, C. M., Bindoff, L. A., Ince, P. G., Chinnery, P. F., et al. (2001). Mutation in the gene encoding ferritin light polypeptide causes dominant adult-onset basal ganglia disease. Nat. Genet. 28, 350–354. doi: 10.1038/ng571 Muhoberac, B. B., Baraibar, M. A., and Vidal, R. (2011). Iron loading-induced aggregation and reduction of iron incorpora- tion in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration. Biochim. Biophys. Acta. 1812, 544–548. Baraibar, M. A., Barbeito, A. G., Muhoberac, B. B., and Vidal, R. (2012). A mutant light-chain fer- ritin that causes neurodegeneration has enhanced propensity toward oxidative damage. Free Radic. Biol. Med. 52, 1692–1697. doi: 10.1016/j. freeradbiomed.2012.02.015 Vidal, R., Delisle, M. B., and Ghetti, B. (2004b). Neurodegeneration caused by proteins with an aberrant carboxyl-terminus. J. Neuropathol. Exp. Neurol. 63, 787–800. Deng, X., Vidal, R., and Englander, E. W. (2010). REFERENCES Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Ory-Magne, F., Brefel-Courbon, C., Payoux, P., Debruxelles, S., Sibon, I., Goizet, C., et al. (2009). Clinical phenotype and neuroimaging ﬁndings in a French family with hereditary ferritinopathy (FTL498- 499InsTC). Mov. Disord. 24, 1676–1683. doi: 10.1002/mds.22669 Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1984). Iron- catalyzed hydroxyl radical forma- tion. J. Biol. Chem. 259, 3620–3624. Barbeito, A. G., Levade, T., Delisle, M. B., Ghetti, B., and Vidal, R. (2010). Abnormal iron metabolism in ﬁbroblasts from a patient with the neurodegenerative dis- ease hereditary ferritinopathy. Mol. Neurodegener. 5, 50. doi: 10.1186/1750-1326-5-50 Kubota, A., Hida, A., Ichikawa, Y., Momose, Y., Goto, J., Igeta, Y., et al. (2009). A novel ferritin light chain gene mutation in a Japanese family with neuroferritinopathy: descrip- tion of clinical features and implica- tions for genotype-phenotype cor- relations. Mov. Disord. 24, 441–445. doi: 10.1002/mds.22435 Received: 30 April 2013; accepted: 22 June 2013; published online: 30 July 2013. Rice, M. E. (2011). H2O2: a dynamic neuromodulator. Neuroscientist 17, 389–406. doi: 10.1177/1073858411404531 Berg, D., and Youdim, M. B. (2006). Role of iron in neurodegenera- tive disorders. Top. Magn. Reson. Imaging 17, 5–17. doi: 10.1097/01. rmr.0000245461.90406.ad Citation: Muhoberac BB and Vidal R (2013) Abnormal iron homeostasis and neurodegeneration. Front. Aging Neurosci. 5:32. doi: 10.3389/fnagi. 2013.00032 Santambrogio, P., Levi, S., Arosio, P., Palagi, L., Vecchio, G., Lawson, et al. (1992). Evidence that a salt bridge in the light chain con- tributes to the physical stability dif- ference between heavy and light human ferritins. J. Biol. Chem. 268, 14077–14083. Lansbury, P. T., and Lashuel, H. A. (2006). A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 443, 774–779. doi: 10.1038/nature05290 Caparros-Lefebvre, D., Destee, A., and Petit, H. (1997). Late onset familial dystonia: could mito- chondrial deﬁcits induce a diffuse lesioning process of the whole basal ganglia system? J. Neurol. Neurosurg. Psychiatry 63, 196–203. doi: 10.1136/jnnp.63.2.196 Copyright © 2013 Muhoberac and Vidal. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). REFERENCES Accumulation of oxidative DNA damage in brain mitochondria in mouse model of hereditary ferritinopathy. Neurosci. Lett. 479, 44–48. doi: 10.1016/j.neulet.2010.05.025 Exp. Neurol. 63, 787–800. Vidal, R., Miravalle, L., Gao, X., Barbeito, A. G., Baraibar, M. A., Hekmatyar, S. K., et al. (2008). Expression of a mutant form of the ferritin light chain gene induces neurodegeneration and iron overload in transgenic mice. J. Neurosci. 28, 60–67. doi: 10.1523/JNEUROSCI.3962-07.2008 Zecca, L., Youdim, M. B. H., Riederer, P., Connor, J. R., and Crichton, R. R. (2004). Iron, brain ageing and neurodegenerative disorders. Nat. Rev. Neurosci. 863–873. doi: 10.1038/nrn1537 p Vidal, R., Miravalle, L., Gao, X., Barbeito, A. G., Baraibar, M. A., Hekmatyar, S. K., et al. (2008). Expression of a mutant form of the ferritin light chain gene induces neurodegeneration and iron overload in transgenic mice. J. Neurosci. 28, 60–67. doi: 10.1523/JNEUROSCI.3962-07.2008 Baraibar, M. A., Muhoberac, B. B., Garringer, H. J., Hurley, T. D., and Vidal, R. (2010). Unraveling of the E helices and disruption of 4-fold pores are associated with iron mishandling in a mutant ferritin causing neurodegeneration. J. Biol. Chem. 285, 1950–1956. doi: 10.1074/jbc.M109.042986 Murphy, M. P., Holmgren, A., Larsson, N. G., Halliwell, B., Chang, C. J., Kalyanaraman, B., et al. (2011). Unraveling the biological roles of reactive oxygen species. Cell Metab. 13, 361–366. doi: 10.1016/j.cmet.2011.03.010 Devos, D., Tchofo, P. J., Vuillaume, I., Destée, A., Batey, S., Burn, J., et al. (2009). Clinical features and natural history of neuroferritinopa- thy caused by the 458dupA FTL mutation. Brain 132, e109. doi: 10.1093/brain/awn274 Zecca, L., Youdim, M. B. H., Riederer, P., Connor, J. R., and Crichton, R. R. (2004). Iron, brain ageing and neurodegenerative disorders. Nat. Rev. Neurosci. 863–873. doi: 10.1038/nrn1537 Ohta, E., Nagasaka, T., Shindo, K., Toma, S., Nagasaka, K., Ohta, K., et al. (2008). Neuroferritinopathy in a Japanese family with a duplication in the ferritin light chain gene. Neurology 70, 1493–1494. Firdaus, W. J. J., Wyttenbach, A., Giuliano, P., Kretz-Remy, C., Currie, R. W., and Arrigo, A.-P. (2006). Huntingtin inclusión bodies are iron-dependent centers of oxidative events. FEBS J. 273, 5428–5441. doi: 10.1111/j.1742-4658.2006.05537.x Barbeito, A. G., Garringer, H. J., Baraibar, M. A., Gao, X., Arredondo, M., Núñez, M. T., et al. (2009). Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene. J. Neurochem. 109, 1067–1078. ACKNOWLEDGMENTS This study was supported by grants from the National Institute on Neurological Disorders and Stroke NS050227 and NS063056 (Ruben Vidal). July 2013 \| Volume 5 \| Article 32 \| 8 www.frontiersin.org www.frontiersin.org Mutant ferritin and neurodegeneration Muhoberac and Vidal REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are cred- ited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, dis- tribution or reproduction is permitted which does not comply with these terms. Maciel, P., Cruz, V. T., Constante, M., Iniesta, I., Costa, M. C., Gallati, S., et al. (2005). Neuroferritinopathy: missense mutation in FTL caus- ing early-onset bilateral pallidal involvement. Neurology 65, 603–605. doi: 10.1212/01.wnl. 0000178224.81169.c2 Santambrogio, P., Levi, S., Cozzi, A., Rovida, E., Albertini, A., and Arosio, P. (1993). Production and char- acterization of recombinant het- eropolymers of human ferritin H and L chains. J. Biol. Chem. 268, 12744–12748. Chinnery, P. F., Crompton, D. E., Birchall, D., Jackson, M. J., Coulthard, A., Lombès, A., et al. (2007). Clinical features and natural July 2013 \| Volume 5 \| Article 32 \| 9 Frontiers in Aging Neuroscience www.frontiersin.org
https://openalex.org/W2955860840	https://zenodo.org/records/3371336/files/Construction%20of%20the%20expert%20system%20of%20geo%C2%ADspatial%20analysis%20that%20employs%20scenarios%20for%20the%20automated%20data%20generation%20for%20a%20digital%20map.pdf	Russian	null	Development of neuralnetwork and fuzzy models of multimass electromechanical systems	Eastern-European journal of enterprise technologies	2,019	cc-by	8,072	Information technology Information technology UDC 004.9 : 623.618 Проведене дослідження по формалізації алгорит мів вирішення задач, генеруванню даних для елек тронної карти і їх реалізації за допомогою набору простих операцій, які інтуїтивно зрозумілі для кори стувача, який не є фахівцем у галузі геоінформаційних технологій. DOI: 10.15587/1729-4061.2019.170620 1. Introduction 1. Introduction CONSTRUCTION OF THE EXPERT SYSTEM OF GEO-SPATIAL ANALYSIS THAT EMPLOYS SCENARIOS FOR THE AUTOMATED DATA GENERATION FOR A DIGITAL MAP Запропоновано підхід, який базується на ство ренні типових сценаріїв виконання моделі. Такі сце нарії редагуються і пристосовуються до використан ня альтернативних електронних карт місцевості. Результатом виконання сценарію стає набір даних – шарів електронної карти, що отримані по вихідних параметрах моделі і алгоритмах вирішення задачі, які створені фахівцем. Це дає змогу створювати бібліо теки типових сценаріїв, централізовано зберігати їх і надавати загальний доступ до цих сценаріїв, а також, виконувати обмін даними між програмними додат ками. У результаті виконання сценарію користувач отримує можливість без написання будь-якого про грамного коду виконувати складні операції оброки гео графічних даних і моделювати різні процеси на елек тронній карті місцевості. G . D r o b a h a Doctor of Military Sciences, Professor Scientific Research Department of the Air Force Scientific Center Ivan Kozhedub Kharkiv National Air Force University Sumska str., 77/79, Kharkiv, Ukraine, 61023 Е-mail: gd.vk.rh.ua@gmail.com V . L i s i t s i n * Е-mail: lisivladimir@gmail.com L . S a f o s h k i n a PhD* Е-mail: saf0705@ukr.net I . M o r o z o v PhD Department of Research and Organization** E-mail: Fozzy_i@ukr.net A . P o b e r e z h n y i * Е-mail: fix086@ukr.net Scientific Research Centre* *National Academy of the National Guard of Ukraine Zakhysnykiv Ukrainy sq., 3, Kharkiv, Ukraine, 61001 Розроблена експертна система геопросторового аналізу, яка містить як базові функції обробки гео графічних даних, так і спеціалізовані моделі висо кого рівневі. У режимі візуальної побудови алгорит му вирішення задачі, створюється дерево рішень. Реалізований конвеєр операцій, коли джерела даних в експертній системі, які отримані під час виконання деякої операції, відсилаються на вхід наступної опе рації. Результати дослідження можуть бути вико ристані в імітаційних моделях військових опера цій, задачах фотограмметрії, під час проектування оптимальних трас обльоту території, та в якості додаткового інструменту аналізу місцевості в геоін формаційних системах. Мається можливість роз ширювання функціональності експертної системи та додавання нових типів операцій. Таким чином, є підстави стверджувати про те, що процес автоматичного створення даних для елек тронних карт, потребує наявності спеціального про грамного забезпечення і високої кваліфікації користу вачів геоінформаційних систем A . P o b e r e z h n y i Е-mail: fix086@ukr.net Scientific Research Centre* *National Academy of the National Guard of Ukraine Zakhysnykiv Ukrainy sq., 3, Kharkiv, Ukraine, 61001 A . G. Drobaha, V. Lisitsin, L. Safoshkina, I. Morozov, A. Poberezhnyi, 2019 Received date: 24.04.2019 Accepted date: 04.06.2019 Published date: 28.06.2019 CONSTRUCTION OF THE EXPERT SYSTEM OF GEO-SPATIAL ANALYSIS THAT EMPLOYS SCENARIOS FOR THE AUTOMATED DATA GENERATION FOR A DIGITAL MAP P o b e r e z h n y i Е-mail: fix086@ukr.net Scientific Research Centre* **National Academy of the National Guard of Ukraine Zakhysnykiv Ukrainy sq., 3, Kharkiv, Ukraine, 61001 Ключові слова: геоінформаційна система, гео просторовий аналіз, електронна карта, оверлейний аналіз, імітаційна модель 1. Introduction – land with no urban facilities (agricultural and park areas); – land with no urban facilities (agricultural and park areas); ) – land with large indicators of slope in the terrain, at which construction is not possible; – reserved land (used for special purpose). Additionally, the model takes into consideration the socio-economic factors affecting the changing boundaries of the urban area. These include the presence of primary and secondary roads, population density for different areas of the city, its distance from the central part, existence of com mercial centers, etc. The preparation of such data on the map was performed using the tools for building buffer zones. It should be noted that the range of tasks that could be solved by modelling the processes of changes to the boundaries of urban territories testifies to a rather narrow specialization of the model, as well as the difficulties to extend it. When performing aerial photography of an urban area, that would make it possible to simulate at a digital map the decoding tasks, the processes for constructing urban infra structure and compiling a land cadaster, to design routes to fly over land plots, etc. [2]. Building such scenarios should be closely linked to geoinformation technologies in order to be implemented as one of the GIS modules. Paper [5] considers a system based on GIS technology and designed to support decision making when modern izing apartment buildings. The decision taken is aimed at saving energy and reducing CO2 emissions. Work on modernization may involve replacement of windows in residential buildings, covering the walls of buildings, re placement of heating systems, etc. A model is built that makes it possible to select, when specifying a building on the map of a city, the optimal scenario for modernization. The model employs WEB technologies and the client-serv er technology, making it possible for the user to obtain information on a particular building from the database on urban facilities. In addition, they use maps of temperature fields in the specified region acquired from a global mete orological database. The decision support system possesses the important property ‒ it has an organized distributed WEB access to the model’s data. 2. Literature review and problem statement Paper [3] outlines approaches to building, as well as de scribes the implementation, of an information system based on ontological principles. A set of tools underlying such a system enables the work with layers of an electronic map and contains a query building component. In addition, the authors propose a script builder, components of logical infer ence that use the principles of description logic and a spatial aspect of the digital map data. By using an example of typical tasks at a land surveying office, the authors describe the principles of script con struction. The thematic aspect of a map is set by the user in the form of a set of semantics in describing the objects. For example: “greening”, “meadow”, “public park”, “lake”. The spatial aspect contains the spatial attributes of the map’s objects (the area of the park or lake, the type of con crete pavement). In addition, the authors introduce spatial relations among the maps’ objects, which are expressed by qualitative (contains, partially overlaps) and quantitative attributes (length, area). Then, for example, a script frag ment that implements the description “public park contain ing a lake” could be recorded to the information system as a predicate: Scenario-based approaches are applied in many known software suites. For example, the functions and algorithms for GIS analysis are performed by using a visual Model Maker. Another example is the Spatial Modeler Scripting Language (SML) in the system of image processing and remote sensing data ERDAS Imagine® [6]. A scenario in these systems refers not only to the verbal description of a certain sequence of actions, aimed at solv ing a task. A scenario implies the formalization of a prob lem-solving algorithm and its implementation through a set of simple user-friendly operations. The result of the im plementation of a SML-script is that the user can, without writing a programming code, process aerial photographs and images, perform a geospatial analysis of terrain. In addition, there is a possibility to choose, based on such an analysis, the optimal routes of movement and the location of objects, to construct new layers on a digital map in an automated mode. public_park_containing_a_lake≡ ≡park_public_∃contains.lake. 1. Introduction GIS, have the skills to work with high-level programming languages and build appropriate algorithms. In addition, a solution constructed in the form of a software tool for certain input data and initial conditions, may prove unsuitable for another type of terrain, different imaging equipment, and for constraints that arise when a model changes. Modern approaches that are used for the automated construction of vector layers at digital maps are inextri cably linked to geoinformation technologies. Specifically, many current geoinformation systems (GIS) contain the sets of tools, procedures, and libraries of standard algo rithms. That makes it possible to perform a geospatial analysis of data, to construct SQL queries and spatial sam plings based on the values for attributes, to edit data under automated or interactive modes [1]. The way to resolve this situation would be to construct typical scenarios for model execution. Such scripts must be edited easily and adapted to the use of an alternative electronic terrain map. The result of scenario execution is a set of data ‒ layers on a digital map that are derived based on certain input attributes and problem-solving algorithms drawn up by an expert. There has been an unresolved issue on training such specialist who, in addition to a thorough knowledge in the subject area of modeling, must possess basic skills of using 43 G. Drobaha, V. Lisitsin, L. Safoshkina, I. Morozov, A. Poberezhnyi, 2019 Received date: 24.04.2019 Accepted date: 04.06.2019 Published date: 28.06.2019 G. Drobaha, V. Lisitsin, L. Safoshkina, I. Morozov, A. Poberezhnyi, 2019 Received date: 24.04.2019 Accepted date: 04.06.2019 Published date: 28.06.2019 3/2 ( 99 ) 2019 3/2 ( 99 ) 2019 – land with urban facilities; Implementing such an approach would significantly simplify and speed up the training of professionals who, by the nature of their principal occupation, would require additional knowledge in the field of geoinformation tech nologies. In addition, specialists in urban area planning may construct libraries of typical scenarios, to store them centralized, and to provide common access to these scenar ios. Military professionals could use generated scripts when planning, conducting, and analyzing military activities. The data exchange between GIS applications is greatly simplified. public_park_containing_a_lake≡ ≡park_public_∃contains.lake. The second issue is the complexity of representation of scripts for processing and construction of geographic data to the end user [3, 6, 8]. Comparison criteria may be: the presence or absence of the feasibility of scripts construction in the system; the presence in a scenario text of predicates and operators, requiring specialized expertise in the fields of mathematical logic and Boolean algebra. Many functions that are embedded into a script are grouped and are poly morphic [6, 8]. That is, while bearing the same name, such functions are redefined and implemented differently depend ing on the type and amount of input arguments. Based on this fact, one more comparison criterion is suggested ‒ the in- tuitive ease of reading a script, expressed by the absence or minimal number of such functions. Fig. 1. Example of processing a bitmap image using Model Maker n4_result n1_m n3_Edge_Detect CONVOLVE The most appropriate solution would be to construct a system where the user is isolated from the process of manu ally editing the script. Such a concept implies extensive use of visual editing tools, as well as scripting. Fig. 1. Example of processing a bitmap image using Model Maker The basic features from ModelBuilder are used to de velop the specialized software. As an example, we consider the system for mapping the inundation zones FloodTools, constructed for the United States air force base at Langley [9]. This base is located at the territory constantly exposed to hurricanes. Prediction and analysis of the effects of these natural phenomena is an important element in the combat readiness of the air fleet. There is an issue related to the representation of simula tion results in the examined systems. When using GIS tech nologies, the output data formats are standardized and well documented. However, the use of modularity when building scenarios raises a series of issues. How, for example, should one submit the data that are generated in the same module to the input of another if the structure of attributes for these data is determined only during the execution of the model. An important criterion for comparing systems is a built-in code tracer (debugger). That makes it possible to monitor the process of compiling and modifying data in the process of design and implementation of the model. public_park_containing_a_lake≡ ≡park_public_∃contains.lake. The downside of this approach is the complexity and low readability of the scenario, even when describing very simple relationships among objects. Study [4] proposed a simulation model for changing the boundaries of urban areas based on the theory of cellular automata and fuzzy sets. The model is integrated into the geoinformation system and was tested on comparative data for 2003 and 2013 at the Yemeni city of Ibb. The validation involved a pixel-to-pixel comparison of modelling results and actual data in the map. The research procedure included three phases: Here is an example of the model constructed using Model Maker. By using visual programming techniques, the origi nal bitmap image was treated with a filter to select the edges with a dimensionality of the kernel of 3×3 [7]. The result is the binary (black and white) outline raster (Fig. 1). Note that the Model Maker focuses on processing raster images and remote sensing data. – preparation of information based on land-use data; A similar approach to scripting is offered by the com pany ESRI, a leading global provider of geo-information technologies and software products, grouped under the heading ArcGIS®. Since the early versions of this system, the toolsets of geoprocessing work together with the vi – construction of map layers using a simulation model; – construction of map layers using a simulation model; – representation of simulation results on a digital map. – representation of simulation results on a digital map. h h b d f f f Changing the boundaries for four types of territory was modeled: 44 Information technology sual editor ModelBuilder. The constructed model could be exported to a script [8]. The format of the script may correspond to one of the three programming languages ‒ Python, Jscript, or VBScript. It is possible to publish a script constructed using ModelBuilder by sending to the ArcGIS Server®. Following the publication, any user with appropriate access rights to the server could connect to the constructed service and use it. – a possibility to construct additional map layers when one changes the initial conditions of the model (for exam ple, simulation of urban traffic in the summer and winter seasons). – a possibility to construct additional map layers when one changes the initial conditions of the model (for exam ple, simulation of urban traffic in the summer and winter seasons). 3. The aim and objectives of the study The aim of this study is to devise principles for the simplified work with geospatial data, for the user-friendly construction of layers for a digital map, to flexibly build the scripts for model execution at a user level. Specialized scripts and simulation principles are widely used in the military sphere. Another example is a tool con structed by using ArcGIS® at the National Defence Uni versity War Game & Simulation Center in Poland [9]. Em ploying it helps train officers at the tactical and operational level, construct the scenarios, simulate a tactical situation at a battlefield. To accomplish the aim, the following tasks have been set: l b ld l h ld – to propose principles for building a toolset that would solve the tasks of intuitively-simple processing and construc tion of geographic data; – to extend the capabilities for simulation modeling at a digital map for tasks in various subject areas, not just for a single, highly specialized solution; The review of the examined solutions allows us to iden tify a series of issues related to geoprocessing, automated generation of data for a digital map, and their application in simulation systems. – to propose a universal technique to exchange the con structed models and geographical data using the mechanism of scenarios; – to propose a universal technique to exchange the con structed models and geographical data using the mechanism of scenarios; The first issue is the extensibility of a simulation model constructed using the selected system. Often, labor costs are associated with building a model in the assigned subject area. As a result, the user is provided with a set of strict ly-defined activities and functions that simulate the aspects of a certain specialized task [4, 5, 9]. Changes in the input parameters for the model require modification of the user interface. A set of criteria to compare systems based on this point could include: –to implement a flexible mechanism to edit scenarios when changing input data and initial conditions for the model. –to implement a flexible mechanism to edit scenarios when changing input data and initial conditions for the model. public_park_containing_a_lake≡ ≡park_public_∃contains.lake. The FloodTools system was designed in the form of a WEB application and makes it possible for the user to choose scenarios when modeling floodplains. Based on the results from running a script, one could plan the activities of rescue teams, flight and command staff at the base. Ten scenarios were constructed for different options of hurricane propagation within the model. The system proved its effec tiveness when analyzing and preventing the consequences of Hurricane Ophelia. 4. Construction principles and features of work of an expert system The most important stages for modeling at a digital map are the preparation of source data for a model and the management of geographic data. Each stage may include an initial analysis of attributive information on a map’s layers, measuring distances, estimating the mutual arrangement – a possibility of scalability of generated models (calcu lations for an urban district, city, region); – possibilities to change the set of procedures used in geoprocessing, when it is necessary to carry out additional analysis of the model’s data; 45 3/2 ( 99 ) 2019 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 a new layer (temporarily or permanently). For example, con sider the following sequence of actions: a new layer (temporarily or permanently). For example, con sider the following sequence of actions: of objects, their shapes, sizes. In addition, in the course of the execution and visualization of the model one may have to construct additional map layers, or remove temporary information. 1. Select point objects of a certain layer on a map using an SQL-query. 1. Select point objects of a certain layer on a map using an SQL-query. We propose an approach that could significantly simplify stages in the preparation and management of geographical data. To solve the set task, we built a module of an expert system of geospatial analysis for GIS. Let us briefly consider the ideas that underlie such an expert system. 2. Construct buffer zones of the predefined depth around the selected points. 3. Construct an integrated buffer zone by merging the buffer zones built at the previous step using a Boolean op eration “OR”. 4. Build a node of results from a sequence of operations that would define the new data source and a polygonal layer with the assigned structure of attributes based on the inte grated buffer zone. The expert system’s main window contains several pan els. The upper panel includes all data sources that are con structed during model execution. The decision tree’s nodes could be of one of the pre defined types: g 5. Add this layer to the map. g 5. Add this layer to the map. – a group of operations, which is a container for multiple nested operations or other groups; – a group of operations, which is a container for multiple nested operations or other groups; Fig. 3. 4. Construction principles and features of work of an expert system Implementation of a hierarchical nesting of nodes and the sequence of operations in an expert system – an operation that performs a fragment of the algorithm; f d h h l d – sources of data that represent the original data ac quired from a digital map or intermediate data obtained from the algorithm execution; – the nodes of results that are stored as permanent or temporary layers of a digital map. Data sources could be added as arguments for unary, bi nary, or special operations, which are the elements of a deci sion tree. The decision tree itself is built, element by element, in a separate panel in the form of a hierarchical structure whose nodes are represented by conditional symbols (Fig. 2). Fig. 3. Implementation of a hierarchical nesting of nodes and the sequence of operations in an expert system Fig. 2. Fragment of a decision tree compiled in an expert system The decision tree that corresponds to the specified se quence of operations is shown in Fig. 2. Three sets of operations, grouped by type, have been implemented in the system: 1. Operations relating to the selected objects on the map. Sampling, in accordance with the parameters for a SQL que ry or a mutual arrangement of objects in the plane of the map (spatial queries), removal of selected objects, changing their attributes’ values or exporting the selected objects to other layers of the map (Fig. 4). Fig. 4. Operations in an expert system with the objects selected on the map Fig. 2. Fragment of a decision tree compiled in an expert system Model execution is carried out with the help of a soft ware expert system shell, which finds in the decision tree a node of the topmost (first) level. Then one sequentially selects, analyzes, and executed each child activity nested in this node of the group. Upon performing all operations in the first group, the interpreter finds the next node in a group, which is a child node of the first-level group. For the found second-level group, the process of finding child nodes of operations, their analysis and execution, is repeated. This is repeated until the interpreter performs the last (terminal) node of operations in the decision tree (Fig. 3). Fig. 4. Operations in an expert system with the objects selected on the map 2. Buffer or overlay operations. 5. Capabilities of modeling on a digital map using an expert system As could be seen from the above examples, the range of modeling areas is quite wide and is not limited to a single specific task. The structure of attributes for geographic data and their content define the initial conditions and data for the model under construction. Then, how does the expert system determine the structure of attributes for objects that were constructed when building the buffer zones? For this purpose, the decision tree’s nodes that generate new data sources (layers of the map) during model execution have three options. The expert system’s capabilities are not limited to the processes of preparing, managing, and generating data for an electronic map. The tools for a visual construction of a decision tree make it possible to implement the algorithm for building many models in a convenient and visible manner. A set of buffer and overlay operations is a cornerstone of modeling in a given expert system. Actually, work at it start ed with an idea to automate the functions for a geospatial analysis, relating to the construction on the digital map of zones where moving objects could be reached. Look at these operations in detail. – Constructing a data source with a simplified, stan dardized structure of attributes. The minimal set includes the required attribute ID, for the map’s objects indexing, and one of the attributes of a specified type ‒ the integer, string, and double precision. Using a standard structure of attributes is appropriate for modeling simple properties and relationships among objects on the map. For example, to cal culate the area of the region covered by the buffer zones built around certain objects. Buffer zones on a map are built around point, linear, or polygonal objects (Fig. 5). Fig. 5. Construction of buffer zones on GIS digital map – The structure of attributes for the constructed data source is copied from the already existing map’s layer. Such an approach is rational to use for cases when existing layers overlap, when automatically selecting and transferring the fragments of geographical data to the specified regions of the map. – A new data source attributes’ structure is formed based on one of the templates, recorded in a GIS database. One could construct the necessary templates and add the attri butes of the selected type to them. 4. Construction principles and features of work of an expert system Capabilities of modeling on a digital map using an expert system 4. Construction principles and features of work of an expert system In this set, the following has been implemented: construction of buffer zones of a cer tain depth around the objects selected on the map. There is a possibility to export buffer zones to the new layer on the map, as well as there are possibilities to perform rudimentary over lay operations based on Boolean logic, that is, merging buffer zones that exist on the map, their overlapping and subtraction. Conditionally, the operations described by using a deci sion tree could be divided into three groups. The first group is the unary operations, which contain a single data source as an input argument. The second group is the binary oper ations that require two input arguments in the form of data sources. The third group is other operations that contain three or more input arguments. The specified groups of oper ations form in the decision tree a corresponding child node of results, build a certain buffer zone on the map, and construct 3. The nodes that implement specific types of opera tions. They represent ready snippets of code with an ex 3. The nodes that implement specific types of opera tions. They represent ready snippets of code with an ex 46 Information technology tended set of attributes and complex relationships among elements. – visualization on the map of the width of paths when building buffer zones around linear segments of roads; – selection of public utilities within the specified radius of availability for each user; – selection of public utilities within the specified radius of availability for each user; Preparation and processing of geographic data in such a system does not require the user to have skills in the program ming languages of high level. New layers on the map are based on the properties of operations for user-selected nodes in the decision tree. The model execution algorithm is designed and edited by the user in the visual environment of a decision tree. – time-dependent change in the initial boundaries of the zone where objects moving with the predefined speed are located; – time-dependent change in the initial boundaries of the zone where objects moving with the predefined speed are located; – time-dependent change in the habitats of natural phenomena (epidemics, floods caused by spring melting of snow, etc.). – time-dependent change in the habitats of natural phenomena (epidemics, floods caused by spring melting of snow, etc.). 5. The city territory’s points that belong to the buffer zones built under the terms of the problem F F F F F F F F F  = ∧  = ∨   = →  (4) (4) There is an issue on the possibility of using the data ob tained as a result of a certain sequence of operations (4) as an input argument for another operation in the decision tree. h h f d h h There is an issue on the possibility of using the data ob tained as a result of a certain sequence of operations (4) as an input argument for another operation in the decision tree. When using the concept of data sources, such an approach is possible. In the expert system we implemented a kind of a conveyor when a data source, obtained while performing a buffer or overlay operations, is sent to the input of the next operation (Fig. 7). The constructed model or its fragments could be saved as a script file. It is advisable to organize storing such scenarios in the form of a library, constructed in the shared directory for users. Using pre-existing and well-established modules would make it possible to avoid the repeatability of the code, and would also ensure the scalability of new simulation models constructed for GIS. Fig. 7. Conveyor of operations implemented in an expert system 5. Capabilities of modeling on a digital map using an expert system Such an approach makes it possible to design new structures of input data and change the initial conditions for modeling. Fig. 5. Construction of buffer zones on GIS digital map Thus, using the simplest operations for building buffer zones one could describe rather complex relationships among objects on the map. However, greater possibilities of the expert system manifest themselves when using buffer and overlay analysis in combination. Depth of the buffer zones could be set as a constant or a value from the table of attributes of the objects selected on the map (Fig. 6). Here is a simple example. At the electronic city map, one selects the polygonal objects ‒ parks and gardens. Buffer zones are built around them with a depth of 300 m, thereby setting region Z1 on the map. Some spatial function F1 is then introduced. The value for this function is equal to 1 if an arbitrary point P(x, y), belonging to the analyzed area M on the city map is also inside one of these buffer zones. Oth erwise, the function is equal to 0. Fig. 6. Setting the value for depth of GIS buffer zones 1, 1 1 . 0, 1 P Z P F P Z ∩ =   =   ∩ = ∅   (1) (1) Next, one selects on the city map the polygonal objects ‒ buildings that house supermarkets. Around them, one con structs buffer zones with a depth of 500 m, thereby setting region Z2 within a walking distance to a respective super market. The spatial function F2 is introduced whose value is 1 if an arbitrary point P(x, y) that is inside the region M on Fig. 6. Setting the value for depth of GIS buffer zones Here are some examples for using such a tool to build buffer zones in the geospatial data analysis: 47 3/2 ( 99 ) 2019 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 the city map is also inside one of the areas within a walking distance to the supermarkets. Otherwise, the function F2 is equal to 0. the city map is also inside one of the areas within a walking distance to the supermarkets. Otherwise, the function F2 is equal to 0. elementary operations. 5. Capabilities of modeling on a digital map using an expert system In addition, in the expert system, one could construct breakpoints at the inputs of conveyor opera tions and to monitor the state of model execution. 1, 2 2 . 0, 2 P Z P F P Z ∩ =   =   ∩ = ∅   (2) (2) 6. Scenarios as a universal technique to exchange data among models The city territory’s points that belong to the buffer zones built under the terms of the problem p F1 0 0 1 1 F2 0 1 0 1 F3 0 0 0 1 Proceed from a tabular description to a formula and de fine a solution to the problem as a Boolean function of two arguments [10]. Thus, we are talking about choosing a format for storing and exchanging data from modeling. By advancing the concept of model’s universality, built in the expert system, we introduce an additional requirement. Storage and exchange format must be known, well described, and documented. In addition, it could be used in other GIS and expert systems as a standard. 3 1 2. F F F = ∧ (3) (3) 3 1 2. F F F = ∧ Thus, in a given example, the solution is represented as overlay operation (3) and is formed as a conjunction of two regions Z1 and Z2. For the buffer zones that form in accor dance with (1) and (2) the arbitrary regions Z1 and Z2, we defined in the expert system a set of three Boolean opera tions, conjunction, disjunction, and negation of implication. Hereafter, the file that contains the structure and data for a decision tree constructed in an expert system is referred to as a script file. A script for model execution could be saved as a data exchange file in XML format to be available to another digital map or at a different computer. When expanded, the script file looks quite difficult. However, a user does not need to understand all the sub tleties of working with XML-format. The only important thing is to understand the hierarchical structure of the data contained in that file. At the top level of the hierarchy is the first node, which unfolds revealing child nodes of the second level. Each of these child nodes could be expended thereby representing the child levels of subsequent nodes. Any node could contain any number of child nodes or not have them at all. In the latter case, the node becomes terminal. Building and editing a script is performed in the main window of an expert system by using visual program ming tools. 3 1 2, 4 1 2, 5 1 2. 6. Scenarios as a universal technique to exchange data among models One must select all residential buildings (a layer of polyg onal objects on the city map) that: The visual expert system builder, described above, makes it possible to construct and edit the required model. A soft ware interpreter starts its execution and adjusts it stepwise over a working cycle of the model. However, modern methods of simulation modeling imply splitting a complex model into several stand-alone modules, each of which performs a specific set of tasks and share fulfillment results with other modules. – are located in a zone within walking distance to at least one supermarket; – are inside at least one of the buffer zones built around parks and gardens. In accordance with the search conditions, we introduce a spatial function F3 assigned in a tabular form (Table 1). Under such a multi-module organization, a series of issues arise related to the overall structure of the model. First, each module must be saved as a standalone file. The following must be implemented: download this module to the expert system, build a decision tree for it, and adjust its performance. Second, the expert system must run the required modules in a user-specified sequence. The results of each module’s execu tion, in the form of layers and data created for the map should be stored and, if necessary, be submitted to the input of other modules. 8. Discussion of results from the automated generation of data for a digital map using the expert system of geospatial analysis possible to construct more flexible and complex models for geographic information systems. The third type is the spe cial operations that implement the high-level fragments of the model. At this stage, the system employs two special operations. The first operation implies building, based on data on topography, the zones of visibility for the objects selected on the map or the center of a cluster of such ob jects (Fig. 8). The expert system is operated by a code interpreter. Such an approach has some drawbacks. Despite the presence of a unit for checking the source data, the user may encounter the run-time errors of the script. For example, when one applies a Boolean operation AND there is a probability of receiving an empty area for an integrated buffer zone (analyzed objects do not intersect). If such an empty area is sent to the input of the subsequent operation, an error may occur and the execution of the script would be suspended. Fig. 8. The script and results of constructing zones of visibility in an expert system of geospatial analysis Thus, there is a significant limitation in the scope of appli cation of this study results. Like most such solutions, managed by threads of input data, a geospatial analysis expert system cannot be applied in real-time control systems. To identify problematic situations, in the process of adjusting the model, we implemented a stepwise tracer that makes it possible to locate a point of the emergence of error within a script. The results obtained make it possible to consider the constructed system as a convenient tool for building data and for GIS operation without a need for special training of the user. Fig. 8. The script and results of constructing zones of visibility in an expert system of geospatial analysis By combining operations on objects identified on the map with the capabilities of buffer and overlay analysis, one can build complex models of the following phenomena on the map: The second specialized operation implies designing a route for an unmanned aerial vehicle (UAV) taking into consideration the altitudes and speeds of flight, the param eters of imaging equipment. A zone of the photographed territory, formed along the route of the flight, is a polyg onal object on a digital map. 7. Testing the possibilities of simulation modeling in an expert system Fig. 7. Conveyor of operations implemented in an expert system Thus, instead of a complex, difficult-to-read chain of predicates [10], we propose a simple and visual sequence of The first two types of operations within an ex pert system are of low level. Using them makes it 48 Information technology – a capability to work with rasters (6); – a capability to work with rasters (6); – construction of zones of flooding; h f h – construction of zones of flooding; h f h – the areas of a phenomenon propagation within cert l d d l – the areas of a phenomenon propagation within certain time intervals (fire spots, disease spread zones, social issues). The expert system of geospatial analysis has been im plemented as one of the modules in the GIS “Instrument” [1]. Thus, by having a source code of the system, developers are provided with a possibility to extend its functionality and to add new types of operations. If required, the modules that implement specialized operations could be used in other geographic information systems. To assess the completeness of solutions to the problem atic part, we shall compare the constructed system with the solutions (Table 2) given in the chapter reporting our analy sis of the scientific literature. The comparison was based on the following criteria: Using the script snippets for specialized operations im plements the required concept of the automated generation of data for a digital map without programming. – the presence of a visual model builder (1); b l – the presence of a visual model builder (1); b l Fig. 9. Results of designing an UAV route in the expert system of geospatial analysis – a possibility to construct scripts to execute models (2); – an intuitively-simple interface for operating the functions and procedures on geoprocessing (3); – extensibility and scalability of the constructed models (4); – possibilities to exchange data with other simu lation models and script modules (5); 8. Discussion of results from the automated generation of data for a digital map using the expert system of geospatial analysis By adjusting the altitude of the flight along a UAV route, or by applying a different type of the camera, one could minimize the flight time. This is subject to continuous coverage of the territory to be photographed and requires the coefficients for longitu dinal and transverse overlapping of imaged zones between the individual frames of the camera [11]. Using a module for the preliminary planning of a UAV route significantly simplifies the photogrammetric problem on compiling the mosaic of aerial photographs [12]. An example of designing an UAV route using the expert system of geospatial analy sis is shown in Fig. 9. – calculation and representation of reachability zones when moving objects at the predefined speed over a spec ified time; – calculation and representation of reachability zones when moving objects at the predefined speed over a spec ified time; – construction of zones of flooding; h f h 9. Conclusions Stvorennia prostorovykh danykh dlia elektronnykh kart heoinformats vnutrishnikh viysk MVS Ukrainy: monohrafiya. Kharkiv: Akad. VV MVS Ukrainy, 2013. 200 p. 2. Tsourdos A., White B., Shanmugavel M. Cooperative Path Planning of Unmanned Aerial Vehicles. Wiley, 2011. 190 p. doi: https:// doi.org/10.1002/9780470974636 3. Wessel M., Möller R. Flexible software architectures for ontology-based information systems // Journal of Applied Logic. 2009. Vol. 7, Issue 1. P. 75–99. doi: https://doi.org/10.1016/j.jal.2007.07.006 4. Predicting the future urban growth and it’s impacts on the surrounding environment using urban simulation models: Case study of Ibb city – Yemen / Al-Darwish Y., Ayad H., Taha D., Saadallah D. // Alexandria Engineering Journal. 2018. Vol. 57, Issue 4. P. 2887–2895. 5. GIS-based Decision Support System for Building Retrofit / Buffat R., Schmid L., Heeren N., Froemelt A., R Energy Procedia. 2017. Vol. 122. P. 403–408. doi: https://doi.org/10.1016/j.egypro.2017.07.433 5. GIS-based Decision Support System for Building Retrofit / Buffat R., Schmid L., Heeren N., Froemelt A Energy Procedia. 2017. Vol. 122. P. 403–408. doi: https://doi.org/10.1016/j.egypro.2017.07.433 5. GIS based Decision Support System for Building Retrofit / Buffat R., Schmid L., Heeren N., Froe Energy Procedia. 2017. Vol. 122. P. 403–408. doi: https://doi.org/10.1016/j.egypro.2017.07.433 6. ERDAS Field Guide. Technical Documentation ERDAS. Norcross, 2010. 811 p. 6. ERDAS Field Guide. Technical Documentation ERDAS. Norcross, 2010. 811 p. 7. Jähne B. Digital Image Processing. Springer, 2002. 585 p. doi: https://doi.org/10.1007/978-3-662-04781-1 7. Jähne B. Digital Image Processing. Springer, 2002. 585 p. doi: https://doi.org/10 8. McCoy J. ArcGIS 9. Geoprocessing in ArcGIS. New York: ESRI, 2004. 370 p. rcGIS 9. Geoprocessing in ArcGIS. New York: ESRI, 2004. 370 8. McCoy J. ArcGIS 9. Geoprocessing in ArcGIS. New York: ESRI, 2004. 370 p. y J g 9. Dangermond J. GIS in the Defence and Intelligence Communities. Vol. 3. New York: ESRI, 2008. 64 p. d J. GIS in the Defence and Intelligence Communities. Vol. 3. 10. Rvachev V. L. Teoriya R-funktsiy i nekotorye ee prilozheniya: monografiya. Kyiv: Nauchnaya mysl’, 198 0. Rvachev V. L. Teoriya R-funktsiy i nekotorye ee prilozheniya: monografiya. Kyiv: Nauchnaya mysl’, 1982. 552 p. 1. Obiralov A. I., Gebgart Ya. I., Il’inskiy N. D. Praktikum po fotogrammetrii i deshifrirovaniyu snimkov: ucheb. pos. Moscow, 1 11. Obiralov A. I., Gebgart Ya. I., Il’inskiy N. D. Praktikum po fotogrammetrii i deshifrirovaniyu snimkov: ucheb. p 12. ER Mapper. Airphoto tutorial. Technical Documentation ERDAS. Norcross, 2010. 169 p. 2. ER Mapper. Airphoto tutorial. Technical Documentation ERDAS. Norcross, 2010. 169 p. 9. Conclusions 1. We have developed principles for constructing a geospa tial analysis expert system, which greatly simplifies work with spatial geographical data for the user. The scientific result of such a solution underlies the proposed method for building buffer zones in GIS and for using methods of overlay analysis for the automation of processes to generate data for digital maps. A data analysis or task modeling on a map are reduced to a set of simple, intuitively-clear actions in the visual envi ronment of a model builder. This is achieved by ensuring that all the complex and polymorphic functions of geoprocessing are hidden from the user. As an alternative, the user is provid ed with a visual and easy-to-build decision tree. Table 2 Comparison between the capabilities of the expert system of geospatial analysis and available solutions Comparison between the capabilities of the expert system of geospatial analysis and available solutions Comparison between the capabilities of the expert system of geospatial analysis and available solutions 1 2 3 4 5 6 7 Expert system of geospatial analysis + + + + + – – Information system based on onto logical principles [3] + + – + + – + Model of changes in the boundaries of urban territories [4] + – – – – – – Decision support system for the ren ovation of residential buildings [5] – + + – + – + Model Maker [6] + + – + + + + ModelBuilder [8] + + – + + + + Flooding zones mapping system [9] + + – – – – – 2. An important aspect of our study’s results is the versa tility of the model builder when using an expert system. The scientific result from solving the set problem is the technique to ensure the standardization of modeling tools on a map. Now one could choose different, even the most unexpected, areas of work with geographic data and build the required models on a map. Such capabilities were demonstrated in the expert sys tem by the implementation of two models in the form of a set of specialized operations. We are talking about building the zones of visibility on a digital map and designing a UAV route. 3. We have proposed and implemented a procedure for ensuring a unified approach to storing and sharing of geographical data, which implies the construction of script libraries. – possibilities for using WEB services (7). – possibilities for using WEB services (7). Another limitation of the expert system follows from Table 2. The software did not implement a WEB-technology for multiuser queries of the cli ent-server type. However, the organization of an ef fective distributed network access to script libraries partially neutralizes this issue. Application of the mechanism of scripts will make it possible for other Fig. 9. Results of designing an UAV route in the expert system of geospatial analysis 49 3/2 ( 99 ) 2019 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 users to employ the devised practices and models. For ex ample, when planning, preparing, conducting and reviewing military activities, it is advisable to have a library of common scenarios that make it possible to analyze the terrain, to ac quire data on the terrain from a digital map and to generate data for the simulation combat models. 9. Conclusions The description of these scenarios in the expert system is based on the XML language standards. Using the expert system presents a possibility to download several common scenarios to the model and to obtain the new set of layers on a digital map. 3. We have proposed and implemented a procedure for ensuring a unified approach to storing and sharing of geographical data, which implies the construction of script libraries. The description of these scenarios in the expert system is based on the XML language standards. Using the expert system presents a possibility to download several common scenarios to the model and to obtain the new set of layers on a digital map. Arguing about the assessment of the completeness of the solution to the set task, it should be noted that, ow ing to the modifications of scripts, the designed system can be extended. The work is under way to expand the functionality of the expert system through capabilities to build scripts for decoding objects on aerial photographs. Such operations include the use of various filters, building contour (binary) image based on images and implementing the signatures for descriptions of template images. The GIS “Instrument” has a module of cluster analysis based on attribute values for layers of a digital map [13]. Trans ferring the functionality of a given module to the expert system would significantly simplify the processing of large amounts of data in GIS related to a digital map and con struct custom scenarios for such fields of human knowledge as sociology and statistics [14]. 4. In the expert system we have implemented a procedure for making simple and easy changes to the input data when one changes the initial conditions for modeling. This results in the unification of the constructed models. Under such an approach, there is no need to delve into the intricacies of the XML data description language. The user no more needs to directly edit the model’s source code. All the changes to the algorithm and to the initial conditions for modeling are performed in the visual environment of the expert system. References 1. Drobakha H. A., Rozanova L. V., Lisitsin V. E. Stvorennia prostorovykh danykh dlia elektronnykh kart heoinformats vnutrishnikh viysk MVS Ukrainy: monohrafiya. Kharkiv: Akad. VV MVS Ukrainy, 2013. 200 p. 1. Drobakha H. A., Rozanova L. V., Lisitsin V. E. 9. Conclusions 13. Han J., Kamber M., Pei J. Data mining: Concepts and techniques. Elsevier, 2012. 744 p. doi: https://doi.org/10.1016/c2009-0-61819-5 14. Murayama Y., Thapa R. B. Spatial analysis and modeling in geographical transformation process. Springer, 2011. 300 p. doi: https:// doi.org/10.1007/978-94-007-0671-2 50
https://openalex.org/W3195522611	http://publikationen.ub.uni-frankfurt.de/files/64541/s41398-021-01609-y.pdf	English	null	SLC25A24 gene methylation and gray matter volume in females with and without conduct disorder: an exploratory epigenetic neuroimaging study	Translational psychiatry	2,021	cc-by	9,130	1School of Psychology and Centre for Human Brain Health, University of Birmingham, Birmingham, UK. 2Department of Child and Adolescent Psychiatry, Psychosomatics, and Psychotherapy, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt am Main, Germany. 3School of Psychology and Institute for Mental Health, University of Birmingham, Birmingham, UK. 4Jacobs Center for Productive Youth Development, University of Zurich, Zurich, Switzerland. 5Department of Child and Adolescent Psychiatry, King’s College London, London, USA. 6Child Development Institute, Toronto, ON, Canada. 7RWTH Aachen University, Aachen, Germany. 8University Psychiatric Clinic Basel, Basel, Switzerland. 9Department of Psychology, University of Bath, Bath, UK. ✉email: exf266@bham.ac.uk; s.a.debrito@bham.ac.uk ARTICLE OPEN SLC25A24 gene methylation and gray matter volume in females with and without conduct disorder: an exploratory epigenetic neuroimaging study Elizabeth Farrow 1✉, Andreas G. Chiocchetti2, Jack C. Rogers3, Ruth Pauli1, Nora M. Raschle4, Karen Gonzalez-Madruga5, Areti Smaragdi6, Anne Martinelli 2, Gregor Kohls7, Christina Stadler 8, Kerstin Konrad 7, Graeme Fairchild9, ✉ ARTICLE OPEN SLC25A24 gene methylation and gray matter volume in female with and without conduct disorder: an exploratory epigenetic neuroimaging study Elizabeth Farrow 1✉, Andreas G. Chiocchetti2, Jack C. Rogers3, Ruth Pauli1, Nora M. Raschle4, Karen Gonzalez-Madruga5, Areti Smaragdi6, Anne Martinelli 2, Gregor Kohls7, Christina Stadler 8, Kerstin Konrad 7, Graeme Fairchild9, Christine M. Freitag 2, Magdalena Chechlacz1 and Stephane A. De Brito 1✉ © The Author(s) 2021, corrected publication 2021 Conduct disorder (CD), a psychiatric disorder characterized by a repetitive pattern of antisocial behaviors, results from a complex interplay between genetic and environmental factors. The clinical presentation of CD varies both according to the individual’s sex and level of callous-unemotional (CU) traits, but it remains unclear how genetic and environmental factors interact at the molecular level to produce these differences. Emerging evidence in males implicates methylation of genes associated with socio-affective processes. Here, we combined an epigenome-wide association study with structural neuroimaging in 51 females with CD and 59 typically developing (TD) females to examine DNA methylation in relation to CD, CU traits, and gray matter volume (GMV). We demonstrate an inverse pattern of correlation between CU traits and methylation of a chromosome 1 region in CD females (positive) as compared to TD females (negative). The identiﬁed region spans exon 1 of the SLC25A24 gene, central to energy metabolism due to its role in mitochondrial function. Increased SLC25A24 methylation was also related to lower GMV in multiple brain regions in the overall cohort. These included the superior frontal gyrus, dorsolateral prefrontal cortex, supramarginal gyrus, secondary visual cortex and ventral posterior cingulate cortex, which are regions that have previously been implicated in CD and CU traits. While our ﬁndings are preliminary and need to be replicated in larger samples, they provide novel evidence that CU traits in females are associated with methylation levels in a fundamentally different way in CD and TD individuals, which in turn may relate to observable variations in GMV across the brain. Translational Psychiatry (2021) 11:492 ; https://doi.org/10.1038/s41398-021-01609-y Translational Psychiatry Translational Psychiatry www.nature.com/tp Received: 11 November 2020 Revised: 29 June 2021 Accepted: 2 September 2021 INTRODUCTION more severe antisocial and aggressive behaviors both in adolescence and adulthood [6]. In this context, understanding the etiology of these CU traits in adolescents with CD is an important step towards identifying risk factors for a subgroup of youths with CD who are particularly susceptible to poorer outcomes in adulthood [7]. Conduct disorder (CD) is a psychiatric disorder of childhood and adolescence characterized by persistent antisocial behaviors (i.e., violence towards others or animals, destruction of property, theft, and serious rule violations), which signiﬁcantly impact the individual’s social, academic, or occupational functioning [1]. There is considerable variation in the possible combinations of symptoms that could lead to a CD diagnosis [2]. Therefore, to identify more homogeneous subgroups of youth with CD, several subtyping approaches are included within the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) [1]. One approach focuses on the ‘Limited Prosocial Emotions’ speciﬁer, which indexes callous-unemotional (CU) traits (i.e., reduced empathy, callousness, a lack of guilt, and shallow effect). This speciﬁer designates a particularly impaired subgroup of youths with CD who are at increased risk of developing psychopathy in adulthood [3, 4]. Levels of CU traits show moderate stability from adolescence to adulthood [5] and are also a predictor of Research shows that both genetic and environmental risk factors are implicated in the development of conduct problems or CD [8, 9], with around 50% of the variance in CD risk attributable to heritable genetic inﬂuences [8]. Crucially, twin studies indicate that youths with CD symptomatology and high versus low levels of CU traits are characterized by different environmental and genetic risk vulnerabilities [4]. Indeed, Viding et al. (2005) demonstrated that antisocial behavior in youths with CD symptomatology and high levels of CU traits is highly heritable (0.76), whereas in youths with CD symptomatology and low levels of CU traits it is moderately heritable (0.64) and more inﬂuenced by environmental factors [10]. Along with CU traits, sex is an E. Farrow et al. 2 inverse association between biomarkers and the level of CU traits in clinical groups as compared to TD populations. Thus, we investigated whether there was a CDxCU traits interaction effect on DNA methylation. The relationship between CU traits and methylation level has been demonstrated in individuals with CD [22, 23] but the nature and direction of this relationship in TD youth is unknown. Genome-wide methylation data pre-processing Genome-wide methylation data pre-processing DNA was extracted from saliva within 7 days of collection using the Oragene OG-500 Kit. DNA quality cutoff was a 260/280 ratio above 1.8. DNA was stored at −80 °C immediately. Genome-wide methylation was measured using the Illumina Inﬁnium HumanMethylationEPIC BeadChip Array at Life & Brain GmbH, Bonn, Germany. Pre-processing was performed in R version 3.6.0 [37]. Raw.idat ﬁles were pre-processed with the minﬁ[38] package (version1.32.0) following standard parameter settings (see Supple- mentary Methods). We removed failed and noisy probes as suggested [39], and also probes spanning an SNP with an SNP147 data-base annotated MAF > 10%. Finally, cross-reactive probes were eliminated. Between-array normalization was completed using the preprocessFunnorm() function [40] included in the minﬁpackage, following standard recommendations. This unsupervised method uses control probes to identify unwanted variation. It then extends the idea of quantile normalization to regresses out components of variation captured by these control probes [40]. This has been shown to be an effective method for removing positional effects [41]. We used ANOVA testing in the normalized methylation data to ensure there were no residual batch effects. As an additional check, we also extracted the ﬁrst principal component of the methylation data and performed pairwise T-tests (with Tukey’s correction for multiple testing) Participants p Fifty-one females with CD (mean age = 14.9, SD = 1.7) and 59 TD females (mean age = 14.7, SD = 2.4), recruited across ﬁve sites, were included as a subsample of the FemNAT-CD study [30] (see Supplementary Tables S1 for details). This study was conducted according to the legal regulations outlined by the European Union, national legislation, and the Declaration of Helsinki. For each site, written informed consent was obtained from all participants and their parents, in accordance with the site-speciﬁc ethical requirements. In addition to standard FemNAT-CD inclusion and exclusion criteria (see Supplementary materials), participants were required to be non-smokers, be medication-free, and have good quality saliva-DNA and structural MRI data. Participants were included in the CD group if they either; (a) met the DSM-5 criteria for a diagnosis of CD; (b) were 9–12 years old, met the criteria for a diagnosis of oppositional deﬁant disorder (ODD) and also had at least one current symptom of CD; or (c) were aged >12 years, met the criteria for ODD and also had at least 2 current CD symptoms. All TD participants had no diagnosable psychiatric disorders and no history of externalizing disorders (ADHD, ODD). The participants were aged 9–18 years and groups were matched on pubertal development status, performance IQ, ethnicity, and data-collection site (Table 1 and Supplementary Table S1). Altered regulation of genes expressed in brain tissues and/or implicated in behavior, may explain how methylation levels mechanistically mediate environmental inﬂuences, e.g. adverse life experiences to subsequent risk for CD [17] and CU traits [18]. A recent study suggests that exposure to adverse prenatal environmental factors has a large effect on the brain epigenome, and that epigenetic effects associated with brain development are also sex-speciﬁc [19]. Translational Psychiatry (2021) 11:492 Gene–environment interplay in CD development p y p A key question in CD research is how genetic and environmental risk factors interact at the molecular level in relation to CU trait phenotypes [13]. One candidate mechanism is via epigenetic changes in the form of DNA methylation, which involves addition of a methyl group at a speciﬁc genomic location [14]. Depending on the pattern, location, and level of methylation within or proximal to the gene’s coding sequence, gene expression may be suppressed or ampliﬁed [14]. The genetic variation of an individual is also an important factor to consider in understanding how environmental factors are translated into methylation signatures. Recent research has highlighted that individual differences in heritable factors may inﬂuence methylation signatures [15] and thus gene regulation. These genetic variants that can affect DNA methylation are known as methylation quantitative trait loci (mQTLs) and may be further useful markers for genetic inﬂuence on gene regulation [16]. Clinical and psychometric measures f p y Detailed information about these measures is provided in our previous work [31]. Brieﬂy, trained staff interviewed the participants and their parents (or caregivers) separately using the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version (K-SADS-PL [32]) to assess for CD and other DSM-IV-TR psychiatric disorders. Supplementary questions from the K-SADS-PL (e.g. for ODD/ ADHD) were completed if key items were endorsed during the initial screening. CU traits were assessed using the parent-version Inventory of Callous-Unemotional Traits (ICU [33]). Total, verbal and performance IQ was assessed using the Wechsler Abbreviated Scale of Intelligence [34] in the UK and the Wechsler Intelligence Scale for Children, Fifth Edition [35] at other sites. Pubertal status was determined using the Pubertal Develop- ment Scale (PDS) [36] completed by the participants (if aged >12 years) or by the parents/caregivers (for participants ≤12 years). p Epigenetic studies of youths with CD or sub-clinical CP have provided initial evidence that DNA methylation patterns may mediate environmental factors associated with antisocial behavior [20, 21]. In males with CD, methylation of the oxytocin receptor gene (OXTR) correlates positively with CU traits [22]. Similarly, in a mixed-sex study, higher methylation of OXTR at birth was associated with higher CU traits in adolescence for participants with low levels of anxiety [23]. Alterations in the expression of genes that govern the oxytocin system, as a result of epigenetic modiﬁcations, may thus play an important biological role in the development of CD and CU traits [22, 23]. A recent small-scale epigenetic neuroimaging study on males with CD showed that OXTR methylation and levels of CU traits interacted to predict frontoparietal hyperactivity and weaker amygdalo-frontoparietal connectivity in males during a face- processing task [24]. This is consistent with previous reports of abnormalities in this circuitry in CD (e.g., [25]) and the fact that OXTR is highly expressed in both limbic and cortical brain tissues [26]. Interestingly, a fundamentally opposite association between brain functional connectivity and level of CU traits was observed in CD as compared to TD youths [24]. INTRODUCTION Finally, to investigate whether these methyla- tion changes co-incidence with altered brain development, we related our methylation data to gray matter volume as measured using voxel-based morphometry (VBM). important factor to consider in youths with CD in relation to genetic vulnerability for this disorder. Indeed, heritability esti- mates for antisocial behavior in youths with CD are higher in males than females [11]. Furthermore, in males with CD and high levels of CU traits, heritable factors explain a high proportion of the variance in antisocial behavior [10]. Conversely, antisocial behavior in females with conduct problems (CP) and high levels of CU traits was shown to be entirely explained by environmental factors in one study [12]. These data suggest sex differences in the biological mechanisms underlying antisocial behavior in youths with CD depending on their levels of CU traits. Study aims d To expand current knowledge on epigenetics in CD and limited research on females with CD, we adopted an exploratory approach and conducted the ﬁrst Epigenome-Wide Association Study (EWAS) with salivary DNA data on females with CD and varying levels of CU traits. As previous research in psychiatric disorders has demonstrated differential methylation according to diagnostic status [27] and level of CU traits [22, 28], we ﬁrst examined the main effects of CD diagnostic status and level of CU traits. Secondly, we [29] and others [24] have demonstrated an Translational Psychiatry (2021) 11:492 E. Farrow et al. 3 Table 1. Demographic and clinical characteristics of the participants. Demographic & Clinical Characteristics CD (n = 51) TD (n = 59) P (t-test) Wilcoxon’s p M SD M SD Demographic Age 14.9 1.73 14.7 2.38 0.670 0.961 PDS 3.98 1.05 4.07 0.98 0.651 0.692 SES −0.540 0.828 0.205 0.902 <0.001 <0.001 Total IQ 94.7 12.2 100.05 10.2 0.013 0.007 Perf. IQ 93.7 14.8 98.83 12.7 0.062 0.091 Verbal IQ 93.6 19.2 101.0 12.9 0.023 0.004 Clinical ADHD symptoms 0.22 0.42 0.14 0.34 0.24 0.28 GAD symptoms 0.24 0.55 0 0.29 0.008 0.004 MDD symptoms 0.5 0.70 0 0 <0.001 <0.001 ICU total 29.6 11.4 17.6 9.02 <0.001 <0.001 ICU callous 10.2 5.38 4.65 3.80 <0.001 <0.001 ICU uncaring 13.1 5.27 8.37 4.54 <0.001 <0.001 ICU unemotional 6.31 3.59 4.93 2.74 0.030 0.040 CD conduct disorder, TD typically developing, PDS Pubertal Development Scale, SES socio-economic status, IQ intelligent quotient, ADHD attention-deﬁcit/ hyperactivity disorder, GAD generalized anxiety disorder, MDD major depressive disorder, ICU inventory of callous-unemotional trait. Table 1. Demographic and clinical characteristics of the participants. Table 1. Demographic and clinical characteristics of CD conduct disorder, TD typically developing, PDS Pubertal Development Scale, SES socio-economic status, IQ intelligent quotient, ADHD attention-deﬁcit/ hyperactivity disorder, GAD generalized anxiety disorder, MDD major depressive disorder, ICU inventory of callous-unemotional trait. conduct disorder, TD typically developing, PDS Pubertal Development Scale, SES socio-economic status, IQ intelligent quo eractivity disorder, GAD generalized anxiety disorder, MDD major depressive disorder, ICU inventory of callous-unemotiona veloping, PDS Pubertal Development Scale, SES socio-economic status, IQ intelligent quotient, ADHD attention-deﬁcit/ ed anxiety disorder, MDD major depressive disorder, ICU inventory of callous-unemotional trait. across the batches to conﬁrm there were no correlations between the batch IDs and M values. VBM analysis Si id iﬁ y Since we identiﬁed a signiﬁcant DMR associated with the group-by-CU traits interaction effect on methylation level, we employed the GLM framework to explore the association between GMV and average M-value across probes within the respective DMR. No DMR associated with main effects for CD or CU-traits was identiﬁed. Study aims d arguments of the Bumphunter function to test separately for: (i) the main effect of CD diagnosis, (ii) the main effect of CU score, and (iii) a CD × CU interaction effect on methylation, while controlling for the main effects of the other two factors. QQ plots were generated to conﬁrm appropriate model ﬁts for each EWAS model (see Supplementary Fig. 2). Correction for multiple testing using the false discovery rate (FDR [44]) was done across the individual probes tested as recommended [45]. Heat maps and hierarchical clustering plots based on the Euclidean distance of the top 2000 loci selected by variance in methylation were generated to visually check for outliers and batch effects (Supplementary Fig. 1). The methylation M-values were calculated based on the log- transformed ratio of methylated to unmethylated signal-intensities for each locus in line with previous research [42] and we ensured these M values were normally distributed across the differentially methylated region (Supplementary Fig. 1). Probes were mapped to their genomic region using the human reference genome hg19. MRI acquisition T1-weighted structural scans were collected at ﬁve research sites using MRI scanners all operating with 3 T ﬁelds (either Siemens or Philips manufactured) and harmonized acquisition sequences (see refs. [29, 31] and Supplementary materials). Speciﬁcally, GMV was analyzed on a voxel-by-voxel basis, via multiple regressions. PDS, SES, total intracranial volume (TIV), scanning site (dummy coded), and total IQ were included as covariates of no interest. Unlike in the epigenetic analysis, we include SES as a covariate here to allow us to investigate the association between methylation and GMV across our full cohort without the potential confounding effects of SES on GMV that are independent of methylation. At a whole-brain level, inferences were made using a statistical threshold of p < 0.05 after family-wise error (FWE) correction for multiple comparisons. We also investigated associations between GMV and M-value in four regions of interest (ROIs, bilaterally) where the identiﬁed gene of interest SLC25A24 is highly expressed (Genotype-Tissue Expression [46] GTEx project database, see supplementary material Fig. 3), namely the amygdala, hippocampus, basal ganglia and cerebellum (Supplementary Fig. 5). Masks of these regions were deﬁned based on the Talairach Daemon database using the WFU PickAtlas tool in SPM12 [47]. The MarsBAR toolbox was used to extract mean-cluster and peak-voxel GMV values from signiﬁcant clusters for each participant. All brain imaging coordinates are reported in the standardized Montreal Neurological Institute (MNI) space. Genome-wide methylation statistical analysis y y To examine the associations between CD diagnostic status, level of CU traits and genome-wide methylation, we employed linear regression modelling: M-values for each CpG site was modelled as a function of CD status, CU traits (total ICU score), and the CDxCU traits interaction effect. Corrections for the effects of age and hormonal contraceptive use were included in the model. Socio-economic status (SES) was not included as a covariate in the DNA methylation analysis on statistical and conceptual grounds (see Supplementary materials for further details). g y To identify components of extraneous variation due to unmodelled or unknown latent variables, surrogate variable analysis in R (sva package, “leek” method selected) was performed and the two factors identiﬁed were included in the ﬁnal model as covariates. The effect sizes and p-value of each predictor (CD-case status, CU-trait levels and CDxCU) were calculated using the suggested Bayesian approach as implemented in the minﬁebayes function. P-values were then submitted to the Bumphunter algorithm [43] to identify differentially methylated regions (DMRs). We speciﬁed different coefﬁcients from the linear regression modelling in the Pre-processing of the neuroimaging data Consistent with our previous work [29], SPM12 (www.ﬁl.ion.ucl.ac.uk/spm), Computational Anatomy 12 (CAT-12: http://dbm.neuro.uni-jena.de/cat/) and template-o-matic (TOM8 [42]) toolboxes were used to pre-process MRI data (see Supplementary materials). Participant characteristics As per matching on PDS and performance IQ, CD and TD females did not differ in terms of age, puberty, ethnicity, site and performance IQ, but the CD group had lower full-scale IQs than the TD group (Table 1). The number of ADHD symptoms did not differ between groups, but individuals with CD had signiﬁcantly more symptoms of a generalized anxiety disorder (GAD) and Translational Psychiatry (2021) 11:492 Fig. 1 Genomic Location of the Chr1. Differentially Methylated Region. (Top) UCSC Genome Browser Illustration showing stacked annotation tracks beneath the genomic coordinates of the region which showed differential methylation according to the CD × CU traits interaction (from the hg19 human reference gene); (bottom) a scatter plot of this differentially methylated region highlighting the opposite relationship between methylation and level of CU traits in CD cases vs. control participants. E. Farrow et al. E. Farrow et al. 4 Fig. 1 Genomic Location of the Chr1. Differentially Methylated Region. (Top) UCSC Genome Browser Illustration showing stacked annotation tracks beneath the genomic coordinates of the region which showed differential methylation according to the CD × CU traits interaction (from the hg19 human reference gene); (bottom) a scatter plot of this differentially methylated region highlighting the opposite relationship between methylation and level of CU traits in CD cases vs. control participants. major depressive disorder (MDD) than the TD participants. Females with CD also had higher total ICU and ICU subscale scores (see Table 1 and Supplementary Fig. 4). slopes of these correlations differed signiﬁcantly (Z = 2.48, p = 0.007). The region identiﬁed includes exon 1 of the solute carrier SLC25A24 gene (see Fig. 1). It is important to note that these methylation ﬁndings do not include the methylation values at common SNPs, as these were removed during the pre-processing stage of our analysis, thus our ﬁndings should be considered in light of this limitation. Identiﬁcation of differentially methylated regions y y g At the single probe level, DNA-methylation was not predicted by case-control status or level of CU traits (at a signiﬁcance level of pFDR < 0.05). However, the CDxCU traits interaction signiﬁcantly predicted differential methylation at one genomic region on chromosome 1 (hg19 chr1: 108,735,312–108,735,893, FDR = 0.004), spanning eight probes. The interaction was driven by a positive association between CU traits and methylation of the respective probes in females with CD (Pearson r(49) = 0.39, p = 0.006), but a negative association between CU traits and methylation in TD females (Pearson r(57) = −0.27, p = 0.042). The Translational Psychiatry (2021) 11:492 Power calculation While we acknowledge that our sample size is rather small for a genome-wide approach, power analysis using the online calcula- tion tool epigenetics.essex.ac.uk/shiny/EPICDNAmPowerCalcs con- ﬁrmed that our analysis with a sample size of n = 110 participants conferred each CpG site tested with ~80% power to detect a difference in methylation at the recommended level for the EPIC array (p < 6.21e−05). Two other recent studies have similarly adopted a genome-wide approach to investigating DNA methyla- tion in relation to aggressive behaviours in youth, both using a sample size <n = 100 [48, 49]. Association between methylation and gray matter volume We then tested whether the SLC25A24 methylation levels observed for the interaction effect of CDxCU traits was also associated with GMV in any brain region. After correction for multiple comparisons, no signiﬁcant (i.e. pFWE < 0.05) positive or negative associations between the average M-value of the SLC25A24-DMR and GMV were detected (in analysis across the whole cohort). However, given the exploratory nature of this study, we report ﬁndings at a more liberal signiﬁcance level of p < 0.001 uncorrected with an extent threshold of k = 72 voxels empirically determined according to random ﬁeld theory [50, 51]. At this level we observed a negative association with SLC25A24 methylation M-value for GMV in several clusters within the brain (please see Supplementary Table s2), indicating that higher SLC25A24 methylation is associated with lower GMV in these regions. We identiﬁed these clusters in multiple brain regions including the superior frontal gyrus (SFG), dorsolateral prefrontal cortex (dlPFC), supramarginal gyrus, the secondary visual cortex in E. Farrow et al. y = 48 x = -41 x = 7 z = -2 z = 37 y = -32 x = -37 z = 3 x = 10 a) Superior Frontal Gyrus g) Dorsolateral PFC b) Ventral Posterior Cingulate Cortex c) Dorsolateral PFC i) R Occipital Pole h) Secondary Visual Cortex d) Supramarginal Gyrus e) Secondary Visual Cortex f) Supramarginal Gyrus Fig. 2 Association between SLC25A24 methylation and gray matter volume. Mean gray matter volume (GMV) values in the cluster signiﬁcantly associated with methylation for p < 0.001, size > 72 voxels were extracted for each participant and then plotted against the average methylation M-value across the DMR on chromosome 1 corresponding to exon 1 of gene SLC25A24. CD participants (red) and TD (blue) participants are differentiated by color. In all clusters there is a negative association between GMV and M value in both CD and TD groups; the difference between groups in the strength of the correlation is not statistically signiﬁcant at p < 0.05. z = 37 10 i) R O i it l P l f) Supramarginal Gyrus z = h) Secondary Visual Cortex x = 10 i) R Occipital Pole Fig. 2 Association between SLC25A24 methylation and gray matter volume. Mean gray matter volume (GMV) values in the cluster signiﬁcantly associated with methylation for p < 0.001, size > 72 voxels were extracted for each participant and then plotted against the average methylation M-value across the DMR on chromosome 1 corresponding to exon 1 of gene SLC25A24. CD participants (red) and TD (blue) participants are differentiated by color. In all clusters there is a negative association between GMV and M value in both CD and TD groups; the difference between groups in the strength of the correlation is not statistically signiﬁcant at p < 0.05. the left hemisphere, and the ventral PCC and secondary visual cortex in the right hemisphere. All coordinates are reported in MNI space. Mean cluster GMV values were extracted for each participant and then plotted against the average methylation M-value across the DMR on chromosome 1 (i.e. exon 1 of gene SLC25A24 (see Fig. 2)). Across all regions, in both CD and TD groups, there was a negative association between GMV and the mean exon 1 SLC25A24 M-value. both in multiple brain regions implicated in CD and CU traits and in a whole-brain exploratory analysis. Genome-wide methylation f ﬁ We found a signiﬁcant CD × CU traits interaction effect on methylation level in exon 1 of the SLC25A24 gene, whereby methylation level was positively correlated with CU traits in CD participants, but negatively correlated with CU traits in TD controls. Elevated methylation at the ﬁrst exon and promoter regions of genes has been demonstrated to decrease the expression of the respective gene [52, 53]. Thus, our results indicate that in adolescent females with CD, higher levels of CU traits are associated with reduced SLC25A24 gene expression, whereas in TD females, CU traits are positively associated with gene expression. Post-hoc testing of OXTR methylation p y g p SLC25A24, a member of a solute-carrier gene family [54], is involved in adenosine triphosphate (ATP)-mediated Calcium buffering at the mitochondrial matrix and is potentially involved in protecting cells against oxidative stress-induced cell death. In mitochondria, ATP production is associated with the production of free oxidative radicals. These cellular redox scavengers, as well as nutrition-derived antioxidants, are crucial to neutralize these free radicals [55]. As the brain accounts for 25% of the body’s total energy expenditure [56], impaired mitochondrial function, as suggested by a reduced expression of SLC25A24, may lead to higher rates of cell death due to oxidative stress [57] and thus leave neuronal cells especially vulnerable to oxidative damage [58]. Increased cell death, due to an impaired redox-scavenger system in the brain’s mitochondria, may also, at least partially, explain the association we observed with GMV. Furthermore, unbalanced energy provision and reduced Calcium homeostasis in neurons may result in impaired functioning and ultimately lead to y We did not observe a signiﬁcant association between CU traits and methylation at any of the 12 CpG sites on the OXTR gene for which we had DNA methylation data. Even when the signiﬁcance threshold was reduced to a nominal level of p < 0.001, uncor- rected, the main effect of CU traits was not signiﬁcant for any of the individual sites, or for this region as a whole. ROI analysis N i iﬁ No signiﬁcant positive or negative association between SLC25A24 methylation and GMV could be detected in the amygdala, hippocampus, basal ganglia or cerebellum ROIs. (Please see Supplementary Fig. 5 for 3D visualization of the four brain regions tested as ROIs.) GMV in regions including the SFG, dlPFC and supramarginal gyrus was negatively correlated with methylation levels, however, these neuroimaging ﬁndings did not reach the minimum threshold for signiﬁcance. DISCUSSION To our knowledge, this is the ﬁrst EWAS and epigenetic neuroimaging study in females with CD. First, we examined the main effects of CD group status, level of CU traits and their interaction on saliva-based DNA methylation. Our analyses revealed that in CD and TD females there is a fundamentally opposite pattern of association between CU traits and methylation at a chromosome 1 genomic region, spanning exon 1 of the SLC25A24 gene. Second, we related the identiﬁed DMR to GMV, Translational Psychiatry (2021) 11:492 E. Farrow et al. 6 neurodegeneration [57]. Accordingly, mitochondrial dysfunction has been suggested to be associated with several neurodevelop- mental disorders, including autism spectrum disorder (ASD) [59, 60] and ADHD [61]. Reduced expression of the SLC25A24 gene has been reported in the thalamus and motor cortex of patients with ASD and hypothesized to be associated with the impairments in sensory processing and response inhibition observed in this population [62]. Epigenetic neuroimaging data Our neuroimaging analysis revealed trend-level negative associa- tions between SLC25A24 methylation values and GMV in several brain regions, namely, the SFG, dlPFC, supramarginal gyrus and secondary visual cortex in the left hemisphere, and the ventral posterior cingulate cortex (PCC) and secondary visual cortex in the right hemisphere. g p These results may suggest that higher levels of SLC25A24 gene methylation is linked to a reduction in GMV in these regions. This ﬁnding would be consistent with the theory that increased methylation has a silencing effect on the gene, leading to impaired mitochondrial function (and thus a reduced capacity for energy production and growth) during brain development. Many of the regions where reduced GMV was observed, such as the SFG, dlPFC, the supramarginal gyrus and the ventral PCC, are involved in higher cognitive functions, such as working memory [76], as well as socio-cognitive processes such as affective empathy, which have been shown to be impaired in CD [13, 77]. For example, in a recent meta-analysis of 13 VBM studies, we found that youths with CP had signiﬁcantly reduced GMV in the left medial SFG [78]. Atypical cortical thickness and functional connectivity have also been reported in adults with psychopathy in several brain regions across the frontal cortices [79] and deﬁcits in cortical folding in these regions are also reported in youths with CD [80]. As discussed, deﬁcient mitochondrial functioning is a possible consequence of increased methylation and the resulting decreased expression of the SLC25A24 gene. Post-hoc testing of OXTR methylation More generally, mitochondrial dysfunction has been linked to exposure to environmental stressors [71]. Mitochondria are key components of the human body’s stress response system, providing intra-cellular energy and synthesizing stress hormones and neurotransmitters central to stress responding [72]. Experi- mental manipulation of mitochondrial function has been shown to inﬂuence physiological and behavioral responses to psychological stress [72]. Crucially, there is evidence that epigenetic markers of stress exposure are mitochondrially regulated [72]. Thus, reduced expression in genes governing mitochondrial function, such as SLC25A24, may arbitrate how environmental factors result in epigenetic modiﬁcations [73]. y The fact that other studies have found an association between CU traits and methylation of the OXTR gene (e.g. refs. [22, 23]), but we did not can be explained by a number of factors. For example, this may be related to methodological differences between our study and previous studies, such as the use of different measures of CU traits (i.e., ICU here, but others [24] have used the Youth Psychopathic Traits Inventory (YPI [81]) or other different investigative approaches, i.e. candidate gene vs. epigenome-wide studies. Additionally, we focused on females only, which contrasts with previous studies that have relied on male-only or mixed-sex samples. Individuals with CD are more likely to have experienced ‘stressful’ early life environments and thus to have elevated stress biomarkers associated with psychiatric symptoms [74]. CU traits may be another factor that moderates the association between environmental risk factors and the individual’s biolo- gical stress response [75]. Consequently, the combination of CD diagnostic status and level of CU traits may inﬂuence epigenetic markers associated with stress exposure. Altered methylation across genes in the energy metabolism system may represent an adaptive response to these variations. Thus, rather than being a unique marker of one stressor, we postulate that SLC25A24 gene methylation may reﬂect the cumulative effect of exposure to multiple early-life environmental factors triggering the biological stress response system. DISCUSSION Given that mitochondria work alongside the mitochondrial-bound mono- amine oxidase A (MAO-A) enzyme to break down catecholami- nergic neurotransmitters [63], altered functioning of either component in the degradation process may contribute to abnormally high or low levels of neurotransmitters in the brain [64]. Importantly, atypical levels of neurotransmitters have previously been associated with both CD [13] and CU traits [28]. Both elevated SLC25A24 methylation and variants of the MAO-A enzyme may contribute to disrupted catecholamine catabolism. This is reported to be the biological means by which variation of the MAOA gene contributes to the affective (e.g., emotion dysregulation) and behavioral (e.g., reactive aggression) features of females with CD [65]. Thus, SLC25A24 gene hypermethylation may also result in behavioral patterns asso- ciated with atypical levels of neurotransmitters in the brain in a similar way to that reported for variants of the MAO-A enzyme, which have previously been linked to aggressive/violent behaviors in both animals [66] and humans [67]. g p y In youths with CD, greater levels of methylation were observed in association with higher CU traits and greater levels of methylation were also related to reductions in GMV at trend- level. In TD youths, we see the inverse pattern (with individuals with higher CU traits having higher GMV in the observed brain regions). We speculate that in individuals with CD and high CU traits this increased methylation and the associated higher levels of oxidative stress during energy production contributes to a higher rate of neuronal death during neuronal pruning, and subsequently leads to a reduction in GMV in the observed brain regions in this group. However, currently, the underlying factors contributing to this mechanism are unknown, and further research with more highly powered studies is needed to determine whether the suggestive negative relationship between GMV and methylation we observed here holds true in larger samples. Environmental risk factors and SLC25A24 methylation Childhood maltreatment, a key factor known to inﬂuence DNA methylation [68], has been shown to interact with MAOA variants to predict aggression in both sexes [69]. In females, the high activity allele has been shown to confer a risk for aggressive behavior following childhood maltreatment [69], but see ref. [70]. Future studies should further investigate the relationship between childhood maltreatment and methylation to determine whether experiences of child maltreatment alter DNA methylation levels and thereby increase the risk for aggressive behaviors. Strengths and limitations As the ﬁrst study integrating epigenetic and neuroimaging data from females with CD, this work is an important contribution to our understanding of the biological factors implicated in CD and CU traits in females. Using multi-site data allowed for a larger sample size than would have been possible at a single site, as CD females are difﬁcult to recruit. Furthermore, as data were collected as part of the FemNAT-CD project, the sample is well- characterized, with all participants undergoing thorough assess- ment for psychiatric disorders and symptoms using a reliable measure based on DSM-IV-TR criteria. Finally, the two groups did not differ on PDS, performance IQ, ADHD symptoms, site and ethnicity, minimizing the potential confounding effects of these factors. Translational Psychiatry (2021) 11:492 E. Farrow et al. 7 Nevertheless, this study has limitations. First, the sample size is relatively small. As mentioned above, power analysis conﬁrmed our analysis with a sample size of n = 110 participants conferred each CpG site tested with ~80% power to detect a difference in methylation at the recommended level for the EPIC array (p < 6.21e−05). This power allows us to detect moderate-to-large effects, however smaller effects (f < 0.35) on genome-wide methylation levels or GMV were not detectable with this study design. Also, we only had data on childhood maltreatment for a small subset of participants (n = 31), so we were unable to include this information in our analysis. Second, while several previous studies report concordance of DNA methylation across saliva and brain tissues (e.g. ref. [82]), tissue-speciﬁc epigenetic modiﬁcations have also been reported [83]. Thus, it is possible that the differential methylation in salivary DNA demonstrated in this study does not accurately reﬂect brain-level methylation and might thus be speciﬁc to buccal cells only. We also did not correct for cell composition in our salivary DNA samples. Third, as the methyla- tion ﬁndings we report do not include the methylation at common SNPs, we do not yet know whether the methylation differences we observe are themselves genetically inﬂuenced. Finally, due to funding limitations, we chose to focus solely on investigating genome-wide methylation in females. We felt this would maximise the novelty of our work and add to the knowledge base in this particularly under-researched group. Strengths and limitations However, as we only included female participants our ﬁndings may not apply to males with CD, as research indicates sex-speciﬁc inﬂuences of environmental and genetic factors on CD and CU traits [10, 12]. Thus similar studies in males and mixed-sex samples will be an important area of future research to investigate whether these mechanisms are sex-speciﬁc. 6. Marcus RF. The development of aggression and violence in adolescence. Springer; 2017. New York, United States p. 141–70. 7. Walters GD. Assessing the proactive and reactive dimensions of criminal thought process: divergent patterns of correlation with variable-and person-level mea- sures of criminal risk and future outcome. J Personal Assess. 2020;102:223–30. 8. Polderman TJ, Benyamin B, de Leeuw CA, Sullivan PF, van Bochoven A, Visscher PM, et al. Meta-analysis of the heritability of human traits based on ﬁfty years of twin studies. Nat Genet. 2015;47:702–9. 9. Salvatore JE, Dick DM. Genetic inﬂuences on conduct disorder. Neurosci Biobehav Rev. 2018;91:91–101. 10. Viding E, Blair RJR, Mofﬁtt TE, Plomin R. Evidence for substantial genetic risk for psychopathy in 7‐year‐olds. J Child Psychol Psychiatry. 2005;46:592–7. 11. Caspi A, Taylor A, Mofﬁtt TE, Plomin R. Neighborhood deprivation affects chil- dren’s mental health: environmental risks identiﬁed in a genetic design. Psychol Sci. 2000;11:338–42. https://doi.org/10.1111/1467-9280.00267 12. Fontaine NM, Rijsdijk FV, McCrory EJ, Viding E. Etiology of different develop- mental trajectories of callous-unemotional traits. J Am Acad Child Adolesc Psy- chiatry. 2010;49:656–64. 13. Fairchild G, Hawes DJ, Frick PJ, Copeland WE, Odgers CL, Franke B, et al. Conduct disorder. Nat Rev Dis Prim. 2019;5:43. https://doi.org/10.1038/s41572-019-0095-y 14. Anastasiadi D, Esteve-Codina A, Piferrer F. Consistent inverse correlation between DNA methylation of the ﬁrst intron and gene expression across tissues and species. Epigenet Chromatin. 2018;11:37. https://doi.org/10.1186/s13072- 018-0205-1 15. van Dongen J, Ehli EA, Jansen R, van Beijsterveldt C, Willemsen G, Hottenga JJ, et al. Genome-wide analysis of DNA methylation in buccal cells: a study of monozygotic twins and mQTLs. Epigenet Chromatin. 2018;11:54. https://doi.org/ 10.1186/s13072-018-0225-x 16. Barker ED, Walton E, Cecil C, Rowe R, Jaffee SR, Maughan B, et al. A Methylome- Wide Association Study of trajectories of oppositional deﬁant behaviors and biological overlap with attention deﬁcit hyperactivity disorder. Child Dev. 2018;89:1839–55. https://doi.org/10.1111/cdev.12957 17. Veroude K, Zhang-James Y, Fernàndez-Castillo N, Bakker MJ, Cormand B, Faraone SV. Genetics of aggressive behavior: an overview. Am J Med Genet B. 2016;171B:3–43. https://doi.org/10.1002/ajmg.b.32364 18. Henry J, Pingault JB, Boivin M, Rijsdijk F, Viding E. CONCLUSIONS M h l i f h 19. Mattern F, Post A, Solger F, O'Leary A, Slattery DA, Reif A, et al. Prenatal and postnatal experiences associated with epigenetic changes in the adult mouse brain. Behav Brain Res. 2019;359:143–8. https://doi.org/10.1016/j.bbr.2018.10.037 Methylation of the SLC25A24 gene was signiﬁcantly associated with CU traits in both females with CD and TD females but in a fundamentally opposing pattern. Given its essential role in energy metabolism, SLC25A24 is a key component of the biological stress response system. We postulate that the combination of the individual’s level of CU traits and the number of stressful early life experiences may epigenetically modify the SLC25A24 gene thus inﬂuencing its functionality. Furthermore, we detected negative trends between SLC25A24 methylation values and GMV in several brain regions, many of which have also been implicated in CD and CU traits. While our ﬁndings are preliminary and need to be replicated in larger samples, they provide novel evidence that CU traits in females are associated with methylation levels in a fundamentally different way in CD and TD groups, which in turn relates to observable variations in GMV in the brain. 20. Gescher DM, Kahl KG, Hillemacher T, Frieling H, Kuhn J, Frodl T. Epigenetics in personality disorders: today’s insights. Front Psychiatry. 2018;9:579. https://doi. org/10.3389/fpsyt.2018.00579 21. Chiocchetti AG. (2020 (under review)). 22. Dadds MR, Moul C, Cauchi A, Dobson-Stone C, Hawes DJ, Brennan J, et al. Methylation of the oxytocin receptor gene and oxytocin blood levels in the development of psychopathy. Dev Psychopathol. 2014;26:33–40. https://doi.org/ 10.1017/S0954579413000497 23. Cecil CA, Lysenko LJ, Jaffee SR, Pingault JB, Smith RG, Relton CL, et al. Environ- mental risk, Oxytocin Receptor Gene (OXTR) methylation and youth callous- unemotional traits: a 13-year longitudinal study. Mol Psychiatry. 2014;19:1071–7. https://doi.org/10.1038/mp.2014.95 24. Aghajani M, Klapwijk ET, Colins OF, Ziegler C, Domschke K, Vermeiren R, et al. Interactions between oxytocin receptor gene methylation and callous- unemotional traits impact socioaffective brain systems in conduct-disordered offenders. Biol Psychiatry Cogn Neurosci Neuroimaging. 2018;3:379–91. https:// doi.org/10.1016/j.bpsc.2017.12.010 Strengths and limitations Genetic and environmental aetiology of the dimensions of callous-unemotional traits. Psychol Med. 2016;46:405–14. https://doi.org/10.1017/S0033291715001919 REFERENCES The complete mitochondrial genome of the Sichuan Hill Partridge (Arborophila ruﬁpectus) and a phylo. Genet Anal Relat Species Gene. 2009;435:23–28. https://doi.org/10.1016/j. gene.2009.01.001 31. Rogers JC, Gonzalez-Madruga K, Kohls G, Baker RH, Clanton RL, Pauli R, et al. White matter microstructure in youths with conduct disorder: effects of sex and variation in callous traits. J Am Acad Child Adolesc Psychiatry. 2019;58:1184–96. https://doi.org/10.1016/j.jaac.2019.02.019 55. Fraunberger EA, Scola G, Laliberte VL, Duong A, Andreazza AC. Redox modula- tions, antioxidants, and neuropsychiatric disorders. Oxid Med Cell Longev. 2016;2016:4729192. https://doi.org/10.1155/2016/4729192 32. Kaufman J, Birmaher B, Brent D, Rao U, Flynn C, Moreci P, et al. Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL): initial reliability and validity data. J Am Acad Child Adolesc Psychiatry. 1997;36:980–8. https://doi.org/10.1097/00004583-199707000-00021 56. Herculano-Houzel S. The remarkable, yet not extraordinary, human brain as a scaled-up primate brain and its associated cost. Proc Natl Acad Sci USA. 2012;109:10661–8. https://doi.org/10.1073/pnas.1201895109 57. Kramer P, Bressan P. Our (mother’s) mitochondria and our mind. Perspect Psychol Sci. 2018;13:88–100. https://doi.org/10.1177/1745691617718356 33. Frick PJ, Cornell AH, Barry CT, Bodin SD, Dane HE. Callous-unemotional traits and conduct problems in the prediction of conduct problem severity, aggression, and self-report of delinquency. J Abnorm Child Psychol. 2003;31:457–70. https://doi. org/10.1023/a:1023899703866 58. de Oliveira DM, Ferreira Lima RM, El-Bacha RS. Brain rust: recent discoveries on the role of oxidative stress in neurodegenerative diseases. Nutr Neurosci. 2012;15:94–102. https://doi.org/10.1179/1476830511Y.0000000029 59. Chiocchetti AG, Haslinger D, Boesch M, Karl T, Wiemann S, Freitag CM, et al. Protein signatures of oxidative stress response in a patient speciﬁc cell line model for autism. Mol Autism. 2014;5:10. https://doi.org/10.1186/2040-392-5-10 34. Wechsler D. Wechsler Abbreviated Scale of Intelligence–2nd Edition. San Antonio, TX: NCS Pearson; 2011. 35. Wechsler D. Wechsler intelligence scale for children. 5th ed (WISC-V). San Anto- nio: Psychological Corporation; 2014. 60. Varga NÁ, Pentelényi K, Balicza P, Gézsi A, Reményi V, Hársfalvi V, et al. Mito- chondrial dysfunction and autism: comprehensive genetic analyses of children with autism and mtDNA deletion. Behav Brain Funct. 2018;14:4. https://doi.org/ 10.1186/s12993-018-0135-x 36. Petersen AC, Crockett L, Richards M, Boxer A. A self-report measure of pubertal status: reliability, validity, and initial norms. J Youth Adolesc. 1988;17:117–33. 37. Team, RCR. A language and environment for statistical computing—version 3.6.0. Team, RCR; Vienna, Austria 2019. 61. Hwang IW, Hong JH, Kwon BN, Kim HJ, Lee NR, Lim MH, et al. REFERENCES 25. Gillespie NA, Neale MC, Hagler DJ Jr, Eyler LT, Fennema-Notestine C, Franz CE, et al. Genetic and environmental inﬂuences on mean diffusivity and volume in subcortical brain regions. Hum Brain Mapp. 2017;38:2589–98. https://doi.org/ 10.1002/hbm.23544 1. American Psychiatric Association. Diagnostic and statistical manual of mental disorders (DSM-5®). American Psychiatric Association Publishing. Washington DC, United States 2013. 2. Nock MK, Kazdin AE, Hiripi E, Kessler RC. Prevalence, subtypes, and correlates of DSM-IV conduct disorder in the National Comorbidity Survey Replication. Psychol Med. 2006;36:699–710. 26. Quintana DS, Rokicki J, van der Meer D, Alnæs D, Kaufmann T, Córdova-Palomera A, et al. Oxytocin pathway gene networks in the human brain. Nat Commun. 2019;10:668. https://doi.org/10.1038/s41467-019-08503-8 3. Frick PJ, Ray JV, Thornton LC, Kahn RE. Annual research review: a developmental psychopathology approach to understanding callous‐unemotional traits in chil- dren and adolescents with serious conduct problems. J Child Psychol Psychiatry. 2014;55:532–48. 27. Lin D, Chen J, Perrone-Bizzozero N, Bustillo JR, Du Y, Calhoun VD, et al. Char- acterization of cross-tissue genetic–epigenetic effects and their patterns in schizophrenia. Genome Med. 2018;10:13. https://doi.org/10.1186/s13073-018- 0519-4 4. Viding E, McCrory EJ. Understanding the development of psychopathy: progress and challenges. Psychol Med. 2018;48:566–77. 28. Moul C, Dobson-Stone C, Brennan J, Hawes D, Dadds M. An exploration of the serotonin system in antisocial boys with high levels of callous-unemotional traits. PLoS ONE. 2013;8:e56619. https://doi.org/10.1371/journal.pone.0056619 5. Loney BR, Huntenburg A, Counts‐Allan C, Schmeelk KM. A preliminary exam- ination of the intergenerational continuity of maternal psychopathic features. Aggress Behav. 2007;33:14–25. 5. Loney BR, Huntenburg A, Counts‐Allan C, Schmeelk KM. A preliminary exam- ination of the intergenerational continuity of maternal psychopathic features. Aggress Behav. 2007;33:14–25. 29. Raschle NM, Menks WM, Fehlbaum LV, Steppan M, Smaragdi A, Gonzalez- Madruga K, et al. Callous-unemotional traits and brain structure: sex-speciﬁc Translational Psychiatry (2021) 11:492 E. Farrow et al. 8 effects in anterior insula of typically-developing youths. Neuroimage Clin. 2018;17:856–64. https://doi.org/10.1016/j.nicl.2017.12.015 53. Brenet F, Moh M, Funk P, Feierstein E, Viale AJ, Socci ND, et al. DNA methylation of the ﬁrst exon is tightly linked to transcriptional silencing. PLoS ONE. 2011;6: e14524. https://doi.org/10.1371/journal.pone.0014524 30. Freitag CM, Konrad K, Stadler C, De Brito SA, Popma A, Herpertz SC, et al. Conduct disorder in adolescent females: current state of research and study design of the FemNAT-CD consortium. Eur Child Adolesc Psychiatry. 2018;27:1077–93. https:// doi.org/10.1007/s00787-018-1172-6 54. He L, Dai B, Zeng B, Zhang X, Chen B, Yue B, et al. REFERENCES Association of mitochondrial DNA 10398 A/G polymorphism with attention deﬁcit and hyper- activity disorder in Korean children. Gene. 2017;630:8–12. https://doi.org/ 10.1016/j.gene.2017.08.004 38. Aryee MJ, Jaffe AE, Corrada-Bravo H, Ladd-Acosta C, Feinberg AP, Hansen KD, et al. Minﬁ: a ﬂexible and comprehensive Bioconductor package for the analysis of Inﬁnium DNA methylation microarrays. Bioinformatics. 2014;30:1363–9. https:// doi.org/10.1093/bioinformatics/btu049 62. Anitha A, Nakamura K, Thanseem I, Yamada K, Iwayama Y, Toyota T, et al. Brain region-speciﬁc altered expression and association of mitochondria-related genes in autism. Mol Autism. 2012;3:12. https://doi.org/10.1186/2040-392-3-12 39. McCall MN, Almudevar A. Affymetrix GeneChip microarray preprocessing for multivariate analyses. Brief Bioinform. 2012;13:536–46. https://doi.org/10.1093/ bib/bbr072 63. Floris G, Cadeddu R, Bortolato M. Handbook of behavioral neuroscience. vol. 31. Elsevier; 2020. p. 267–78. 40. Fortin JP, Labbe A, Lemire M, Zanke BW, Hudson TJ, Fertig EJ, et al. Functional normalization of 450k methylation array data improves replication in large cancer studies. Genome Biol. 2014;15:503. https://doi.org/10.1186/s13059-014-0503-2 64. Godar SC, Fite PJ, McFarlin KM, Bortolato M. The role of monoamine oxidase A in aggression: current translational developments and future challenges. Prog Neuropsychopharmacol Biol Psychiatry. 2016;69:90–100. https://doi.org/10.1016/ j.pnpbp.2016.01.001 41. Jiao C, Zhang C, Dai R, Xia Y, Wang K, Giase G, et al. Positional effects revealed in Illumina methylation array and the impact on analysis. Epigenomics. 2018;10:643–59. https://doi.org/10.2217/epi-2017-0105 65. Holz N, Boecker R, Buchmann AF, Blomeyer D, Baumeister S, Hohmann S, et al. Evidence for a sex-dependent MAOAx childhood stress interaction in the neural circuitry of aggression. Cereb Cortex. 2016;26:904–14. https://doi.org/10.1093/ cercor/bhu249 42. Wilke M, Holland SK, Altaye M, Gaser C. Template-O-Matic: a toolbox for creating customized pediatric templates. Neuroimage. 2008;41:903–13. https://doi.org/ 10.1016/j.neuroimage.2008.02.056 43. Jaffe AE, Murakami P, Lee H, Leek JT, Fallin MD, Feinberg AP, et al. Bump hunting to identify differentially methylated regions in epigenetic epidemiology studies. Int J Epidemiol. 2012;41:200–9. https://doi.org/10.1093/ije/dyr238 66. Bortolato M, Floris G, Shih JC. From aggression to autism: new perspectives on the behavioral sequelae of monoamine oxidase deﬁciency. J Neural Transm. 2018;125:1589–99. https://doi.org/10.1007/s00702-018-1888-y 44. Genovese CR, Wasserman L. Exceedance control of the false discovery propor- tion. J Am Stat Assoc. 2006;101:1408–17. 67. Kolla NJ, Houle S. Single-photon emission computed tomography and positron emission tomography studies of antisocial personality disorder and aggression: a targeted review. Curr Psychiatry Rep. 2019;21:24. https://doi.org/10.1007/s11920- 019-1011-6 45. Li D, Xie Z, Pape ML, Dye T. An evaluation of statistical methods for DNA methylation microarray data analysis. BMC Bioinforma. 2015;16:217. https://doi. org/10.1186/s12859-015-0641-x 68. REFERENCES Cecil CAM, Zhang Y, Nolte T. Childhood maltreatment and DNA methylation: a systematic review. Neurosci Biobehav Rev. 2020;112:392–409. https://doi.org/ 10.1016/j.neubiorev.2020.02.019 46. Consortium GT. The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013;45:580–5. https://doi.org/10.1038/ng.2653 69. Byrd AL, Manuck SB. MAOA, childhood maltreatment, and antisocial behavior: meta-analysis of a gene-environment interaction. Biol Psychiatry. 2014;75:9–17. 47. Lancaster JL, Woldorff MG, Parsons LM, Liotti M, Freitas CS, Rainey L, et al. Automated Talairach atlas labels for functional brain mapping. Hum Brain Mapp. 2000;10:120–31. https://doi.org/10.1002/1097-0193(200007)10:3<120:: aid-hbm30>3.0.co;2-8 70. Ducci F, Enoch MA, Hodgkinson C, Xu K, Catena M, Robin RW, et al. Interaction between a functional MAOA locus and childhood sexual abuse predicts alco- holism and antisocial personality disorder in adult women. Mol Psychiatry. 2008;13:334–47. https://doi.org/10.1038/sj.mp.4002034 48. Guillemin C, Provençal N, Suderman M, Côté SM, Vitaro F, Hallett M, et al. DNA methylation signature of childhood chronic physical aggression in T cells of both men and women. PLoS ONE. 2014;9:e86822. https://doi.org/10.1371/journal. pone.0086822 71. Bennuri SC, Rose S, Frye RE. Mitochondrial dysfunction is inducible in lympho- blastoid cell lines from children with autism and may involve the TORC1 pathway. Front Psychiatry. 2019;10:269. https://doi.org/10.3389/fpsyt.2019.00269 49. Provençal N, Suderman MJ, Guillemin C, Vitaro F, Côté SM, Hallett M, et al. Association of childhood chronic physical aggression with a DNA methylation signature in adult human T cells. PLoS ONE. 2014;9:e89839. https://doi.org/ 10.1371/journal.pone.0089839 72. Picard M, McEwen BS, Epel ES, Sandi C. An energetic view of stress: focus on mitochondria. Front Neuroendocrinol. 2018;49:72–85. https://doi.org/10.1016/j. yfrne.2018.01.001 50. Hayasaka S, Nichols TE. Combining voxel intensity and cluster extent with per- mutation test framework. Neuroimage. 2004;23:54–63. https://doi.org/10.1016/j. neuroimage.2004.04.035 73. Aon MA, Cortassa S, Juhaszova M, Sollott SJ. Mitochondrial health, the epigen- ome and healthspan. Clin Sci. 2016;130:1285–305. https://doi.org/10.1042/ CS20160002 51. Worsley KJ, Marrett S, Neelin P, Vandal AC, Friston KJ, Evans AC. A uniﬁed sta- tistical approach for determining signiﬁcant signals in images of cerebral acti- vation. Hum Brain Mapp. 1996;4:58–73. https://doi.org/10.1002/(SICI)1097-0193 (1996)4:1<58::AID-HBM4>3.0.CO;2-O 74. Horn SR, Leve LD, Levitt P, Fisher PA. Childhood adversity, mental health, and oxidative stress: a pilot study. PLoS ONE. 2019;14:e0215085. https://doi.org/ 10.1371/journal.pone.0215085 52. Li S, Li Y, Chen B, Zhao J, Yu S, Tang Y, et al. exoRBase: a database of circRNA, lncRNA and mRNA in human blood exosomes. Nucleic Acids Res. 2018;46: D106–D112. 75. Hawes DJ, Brennan J, Dadds MR. Cortisol, callous-unemotional traits, and path- ways to antisocial behavior. Curr Opin Psychiatry. 2009;22:357–62. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41398-021-01609-y. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41398-021-01609-y. 82. Braun PR, Han S, Hing B, Nagahama Y, Gaul LN, Heinzman JT, et al. Genome-wide DNA methylation comparison between live human brain and peripheral tissues within individuals. Transl Psychiatry. 2019;9:47. https://doi.org/10.1038/s41398- 019-0376-y Correspondence and requests for materials should be addressed to Elizabeth Farrow or Stephane A. De Brito. 83. Gutierrez-Arcelus M, Ongen H, Lappalainen T, Montgomery SB, Buil A, Yurovsky A, et al. Tissue-speciﬁc effects of genetic and epigenetic variation on gene regula- tion and splicing. PLoS Genet. 2015;11:e1004958. https://doi.org/10.1371/journal. pgen.1004958 Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. REFERENCES https://doi.org/ 10.1097/YCO.0b013e32832bfa6d Translational Psychiatry (2021) 11:492 E. Farrow et al. 9 AUTHOR CONTRIBUTIONS 76. du Boisgueheneuc F, Levy R, Volle E, Seassau M, Duffau H, Kinkingnehun S, et al. Functions of the left superior frontal gyrus in humans: a lesion study. Brain. 2006;129:3315–28. https://doi.org/10.1093/brain/awl244 The authors conﬁrm that all individuals listed as authors meet authorship criteria. Authors who were also members of the FemNAT-CD Consortium steering committee were involved in the design of the FEMNAT-CD project methods and organisation of data collection. All listed authors reviewed the manuscript and provided critical inputs prior to submission for publication. 77. Martin-Key N, Brown T, Fairchild G. Empathic accuracy in male adolescents with conduct disorder and higher versus lower levels of callous-unemotional traits. J Abnorm Child Psychol. 2017;45:1385–97. https://doi.org/10.1007/ s10802-016-0243-8 78. Rogers JC, De Brito SA. Cortical and subcortical gray matter volume in youths with conduct problems: a meta-analysis. JAMA Psychiatry. 2016;73:64–72. https:// doi.org/10.1001/jamapsychiatry.2015.2423 COMPETING INTERESTS As stated above, all the authors of this paper were involved in the FemNAT-CD project, which was funded by the European Commission under the 7th Framework Health Program with Grant Agreement no. 602407. To the authors’ knowledge, there are no other biomedical ﬁnancial interests or potential competing interests. 79. Yang Y, Raine A, Joshi AA, Joshi S, Chang YT, Schug RA, et al. Frontal information ﬂow and connectivity in psychopathy. Br J Psychiatry. 2012;201:408–9. https:// doi.org/10.1192/bjp.bp.111.107128 80. Hyatt CJ, Haney-Caron E, Stevens MC. Cortical thickness and folding deﬁcits in conduct-disordered adolescents. Biol Psychiatry. 2012;72:207–14. 81. Pechorro P, Ribeiro da Silva D, Andershed H, Rijo D, Abrunhosa Goncalves R. The Youth Psychopathic Traits Inventory: measurement invariance and psychometric properties among Portuguese youths. Int J Environ Res Public Health 2016;13. https://doi.org/10.3390/ijerph13090852 Translational Psychiatry (2021) 11:492 © The Author(s) 2021, corrected publication 2021 ACKNOWLEDGEMENTS This study was conducted as part of the FemNAT-CD consortium (Neurobiology and treatment of adolescent female conduct disorder: The Central Role of Emotion Processing, coordinator Christine M. Freitag). This collaborative project is funded by the European Commission under the 7th Framework Health Program with Grant Agreement no. 602407. We would like to thank all members of the FemNAT-CD consortium for their contributions to the project. We would also like to thank our participants, their families, and the numerous teachers, clinicians, social workers, foster carers and other professionals who gave their time generously to assist with recruitment and participate in the study. Elizabeth Farrow is a Ph.D. student funded by the Biotechnology and Biological Sciences Research Council (BBSRC)’s Midlands Integrative Biosciences Training Partnership (MIBTP). During the writing of the manuscript, Stephane A. De Brito was supported by a short-term Invitational Fellowship from the Japanese Society for the Promotion of Science (JSPS - S19103) and an International Academic Fellowship from the Leverhulme Trust (IAF-2019-032). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. © The Author(s) 2021, corrected publication 2021 Translational Psychiatry (2021) 11:492
W4321603267.txt	https://www.nss-journal.org/articles/nss/pdf/first/nss230008.pdf	fr		Ouvrages en débat	Natures sciences sociétés/Natures sciences sociétés	2,022	cc-by	15,873	Natures Sciences Sociétés © NSS-Dialogues, Hosted by EDP Sciences, 2023 https://doi.org/10.1051/nss/2023008 Natures S ciences S ociétés Disponible en ligne : www.nss-journal.org Repères Events & books Ouvrages en débat La systémique agraire à l’INRA. Histoire d’une dissidence Pierre Cornu Quæ, 2021, 183 p. de la cybernétique des années 1950. Il veut nous montrer que l’émergence du SAD à l’INRA est le marqueur d’une évolution de fond qui bouleverse simultanément les systèmes agricoles et les connaissances nécessaires à leur fonctionnement. « Ce livre est le produit d’une enquête historique menée pendant près de dix ans dans les archives et les mémoires de la recherche agronomique publique française et plus particulièrement au sein du département de recherche “systèmes agraires et développement (SAD)” ». Ces premières lignes décrivent bien l’ouvrage de Pierre Cornu, La systémique agraire à l’INRA. Histoire d’une dissidence, mais avant d’en rendre compte dans cette note, je dois préciser que, en tant que chercheur, j’ai beaucoup croisé la route de l’INRA et celle du SAD1. Cependant, je me suis efforcé de lire ce livre comme un lecteur sensible à l’histoire du monde agricole et à l’histoire de la recherche. Ce travail propose donc une histoire des sciences qui est inséparable de l’histoire de la civilisation et inséparable des institutions qui, depuis l’après Seconde Guerre mondiale, orientent et structurent les collectifs de chercheurs. On dispose d’histoires institutionnelles du CNRS, du CEA ou de l’INRA. Mais l’approche de P. Cornu traverse les échelles en allant des grandes lois de développement agricole au travail des agronomes, en passant par la question des ancrages disciplinaires et celle des structures de l’INRA. Cette analyse multiniveaux est devenue indispensable à toute politique de la recherche. Et ce livre devrait d’abord éclairer ceux qui réﬂéchissent à la capacité des grands organismes scientiﬁques à soutenir une recherche diverse et nécessairement... dissidente. Et de ce point de vue, l’ampleur de l’enquête menée par P. Cornu (Université Lumière-Lyon-II) ne manque pas d’intriguer : pourquoi dix ans d’efforts consacrés à un seul département de recherche ? Et de quoi témoigne l’histoire singulière de ce département ? Le sous-titre du livre répond à ces questions : il s’agit de raconter l’histoire d’une « dissidence » et ce terme ne désigne pas des faits anodins. Il ne s’agit pas de suivre des controverses comme il en existe continuellement dans la science. Il s’agit de comprendre comment est née, puis s’est développée, au sein de l’INRA, une conception divergente de la recherche, de son objet, de ses méthodes et de ses ﬁnalités. Car le département « Systèmes agraires et développement » (SAD), dont le livre retrace la genèse et le parcours, a voulu incarner une alternative à la recherche agronomique dominante. P. Cornu ne prend pas fait et cause pour cette alternative. Il ne lui oppose pas non plus sa propre critique. Il sait aussi que cette dissidence – serait-elle « systémique » – ne se limite pas à l’application à l’activité agricole d’une « théorie des systèmes » venue 1 Comme chercheur en sciences de gestion, j’ai été membre de commissions chargées des promotions à l’INRA, j’ai participé à des missions de prospective et d’évaluation, notamment du SAD. Et mon laboratoire a mené de multiples recherches avec l’INRA et le SAD. La genèse du modernisme agricole et ses premières critiques L’ouvrage décrit d’abord la fondation de l’INRA en 1946 comme l’installation d’un modèle de « mise à niveau technique » de l’agriculture nationale. Les savoirs pratiques et ancestraux doivent céder la place à la recherche scientiﬁque, au machinisme et à l’expérimentation. Le succès productif de ce mouvement est bien connu. En outre, à travers les lunettes d’une histoire industrielle souvent caricaturale, l’agriculture semble suivre, avec retard, la voie du progrès technique et de la grande concentration que l’industrie aurait suivie avant elle. Le mouvement coïncide aussi avec la révolution scientiﬁque de la biologie qui, au cours des années 1960, éclaire les mécanismes du vivant. Reste qu’entre le modernisme scientiste et l’obscurantisme que l’on prête à la tradition agricole, on voit naître un modernisme critique chez ceux qui « ne désespèrent pas de sauver le monde social de l’agriculture familiale, aussi bien en métropole que dans les jeunes nations issues de la décolonisation et tutorées par la recherche agronomique française » (p. 17). En effet, c’est dans le creuset des expériences en Afrique This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC-BY (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, except for commercial purposes, provided the original work is properly cited. 326 Nat. Sci. Soc. francophone et en Amérique latine que se recrutent les pionniers de cette « systémique agraire » qui donnera naissance, plus tard, au département SAD. Un second afﬂuent de ce modernisme critique est issu du nouveau « discours de la méthode » de l’agronomie française. Celle-ci défend une ingénierie scientiﬁque qui ne se contente plus des modèles de laboratoire ou des essais à la parcelle. De même que la conception des systèmes industriels passe par de multiples échelles et suit les complémentarités techniques, la nouvelle agronomie – dont Michel Sébillotte fut l’une des grandes voix – veut replacer l’exploitation agricole dans l’ensemble des grandes interdépendances sociales, économiques et biologiques qui déterminent sa survie. Cette nouvelle agronomie porte en outre la critique d’une économie rurale qui impose des rationalités universelles là où, au contraire, l’agronome doit inventer des modèles de revenus et des solidarités en lien avec les interdépendances indispensables. Enﬁn, dans le domaine de l’élevage et de la sélection génétique s’opère une prise de conscience en faveur des races rustiques. Elle provoque une recherche en rupture avec une zootechnie parfois aveugle à ses propres effets pervers. De ces différents courants, ainsi que de multiples expériences dans l’Aubrac, en Corse et dans les Vosges, émerge un groupe de chercheurs qui partagent une approche subversive de la science des systèmes agraires et « cette nouvelle pensée les éloigne de leurs collègues inscrits dans la logique de “laboratorisation” et de spécialisation des sciences agronomiques, alors triomphante à l’INRA ». Un département de recherche hors norme Le cheminement qui va de ces mouvements critiques et épistémologiques à la création « d’un département de recherche hors norme », le futur SAD, est trop inscrit dans les questionnements et les mécanismes organisationnels de l’INRA pour être ici résumé. Mais pour celui qui s’intéresse à la gestion de la science, ce livre éclaire avec grande ﬁnesse ces mécanismes et témoigne d’une réelle capacité de réﬂexivité collective et d’innovation au sein de l’INRA. Il est vrai qu’il ne s’agissait que d’un petit nombre de chercheurs, mais le signal envoyé par la création du SAD ne manquait pas d’audace. On rêve d’une École d’Administration de la recherche dans laquelle, grâce à ce livre, cette histoire serait étudiée pour réﬂéchir à ce qu’est la « bonne gestion » de la recherche scientiﬁque. Le premier janvier 1980, l’INRA crée le SAD. Reste à le faire vivre, à attirer des chercheurs, à les promouvoir et à développer les partenariats indispensables à la conception holistique, ingénierique et pluridisciplinaire promise par la feuille de route du nouveau département. Les difﬁcultés n’allaient pas manquer. D’abord, et sans surprise, avec le département d’Économie et de sociologie rurales qui défend une recherche plus disciplinaire. Ensuite, et c’est moins attendu, parce que les recherches de terrain sont longues, difﬁciles, et nécessitent une capitalisation des résultats exigeante pour en dégager des enseignements robustes. Pour affronter ces épreuves, le SAD s’engage dans un double effort : construire une doctrine fédératrice, s’ouvrir à des disciplines et à des formes de recherches hors du monde agricole. À cette étape du récit (vers la page 110), on mesure l’effort d’investigation de l’auteur et sa familiarité avec son objet d’études. Il nous permet de suivre pas à pas les explorations conceptuelles et méthodologiques que développent les chercheurs du SAD ainsi que leurs nouvelles thématiques : la qualité des produits, les enjeux environnementaux, les nouvelles ruralités... In ﬁne, les chercheurs du SAD n’ont pas seulement développé une nouvelle agronomie de terrain, ils ont questionné, avec une belle précocité, les rapports entre agriculture et société, agriculture et environnement, alimentation et santé, agriculture et aménagement du territoire, etc... Une reconnaissance paradoxale Hier respectueux des savoirs traditionnels et adeptes d’une science attentive aux interdépendances du vivant et du social, les anciens modernistes critiques allaient ainsi se retrouver aux avant-postes d’une mutation paradigmatique majeure : celle qu’impose la crise devenue ouverte et notoire du modèle agricole dominant. Les travaux du SAD n’auraient pas sufﬁ à provoquer cette rupture. Mais une fois celle-ci avérée, les chercheurs du SAD ont pu bénéﬁcier des « voies de la reconnaissance » que méritent ceux qui par leur dissidence ont su préparer des principes et des méthodes désormais adaptées aux réalités nouvelles. L’auteur résume remarquablement leur itinéraire paradoxal : « Dissidents revendiqués, victimes à l’occasion de moqueries et de rejets, les porteurs de la systémique agraire ont, pour une partie d’entre eux du moins, fait de belles carrières dans la recherche publique, jusque dans sa gouvernance : c’est que la prise de risque et l’inactualité qu’ils avaient assumées avaient rendu possible une requaliﬁcation spectaculaire de l’ingénierie des bioressources au nom du bien commun, au moment où aussi bien la big science que le volontarisme d’État étaient partout battus en brèche » (p. 170). Pourtant, au moment de conclure cette histoire, l’auteur s’inquiète pour l’avenir de cette dissidence « qui n’a modiﬁé qu’à la marge l’intégration de l’agriculture française dans la mondialisation ». Certes, la « crise du système terre » n’est plus contestée – du moins scientiﬁquement – mais dans le monde de la recherche les Nat. Sci. Soc. modèles alternatifs sont toujours vulnérables. En outre, la fusion récente entre INRA et IRSTEA a plongé le SAD dans une nouvelle unité qui reprend l’héritage. Le nouvel INRAE devrait trouver un grand intérêt dans ce travail, car les déﬁs sont immenses et il lui faudra démontrer une inventivité scientiﬁque et institutionnelle au moins égale à celle des dirigeants et des chercheurs qui ont porté le SAD sur les fonts baptismaux. Ce travail montre aussi les apports d’une histoire conjointe des sciences et des institutions de la recherche qui mériterait d’être plus répandue. Elle offre à la fois une bonne compréhension des objets de la science et une connaissance ﬁne des systèmes de gestion qui ont façonné, protégé, ou parfois empêché le développement de ces objets. En terminant cette note, il me faut nuancer l’idée (p. 150) que le SAD apparaissait « trop agricole pour espérer se rattacher à la recherche [...] en sciences de gestion ». Je peux témoigner que les partenariats avec les chercheurs en sciences de gestion n’ont jamais cessé et, comme le signale l’auteur, l’INRA a recruté des chercheurs de cette discipline. Plus important : les principes de la recherche-intervention ainsi que la théorie de la conception innovante, deux acquis des sciences de gestion, ont été réappropriés avec succès par les chercheurs de l’INRAE. Avec le recul, le parcours du SAD s’inscrit dans un aggiornamento scientiﬁque plus large qu’explorent aussi les sciences de gestion contemporaines car il s’agit d’inventer des modèles de l’agir collectif, qui renouvellent autant les sciences de l’ingénieur que les sciences économiques et sociales. Par son travail d’investigation et par sa grande lisibilité, le livre de P. Cornu est un ouvrage remarquable qui, en permettant à cette histoire d’être mieux connue, offre un éclairage précieux sur des mouvements sociétaux et scientiﬁques cruciaux pour notre époque. Armand Hatchuel (Professeur émérite, Mines Paris, Université PSL, membre de l’Académie des technologies) armand.hatchuel@mines-paristech.fr Un monde sans faim. Gouverner la sécurité alimentaire Antoine Bernard de Raymond, Delphine Thivet (Eds) Presses de Sciences Po, 2021, 304 p. L’invasion de l’Ukraine par la Russie le 24 février 2022 a remis à l’agenda la question de la sécurité alimentaire. Ensemble, la Russie et l’Ukraine sont des acteurs agricoles majeurs avec, notamment, près du tiers des exportations mondiales de blé. Le blocage des ports de la mer Noire et la destruction des capacités de production d’une part importante du territoire ukrainien ont affolé les marchés internationaux, renforçant une tendance à la hausse des 327 matières premières agricoles. Le cours du blé a alors atteint 450 € par tonne contre 300 € les jours précédant le conﬂit, un niveau déjà très élevé par rapport au cours historique qui s’établit plutôt autour de 150 € par tonne. Le spectre de la pénurie alimentaire mondiale fut alors invoqué par de nombreux acteurs français pour renouer avec le productivisme et les politiques agro-exportatrices. Pour d’autres, il est au contraire impératif d’augmenter la production dans les pays structurellement déﬁcitaires, notamment en Afrique. Dans cette guerre se joue à nouveau l’opposition entre ceux pour qui la solution à l’insécurité alimentaire réside dans l’augmentation de la production par la technique et le libre-échange et ceux pour qui il faut avant tout renforcer la production locale et s’engager dans des modes de production et de consommation plus sobres, d’où l’impératif de la transformation des régimes alimentaires. Au fond, de tels débats n’ont rien de nouveau. C’est l’un des mérites de ce livre, coordonné par Antoine Bernard de Raymond (INRAE) et de Delphine Thivet (Université de Bordeaux) que de donner des clés de compréhension fondées sur une analyse historique des façons de penser le problème de la faim dans le monde ainsi que ses solutions. La crise de 2008 offre aux auteurs un point de départ idéal pour une enquête approfondie. Malgré une production agricole très élevée, de nombreux pays ont alors connu une crise alimentaire de grande ampleur, provoquant des émeutes qui ont parfois conduit au renversement des régimes politiques. Le « moment 2008 » constitue ainsi un point de bifurcation important. C’est d’une part le retour des questions agricoles et alimentaires au premier plan des préoccupations des acteurs de la gouvernance mondiale, alors qu’elles avaient été plutôt oubliées, comme si la question allait de soi dans une période tournée vers les nouvelles technologies et la dématérialisation. C’est, d’autre part, comme toute période de crise, un moment de déstabilisation des structures dominantes et d’ouverture d’alternatives. Plus de dix ans après le moment 2008, l’un des enjeux de cet ouvrage collectif est de faire un bilan des initiatives et des ouvertures. Aﬁn de démêler les ﬁls d’une histoire complexe et multi-échelles où les forces de changement se confrontent à l’inertie des systèmes en place, les coordinateurs de l’ouvrage ont rassemblé un ensemble d’analyses qui abordent la question de la sécurité alimentaire dans des perspectives très différentes. L’enquête sur les dimensions cognitives (les idéologies et outils de connaissance pour percevoir et agir sur le problème) conduit les auteurs à aborder le rôle des modèles et des statistiques ainsi que les batailles idéologiques sur le « productivisme ». Il en ressort une forte dépendance au sentier. Au Royaume-Uni, le moment 2008 se traduit par la 328 Nat. Sci. Soc. conception et à la mise en œuvre de la politique de la global food security qui considère que la sécurité alimentaire est un problème de déséquilibre global entre offre et demande et que la recherche de solutions passe par l’augmentation de la production grâce à la technologie. C’est ainsi qu’un soutien aux « agri-tech » s’impose comme un moyen permettant d’augmenter les rendements sans impact environnemental négatif. Le motif n’est pas sans rappeler le slogan de la révolution doublement verte lancé par Gordon Conway dès la ﬁn des années 1990. Cette vision de la sécurité mondiale permet d’esquisser une stratégie nationale qui vise à positionner le Royaume-Uni comme leader mondial des technologies et de l’innovation de la durabilité agricole en s’appuyant sur des percées dans des domaines aussi variés que la nutrition, l’amélioration des plantes, le numérique, l’imagerie satellitaire, les capteurs à distance... Ainsi, la global food security intègre les critiques sur les dégâts environnementaux du productivisme pour renouveler les promesses du solutionnisme technologique. L’inertie est aussi liée aux rapports de force entre les acteurs. Si la réforme du Conseil de la sécurité alimentaire est porteuse d’un élan de démocratisation de la gouvernance mondiale, elle se heurte au pouvoir des institutions telles que la Banque mondiale, l’OMC, le G7 ou le G20 en matière de ﬁnancement et d’édiction des règles. Ces institutions inscrivent principalement leurs réactions à la crise dans le cadre du paradigme traditionnel de la sécurité alimentaire. Le livre pousse l’analyse un cran plus loin en analysant le rôle croissant des plateformes multi-acteurs (public-privé) [PMA] depuis les années 2000. S’inscrivant dans une vision du capitalisme vert comme clé du développement durable, ces plateformes ou « alliances » réunissent de grandes entreprises transnationales, des organisations internationales, des fondations, des chercheurs et des ONG. Elles se constituent autour d’objectifs ciblés : biofortiﬁcation des aliments, climate smart agriculture, accès des petits paysans aux intrants... Ces plateformes traduisent la consolidation du pouvoir économique et discursif d’un « bloc historique agro-industriel » qui promeut des actions coordonnées pour résoudre le problème de la faim, mais allant systématiquement dans le sens du productivisme et du libre-échange. L’une des forces de l’ouvrage est de proposer des analyses à différentes échelles, pas seulement au niveau global. Nous l’avons évoqué avec la construction de la global food security au Royaume-Uni. Deux autres cas nationaux particulièrement intéressants sont abordés, celui de l’expérience du programme « faim zéro » au Brésil et celui de la constitutionnalisation du droit à l’alimentation en Inde. Les auteurs rappellent qu’en 2004, le président Lula créait le ministère du Développement social et du Combat contre la faim, conﬁé à José Graziano da Silva, aﬁn de coordonner un large ensemble d’actions conduites dans le cadre du programme « faim zéro » : allocations familiales conditionnelles, distribution de repas, activités génératrices de revenus pour les agriculteurs, connexion entre l’urbain et le rural à travers des systèmes d’approvisionnement locaux... Dans un pays où des excédents agricoles commerciaux considérables côtoient la sous-nutrition endémique d’une partie importante de la population, cette politique volontariste est venue renforcer le soutien à la petite agriculture familiale. Cette politique très efﬁcace de lutte contre la faim, fortement soutenue par la base sociale de Lula et ses proximités avec le Mouvement des « sans-terre », n’a pas pour autant contraint les stratégies de l’agrobusiness. Le compromis fut ainsi trouvé avec une approche duale des politiques agricoles. L’élection de Jair Bolonaro en 2018 mit ﬁn à cette expérience et enclencha une désastreuse marche arrière avec la remise en cause systématique du droit des plus faibles. À contre-courant de l’analyse de cette dialectique qui montre que, encore une fois, la promesse d’un monde d’après n’est pas tenue, François Collart Dutilleul plaide dans la postface à l’ouvrage pour un exercice effectif du droit à l’alimentation. Il faut reprendre à nouveaux frais les réﬂexions sur l’exception agricole et alimentaire et sur ses implications dans trois grands domaines : les règles internationales qui structurent la mondialisation (en remettant en cause la priorité du principe de libre commerce), la nécessaire souveraineté des États sur les produits agricoles (en reconnaissant que ce ne sont pas des marchandises comme les autres) et les formes d’une démocratie alimentaire qui imposent une information des consommateurs aﬁn qu’ils puissent exercer librement leurs droits et leurs responsabilités. Ajoutons à cela que le droit à l’alimentation concerne en premier lieu ceux qui n’y ont pas accès, notamment les petits paysans, ce qui pose la question du juste prix, incompatible avec un prix de marché unique, et celle de l’accès à des techniques appropriées. Autant de considérations qui, sans être totalement nouvelles, n’en sont pas moins d’une actualité brûlante. Le paradigme dominant de la sécurité alimentaire fondé sur le couple libre-échange et productivisme est confronté au renforcement de la critique qui se fonde aujourd’hui sur de nouveaux fondamentaux : changement climatique, démographie et transition nutritionnelle, épuisement des ressources naturelles, effondrement de la biodiversité... La critique se situe désormais au niveau des systèmes agricoles et alimentaires et conduit à interroger l’évolution des régimes alimentaires. Elle s’appuie sur les recherches qui mettent notamment en évidence les problèmes de maladies chroniques et d’empreinte environnementale résultant d’une alimentation riche en protéines animales. Elle nourrit des politiques qui, à différentes échelles, Nat. Sci. Soc. visent à accompagner les nécessaires transitions des systèmes agricoles et alimentaires. Dans les débats liés à la guerre en Ukraine, il ne faudrait pas que le spectre de la pénurie alimentaire conduise à ne répondre aux inquiétudes légitimes qu’avec des solutions périmées. Pierre-Benoît Joly (INRAE, Centre de recherche Occitanie-Toulouse, Castanet-Tolosan, France) pierre-benoit.joly@inrae.fr Philosophie d’une écologie anticapitaliste. Pour un nouveau modèle de gestion écologique Alexandre Rambaud, Jacques Richard Hermann, 2021, 324 p. Comme le titre de leur ouvrage l’indique, Alexandre Rambaud (AgroPariTech) et Jacques Richard (Université Paris Dauphine) nous invitent à explorer la thématique écologique sous un jour nouveau en croisant les approches philosophique, juridique, économique, historique et comptable aﬁn de proposer un modèle de gestion qui rompt avec le modèle capitaliste moderne au sein duquel nous évoluons. En particulier, les deux auteurs fournissent une théorie mais aussi une pratique de l’action écologique reposant sur une remise en cause radicale de ce qu’ils considèrent être au cœur du système capitaliste, à savoir le droit des sociétés et, peut-être plus encore, le système comptable actuel. En s’éloignant quelque peu des approches classiques en matière de gestion écologique qui privilégient une explication aux atteintes à notre environnement naturel par le développement d’une modernité souvent circonscrite à sa dimension mécaniste cartésienne, A. Rambaud et J. Richard proposent une vision qui place au centre de ces atteintes l’inﬂuence du capitalisme moderne et de ce qui, selon eux, en est sa marque fondamentale : la comptabilité. Ils nous proposent ainsi une exploration historique, philosophie, juridique, économique, ﬁnancière et comptable de l’apparition du capitalisme moderne et de son évolution permettant d’éclairer d’un jour plus complexe la question de l’impact écologique de l’activité humaine, ce qui les amène à davantage parler de Capitalocène que d’Anthropocène. Ils mènent cette exploration en structurant leur ouvrage en quatre grandes parties et nous proposent en premier lieu une histoire de la modernité et du capitalisme moderne en revenant successivement sur : – La notion de modernité et ses différentes conceptions (conception très générale qui qualiﬁe de moderne la période s’ouvrant avec la Renaissance et qui voit apparaître des transformations de grande ampleur 329 affectant tout à la fois les structures sociales, les idées, les modes de vie et la politique, ou approche mettant davantage l’accent sur la distinction entre l’hommesujet et son environnement-objet) ; – L’histoire de l’apparition de cette modernité qui, selon les auteurs, doit être datée non pas à l’époque de la Renaissance mais plutôt à l’époque du haut Moyen Âge, marquée par l’émergence d’un type de gestion des entreprises inédit et caractéristique du capitalisme moderne ; – La naissance de ce capitalisme moderne en Italie et sa traduction comptable permise par le développement de la comptabilité en partie double consacrant la dichotomie entre le sujet (capitaliste) positionné du côté du passif (des dettes) et des objets soumis à la volonté de ces sujets (du côté des actifs), et reposant sur une vision « prudente » de préservation d’un seul type de capital (le capital ﬁnancier) ; – Le dépassement de ce capitalisme moderne au proﬁt d’un capitalisme ﬁnancier avec le développement et la quasi-généralisation du cadre conceptuel des normes comptables internationales (IFRS) aboutissant à un déplacement de la conservation du capital de la ﬁrme vers le patrimoine des capitalistes euxmêmes et ses corollaires : « l’effacement comptable » de la ﬁrme, la remise en cause du concept d’amortissement systématique et l’affaiblissement du principe de prudence. Ils reviennent dans un deuxième temps sur les raisons expliquant qu’il ait fallu attendre la période du haut Moyen Âge pour voir se développer le capitalisme moderne. Ils sont ainsi amenés à réexaminer les concepts d’intérêt et de capital développés au moment de l’Antiquité ainsi que la séparation entre les sphères commerciales et ﬁnancières, d’une part, et l’activité agricole, d’autre part, cette dernière caractérisant le monde grec ancien et obéissant à des principes économiques et moraux encore très éloignés de ce que sera le capitalisme moderne. Ils expliquent ensuite que les Romains, bien que plus avancés que les Grecs sur les questions économiques et juridiques, ne seront pour autant pas davantage les promoteurs d’un tel type de capitalisme dans la mesure où leur vision de l’entreprise reste ancrée dans un cadre agricole et familial qui ne nécessite pas une séparation sujet/objet. C’est ﬁnalement l’émergence de nouvelles conceptions du capital et de l’intérêt qui autorisera l’apparition du capitalisme moderne. En effet, les deux auteurs montrent qu’à la ﬁn du Moyen Âge va s’opérer un glissement de la notion de capital. Ce dernier n’est plus stérile – conformément à la doctrine chrétienne héritée des conceptions de l’Antiquité qui condamne la reproduction de l’argent (donc l’intérêt) –, mais devient au contraire de l’argent productif, c’est-à-dire autorisant la production d’un 330 Nat. Sci. Soc. proﬁt. Parallèlement à cette révolution va s’afﬁrmer un autre rapport au temps (le temps n’appartient plus désormais seulement à Dieu mais les hommes peuvent d’une certaine façon le maîtriser) débouchant sur la découverte de la technique de l’actualisation qui deviendra quelques siècles plus tard l’outil de référence des capitalistes ﬁnanciers. La troisième partie de leur ouvrage glisse de l’approche historique caractérisant les deux parties précédentes vers une analyse des visions philosophiques et économiques se donnant pour objectif de contrer les effets nocifs du développement du capitalisme moderne sur la nature. Sont ainsi successivement examinés les développements économiques autour des concepts de soutenabilité (promue par les économistes forestiers des XVIe et XVIIe siècles), d’externalités (développées par l’approche néoclassique) et de valeur d’existence qui ne parviennent pas, selon les auteurs, à se démarquer d’une vision utilitariste de la nature. Les différentes approches philosophiques de l’éthique environnementale (pathocentrisme, biocentrisme, écocentrisme) sont ensuite présentées et analysées au prisme de leur rupture avec cette vision. Si les auteurs reconnaissent que ces approches élargissent la notion de sujet aux nonhumains, ils montrent que la séparation entre sujet et objet subsiste néanmoins et qu’elles n’offrent guère de solution concrète aux méfaits du capitalisme. Pour cette raison, ils proposent dans la dernière partie de leur ouvrage de rompre de façon radicale avec les conceptions précédentes en développant une vision écologique mobilisant une ontologie relationnelle forte du monde dans laquelle les relations entre les sujets importent ﬁnalement davantage que les sujets eux-mêmes. La dernière partie de l’ouvrage propose ainsi une piste concrète d’évolution du mode de gouvernance des entreprises vers une cogestion écologique associant les représentants de toutes les formes de capital et non plus seulement les détenteurs du capital ﬁnancier. Cette cogestion prendrait appui sur le développement et la généralisation d’un nouveau modèle comptable : le modèle CARE/TDL (comprehensive accounting in respect of ecology/triple depreciation line) développé par les deux auteurs et fondé sur une éthique de préservation étendue aux trois types de capital (ﬁnancier, naturel et humain). Dans cette conception, un nouveau bilan apparaît avec la présence de trois capitaux-dettes qui sont à préserver (par la technique de l’amortissement) séparément, conformément à une vision de soutenabilité forte. Les auteurs expliquent que le développement de ce nouveau modèle comptable repose sur une participation des représentants des trois types de capital à la déﬁnition de ce qui doit être préservé et débouche sur une modiﬁcation des concepts de coûts complets et de juste prix (intégrant le coût de préservation des trois types de capital) autorisant la détermination d’un « proﬁt écologique ». Les apports de cet ouvrage sont multiples : Il permet tout d’abord d’éclairer d’un jour nouveau la question de la responsabilité de l’Homme dans l’atteinte à son environnement. En effet, si les auteurs ne remettent nullement en question l’impact de l’Homme en tant que tel, ils insistent sur le fait que certains agents, au premier rang desquels les capitalistes, sont plus responsables que d’autres dans la dégradation de la nature. Cette position, si elle peut, bien évidemment, ne pas être partagée par tous, a au moins le mérite de recontextualiser le débat autour du rôle de la modernité dans le développement d’actions aboutissant à la dégradation de l’environnement. La modernité est ainsi replacée dans le cadre de l’activité économique et de sa régulation par un mode particulier : le capitalisme moderne. Il offre ensuite une analyse pluridisciplinaire de la gestion écologique qui fait cohabiter les dimensions économique, juridique, philosophique, religieuse et ﬁnancière. Il permet ainsi de montrer en ﬁligrane que le traitement du problème environnemental ne pourra se trouver que dans l’abandon d’une vision étroite et la participation de tous. Il replace le fait comptable au cœur des problématiques non seulement économiques et ﬁnancières mais également politiques et sociétales. Ce faisant, il montre que loin d’être un outil purement technique, la comptabilité doit être vue comme relevant d’une cosmologie particulière constituant par conséquent un support non neutre de toute action, ce qui peut constituer tout à la fois une menace pour la préservation du capital humain et naturel (dans le cas de la comptabilité en partie double classique) mais également une formidable opportunité dès lors qu’on accepte que l’information comptable traduise la nécessité de prendre en compte la préservation de toutes les formes de capital. Il propose enﬁn une piste concrète d’évolution des formes de gouvernance qui repose sur une participation équitable des représentants de l’ensemble des formes de capital et le développement d’une nouvelle méthode comptable. Finalement, avec cet ouvrage les deux auteurs nous offrent l’opportunité salutaire de redonner un nouveau soufﬂe au débat de la question écologique reposant sur une réﬂexion riche et nourrie à propos de nos modes de régulation économiques et sociaux et à même de déboucher sur des pistes qui, à défaut d’être partagées par tous, permettraient néanmoins à coup sûr de redonner Nat. Sci. Soc. un nouvel élan à la question de la gouvernance en général et à la gouvernance responsable en particulier. Jean-Luc Petitjean (Université Reims Champagne-Ardenne, EA REGARDS, Reims, France) jl.petitjean@univ-reims.fr La fabrique d’un droit climatique au service de la trajectoire « 1.5 » Christel Cournil (Ed.) A. Pedone, 2021, 510 p. La fabrique d’un droit climatique au service de la trajectoire « 1.5 » est le produit d’un travail collectif d’une vingtaine d’enseignants-chercheurs et chercheurs, quasiment tous juristes, à l’instar de Christel Cournil, professeur de droit à Sciences Po Toulouse, qui en a assuré la direction. Cette dernière avait également organisé en novembre 2020 le colloque dont cet ouvrage constitue les actes. Sur le plan formel, l’ouvrage comprend 21 chapitres, chacun correspondant à une contribution. La présence d’une table des matières avec le plan détaillé de chaque chapitre est d’ailleurs très utile pour s’y repérer. Deux grandes parties le structurent ; la première est consacrée aux instruments juridiques, tandis que la seconde traite des acteurs, qu’ils soient producteurs ou sujets de ce droit climatique. Quant à la première, il s’agit de « Construire des instruments juridiques pour l’horizon 2050 », ce qui implique, selon les auteurs, de « polycentrer le droit » en mobilisant des instruments multi-échelles, mais aussi de le « décloisonner » en « climatisant » différents secteurs du droit relativement indépendants les uns des autres (agriculture, énergie, etc.) et enﬁn de « repenser » les instruments transversaux, comme le droit des investissements ou celui des accords commerciaux. Le plan de la seconde partie, consacrée aux acteurs qui vont devoir « tenir » le réchauffement à 1.5, surprend un peu. On y trouve une première catégorie constituée des « acteurs privés » que sont les entreprises et les salariés. Vient ensuite un bloc assez disparate qui comprend les « individus », étudiés à travers la Convention citoyenne pour le climat, les ONG et les collectivités territoriales et leurs groupements, acteurs centraux d’un droit climatique territorialisé. Un dernier titre traite de manière tout à fait bienvenue, et en soulignant leur singularité, des acteurs que sont l’expert et les juges. L’absence des États parmi les acteurs peut étonner, mais on comprend rapidement qu’ils sont omniprésents, principalement dans la première partie consacrée aux instruments juridiques, dans la mesure où ils en sont les principaux concepteurs et destinataires. 331 Enﬁn, et c’est là l’un de ses intérêts, l’ouvrage se clôt par une liste de 60 « Propositions de pistes prospectives », reprises des différents chapitres et ordonnées dans cette partie ﬁnale. Les propositions peuvent être très précises et ciblées comme l’instauration d’un rapport de compatibilité entre différents instruments de planiﬁcation territoriale (SCoT, PLU, PCAET, etc.), ou une refonte de l’action de groupe en matière environnementale pour favoriser l’accès au juge des individus. Certaines de ces propositions sont opérationnelles au sens où le gouvernement français ou des parlementaires pourraient les intégrer dans notre droit relativement facilement. D’autres ne sont évidemment pas à la seule portée des élus nationaux, comme, dans le domaine des accords commerciaux, l’abandon du concept de similarité des marchandises et l’incorporation dans les classements douaniers des émissions de GES de l’analyse du cycle de vie (ACV) des marchandises ou la réécriture des standards de protection des investissements aﬁn que les investisseurs étrangers ne bénéﬁcient plus d’une protection exorbitante qui leur permet de remettre en cause les politiques climatiques des États hôtes. Dans leur grande majorité, ces propositions, comme l’ensemble de l’ouvrage, nous semblent devoir intéresser et être accessibles au lectorat visé, à savoir les universitaires, juristes ou non, ainsi que des décideurs publics – on pense ici notamment aux collectivités territoriales – ou des praticiens du droit, par exemple des juristes d’ONG, mais aussi toute personne désireuse « d’apprendre davantage sur les aspects juridiques de la gouvernance climatique ». Un autre intérêt, et non des moindres, de cet ouvrage collectif, est de ne pas se limiter à une acception étroite, et classique, du « droit climatique » (à savoir le droit découlant du régime international du climat lui-même, composé de plusieurs accords internationaux, du droit de l’Union européenne et du droit français, qui réglemente tant l’atténuation que l’adaptation au changement climatique) pour ouvrir sur d’autres domaines et outils du droit qui jouent un rôle absolument déterminant dans notre possibilité de limiter le réchauffement climatique. La plongée dans cette fabrique d’un droit climatique, nous montre, pour reprendre les mots de C. Cournil dans son introduction, « un puzzle que l’on assemble progressivement » et dont on peut mieux aussi repérer les pièces manquantes. De plus, l’analyse de la fabrique de ce droit se fait en quelque sorte de l’intérieur, par un collectif de juristes qui nous guide dans des domaines juridiques très divers et souvent très techniques (comme le droit des bâtiments ou les règles relatives aux informations environnementales des entreprises) et nous montre, en les étayant scientiﬁquement, les modiﬁcations nécessaires pour 332 Nat. Sci. Soc. rester dans la trajectoire « 1.5 ». C’est d’ailleurs en considérant ce point de vue que le choix d’un ouvrage mono-disciplinaire (parmi les 23 contributeurs, on compte 21 juristes et seulement une sociologue et un agronome) nous semble pertinent. Car l’objectif, qualiﬁé de modeste dans l’introduction, est en réalité très ambitieux, qui consiste à proposer un éclairage sur les instruments juridiques existants et potentiels pour construire un droit climatique à la hauteur de l’urgence écologique. Mais surtout la palette est très large. Tantôt, les auteurs nous offrent une vision micro du droit (un bâtiment qui émet plus de GES que la réglementation ne le prévoit sera-t-il conforme à sa destination au sens du code civil ou entraînera-t-il la responsabilité de plein droit de son constructeur ?), nous montrent des « petites victoires » aux résultats « relativement ténus » à propos des actions juridiques des ONG, mais qui peuvent faire du bruit comme « caisses de résonance ». Tantôt, c’est une vision macro, systémique, du droit qui prévaut et permet d’identiﬁer les obstacles structurels de notre ordre juridique, mais aussi sa capacité à modiﬁer nos sociétés. Et nous en aurons besoin. Car la question est cruciale aujourd’hui de savoir comment mettre le monde sur une trajectoire de 1.5°C de réchauffement, sachant que ce seuil, en tant qu’objectif, ne sera vraisemblablement pas respecté, comme cela est très clairement expliqué dès le premier chapitre. C’est donc toute la question de la fonction et de la force du droit qui est posée. Compte tenu de l’urgence et de l’ampleur des changements à opérer, il faut faire appel aux « forces imaginantes du droit », et à sa capacité à servir de boussole dans un monde en quête de repères, comme la grande juriste Mireille DelmasMarty, récemment disparue, nous l’a appris. Mais aussi, comme le montre l’analyse des actions juridiques des ONG, il faut penser le droit comme une arme, dans ses principes, ces « garde-fous » et ses procédures, notamment le recours au juge. Dans le chapitre conclusif de l’ouvrage, l’autrice souligne l’ampleur de la tâche (repenser notre rapport à la nature et aux autres) et le fait que les changements à impulser sont de véritables changements de fond. Et ce qui la frappe, c’est la conﬁance dans les possibilités de l’outil juridique, comme élément central des politiques publiques, qui émane des différentes contributions. Cette conﬁance – ou parfois cet espoir – s’appuie pourtant sur une vision pragmatique du droit. Plusieurs contributions montrent en effet comment certains pans du droit, qu’il s’agisse du droit agricole ou du droit du commerce international, ont intégré tardivement et souvent marginalement les contraintes environnementales, continuent parfois à rester muets sur la question climatique ou encore, comme le droit des investissements, sont construits et commandés par une « rationalité strictement économique ». Ce dernier est d’ailleurs très parlant, qui protège les investisseurs étrangers en leur donnant la main sur les réglementations environnementales des États hôtes. Le droit apparaît ici comme un obstacle aux changements, à la nécessaire transition climatique et écologique. Mais montrer le rôle et les mécaniques du droit dans la construction et le fonctionnement d’un ordre économique et ﬁnancier aux effets terriblement destructeurs, c’est aussi montrer la force et la puissance dont il est porteur. À condition que nous réussissions à les inverser pour les mettre au service de la trajectoire « 1.5 ». Isabelle Doussan (INRAE, UMR Gredeg, Valbonne, France) isabelle.doussan@inrae.fr Sustainable futures. An agenda for action Raphael Kaplinsky Polity, 2021, 264 p. Avec ce remarquable Sustainable futures. An agenda for action, Raphael Kaplinsky propose un ouvrage qui intéressera celles et ceux qui souhaitent penser l’entrée dans la formation d’un nouvel agenda de transition de durabilité (imbriquant donc l’économique, le social et l’environnement) en s’appuyant sur une relecture historique du capitalisme d’après-guerre et sur les travaux de l’économie évolutionnaire augmentés d’une fréquentation des études du développement et des études d’innovation. L’auteur est une des ﬁgures emblématiques du Science Policy Research Unit (SPRU) – et plus particulièrement de l’Institut des études sur le développement (Institute of Development Studies), de l’Université du Sussex, dont il est aujourd’hui un professeur émérite et néanmoins actif. Il doit ainsi à Chris Freeman, reconnu comme son mentor, une pensée interdisciplinaire sur le temps long de ce qu’il appelle les paradigmes technico-économiques. Comme l’indiquent les remerciements et sa biographie, il doit aussi beaucoup à sa rencontre avec Robin Murray qui lui a apporté un regard plus politique sur la question de la redistribution du pouvoir et de l’inclusivité ainsi que sur les fondements de la durabilité environnementale et de l’économie circulaire. L’autre « dette » est celle qu’il situe chez Carlotta Perez, autre « disciple » de Freeman, avec le renforcement de l’idée qu’un monde plus durable ne peut se penser sans l’accomplissement d’un nouveau paradigme technico-économique fondé sur les nouvelles techniques d’information et de communication, et dans leurs usages des effets politiques qu’elles engendrent. Mais une autre dimension doit être précisée pour situer cet ouvrage dans la fondation de ce travail d’intellectuel poussé à l’action. C’est celle que R. Kaplinsky évoque quand il souligne l’importance du travail de recherche-action collectif, comme avec ce programme de fondation d’une politique industrielle post-apartheid en Afrique du Sud. Nat. Sci. Soc. En effet, ce livre témoigne aussi d’une longue expérience des tentatives et des accomplissements tâtonnants pour faire passer des visions, des idées, des méthodes et des façons de faire de politique publique d’innovation et de développement au sein même des appareils de gouvernement, des milieux industriels mais aussi des syndicats et des acteurs de la société civile. Cet ouvrage rend donc justice à cette accumulation d’expériences dans certains pays du monde, tout autant qu’il cherche à justiﬁer et à fonder, de façon réﬂéchie, non pas une troisième voie mais une bifurcation dans le capitalisme néolibéral contemporain (chapitre 1 : « A fork in the road »). Au moment d’en achever l’écriture, la crise de la COVID-19 a apporté à l’auteur un supplément à sa démonstration initialement construite autour de l’analyse de la crise ﬁnancière de 2008. La guerre totale déclenchée contre l’Ukraine, et ses effets directs comme ceux attendus, vient ajouter incidemment matière à penser le rapprochement de la première et de la dernière phrase de cet ouvrage : « We live in perilous times. » ... « However, at this crucial turning point in history, we need a greater sense of urgency ‘To choose not to act is in fact to choose’ ». Ce livre propose ainsi une relecture de ces paradigmes technico-économiques successifs mais « emboîtés » qui ont organisé conjointement la production industrielle et les formes sociales et économiques spéciﬁques au régime de régulation du capitalisme d’après-guerre. L’auteur pose ainsi un diagnostic quasi clinique de la création et de la ﬁn de l’économie de production de masse (chapitre 2 : « The rise and fall of the mass production economy »), puis des effets des mécanismes de la rationalisation néolibérale sur la société avec l’accumulation conjointe de différentes formes de dettes et de tensions et conﬂits sociaux portés par des inégalités de revenus et de distribution de la richesse au niveau national comme international (chapitre 3 : « The bumpy ride to social decay »). Il passe ensuite à l’analyse de l’effondrement du développement durable, réquisitoire un peu rapide qui rejoint des travaux en sciences sociales sur les ﬁgures de l’anthropocène (chapitre 4 : « The collapse of environmental sustainability »). Cette relecture en termes de temps long ayant été établie, voire démontrée (car l’ouvrage n’est pas avare de données et d’analyse de séries économiques), R. Kaplinsky expose ensuite dans le chapitre 5 (« Mass production runs out of steam ») le pivot de sa réﬂexion : d’une part pour rappeler ce que signiﬁe cette notion de paradigme technico-économique puis pour en nommer les cinq principaux, qui s’accompagnent d’un récapitulatif des travaux du SPRU sur une cinquantaine d’années que l’on trouvera en note 1 du chapitre 5 (un très intéressant programme de lecture qui s’étend de Chris 333 Freeman jusqu’à la période plus récente d’émergence des Transitions Studies à la Johan Schot, et Frank Geels) : la force motrice de l’eau, la machine à vapeur (et à charbon...), l’âge industriel du fer et de l’électricité, la civilisation du pétrole, du transport et de la chimie, et enﬁn ce nouveau paradigme des technologies d’information et de communication (TIC). Cette caractérisation s’accompagne de la restitution d’un fait majeur : tous ces paradigmes ont pris ﬁn dans des crises ﬁnancières, signiﬁant également des crises pour les institutions de régulation, jusqu’aux moments plus actuels de néopopulismes, de conﬂits locaux et de guerres régionales de civilisation que vivent aujourd’hui un grand nombre de peuples. L’expérience du Thatchérisme – et peut-être de son prolongement avec le Brexit – pèse ici sur l’auteur dans son travail de décorticage de ces phénomènes de crises dans la façon dont ils affectent autant l’économique que le social. Le lecteur français connaisseur des théories de la régulation trouvera ici le trait un peu rapide mais il s’agit bien pour l’auteur de ne pas en rester à la conjugaison des techniques, des marchés et du capital mais bien de comprendre les formes sociales et organisationnelles qui rendent possible autant que critiquable cette « conjugaison », sans oublier le moment anthropocène dans lequel nous sommes rentrés jusqu’à la dernière tonne de charbon et goutte de pétrole... Mais il ne s’agit pas d’un plaidoyer à charge à la fois marxiste et luddite, car R. Kaplinsky conserve – sans s’étendre – une forme de lucidité sur les bienfaits et les infrastructures apportés par la modernité dans cet âge d’or des démocraties libérales, position relevant d’un évolutionnisme néo-schumpterien vigilant et critique, restant pourtant optimiste, même s’il n’est « réservé » qu’à une certaine partie de l’humanité. Le chapitre sur le paradigme des NTIC qui suit (Chapitre 6 : « Information and communication technologies: the motor of the new paradgim ») ouvre la suite de l’histoire en train de se faire. Il peut paraître risqué d’écrire sur la confrontation de ce paradigme naissant suite à la seconde cybernétique des années 1970 à l’histoire longue du capitalisme. Ce n’est certainement pas le chapitre le plus fort de cet ouvrage, mais il fait le point sur les grandes tendances de l’essor des TIC : data et serveurs, logiciels et plateformes, réseaux sociaux et objets connectés, big data et machines apprenantes. S’ensuit une liste d’effets en cours ou attendus de ce nouveau paradigme technico-économique pouvant provoquer le déclin gouverné de la production de masse : nouveaux chemins de productivité, relocalisation et décentralisation de la production, customisation de la rencontre des besoins et des offres, partage et durabilité de la consommation, sobriété énergétique et capacitation de la société civile. L’auteur n’est pour autant pas dupe de cet élan que d’aucuns pourront trouver par trop généreux, 334 Nat. Sci. Soc. car il atteste en connaître la face sombre. Le chapitre 7 (« Transformative change in practice ») est dédié à des études de cas illustratives : le rôle des smartphones dans différentes activités économiques et sociales décentralisées ; le pilotage de la consommation électrique pour viser le retrait des grands barrages hydroélectriques ; l’agriculture dite de précision et les robots agricoles. Il est heureux que cette vision des jours heureux potentiels soit contrebalancée par l’analyse des inégalités économiques et de redistribution ainsi que par la prise en compte des effets de la pandémie, car le livre frise parfois une forme redoutée de techno-positivisme béat. Une fois les bonnes raisons établies du pourquoi agir que fondent les sept premiers chapitres, les trois suivants adoptent la perspective annoncée d’une mise à l’agenda pour l’action, et cela à l’aune de l’expérience de l’intellectuel embarqué. Il est construit autour de deux questions essentielles auxquelles l’auteur tente d’apporter des réponses. Que faire (Chapitre 8 : « What to be done? ») ? Ce à quoi l’auteur répond en substance : un smart green deal associant durabilité forte et déploiement du nouveau paradigme technico-économique de l’information and communications technology (ICT) ! Qui doit mettre en œuvre et piloter la transformation (Chapitre 10 : « Who will do it? Making change happen ») ? Ce à quoi l’auteur répond : des entrepreneurs, des acteurs de la société civile et des coalitions éclairées pour piloter et organiser les transformations souhaitables avec un État régulateur et visionnaire aux commandes. On pourrait reprocher à l’auteur un certain angélisme en raison de cette vision techno-utopique du déploiement du paradigme des TIC tout comme de son progressisme social-démocrate et écologique. On pourra aussi reprocher à cet ouvrage de ne pas livrer un programme bien organisé de prescriptions et de mesures. Ce n’est pourtant pas la meilleure façon de comprendre ce qui se joue du point de vue de l’intellectuel engagé dans la fabrique des politiques de développement : parvenir à tisser un continuum – robuste sur le plan intellectuel et praticable sur le plan de l’action – entre l’exercice de la pensée critique et réﬂexive dans le temps long et celui de la formation d’un agenda visionnaire, directionnel et socialement portée pour asseoir ce smart green deal. Pris sous cet angle, l’ouvrage est éclairant car toujours bien construit dans son argumentation et ses « démonstrations », pétillant parfois quand il rend compte de l’expérience vécue, et toujours très honnête dans la façon d’établir un rapport à l’histoire à travers ce prisme constamment maintenu des paradigmes technico-économiques. Marc Barbier (INRAE, UMR LISIS, Marne-la-Vallée, France) marc.barbier@inrae.fr Le puritanisme vert. Aux origines de l’écologisme Philippe Pelletier Le Pommier, 2021, 428 p. Philippe Pelletier, géographe qui a notamment travaillé sur le Japon, s’est également intéressé dans ses recherches à des courants de pensée comme l’anarchisme ou encore l’écologisme. Dans un ouvrage paru en janvier 20212, il proposait d’ailleurs son analyse des liens historiques et idéologiques existants selon lui entre anarchie et écologie. Dans son dernier essai, Le puritanisme vert. Aux origines de l’écologisme, paru en octobre 2021, l’auteur développe une démarche similaire, mais il essaye ici de montrer les liens entre l’écologisme et le religieux à travers le puritanisme protestant. P. Pelletier entend s’inscrire dans la continuité des travaux de Max Weber dans L’éthique protestante et l’esprit du capitalisme, où le sociologue défend l’idée selon laquelle la Réforme protestante et le système de valeurs que cela a engendré ont joué un rôle central dans l’émergence du capitalisme. Pour l’auteur, il convient d’élargir la relation entre protestantisme et capitalisme à une troisième dimension : « l’environnementalisme ». P. Pelletier donne le ton dès le début de l’introduction : il souhaite aller à rebours de l’image d’une écologie « hédoniste, affranchie et libertaire » et démontrer au contraire la dimension austère, voire autoritaire de cette dernière. P. Pelletier expose ainsi rapidement l’image qu’il se fait de l’écologie : une écologie dite punitive caractérisée par des injonctions, voire des interdits (ne pas prendre l’avion, ne pas manger de viande, etc.). On peut ainsi lire page 6 : « Ces caractères austères et autoritaires renouent en réalité – et c’est la thèse de ce livre – avec les prémices de l’écologie et de l’écologisme qui s’ancrent dans un puritanisme originel ». Toujours dans l’introduction, l’auteur postule que « le fait religieux constitue, avec la nature, l’un des grands enjeux de ce siècle ». Le fait de mettre le religieux et la nature sur le même plan prête à débat. L’afﬁrmation, qui mériterait d’être précisée, invite du moins à interroger les liens entre les deux. L’ouvrage comporte une bibliographie bien fournie référencée en notes de bas de page et il n’y a pas d’iconographie. L’essai se compose de huit chapitres de taille à peu près équivalente. Ces chapitres suivent globalement une logique chronologique sans que cela soit au cœur de leur structuration. La démonstration passe en effet par une exploration historique et géographique des pensées écologistes analysées au prisme du religieux, ou, pour être plus précis, du puritanisme considéré comme un ensemble de valeurs et de comportements issus de la foi monothéiste protestante, qui se sont diffusés à travers le 2 Pelletier P., 2021. Noir & vert. Anarchie et écologie, une histoire croisée, Paris, Le Cavalier Bleu. Nat. Sci. Soc. monde à partir de la culture WASP (white anglo-saxon protestant) des États-Unis. Certains chapitres portent sur une période particulière ou encore sur une région du monde spéciﬁque, et tous alimentent la thèse principale du livre : celle du puritanisme vert. Le propos reste cohérent dans l’ensemble, l’auteur mobilise des références et des sources variées, cependant les exemples et les arguments développés sont inégalement convaincants. Nous reviendrons sur ce point un peu plus loin. Pour appuyer ses analyses, P. Pelletier s’attarde avant tout sur les parcours et les proﬁls de plusieurs ﬁgures inﬂuentes de l’écologisme à partir du milieu du XIXe siècle. Il revient ainsi sur des personnalités historiques telles que John Muir, Aldo Leopold, Henry David Thoreau ou encore George Perkins Marsh, Ernst Haeckel, Jacques Ellul et Rachel Carson. Le tour d’horizon qu’il dresse des penseurs et penseuses de l’écologie est très intéressant et assez diversiﬁé. Son prisme de lecture étant le puritanisme vert, on constate toutefois une sélection essentiellement tournée vers le monde anglosaxon (notamment Amérique du Nord), mais aussi européen (France, Suisse, Allemagne), avec quelques rares exemples pris ailleurs (Japon). Si ce choix peut paraître évident au regard de l’histoire de l’écologisme et de ses formes contemporaines, on peut toutefois regretter que les critères de sélection des idées et des personnes censées rendre compte de cette histoire n’aient pas été davantage explicités dans le livre. Une des idées fortes de l’essai défend que l’écologie savante est née et s’est développée dans un contexte puritain, et que cela a fortement imprégné les modèles théoriques, les interprétations et jusqu’aux concepts de la discipline (biome, écosystème, etc.). Le chapitre 2, par exemple, cherche à montrer comment l’écologie savante est initialement imprégnée par le monisme haeckelien. Ernst Haeckel est un des précurseurs de l’écologie et c’est d’ailleurs lui qui a inventé le terme. P. Pelletier dépeint Haeckel comme un puritain inventeur d’une nouvelle religion : le monisme. Il écrit à ce propos p. 111 : « Le monisme haeckelien est un déterminisme vitaliste, où l’élite s’en sort tautologiquement parce qu’elle est l’élite, c’est-à-dire supérieure. Nulle place au libre arbitre ». Au ﬁl des chapitres, P. Pelletier développe des analyses intéressantes et foisonnantes à propos de thématiques diverses dont on ne donnera ici que quelques exemples. Il réﬂéchit dans le chapitre 3 sur les nuances de l’environnementalisme entre conservationnisme et préservationnisme et les associe à la fracture idéologique entre écologie superﬁcielle et écologie profonde. P. Pelletier propose aussi une critique forte et argumentée des dimensions moins glorieuses de certains courants écologistes qui ont entretenu des liens intimes avec l’eugénisme, le racisme, les totalitarismes ou encore le malthusianisme radical et l’anti-immigration. L’auteur s’attache également à analyser l’émergence de 335 l’écologie politique qui constitue un réel tournant dans la période après 1945 ; puis la maturation que connaît l’écologisme en Europe entre les années 1950 et 1970. P. Pelletier propose ainsi un essai riche en exemples et en idées stimulantes tout en développant une lecture originale de l’histoire de l’écologisme. Certains éléments viennent toutefois desservir son propos, au point parfois de remettre en cause la pertinence même de sa démarche. L’une des principales faiblesses dans l’argumentation de l’auteur nous semble être sa vision parfois trop orientée des choses. P. Pelletier privilégie les éléments qui vont dans le sens de sa thèse sans accorder assez de crédit aux éléments qui vont plutôt à l’encontre. Et pourtant l’auteur lui-même pointe cet écueil potentiel de sa démarche lorsqu’il mentionne dans l’introduction le double risque de l’anachronisme et de la surinterprétation. Il semble malheureusement que cette auto-mise en garde n’ait pas été sufﬁsante. L’auteur apporte bien sûr de la nuance par endroits, mais la lecture laisse souvent l’impression qu’il prend une certaine distance avec les données dont il dispose. Il s’applique à mettre en exergue la moindre chose qui relie la pensée ou la vie de telle personne avec le puritanisme, sans que le lien direct sur la manière dont le puritanisme a réellement inﬂuencé la pensée écologiste de l’auteur ou l’autrice en question ne soit véritablement démontré (on peut penser à la partie consacrée à Bernard Charbonneau dans le chapitre 4). L’inﬂuence du puritanisme sur les premiers penseurs de l’écologie au XIXe siècle apparaît plus nettement étant donné le poids du contexte religieux, mais cela n’est plus vraiment le cas en ce qui concerne le XXe siècle et en particulier les dernières décennies. On pourra aussi reprocher à l’auteur d’avoir une vision trop unitaire de l’écologisme, mais aussi, et surtout, d’alimenter l’ambiguïté entre écologisme et écologie. L’écologisme semble d’ailleurs être de longue date un cheval de bataille pour P. Pelletier : dès 1993, il publie un essai3 dans lequel il dénonce les travers de l’écologisme en tant qu’idéologie (qui peut prendre également la forme d’un engagement politique) mais qu’il distingue alors de l’écologie dite savante (c’est-à-dire en tant que science qui recourt à des méthodes pour produire de la connaissance). Or, c’est bien cette distinction, pourtant pertinente, que P. Pelletier tend à effacer dans Le puritanisme vert. L’auteur associe l’écologie à la religion, en même temps qu’il l’associe à la science, ce qui a pour effet de mettre science et religion sur un même niveau. Cette grande confusion est entretenue tout au long du livre, mais elle est perceptible dès l’introduction, lorsqu’à la page 10, P. Pelletier dénonce les discours écologistes de mise en garde vis-à-vis des catastrophes à venir. Ces derniers relèvent selon lui du prophétisme, un terme très 3 Pelletier P., 1993. L’imposture écologiste, Montpellier, Reclus. 336 Nat. Sci. Soc. connoté religieusement, ce qui lui permet au passage de consolider le parallèle qu’il établit entre l’écologisme et le puritanisme. Cette critique de prophétisme écologiste n’est pas totalement infondée, mais l’auteur a tort de la généraliser au point de discréditer les discours de mise en garde de la communauté scientiﬁque notamment vis-àvis du réchauffement climatique. Aucun scientiﬁque a priori honnête ne prétendra jouer les Cassandre en livrant des prophéties, cependant ce n’est pas de cela qu’il s’agit ici, et P. Pelletier passe à côté des travaux scientiﬁques qui ont permis d’établir des modèles prévisionnels d’évolution, tout particulièrement en ce qui concerne le climat via les travaux du GIEC (Groupe d’experts intergouvernemental sur l’évolution du climat). À plusieurs reprises dans son essai, P. Pelletier remet en cause les rapports du GIEC sans démontrer quelles sont les erreurs qu’ils contiendraient tant au niveau des méthodes de collecte des données que dans les analyses et les conclusions. L’auteur laisse même entendre que les experts du GIEC servent les instances et les intérêts des puissants et qu’il faut donc faire preuve de méﬁance vis-à-vis des rapports et des alarmes répétées concernant les crises écologiques en cours. Il s’agit d’une afﬁrmation sans preuve, ce qui revient à souscrire à une forme de climatoscepticisme. Il nous paraît ainsi dangereux et irresponsable de la part d’un universitaire comme P. Pelletier de décrédibiliser un travail collectif, scientiﬁque aussi rigoureux que celui du GIEC (qui, faut-il le rappeler, rassemble les résultats de milliers de publications scientiﬁques et techniques sur les questions climatiques à travers de nombreuses institutions dans le monde). Certes, l’appel que lance P. Pelletier à être critique et à douter est sain, mais le faire sans méthode et discernement risque de conduire au complotisme et au refus du réel (du moins au refus d’interpréter les données les plus ﬁables et récentes qui permettent d’appréhender le réel). La lecture du livre laisse donc une impression très mitigée entre, d’une part, l’intérêt réel d’un travail historique bien mené et agréable à lire, et, d’autre part, l’expression d’opinions tout à fait discutables et contestables mais qui ne sont malheureusement pas assumées telles quelles (en particulier dans le dernier chapitre où l’auteur semble rendre des comptes plutôt que d’argumenter). En conclusion, l’apport principal de cet essai est sans doute d’inviter les lecteurs et lectrices à approfondir la question des liens historiques et idéologiques entre le puritanisme et l’écologie, et ainsi à questionner les courants de pensée de l’écologie contemporaine à l’aune du religieux. Hugo Mazzero (Université Bordeaux Montaigne, UMR Passages, Pessac, France) hugo.mazzero@u-bordeaux-montaigne.fr La société des eaux cachées du Saïss. Ethnographie d’un basculement hydrotechnique Rhoda Fofack-Garcia Peter lang, 2021, 248 p. Une anthropologie chez les hydrologues. Penser le dialogue interdisciplinaire Jeanne Riaux Quæ, 2022, 165 p. Il était difﬁcile de ne pas proposer un compte rendu parallèle de ces ouvrages portant sur le domaine de l’irrigation par eau souterraine au Maghreb et parus à quelques mois d’intervalle. Tous deux renvoient dès le titre à l’ethnologie, ou plus précisément à l’ethnographie pour celui (issu d’une thèse de sociologie) de Rhoda Fofack-Garcia et à l’anthropologie pour celui (issu d’une HDR dans cette discipline) de Jeanne Riaux. Tous deux sont le fruit de recherches menées par des femmes dans un monde masculin. Tous deux défendent une approche qui prend les objets hydrauliques comme point de départ pour analyser des réseaux sociotechniques pour l’une, et construire une démarche sociohydrologique pour l’autre. Tous deux s’intéressent aux savoirs associés aux eaux souterraines, et tous deux se lisent avec plaisir. Toutefois, les objectifs sont différents, et les cadres analytiques également. Il en résulte des ouvrages qui nous offrent une complémentarité de regards sur ces « sociétés des eaux cachées », sur la multiplicité d’acteurs, techniques et savoirs impliqués, et sur la complexité des situations étudiées. L’ouvrage de R. Fofack-Garcia porte sur les dynamiques sociales (et techniques) qui permettent d’expliquer le basculement, dans la plaine du Saïss au Maroc, d’une agriculture pluviale vers une agriculture dorénavant dépendante de l’irrigation par eau souterraine (91 % des terres irriguées le sont par pompage dans les nappes). Ce basculement s’accompagne d’un deuxième basculement, à partir des années 2000, du puits vers les forages profonds. L’auteure propose un positionnement original pour analyser l’utilisation de ces ressources souterraines, dépendantes de techniques d’exhaure individuelles : montrer qu’il existe un « commun socio-territorial » (p. 41), un « socle social qui guide, oriente, explique et éclaire les mécanismes et processus sociaux qui se déploient autour de l’exploitation des eaux souterraines » (p. 20). Elle s’appuie pour cela sur la théorie de l’acteurréseau et la sociologie de l’environnement, en prenant pour terrain d’étude plusieurs villages et centres urbains de la vallée. Elle y interroge agriculteurs, institutions publiques et acteurs privés (garagistes, revendeurs de moteurs et pompes, etc.), les divers proﬁls d’acteurs étant systématiquement décrits ce qui est bien la signature Nat. Sci. Soc. d’un travail de sociologue ! L’auteure nous donne ainsi à comprendre les interactions qui se tissent autour de l’exploitation des eaux souterraines, de même que les représentations et discours qui soutiennent les pratiques. Le lecteur est très bien guidé dans l’évolution de la démonstration. De nombreuses illustrations (photos, schémas, graphes, cartes et tableaux) viennent compléter et agrémenter la lecture. Le premier chapitre retrace l’historique de l’utilisation de l’eau souterraine en la combinant avec l’histoire agraire de la plaine du Saïss depuis l’époque coloniale, période à l’origine d’une transformation de l’agriculture pluviale extensive vers une agriculture plus intensive et commerciale. L’analyse nous présente les registres de pensée et les savoirs relatifs à deux mondes de l’eau souterraine, non exclusifs l’un de l’autre : le « monde de la rareté » associé aux puits, et le « monde de l’abondance » lié aux forages dans la nappe profonde et dont le développement implique des savoirs, techniques et acteurs qui relèvent de l’international (diverses techniques importées, foreurs provenant de Syrie). Que le basculement ait eu lieu dans les années 2000 s’explique par le contexte : levée de boucliers juridiques et administratifs, instauration de nouveaux programmes, implication d’acteurs différents, le tout soutenant l’installation de forages tout en disqualiﬁant l’utilisation des puits. En raison de la place centrale des techniques et de leur évolution dans la compréhension des dynamiques à l’œuvre, le deuxième chapitre se penche sur l’étude des moteurs et des pompes, tandis que le troisième se concentre sur le monde des moteurs de Renault 25, les plus utilisés – après transformation – pour les pompages dans le Saïss. Tous ces objets sont étudiés comme des éléments de réseaux qui connectent les acteurs et créent entre eux des interdépendances. Ils participent à la coévolution de la technique et du social dans des processus sociotechniques en perpétuel réagencement (p. 155). R. Fofack-Garcia décortique les composants, la structure, la répartition sociospatiale des trois réseaux sociotechniques (mécanique, électrique et solaire) qu’elle distingue, et explicite les interactions entre objets et acteurs. À ces eaux cachées est ainsi associée toute une « société minière » qui partage un même objectif : répondre à la demande en eau d’irrigation. Cette société réagit à la baisse du niveau des aquifères en cherchant des solutions techniques pour puiser plus profondément, solutions qui permettent en outre la survie de l’économie locale (production agricole et métiers artisanaux). Ces deux chapitres démontrent qu’un commun se forge au ﬁl du temps dans cette société minière. Ce commun relève des partages d’expériences et de valeurs, des savoirs et savoir-faire au sein des réseaux sociotechniques, et non d’une gestion coordonnée (comme c’est le cas en irrigation communautaire) puisqu’agriculteurs et/ou artisans ne se coordonnent pas pour gérer les ressources en eaux souterraines. 337 La conclusion ouvre l’analyse sur la place du capitalisme dans l’évolution des modes d’exploitation des eaux souterraines. Une brève référence aux diverses phases que connaissent les sociétés minières ailleurs dans le monde est donnée, mais une mise en perspective plus conséquente aurait pu être faite. On regrettera certaines imprécisions (par exemple l’absence de déﬁnition d’une motopompe alors que l’auteure fait l’effort de distinguer pompes et moteurs dans son analyse) dans un ouvrage détaillant les techniques et leurs processus opératoires. Quelles sont par ailleurs les connaissances sur le fonctionnement des aquifères pour décider de la profondeur de la pompe ? Comment sont-elles déﬁnies ? Car à Haj Kaddour (graphique 9) l’eau est à 70 m de profondeur aujourd’hui – ce qui permet sans doute de la dénommer nappe profonde – mais dans les années 1970, elle était proche de la surface. L’arrivée du projet d’irrigation via un barrage (p. 148) contribuera sans doute à la recharge des aquifères et pourrait ainsi être analysée comme un soutien à l’utilisation des eaux souterraines. Ces critiques sont toutefois minimes et renvoient à la nécessité d’une inter- ou pluridisciplinarité pour aborder ces questions (cf. ciaprès). Elles ne doivent pas masquer les qualités indéniables de l’ouvrage, notamment ses apports sur la connaissance d’une société des eaux cachées et un angle d’approche original. En effet, après avoir lu ce livre, on ne peut plus regarder un moteur ou un puits de la même façon. On ne peut plus les considérer comme de « simples » techniques, indépendantes de tout lien social ou choix politique, qui ne participent pas à structurer la société. R. Fofack-Garcia réussit très bien à convaincre de cette coconstruction techniques-société autour de l’irrigation par eau souterraine, de l’importance des liens sociaux qui se nouent et évoluent à travers les objets, et de la légitimité méthodologique d’aborder une société par ses techniques. L’entrée par la matérialité et le terrain se retrouve chez Jeanne Riaux. Son livre porte moins sur l’analyse de cette coconstruction techniques-société, ici dans la région de Kairouan – même si les hypothèses et résultats de la recherche sont présentés –, que sur le processus de coconstruction d’une interdisciplinarité sur l’eau, impliquant hydrologues, hydrogéologues (parfois agronomes et pédologues) et elle-même comme anthropologue. L’auteure, bien connue de NSS, retrace l’évolution de sa pratique de recherche dans une réﬂexivité exemplaire et révèle l’arrière-cuisine de la fabrique de cette interdisciplinarité « grand écart4 » qui est loin d’être aisée. 4 S’inspirant de l’expression interdisciplinarité « étendue » ou « élargie » de Marcel Jollivet et Jean-Marie Legay, 2005. « Canevas pour une réﬂexion sur une interdisciplinarité entre sciences de la nature et sciences sociales », Natures Sciences Sociétés, 13, 2, 184-188, https://doi.org/10.1051/nss:2005030. 338 Nat. Sci. Soc. L’expérience restituée, même si elle revêt un caractère autobiographique, dépasse largement l’exemple individuel. En effet, elle est très bien replacée dans les limites des divers cadres théoriques utilisés ; les controverses scientiﬁques des recherches sur l’eau sont efﬁcacement synthétisées et l’auteure explicite le décentrement méthodologique et épistémologique, individuel et collectif, nécessaire aux chercheurs pratiquant l’interdisciplinarité. En outre, cette rétrospective est relatée avec beaucoup de sincérité et d’humour, incluant sentiments et difﬁcultés rencontrés. Dès l’introduction et tout au long de l’ouvrage, J. Riaux afﬁrme sa posture d’anthropologue qui prend pour terrain le monde des hydrologues. Elle travaille avec eux – l’interdisciplinarité se construisant au moins en binôme –, mais aussi « chez5 » les hydrologues : elle analyse comment leurs savoirs se construisent, évoluent, se diffusent (via les services techniques des administrations hydrauliques auxquels l’auteure a eu plus facilement accès en accompagnant ses collègues hydrologues) et donnent aux hydrologues des positions de pouvoir. Dans le chapitre premier, J. Riaux expose les différentes façons de concevoir la recherche sur l’eau et les diverses familles épistémiques qui en découlent. Elle montre aussi comment ses propres connaissances et cadres initiaux d’analyse (issus de l’anthropologie et du courant de la Gestion sociale de l’eau6) ne lui permettaient pas d’aborder de façon fructueuse un travail interdisciplinaire avec les hydrologues. Elle a alors ouvert son champ théorique à la socioanthropologie du développement. Dans le deuxième chapitre, l’auteure montre comment elle a inﬂuencé la construction de l’interdisciplinarité hydrosociologique au sein de l’UMR G-Eau, qui pratiquait déjà la pluridisciplinarité sur l’eau. Les conditions de réussite de l’interdisciplinarité sont avancées. Les autres chapitres se réfèrent à la pratique même de cette interdisciplinarité : le rôle central du terrain pour construire les interactions entre chercheurs (chapitre 3), la fabrique collective de la forme narrative choisie pour restituer les résultats (chapitre 4) et l’interrogation collective des savoirs produits et de la place du chercheur vis-à-vis de la société (chapitre 5). 5 Le titre de l’ouvrage peut surprendre. Pourquoi ne pas parler d’une anthropologue chez les hydrologues (comme Gentelle P., 2003. Traces d’eau. Un géographe chez les archéologues, Paris, Belin) ? En proposant un terme plus généralisant, l’auteure défend un type d’anthropologie, inductif, réﬂexif et de terrain, qui prend pour objet d’étude le savoir des hydrologues, ce qui a permis la coconstruction d’une recherche interdisciplinaire. 6 La GSE était à la fois un groupe de travail né au début des années 1990 à Montpellier autour de deux agronomes, JeanLuc Sabatier (CIRAD) et Thierry Ruf (ORSTOM, maintenant IRD), et une formation dispensée au CNEARC, devenu Institut des régions chaudes. J. Riaux prône une interdisciplinarité « disciplinée » (c’est-à-dire ancrée dans chaque discipline) et montre qu’elle est le produit de négociations, de points de tension qui obligent chacun des collaborateurs à être explicite dans ses attentes de résultats de la part de l’autre discipline, sur ses a priori (sur l’autre), ses propres hypothèses de travail, objectifs de recherche et méthodes d’investigation. Il faut se battre (p. 40) pour faire reconnaître sa discipline (en l’occurrence l’ethnologie, dont elle est la seule représentante) aux yeux des autres. Cela passe notamment par une explicitation des éléments d’argumentation et de démonstration car les critères de validation d’un résultat ne sont pas les mêmes selon les disciplines. Il faut également un travail de traduction des notions, voire des termes issus du langage courant employés par chaque discipline, car sinon une incompréhension et un décalage de perception de l’objet de recherche peuvent venir entraver le bon déroulement de la collaboration. L’un des exemples développés est celui « d’usages » de l’eau. La réﬂexivité, individuelle et collective, est indispensable pour construire l’interdisciplinarité. L’écriture occupe une place essentiellement dans le processus. Notons au passage le rôle de soutien qu’a joué la revue NSS, donnant reconnaissance et légitimité aux auteurs pour poursuivre leur travail interdisciplinaire. Une autre difﬁculté pour le dialogue interdisciplinaire est la démarche inductive employée, car il n’est pas possible de délimiter clairement l’objet de recherche en amont du terrain, ni d’avoir des questionnements a priori, ou des outils et des approches prédéﬁnis. Tout est à construire, avec l’inattendu qui occupe une place non négligeable dans le processus. Cela complique la transmission de la méthode et empêche une quelconque généralisation. Cette démarche est d’ailleurs incompatible avec la socio-hydrology, en vogue chez certains hydrologues. Ce courant reste centré sur la modélisation mathématique, les dimensions sociales étant simpliﬁées et quantiﬁées, les singularités du terrain gommées (ce qui va à l’encontre de la posture de notre auteure et de ses collègues, p. 57 et p. 145). D’un point de vue ontologique, la sociohydrologie proposée (malgré l’absence de trait d’union) reste toutefois (comme la socio-hydrology) dans un parallélisme du naturel et du social et on ne retrouve pas les imbrications de ces domaines présentes dans le cycle hydrosocial (du courant de la political ecology) ou dans le dépassement de la dichotomie proposé par des anthropologues tel Philippe Descola. On n’y voit pas non plus l’imbrication du technique et du social chère à des auteurs inﬂuents (par exemple Geneviève Bédoucha) de la formation Gestion sociale de l’eau. On peut être étonné de lire que cette formation initiale ne permettait pas de sortir de l’échelle locale (de l’anthropologie) puisque l’un de ses instigateurs, Thierry Nat. Sci. Soc. Ruf, a de longue date changé d’échelle d’analyse par rapport à celle des réseaux d’irrigation (certes via la géographie) pour montrer les relations entre amont et aval d’un bassin versant. Mais peut-être interagir avec des disciplines épistémologiquement éloignées facilite-til certains décentrements ? On regrettera que la notion de ressource ne soit pas discutée, alors qu’elle apparaît dans le triptyque « ressources, sociétés, techniques » partagé avec les hydrologues. Mais il est vrai que cette notion est davantage discutée en géographie qu’en anthropologie. Ces quelques critiques ne font qu’aller dans le sens de l’auteure : l’interdisciplinarité est indispensable pour étudier l’eau (et ses « usages ») et peut se décliner de multiples façons. Dans les deux ouvrages, la société locale a pleinement conscience de la baisse progressive du niveau des aquifères et de sa participation à la raréfaction de la ressource. Et l’État, avec ses politiques publiques au rôle ambigu, évite le débat et participe pleinement à promouvoir l’utilisation des eaux profondes. L’« environnement [est ainsi] un instrument de légitimation d’une politique anti-environnementale » (Fofack-Garcia, p. 146). Olivia Aubriot (CNRS, UPR CEH, Aubervilliers, France) olivia.aubriot@cnrs.fr L’action paysagère. Construire la controverse Hervé Davodeau Quæ, 2021, 168 p. À partir d’un travail inédit réalisé pour l’obtention d’une Habilitation à diriger des recherches, Hervé Davodeau, maître de conférences en géographie au pôle Paysage de l’Institut Agro d’Angers livre une contribution ambitieuse sur l’action paysagère et ses controverses. Son envoi, en guise de préface, donne le ton : loin « d’un esthétisme paysager aujourd’hui daté », la description d’une photo de trois adolescents jouant sur les rives d’un ﬂeuve soulève toute une série de questions débordant le cadre même du paysage donné à voir. Le propos est déjà là et l’hypothèse posée : celle d’une connivence entre une action ludique ici un jeu de pingpong – et le lieu où elle prend place, un bord de la Loire. Ce « portrait de Loire ordinaire », loin de la patrimonialisation, loin des images d’Épinal du « ﬂeuve sauvage » et de la « Loire des châteaux » exempliﬁe ce qui cherche à être théorisé dans cet ouvrage. Mettant volontairement de côté la dimension esthétique et la posture de contemplation qu’il suscite, l’auteur aborde le paysage « en tant qu’action » et plus encore à partir de l’idée d’action paysagère au singulier, ce qui renvoie à son principe et non à la multiplicité de ses déclinaisons possibles. 339 Le chercheur-géographe qu’est H. Davodeau travaille la question du paysage depuis plus de 10 ans et produit là une réﬂexion où le processus même de l’action paysagère se situe aux croisements de trois ensembles : les politiques publiques paysagères, les pratiques des paysagistes et ce qu’il nomme « les conﬂictualités paysagères » issues de la société civile. Pour que la rencontre entre ces trois registres puisse avoir lieu, chacun doit s’ouvrir aux deux autres. Pour cela, il est nécessaire que les politiques publiques paysagères se déploient au-delà de la seule patrimonialisation (chapitre 1) ; que les pratiques paysagistes quittent le domaine des parcs et des jardins (chapitre 2) ; que la « Logique Nimby » (Not in my backyard, en français pas près de chez moi) ne soit pas ou plus celle qui règle les regards citoyens sur la transformation de leur lieu de vie (chapitre 3). Ces trois ensembles constituent le corpus de l’action paysagère (1re partie) et chacun des trois chapitres cerne « le seuil au-delà duquel l’action paysagère peut s’appréhender » (p. 57). À partir d’expériences et d’analyses de situations, les conditions dans lesquelles il devrait être possible d’assurer ces dépassements y sont développées. C’est la première pierre d’un édiﬁce élaborant si ce n’est une théorie tout au moins le cadre théorique de l’action paysagère. La deuxième partie, « L’action paysagère : le cadre théorique », s’attelle à cette élaboration, en la situant d’emblée à l’intérieur « d’une théorie principalement géographique de la spatialité » entendue selon les termes de Michel Lussault comme « action spatiale des opérateurs sociaux » (p. 72). Mais cela ne va pas de soi dès lors que les deux concepts ainsi rapprochés – celui d’action et celui de paysage – semblent à première vue antinomiques. Extraire les dimensions contemplatives, voire passives du paysage, tout comme sa réduction à la morphologie physique, est dès lors un préalable à toute pensée de l’action paysagère. Cette deuxième partie est développée en trois chapitres dont le troisième, « Agir avec le paysage », est le plus conséquent. Le premier, « Modéliser l’action paysagère », tente de « saisir l’articulation entre action et représentation » (p. 73) de deux manières : en explorant les représentations dans l’action d’une part, en cherchant à catégoriser l’action paysagère elle-même d’autre part. Sur l’un et l’autre de ces deux versants, l’analyse de plusieurs travaux de chercheurs permet d’identiﬁer leurs apports et leurs limites. Ces dernières aident à préciser vers où aller pour parvenir à construire l’idée d’action paysagère au croisement des trois sphères de l’action publique, professionnelle et citoyenne. Mais aujourd’hui, l’excès de contraintes dans chacune de ces trois sphères fait que « l’action paysagère est sujette à des codiﬁcations successives qui permettent le passage à l’action mais produisent inévitablement aussi en retour une normalisation des paysages à laquelle s’oppose paradoxalement l’action paysagère » (p. 83). 340 Nat. Sci. Soc. Pour tenter de sortir de ces effets, les deux chapitres suivants visent à conceptualiser le paysage opérant pour l’action (chapitre 5), tout comme l’action opérante avec le paysage (chapitre 6). Sans détour, c’est la compréhension relationnelle du paysage développée par Augustin Berque qui s’avère la plus pertinente. Cela suppose de distinguer le paysage de l’environnement ou « monde extérieur objectif » tout comme du « monde intérieur subjectif », pour le construire dans un mouvement d’aller-retour de l’un à l’autre. Ni seulement extérieur (la matérialité), ni seulement intérieur à l’observateur, le paysage est « saisi de façon dynamique par le double mouvement de l’action paysagère qui déploie le paysage en nous et hors de nous » (p. 87). Appréhender ainsi l’espace ou l’environnement « en tant que paysage » révèle les dimensions esthétiques et sensibles de ce dernier. Un pas de plus est nécessaire pour en faire un concept opératoire pour l’action. C’est ce que se propose d’explorer le chapitre six, « Agir avec le paysage », en situant le paysage dans la triade acteur-agent-actant, et en afﬁrmant l’action paysagère comme une des modalités de l’action spatiale telle que pensée en géographie sociale. Le paysage est du côté de l’actant, car dénué de la passivité de l’agent et de l’intentionnalité de l’acteur. Cette deuxième partie, centrale, pose le cadre théorique de l’action paysagère où la relation de l’homme sensible à son environnement qu’est le paysage (Berque) active le principe d’agentivité – discutée avec Bruno Latour et Tim Ingold –, dès lors qu’il a « le pouvoir de déﬁnir des règles d’organisation des pensées, des conduites, des actions dans la société et vis-à-vis du monde environnant », comme l’énonce Jean-Marc Besse. Mais cette agentivité du « paysage-actant » peut être en excès si l’on se laisse prendre par l’idée d’harmonie et d’éthique qui serait intrinsèque au paysage. Pour s’en garder, la prudence du chercheur en appelle au doute : « douter de cette éthique du paysage, tout en prenant au sérieux l’utopie des paysagistes et la foi qui les anime pour porter, citant Denis Delbaere, “l’évangélisme du projet de paysage” » (p. 115). Mieux vaut s’en tenir à l’« impureté dans l’action paysagère et paysagiste » et chercher à comprendre la ou les controverses qu’elle suscite. Ces controverses sont explorées en trois temps, mettant en œuvre le doute méthodique du chercheur. Il s’agit de « Mettre à l’épreuve l’action paysagère » (chapitre 7), de souligner « Les ambiguïtés du principe d’agentivité appliqué au paysage » (chapitre 8) jusqu’au point de se demander : « Faut-il abandonner le paysage ? » (chapitre 9) en occupant cette fois-ci le parti des « adversaires du paysage » pour parvenir à entendre et à prendre au sérieux leurs objections (p. 141). En fait, l’objectif de cette dernière partie est bien de « Construire la controverse de l’action paysagère » en reconnaissant les divergences de points de vue y compris à l’intérieur de la formation et de la profession de paysagiste aﬁn de pouvoir « en discuter et en faire quelque chose », au-delà des conﬂits ; et aussi construire la controverse autour de l’action paysagère pour parvenir à la faire reconnaître en tant que telle, distincte de l’action environnementale ou de l’action spatiale. Sur quatre pages, un tableau présente les arguments en alternant les « Il faut abandonner le paysage car... » avec les « Il faut défendre le paysage car... ». Les thématiques portent sur les rapports du paysage avec l’environnement, l’attractivité des territoires, le bienêtre, l’aménagement du territoire, les démarches participatives, les politiques publiques, les métiers et enﬁn son efﬁcacité ou non dans les conﬂits d’aménagements. On ne sera pas étonné de la prise de position de l’auteur en cette ﬁn de partie en faveur d’une éthique de l’action paysagère, mais pas n’importe laquelle : celle répondant aux catégories du bien, du juste et du beau à partir desquelles déployer des valeurs environnementales de soin, de ménagement (le bien), des valeurs démocratiques selon une éthique de l’espace public et de la participation (le juste), et aussi une esthétique (le beau) mais portée par une éthique relationnelle fondée sur « une pleine présence polysensorielle au paysage » (p. 146). Au ﬁl de cet essai, étayé par de nombreuses analyses de situations et de travaux d’auteurs de références, et dans une conclusion résumant le mouvement de pensée à l’œuvre dans cet ouvrage (le corpus, le cadre théorique, la controverse de l’action paysagère), la visée déconstructiviste de la recherche s’afﬁrme et avec elle la position de l’auteur. Ni désabusé ni enthousiaste, ni croyant ni athée, ni conservateur ni progressiste, mais en un lieu en tension entre toutes ces tendances portées par le paysage : « Ce travail de déconstruction n’est pas une entreprise de démolition, car ma conviction profonde n’est pas que nous devons refermer la parenthèse du paysage au motif qu’il en existe de mauvais usages, mais de décrire et de faire avec l’action paysagère telle qu’elle est, sous tension donc controversée » (p. 154). Si l’on peut être dérouté à la lecture de cet ouvrage du fait d’un parcours sinueux, d’un argumentaire ﬂottant dès lors que les conclusions des analyses conduites sont immédiatement déconstruites, il n’en ﬁnit pas moins par mener le lecteur en un lieu qui est loin d’être inintéressant : il conﬁrme le caractère insaisissable, labile, fuyant de la notion de paysage et de l’action qu’il suscite, et de ce fait sujet à tous les mésusages, des plus délétères lorsqu’il justiﬁe les attitudes de repli sur soi, de rejet de l’autre et des actions paysagères d’exaltation nationale comme ce fut le cas sous l’Allemagne nazie... aux plus enthousiastes lorsqu’il est porteur et est porté par un désintéressement fondamental, par la possibilité d’un « partage du Nat. Sci. Soc. sensible », d’un soin mis en œuvre dans les transformations des territoires. Dès lors qu’un homme averti en vaut deux, agir avec et par le paysage, c’est savoir se tenir en équilibre, sur le tranchant d’un couteau ou plus géographiquement sur la ligne d’une crête aiguisée, en déployant une qualité d’écoute avertie des controverses, guidée par une éthique personnelle capable de prendre soin et des gens et des choses, au croisement des politiques publiques, de compétences professionnelles et de conﬂits citoyens. Cet ouvrage y invite. Catherine Franceschi-Zaharia (ENSAPLV, UR AMP-ENSAPLV-HESAM, Paris, France) catherine.zaharia@paris-lavillette.archi.fr L’homme et sa maison Pierre Deffontaines Parenthèses, 2021, 301 p. Quelle chance d’avoir à faire pour NSS le compte rendu de cette réédition du livre de Pierre Deffontaines presque 50 ans après sa publication originale. Je n’avais pas pu exprimer au sein de la revue mon enthousiasme pour cet ouvrage – un exemplaire jauni par le temps de L’homme et sa maison publiée en 1972 par Gallimard dans la collection NRF – découvert à la ﬁn des années 1990 alors que je m’appliquais à approfondir le concept de « mode d’habiter » pour moi indissociablement lié à celui de « maison ». Étonnée de n’avoir jamais entendu parler de P. Deffontaines ni dans les cours que je suivais à l’Institut de géographie, ni, par la suite, dans mes relations au sein de l’équipe rurale du LA 142 parisien ou dans les échanges avec mes collègues ruralistes ou non, je brûlais de le faire connaître au-delà même de mon intérêt pour une géographie qui mettait l’« habitant » et l’« habiter un milieu » au cœur de la géographie. J’admirais cette écriture aussi précise que celle des célèbres anthropo-ethnographes cités dans l’ouvrage et tellement ambitieuse dans sa volonté de révéler à la fois la diversité des cultures de la nature des peuples du monde et les caractères « ordinaires » et communs aux humains quand il s’agit de couvrir leurs besoins élémentaires : être à l’abri, affronter le froid et le chaud, se nourrir en ayant accès à l’eau, aux sols et aux matériaux, marquer ses lieux de vie de spiritualité, etc. Aujourd’hui que je suis en charge d’en rendre compte dans l’édition Parenthèses, préfacée par Germain Viatte, il me sera possible d’aller au-delà de la brève référence que je ﬁs à cet ouvrage dans le chapitre « À l’origine du concept » de La fabrique des modes d’habiter7. Et surtout 341 il me faut convaincre toute personne s’intéressant aux démarches mettant en relation natures, sciences et sociétés qu’il lui faut lire du Pierre Deffontaines (1894-1978) et en particulier cet ouvrage magistral L’homme et sa maison. Car cette géographie, « naturellement » interdisciplinaire, mérite d’être connue du point de vue et conceptuel et méthodologique. De plus, n’estce pas l’occasion de rendre hommage aussi à son ﬁls Jean-Pierre Deffontaines, activement engagé dans NSS, si doué pour conjuguer cette géographie à sa discipline, l’agronomie ? Avec sous les yeux les deux exemplaires (1972, 2021), je ne peux m’empêcher dans un premier temps de les associer dans ce compte rendu pour tenter de comprendre pourquoi le premier passa inaperçu tandis que le second répond incontestablement aux interrogations qui traversent cette période de « désorientation du monde8 ». Ils se distinguent d’abord formellement. Au contraire d’un lettré serré façon édition Proust du premier, le second aéré, limpide, met en corps gras les titres des chapitres et des sous-chapitres et rend incontestablement la lecture de ces 300 pages plus aisée. Certes, la ligne générale est la même : « La marque la plus visible de la présence de l’homme à la surface de la terre est la maison » (4e de couverture Gallimard) ; « La maison est le reﬂet de la vie des hommes, de leur effort physique, de leur pensée, de leur état social, de leur degré d’évolution » (4e de couverture Parenthèses). Mais le volume réédité facilite et augmente le plaisir de savourer, selon sa préférence (féminine ou masculine ?) ou sa curiosité, tel ou tel des « dispositifs » qui révèle et marque la relation des humains à la « maison » dans toutes les dimensions de leur vie : relation aux lieux, au chaud et au froid, aux animaux désirés ou indésirables, aux autres êtres vivants, voire aux morts, etc. S’il est plutôt matérialiste ou naturaliste, le lecteur butinera les chapitres « dispositifs » pour l’eau ou le feu (p. 133-166), le « contact avec le sol, caves et pilotis » (p. 199-206) ou « les ouvertures, l’accès à l’air, la lumière » (p. 209-222). S’il a un penchant d’architecte, ce seront plutôt « La maison construite » avec ses « matériaux » et ses « toitures », la « maison mobile » ou celle à « nombreux occupants » que, – clin d’œil aux architectes d’aujourd’hui –, P. Deffontaines met toujours en relation avec le dedans et le dehors, la richesse et la pauvreté de ses habitants, leur bien-être et en premier lieu leur santé. Quant à moi, toujours obsédée par l’évaluation de la capacité prospective du concept de mode d’habiter, je me plais à relire les chapitres qui décrivent l’ordonnancement du lieu-maison pour tous nos gestes et besoins élémentaires : un abri qui protège (la maison refuge et les « camps refuges » [p. 266], une place « pour le sommeil 7 Morel-Brochet A., Ortar N. (Eds), 2012. La fabrique des modes d’habiter. Homme, lieux et milieux de vie, Paris, L’Harmattan. 8 Badiou A., 2022. Remarques sur la désorientation du monde, Paris, Gallimard. 342 Nat. Sci. Soc. et les repas » [p. 259-266], celle convenant au « travail » dans et autour de la maison, enﬁn les formes et les « endroits » réservés à la spiritualité, au religieux et aux morts [p. 267-276]). Ce qui est décrit pour les pays asiatiques, de l’Amérique latine ou du Maghreb ne se retrouve-t-il pas dans quelques restaurants en France, voire même dans l’ordonnancement intérieur des F4 d’une tour de la ZUP sud de Rennes9 ? Dessiner ce qu’il observe dans tous les continents qu’il a traversés, ﬁxer les détails qui marquent les diversités et lui permettent d’écrire les « communs », tels sont les gestes qui font partie intrinsèque de la méthode de P. Deffontaines, le dessin renforçant le caractère d’« universel-concret » de cette géographie. Une autre différence formelle rend encore plus attachante la lecture de l’ouvrage réédité. L’ouvrage de 1972 comportait certes un ensemble de planches format ﬁgure ou paysage. Dans sa table des matières, on en compte 14 « dans le texte » auquel s’ajoute en ﬁn du volume, et avant la copieuse bibliographie mêlant ethnographes, historiens, sociologues et géographes, un cahier de 29 « illustrations » intitulées « Planches hors textes » toutes localisées et signées P. Deffontaines. Au total, ce sont 43 photographies sur papier jauni qu’on qualiﬁerait aujourd’hui « à faible résolution ». Une facture qui tend à renvoyer à un passé perdu et pourrait engendrer un sentiment de nostalgie en contradiction avec le texte et les légendes de l’auteur. En revanche, dans l’ouvrage réédité, non seulement les dessins reproduits sont plus nombreux (j’en ai compté 67) mais, du fait du format (30 x 25 cm contre 26 x 20 cm) et du grammage blanc mat du papier, ils ont une qualité visuelle qui rivalise presque avec un livre d’art. Quelques dessins sont en pleine page, ce qui permet de mieux en contempler les traits (Cf. Palmeraie dans une oasis, p. 97). Tout est fait pour que les « gestes du regard » de P. Deffontaines soient superbement mis en valeur du point de vue esthétique. On comprend alors que la supériorité artistique de cet ouvrage sur celui de 1972 repose sur celui qui en a assuré la réédition : Germain Viatte, le préfacier, grand connaisseur de l’art moderne, des arts premiers et de la photographie, naguère à la tête de l’Inspection générale des musées de France. Mais en réinsérant chacun de ces dessins à une place précise du texte – travail méticuleux semblable à celui d’un historien –, le supposé ré-éditeur y ajoute une valeur scientiﬁque incontestable. Ces dessins de la main de P. Deffontaines, avec leurs légendes rigoureusement explicites, apportent une preuve concrète à la démonstration générale voulue 9 Mathieu N., Rivault C., Blanc N., Cloarec A., 1997. Le dialogue interdisciplinaire mis à l’épreuve : réﬂexions à partir d’une recherche sur les blattes urbaines, Natures Sciences Sociétés, 1997, 5, 1, 18-30, https://doi.org/10.1051/nss/ 19970501018. par l’auteur : sous la diversité géographique et historique des cultures et des peuples (les humains ?), un système commun que l’on appellerait aujourd’hui « adaptation » s’effectue à toutes les échelles, locales, individuelles et collectives. Le concept de « maison » (proche de celui d’« habitat » pour les animaux) en est le cœur. Il émerge de l’intelligence humaine des rapports entre natures et sociétés et se renouvelle dans le temps et selon les milieux. Il procède d’une capacité d’observation et d’invention elles-mêmes diverses et spéciﬁques. Mais, pour moi, la différence essentielle de valeur qu’apporte la réédition de L’homme et sa maison est incontestablement la longue (33 pages) et passionnante préface de G. Viatte, qu’il intitule « L’homme et ses maisons ». On y découvre le cheminement intellectuel et la carrière de P. Deffontaines dans sa famille depuis la Première Guerre mondiale jusqu’au début des années 1980, les moments historiques qu’il a traversés, les villes et les pays où il a exercé des fonctions scientiﬁques toujours pionnières. Il serait trop long d’énumérer ici toutes les informations qu’elle fournit sur le milieu scientiﬁque et interdisciplinaire de l’époque. C’est en somme une biographie dont les qualités sont celles d’un historien rigoureux recourant à toutes les formes d’archives, notamment des illustrations à nouveau source d’émotions : dessins inédits de P. Deffontaines, photos de l’auteur dans ses lieux de travail et dans sa famille, couvertures de livres, etc. Elle restitue le mode de fonctionnement de l’époque où géographes, ethnologues, historiens, voire sociologues, étaient en constant dialogue. Mais c’est aussi une biographie qui touche à l’intime d’une personne, un géographe avec ses hésitations, ses conﬂits, ses convictions humanistes, ses espérances. En somme, une préface dont l’originalité et la profondeur tiennent à son auteur G. Viatte. En effet le regard porté sur l’ouvrage est bien celui d’un historien de l’art, grand connaisseur de l’art moderne, des arts premiers et de la photographie. Mais son acuité tient aussi à ce non-dit que je découvre en déchiffrant les légendes des photos de la famille Deffontaines où Germain apparaît aux côtés de Jean-Pierre dont il est le cousin. C’est pourquoi tout en reconnaissant le caractère scientiﬁque de cette biographie, le lecteur est saisi par cette écriture qui sait faire vibrer le vécu et l’affection portée à « l’oncle Pierre ». Pour conclure, je ne peux m’empêcher de revenir aux chapitres que je relis une fois de plus dans la belle réédition de Parenthèses : le premier, « Introduction », et le ﬁnal, « Réﬂexion sur l’histoire de la maison ». On y relève le difﬁcile effort de théorisation des produits d’une si longue carrière consacrée à l’observation et à l’identiﬁcation des rapports des humains – et de leurs « genres » – à tous leurs lieux et milieux de vie sur Terre. Quelle accumulation de connaissances ! Quelle ambition Nat. Sci. Soc. de vouloir rendre compte, dans le détail et sans généralisation imposée depuis la culture qui est la sienne, de presque toutes les cultures locales du monde ! Quel respect des gens et des choses se lit dans le souci d’utiliser pour chaque détail des maisons décrites les mots de la langue de celles et ceux qu’il observe. P. Deffontaines ne cherche pas à en faire la synthèse comme on l’a souvent reproché aux géographes de cette époque. Son insistance sur la diversité n’est qu’une façon de réveiller en chacun de nous le goût de la compréhension de l’autre ainsi que, dans le même mouvement, une reconnaissance de ce qui est semblable en tous. P. Deffontaines nous incite à repérer « l’ordinaire » sous la « différence ». La maison, son idée et son 343 faire, c’est ce qui nous rend proches des animaux (cf. « Les logis d’animaux », p. 44-45) et du vivant, c’est aussi ce qui réveille notre sentiment d’injustice envers les « sans-abri », les sans-refuges et ceux qui ont faim (cf. « Un homme sans abri nous apparaît comme un cas presqu’inhumain », p. 4910). C’est peut-être le terme qui redonne sens à celui d’hospitalité et nous encourage à « faire de tous les lieux une maison 11». Nicole Mathieu (Directrice de recherche honoraire, CNRS, UMR Ladyss, Nanterre, France) Nicole.Mathieu@univ-paris1.fr 10 Mathieu N., 2008. L’utopie féminine : faire de tous les lieux une maison, Écologie & politique, 3, 37, 93-101, www.cairn. info/revue-ecologie-et-politique-sciences-cultures-societes2008-3-page-93.htm. 11 Ibid.
https://openalex.org/W2983473682	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/C54FBB7015249A9827FC93AAF166677C/S0140078900011081a.pdf/div-class-title-job-stress-and-burnout-div.pdf	English	null	Job stress and burnout	The bulletin of the Royal College of Psychiatrists/Bulletin of the Royal College of Psychiatrists	1,984	cc-by	1,576	TIle App"",alEurdse-eDlIStipated c1uuJ,1 DEAR SIRS Dr Launer (Bulletin, April 1984, 8, 74-5) brings serious charges to bear against the Approval Exercise, claiming that it is 'bringing units all over the country to their knees'; 'lead- ing to real suffering among patients'; 'morally wrong'; and 'undermining patients' rights to care and treatment'. He attributes this to widespread anal fixation among those responsible, leading to progressive costiveness (metaphorically speaking, I presume). p p y To' explore these misgivings, we repeated part of the Morrice experiment with one addition to the questionnaire: the psychiatrist was asked to explain why he answered 'yes' or 'no' to question (e)-see Table I. TABLE I Responses from psychiatrists (Aberdeen-Eb: Eastern Health Board-EHB) expressed as percentages ofeach group Eb EHB Question (n = 11) (n = 22) (a) Lack of work enjoyment 9 91 (b) Periods ofexhaustion 54 68 (c) Work stressful 63 55 (d) Enduring boredom 27 41 (e) Depression 0 14 TABLE I The evidence which I have collected from many parts of the country runs counter to this view. Most have found the Exercise to be an excellent aperient-perhaps resulting in a little painful colic at times, but usually constructively productive. Responses from psychiatrists (Aberdeen-Eb: Eastern Health Board-EHB) expressed as percentages ofeach group Eb EHB Question (n = 11) (n = 22) (a) Lack of work enjoyment 9 91 (b) Periods ofexhaustion 54 68 (c) Work stressful 63 55 (d) Enduring boredom 27 41 (e) Depression 0 14 I believe the Exercise to be an important instrument of College educational policy. There are good grounds for con- cluding that it has promoted an improvement of educational standards throughout Great Britain and Ireland and this, in the long run, must bear fruit in terms of better clinical prac- tice. I would like to pay tribute to the large number of members who have taken part in visits, to the Panel Con- veners who carry a heavy burden, and to the Dean, who is responsible for the Exercise to the Court. Seventy-eight questionnaires were sent by post to consul- tant psychiatrists, psychiatrist grade practitioners, and senior registrars in the Irish Eastern Health Board area. The names were obtained from the comprehensive mailing list of the Irish Journal of Psychotherapy. Twenty-two (28 per cent) completed questionnaires were returned-21 were from consultants, one came from a senior registrar. K. TIle App"",alEurdse-eDlIStipated c1uuJ,1 DEAR SIRS RAWNSLEY Royal College ofPsychiatrists 17 Belgrave Square, London SW1 When these results are compared with those of Morrice, psychiatrists emerge as the group most lacking in work enjoyment and as the group reporting the highest incidence of'clinical depression' (Table I). Since this is a reversal ofthe original findings, and despite the different geographical set- ting and the lack of controls, doubt is cast on whether Morrice's questionnaire measured what was intended. Halton Hospital Runcom, Cheshire Halton Hospital Runcom, Cheshire The 'Remarks' section was filled in by most respondents. Many of the entries were remarkably candid and personal. The greatest stressors were perceived as 'lack of an under- standing colleague to confide in'; 'lack of sabbatical and educational breaks'; 'the obstructionism of administrators'; Correspondence we wondered if the questionnaire employed by Dr Morrice really measured what it was intended to measure. In particular, we were concerned over the possibility that gen- eral practitioners and hospital doctors would use a less exact definition of'clinical depression' than psychiatrists. TIle App"",alEurdse-eDlIStipated c1uuJ,1 DEAR SIRS DEAR SIRS Professor Gunn's letter about 'Section jargon' (Bulletin, April 1984, 8, 74) is most timely, but the headings could be even more coherenL I would suggest the following for the sections most relevant to general psychiatrists: S 2-assess- ment order (civil); S 3-treatment order (civil); S 4-emer- gency order; S 5-detention order; S 35-assessment remand; S 36-treatment remand; S 37-treatment order (judicial); S 41-restriction order; S 57-irreversible treat- ment certificate; S 58-hazardous treatment certificate; S 78-assessment order (judicial); S 136-police order. In answer to the question 'Please explain the basis for your answer to question (e)' (i.e. 'Would you say this has amounted to clinical depression?'), the expected explana- tions were received: psychiatrists will not use such a term unless they perceive certain symptoms (e.g. sleep dis- turbances, loss of interest, or reduced vegetative functions) which would have to last for at least a number of weeks. By contrast, one may gather that non-psychiatrists employ the term in the same way as we would use terms like 'unhappy' or 'bored'. JOHN MARKs A 'CoUege recommended'textbook 01psychiatry? DEAR SIRS During our preparations for the second part of the examination for membership of the College, my friends and I noted the large number of books we had to refer to, in order to gather what information we felt would meet the require- ments of the examination. Even then, depending on our sources, we held different views on im"portant issues. D. V. CARPY The Tavistock Clinic 120 Be/size Lane, London NW3 The Tavistock Clinic 120 Be/size Lane, London NW3 I suggest that the College considers appointing a special Task Force to produce a Standard Textbook of Psychiatry, in two volumes. Volume I would cover the theoretical requirements for the MRCPsych Preliminary Test, and Volume II would do the same for the Membership Examina- tion. DEAR SIRS lPRmST, R. G. (1979) Aggression and suicide. In Aetiology and Management of Affective Disorders (00. A. M. Jukes). Horsham: Ciba. lPRmST, R. G. (1979) Aggression and suicide. In Aetiology and Management of Affective Disorders (00. A. M. Jukes). Horsham: Ciba. lPRmST, R. G. (1979) Aggression and suicide. In Aetiology and Management of Affective Disorders (00. A. M. Jukes). Horsham: Ciba. I should like to take up Dr Holmshaw's invitation to comment on his 'Clomipramine Challenge Test' (Bulletin, April 1984, 8, 76). __ & WOOLPSON, G. (1978) Minski's Handbook of Psychiatry. London: Heinemann. _ & WOOLPSON, G. (1978) Minski's Handbook of Psychiatry. London: Heinemann. I am extremely concerned that Dr Holmshaw should feel it is appropriate for a psychiatrist deliberately to induce 'florid schizophrenic psychosis' in susceptible people by neuropharmacological methods. In addition, I wonder whether I might assume that Dr Holmshaw follows usual medical practice by informing his patients of the desired effects of the drugs he gives them, and also of any likely side-effects. DEAR Sols DEAR Sols We found Dr Morrice's article on job stress and burnout (Bulletin, March 1984, 8, 45-6) very interesting. However, 139 https://doi.org/10.1192/pb.8.7.139-b Published online by Cambridge University Press features. The younger one is quite intelligent, and the older one has an intelligence in the dull-normal range intelligence. Both have taken overdoses, but the younger has swallowed razor blades, and abused alcohol and other drugs. and 'psychotherapy'. Personal tragedies were also blamed for difficulties in maintaining interest in one's work by a sig- nificant minority. Several coping strategies were reported as being helpful, especially: 'turning oW after work, maintain- ing outside interests, research, and 'devaluation' of per- ceived opponents. Most respondents felt that the question- naire was not detailed enough to tap the essence of burnout. Two respondents questioned its validity as an entity. In con- trast to Morrice, we were struck by the openness and honesty of the respondents, many of whom signed their questionnaires. We are indeed grateful to them. features. The younger one is quite intelligent, and the older one has an intelligence in the dull-normal range intelligence. Both have taken overdoses, but the younger has swallowed razor blades, and abused alcohol and other drugs. Although self-cutting is well known to occur in patients of definitely subnormal intelligence, it is not so common in adults in the normal intellectual range. In the patients described here it seems to be more related to their abnormal personalities.1) We are wondering if other psychiatrists have encountered this combination of Klinefelter's syndrome and persistent self-mutilation. If so, through the courtesy of your columns we should like to invite them to write to us so that we may build up a picture of the resulting syndrome, if such an entity exists. In conclusion, whilst we do not feel that Dr Morrice's questionnaire measured a specific syndrome, we do believe that we tapped, ifnot lanced, a boil. BRIAN O'SHEA AmAN McGENNls MICHELE CAIDLL D. A. ADDALA St Brendan's Hospital Dublin St Brendan's Hospital Dublin St Brendan's Hospital Dublin St Brendan's Hospital Dublin https://doi.org/10.1192/pb.8.7.139-b Published online by Cambridge University Press Royal Free Hospital Hampstead, London NW3 DEAR SIRs Two of us (Prof R. G. Priest and Dr G. G. Wallis) found out by chance that we had a patient each who both showed striking features in common. They both have been diagnosed as Klinefelter's syndrome and XXV constitution has been confirmed on chromosomal assay. They are both persistent self-mutilators of an extreme degree, and show extensive scarring from skin cuts. Both have been diagnosed as having gross personality disorder. They are single, with scanty facial hair and atrophic testes. One is 2S years old and the other is 33. The general idea is that the two volumes of the Standard Textbook of Psychiatry would represent views generally agreed by the College on the essential theoretical material of psychological medicine. As such, they will serve not only to facilitate the training of psychiatrists, but also (much more importantly) help to bring into coordination what the College sees as psychiatry. IKECHUKWU AzUONYE Royal Free Hospital Hampstead, London NW3 There are also differences. The younger one has diffuse slow waves in the EEG, and the older one has had psychotic 140 https://doi.org/10.1192/pb.8.7.139-b Published online by Cambridge University Press
https://openalex.org/W2153967508	https://www.animbiosci.org/upload/pdf/15_126.pdf	English	null	Effects of Rumen Undegradable Protein and Minerals Proteinate on Early Lactation Performance and Ovarian Functions of Dairy Cows in the Tropics	Asian-Australasian journal of animal sciences	2,002	cc-by	4,668	Effects of Rumen Undegradable Protein and Minerals Proteinate on Early Lactation Performance and Ovarian Functions of Dairy Cows in the Tropics** J. Kanjanapruthipong,1 and N. Buatong1 Department of Animal Sciences, Kasetsart University, Kampaengsaen, Nakornpathom 73140, Thailand ABSTRACT : A 90 d study was designed to investigate the effects of rumen undegradable protein (RUP) and a mixture of Cu, Zn and Mn proteinate (CZMP) on milk yield and composition and ovarian functions during rainy months. Twenty four Holstein× indigenous cows in their 2nd and 3rd lactation were randomly allocated to total mixed rations (TMR) containing soy bean meal (SBM) as a source of rumen degradable protein (RDP), SBM plus CZMP, and formalin treated SBM (FSBM) as a source of RUP. Maximum and minimum temperature humidity index during the experimental period were 83.6-84.7 and 75.4-76.1. There were no differences (p>0.05) in intakes of dry matter, crude protein and net energy and in contents of butterfat, lactose and minerals. Cows on TMR containing FSBM not only lost less weight (-278, -467 and -433 g/d) with more intake of RUP (0.92, 0.58 and 0.59 kg/d) but also produced more milk (19.27, 18.23 and 18.13 kg/d) and 4% fat corrected milk (18.57, 17.57 and 17.51 kg/d) with more protein (3.06, 2.81 and 2.80%), solids-not-fat (8.69, 8.38 and 8.38%) and less milk urea N (9.3, 15.4 and 15.0 mg/dl) compared with those on TMR containing SBM and SBM+CZMP, respectively (p<0.01). However, cows on TMR containing SBM and SBM+CZMP did not differ in these respects (p>0.05). Whereas incidence of cystic ovaries at 20 and 90 d pospartum was less (p<0.01) in cows on TMR containing SBM+CZMP (37.3 and 12.5%) than those on TMR containing SBM (62.5 and 25%), it was nil for cows on TMR containing FSBM. Cows in all three group differed (p<0.01) from each other for the recurrence of first observed estrus with those on TMR containing FSBM having least days (22, 36 and 47 d) compared with their counterpart on TMR containing SBM+CZMP and SBM, repectively. The results suggest that RUP is one of the limiting factors affecting milk yield and its composition and ovarian functions during early lactation of dairy cows in the tropics. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 6 : 806-811) Key Words : Rumen Undegradable Protein, Minerals Proteinate, Early Lactation, Ovarian Functions, Tropics 806 806 * Supported by Kasetsart University Research and Development Institute, Kasetsart University, Bangkok, Thailand. * Corresponding Author: J. Kanjanapruthipong. Tel: +66-34- 351033, Fax: +66-34-351892, E-mail: agrjck@nontri.ku.ac.th 1 Dairy Research and Development Center, Kasetsart University, Kampaengsaen, Nakornpathom 73140, Thailand. Received November 1, 2001; Accepted January 2, 2002 ** Supported by Kasetsart University Research and Development Institute, Kasetsart University, Bangkok, Thailand. * Corresponding Author: J. Kanjanapruthipong. Tel: +66-34- 351033, Fax: +66-34-351892, E-mail: agrjck@nontri.ku.ac.th 1 Dairy Research and Development Center, Kasetsart University, Kampaengsaen, Nakornpathom 73140, Thailand. Received November 1, 2001; Accepted January 2, 2002 MATERIALS AND METHODS Before the study was started, all cows were examined with particular reference to pathologic features of reproductive tracts and only normal and healthy cows were used. Twenty four Holstein×indigenous (93.75×6.25%) cows in their 2nd and 3rd lactation were randomly allocated to dietary treatments according to completely randomized design. All the cows were in early lactation. Experimental cows were hold at the holding area for 30 min. at before and after a.m. (4:30 h) and p.m. (15:30 h) milking for observation for signs of estrus. The observation was done again at 11:00 and 22:00 h. Signs of estrus were determined by observing “mounting” behavior or “standing-for-mounting” nervousness and increased physical activity, swelling of the vulva, and clear stringy mucous discharge from the vulva and then rectal palpation were done. Cystic ovaries were determined at 20 d and 90 d postpartum by rectal palpation. Dietary treatments consisted of soybean meal (SBM) as a source of RDP, SBM plus a mixture of Cu, Zn and Mn proteinate (CZMP; Applied Force Ltd., Thailand) and formalin treated SBM (FSBM; Cows Friend Ltd., Thailand) as a source of RUP. Paragrass hay was made from perennial paragrass. The paragrass was cut, chopped into 2-3 cm length and sun dried for total mixed rations (TMR). Diets contained CP approximately 10% above the NRC (1988) requirement and were isonitrogenous and isocaloric as shown in table 1. Crude protein (CP), ether extract (EE), ash and DM contents of the experimental diets were determined according to the AOAC (1980). Neutral detergent fiber (NDF) and neutral detergent insoluble nitrogen (NDIN) were measured following the method of Van Soest et al. (1991). Total non-fiber carbohydrates (TNFC) is calculated following the equation; TNFC=100-CP-EE-(NDF-NDIN) -ash. Milk compositions were measured with MilkoScan (Foss Electric, Denmark). Milk (0.2 ml) was deproteinized with 1.8 ml of TCA (3%), allowed to stand for 5 min, and centrifuged at 3,500×g for 30 min. at 2ºC. The clear supernatant was pipetted carefully from the solidified milk fat and analyzed at 535 nm for milk urea nitrogen (MUN) using a colorimetric diacetyl monoxine procedure (number 535; Sigma Diagnostic, St. Louis, MO). Statistical analysis was carried out by SAS (1989). Experimental diets were offered ad libitum at 06:00, 10:00 and 18:00 h and were sampled weekly and bulked for later analysis of chemical composition. Feed intake and milk yield were daily measured. Milk samples of 50 ml were weekly collected at consecutive a.m. INTRODUCTION 1989) and low (Taylor et al., 1991) in rumen degradable protein (RDP). Intakes of dietary protein that maximize milk yield can cause changes of reproduction and fertility unless release of ammonia within the rumen is controlled (Visek, 1984). However, RDP or rumen undegradable protein (RUP) in excess of requirement can contribute to reduced fertility (Bulter, 1998) resulting economic losses approximately 50 million US$ a year in Thailand. Ferguson and Chalupa (1989) suggest that amount and degradability of dietary protein can explain protein-fertility relationships. The biological value of protein for dairy cows is directly related to the energy status of the dairy cows and the balance of absorbed amino acids relative to their requirement (Van Straalen et al., 1994). Reduced energy consumption and increased energy maintenance requirement during heat stress often results in considerably more protein being metabolized to meet energy requirements of dairy cows (Beede and Collier, 1986). Dairy cows under heat stress often have negative nitrogen balance (Kamal and Johnson, 1970; Hassan and Roussel, 1975) and thus less protein is available for productive functions. An adequate supply of dietary protein to dairy cows in the tropics is necessary in order to optimize milk yield and fertility. The ability to provide adequate protein to dairy cows depends on moderate the balance between the availability of nitrogen for microbial growth in the rumen and for productive functions (Ferguson and Chalupa, 1989). Too little RDP reduce microbial growth in the rumen while increasing availability of protein for digestion and absorption at the small intestine, whereas too much RDP increase ruminal ammonia (NH3) and blood urea resulting in less protein available for productive functions (Ferguson et al., 1988). However, information is limited on the influence of amount and degradability of protein as well as trace minerals proteinate on yield and composition of milk and reproduction for dairy cows in the tropics. An objective of this study was to evaluate diets containing two protein sources of different degradabilities and a mixture of trace It has been observed that under thermal stress, dairy cows produce more milk in high protein diets (Hassan and Roussel, 1975) and in diets medium (Higginbotham et al., RUP, MINERAL PROTEINATE AND DAIRY PERFORMANCE IN THE TROPICS 807 mineral proteinate on early lactation performance and ovarian functions of dairy cows in the tropics. during rainy months from the end of May to the beginning of September (90 d). MATERIALS AND METHODS and p.m. milking in bottles containing 2-bromo-2-nitro-1, 3-propadiol and stored at 5ºC for composition analysis. Cows were weighed once each week immediately following the a.m. milking prior to accessing feed and water. The trial was conducted Table 1. Ingredients of total mixed rations (TMR) TMR containing SBM SBM+ CZMP FSBM Whole cotton seed 11 11 11 Soy bean meal (SBM) 18.6 18.6 - Formalin treated soybean meal (FSBM) - - 18.6 Cassava chips 36.8 36.5 36.8 Molasses 2.8 2.8 2.8 Urea 0.9 0.9 0.9 Monocalcium phosphate 0.7 0.7 0.7 Dicalcium phosphate 0.7 0.7 0.7 CaCO3 0.3 0.3 0.3 NaHCO3 0.6 0.6 0.6 NaCl 0.4 0.4 0.4 Premix 0.2 0.2 0.2 Cu, Zn and Mn proteinate (CZMP) - 0.3 - Paragrass hay 27 27 27 Total 100 100 100 Ingredients Table 1. Ingredients of total mixed rations (TMR) TMR containing SBM I di t Table 1. Ingredients of total mixed rations (TMR) ble 1. Ingredients of total mixed rations (TMR) INTRODUCTION Ambient temperature and relative humidity were recorded before morning feeding with thermograph and hygrograph (Classella, London). The temperature humidity index (THI) was calculated following the equation: THI=td-(0.55-0.55RH) (td-58), where td is the dry bulb temperature (ºF) and RH is the relative humidity expressed as a decimal (NOAA, 1976). RESULTS However, incidence of cystic ovaries and days to first observed estrus were significantly improved for dairy cows fed TMR containing SBM with supplementation of a mixture of Cu, Zn and Mn proteinate, compared to Dietary treatment effects on milk composition, yield and 4% fat corrected milk are shown in table 4. With an exception of fat, lactose and mineral contents, protein, solids-not-fat and total solids were significantly lower for dairy cows fed TMR containing SBM and SBM plus CZMP than those fed TMR containing FSBM (p<0.01). Milk yield and 4% fat corrected milk were also significantly lower for dairy cows fed TMR containing SBM and SBM plus CZMP than those fed TMR containing FSBM (p<0.01). Milk urea nitrogen in dairy cows fed TMR containing SBM and SBM plus CZMP were significantly higher than those fed TMR Table 4. Composition and yield of milk in dairy cows fed different total mixed ration (TMR) Table 4. Composition and yield of milk in dairy cows fed different total mixed ration (TMR) TMR containing Item SBM* SBM + CZMP* FSBM* SE Butter fat % 3.76 3.78 3.75 0.06 kg/d 0.69 0.68 0.72 <0.01 Milk protein % 2.81 a 2.80 a 3.06 b 0.02 kg/d 0.52 a 0.51 a 0.59 b <0.01 Lactose % 4.87 4.88 4.93 0.08 kg/d 0.89 0.88 0.95 0.01 Minerals % 0.70 0.70 0.70 <0.01 kg/d 0.13 0.13 0.14 <0.01 Solids-not-fat % 8.38 a 8.38 a 8.69 b 0.02 kg/d 1.53 a 1.52 a 1.67 b 0.02 Total solids % 12.14 a 12.16 a 12.44 b 0.10 kg/d 2.21 a 2.20 a 2.40 b 0.05 Milk yield, kg/d 18.23 a 18.13 a 19.27 b 2.10 Fat corrected milk (4%), kg/d 17.57 a 17.51 a 18.57 b 2.75 Milk urea N, mg/dl 15.4 a 15.0 a 9.3 b 0.3 * SBM-soy bean meal; CZMP-Cu, Zn and Mn proteinate mixture; FSBM-formalin treated SBM. a,b Means within a row without a common superscript letter differ (p<0.01). Table 3. RESULTS Chemical composition of the dietary treatments was similar. Values of nutrient composition on a DM basis for total mixed rations (TMR) were as follows: CP, 15.87%; NDF, 28.11%; EE, 3.60%; TNFC, 41.27%; and calculated net energy for lactation (NEl), 1.57 (Mcal/kgDM). Calculated RUP for TMR containing SBM, SBM plus CZMP and FSBM was 24.1, 24.1 and 38.5% of CP, respectively. Means for environmental conditions during the experimental period are presented in table 2. Daytime temperature was much higher than the nighttime while daytime relative humidity was much lower than the nighttime. The combination of moderately high temperature and extremely high humidity led to high temperature humidity index (THI) during rainy months. Dietary treatment effects on nutrient intakes and live KANJANAPRUTHIPONG AND BUATONG 808 Table 2. Means for environmental conditions during the experimental period Value Item May Jun. Jul. Aug. Sep. Maximum temperature, ºC 33.5±2.4 33.2±2.4 33.6±2.4 33.1±2.3 33.4±2.1 Minimum temperature, ºC 24.7±1.0 24.4±0.9 24.7±1.2 24.3±0.4 24.1±0.2 Maximum relative Humidity, % 96.0±4.0 95.0±3.0 96.0±4.0 98.0±2.0 97.0±2.0 Minimum relative humidity, % 53.0±8.0 57.0±9.0 59.0±10.0 60.0±8.0 60.0±10.0 Maximum THI* 83.6±1.3 83.8±2.0 84.7±1.2 84.2±1.6 84.6±0.9 Minimum THI* 76.1±1.3 75.4±1.1 76.1±1.6 75.5±0.6 75.9±0.2 THI-Temperature humidity index. Table 2. Means for environmental conditions during the experimental period containing FSBM (p<0.01). weight are presented in table 3. Daily intakes of DM, CP and NEl for all cows were not significantly different (p>0.05). However, daily intakes of RUP for dairy cows fed TMR containing SBM and SBM plus CZMP were significantly lower than those fed TMR containing FSBM (p<0.01). Dairy cows fed TMR containing SBM and SBM plus CZMP significantly lost more weight than those fed TMR containing FSBM (p<0.01). weight are presented in table 3. Daily intakes of DM, CP and NEl for all cows were not significantly different (p>0.05). However, daily intakes of RUP for dairy cows fed TMR containing SBM and SBM plus CZMP were significantly lower than those fed TMR containing FSBM (p<0.01). Dairy cows fed TMR containing SBM and SBM plus CZMP significantly lost more weight than those fed TMR containing FSBM (p<0.01). Dietary treatment effects on ovarian functions are presented in table 5. Incidence of cystic ovaries and days to first observed estrus were significantly higher (p<0.01) for dairy cows fed TMR containing SBM than those fed TMR containing FSBM. DISCUSSION Protein nutrition can affect milk yield through the availability of NH3, peptides and amino acids for microbial growth in the rumen and of amino acids for milk protein synthesis (Bequette et al., 1998). It can also affect reproduction through toxic effects of ammonia and its metabolites on gametes and early embryos (Ferguson and Chalupa, 1989) and by exacerbation of negative balance of energy (Oldham, 1984). In this study, CP content of dietary treatments was approximately 10% above the NRC (1988) recommendation and undegraded intake protein (UIP) calculated from data of NRC (1988) were 24.1, 24.1 and 38.5% of CP for TMR containing SBM, SBM plus CZMP and FSBM, respectively. In this study, UIP for TMR containing FSBM slightly exceeded the upper level of 37% of CP recommended by NRC (1988), whereas it was well below the NRC (1988) recommendation of 34% for TMR containing SBM and SBM plus CZMP. It is likely that TMR containing FSBM, in this study, will adequate provide RDP for microbial growth and RUP for productive functions of dairy cows. During an early stage of lactation, dairy cows are in a negative energy balance. A negative energy balance can be exacerbated by feeding excess RDP and RUP as it requires energy to metabolize excess protein (Oldham, 1984). Diets high in RDP increase ruminal NH3 (Visek, 1984; Stokes et al., 1991) and blood urea-N (Higginbotham et al., 1989) and thus increase energy cost of synthesizing and excreting the RDP as urea (Oldham, 1984). In the present study, dairy cows fed TMR containing SBM and SBM plus CZMP lost more weight than those fed TMR containing FSBM, though net energy intake were similar. Therefore, dairy cows in particular in early lactation on high RDP diets in the tropics can be in a severe negative energy balance. During rainy months in the tropics, ambient temperature, typically, is 2-4ºC lower than summer months while relative humidity is 8-16% higher. The combinations of moderately high ambient temperature and extremely high relative humidity during rainy months contributes to extremely high THI (Johnson, 1987) which is close to the THI during summer months (Kanjanapruthipong et al., 2001). The maximum THI of 84.7 reported, in this study, exceeded the lower range of danger zone of 78 for survival of Holstein cows (Johnson, 1987) and the minimum THI of 75.4 exceeded the upper critical point of 72 for optimal productivity (Johnson, 1987). RESULTS Ovarian activities in dairy cows fed different total mixed rations (TMR) a,b,c Means within a row without a common superscript letter differ (p<0.01). Circulating NH3 can originate from both RDP and RUP (Ferguson and Chalupa, 1989). The quantity of NH3 that is produced and the amount that escapes from the rumen directly reflects both dietary RDP and the availability of the fermentable carbohydrates to support microbial growth and protein synthesis (Firkins, 1996). The available amino acids that are not taken up for milk protein synthesis or deposited elsewhere are deaminated by the liver to yield energy substrates and NH3 (Oldham, 1984). As urea that is synthesized from the NH3 by the liver circulates through the blood, it equilibrates all tissue (Chalmers et al., 1971). Plasma urea nitrogen (PUN) and MUN are useful indicators of protein metabolism and status in dairy cows (Roseler et al., 1993). Higher MUN in dairy cows fed TMR containing SBM and SBM plus CZMN in this study may be due to excess RDP, while a good value of MUN in dairy cows fed TMR containing FSBM is likely to reflect the balance between the amount and degradability of CP in the diets. those fed TMR containing SBM (p<0.01). those fed TMR containing SBM (p<0.01). RESULTS Live weight changes and voluntary intake of total mixed rations (TMR) TMR containing Item SBM SBM + CZMP* FSBM* SE Nutrient intake Dry matter, kg/d 15.06 15.52 14.96 2.35 Dry matter, %BM 3.32 3.36 3.19 0.20 Crude protein, kg/d 2.39 2.46 2.37 0.05 Undegraded intake protein, kg/d 0.58 a 0.59 a 0.92 b 0.03 Net energy*, Mcal/d 23.64 24.37 23.49 5.76 Live weight change Initial weight, kg 474 481 482 5.69 Final weight, kg 432 442 457 5.97 Average weight, kg 453 462 469 8.68 Daily gain, g/d -467a -433 a -278b 20.1 SBM-soy bean meal; CZMP-Cu, Zn and Mn proteinate mixture; FSBM-formalin treated SBM. ** Net energy for lactation (Mcal/kgDM)=(0.0245×TDN)-0.12. a,b Means within a row without a common superscript letter differ (p<0.01). Table 3. Live weight changes and voluntary intake of total i d i (TMR) Table 3. Live weight changes and voluntary intake of total mixed rations (TMR) RUP, MINERAL PROTEINATE AND DAIRY PERFORMANCE IN THE TROPICS 809 Table 5. Ovarian activities in dairy cows fed different total mixed rations (TMR) TMR containing Item SBM* SBM + CZMP* FSBM* SE Incidence of cystic ovaries, % at 20 d postpartum at 90 d postpartum 62.5 a 25 a 37.3 b 12.5 b nilc nil c 5.4 1.9 Days to first observed estrus** 47 a 36 b 22 c 4.09 * SBM-soy bean meal; CZMP-Cu, Zn and Mn proteinate mixture; FSBM-formalin treated SBM. ** Excluding cystic ovary cows. a,b,c Means within a row without a common superscript letter differ (p<0.01). Hassan and Roussel (1975) observed increased DMI in dairy cows fed 21% CP, compared to those fed 14% CP. Similarly, Higginbotham et al. (1989) compared diets with two CP contents (18.4 and 16.1%) and degradabilities (65 and 58%) and observed increased DMI in dairy cows fed diets high in CP than those fed diets medium in CP, irrespective of degradability. Whereas, Taylor et al. (1991) reported increased DMI in dairy cows fed diets high in CP (17.8%) and low in degradability (47%) than those fed diets high in CP (18.1%) and medium in degradability (61%). In this study, DMI did not differ in dairy cows fed diets medium in CP (15.9%) and two degradabilities (75.9 and 61.5%). These results suggest that under heat stress diets high in CP stimulate DMI of dairy cows and effect of degradability in diets on DMI is not consistent. Table 5. REFERENCES Association of Official Analytical Chemists. 1980. Official Methods of Analysis 13th Ed. AOAC, Washington, DC. Beede, D. K. and R. J. Coller. 1986. Potential nutritional strategies for intensively managed cattle during thermal stress. J. Anim. Sci. 62:543-554. Bequette, B. J., F. R. C. Backwell and L. A. Crompton. 1998. Current concepts of amino acid and protein metabolism in the mammary gland of the lactating ruminant. J. Dairy Sci. 81:2540-2559. Cystic ovaries can be a serious cause of reproductive failure in dairy cows because it prolongs the postpartum interval to first estrus and conception in probably 10 to 30% of the dairy cows (Kesler and Garverick, 1982). The frequency of detecting cystic ovaries is higher in early postpartum cows. Whitmore et al. (1974) reported that 71% of cystic ovaries developed within 45 d postpartum. Delays in onset of normal ovarian activities thus limiting the number of estrus cycles before breeding, may account for the observed decrease in fertility (Butler and Smith, 1989). In this study, higher incidence of cystic ovaries and longer days to first estrus were observed in dairy cows fed TMR containing SBM and SBM plus CZMP than those fed TMR containing FSBM. It is likely that for dairy cows in the tropics, excess RDP can reduce fertility possibly via the exacerbation of negative energy balance (Butler and Smith, 1989), the detrimental effects of NH3 and its metabolites on ovarian functions (Swanson, 1989) and the deficiency of amino acids relative to requirement (Ferguson and Chalupa, 1989). Butler, W. R. 1998. Effect of protein nutrition on ovarian and uterine physiology in dairy cattle. J. Dairy Sci. 81:2533-2539. Butler, W. R. and R. D. Smith. 1989. Interrelation ship between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72:767-783. Chalmers, M. E., A. E. Jeffray and F. White. 1971. Movements of ammonia following intraruminal administration of urea or casein. Proc. Nutr. Soc. 30:7-17. Ferguson, J. D. and W. Chalupa. 1989. Impact of protein nutrition on reproduction in dairy cows. J. Dairy Sci. 72:746-766. Ferguson, J. D., T. Blandchart, D. T. Galligan, D. C. Hoshall and W. Chalupa. 1988. Infertility in dairy cattle fed a high percentage of protein degradable in the rumen. J. Am. Vet. Med. Assoc. 192:659-662. Firkins, J. L. 1996. Maximizing microbial protein synthesis in the rumen. J. Nutr. 126:1347s-1354s. Hassan, A. and J. D. Roussel. 1975. DISCUSSION These environmental conditions suggest the extremely stressful conditions to dairy cows under rainy months in the tropics. An increased supply of RUP for digestion and absorption at the small intestine in ruminants will increase glucose availability in circulation due to increased gluconeogenesis, sparing of glucose from oxidation and increased glucose from glycerol released from adipose tissue breakdown (Oldham, 1984). It is evident that dairy cows under heat stress produce more milk on a diet high in KANJANAPRUTHIPONG AND BUATONG 810 of this study suggest that RUP is one of the limiting factors affecting productivity of dairy cows in the tropics. RUP, when the dietary protein fed to dairy cows in hot environments should not exceed the NRC (1988) requirement by more than 10-15% CP (Huber et al., 1994). Taylor et al. (1991) reported that on diets averaged 18.3% CP, dairy cows fed 51% RUP yielded more milk (3.4 kg/d) than those fed 37.8% RUP. On diets averaged 15.9% CP in this study, dairy cows fed 38.5% RUP produced more milk (1.1 kg/d) than those fed 24.1% RUP. These results suggest that increased RUP in the diets will increase glucose availability for milk secretion. ACKNOWLEDGMENTS The research was supported by the Kasetsart University Research and Development Institute, Kasetsart University, Bangkhan, Bangkok, Thailand. The authors appreciate the assistance of the Dairy Research and Development Center staffs. McGuire et al. (1989) reported that net flux of ∝ amino N was reduced 35% by thermal stress compared with thermal comfort and hence less protein is available for productive functions. Increased intake of RUP from 580 g/d to 920 g/d as presented in table 3 will increase protein for digestion and absorption at the small intestine. Increased milk protein as a result of increased intake of RUP reported in this study is likely due to increase amino acids available for mammary protein secretion. REFERENCES Effect of protein concentration in the diet on blood composition and productivity of lactating Holstein cows under thermal stress. J. Agric. Sci. Camb. 85:409-415. Higginbotham, G. E., M. Torabi and J. T. Huber. 1989. Influence of dietary protein concentration and degradability on performance of lactation cows during hot environmental temperature. J. Dairy Sci. 72:2554-2564. In addition, supplementation with complexed trace minerals appeared to improve ovarian functions. O’Donoghue et al. (1995) reported that days to first estrus reduced from 25 to 20 days postpartum in dairy cows supplemented with a mixture of CU, Zn and Se proteinate. Similar results were observed in this study in dairy cows fed TMR containing SBM supplemented with a mixture of Cu, Zn and Mn proteinate. Huber, J. T., G. E. Higginbotham, R. A. Gomez-Alarcon, R. B. Taylor, K. H. Chen, S. C. Chan and Z. Wu. 1994. Heat stress interactions with protein, supplemental fat, and fungal cultures J. Dairy Sci. 77:2080-2090. Johnson, H. D. 1987. Bioclimates and livestock. In: Bioclimatology and the Adaptation of Livestock. (Ed. H. D. Johnson). Elsevier Science publishers. Amsterdam, Netherlands. pp. 3-16. Milk yield and its compositions and ovarian functions during early lactation can be significantly improved with diets high in RUP for dairy cows in the tropics. Additionally supplementation of a mixture of Cu, Zn and Mn proteinate to diets high in RDP appeared to improve ovarian functions during early lactation in dairy cows in the tropics. Results Kamal, T. H. and H. D. Johnson. 1970. Whole body 40k loss as a predictor of heat tolerance in cattle. J. Dairy Sci. 53:1734- 1738. RUP, MINERAL PROTEINATE AND DAIRY PERFORMANCE IN THE TROPICS 811 Kanjanapruthipong, J., N. Buatong and S. Buaphan. 2001. Effects of roughage neutral detergent fiber on dairy performance under tropical conditions. Asian-Aust. J. Anim. Sci. 14(10):1400- 1404. Roseler, D. K., J. D. Ferguson, C. J. Sniffen and J. Herrema. 1993. Dietary protein degradability effects on plasma and milk urea nitrogen and milk nonprotein nitrogen in Holstein Cows. J. Dairy Sci. 76:525-534. Kesler, D. J. and H. A. Garverick. 1982. Ovarian cysts in dairy cattle: A review. J. Anim. Sci. 55(5):1147-1159. SAS/STAT User’ Guide, Version 6, 4th Edition. Vol 2. 1989. SAS Inst., Cary, NC. Stokes, S. R., W. H. Hoover, T. K. Milker and R. Blauweikel. 1991. Ruminal digestion and microbial utilization of diets varying in type of carbohydrate and protein. J. Dairy Sci. 74:871-881. McGuire, M. REFERENCES A., D. K. Beede, M. A. Delorenzo, C. J. Wilcox, G. B. Huntington, C. K. Reynolds and R. J. Collier. 1989. Effects of thermal stress and level of feed intake on portal plasma flow and net fluxes of metabolites in lactation Holstein cows. J. Anim. Sci. 67:1050-1060. Stokes, S. R., W. H. Hoover, T. K. Milker and R. Blauweikel. 1991. Ruminal digestion and microbial utilization of diets varying in Ruminal digestion and microbial utilization of diets varying in type of carbohydrate and protein. J. Dairy Sci. 74:871-881. type of carbohydrate and protein. J. Dairy Sci. 74:871-881. Swanson, L. V. 1989. Interactions of nutrition and reproduction. J. Dairy Sci. 72:805-814. National Oceanic and Atmospheric Administration. 1976. Livestock hot weather stress. US Dept. Commerce. National Weather Serv. Central Reg., Reg. Operations manual Lett. C- 31-36. Taylor, R. B., J. T. Huber, R. A. Gomez-Alarcon, F. Wiersma and X. Pang. 1991. Influence of protein degradability and evaporative cooling on performance of dairy cows during hot environmental temperatures. J. Dairy Sci. 74:243-249. National Research Council. 1988 Nutrient Requirements of Dairy Cattle. 6 th Rev. Ed., National Academic Press, Washington. DC. p. 157. Van Straalen, W. M., C. Salaum, W. A. G. Veen, Y. S. Rijpkema, G. Hof and T. J. Boxem. 1994. Validation of protein evaluation systems by means of milk production experiments with dairy cows. Neth. J. Agric. Sci. 42:89-104. O’Donoghue, D., P. O. Brophy, M. Rath and M. P. Boland. 1995. The effect of proteinated minerals added to the diet on the performance of post-partum dairy cows. In: Proceedings of Alltech’ Eleventh Annual Symposium. (Ed. T. P. Lyons and K. A. Jacques). pp. 293-297. Visek, W. J. 1984. Ammonia: Its effects on biological systems, metabolic hormone, and reproduction. J. Dairy Sci. 67:481- 498. Whitmore, H. L., Tyler and L. E. Casida. 1974. Incidence of cystic ovaries in Holstein-Frisian cows. J. Am. Vet. Med. Assoc. 165:693-694. Oldham, J. D. 1984. Protein-energy interrelationships in dairy cows. J. Dairy Sci. 67:1090-1114. KANJANAPRUTHIPONG AND BUATONG 812 RUP, MINERAL PROTEINATE AND DAIRY PERFORMANCE IN THE TROPICS 813 KANJANAPRUTHIPONG AND BUATONG 814 RUP, MINERAL PROTEINATE AND DAIRY PERFORMANCE IN THE TROPICS 815 KANJANAPRUTHIPONG AND BUATONG 816 RUP, MINERAL PROTEINATE AND DAIRY PERFORMANCE IN THE TROPICS 817
https://openalex.org/W3037338809	https://ajopa.org/index.php/ajopa/article/download/20/107	English	null	Time limits and English proficiency tests: Predicting academic performance	African journal of psychological assessment	2,020	cc-by	7,816	African Journal of Psychological Assessment ISSN: (Online) 2617-2798, (Print) 2707-1618 Page 1 of 9 Original Research Page 1 of 9 Original Research Original Research Page 1 of 9 How to cite this article: How to cite this article: Opperman, I. (2020). Time limits and English proficiency tests: Predicting academic performance. African Journal of Psychological Assessment, 2(0), a20. https://doi.org/ 10.4102/ajopa.v2i0.20 Time limits and English proficiency tests: Predicting academic performance English is the primary language of instruction in South African higher education, but entering students of first year are often not sufficiently proficient. Therefore, a need is evident for proficiency testing to guide intervention initiatives. International proficiency tests are lengthy and expensive, but Cloze procedure and vocabulary tests have been used as effective alternatives. However, time limits may affect observed reliability and predictive validity in the context of higher education. The present research assessed a cohort of first-year tourism management students using versions of the English Literacy Skills Assessment (ELSA) Cloze procedure and Vocabulary in Context tests under three time-limit conditions: normal, double and no time limits. Students in double and no time-limit conditions performed significantly better than the normal time-limit group. Group scores were correlated with, and significant predictors of, academic subject first-test scores. Better performance and more accurate prediction under extended time limits may be related to students attempting more questions. As the ELSA Vocabulary in Context was the better predictor in this research, the importance of non-technical vocabulary, as opposed to semantic and contextual understandings in Cloze procedure, is highlighted. Therefore, screening the English proficiency levels of students admitted to higher education institutions may be useful to flag likelihood of success and guide interventions. Corresponding author: Ingrid Opperman, ingrid258@gmail.com Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Dates: Keywords: higher education; English proficiency; Cloze procedure; vocabulary; time limits. Introduction English has become the dominant language of business, public life and higher education (Benzie, 2010; Casale & Posel, 2011; Coleman, 2006; Nunan, 2003). Therefore, formal acquisition of English language skills has become essential for success in both higher education and business contexts to enhance economic opportunities in a multinational and international economy (Bedenlier & Zawacki-Richter, 2015; Prinsloo & Heugh, 2013). Higher education serves an essential role in enhancing the future career prospects in a competitive social and economic framework, making success integral for many young people (Coleman, 2006; Cross & Carpentier, 2009; Prinsloo & Heugh, 2013). Although higher academic success has become essential for entry into the 21st century economy (Jackson, 2015), academic English language proficiency remains a challenge for the majority of South African students in a linguistically diverse society (Andrade, 2006; Cross & Carpentier, 2009; Murray, 2010; Trenkic & Warmington, 2018). Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. Academic English proficiency in higher education encompasses formal and functional control of the properties of English language, including vocabulary, grammar and contextual understanding (Bridgeman, McBride, & Monaghan, 2004; Masrai & Milton, 2018; Murray, 2010). Limited English proficiency on entry may lead to academic vulnerability, characterised by unsuccessful adaptation to higher education demands, which could be detrimental to academic literacy, problem-solving techniques, constructive engagement in learning processes (Murray, 2010; Taylor & Von Fintel, 2016) and communications (Benzie, 2010; Murray, 2010; Trenkic & Warmington, 2018; Webb, 2002). Concomitantly, lack of capability in basic interpersonal communication skills (BISC; expression of conversational fluency) alongside cognitive academic language proficiency (CALP; decontextualised language proficiency) may synergistically impact the expression of general English language proficiency in multiple contexts (Bruton, Wisessuwan, & Tubsree, 2018; Cummins, 2000). This disadvantage is displayed where decontextualised language learning experiences in everyday learning and communications, linked to BISC, impact the learning of academic concepts, and thereby result in less than optimal CALP (Abriam-Yago, Yoder, & Kataoka-Yahiro, 1999; Tomasello, 2014). Thus, students lacking English language skills sufficient for the tertiary academic environment are placed at a disadvantage, even if basic literacy skills are sufficient. Read online: Scan this QR code with your smart phone or mobile device to read online. Introduction Thus, other research has indicated that briefer, basic ability tests, including Cloze procedure protocols and vocabulary assessments, are time- and cost-effective whilst retaining sufficient psychometric properties (Goto et al., 2010; Sun & Henrichsen, 2010). Although research has demonstrated that both Cloze procedure protocols and contextually based vocabulary tests may be used as proxies to understand English proficiency, these assessments are often conducted under time constraints, potentially confounding content performance with response time (e.g. Goto et al., 2010; Harrington & Roche, 2014; Masrai & Milton, 2018). Administration under time-constrained conditions remains a common practice for a variety of reasons but may result in decreased validity and reliability values (Van der Linden, 2011). Concomitantly, the test may then lack accuracy for its stated purpose, which is problematic for both selections and post-admission competency identification contexts. Therefore, a balance between internal consistency, predictive validity, length of assessment and other administration factors is required to enhance identification of the status of English language skills. The question then arises as to whether a sufficient balance of time-effectiveness, practicality and predictive validity is present when time constraints are implemented. Researchers have reported improvements in performance on various English language tests with additional time allocations (Bridgeman et al., 2004; Powers & Fowles, 1997), suggesting a focus on performance in complex understandings may be more important for academic outcomes than time- constrained responses (Daly & Stahmann, 1968; Harrington & Roche, 2014; MacIntyre & Gardner, 1994). The removal of time constraints may also mitigate other factors associated with poorer performance, including inadequate test-taking strategies, test anxiety and familiarity with testing contexts (Anderson, 1991; Fairbairn, 2007; Solano-Flores, 2008). Similar findings are present in the context of higher education, for which increased predictive validity, reliability and construct validity of Cloze procedure protocols and vocabulary tests have been reported when time constraints are removed (Hajebi, Taheri, & Allami, 2018; Snow et al., 2009; Trace et al., 2017). Cloze procedure protocols require the reader to insert missing words or phrases, illustrating semantic and contextual understanding linked to reading comprehension and writing skills (Gellert & Elbro, 2013; Trace, Brown, Janssen, & Kozhevnikova, 2017). Such skills are considered essential in higher education and significantly vulnerable for second-language English speakers, perhaps because of inability to decode new information and translate key words within specific contexts (Escamilla, 2009; Huettig, 2015; Staub, Grant, Astheimer, & Cohen, 2015). Introduction Read online: Scan this QR code with your smart phone or mobile device to read online. Open Access http://www.ajopa.org Page 2 of 9 Page 2 of 9 Original Research Acquired vocabulary has often been used as a proxy for general proficiency, demonstrating predictive power (Masrai & Milton, 2018; Trenkic & Warmington, 2018). Non-technical vocabulary levels have been further linked to academic writing, reading comprehension and general academic performance (Harrington & Roche, 2014; Qian, 2002; Schmitt, Jiang, & Grabe, 2011; Snow, Lawrence, & White, 2009; Trenkic & Warmington, 2018). These findings are supportive of the inclusion of vocabulary components in traditional gate-keeping tests, lending support for the use of these tests as a proxy for proficiency even post-admissions in first-year students. In both cases, the feasibility of reduction in time and cost is a significant benefit. Apart from basic literacy, the context of higher education often requires content-specific skills (linked to CALP; Cummins, 2000), which are reliant on technical vocabulary beside general contextual identification and understanding (Dalton-Puffer, 2011; Fenton-Smith, Humphreys, & Walkinshaw, 2018; Millin & Millin, 2018). Global research has implied that basic skills are a necessary component for developing technical/academic language (Birrell, 2006; Coleman, 2006). Consequentially, students lacking English proficiency skills, or exhibiting competency gaps, may be at an academic disadvantage on entering English language institutions. Internationally, English proficiency tests are frequently conducted pre-admission for selection purposes. Although these tests could be utilised for admitting students in first year, they are often time-consuming, expensive and focused on overall proficiency rather than critical basic skills more relevant to post-admissions phase (Arrigoni & Clark, 2015; Feast, 2002; Goto, Maki, & Kasai, 2010; Murray, 2010). These traditional gate-keeping tests include the International English Language Testing System (IETLS) and the Test of English as a Foreign Language (TOEFL). The viability and financial feasibility of utilising these assessments post-admissions to identify competency gaps is insufficient. Post-admissions, other options, including the Diagnostic English Language Test and Diagnostic English Language Needs Assessment, have been used globally for screening and diagnosis with good predictive and diagnostic validity (Doe, 2014; Read, 2008). Similar to pre-admission tests, the foci include vocabulary, speed- reading, listening and interpretation of texts. In both cases, complex, rather than base skills are inherent to the tests. Original Research Page 3 of 9 Original Research Page 3 of 9 semantics and syntax, although this may only be true for lengthy fragments in Cloze procedure protocols or when a wide range of possible responses is presented (Hajebi et al., 2018; Staub et al., 2015). Although this research has considered Cloze procedure protocols, vocabulary and other English proficiency tests without time constraints, limited published work (e.g. Goto, Maki, & Kasai, 2010) has considered different predictive validity of short assessments under various time constraints. Vocabulary in Context comprises 30 questions, each with four possible responses, of which one is correct (max = 30). No penalty scoring is implemented for either test. In this study, academic performance was assessed using percentages for the first-test marks for first-year subjects of national diploma courses in the department of tourism management (min = 1%, max = 100%). All marks obtained were above 0%. semantics and syntax, although this may only be true for lengthy fragments in Cloze procedure protocols or when a wide range of possible responses is presented (Hajebi et al., 2018; Staub et al., 2015). Although this research has considered Cloze procedure protocols, vocabulary and other English proficiency tests without time constraints, limited published work (e.g. Goto, Maki, & Kasai, 2010) has considered different predictive validity of short assessments under various time constraints. Instruments Kaleidoprax (2014) developed English Literacy Skills Assessment (ELSA) as two modified tests for the institute conducting the study: the Cloze procedure and the Vocabulary in Context tests. At present, no psychometric properties have been made available for the tests (Kaleidoprax, 2014). The Cloze procedure test requires the insertion of missing words within the context of a sentence. Cloze procedure comprises 20 questions, each with four possible responses, of which one is correct (max = 20). The Vocabulary in Context test identifies words in the context of a full sentence to require extrapolation of meaningof definitions, synonyms, antonyms and usage. Participants Participants comprised commencing first-year students (n = 81) enrolled in an institute for a tourism management national diploma course with common first-year academic subjects and admission requirements. The restriction for course enrolment was intended to indirectly standardise minimum English language entry criteria. The majority of enrolled first-year students at the institute were aged between 18 and 20 years, with a vast majority being of black ethnicity equally split between males and females. Examples were administered, and the test methods were explained, including the use of multiple-choice answer sheet, demands of the assessment and use of examples for familiarity and understanding. Participants were informed about relevant time limit and provided with a clock to monitor timings. Completed answer sheets were collected and checked for clarity of response prior to optical scanning and passing through a software program. Electronic data scores were collated with first-test subject performance marks from the institute’s management information systems. Data were anonymised and stored appropriately and securely for analysis. Research design The present research made use of a cross-sectional, quasi- experimental design to assess the impact of different time limits on performance of both Cloze procedure protocol and contextual vocabulary assessment. Procedure Data on the ELSA were generated as part of administration of a battery which took place after English language portion. The battery was solicited by the academic departments of the institute as part of a post-admissions first-year student assessment. Academic departments granted permission to modify the English language portion for research purposes, and all participants gave informed consent. No data were used for exclusionary, probationary or placement purposes. The present study assessed the relative influence of time limits on two English language proficiency tests, that is, a Cloze procedure protocol and contextual vocabulary assessment, to understand differences in the predictive validity under each time limit in determining first-test academic outcomes. The importance of this study lies in differentiation between English proficiency itself and the impact of time constraints on the expression of that proficiency in predicting academic outcomes. Thus, the study intends to contribute through further understanding English proficiency testing in terms of the potentially detrimental impact of time limitations on test outcomes. These findings are potentially useful in enhancing mass language post- admission screening to improve skills-targeted interventions which are time-efficient and effective. The full sample (n = 81) was broken down into three groups: Normal time limit (n = 44), double time limit (n = 23) and no time limit (n = 15). Separate test sessions took place for each group. Participants had freedom to join the group of their choice. Participation in the experimental group was voluntary, and verbal informed consent was obtained with written signatory. Because of the voluntary nature of participation, a convenience sample was produced. Resultantly, control for Grade-12 English performance and the size of groups were not possible. Voluntariness of participation, however, was essential because of the testing (personal development) and deviation from the normal quasi-experimental protocol. Thus, it was not possible to specifically split students in experimental and control groups whilst retaining the intent of the testing session and considering the autonomy. Introduction Decoding, recognition and translation to English (in the case of non- native speakers) have been closely related to Cloze procedure protocol performance in children and adults (Gellert & Elbro, 2013; Keenan, Betjemann, & Olson, 2008). These findings suggest that background and fundamental learning could play a role in developing essential skills which are transferable to higher education English language requirements. Similarly, vocabulary acquisition has been linked to success in the context of higher education. Researchers have hypothesised that changes in performance under different time constraints may be linked to the number of items attempted, changes to item structures or content functions operating differently (Luke & Christianson, 2016; Talento-Miller, Guo, & Han, 2013; Van der Linden, 2011). Other research has suggested that increased time may allow for better translation and internal reconstructions of http://www.ajopa.org Open Access Results The no time-limit group performed best on the Vocabulary in Context subtest (M = 12.07, SD = 4.98), whilst the double time-limit group’s scores were slightly lower (M = 11.13, SD = 5.36) and the normal time-limit group’s scores were considerably lower (M = 6.48, SD = 4.61). The one-way analysis of variance revealed that the groups differed significantly (F = 10.902, p = 0.000) and the requirement of homogeneity of variance was satisfied (F = 0.666, p = 0.517). Examination of Tukey’s HSD post hoc showed that the statistically significant differences were present between the normal time-limit group and the double time-limit group (MDifference = 4.653, p = 0.001) as well as the no time-limit group (MDifference = 5.589, p = 0.001). The no time-limit and double time-limit groups did not differ significantly (MDifference = 0.936, p = 0.833). Therefore, significant differences were observed between the three time-limit groups, suggesting that time limitations influenced measuring English language skills by these tests. As a result, the timed conditions may also have affected the predictive power of each test. The Cloze procedure subtest yielded a maximum score of 20, whilst the Vocabulary in Context subtest score was out of a possible 30. First-test subject marks were expressed as a percentage value out of 100 possible points. Table 1 shows the mean values (M) and standard deviations (SD) of variables. Table 1 shows similar levels of dispersion across different groups and subjects. Performance on the ELSA tests improved when time constraints were reduced but levels of dispersion remained stable despite differing sample sizes. No substantial differences in academic marks were present between the three time-limit groups. Differences between the time-limit groups The one-way analysis of variance with Tukey’s HSD post hoc revealed that the three time-limit groups differed significantly. The group without a time limit had higher scores on the Cloze procedure subtest (M = 13.93, SD = 4.30) than the double time-limit group (M = 12.22, SD = 4.40) or the normal time-limit group (M = 6.80, SD = 4.08). The one-way analysis of variance demonstrated that the groups differed significantly (F = 22.156, p = 0.000) and the Levene’s test of homogeneity of variance met the required assumption of equal variances (F = 0.100, p = 0.905). The significant differences were identified as involving the normal time- limit group, for which scores were significantly lower than that of the double time-limit group (MDifference = 5.442, Data analyses Data analyses were conducted on SPSS® version 25. Comparisons of the three time-limit groups were conducted using a one-way analysis of variance and Tukey’s Honest Significant Difference (HSD) post hoc test of mean differences and significances. Pearson’s r correlation coefficients and standard linear regression models (standardised beta weights because of range discrepancies) were used to assess the relationship between scores of tests and first-test marks. http://www.ajopa.org Open Access Page 4 of 9 Original Research p = 0.000) and the no time-limit group (MDifference = 7.138, p = 0.000). However, the no time-limit and double time-limit groups did not differ significantly, despite slightly better performance by the no time-limit group (MDifference = 1.716, p = 0.440). Similar findings were observed for the Vocabulary in Context subtest. Ethical consideration This study received ethical clearance from the Tshwane University of Technology Research Ethics Committee (No. REC/2016/09/001) Original Research Statistically significant positive correlations were present between the Cloze procedure subtest and the subject of ‘Communications’, which had a strong emphasis on English language. Similar coefficients were observed for the normal time-limit group (r = 0.437, p = 0.003) and the double time- limit group (r = 0.473, p = 0.023). A stronger statistically significant correlation was observed between the no time- limit group and the scores of the subject of ‘Communications’ (r = 0.706, p = 0.003). The no time-limit group scores were also significantly correlated with scores of the first-test of tourism development (r = 0.574, p = 0.025), whilst the normal time-limit group was less strongly, but more significantly, correlated (r = 0.373, p = 0.013). The same is true about correlations between travel and tourism practice and Cloze procedure for the normal time-limit group (r = 0.450, p = 0.002) and the no time-limit group (r = 0.656, p = 0.008). No other statistically significant correlation coefficients were present. The correlational findings tentatively suggested that higher scores on the Cloze procedure test were associated with better performance on the subjects of ‘Communications’, ‘Tourism Development’ and ‘Travel and Tourism Practice’. In most of the cases, the relationship between the scores and academic performance was strongest when no time limit was present, although the double time-limit coefficients were frequently similar. Significant positive correlation coefficients were also observed between the Vocabulary in Context test scores and the first-test subject marks, particularly if no time limit was implemented. Vocabulary in Context was more strongly associated with academic performance than the Cloze procedure. with performance on the first-test of various subjects of tourism management, particularly the subject of ‘Communications’. with performance on the first-test of various subjects of tourism management, particularly the subject of ‘Communications’. Regression models were used to understand the relative predictive power of different time limit groups of each subject. Table 3 shows the standardised beta weights, statistically significant levels of the Cloze procedure subtest and coefficients of determination reporting the amount of variance explained. When Cloze procedure is used as a predictor of the first-test marks, the regression on the subject of ‘Communications’ was strong, but the ‘Marketing for Tourism’ and ‘Travel and Tourism Management’ scores were not well predicted. Original Research Statistically significant increase in the SDs of first-test scores were associated with a single SD increase in Cloze procedure for the no time-limit group for the subjects of ‘Communications’ (β = 0.706, p = 0.003), ‘Tourism Development’ (β = 0.574, p = 0.025) and ‘Travel and Tourism Practice’ (β = 0.656, p = 0.008). However, a slight inverse predictive function was observed for ‘Travel and Tourism Management’ (β = -0.265, p = 0.013). The first-test scores for the subject of ‘Communications’ were also predicted by scores on the Cloze procedure for the normal time-limit group (β = 0.437, p = 0.003) and the double time-limit condition (β = 0.473, p = 0.023). The same was true for the subject of ‘Travel and Tourism Practice’ for the no time-limit (β = 0.656, p = 0.008), double time-limit (β = 0.407, p = 0.054) and normal time-limit groups (β = 0.450, p = 0.002). For the subject of ‘Travel and Tourism Practice’, all three conditions had similar predictive power. For the Cloze procedure, no time limits resulted in stronger strength of prediction than doubling the time limits or implementing the normal time limit. Similar findings were present for the Vocabulary in Context test. The coefficients of determination, standardised regression values and probability values for Vocabulary in Context are shown in Table 4. Correlations between the subject of ‘Communications’ scores and Vocabulary in Context scores were statistically significant for the normal time-limit (r = 0.313, p = 0.038), double time-limit (r = 0.600, p = 0.002) and no time-limit groups (r = 0.634, p = 0.011). The double time-limit group was also significantly correlated with ‘Travel and Tourism Practice’ scores (r = 0.544, p = 0.007). However, only the no time-limit group was statistically significantly correlated with the first-test marks on ‘Marketing for Tourism’ (r = 0.648, p = 0.009), ‘Tourism Development’ (r = 0.708, p = 0.003) and ‘Travel and Tourism Practice’ (r = 0.590, p = 0.210). For the Cloze procedure subtest, no statistically significant correlations were present with the first-test marks on ‘Travel and Tourism Management’. Prediction of first-test subject marks Pearson’s r correlation coefficients were calculated to examine the association between performance on the ELSA tests and performance in the first-test of each subject, followed by separate regression models for each group. Table 2 shows the correlation coefficients between the three time-limit mean values and subject performance. TABLE 1: Descriptive statistics for the English Literacy Skills Assessment tests and first-test subject marks by time-limit group. Assessment All groups Normal time limit Double time limit No time limit Mean (M) SD Mean (M) SD Mean (M) SD Mean (M) SD Cloze procedure 9.62 5.19 6.80 4.08 12.22 4.40 13.93 4.30 Vocabulary in Context 8.80 5.46 6.48 4.61 11.13 5.36 12.07 4.98 Communications 52.38 13.63 51.80 15.19 54.57 11.04 50.73 12.76 Marketing for Tourism 70.13 20.37 68.80 20.60 70.13 21.26 74.07 19.10 Tourism Development 70.63 15.10 68.41 15.45 71.57 15.91 75.73 11.98 Travel and Tourism Management 63.36 18.80 65.00 17.08 60.17 22.25 63.20 18.59 Travel and Tourism Practice 59.95 11.80 60.11 11.30 57.39 11.82 63.40 13.02 M, mean; SD, standard deviation. http://www.ajopa.org Open Access TABLE 2: Pearson’s r correlations between the English Literacy Skills Assessment tests and first-test subject marks by time-limit group. Academic subject Cloze procedure Vocabulary in context Normal time limit Double time limit No time limit Normal time limit Double time limit No time limit Communications 0.437** 0.473* 0.706** 0.313* 0.600** 0.634* Marketing for Tourism 0.252 0.318 0.331 0.131 0.393 0.648** Tourism Development 0.373* 0.359 0.574* 0.164 0.283 0.708 Travel and Tourism Management −0.049 0.104 −0.265 0.047 0.050 −0.138 Travel and Tourism Practice 0.450 0.407 0.656 0.265 0.544 0.590* , Significant at the 5% level. *, Significant at the 1% level. http://www.ajopa.org Open Access Page 5 of 9 Original Research Page 5 of 9 Page 6 of 9 Original Research TABLE 4: Regression values for the Vocabulary in Context test on the first-test subject marks by time-limit group. Academic subject Normal time limit Double time limit No time limit Constant R2 β p Constant R2 β p Constant R2 β p Communications 45.10 0.098 0.313 0.038 40.82 0.360 0.600 0.002 31.11 0.402 0.634 0.011 Marketing for Tourism 65.00 0.017 0.131 0.396 52.80 0.154 0.393 0.064 44.09 0.419 0.648 0.009 Tourism Development 64.85 0.027 0.164 0.287 62.24 0.080 0.283 0.191 55.17 0.502 0.708 0.003 Travel and Tourism Management 63.87 0.002 0.047 0.762 57.86 0.003 0.050 0.820 69.43 0.019 0.138 0.623 Travel and Tourism Practice 55.90 0.070 0.265 0.082 44.04 0.296 0.544 0.007 44.77 0.348 0.590 0.021 Note: R2 is the coefficient of determination representing the proportion of variance explained by the model. TABLE 4: Regression values for the Vocabulary in Context test on the first-test subject marks by time-limit group. Academic subject Normal time limit Double time limit constructs (Lu & Sireci, 2007), resulting in the absence of equivalency across instruments (Cronbach & Warrington, 1951). Additionally, a biased presentation of English language ability is present if response levels below certain thresholds occur, or without readjustment of item functions (e.g. Van der Linden, 2011). The present research findings of improved performance without time constraints cannot necessarily be equated to changes in reliability or validity per se because of the absence of measurement of item response functions, despite studies such as those performed by Harrington and Roche (2014) being focused on similar assessment types. Nonetheless, Talento-Miller et al. (2013) also suggested that increasing the number of items attempted influenced the outcome of English language tests because of the varying difficulties and types of items rather than processing speed. The evidence suggests that inherent, internal test-structure issues under time-constrained conditions are influential, and the present findings concurred that working under time constraints could have negatively affected performance on both ELSA tests for this cohort. Although some other research has explored the inherent reliability issues surrounding time limits on English tests, the reviewed literature has not extensively explored the relative impact of differing time limits on predictive validity in the context of higher education. The regression analyses in the present research provided evidence of a predictive component for the two ELSA tests utilised, which strengthened when time limitations were extended or nulled. Page 6 of 9 Original Research predictor, also having statistically significant power for the subjects of ‘Marketing for Tourism’ (β = 0.648, p = 0.009) and ‘Tourism Development’ (β = 0.708, p = 0.003). Like the Cloze procedure test, the regression of Vocabulary in Context test on the subject of ‘Travel and Tourism Management’ was poor and not statistically significant (p > 0.05). Both ELSA tests showed predictive power for the majority of the first-year subjects of tourism management based on statistically significant correlation coefficients and regression models. However, the no time-limit condition exhibited the strongest predictive power. Variance between ~33% and ~50% in academic first-test subject performance was explicable by English language proficiency measured on each of the two ELSA tests. In spite of not being significantly different from the no time-limit group, the double time-limit group did not show the same predictive relationship, potentially because of a truncated range of scores. The subject of ‘Travel and Tourism Management’, however, was not sufficiently associated with scores on either of the ELSA tests in terms of correlation or prediction. Original Research For both tests, the no time-limit group appeared to be the most strongly associated group The Vocabulary in Context scores had statistically significant regression values for the subject of ‘Communications’ for the normal time-limit group (β = 0.313, p = 0.038), double time- limit group (β = 0.600, p = 0.002) and no time-limit group (β = 0.634, p = 0.011). Standard deviation values of subjects were substantially increased with subtest increase for ‘Travel and Tourism Practice’ for both the double time-limit (β = 0.544, p = 0.007) and the no time-limit groups (β = 0.590, p = 0.021). However, the no time-limit group proved to be the strongest TABLE 3: Regression values for the Cloze procedure test on first-test subject marks by time-limit group. Academic subject Normal time limit Double time limit No time limit Constant R2 β p Constant R2 β p Constant R2 β p Communications 40.73 0.191 0.437 0.003 40.07 0.224 0.473 0.023 21.53 0.499 0.706 0.003 Marketing for Tourism 60.14 0.064 0.252 0.099 51.36 0.101 0.318 0.139 53.56 0.110 0.331 0.228 Tourism Development 58.81 0.139 0.373 0.013 55.70 0.129 0.359 0.092 53.47 0.329 0.574 0.025 Travel and Tourism Management 66.40 0.002 0.049 0.752 66.62 0.011 −0.104 0.635 79.17 0.139 −0.265 0.013 Travel and Tourism Practice 51.64 0.203 0.450 0.002 44.02 0.166 0.407 0.054 35.72 0.431 0.656 0.008 Note: R2 is the coefficient of determination representing the proportion of variance explained by the model. Open Access http://www.ajopa.org Page 6 of 9 Original Research Original Research Non-technical vocabulary levels have been successfully used as predictors in higher education institutions (HEIs) as well as a proxy for general English proficiency and Cloze procedures (Daller & Wang, 2014; Masrai & Milton, 2018; Qian, 2002; Schmitt et al., 2011; Snow et al., 2009; Trenkic & Warmington, 2018). The present study’s findings suggest that vocabulary levels were more important in accurately predicting academic success than the Cloze procedure test, which required semantic manipulation and decision-making within the context of a passage. However, vocabulary ability could be subsumed into a variety of English functions present in the HEI performance requirements, such as lecture participation and development of text understanding and technical vocabulary. Vocabulary may be linked to other aspects of English language performance related to higher education, including deliberate performance and response selection (Macalister, 2010), improved heuristic learning of phrases and lexical translation (Koehn, Och, & Marcu, 2003), speed of translation and decoding within a finite memory capacity system (Sakurai, 2015), and meta-cognitive focus on syntactical awareness beside reformulation between languages in an attempt at better understanding (Jiménez et al., 2015). Known to be influenced by time constraints, some of these factors directly relate to essential skills measured in vocabulary and Cloze procedure tests, including semantic representations, understanding of words in context, reading speed and quality and the ability to manipulate syntactical arguments. In spite of important findings in the present research, the study carried some limitations which created some uncertainty in the interpretation of the results. Groups of unequal sizes, because of the voluntary nature of participation, may have resulted in misrepresentation of values because of the use of parametric statistics in such a case (Rosenthal & Rosnow, 2008). Similarly, small groups and lack of randomisation could have affected the statistical outcomes. An example of this issue could be the negative correlations seen in for the subject of ‘Travel and Tourism Management’, although alternate explanations such as subject content could also account for this anomaly. Nonetheless, inequality in ranges of scores between different variables still resulted in Pearson’s r and a linear regression model being the most suitable choice, albeit imperfect. In addition, it was not possible to fully standardise the English language pre-entry (Grade 12) performance in this case.Therefore, this criterion was only passively standardised as a minimum level through the use of a specific qualification grouping of students. Original Research Pre-entry English ability could have impacted the outcome on either of the English proficiency tests, thus introducing bias in the results or impacting the selection of groups in an attempt by the participants to maximise their performance. Nonetheless, it is believed that present language ability, regardless of prior ability, is the most important factor in interpreting the findings, because the intention is to predict academic performance rather than investigate validity of the assessments in question. Furthermore, the results appear to indicate that time limitations imposed on English proficiency tests are of importance in fully applying the concept of language proficiency to higher education outcomes. Reported findings that English proficiency tests encompassing vocabulary, grammar and contextual representation are affected by time limitations (e.g. Bridgeman et al., 2004; Murray, 2010) were confirmed by the present research using two ELSA tests. Such content-specific skill development for understanding may require measurement outside of what could generally be considered as the normal, time-constrained and psychometrically focused framework. Furthermore, development of content/subject-specific technical language could also play a role in academic outcomes, particularly if basic levels have not been fully developed as a foundation (Birrell, 2006). The findings of the present research also suggest that time limitations play an important role in performance and predictive validity, beside choice of test for predictive purposes. Removal of time limitations resulted in more accurate prediction of academic success outcomes, and use of the Vocabulary in Context test resulted in the strongest predictive power. These findings suggested that appropriate English proficiency assessment could hinge more on the determination of specific academic weaknesses within English language whilst reducing the role of time limitation as an essential factor in predicting performance. In spite of the various findings suggesting that time constraints impact a variety of factors concerning English proficiency tests, from a practical perspective, it is unlikely that performing lengthy tests without time limits would be practical in the context of real world. Nonetheless, studies suggest that time constraints could alter the psychometric properties of tests in a variety of ways. Discussion The findings indicated that performance on both ELSA tests improved relatively to increase in time limitations. The statistics demonstrated that increased time limits resulted in a statistically significant improvement in performance, whilst the SD levels of mean values remained stable, suggesting that a consistent dispersion in scores was retained. Therefore, the findings reflected improvements in test outcome predictive quality when time limits are removed, despite the inherent limitations of comparing groups of differing sizes (Rosenthal & Rosnow, 2008). Nonetheless, similar improvements in English test outcomes were found by Hajebi et al. (2018) and Snow et al. (2009). In this regard, Harrington and Roche (2014) and Van der Linden (2011) also suggested that improvements in performance could be related to the more accurate assessment of constructs in the English language, rather than the ability to perform under time constraints. This disparity could be partially because of long-held notion of the influence of time constraints on the number of item responses and internal reliability of English proficiency tests themselves (Evans & Reilly, 1972). The double time-limit and no time-limit groups’ academic performance was positively and significantly correlated with performance on both ELSA tests, whilst the normal time- limit group demonstrated limited predictive power. Interestingly, predictive performance was similar for both double time-limit and no time-limit groups in most of the cases. This finding suggested that item response thresholds, such as those discussed by Van der Linden (2011) and Talento- Miller et al. (2013), could be important for predictive power as well as for internal consistency and reliability of measurement. Therefore, the present academic first-test performance could have been at least a partial function of English language ability, as measured by the ELSA tests. Several English language performance actions applied to the Cloze procedure protocol were used as one of the ELSA tests. However, in the present research, non-technical vocabulary levels measured in the context were found to be better predictors of academic performance. Similar studies have suggested that implementing time constraints could reduce the reliability and validity of psychometric and language tests for a wide variety of http://www.ajopa.org Open Access Page 7 of 9 Original Research Page 7 of 9 Conclusion The present research findings demonstrate that performance and predictive power on the modified ELSA versions of Cloze procedure and Vocabulary in Context improves when time limits are increased or removed. The findings imply that factors such as item completion thresholds, reading speed, semantic understanding, and translation for decision-making requirements could contribute to negative changes in performance under time-constrained conditions. Therefore, students may possess some of the English language skills associated with academic performance but are unable to demonstrate these skills within the imposed time constraints. Although these findings are useful, they should be treated with caution as current internal reliability and predictive validity data are not available for full assessment and this pilot study was conducted on smaller, unequal sample groups. Nonetheless, it is apparent that the English proficiency as measured by the ELSA could be inaccurately reflected under time-constrained conditions, limiting the ability of the test to serve as a predictor of academic performance in tertiary education. These findings imply that further investigations are required to develop sufficiently http://www.ajopa.org Open Access Page 8 of 9 Original Research competency gap-targeted English interventions, and the future research should consider larger-scale studies to identify specific components within the tests which contribute to academic success in South African HEIs. Cummins, J. (2000). Language, power, and pedagogy: Bilingual children in the crossfire. New York, NY: Multilingual Matters. ISBN: 9781853594731 Daller, M., & Wang, Y. (2017), Predicting study success of international students. Applied Linguistics Review, 8(4), 355–374. https://doi.org/10.1515/ applirev20162013 Dalton-Puffer, C. (2011). Content-and-language integrated learning: From practice to principles? Annual Review of Applied Linguistics, 31, 182–204. https://doi. org/10.1017/S0267190511000092 The author has declared that no competing interest exists. Escamilla, K. (2009). English language learners: Developing literacy in second-language learners – Report of the national literacy Panel on language-minority children and youth (D. August, & T. Shanahan, Eds.). Journal of Literacy Research, 41, 432–452. https://doi.org/10.1080/10862960903340165 References Huettig, F. (2015). Four central questions about prediction in language processing. Brain Research, 1626, 118–135. https://doi.org/10.1016/j.brainres.2015.02.014 Jackson, D. (2015). Employability skill development in work-integrated learning: Barriers and best practice. Studies in Higher Education, 40(2), 350–367. https:// doi.org/10.1080/03075079.2013.842221 Abriam-Yago, K., Yoder, M., & Kataoka-Yahiro, M. (1999). The Cummins model: A framework for teaching nursing students for whom English is a second language. Journal of Transcultural Nursing, 10(2), 143–149. https://doi.org/10.1177/10436 5969901000208 Jiménez, R.T., David, S., Fagan, K., Risko, V.J., Pacheco, M., Pray, L., et al. (2015). Using translation to drive conceptual development for students becoming literate in English as an additional language. Research in the Teaching of English, 49, 248–271. Anderson, N.J. (1991). Individual differences in strategy use in second language reading and testing. The Modern Language Journal, 75(4), 460–472. https://doi. org/10.1111/j.1540-4781.1991.tb05384.x Kaleidoprax. (2014).What ELSA measures? Retrieved n.d. from https://www. kaleidoprax.co.za/english-literacy-skills-assessment.html Andrade, M.S. (2006). International students in English-speaking universities. Journal of Research in International Education, 5(2), 131–154. https://doi.org/10. 1177/1475240906065589 Keenan, J.M., Betjemann, R.S., & Olson, R.K. (2008). Reading comprehension tests vary in the skills they assess: Differential dependence on decoding and oral comprehension. Scientific Studies of Reading, 12(3), 281–300. https://doi. org/10.1080/10888430802132279 Arrigoni, E., & Clark, V. (2015). Investigating the appropriateness of IELTS cut-off scores for admissions and placement decisions at an English-medium university in Egypt. IELTS Research Report Series. Retrieved from https://www.ielts.org/teaching- and-research/research-reports Koehn, P., Och, F.J., & Marcu, D. (2003). Statistical phrase-based translation. In Proceedings of the 2003 Conference of the North American Chapter of the Association for Computational Linguistics on Human Language Technology (NAACL ‘03) (vol. 1, pp. 48–54). Edmonton: Association for Computational Linguistics. https://doi.org/10.3115/1073445.1073462 Bedenlier, S., & Zawacki-Richter, O. (2015). Internationalization of higher education and the impacts on academic faculty members. Research in Comparative & International Education, 10(2), 185–201. https://doi.org/10.1177/174549 9915571707 Lu, Y., & Sireci, S.G. (2007). Validity issues in test speededness. Educational Measurement: Issues and Practice, 26(4), 29–37. https://doi.org/10.1111/j.1745- 3992.2007.00106.x Benzie, H.J. (2010). Graduating as a ‘native speaker’: International students and English language proficiency in higher education. Higher Education Research & Development, 29(4), 447–459. https://doi.org/10.1080/07294361003598824 Luke, S.G., & Christianson, K. (2016). Limits on lexical prediction during reading. Cognitive Psychology, 88, 22–60. https://doi.org/10.1016/j.cogpsych.2016.06.002 Birrell, B. (2006). Implication of low English standards among overseas students at Australian universities. People and Place, 14(4), 53–64. Macalister, J. (2010). Investigating teacher attitudes to extensive reading practices in higher education: Why isn’t everyone doing it? RELC Journal, 41(1), 59–75. Funding information Fairbairn, S. (2007). Facilitating greater test success for English language learners. Practical Assessment, Research & Evaluation, 12(11), 1–7. The research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Feast, V. (2002). The impact of IETLS scores on performance at university. International Education Journal, 3(4), 70–85. Fenton-Smith, B., Humphreys, P., & Walkinshaw, I. (2018). On evaluating the effectiveness of university-wide credit-bearing English language enhancement courses. Journal of English for Academic Purposes, 31, 72–83. https://doi. org/10.1111/j.1745-3984.1972.tb00767.x Disclaimer Hajebi, M., Taheri, S.Q., & Allami, H. (2018). A comparative study of cloze test and C-test in assessing collocational competence of Iranian EFL learners. European Online Journal of Natural and Social Sciences, 7(1), 225–234. The views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. Harrington, M., & Roche, T. (2014). Identifying academically at-risk students at a English-as-a-Lingua-Franca university setting. Journal of English for Academic Purposes, 15, 37–47. https://doi.org/10.1016/j.jeap.2014.05.003 I declare that I am the sole author of this research article. I declare that I am the sole author of this research article. Evans, F., & Reilly, R. (1972). A study of speededness as a source of test bias. Journal of Educational Measurement, 9(2), 123–131. https://doi.org/10.1111/j.1745-3984. 1972.tb00767.x Data availability statement Gellert, A.S., & Elbro, C. (2013). Cloze tests may be quick, but are they dirty? Development and preliminary validation of a Cloze test of reading comprehension. Journal of Psychoeducational Assessment, 31(1), 16–28. https://doi.org/ 10.1177/0734282912451971 Data sharing is negotiable by request. Sharing of data cannot Data sharing is negotiable by request. Sharing of data cannot be guaranteed and will depend on the nature of the request Data sharing is negotiable by request. Sharing of data cannot be guaranteed and will depend on the nature of the request. be guaranteed and will depend on the nature of the request. Goto, K., Maki, H., & Kasai, C. (2010). The minimal English test: A new method to measure English as a second language proficiency. Evaluation & Research in Education, 23(2), 91–104. https://doi.org/10.1080/09500791003734670 Acknowledgements Competing interests Daly, J.L., & Stahmann, R.F. (1968). The effect of time-limits on a university placement test. The Journal of Educational Research, 62(3), 103–104. https://doi.org/10.108 0/00220671.1968.10883779 Doe, C. (2014). Diagnostic English language needs assessment. Language Testing, 31(4), 537–543. https://doi.org/10.1177/0265532214538225 The author has declared that no competing interest exists. References Bridgeman, B., McBride, A., & Monaghan, W. (2004). Testing and time-limits. Princeton, NJ: Educational Testing Services. MacIntyre, P.D., & Gardner, R.C. (1994). The subtle effects of language anxiety on cognitive processing in the second language. Language Learning, 44(2), 283–305. Bruton, C., Wisessuwan, A., & Tubsree, C. (2018). Praxial interlanguage experience: Developing communicative intentionality through experiential and contemplative inquiry in international education. HRD Journal, 9(1), 27–36. Masrai, A., & Milton, J. (2018). Measuring the contribution of academic and general vocabulary knowledge to learners’ academic achievement. Journal of English for Academic Purposes, 31, 44–57. https://doi.org/10.1177/0033688210362609 Casale, D., & Posel, D. (2011). English language proficiency and earnings in a developing country: The case of South Africa. Journal of Behavioral and Experimental Economics 40(4), 385–393. Millin, T., & Millin, M. (2018). English academic writing convergence for academically weaker senior secondary school students: Possibility or pipe-dream? Journal of English for Academic Purposes, 31, 1–17. https://doi.org/10.1016/j.jeap.2017. 12.002 Coleman, J.A. (2006). English-medium teaching in European higher education. Language Teaching, 39(1), 1–14. https://doi.org/10.1017/S026144480600320X Murray, N.L. (2010). Conceptualising the English language needs of first year university students. The International Journal of the First Year in Higher Education, 1(1), 55–64. https://doi.org/10.5204/intjfyhe.v1i1.19 Cronbach, L.J., & Warrington, W.G. (1951). Time-limit tests: Estimating their reliability and degree of speeding. Psychometrika, 16(2), 167–188. https://doi.org/10.1007/ BF02289113 Nunan, D. (2003). The impact of English as a global language on educational policies and practices in the Asia-Pacific region. TESOL Quarterly, 37(4), 589–613. https:// doi.org/10.5204/intjfyhe.v1i1.19 Cross, M., & Carpentier, C. (2009). ‘New students’ in South African higher education: Institutional culture, student performance and the challenge of democratisation. Perspectives in Education, 27(1), 6–18. Open Access http://www.ajopa.org Original Research Page 9 of 9 Page 9 of 9 Powers, D.E., & Fowles, M.E. (1997). Effects of applying different time-limits to a proposed GRE writing test. Princeton, NJ: Educational Testing Service. Staub, A., Grant, M., Astheimer, L., & Cohen, A. (2015). The influence of cloze probability and item constraint on cloze task response time. Journal of Memory and Language, 82, 1–17. https://doi.org/10.3102/0013189X08319569 Prinsloo, C.H., & Heugh, K. (2013). The role of language and literacy in preparing South African learners for educational success: Lessons learnt from a classroom study in Limpopo province. Pretoria: Human Sciences Research Council. Sun, C., & Henrichsen, L. (2010). Major university English tests in China: Their importance, nature and development. TESL Reporter, 44, 1–24. Qian, D. (2002). Webb, V. (2002). English as a second language in South Africa’s tertiary institutions: A case study at the University of Pretoria. World Englishes, 21(1), 49–61. https://doi. org/10.1111/1467-971X.00231 References Investigating the relationship between vocabulary knowledge and academic reading performance: An assessment perspective. Language Learning, 52(3), 513–536. https://doi.org/10.1111/1467-9922.00193 Talento-Miller, E., Guo, F., & Han, K.T. (2013). Examining test speededness by native language. International Journal of Testing, 13, 89–104. https://doi.org/10.1080/1 5305058.2011.653021 Read, J. (2008). Identifying academic language needs through diagnostic assessment. Journal of English for Academic Purposes, 7(3), 180–190. https://doi.org/ 10.1016/j.jeap.2008.02.001 Taylor, S., & von Fintel, M. (2016). Estimating the impact of language instruction in South African primary schools: A fixed effects approach. Economics of Education Review, 50, 75–89. https://doi.org/10.1016/j.econedurev.2016.01.003 Rosenthal, R., & Rosnow, R.L. (2008). Essentials of behavioral research (3rd edn.). New York, NY: McGraw Hill. Tomasello, M. (2014). A natural history of human thinking. Boston, MA: Harvard University Press. ISBN: 9780674724778 Sakurai, N. (2015). The influence of translation on reading amount, proficiency, and speed in extensive reading. Reading in a Foreign Language, 27(1), 96–112. ISSN 1539-0578. Trace, J., Brown, J.D., Janssen, G., & Kozhevnikova, L. (2017). Determining cloze item difficulty from item passage characteristics across different learner backgrounds. Language Testing, 34(2), 151–174. https://doi.org/10.1177/ 0265532215623581 Schmitt, N., Jiang, X., & Grabe, W. (2011). The percentage of words known in a text and reading comprehension. The Modern Language Journal, 95, 26–43. https:// doi.org/10.1111/j.1540-4781.2011.01146.x Trenkic, D., & Warmington, M. (2018). Language and literacy skills of home and international university students: How different are they, and does it matter? Bilingualism: Language and Cognition, 22(2), 349–365. https://doi.org/10.1017/ S136672891700075X Snow, C.E., Lawrence, J.F., & White, C. (2009). Generating knowledge of academic language among urban middle school students. Journal of Research on Educational Effectiveness, 2(4), 325–344. https://doi.org/10.1080/19345740 903167042 Van der Linden, W.J. (2011). Test design and speededness. Journal of Educational Measurement, 48(1), 44–60. https://doi.org/10.1111/j.1745-3984.2010.00130.x Solano-Flores, G. (2008). Who is given tests in what language by whom, when and where? The need for probabilistic views of language in the testing of English language learners. Educational Researcher, 37(4), 189–199. https://doi. org/10.3102/0013189X08319569 Webb, V. (2002). English as a second language in South Africa’s tertiary institutions: A case study at the University of Pretoria. World Englishes, 21(1), 49–61. https://doi. org/10.1111/1467-971X.00231 http://www.ajopa.org Open Access
https://openalex.org/W3197539777	https://europepmc.org/articles/pmc8467015?pdf=render	English	null	System for Monitoring Progress in a Mixing and Grinding Machine Using Sound Signal Processing	Micromachines	2,021	cc-by	11,749	Citation: Wangkanklang, E.; Koike, Y. System for Monitoring Progress in a Mixing and Grinding Machine Using Sound Signal Processing. Micromachines 2021, 12, 1041. https://doi.org/10.3390/mi12091041 Article System for Monitoring Progress in a Mixing and Grinding Machine Using Sound Signal Processing Ekkawit Wangkanklang and Yoshikazu Koike * Ekkawit Wangkanklang and Yoshikazu Koike * Electro-Mechanical System Laboratory, Department of Electronics Engineering, Shibaura Institute of Technology, Tokyo 135-8548, Japan; nb17102@shibaura-it.ac.jp * Correspondence: koikey@shibaura-it.ac.jp Abstract: In this paper we present a system for monitoring progress in a mixing and grinding ma- chine via the signal processing of sound emitted by the machine. Our low-cost, low-maintenance system may improve automatic machines and the industrial Internet of Things. We used the Pump- kin Pi board and Raspberry Pi, which are low-cost hardware devices, for recording sounds via a microphone and analyzing the sound signals, respectively. Sound data obtained at regular intervals were compressed. The estimated power spectral density (PSD) values calculated from the sound signals were related to the status of the material during mixing and grinding. We examined the correlation between the PSD obtained by the STFT and the particle distributions in detail. We found that PSD values had both repeatability and a strong relation with the particle distributions that were created by the mixing and grinding machine, although the relation between the PSD and the particle size distributions was not merely linear. We used the PSD values to estimate the progress remotely during the operation of the machine. Citation: Wangkanklang, E.; Koike, Y. System for Monitoring Progress in a Mixing and Grinding Machine Using Sound Signal Processing. Micromachines 2021, 12, 1041. https://doi.org/10.3390/mi12091041 Academic Editor: Aiqun Liu Received: 29 July 2021 Accepted: 28 August 2021 Published: 29 August 2021 Keywords: Internet of Things; mixing and grinding machine; power spectral density; short-time Fourier transform; sound signal processing micromachines micromachines 1. Introduction Mortars and pestles have traditionally been used to mix and grind materials, such as rice powder, peanuts, and pottery. However, machines are now used for mixing and grinding to produce large quantities of high-quality ground materials. In particular, the Ishikawa mixing and grinding machine is used in industrial manufacturing processes [1] for various types of materials, including those for electronic parts, chemical products, and art supplies, as well as some special materials, such as materials for solar batteries and ﬂuorescent paint or gold powder. Received: 29 July 2021 Accepted: 28 August 2021 Published: 29 August 2021 p g p Systems for monitoring mixing and grinding while the machine operates are critical. However, currently, operators cannot remotely observe the progress of mixing and grinding. Methods of checking the status of the material during the process, such as by using a microscope or conducting a sieving analysis, require the machine to be stopped to collect samples at regular intervals and are inefﬁcient due to the increase in processing time and high cost of operation. Thus, the development of methods for remotely monitoring progress is highly desirable. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Advancements in the Internet of Things (IoT) have led to the development of online monitoring systems. For instance, Dhingra et al. proposed real-time monitoring using the Arduino integrated development environment software with a Wi-Fi communication module. The sensor network monitors air pollution or particles, and data are sent to a cloud system. Users can check pollution levels via an Android application, IoT-Mobair [2]. López-Vargas et al. proposed an online monitoring system on a website and a mobile application for solar measurement based on Arduino [3]. Lv et al. designed a monitoring system with an IoT technology for a smart city using a Zigbee wireless network for moni- toring and controlling temperature, lighting, air conditioning, and various appliances [4]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/micromachines Micromachines 2021, 12, 1041. https://doi.org/10.3390/mi12091041 2 of 13 Micromachines 2021, 12, 1041 Kumar et al. reported a lightweight, secure session key establishment scheme for smart home environments. 1. Introduction They used a short authentication token and established a secure session key between a gateway network and smart devices [5]. To reduce home energy use, Han et al. monitored the energy consumption of home appliances with the Zigbee module and monitored the generation of renewable energy via power line communication [6]. For industrial manufacturing, Industry 4.0 is focusing on smart hardware and real-time data. Han et al. proposed a system for monitoring and predicting air pollution in the manufacturing industry. They designed sensors for measuring parameters such as carbon monoxide, nitrogen dioxide, sulfur dioxide, ozone, particulate matter, temperature, and humidity, and they collected the sensor data with the Zigbee network [7]. Zhang et al. investigated IoT technologies in manufacturing in order to obtain a system with an online monitoring architecture for steel casting [8]. Sung and Hsu reported an industrial real-time measurement and monitoring system using Zigbee and a data acquisition application. They designed and developed a linear variable differential transformer sensor, current sensor, carbon dioxide sensor, and energy monitor [9]. Deep learning can be utilized in future work, as claimed by [10]. This study proved that AI can be used to analyze and classify types of sound in a sample. Various sensor technologies have been used to measure particle size. For example, Hu et al. used acoustic emission signals and signal analysis to measure the particle size of a pneumatic conveyer [11], and Carter and Yan [12] evaluated an electrostatic sensor and imaging sensor in order to measure the mass ﬂow rate and size distribution of particles in a pneumatic suspension. Mao and Towhata monitored the particle size during the crushing process using acoustic emissions [13]. In addition, Carter et al. [14] measured particle size in a pneumatic suspension using digital image processing. However, these methods are complicated and inconvenient for monitoring progress in mixing and grinding machines. p g p g g g g Sound analysis has been widely used in many ﬁelds. For example, Takamichi et al. [15] created the CogKnife to identify types of food using a microphone sensor to record the cutting sound, which was analyzed by the system using spectrograms. Although their method could be used to evaluate the type of food with high accuracy, it could not be used to identify foods that produced little noise, such as tofu or jelly. Chitnaont et al. 2. Pumpkin Pi 2.2. Pumpkin Pi 2 2 Pumpkin Pi The Pumpkin Pi expansion board was developed by Marutsu, Japan, and is intended r experimenting with and measuring high-resolution audio signals. The Raspberry Pi odel B+ does not have an analog input signal function and is disabled for voice recogni- on and measurement; therefore, the Pumpkin Pi board was developed to solve this prob- m. The Pumpkin Pi is an analog-to-digital (A-D) converter that consists of an 18-bit A- converter (MCP3422, Microchip Technology Inc., Chandler, AZ, USA) for sensor meas- rements and an A-D converter (PCM1808, Texas Instruments, Dallas, TX, USA) for the nalog input of 24 bit/s and 96 kHz used for audio measurements. The analog audio input as a maximum bit rate of 24 bit/s and a sampling frequency of 48 or 96 kHz via pulse ode modulation (PCM). Figure 2 shows a diagram of the structure of the Pumpkin Pi. The Pumpkin Pi expansion board was developed by Marutsu, Japan, and is intended for experimenting with and measuring high-resolution audio signals. The Raspberry Pi model B+ does not have an analog input signal function and is disabled for voice recognition and measurement; therefore, the Pumpkin Pi board was developed to solve this problem. The Pumpkin Pi is an analog-to-digital (A-D) converter that consists of an 18-bit A-D converter (MCP3422, Microchip Technology Inc., Chandler, AZ, USA) for sensor measurements and an A-D converter (PCM1808, Texas Instruments, Dallas, TX, USA) for the analog input of 24 bit/s and 96 kHz used for audio measurements. The analog audio input has a maximum bit rate of 24 bit/s and a sampling frequency of 48 or 96 kHz via pulse code modulation (PCM). Figure 2 shows a diagram of the structure of the Pumpkin Pi. 2.2. Pumpkin Pi The Pumpkin Pi expansion board was developed by Marutsu, Japan, and is intended for experimenting with and measuring high-resolution audio signals. The Raspberry Pi model B+ does not have an analog input signal function and is disabled for voice recogni- tion and measurement; therefore, the Pumpkin Pi board was developed to solve this prob- lem. The Pumpkin Pi is an analog-to-digital (A-D) converter that consists of an 18-bit A- D converter (MCP3422, Microchip Technology Inc., Chandler, AZ, USA) for sensor meas- urements and an A-D converter (PCM1808, Texas Instruments, Dallas, TX, USA) for the analog input of 24 bit/s and 96 kHz used for audio measurements. 2.1. Mixing and Grinding Machine 1. Mixing and Grinding Machine 2. Materials and Methodology 2 1 Mixing and Grinding Machine Figure 1 shows the mixing and grinding machine (model D101S, Ishikawa Kojo Co., Ltd., Tokyo, Japan), which simultaneously mixes and grinds and consists of a mortar, two pestles, a stirring bar, and an electrical motor. The machine rotates the pestles around the mortar with a gear rotation. The materials are mixed with the stirring bar and ground by the pestle tip as it spins around the bottom of the mortar. There are many types of ceramic mortars and pestles depending on the material being ground. For instance, a stone mortar and wood pestle are used for food processing, and a porcelain mortar and pestle are used for hard materials, such as those used in semiconductors. In this experiment, we used a porcelain mortar and pestle. The inverter in the machine for setting the motor speed could be set to 8–40 rpm by a controller, and the timer relay could be set to run the machine for periods of up to 12 h. Figure 1 shows the mixing and grinding machine (model D101S, Ishikawa Kojo Co., td., Tokyo, Japan), which simultaneously mixes and grinds and consists of a mortar, two estles, a stirring bar, and an electrical motor. The machine rotates the pestles around the ortar with a gear rotation. The materials are mixed with the stirring bar and ground by e pestle tip as it spins around the bottom of the mortar. There are many types of ceramic ortars and pestles depending on the material being ground. For instance, a stone mortar nd wood pestle are used for food processing, and a porcelain mortar and pestle are used r hard materials, such as those used in semiconductors. In this experiment, we used a orcelain mortar and pestle. The inverter in the machine for setting the motor speed could e set to 8–40 rpm by a controller, and the timer relay could be set to run the machine for eriods of up to 12 h. 2.1. Mixing and Grinding Machine Figure 1 shows the mixing and grinding machine (model D101S, Ishikawa Kojo Co., Ltd., Tokyo, Japan), which simultaneously mixes and grinds and consists of a mortar, two pestles, a stirring bar, and an electrical motor. The machine rotates the pestles around the mortar with a gear rotation. The materials are mixed with the stirring bar and ground by the pestle tip as it spins around the bottom of the mortar. 1. Introduction classiﬁed the sounds of a type of Thai ﬂute called a khlui made from bamboo, pradu wood, and plastic [16]. The sounds were recorded by using a microphone and analyzed using spectral centroids. Bastari et al. proposed an acoustic signal processing system for measuring the particle size of coal powder. They studied the relationship between acoustic emission signals and powder particle size distribution [17]. Quino et al. monitored the sound of ﬁbers breaking in E-glass ﬁber bundles by using Matlab and found that the system could detect the initiation and progression of failures [18]. De Cola et al. studied three types of sand grain shapes using sound measurements combined with microscopy [19]. Sen and Kumar Bhaumik reported a mathematical model of the sound emissions created during ball milling; however, the sound model is not suitable for mixing and grinding machines because it is limited to hard materials [20]. The mixing and grinding process has been employed for a long time in manufacturing processes. Recently, the demand for the application of IoT or deep learning to the mixing and grinding process has increased. For such extensions, a new and simple method of knowing the progress of manufacturing must be proposed, and it can be implemented in an existing mixing and grinding process without complicated remodeling or high costs. Here, we describe a method for estimating the progress in Ishikawa mixing and grinding machines using sound signal processing. The sound emissions are similar to noise; however, the noise level changes with the status of the grinding and mixing material. The sound is recorded and signal processing is performed. To validate the method, sieve analysis is used to determine the particle size distribution. The progress data are then compressed and sent through a wireless network to monitor the progress remotely. To extend the application to machine learning, we also prepared detailed data as training data. 3 of 13 Micromachines 2021, 12, 1041 Therefore, we propose the combination of the STFT and PSD in the sound waveforms of the mixing and grinding process. ata. Therefore, we propose the combination of the STFT and PSD in the sound wave- rms of the mixing and grinding process. To extend the application to machine learning, we also prepared detailed data as training data. Therefore, we propose the combination of the STFT and PSD in the sound wave- forms of the mixing and grinding process. 1. Introduction Therefore, we propose the combination of the STFT and PSD in the sound waveforms of the mixing and grinding process. ata. Therefore, we propose the combination of the STFT and PSD in the sound wave- rms of the mixing and grinding process. To extend the application to machine learning, we also prepared detailed data as training data. Therefore, we propose the combination of the STFT and PSD in the sound wave- forms of the mixing and grinding process. 2.1. Mixing and Grinding Machine 1. Mixing and Grinding Machine 2. Materials and Methodology 2 1 Mixing and Grinding Machine There are many types of ceramic mortars and pestles depending on the material being ground. For instance, a stone mortar and wood pestle are used for food processing, and a porcelain mortar and pestle are used for hard materials, such as those used in semiconductors. In this experiment, we used a porcelain mortar and pestle. The inverter in the machine for setting the motor speed could be set to 8–40 rpm by a controller, and the timer relay could be set to run the machine for i d f t 12 h (a) (b) gure 1. Mixing and grinding machine. (a) Schematic of the machine, (b) Whole picture of the achine. Figure 1. Mixing and grinding machine. (a) Schematic of the machine, (b) Whole picture of the machine. periods of up to 12 h. (a) (b) Figure 1. Mixing and grinding machine. (a) Schematic of the machine, (b) Whole picture of the machine. (a) p p (a) (b) (b) (b) (b (a) (a gure 1. Mixing and grinding machine. (a) Schematic of the machine, (b) Whole picture of the achine. Figure 1. Mixing and grinding machine. (a) Schematic of the machine, (b) Whole picture of the machine. Figure 1. Mixing and grinding machine. (a) Schematic of the machine, (b) Whole picture of the machine. 2.3. Methodology We used soun We used sound signal processing to monitor the progress of the mixing and grinding machine. A block diagram of the sound signal analysis is shown in Figure 3. The system consisted of recorded sound data, analysis signals, and average power spectral density (PSD) data. First, the sound level from the mixing and grinding process was recorded with an electric condenser microphone (ECMPC60, Sony Corp., Tokyo, Japan) with frequency responses between 50 and 15,000 Hz, low noise, and high sensitivity (−38 ± 3.5 dB). The microphone was installed in a cylinder to reduce powder adhesion and was placed on the back cover of the machine, 200 mm from the bottom mortar. The microphone recorded audio data and was connected via a 3.5 mm jack to the ampliﬁer. Analog signals that passed through the ampliﬁer (20–50 dB, AT-MA2, Audio-Technica Corp., Tokyo, Japan) were ampliﬁed with a gain of 35 dB. Second, we used the A-D convertor on the Pumpkin Pi board to convert the signal into a digital signal at a bit rate of 16 bit/s and a sampling frequency of 48 kHz. The Raspberry Pi board [21] was combined with the Pumpkin Pi board [22] to save data logs by recording the sound at regular intervals and analyzing the signal. We saved the data as WAV ﬁles and calculated the PSD using the short-time Fourier transform (STFT) [23] in Python on the Raspberry Pi. Finally, we compressed the data by calculating the average PSD value, which was shown in the real-time monitor, by using a transistor–transistor logic serial cable (FTDI-232R, Future Technology Devices International Ltd., Singapore). Owing to the compression of the data, we could remotely monitor the progress of the mixing and grinding process by using a wireless communication technology, such as WiFi. The PSD data that were received with a timestamp were employed a as database for the estimation of progress. Therefore, the conventional mixing and grinding machine, which did not have a sensing device for monitoring, could be connected to the IoT. machine. A block diagram of the sound signal analysis is shown in Figure 3. The system consisted of recorded sound data, analysis signals, and average power spectral density PSD) data. 2.3. Methodology We used soun First, the sound level from the mixing and grinding process was recorded with an electric condenser microphone (ECMPC60, Sony Corp., Tokyo, Japan) with frequency esponses between 50 and 15,000 Hz, low noise, and high sensitivity (‒38 ± 3.5 dB). The microphone was installed in a cylinder to reduce powder adhesion and was placed on the back cover of the machine, 200 mm from the bottom mortar. The microphone recorded audio data and was connected via a 3.5 mm jack to the amplifier. Analog signals that passed through the amplifier (20–50 dB, AT-MA2, Audio-Technica Corp., Tokyo, Japan) were amplified with a gain of 35 dB. Second, we used the A-D convertor on the Pumpkin Pi board to convert the signal into a digital signal at a bit rate of 16 bit/s and a sampling requency of 48 kHz. The Raspberry Pi board [21] was combined with the Pumpkin Pi board [22] to save data logs by recording the sound at regular intervals and analyzing the ignal. We saved the data as WAV files and calculated the PSD using the short-time Fou- ier transform (STFT) [23] in Python on the Raspberry Pi. Finally, we compressed the data by calculating the average PSD value, which was shown in the real-time monitor, by using a transistor–transistor logic serial cable (FTDI-232R, Future Technology Devices Interna- ional Ltd., Singapore). Owing to the compression of the data, we could remotely monitor he progress of the mixing and grinding process by using a wireless communication tech- nology, such as WiFi. The PSD data that were received with a timestamp were employed a as database for the estimation of progress. Therefore, the conventional mixing and grind- ng machine, which did not have a sensing device for monitoring, could be connected to he IoT. Figure 3. Block diagram for sound signal analysis. Figure 3. Block diagram for sound signal analysis. Figure 3. Block diagram for sound signal analysis Figure 3. Block diagram for sound signal analysis. 2. Pumpkin Pi 2.2. Pumpkin Pi 2 2 Pumpkin Pi The analog audio input has a maximum bit rate of 24 bit/s and a sampling frequency of 48 or 96 kHz via pulse code modulation (PCM). Figure 2 shows a diagram of the structure of the Pumpkin Pi. igure 2. The structure of the developed Pumpkin Pi. Figure 2. The structure of the developed Pumpkin Pi. Figure 2. The structure of the developed Pumpkin Pi. gure 2. The structure of the developed Pumpkin Pi. Figure 2. The structure of the developed Pumpkin Pi. Figure 2. The structure of the developed Pumpkin Pi. 4 of 13 of 14 Micromachines 2021, 12, 1041 , , 4 E ti ti PSD 2.4. Estimating PSD 2.4. Estimating PSD PSD estimation was used to analyze the sounds emitted from the mixing and grind- ng machine. The PSD is usually calculated with the Fourier transform by using paramet- ric methods, such as an autoregressive model or moving average model, or by using non- parametric methods, such as a periodogram, Bartlett’s method, or Welch’s method [24]. We calculated the estimated PSD through a spectrogram by using the STFT to divide the analyzed signal into particular segments, such as frame lengths. Then, the Fourier trans- orm was calculated and multiplied by a window function that overlapped at 50% to avoid spectral leakage. Many window function methods have been used, such as the Hanning window, Hamming window, Blackman, Gaussian, and Kaiser–Bessel methods. In gen- eral, the Hanning window has a good frequency resolution and reduced spectra. How- ever, the Hamming window does a better job of cancelling the nearest side lobe [25]. It was confirmed that the PSD values obtained with the Hamming window were almost the same as the values obtained with the Hanning window. Therefore, we chose the PSD estimation was used to analyze the sounds emitted from the mixing and grinding machine. The PSD is usually calculated with the Fourier transform by using parametric methods, such as an autoregressive model or moving average model, or by using non- parametric methods, such as a periodogram, Bartlett’s method, or Welch’s method [24]. We calculated the estimated PSD through a spectrogram by using the STFT to divide the analyzed signal into particular segments, such as frame lengths. Then, the Fourier transform was calculated and multiplied by a window function that overlapped at 50% to avoid spectral leakage. Many window function methods have been used, such as the Hanning window, Hamming window, Blackman, Gaussian, and Kaiser–Bessel methods. In general, the Hanning window has a good frequency resolution and reduced spectra. However, the Hamming window does a better job of cancelling the nearest side lobe [25]. It was conﬁrmed that the PSD values obtained with the Hamming window were almost the same as the values obtained with the Hanning window. Therefore, we chose the Hamming window for the STFT and calculated the PSD values. 3.1. Peanut Butter 3.1. Peanut Butter We mixed and We mixed and ground 80 g of roasted peanuts with 50 g of sugar. We observed the peanut butter from the beginning until it became creamy or until the end of the processing time. The machine ran continuously for 200 min. Figure 4a–d show the status of the material during mixing and grinding. We mixed and ground 80 g of roasted peanuts with 50 g of sugar. We observed the peanut butter from the beginning until it became creamy or until the end of the processing time. The machine ran continuously for 200 min. Figure 4a–d show the status of the ma- terial during mixing and grinding. Figure 4. The progress of peanut butter during the experiment: (a) 0, (b) 20, (c) 40, and (d) 180 min. Figure 4. The progress of peanut butter during the experiment: (a) 0, (b) 20, (c) 40, and (d) 180 min. Figure 4. The progress of peanut butter during the experiment: (a) 0, (b) 20, (c) 40, and (d) 180 min. Figure 4. The progress of peanut butter during the experiment: (a) 0, (b) 20, (c) 40, and (d) 180 min. Figure 4. The progress of peanut butter during the experiment: (a) 0, (b) 20, (c) 40, and (d) 180 min Figure 4. The progress of peanut butter during the experiment: (a) 0, (b) 20, (c) 40, and (d) 180 min 3. Experiment 3. Experiment Various ty Various types of materials can be processed by mixing and grinding. To test the sound signal processing, we used peanut butter, Japanese rice, and Japanese green tea. The conditions were as follows: the motor speed of the mixing and grinding machine was set at 20 rpm; the sound signal was recorded via a microphone for 5 s every 2 min; the wave ﬁle’s bit rate was 16 bit/s with a sampling frequency of 48 kHz; the average PSD value was calculated for the recorded data every 5 s. yp p y g g g sound signal processing, we used peanut butter, Japanese rice, and Japanese green tea. The conditions were as follows: the motor speed of the mixing and grinding machine was set at 20 rpm; the sound signal was recorded via a microphone for 5 s every 2 min; the wave file’s bit rate was 16 bit/s with a sampling frequency of 48 kHz; the average PSD value was calculated for the recorded data every 5 s. 4 E ti ti PSD 2.4. Estimating PSD The general equation for the STFT is: xm( f ) = ∑ ∞ n=−∞x(n)g(n −mR)e−j2π f n xm( f ) = ∑ ∞ n=−∞x(n)g(n −mR)e−j2π f n (1) Micromachines 2021, 12, 1041 5 of 13 on for where xm(f) is the STFT, x(n) is a signal, g(n) is a window function, and R is the hop size. The hop size is the difference between the window length and the overlap length. The square of the magnitude of the STFT is a spectrogram, and we calculate the average PSD in decibels (dB) as: where xm(f) is the STFT, x(n) is a signal, g(n) is a window function, and R is the hop size. The hop size is the difference between the window length and the overlap length. The square of the magnitude of the STFT is a spectrogram, and we calculate the average PSD in decibels (dB) as: average PSD(n) = mean(10 × log10(mean\|xm(n, f )\|2)) (2) 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑃𝑆𝐷(𝑛) = 𝑚𝑒𝑎𝑛(10 × 𝑙𝑜𝑔ଵ଴(𝑚𝑒𝑎𝑛\|𝑥௠(𝑛, 𝑓)\|ଶ)) (2) ion (2) we calculated the average frequencies and average times of the (2) (2) the With Equation (2), we calculated the average frequencies and average times of the STFT. Finally, we calculated the average PSD values and collected the data in one ﬁle. q ( ) g q g STFT. Finally, we calculated the average PSD values and collected the data in one file. With Equation (2), we calculated the average frequencies and average times of the STFT. Finally, we calculated the average PSD values and collected the data in one ﬁle. q g q g STFT. Finally, we calculated the average PSD values and collected the data in one file. 3.3. Rice Powder 3.3. Rice Powder We used 200 3.3. Rice Powder 3.3. Rice Powder We used 200 We used 200 and 500 g of Japanese rice as a hard material. The mixing and grinding machine was operated continuously to grind the rice into powder. Figure 6a–d show the status of the 500 g of the material during the mixing and grinding operation. We used 200 and 500 g of Japanese rice as a hard material. The mixing and grinding machine was operated continuously to grind the rice into powder. Figure 6a–d show the status of the 500 g of the material during the mixing and grinding operation. Figure 6. Progress of rice powder during the experiment: (a) 4, (b) 16, (c) 32, and (d) 48 h. Figure 6. Progress of rice powder during the experiment: (a) 4, (b) 16, (c) 32, and (d) 48 h. Figure 6. Progress of rice powder during the experiment: (a) 4, (b) 16, (c) 32, and (d) 48 h. Figure 6. Progress of rice powder during the experiment: (a) 4, (b) 16, (c) 32, and (d) 48 h. 4. Analysis and Results 4.1. Setup Position of Microphone Microphone position (a), setup the microphone, (b) sound signals between behind and beside of the machine, and (c) spectrogram of motor with the pestle in the machine. 4.2. Waveform Analysis Figure 8a,b show examples of the waveforms recorded for peanut butter at 30 and 60 min. The wave files contained 120,000 data points and had a sampling frequency of 48 kHz. The vertical axis represents sound pressure. The waveform graphs changed between 30 and 60 min. It was difficult to quantitatively analyze the differences in the signals be- We also measured the machine’s noise at a rotation speed of 20 rpm. The spectrogram of the machine noise is shown in Figure 7c. The stationary components appeared around 12 kHz. Additionally, impulses appears around 1.5 and 2 s due to the motion of the pestle. However, the progress of the grinding and mixing affected such components as machine noise. 3 2 Japanese Green Tea 3.2. Japanese Green Tea 3.2. Japanese Green Tea We mixed and ground 200 g of Japanese green tea. The mixing and grinding machine ran continuously for 180 min. Figure 5a–d show the status of the material during the mix- ing and grinding We mixed and ground 200 g of Japanese green tea. The mixing and grinding machine ran continuously for 180 min. Figure 5a–d show the status of the material during the mixing and grinding. 6 of 14 ing and grinding. Figure 5. Progress of Japanese green tea during the experiment: (a) 0, (b) 60, (c) 120, and (d) 180 min. Figure 5. Progress of Japanese green tea during the experiment: (a) 0, (b) 60, (c) 120, and (d) 180 min. g g g Figure 5. Progress of Japanese green tea during the experiment: (a) 0, (b) 60, (c) 120, and (d) 180 min. Figure 5. Progress of Japanese green tea during the experiment: (a) 0, (b) 60, (c) 120, and (d) 180 min. 6 of 13 Micromachines 2021, 12, 1041 i 4. Analysis and Results 4.1. Setup Position of Microphone 4.1. Setup Position of Microphone To set the position of the microphone, we measured the sound signal from the mi- crophone with an oscilloscope and compared the signals from beside and behind the mix- ing and grinding machine with an empty crucible. The PSD values beside the machine were calculated as –5.0 dB/Hz, and those behind were found to be –4.2 dB/Hz. Because the signals were similar, we placed the microphone behind the cover of the machine (Fig- To set the position of the microphone, we measured the sound signal from the mi- crophone with an oscilloscope and compared the signals from beside and behind the mixing and grinding machine with an empty crucible. The PSD values beside the machine were calculated as −5.0 dB/Hz, and those behind were found to be −4.2 dB/Hz. Be- cause the signals were similar, we placed the microphone behind the cover of the machine (Figure 7b). 7 of 14 (a) ure 7b). We also measured the machine’s noise at a rotation speed of 20 rpm. The spectrogram of the machine noise is shown in Figure 7c. The stationary components appeared around 12 kHz. Additionally, impulses appears around 1.5 and 2 s due to the motion of the pestle However, the progress of the grinding and mixing affected such components as machine noise. (a) (b) (c) Figure 7. Microphone position (a), setup the microphone, (b) sound signals between behind and beside of the machine, and (c) spectrogram of motor with the pestle in the machine. Figure 7. Microphone position (a), setup the microphone, (b) sound signals between behind and beside of the machine, and (c) spectrogram of motor with the pestle in the machine. ) We also measured the machine’s no of the machine noise is shown in Figure 12 kHz. Additionally, impulses appears However, the progress of the grinding a noise. (b) e at a rotation speed of 20 rpm. The spectrogr c. The stationary components appeared arou ound 1.5 and 2 s due to the motion of the pes d mixing affected such components as mach (c) (b) (c) (a) Figure 7. Microphone position (a), setup the microphone, (b) sound signals between behind and beside of the machine, and (c) spectrogram of motor with the pestle in the machine. Figure 7. 4.4. PSD Estimation We used the average PSD values to monitor the progress of the mixing and grinding machine. To make the PSD variations clear, we applied smoothing splines to the original curves. In the PSD variation graphs, both the original curves and those with the smoothing splines are shown. We examined the characteristics of the PSD values during grinding and mixing. We used peanuts, green tea, and Japanese rice and performed each experiment in duplicate (experiments 1 and 2) to verify the accuracy and reproducibility of the method. Figure 10 shows the average PSD value for roasted peanuts and sugar. In experiment 1, the PSD value decreased to −18 dB/Hz, and the ground peanuts gathered into a ball. The PSD value increased to a maximum of −10 dB/Hz, and then decreased. After 40 min, the ball became bound together and became a large, sticky lump. Finally, the average PSD value decreased slowly until it approached a straight line as the lump of peanuts became creamy. In experiment 2, the PSD value decreased to −17 dB/Hz at 10 min, increased to a maximum of −9 dB/Hz at 40 min, and then decreased slowly until forming a straight line. The waveform graph for experiment 1 was similar to that for experiment 2 from the start until the maximum values, although subsequently, the PSD values were different. The difference arose because the temperature affected the viscosity and friction of the peanuts after the maximum PSD value was reached. 9 of 14 The waveform graph for experiment 1 was similar to that for experiment 2 from the start until the maximum values, although subsequently, the PSD values were different. The difference arose because the temperature affected the viscosity and friction of the peanuts after the maximum PSD value was reached. Figure 10. Variation of the PSD for peanuts. Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from ‒16 to ‒20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between ‒20 and ‒22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was con- verted into powder. 4.3. Spectrogram Analysis The relationship between the frequency and magnitude of the sound signals was analyzed. The color of the spectrogram shows the power level of the frequency: Yellow indicates a high value and blue indicates a low value. Figure 9a,b show spectrogram graphs of peanut butter at 30 and 60 min with a frequency between 0 and 15 kHz. The spectrogram is yellow between 0 and 2 kHz, which means that there was some noise while the microphone was recording. This is complicated to observe and monitor remotely. Therefore, the spectrograms were converted into PSD values with a frequency range of 2–15 kHz to estimate the PSD values. tween the recorded wa grams (Figure 9) 4.2. Waveform Analysis grams (Figure 9). Figure 8a,b show examples of the waveforms recorded for peanut butter at 30 and 60 min. The wave ﬁles contained 120,000 data points and had a sampling frequency of 48 kHz. The vertical axis represents sound pressure. The waveform graphs changed between 30 and 60 min. It was difﬁcult to quantitatively analyze the differences in the signals between the recorded waveforms; therefore, the waveforms were converted into spectrograms (Figure 9). 7 of 13 Micromachines 2021, 12, 1041 q y y g tween the recorded waveforms; therefore, the waveforms were converted into s grams (Figure 9). (a) (b) Figure 8. Recorded waveforms of peanut butter at (a) 30 and (b) 60 min. Figure 8. Recorded waveforms of peanut butter at (a) 30 and (b) 60 min. 8 (a) (b) Figure 9. Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 min. 4.3. Spectrogram Analysis The relationship between the frequency and magnitude of the sound signals w alyzed. The color of the spectrogram shows the power level of the frequency: Yello Figure 9. Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 m (a) (b) Figure 8. Recorded waveforms of peanut butter at (a) 30 and (b) 60 min. Figure 8. Recorded waveforms of peanut butter at (a) 30 and (b) 60 min. 8 of 14 (a) (b) (b) gure 8. Recorded waveforms of peanut butter at (a) 30 and (b) 60 min. Figure 8. Recorded waveforms of peanut butter at (a) 30 and (b) 60 min. (a) (b) gure 9. Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 min. 3 S e t o a A aly i Figure 9. Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 min. (a) (b) ure 9 Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 min Fi 9 C t d t h f t b tt t ( ) 30 d (b) 60 i ure 9. Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 min. Figure 9. Converted spectrogram graphs of peanut butter at (a) 30 and (b) 60 min. 8 of 13 Micromachines 2021, 12, 1041 4.3. Spectrogram Analysis 4.4. PSD Estimation In experiment 2, the PSD value decreased to between –15 and –20 dB/Hz within 60 min, and then the value varied between ‒20 and ‒21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between ‒21 and ‒21.5 dB/Hz, which is the downward trend observed in the figure. The waveform graphs were similar for both experiments because the experiments were short Figure 10. Variation of the PSD for peanuts. Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from −16 to −20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between −20 and −22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was converted into powder. In experiment 2, the PSD value decreased to between −15 and −20 dB/Hz within 60 min, and then the value varied between −20 and −21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between −21 and −21.5 dB/Hz, which is the downward trend observed in the ﬁgure. Figure 10. Variation of the PSD for peanuts. h h l f I h Figure 10. Variation of the PSD for peanuts. Figure 11 shows the average PSD values for Japanese green tea In experiment 1 the Figure 10. Variation of the PSD for peanuts. Figure 10. Variation of the PSD for peanuts. Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from ‒16 to ‒20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between ‒20 and ‒22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was con- verted into powder. In experiment 2, the PSD value decreased to between –15 and –20 dB/Hz within 60 min, and then the value varied between ‒20 and ‒21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between ‒21 and ‒21.5 dB/Hz, which is the downward trend observed in the figure. The Figure 11 shows the average PSD values for Japanese green tea. 4.4. PSD Estimation In experiment 1, the estimated PSD value decreased from −16 to −20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between −20 and −22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was converted into powder. In experiment 2, the PSD value decreased to between −15 and −20 dB/Hz within 60 min, and then the value varied between −20 and −21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between −21 and −21.5 dB/Hz, which is the downward trend observed in the ﬁgure. Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from ‒16 to ‒20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between ‒20 and ‒22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was con- verted into powder. In experiment 2, the PSD value decreased to between –15 and –20 dB/Hz within 60 min, and then the value varied between ‒20 and ‒21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between ‒21 and ‒21.5 dB/Hz, which is the downward trend observed in the figure. The Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from −16 to −20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between −20 and −22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was converted into powder. In experiment 2, the PSD value decreased to between −15 and −20 dB/Hz within 60 min, and then the value varied between −20 and −21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between −21 and −21.5 dB/Hz, which is the downward trend observed in the ﬁgure. Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from ‒16 to ‒20 dB/Hz within 60 min. 4.4. PSD Estimation The particle size of the green tea leaves decreased and the PSD value varied between ‒20 and ‒22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was con- verted into powder. In experiment 2, the PSD value decreased to between –15 and –20 dB/Hz within 60 min, and then the value varied between ‒20 and ‒21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between ‒21 and ‒21.5 dB/Hz, which is the downward trend observed in the figure. The Figure 11 shows the average PSD values for Japanese green tea. In experiment 1, the estimated PSD value decreased from −16 to −20 dB/Hz within 60 min. The particle size of the green tea leaves decreased and the PSD value varied between −20 and −22 dB/Hz from 60 to 120 min, and some leaves became powder. After 120 min, the green tea was converted into powder. In experiment 2, the PSD value decreased to between −15 and −20 dB/Hz within 60 min, and then the value varied between −20 and −21 dB/Hz from 60 to 120 min, and some tea leaves became powder. After 120 min, the PSD value decreased to between −21 and −21.5 dB/Hz, which is the downward trend observed in the ﬁgure. 9 of 13 nd 20 m 60 to Micromachines 2021, 12, 1041 The waveform graphs were similar for both experiments because the experiments were short and the PSD values decreased when the particle size decreased. / , g waveform graphs were similar for both experiments because the experiments were short and the PSD values decreased when the particle size decreased. The waveform graphs were similar for both experiments because the experiments were short and the PSD values decreased when the particle size decreased. g waveform graphs were similar for both experiments because the experiments were short and the PSD values decreased when the particle size decreased. Figure 11. Variation of the PSD for Japanese green tea. Figure 11. Variation of the PSD for Japanese green tea. Figure 11. Variation of the PSD for Japanese green tea. Figure 11. Variation of the PSD for Japanese green tea. Figure 12 shows the average PSD values for 200 g of rice powder. To avoid small variations, we used the fifth-point-averaging filter curve to reduce the impulse signals. 4.4. PSD Estimation In experiment 1, the PSD value varied between ‒2 and ‒5 dB/Hz up to 8 h, and then increased to a maximum value of 1 dB/Hz at 8 h. The particle size of the rice decreased, and some of it became powder. The PSD values decreased to ‒8 dB/Hz at 12 h. In experiment 2, the PSD value varied between ‒3 and ‒6 dB/Hz. The PSD value increased to a maximum of ‒ Figure 12 shows the average PSD values for 200 g of rice powder. To avoid small variations, we used the ﬁfth-point-averaging ﬁlter curve to reduce the impulse signals. In experiment 1, the PSD value varied between −2 and −5 dB/Hz up to 8 h, and then increased to a maximum value of 1 dB/Hz at 8 h. The particle size of the rice decreased, and some of it became powder. The PSD values decreased to −8 dB/Hz at 12 h. In experiment 2, the PSD value varied between −3 and −6 dB/Hz. The PSD value increased to a maximum of −0.5 dB/Hz at 8 h, at which point the rice had mainly become powder. Finally, the PSD value decreased to −12 dB/Hz at 12 h, at which point all of the rice was powder. The waveform graphs were similar in both experiments from the start until the maximum value, and subsequently, the PSD values were different because the rice powder was affected by humidity. 10 of 14 0.5 dB/Hz at 8 h, at which point the rice had mainly become powder. Finally, the PSD value decreased to ‒12 dB/Hz at 12 h, at which point all of the rice was powder. The wave- form graphs were similar in both experiments from the start until the maximum value, and subsequently, the PSD values were different because the rice powder was affected by humidity. Figure 12. Variation of the PSD for 200 g of rice powder. Precision is quantified by repeatability and reproducibility; thus, we performed the Japanese rice powder experiment using 500 g of rice (Figure 13). The PSD values varied between −10 and −15 dB/Hz, and after 32 h, the rice particle size was smaller, and some rice had become powder. The PSD value increased to a maximum of 8.3 dB/Hz at 40 h, at which time the rice had mostly become powder. 4.4. PSD Estimation Finally, the PSD value decreased to −16 dB/Hz at 44 h, and all of the rice was transformed into powder. We found that the wave- form graph for 500 g of rice powder (Figure 13) was similar to that for 200 g of rice powder (Figure 12). Figure 12. Variation of the PSD for 200 g of rice powder. Precision is quantiﬁed by repeatability and reproducibility; thus, we performed the Japanese rice powder experiment using 500 g of rice (Figure 13). The PSD values varied between −10 and −15 dB/Hz, and after 32 h, the rice particle size was smaller, and some rice had become powder. The PSD value increased to a maximum of 8.3 dB/Hz at 40 h, at which time the rice had mostly become powder. Finally, the PSD value decreased to −16 dB/Hz at 44 h, and all of the rice was transformed into powder. We found that the waveform graph for 500 g of rice powder (Figure 13) was similar to that for 200 g of rice powder (Figure 12). Figure 12. Variation of the PSD for 200 g of rice powder. Figure 12. Variation of the PSD for 200 g of rice powder. Figure 12. Variation of the PSD for 200 g of rice powder. Figure 12. Variation of the PSD for 200 g of rice powder. Precision is quantified by repeatability and reproducibility; thus, we performed the Japanese rice powder experiment using 500 g of rice (Figure 13). The PSD values varied between −10 and −15 dB/Hz, and after 32 h, the rice particle size was smaller, and some rice had become powder. The PSD value increased to a maximum of 8.3 dB/Hz at 40 h, at which time the rice had mostly become powder. Finally, the PSD value decreased to −16 dB/Hz at 44 h, and all of the rice was transformed into powder. We found that the wave- form graph for 500 g of rice powder (Figure 13) was similar to that for 200 g of rice powder (Figure 12). Precision is quantiﬁed by repeatability and reproducibility; thus, we performed the Japanese rice powder experiment using 500 g of rice (Figure 13). The PSD values varied between −10 and −15 dB/Hz, and after 32 h, the rice particle size was smaller, and some rice had become powder. 4.4. PSD Estimation Variation of the PSD for 500 g of rice powder. Figure 13. Variation of the PSD for 500 g of rice powder. 4 5 Correlation between Particle Size and Sound Signal Processing 4.5. Correlation between Particle Size and Sound Signal Processing 4 5 Correlation between Particle Size and Sound Signal Processing 4.5. Correlation between Particle Size and Sound Signal Processing 4.5. Correlation between Particle Size and Sound Signal Processing The particle sizes of the Japanese rice and green tea leaves were measured visually or with a microscope to determine the particle size distribution in order to acquire further information about the mixing and grinding process. The particle size distributions were obtained through a sieve analysis with sieve sizes of 250, 180, and 75 µm. The weights for each sieve size were recorded and the percentages were calculated. We calculated the cor- relation coefficient between the particle size distribution and the sound signal analysis by stopping the machine and collecting samples for calculating the size distribution at every 4 h f th J i d 1 h f th t The particle sizes of the Japanese rice and green tea leaves were measured visually or with a microscope to determine the particle size distribution in order to acquire further information about the mixing and grinding process. The particle size distributions were obtained through a sieve analysis with sieve sizes of 250, 180, and 75 µm. The weights for each sieve size were recorded and the percentages were calculated. We calculated the correlation coefﬁcient between the particle size distribution and the sound signal analysis by stopping the machine and collecting samples for calculating the size distribution at every 4 h for the Japanese rice and every 1 h for the green tea. 4 h for the Japanese rice and every 1 h for the green tea. Th ti f l l ti th l ti ffi i t i y p y g The equation for calculating the correlation coefﬁcient is: culating the correlation coefficient is: r = n(∑xy) −(∑x)(∑y) p [∑x2 −(∑x)2][n∑y2 −(∑y)2] (3) (3) where r is the correlation coefﬁcient, n is the number of pairs of data, x is the value of all sieve sizes, and y is the value of all average PSD values. We analyzed the correlation coefﬁcient between the sieve sizes of 250, 180, and 75 µm and the average PSD values. 4.4. PSD Estimation The PSD value increased to a maximum of 8.3 dB/Hz at 40 h, at which time the rice had mostly become powder. Finally, the PSD value decreased to −16 dB/Hz at 44 h, and all of the rice was transformed into powder. We found that the waveform graph for 500 g of rice powder (Figure 13) was similar to that for 200 g of rice powder (Figure 12). Precision is quantified by repeatability and reproducibility; thus, we performed the Japanese rice powder experiment using 500 g of rice (Figure 13). The PSD values varied between −10 and −15 dB/Hz, and after 32 h, the rice particle size was smaller, and some rice had become powder. The PSD value increased to a maximum of 8.3 dB/Hz at 40 h, at which time the rice had mostly become powder. Finally, the PSD value decreased to −16 dB/Hz at 44 h, and all of the rice was transformed into powder. We found that the wave- form graph for 500 g of rice powder (Figure 13) was similar to that for 200 g of rice powder (Figure 12). Precision is quantiﬁed by repeatability and reproducibility; thus, we performed the Japanese rice powder experiment using 500 g of rice (Figure 13). The PSD values varied between −10 and −15 dB/Hz, and after 32 h, the rice particle size was smaller, and some rice had become powder. The PSD value increased to a maximum of 8.3 dB/Hz at 40 h, at which time the rice had mostly become powder. Finally, the PSD value decreased to −16 dB/Hz at 44 h, and all of the rice was transformed into powder. We found that the waveform graph for 500 g of rice powder (Figure 13) was similar to that for 200 g of rice powder (Figure 12). 10 of 13 e wave- powder Micromachines 2021, 12, 1041 Figure 13 Variation of the PSD for 500 g of rice powder Figure 13. Variation of the PSD for 500 g of rice powder. Figure 13 Variation of the PSD for 500 g of rice powder Figure 13. Variation of the PSD for 500 g of rice powder. Figure 13. Variation of the PSD for 500 g of rice powder. Figure 13. Variation of the PSD for 500 g of rice powder. Figure 13. Variation of the PSD for 500 g of rice powder. Figure 13. 4.4. PSD Estimation Time (h) Sieve Size 250 µm (%) Sieve Size 180 µm (%) Sieve Size 75 µm (%) Average PSD Value (dB/Hz) 0 100 0 0 −1.8 1 97.5 2.5 0.0 −3.6 2 96.6 3.4 0.1 −5.7 3 95.6 4.3 0.1 −5.8 4 94.7 5.0 0.3 −5.8 5 93.9 5.9 0.2 −5.3 6 91.8 7.8 0.4 −3.6 7 83.3 16.4 0.4 −1.0 8 61.5 37.2 1.3 −0.5 9 49.9 50.0 0.1 −0.9 10 28.9 56.2 14.9 −3.4 11 25.3 49.6 25.1 −8.3 12 21.7 61.1 17.2 −9.8 13 20.0 67.6 12.4 −10.7 14 17.3 66.0 16.8 −10.7 15 14.3 52.6 33.1 −11.1 16 10.9 59.9 29.2 −11.0 Table 3 shows the particle size distribution compared with the average PSD values for 500 g of Japanese rice shown in Figure 13. The correlation coefﬁcient of rice with a particle size greater than 250 µm had an average PSD value of 0.68; thus, the variables had a positive relationship. Particle sizes between 181 and 250 µm had an average PSD value of −0.61, and particle sizes smaller than 180 µm had an average PSD value of −0.58, indicating that the variables had a negative relationship. The experiments with 200 and 500 g of rice (Figures 12 and 13) had similar correlation coefﬁcients, indicating that the method was repeatable. of −0.68. Table 2. Particle size percentage and average PSD values for Japanese rice (200 g). Time (h) Sieve Size 250 µm (%) Sieve Size 180 µm (%) Sieve Size 75 µm (%) Average PSD Value (dB/Hz) 0 100 0 0 −1.8 1 97.5 2.5 0.0 −3.6 2 96.6 3.4 0.1 −5.7 3 95.6 4.3 0.1 −5.8 4 94.7 5.0 0.3 −5.8 5 93.9 5.9 0.2 −5.3 6 91.8 7.8 0.4 −3.6 7 83.3 16.4 0.4 −1.0 8 61.5 37.2 1.3 −0.5 9 49.9 50.0 0.1 −0.9 10 28.9 56.2 14.9 −3.4 11 25.3 49.6 25.1 −8.3 12 21.7 61.1 17.2 −9.8 13 20.0 67.6 12.4 −10.7 14 17.3 66.0 16.8 −10.7 15 14.3 52.6 33.1 −11.1 16 10.9 59.9 29.2 −11.0 of −0.68. Table 2. Particle size percentage and average PSD values for Japanese rice (200 g). Table 3 shows the particle size distribution compared with the average PSD values for 500 g of Japanese rice shown in Figure 13. 4.4. PSD Estimation g Table 1 shows the particle size distribution compared with the average PSD values of green tea leaves shown in Figure 14. For green tea leaves, the correlation coefﬁcient between particle sizes of more than 250 µm and the average PSD value was 0.96; thus, the variables had a highly positive relationship. Particle sizes between 181 and 250 µm had a correlation coefﬁcient of −0.96, and particle sizes smaller than 180 µm had a correlation coefﬁcient of −0.88, meaning that the variables had a highly negative relationship. Table 1. Particle size percentage and average PSD values for Japanese green tea. Table 1. Particle size percentage and average PSD values for Japanese green tea. Time (h) Sieve Size 250 µm (%) Sieve Size 180 µm (%) Sieve Size 75 µm (%) Average PSD Value (dB/Hz) 0 99 0.3 0.25 −14.58 1 69.5 26.6 3.5 −19.72 2 50.9 41 4.4 −20.44 3 48.9 45 5.4 −21.13 4 41 50.5 8.2 −21.21 11 of 13 Micromachines 2021, 12, 1041 Figure 14. Sieving equipme Figure 14. Sieving equipment. Figure 14. Sieving equipm Figure 14. Sieving equipment. Figure 14. Sieving equipm Figure 14. Sieving equipment. Table 2 shows the particle size distribution compared with the averag for 200 g of Japanese rice shown in Figure 12 (experiment 2). The correlation rice with a particle size greater than 250 µm had an average PSD value of variables had a positive relationship. Particle sizes between 181 and 250 µm age PSD value of −0.41, and particle sizes smaller than 180 µm had an aver Table 2 shows the particle size distribution compared with the average PSD values for 200 g of Japanese rice shown in Figure 12 (experiment 2). The correlation coefﬁcient of rice with a particle size greater than 250 µm had an average PSD value of 0.65; thus, the variables had a positive relationship. Particle sizes between 181 and 250 µm had an average PSD value of −0.41, and particle sizes smaller than 180 µm had an average PSD value of −0.68. g , p µ of −0.68. Table 2. Particle size percentage and average PSD values for Japanese rice (200 g). 4.4. PSD Estimation The correlation coefﬁcient of rice with a particle size greater than 250 µm had an average PSD value of 0.68; thus, the variables had a positive relationship. Particle sizes between 181 and 250 µm had an average PSD value of −0.61, and particle sizes smaller than 180 µm had an average PSD value of −0.58, indicating that the variables had a negative relationship. The experiments with 200 and 500 g of rice (Figures 12 and 13) had similar correlation coefﬁcients, indicating that the method was repeatable. 12 of 13 Micromachines 2021, 12, 1041 Table 3. Particle size percentages and average PSD values for Japanese rice (500 g). Time (h) Sieve Size 250 µm (%) Sieve Size 180 µm (%) Sieve Size 75 µm (%) Average PSD Value (dB/Hz) 0 100 0 0 −9.1 4 97 2 0 −13.1 8 96 2 0 −13.8 12 96 2 1 −13.4 16 94 4 2 −13.1 20 92 5 2 −15.1 24 90 4 3 −16.3 28 88 6 4 −14.8 32 86 8 4 −10.8 36 62 25 11 2.6 40 37 38 21 6.7 44 28 45 26 −12.7 48 21 51 26 −15.9 Table 3. Particle size percentages and average PSD values for Japanese rice (500 g). To employ the combination of the image of the curve of the PSD at a certain time, as shown in Figures 10–13, machine learning could also be applied in our proposed method in order to make the accuracy of the estimation of progress the output. We plan to combine the images of the curve of the PSD with the measured particle distributions as training data in future. Author Contributions: Conceptualization, Y.K. and E.W.; formal analysis, Y.K. and E.W.; investiga- tion, Y.K. and E.W.; writing—original draft preparation, E.W.; writing—review and editing, Y.K. and E.W. Both authors have read and agreed to the published version of the manuscript. 5. Conclusions 13 of 13 13 of 13 Micromachines 2021, 12, 1041 Funding: This research was performed by the Environment Research and Technology Development Fund JPMEERF21356447 of the Environmental Restoration and Conservation Agency of Japan and by JSPS KAKENHI Grant Numbers JP21H01554. Funding: This research was performed by the Environment Research and Technology Development Fund JPMEERF21356447 of the Environmental Restoration and Conservation Agency of Japan and by JSPS KAKENHI Grant Numbers JP21H01554. Acknowledgments: We thank Masakazu Nakamura, CEO of Ishikawa Kojo Co., Ltd., Koto-ku, Tokyo, Japan, for supplying the mixing and grinding machine. Acknowledgments: We thank Masakazu Nakamura, CEO of Ishikawa Kojo Co., Ltd., Koto-ku, Tokyo, Japan, for supplying the mixing and grinding machine. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Ishikawa of Mixing and Grinding Machine Introduction. Available online: http://www.ishikawakojo.jp/english/ (accessed on 20 September 2020). p ) 2. Dhingra, S.; Madda, R.B.; Gandomi, A.H.; Patan, R.; Daneshmand, M. Internet of Things Mobile—Air Pollution Monitoring System (IoT-Mobair). IEEE Internet Things J. 2019, 6, 5577–5584. [CrossRef] TM 3. López-Vargas, A.; Fuentes, M.; Vivar, M. IoT Application for Real-Time Monitoring of Solar Home Systems Based on ArduinoTM with 3G Connectivity. IEEE Sens. J. 2019, 19, 679–691. [CrossRef] 4. Lv, Z.; Hu, B.; Lv, H. Infrastructure Monitoring and Operation for Smart Cities Based on IoT System. IEEE Trans. Ind. Inf. 2020, 16, 1957–1962. [CrossRef] A.; Iinatti, J.; Ylianttila, M.; Sain, M. Lightweight and Secure Session-Key Establishment Scheme in Smart 2016, 16, 254–264. [CrossRef] 5. Kumar, P.; Gurtov, A.; Iinatti, J.; Ylianttila, M.; Sain, M. Lightweight and Secure Session-Key Establis Home. IEEE Sens. J. 2016, 16, 254–264. [CrossRef] 6. Han, J.; Choi, C.-S.; Park, W.-K.; Lee, I.; Kim, S.-H. Smart Home Energy Management System Including Renewable Energy Based on ZigBee and PLC. IEEE Trans. Consum. Electron. 2014, 60, 198–202. [CrossRef] g 7. Han, Q.; Liu, P.; Zhang, H.; Cai, Z. A Wireless Sensor Network for Monitoring Environmental Quality in the Manufacturing Industry. IEEE Access 2019, 7, 78108–78119. [CrossRef] 8. Zhang, F.; Liu, M.; Zhou, Z.; Shen, W. An IoT-Based Online Monitoring System for Continuous Steel Casting. IEEE Internet Things J. 2016, 3, 1355–1363. [CrossRef] 9. Sung, W.T.; Hsu, Y.C. Designing an industrial real-time measurement and monitoring system based on embedded system and ZigBee. Expert Syst. Appl. 2011, 38, 4522–4529. [CrossRef] pp amora, M.; Miron, M.; Font, F.; Serra, X. An Interpretable Deep Learning Model for Automatic Sound onics 2021, 10, 850. [CrossRef] g 10. Zinemanas, P.; Rocamora, M.; Miron, M.; Font, F.; Serra, X. An Interpretable Deep Learning Mod Classiﬁcation. Electronics 2021, 10, 850. [CrossRef] 11. Hu, Y.; Wang, L.; Huang, X.; Qian, X.; Gao, L.; Yan, Y. On-line Sizing of Pneumatically Conveyed Particles Through Acoustic Emission Detection and Signal Analysis. IEEE Trans. Instrum. Meas. 2015, 64, 1100–1109. g y 12. Carter, R.M.; Yan, Y. An Instrumentation System Using Combined Sensing Strategies for Online Mass Flow Rate Measurement and Particle Sizing. IEEE Trans. Instrum. Meas. 2005, 54, 1433–1437. [CrossRef] g 13. Mao, W.; Towhata, I. Monitoring of single-particle fragmentation process under static loading using acoustic emission. Appl. Acoust. 2015, 94, 39–45. [CrossRef] 14. Carter, R.M.; Yan, Y.; Lee, P. References On-line Nonintrusive Measurement of Particle Size Distribution Through Digital. IEEE Trans. Instrum. Meas. 2006, 55, 2034–2038. [CrossRef] 15. Kojima, T.; Ijiri, T.; White, J.; Kataoka, H.; Hirabayashi, A. CogKnife: Food recognition from their cutting sounds. In Proceedings of the 2016 IEEE International Conference on Multimedia & Expo Workshops (ICMEW), Seattle, WA, USA, 11–15 June 2016. 15. Kojima, T.; Ijiri, T.; White, J.; Kataoka, H.; Hirabayashi, A. CogKnife: Food recognition from their cutting sounds. In Proceedings of the 2016 IEEE International Conference on Multimedia & Expo Workshops (ICMEW), Seattle, WA, USA, 11–15 June 2016. 16. Chitnaont, N.; Thumwarin, P.; Prasit, C. Sound analysis of Thai musical instrument: Khlui. In Proceedings of the International of the 2016 IEEE International Conference on Multimedia & Expo Workshops (ICMEW), Seattle, WA, USA, 11–15 June 2016. 16. Chitnaont, N.; Thumwarin, P.; Prasit, C. Sound analysis of Thai musical instrument: Khlui. In Proceedings of the International 16. Chitnaont, N.; Thumwarin, P.; Prasit, C. Sound analysis of Thai musical instrument: Khlui. In Proceed Conference on Digital Arts, Media and Technology (ICDAMT), Chiangrai, Thailand, 25–28 February 2 17. Bastari, A.; Cristalli, C.; Morlacchi, R.; Pomponi, E. Acoustic emissions for particle sizing of powders through signal processing techniques. J. Mech. Syst. Signal Process. 2011, 25, 901–916. [CrossRef] q J y g 18. Quino, G.; De Cola, F.; Tagarielli, V.; Petrinic, N. Exploring the application of sound measurements to assess the structural integrity of ﬁbre bundles. Procedia Struct. Integr. 2019, 18, 507–515. [CrossRef] 19. De Cola, F.; Quino, G.; Dragnevski, K.; Petrinic, N. An extended in-situ method to improve the understanding of fracture mechanics of granular materials using sound measurements. Eur. J. Mech.—A Solids 2019, 76, 1–12. [CrossRef] 20. Sen, S.; Bhaumik, A.K. Mathematical Modeling of Predictive Grinding for Ball Mill. In Proceedings of the IEEE Region 10 Conference (TENCON) Singapore 22 25 November 2016; pp 1180 1184 19. De Cola, F.; Quino, G.; Dragnevski, K.; Petrinic, N. An extended in-situ method to improve the understanding of fracture mechanics of granular materials using sound measurements. Eur. J. Mech.—A Solids 2019, 76, 1–12. [CrossRef] g p g mechanics of granular materials using sound measurements. Eur. J. Mech.—A Solids 2019, 76, 1–12. [CrossRef] 20. Sen, S.; Bhaumik, A.K. Mathematical Modeling of Predictive Grinding for Ball Mill. In Proceedings of the IEEE Region 10 Conference (TENCON), Singapore, 22–25 November 2016; pp. 1180–1184. 20. Sen, S.; Bhaumik, A.K. 5. Conclusions We proposed a method for monitoring the progress in a mixing and grinding machine. To monitor data remotely, we used sound analysis and estimated the PSD. The PSD values for peanuts, green tea leaves, and Japanese rice were different. The PSD values for peanuts decreased at the beginning of the process and increased when the peanuts and sugar formed a lump, and then decreased again when the mixture became creamy. The process of mixing and grinding Japanese rice took a long time. The PSD values of Japanese rice did not change much until the rice grains began to crack and became powder; then, the PSD values began to increase. The PSD values increased to the maximum when the rice became mainly powder, and then the values decreased until all of the rice became powder. The PSD values of green tea leaves decreased as the particle size decreased. Regardless of the material and the mixing and grinding process, the PSD values decreased until the steady state, at which point the PSD did not change. Therefore, the PSD values were estimated as the materials were ground, and temperature and humidity were found to be important. We measured the particle size distribution for Japanese rice and green tea and calcu- lated the correlation between the particle distribution and the processed sound signal. The average PSD values depended directly on the particle size for Japanese green tea. However, for Japanese rice, the average PSD values increased to the maximum, and then decreased as the particle size decreased until it became a powder. The sound signal was able to indicate the progress in the mixing and grinding machine, and we demonstrated that the results were repeatable. p Our paper contributes greatly to the extension of the Ishikawa mixing and grinding machine with respect to automatically knowing the progress in manufacturing made by such a mixing and grinding machine. Additionally, the parameters that we obtained have possibilities for applications not only in the IoT, but also in machine learning. In the future, we plan to measure more of the same data indicated in this paper and to apply machine learning to the estimation of progress. Author Contributions: Conceptualization, Y.K. and E.W.; formal analysis, Y.K. and E.W.; investiga- tion, Y.K. and E.W.; writing—original draft preparation, E.W.; writing—review and editing, Y.K. and E.W. Both authors have read and agreed to the published version of the manuscript. pp Prabhu, K.M.M. Window Functions and Their Applications in Signal Processing; CRC Press: Boca Raton, FL, U Roy, M. Howard Principles of Random Signal Analysis and Low Noise Design: The Power Spectral Density and Its Ap Press: New York, NY, USA, 2002; pp. 59–91. References Mathematical Modeling of Predictive Grinding for Ball Mill. In Proceedings of the IEEE Region 10 Conference (TENCON), Singapore, 22–25 November 2016; pp. 1180–1184. 21. Raspberry Pi—Teach, Learn, and Make with Raspberry Pi. Available online: http://www.raspberr September 2020). 21. Raspberry Pi Teach, Learn, and Make with Raspberry Pi. Available online: http://www.raspberrypi.org/ (accessed on 20 September 2020). 22 Pumpkin Pi Introduction Available online: http://select marutsu co jp/list/detail php?id 258 (accessed on 20 September 2020) p 22. Pumpkin Pi Introduction. Available online: http://select.marutsu.co.jp/list/detail.php?id=258 (accesse 22. Pumpkin Pi Introduction. Available online: http://select.marutsu.co.jp/list/detail.php?id=258 (accessed on 20 September 2020). 23. Wang, X.; Shi, X.; Yang, D.; Zhou, Y. Research on the Application of 3D Spectrogram in Bird Tweet and Speech Signals. In Proceedings of the IEEE Chinese Control and Decision Conference (CCDC), Chongqing, China, 28–30 May 2017; pp. 7744–7747. 24. Roy, M. Howard Principles of Random Signal Analysis and Low Noise Design: The Power Spectral Density and Its Applications; Wiley-IEEE Press: New York, NY, USA, 2002; pp. 59–91. 22. Pumpkin Pi Introduction. Available online: http://select.marutsu.co.jp/list/detail.php?id 258 (accessed on 20 September 2020). 23. Wang, X.; Shi, X.; Yang, D.; Zhou, Y. Research on the Application of 3D Spectrogram in Bird Tweet and Speech Signals. In Proceedings of the IEEE Chinese Control and Decision Conference (CCDC), Chongqing, China, 28–30 May 2017; pp. 7744–7747. 24. Roy, M. Howard Principles of Random Signal Analysis and Low Noise Design: The Power Spectral Density and Its Applications; Wiley-IEEE 23. Wang, X.; Shi, X.; Yang, D.; Zhou, Y. Research on the Application of 3D Spectrogram in Bird Tweet and Speech Signals. In Proceedings of the IEEE Chinese Control and Decision Conference (CCDC), Chongqing, China, 28–30 May 2017; pp. 7744–7747. 24. Roy, M. Howard Principles of Random Signal Analysis and Low Noise Design: The Power Spectral Density and Its Applications; Wiley-IEEE Press: New York, NY, USA, 2002; pp. 59–91.
https://openalex.org/W4381033050	https://zenodo.org/records/8050695/files/IJISRT23JUN622.pdf	English	null	Design Alchemy: Streamlining Design Implementation	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,641	Volume 8, Issue 6, June 2023 Volume 8, Issue 6, June 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Design Alchemy: Streamlining Design Implementation Prasham Kamlesh Mehta, ShrutiKumathekar, Information Technology, Atharva College of Engineering, Mumbai, India Keval Kaushal Shah Computer Science (A.I),NMIMS, Mumbai,India Abstract:- In this article we present a website that offers snippets that we used for our web development project. The snippets are created using HTML, CSS and JavaScript. When creating complex designs like skeuomorphism, flat design, glass morphism, Bauhaus style and neomorphism, these strategies prove useful for web designers. The snippets are responsive and work on both large and small devices, including desktop computers. mimic the soft, physical appearance associated with neomorphism. Neomorphism is mostly used in mobile and websites. It makes it easy for the users to interact and understand with the elements of a screen to improve its usability. It can be used to create a depth and dimension in a design for a quality product to the user’s expectations. B. Glass Morphism It consists of emerging design trends, and provides a translucent, glass-like appearance to user interfaces. WebCode Snippets enables developers with code snippets that generate the necessary HTML, CSS, and JavaScript elements to create visually glass morphism effects. It uses a semi-transparent background with a subtle shadow to create a sense of depth and dimension.By utilizing the above snippet tools, developers can achieve the illusion of depth, blurring, and refraction, thereby enhancing the overall user experience. Glass morphism is often used in mobile and web applications. Skeuomorphism-specific code snippets are also considered, highlighting how well they work to incorporate skeuomorphic aspects into web designs. These code snippets foster a sense of familiarity and improve user engagement by accurately reproducing textures, shadows, fonts, backgrounds, and 3D effects that mimic real things. This endeavor can help designers create intricate designs that are both aesthetically pleasing and useful. Keywords:- Flat design, neomorphism, glass morphism, Bauhaus style, skeuomorphism. C. Skeuomorphism Sk hi Skeuomorphism is a design approach that uses visual metaphors and mimicking for the representation of the real-world objects in a digital environment. Web Code Snippets have a series of code snippets that allow developers to integrate skeuomorphic elements into their web designs or projects. It enables the recreation of textures, shadows, and other 3D effects that resemble physical objects, thereby creating a sense of familiarity and it enhances the user's interaction with the interface. it can be seen as cluttering the user interface. It is difficult for users to quickly understand what an object does and also difficult to implement skeuomorphism consistently across a variety of devices. Skeuomorphism also can be used for creating a sense of familiarity and comfort for the users. It can also be used to convey information about the function of an object. However, it is also very important to use skeuomorphism sparingly and to make sure that it does not detract from the overall usability of the user interface. I. Web development is a complex process that requires a lot of skills and expertise. It is the core component of a website. Writing and executing the code can be complex ,challenging and time consuming[3]. In the rapid evolution of web technology, the demand for innovative and visually appealing designs continues to grow continuously. These Code Snippets provide HTML, CSS, and JavaScript code that are useful for web developers and it provides an extensive library of code snippets that can be effortlessly integrated into their projects using any Integrated Development Environment (IDE). The snippets are organized by design style, so web developers can quickly find the code they need to create a specific look and feel for their website. The snippets are quite responsive[1]. This paper highlights the project's focus on neomorphism, glass morphism, Bauhaus style, skeuomorphism, and flat design, as well as its commitment to responsiveness. Keywords:- Flat design, neomorphism, glass morphism, Bauhaus style, skeuomorphism. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ISSN No:-2456-2165 in a wide variety of applications such as websites, mobile applications and software. Some of the benefits of using flat design are such as simplicity, clarity ,modernity and flexibility. B. Slider Sliders have become a popular and versatile component in modern GUI, allowing the view of dynamic content in a visually engaging and interactive manner. This research paper explores the design and implementation of sliders in the modern website, examining their significance, functionalities, and various approaches. By analyzing different slider types, navigation options, responsive techniques, and usability considerations, this paper aims to provide insights and recommendations for creating effective and captivating sliders that enhance user experience. A. Navigation Bar The navigation bar is a critical component of web design, providing users with a means to navigate through different sections and pages of a website. By examining various design styles, responsive techniques, and usability considerations, this paper aims to provide insights and recommendations for creating effective and visually appealing navigation bars. The paper begins by highlighting the importance of navigation bars in web design, emphasizing their role in enhancing user experience and facilitating easy access to website content[1]. It discusses the key objectives of a navigation bar, including clear organization, intuitive navigation, and seamless interaction. D. Footer The footer is an essential component of web design, located at the bottom of a web page, providing users with supplementary information, navigation options, and contact details. This research paper explores the design and implementation of footers in web development, examining their significance, best practices, and innovative approaches[4]. By analyzing various footer styles, content considerations, usability factors, and responsive techniques . The footer section of a web page serves as a container for various elements that offer additional context, functionality, and navigational options to users. Positioned at the bottom of the page, the footer is designed to be consistently present across all pages of a website, providing continuity and easy access to important information. C. Header The header is a critical component of web design, serving as the topmost section of a website that often includes the site logo, navigation menu, and key information. Usability factors are examined, including the placement and visibility of important elements within headers, efficient navigation menus, and effective call-to- action buttons. The head section of an HTML document holds critical information that is necessary for the proper functioning and presentation of the web page. While it doesn't contain visible content, it plays a significant role in defining the document's characteristics and providing instructions to browsers and search engines. A. Neomorphism Flat designs are characterized by minimalism, simplicity that emphasizes usability, and the absence of 3D effects, which has become increasingly popular in modern web development. This design focuses on clean typography, vibrant colors, dimensional illustration and crisp shapes, resulting in aesthetically pleasing, high quality user-friendly experience. Flat design has become a popular design trend over the past few years, and it is used A. Neomorphism It is visually appealing and can make a design more engaging. Neomorphism is a design trend that is a combination of skeuomorphism and flat design. It uses soft shadows and gradients to create a 3D effect. It has a significant growth in recent years. Web Code Snippets that enable developers to effortlessly incorporate neomorphic design elements into their web related projects. These snippet tools include shadows, gradients, and color schemes that www.ijisrt.com IJISRT23JUN622 261 Volume 8, Issue 6, June 2023 III. As web design trends continue to evolve, future work can involve the inclusion of additional design styles in DesignVerse. By staying up to date with emerging trends and incorporating new design approaches, the platform can provide web developers with an even broader range of code snippets to create innovative and cutting-edge designs[3]. Enhancing DesignVerse with collaborative features can foster a sense of community among web developers. Implementing functionalities such as user-contributed snippets, forums for sharing design tips and tricks, and the ability to collaborate on design projects can further enrich the platform and encourage knowledge exchange and creativity[5]. Future work can focus on developing snippets that enable animations, transitions, and interactive user interfaces, empowering web developers to create more immersive and engaging experiences.Leveraging advancements in artificial intelligence, future work can explore the potential of generating AI-assisted design snippets. AI algorithms could analyze design preferences, user input, and industry trends to automatically generate code snippets tailored to specific requirements, saving time and offering personalized design solutions. Fig. 1: Slider Fig. 1: Slider Fig. 1: Slider 262 IJISRT23JUN622 IJISRT23JUN622 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 6, June 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 REFERENCES [1.] [1.] G. Petrosyan, M. P. Robillard, and R. De Mori, “Discovering information explaining API types using text classification,” in Proc. 37th Int’l Conf. Software Engineering, 2015, pp. 869–879. [2.] [2.] Ricky Gunawan; Geraldi Anthony; Vendly; Maria Susan Anggreainy "The Effect of Design User Interface (UI) E-Commerce on User Experience (UX)" The Effect of Design User Interface (UI) E- Commerce on User Experience (UX) Review 14.1 (2021): 265-285. [3.] [3.] Zhafirah Indira ParamariniHardianto; Karmilasar. "Analysis and Design of User Interface and User Experience (UI / UX) E-Commerce Website PT PentasadaAndalanKelola Using Task System Centered Design (TCSD) Method'' 2022 International Conference on Applied Artificial Intelligence and Computing. IEEE, 2018. [4.] IEEE, “Introduction to a user interface design/information architecture process for Web sites" IEEE 2001 [5.] [5.] V. Flanders and M. Willis, "Web Pages that Suck: Leam Good Design by Looking at Bad Design", Entertaining and accessible advice for anyone who creates web pages. Includes HTML snippets so you can see exactly how to code (or not code) things. Good as a nontechnical introduction to usability/IA issues or when you're too tired to read anything else" IEEE March 2000. IJISRT23JUN622 IJISRT23JUN622 263 www.ijisrt.com
https://openalex.org/W4220869237	https://hal.science/hal-03607369/file/cp-18-435-2022.pdf	English	null	Clumped isotope evidence for Early Jurassic extreme polar warmth and high climate sensitivity	Climate of the past	2,022	cc-by	12,274	To cite this version: Thomas Letulle, Guillaume Suan, Mathieu Daëron, Mikhail Rogov, Christophe Lécuyer, et al.. Clumped isotope evidence for Early Jurassic extreme polar warmth and high climate sensitivity. Climate of the Past, 2022, 18 (3), pp.435 - 448. 10.5194/cp-18-435-2022. hal-03607369 Clumped isotope evidence for Early Jurassic extreme polar warmth and high climate sensitivity Thomas Letulle, Guillaume Suan, Mathieu Daëron, Mikhail Rogov, Christophe Lécuyer, Arnauld Vinçon-Laugier, Bruno Reynard, Gilles Montagnac, Oleg Lutikov, Jan Schlögl 1 Introduction Abstract. Periods of high atmospheric CO2 levels during the Cretaceous–early Paleogene (∼140 to 34 Myr ago) were marked by very high polar temperatures and reduced lat- itudinal gradients relative to the Holocene. These features represent a challenge for most climate models, implying ei- ther higher-than-predicted climate sensitivity to atmospheric CO2 or systematic biases or misinterpretations in proxy data. Here, we present a reconstruction of marine temperatures at polar (> 80◦) and middle (∼40◦) paleolatitudes during the Early Jurassic (∼180 Myr ago) based on the clumped isotope (147) and oxygen isotope (δ18Oc) analyses of shal- low buried pristine mollusc shells. Reconstructed calciﬁca- tion temperatures range from ∼8 to ∼18 ◦C in the Toar- cian Arctic and from ∼24 to ∼28 ◦C in Pliensbachian mid- paleolatitudes. These polar temperatures were ∼10–20 ◦C higher than present along with reduced latitudinal gradients. Reconstructed seawater oxygen isotope values (δ18Ow) of −1.5 ‰ to 0.5 ‰ VSMOW and of −5 ‰ to −2.5 ‰ VS- MOW at middle and polar paleolatitudes, respectively, point to a signiﬁcant freshwater contribution in Arctic regions. These data highlight the risk of assuming the same δ18Osw value for δ18O-derived temperature from different oceanic regions. These ﬁndings provide critical new constraints for model simulations of Jurassic temperatures and δ18Osw val- ues and suggest that high climate sensitivity has been a hall- mark of greenhouse climates for at least 180 Myr. Proxy data indicate that the Cretaceous–early Paleogene (∼140 to 34 Myr ago) was characterized by high atmo- spheric CO2 concentrations, extreme polar warmth, and re- duced latitudinal temperature gradients (Sluijs et al., 2006; Suan et al., 2017; Evans et al., 2018). Most state-of-the-art climate models hardly reproduce such features, implying ei- ther a higher climate sensitivity under greenhouse conditions or systematic biases in proxy data interpretation (Huber and Caballero, 2011; Laugié et al., 2020; Zhu et al., 2020). It re- mains unclear whether higher climate sensitivity is unique to the Cretaceous–early Paleogene world or is rather a hallmark of Earth’s climate under high atmospheric pCO2. Tempera- ture proxies sensitive to overwriting under important burial, such as molecular or clumped isotope thermometry (Henkes et al., 2014; Fernandez et al., 2021; Hemingway and Henkes, 2021), have seldom been applied to older sediments owing to their generally higher thermal maturity (Robinson et al., 2017; Ruebsam et al., 2020; Fernandez et al., 2021). Clumped isotope evidence for Early Jurassic extreme polar warmth and high climate sensitivity Thomas Letulle1, Guillaume Suan1, Mathieu Daëron2, Mikhail Rogov3, Christophe Lécuyer1, Arnauld Vinçon-Laugier1, Bruno Reynard1, Gilles Montagnac1, Oleg Lutikov3, and Jan Schlögl4 homas Letulle1, Guillaume Suan1, Mathieu Daëron2, Mikhail Rogov3, Christophe Lécuyer1, 1 1 1 3 Thomas Letulle1, Guillaume Suan1, Mathieu Daëron2, Mikhail Rogov3, Christophe Lécuyer Arnauld Vinçon-Laugier1, Bruno Reynard1, Gilles Montagnac1, Oleg Lutikov3, and Jan Sch , , , g , p y , rnauld Vinçon-Laugier1, Bruno Reynard1, Gilles Montagnac1, Oleg Lutikov3, and Jan Schlög 1Univ Lyon, UCBL, ENSL, UJM, CNRS, LGL-TPE, 69622, Villeurbanne, France 2Laboratoire des Sciences du Climat et de l’Environnement, LSCE/IPSL, CEA-CNRS-UVSQ, Université Paris-Saclay, Orme des Merisiers, 91191 Gif-sur-Yvette CEDEX, France 3Laboratory of Phanerozoic Stratigraphy, Geological Institute of Russian Academy of Sciences, 119017 Moscow, Russia 4Department of Geology and Palaeontology, Faculty of Natural Sciences, Comenius University, 3Laboratory of Phanerozoic Stratigraphy, Geological Institute of Russian Academy of Sciences, 119017 Moscow, Russia 4Department of Geology and Palaeontology, Faculty of Natural Sciences, Comenius University, Mlynská dolina G, 842 15 Bratislava, Slovak Republik Correspondence: Thomas Letulle (thomas.letulle@univ-lyon1.fr) Correspondence: Thomas Letulle (thomas.letulle@univ-lyon1.fr) Received: 29 June 2021 – Discussion started: 29 July 2021 Revised: 21 January 2022 – Accepted: 25 January 2022 – Published: 4 March 2022 Received: 29 June 2021 – Discussion started: 29 July 2021 Revised: 21 January 2022 – Accepted: 25 January 2022 – Published: 4 March 2022 HAL Id: hal-03607369 https://hal.science/hal-03607369v1 Submitted on 13 Mar 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Clim. Past, 18, 435–448, 2022 https://doi.org/10.5194/cp-18-435-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Clim. Past, 18, 435–448, 2022 https://doi.org/10.5194/cp-18-435-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. 2.2 Warcq Here, we use carbonate clumped isotope thermometry (147) to simultaneously constrain the calciﬁcation temper- atures and associated δ18Ow values of marine carbonate shells (mostly aragonite) collected from Lower Jurassic sed- imentary successions with exceptionally shallow to moder- ate burial depths spanning subtropical to polar paleolatitudes. We compare our results to existing Jurassic to Eocene climate proxy data and simulations and discuss their implications for climate sensitivity under greenhouse conditions. Samples from the north-eastern Paris Basin were collected in 2014 from a temporary road cutting located near Warcq, Ar- dennes, France (49◦45′21.6′′ N, 4◦39′28.8′′ E). They consist of grey silty claystone with lenses of packed carbonated shell fragments, mainly from a variety of bivalves (Grammatodon, Malletia, Limea, Oxytoma) and a few ammonoids (Bean- iceras, Aegoceras ?, Dactylioceratidae) (Fig. 2). The lithol- ogy, fossil preservation, and assemblages of the sampled beds are similar to those described by Thuy et al. (2011) from a nearby site in Sedan dated from the Pliensbachian Davoei zone. The sampled levels are therefore tentatively attributed to the lower Pliensbachian Davoei ammonite zone. Mean Tmax values of 425 ◦C and maximum burial temperatures near 60 ◦C have been reported for Pliensbachian sediments from NE Paris Basin boreholes where the Davoei zone is ∼1100 m deep in the EST 433 borehole, some 150 km south from Warcq (Blaise et al., 2014), and at ∼860 m in the Mont- cornet borehole, some 50 km west from Warcq (Disnar et al., 1996; Bougeault et al., 2017). These burial temperatures and depth should be regarded as an upper limit, as the very prox- imal sampling area near Warcq repeatedly emerged during the Mesozoic and hence shows a much thinner Mesozoic cover of ∼500 m compared to these more distal sites (Wa- terlot et al., 1960). Assuming 860 m of overburden, ∼300 m of Cretaceous overburden eroded during the Cenozoic based on Paris Basin thermal history (Brigaud et al., 2020), a Meso- zoic surface temperature of ∼20 ◦C, and a canonical conti- nental geothermal gradient of ∼35 ◦Ckm−1, maximal burial temperature of ∼60 ◦C can be estimated for the studied spec- imens from Warcq. 2.1 Polovinnaya River The Polovinnaya River section is located in northern Siberia (72◦36′05′′ N, 107◦58′52.2′′ E) and was located near the North Pole during the Early Jurassic (Fig. 1). Our bivalve samples come from between 0 and 14 m in the section and belong to the Toarcian (Suan et al., 2011). This interval con- sists of silty shale slightly enriched in organic carbon (TOC ∼0.5 %). Except for localized carbonate concretions, there is no carbonate fraction in the sediment. The studied in- terval has been previously correlated with the lower Toar- cian serpentinum ammonite zone based on biostratigraphy of foraminifera as well as dinoﬂagellate cyst and lithostrati- graphic correlation with other sections of the basin (Suan et al., 2011). This section records very abundant Dacry- omya bivalve shells (Fig. S1 in the Supplement), an op- portunistic suspension-feeder genus tolerant to poorly oxy- genated waters, which preferred conditions with weak hy- drodynamics (Zakharov and Shurygin, 1978). A few belem- nite rostra were also recorded, as were isolated ﬁsh scales and teeth. Overall the fossil assemblage indicates fully ma- rine conditions within proximity of the continents as evi- denced by the occurrence of wood debris. The section has undergone low burial as suggested by the low values of Rock-Eval pyrolysis Tmax (mean = 420 ◦C) previously mea- sured in the host sediments (Suan et al., 2011). Regional stratigraphy from the more distal Anabar area suggests local overburden not exceeding 1000 m: a total overburden (lower Toarcian to Valanginian) of about 380 m is recorded in the Anabar River area (Nikitenko et al., 2013) located 200 km east of the Polovinnaya section, which may be extended to about 1000 m when adding Valanginian–Cenomanian over- burden from the more distal Bol’shoi Begichev islands. Mod- ern local geothermal ﬂuxes are lower than 50 mW m−2 (Ke- rimov et al., 2020), indicative of a low geothermal gradient (< 25 ◦Ckm−1). Assuming a warm mean surface tempera- ture of 10 ◦C, 1000 m of overburden, and that the geothermal gradient of the Siberian craton did not signiﬁcantly change in the last 200 Myr, maximum burial temperatures of around 35 ◦C can be estimated for the studied specimens from the Polovinnaya River. 1 Introduction Con- sequently, current temperature estimates predating the Cre- taceous period are mostly derived from the oxygen isotope composition of marine carbonate fossils (δ18Oc), with well- known limitations related to uncertainties in the past δ18O signature of seawater (δ18Ow) (Epstein et al., 1953; Roche et al., 2006; Laugié et al., 2020). Published by Copernicus Publications on behalf of the European Geosciences Union. 436 3.1 Sampled material The two studied sites present exceptionally rare records of aragonite preservation for the Lower Jurassic interval. Dacryomya shells are the most abundant macrofossil and the only bivalve genus to occur in the Polovinnaya River sec- tion. They are very abundant in the lower part of the sec- tion (0 to 8 m). They are mainly represented by adult shells, while juveniles are common in only a few levels. They appear as ∼1 cm distinct individual or detached valves, sometimes close to each other (Fig. 2). The carbonate shells, commonly ﬂattened and partially to entirely preserved, are a few mil- limetres thick but brittle and detached easily from their inner and outer mould. Their cream to white colour contrasts with the dark aspect of the sediment, and a few thicker individuals are iridescent. Mollusc shells from Warcq clearly show a more energetic environment as they mostly appear as packed shell fragments with higher taxonomic diversity relative to the other site. A few complete individuals and separated valves can be ob- Clim. Past, 18, 435–448, 2022 https://doi.org/10.5194/cp-18-435-2022 T. Letulle et al.: Early Jurassic extreme polar warmth T. Letulle et al.: Early Jurassic extreme polar warmth 437 Figure 1. Location of the studied sites with regard to Toarcian (Early Jurassic) geography. (a) Global map modiﬁed from Dera et al. (2009) (b) Arctic map modiﬁed from Nikitenko and Mickey (2004). (c) Tethyan map modiﬁed from Thierry (2000). Localities: PR – Polovinnay Figure 1. Location of the studied sites with regard to Toarcian (Early Jurassic) geography. (a) Global map modiﬁed from Dera et al. (2009). (b) Arctic map modiﬁed from Nikitenko and Mickey (2004). (c) Tethyan map modiﬁed from Thierry (2000). Localities: PR – Polovinnaya River; Wq – Warcq. Figure 1. Location of the studied sites with regard to Toarcian (Early Jurassic) geography. (a) Global map modiﬁed from Dera et al. (2009). (b) Arctic map modiﬁed from Nikitenko and Mickey (2004). (c) Tethyan map modiﬁed from Thierry (2000). Localities: PR – Polovinnaya River; Wq – Warcq. 3.2 Geochemical analysis and data processing served among the debris with their associated mould in or around the remaining shell. Shells are cream to clear white, with some showing iridescence. The 147 and δ18O values of 15 samples were measured (one to ﬁve replicates each) using methods described by Daëron et al. (2016). Carbonate samples were converted to CO2 by phosphoric acid reaction at 90 ◦C in a common, stirred acid bath for 15 min. Initial phosphoric acid concen- tration was 103 % (1.91 gcm−3), and each batch of acid was used for 7 d. After cryogenic removal of water, the evolved CO2 was helium-ﬂushed at 25 mLmin−1 through a puriﬁ- cation column packed with Porapak Q (50/80 mesh, 1 m length, 2.1 mm i.d.) and held at −20 ◦C, then quantitatively recollected by cryogenic trapping and transferred into an Iso- prime 100 dual-inlet mass spectrometer equipped with six Faraday collectors (m/z 44–49). Each analysis took about 2.5 h, during which analyte gas and working reference gas were allowed to ﬂow from matching 10 mL reservoirs into the source through deactivated fused silica capillaries (65 cm length, 110 µm i.d.). Every 20 min, gas pressures were ad- justed to achieve m/z = 44 current of 80 nA, with differ- ences between analyte gas and working gas generally be- low 0.1 nA. Pressure-dependent background current correc- The remnants of carbonate shells were sampled as a whole using dental tools under a binocular microscope. A carbon- ate vein and matrix from the carbonate nodule POL-29 were also sampled to constrain the geochemistry of this potential diagenetic phase. The microstructural preservation state and mineralogy of the analysed bivalve and ammonite shells were investigated using a Phenom Pure G2 scanning electron microscope (SEM) in backscatter mode and Raman spectroscopy using an XploRA Raman microscope in the Laboratoire de Géolo- gie de Lyon (LGL-TPE). SEM observations were performed on relatively large fragments of the most complete speci- mens. Raman spectra were acquired either directly on the fossil specimens partly enclosed in the sedimentary matrix or on several grains of the sampled powders. https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 438 T. Letulle et al.: Early Jurassic extreme polar warmth Figure 2. Selected specimens from the sampled successions. (a–d) Polovinnaya River section (Toarcian). (e–l) Warcq section (Pliens- bachian). (a) Dacryomya jacutica, specimen POL-29 (on the surface of a carbonate concretion). (b) Dacryomya jacutica, specimen POL-13. (c, d) Dacryomya jacutica, specimen POL-05. 5.1 Sample preservation The SEM and Raman observations reveal that the analysed mollusc shells from both sites retain pristine aragonite miner- alogy and microstructures with evidence of neither recrystal- lization nor mineralogical conversion (Fig. 3). Despite their aragonite mineralogy, the Dacryomya shells from sample POL-29 record unusually low δ13C values that are ∼8 ‰ lower than the other Dacryomya shells analysed from the same succession. The carbonate matrix of the nodule where these shells are embedded also records a very low δ13C value (−21.43 ‰) but a δ18O value within the range of the bivalve shells. We therefore attribute the extremely low δ13C values of bivalves shells of this level to an early diagenetic phase resulting in the formation of carbonate nodules derived from respiratory CO2 that locally altered the bivalve shell geo- chemistry. 3.2 Geochemical analysis and data processing Letulle et al.: Early Jurassic extreme polar warmth 439 much lower value of −10.6 ± 0.9 ‰ is obtained for the fracture-inﬁlling calcite vein from the Polovinnaya River. prime™mass spectrometer. Samples were reacted with an- hydrous phosphoric acid at 90 ◦C. Duplicated samples were adjusted to the international references NIST NBS 18 and NBS 19 as well as in-house standard Carrara Marble. Since 2019 overall reproducibility of the in-house standard Car- rara Marble has been ± 0.088 ‰ for δ18O (2 SE, n = 649) and ± 0.064 ‰ for δ13C (2 SE, n = 649) with mean δ18O and δ13C values of −1.041 ‰ and +2.025 ‰ (VPDB), re- spectively. All carbonate isotopic values (δ13C, δ18Oc) are reported in ‰ VPDB. 4.2 Warcq, France Mollusc shells from Warcq showing an aragonite mineral- ogy revealed microstructures similar to those observed in Dacryomya jacutica from Siberia, the main differences be- ing that sheet nacreous structures of the studied ammonite shell (ARD-05) show thinner tablets than those of bivalve shells (Fig. 3). Both SEM and Raman data indicate that the sample ARD-03 (bivalve fragment) is in calcite, showing a darker colour and no iridescence, with a much simpler and massive structure observed in SEM (Fig. 3). p Clumped isotope temperatures were computed based on the I-CDES calibration of Anderson et al. (2021). Temper- ature uncertainties correspond to the fully propagated 95 % conﬁdence intervals from 147 measurements of each sample (Daëron, 2021), neglecting the much smaller uncertainties in the calibration. The δ18O values from aragonite samples were adjusted considering the different phosphoric acid frac- tionation factors for calcite and aragonite (Kim et al., 2007). The δ18Ow values relative to VSMOW were estimated us- ing 147-derived temperatures and the equations of Grossman and Ku (1986) and Kim and O’Neil (1997) for mollusc shells and calcite vein, respectively. The 147 values of measured bivalve and ammonite shells from Warcq range from 0.5851 ± 0.0095 ‰ to 0.5955 ± 0.0130 ‰ I-CDES. Reconstructed 147 tempera- tures range from 24.4 ± 4.4 to 28.0 ± 3.3 ◦C. Mean δ18Oc values are −2.30 ± 0.76 ‰ (1 SD, n = 6, max = −0.83 ‰, min = −2.79 ‰), and δ13C ranges from 0.37 ‰ to 2.82 ‰. The calculated δ18Ow values range from 0.6 ± 0.7 ‰ to −1.4 ± 1.0 ‰. Paleolatitude of the studied sites was computed using the online paleolatitude calculator paleolatitude.org (van Hins- bergen et al., 2015) computed with the model of Torsvik et al. (2012). 3.2 Geochemical analysis and data processing (e, f) Grammatodon sp., specimen ARD-01 (inner e and outer f mould after sampling of the shell). (g, h) Malletia sp. (i) Limea sp., specimen ARD-03 (inner mould after sampling of the shell). (j) Oxytoma sp. ind. (inner mould with remains of a calcite shell). (k) Aegoceras ?, specimen ARD-06. (l) Dactylioceratidae indet., specimen ARD-07. Figure 2. Selected specimens from the sampled successions. (a–d) Polovinnaya River section (Toarcian). (e–l) Warcq section (Pliens- bachian). (a) Dacryomya jacutica, specimen POL-29 (on the surface of a carbonate concretion). (b) Dacryomya jacutica, specimen POL-13. (c, d) Dacryomya jacutica, specimen POL-05. (e, f) Grammatodon sp., specimen ARD-01 (inner e and outer f mould after sampling of the shell). (g, h) Malletia sp. (i) Limea sp., specimen ARD-03 (inner mould after sampling of the shell). (j) Oxytoma sp. ind. (inner mould with remains of a calcite shell). (k) Aegoceras ?, specimen ARD-06. (l) Dactylioceratidae indet., specimen ARD-07. et al., 2007). Raw 147 values were then converted to the Intercarb-Carbon Dioxide Equilibrium Scale (I-CDES) 147 reference frame by comparison with four “ETH” carbonate standards (Bernasconi et al., 2021) using a pooled regression approach (Daëron, 2021). Full analytical errors are derived from the external reproducibility of unknowns and standards (Nf = 89) and conservatively account for the uncertainties in raw 147 measurements as well as those associated with the conversion to the “absolute” 147 reference frame. tions were measured 12 times for each analysis. All back- ground measurements from a given session are then used to determine a mass-speciﬁc relationship linking background intensity (Zm), total m/z = 44 intensity (I44), and time (t): Zm = a + bI44 + ct + dt2. Background-corrected ion cur- rent ratios (δ45 to δ49) were converted to δ13C, δ18O, and “raw” 147 values as described by Daëron et al. (2016) us- ing the IUPAC oxygen-17 correction parameters. The iso- topic composition (δ13C, δ18O) of our working reference gas was computed based on the nominal isotopic composi- tion of carbonate standard ETH-3 (Bernasconi et al., 2018) and an oxygen-18 acid fractionation factor of 1.00813 (Kim Complementary δ13C and δ18O analyses of the smallest Arctic shells were performed at LGL-TPE using a Multi- prep™automated sampler coupled to a dual-inlet GV Iso- https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. 4.1 Polovinnaya River, Siberia The SEM observations of shell fragments of Dacryomya ja- cutica revealed well-preserved sheet nacreous microstruc- tures underlying a prismatic layer we interpret as the outer shell layer (Fig. 3). All Raman spectra gathered from Dacry- omya jacutica shells conﬁrm that the original aragonite min- eralogy is preserved. The 147 values range from 0.6151 ± 0.0108 ‰ to 0.6457 ± 0.0182 ‰ I-CDES for Siberian aragonite bivalves, with a 147 of 0.5752 ± 0.0134 for the fracture-inﬁlling cal- cite vein. Reconstructed 147 temperatures, applying the equation of Anderson et al. (2021), range from 8.8 ± 5.2 to 18.0 ± 3.4 ◦C for Siberian bivalves and 31.5 ± 4.8 ◦C for the calcite vein. Organic matter maturity, mineralogical, and sedimento- logical data all imply exceptionally shallow burial depth (< 1 km) for the samples investigated here. Maximum burial temperature (Tburial) remained well below the commonly as- sumed minimum temperature (80–120 ◦C) of solid-state re- ordering of C–O bonds in calcite at geological timescales (Henkes et al., 2014; Stolper and Eiler, 2015; Hemingway and Henkes, 2021). Recent experiments show that arago- nite is more susceptible to solid-state bond reordering (Chen et al., 2019), but to our knowledge there is no existing model constraining the temperatures at which this process would markedly overwrite the 147 value of this mineral at such timescales. The exceptional preservation of aragonite nacre- Mean δ18Oc values are −2.73 ± 0.71 ‰ (1 SD, n = 31, max = 0.36, min = −5.08 ‰) for Siberian bivalves and −14.21 ± 0.02 ‰ for the fracture-inﬁlling calcite vein, while the carbon isotope values (δ13C) range from 3.47 ‰ to 5.09 ‰ in bivalve shells and reach values down to −21.43 ‰ and −4.67 ‰ for the carbonate nodule matrix and embed- ded bivalve shells (sample POL-29), respectively. Using the 147-derived temperature to estimate the oxygen isotope fractionation factor results in δ18Ow values ranging from −4.88 ± 1.20 ‰ to −2.52 ± 0.78 ‰ in Siberian bivalves. A https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. Letulle et al.: Early Jurassic extreme polar warmth 440 Figure 3. SEM images and Raman spectra for a selection of samples. (a, c, e, and g) SEM images of samples POL-8, POL-12, ARD-05, and ARD-03, respectively. (b, d, f, and g) Raman spectra of samples POL-8, POL-12, ARD-05, and ARD-03 compared to the reference spectra of calcite and aragonite. Figure 3. 4.1 Polovinnaya River, Siberia SEM images and Raman spectra for a selection of samples. (a, c, e, and g) SEM images of samples POL-8, POL-12, ARD-05, and ARD-03, respectively. (b, d, f, and g) Raman spectra of samples POL-8, POL-12, ARD-05, and ARD-03 compared to the reference spectra of calcite and aragonite. ingway and Henkes, 2021). Therefore, both the temperature and δ18Ow reported here would be overestimated and should be taken as an upper limit of original environmental parame- ters. ous sheet microstructures in these samples implies minimal amounts of ﬂuid circulation and recrystallization, if any. Ex- change between ﬂuid inclusions in mollusc shells and the surrounding carbonate minerals was recently suggested as an alternative process that may alter the clumped isotope sig- nature of biogenic carbonates without substantially affect- ing the stable isotope signature of the shell or its mineralogy (Nooitgedacht et al., 2021). In heating experiments, these ex- changes resulted in a signiﬁcant decrease in the 147 value of the bivalve shells compared to the original shell and a minor (∼0.1 ‰) decrease in δ18O of the heated shell. We cannot exclude the possibility that this process has altered the fos- sils studied here even at low temperature, nor do we have evi- dence that it occurred. The 147 temperature of 31.1 ± 4.8 ◦C for the fracture-inﬁlling calcite vein in Arctic Russia is sig- niﬁcantly higher than those inferred from bivalves and is con- sistent with a formation depth < 1 km assuming a geothermal gradient of 25 ◦Ckm−1. The reconstructed δ18Ow value of −10.7 ± 0.9 ‰ for this calcite vein is also substantially lower than those inferred from associated bivalves, consistent with a late-phase meteoric source for the mineralizing ﬂuid. The precise depth and date at which this vein formed, however, remain uncertain. T. Letulle et al.: Early Jurassic extreme polar warmth A comparable seasonal δ18Oc record could not be generated from our Russian Arc- tic material owing to the very small size of the available Dacryomya shells (1 to 2 cm). In any case, the temperate data from NE France should be minimally affected by sea- sonal biases as shell precipitation occurs more continuously throughout the year in modern temperate molluscs (Killam and Clapham, 2018). Besides, both sites were deposited in nearshore environments at very shallow depths likely not ex- ceeding a few tens of metres (Suan et al., 2011; Thuy et al., period of maximum growth rate. Shell growth rate can be controlled by environmental parameters (temperature, salin- ity, food availability), biological processes such as spawning, and changes during the ontogeny (Schöne, 2008). One major aspect of shell growth that may bias the geochemical sig- nal data is seasonal shell growth cessation. In modern high- latitude bivalves, seasonal shell growth cessation generally occurs during the winter, triggered by low temperatures or low food availability (Peck et al., 2000; Vihtakari et al., 2016; Killam and Clapham, 2018). In the present-day Nucula an- nulata, an aragonite bivalve with ecology similar to the anal- ysed Dacryomya jacutica, growth cessation occurs in win- ter and during spawning at peak local temperatures, with its average δ18Oc hence recording late spring to early fall SST (Craig, 1994). By contrast, growth band δ18Oc offers evi- dence for summertime-only growth cessation in high-latitude Eocene bivalves from Antarctica, with inferred winter SST of 11.1 ± 0.6 and summer SST of 17.6 ± 1.3 ◦C (Buick and Ivany, 2004; Douglas et al., 2014). A comparable seasonal δ18Oc record could not be generated from our Russian Arc- tic material owing to the very small size of the available Dacryomya shells (1 to 2 cm). In any case, the temperate data from NE France should be minimally affected by sea- sonal biases as shell precipitation occurs more continuously throughout the year in modern temperate molluscs (Killam and Clapham, 2018). Besides, both sites were deposited in nearshore environments at very shallow depths likely not ex- ceeding a few tens of metres (Suan et al., 2011; Thuy et al., The reconstructed polar δ18Ow values ranging from −4.9 ± 1.2 ‰ to −2.5 ± 0.8 ‰ VSMOW during the T-OAE are signiﬁcantly lower than the value of −1 ‰ VSMOW expected for an ice-free world mean open ocean (Shackle- ton and Kennett, 1975). 5.2 Evidence for extreme warmth and reduced salinity in the Arctic during the Toarcian Oceanic Anoxic Event (T-OAE) Bivalve shells record a marked rise in δ13C along the section up to ∼5 ‰ that parallels that recorded by organic carbon δ13C data (Fig. 4; Suan et al., 2011). These results strengthen the correlation of the corresponding part of the succession with the rising limb of the positive carbon isotope excursion commonly used to characterize the termination of the T-OAE interval at coeval sites in Europe and North Africa (Jenkyns and Clayton, 1986; Suan et al., 2010; Krencker et al., 2014; Baghli et al., 2020; Ullmann et al., 2020). Bivalve shell δ18Oc values, however, show no stratigraphic trend as opposed to brachiopod shell T-OAE records from the western Tethys at mid-latitudes (Suan et al., 2010; Krencker et al., 2014; Baghli et al., 2020; Ullmann et al., 2020). Our 147 results yield polar temperatures ranging from 8.8 ± 5.2 to 18.0 ± 3.4 ◦C (mean = 14.7 ◦C). As occurs with most 147-derived temper- ature datasets, the relatively large uncertainties of the present estimates of Siberian sea surface temperature (SST) hamper the identiﬁcation of distinctive stratigraphic trends. Based on the geological setting of the samples and their preservation, we consider any substantial alteration of their original geochemical signature unlikely. In the remote sce- nario that the studied material has been slightly modiﬁed by solid-state bond reordering, one would expect the 147 of the samples to be lower than their original values (Henkes et al., 2014; Stolper and Eiler, 2015; Fernandez et al., 2021; Hem- Bivalve shell growth can be highly variable during the an- imal’s life (Schöne, 2008), making any paleoenvironmental record derived from the bivalve shell either incomplete (be- cause of growth cessation) or at least biased towards the https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. Letulle et al.: Early Jurassic extreme polar warmth 441 Figure 4. Geochemical record of the T-OAE at the Polovinnaya River, Arctic Siberia. Stratigraphy and biostratigraphic zones are for the Arctic realm (falc.: falciferum zone, com.: commune zone). The organic carbon isotope (δ13CTOC) data (black squares) and wood debris δ13C data (white diamonds) are from Suan et al. (2011); the bivalve shell δ13C data (blue squares), bivalve shell δ18O (red squares), 147 temperatures (blue stars), and δ18Ow estimates (blue circles) inferred from bivalve shell 147 values are from this study. The analysed bivalve samples all belong to the species Dacryomya jacutica. 147-derived temperatures were computed using the equation of Anderson et al. (2021). T. Letulle et al.: Early Jurassic extreme polar warmth T. Letulle et al.: Early Jurassic extreme polar warmth T. Letulle et al.: Early Jurassic extreme polar warmth 441 Figure 4. Geochemical record of the T-OAE at the Polovinnaya River, Arctic Siberia. Stratigraphy and biostratigraphic zones are for the Arctic realm (falc.: falciferum zone, com.: commune zone). The organic carbon isotope (δ13CTOC) data (black squares) and wood debris δ13C data (white diamonds) are from Suan et al. (2011); the bivalve shell δ13C data (blue squares), bivalve shell δ18O (red squares), 147 temperatures (blue stars), and δ18Ow estimates (blue circles) inferred from bivalve shell 147 values are from this study. The analysed bivalve samples all belong to the species Dacryomya jacutica. 147-derived temperatures were computed using the equation of Anderson et al. (2021). δ18Ow was calculated using the oxygen isotope fractionation equation of Grossman and Ku (1986). 2011). Although bivalves from both sections record temper- atures near the sea bottom that were likely slightly cooler than the sea surface, the difference should not have exceeded a few degrees; owing to their shallow living depth we ex- pect the studied bivalves to have lived within the thermo- cline. We therefore conservatively interpret the reconstructed temperatures as reﬂecting polar warm-season SST (summer; SSTPWS) in Arctic Russia and low-latitude annual SST in NE France. These SSTPWS values for the T-OAE are still 10– 20 ◦C higher than present-day SSTPWS (Fig. 5). period of maximum growth rate. Shell growth rate can be controlled by environmental parameters (temperature, salin- ity, food availability), biological processes such as spawning, and changes during the ontogeny (Schöne, 2008). One major aspect of shell growth that may bias the geochemical sig- nal data is seasonal shell growth cessation. In modern high- latitude bivalves, seasonal shell growth cessation generally occurs during the winter, triggered by low temperatures or low food availability (Peck et al., 2000; Vihtakari et al., 2016; Killam and Clapham, 2018). In the present-day Nucula an- nulata, an aragonite bivalve with ecology similar to the anal- ysed Dacryomya jacutica, growth cessation occurs in win- ter and during spawning at peak local temperatures, with its average δ18Oc hence recording late spring to early fall SST (Craig, 1994). By contrast, growth band δ18Oc offers evi- dence for summertime-only growth cessation in high-latitude Eocene bivalves from Antarctica, with inferred winter SST of 11.1 ± 0.6 and summer SST of 17.6 ± 1.3 ◦C (Buick and Ivany, 2004; Douglas et al., 2014). 5.2 Evidence for extreme warmth and reduced salinity in the Arctic during the Toarcian Oceanic Anoxic Event (T-OAE) δ18Ow was calculated using the oxygen isotope fractionation equation of Grossman and Ku (1986). T. Letulle et al.: Early Jurassic extreme polar warmth 147 data are from the following: Keating-Bitonti et al., 2011; Douglas et al., 2014; Petersen et al., 2016b, a; Evans et al., 2018; Wierzbowski et al., 2018; Meyer et al., 2018; Vickers et al., 2019, 2020, 2021; Price et al., 2020; Brigaud et al., 2020; Fernandez et al., 2021; de Winter et al., 2021. TEXH 86 paleothermometry data are from the following: Jenkyns et al., 2012; Lunt et al., 2012; Douglas et al., 2014; Frieling et al., 2014; O’Brien et al., 2017; Robinson et al., 2017; Cramwinckel et al., 2018; O’Connor et al., 2019; Ruebsam et al., 2020; Cavalheiro et al., 2021 . Marine turtle phosphate δ18O data are from Billon- Bruyat et al. (2005), Coulson et al. (2011), and van Baal et al. (2013). The FOAM 6× simulation is from Dera and Donnadieu (2012), the Genesis-MOM 8× simulation is from Zhou et al. (2008), IPSL-CM5A2 4× is from Laugié et al. (2020), CESMA.2 CAM5 6× is from Zhu et al. (2020), HadCM3 6× is from Tindall et al. (2010), and CCSM3 16× is from Huber and Caballero (2011), with Nx indicating CO2 levels used in the simulations as a multiple of pre-industrial level (i.e. 280 ppm). More detailed information on the building of this ﬁgure and the data compilation is available in the Supplement. Figure 5. Comparison of the new (bold outline) reconstructed Early Jurassic SST and δ18Osw with published Jurassic–Eocene proxy-based reconstructions (thin outline) and Earth system simulations. Proxy–model comparisons of SST and δ18Osw are shown for the Jurassic (a, b), the Cretaceous (c, d), and the early Paleogene (e, f). Proxy data are divided between eight time slices based on their deﬁnition in the Interna- tional Chronostratigraphic Chart v2020/03 (Cohen et al., 2013; updated); early Late Cretaceous: Cenomanian to Santonian; latest Cretaceous: Campanian–Maastrichtian. Marker colour shows sample age accordingly (see key). New data are displayed as the reported value and its as- sociated uncertainties for each samples. Datasets from the literature, from the same proxy and location, were regrouped within each time slice. The marker displays the mean of available data and the error bar the extent of the data (minimal to maximal value). Both 147 and TEXH 86 temperatures are published temperatures. δ18Ow values were recomputed using published 147 temperatures and δ18Oc with the fol- lowing fractionation equations – belemnite calcite: Coplen, (2007), aragonite: Grossman and Ku(1986), bivalve calcite: Epstein et al. T. Letulle et al.: Early Jurassic extreme polar warmth These results imply a substantial freshwater contribution to the studied basin during the T- OAE, probably resulting from coastal runoff at this rela- tively proximal site (Suan et al., 2011). High temperatures and reduced salinity are in broad agreement with paleonto- logical evidence for warm and humid temperate conditions during the T-OAE interval in Arctic Siberia (Rogov et al., 2019). Brackish conditions are also consistent with the fos- sil assemblages of the succession that includes abundant ter- restrial organic matter and wood debris, marine to brack- ish elements such as abundant dinoﬂagellate cysts, benthic foraminifera (preserved as organic linings and agglutinate forms), and typically marine elements that are represented by a few belemnite rostra and unidentiﬁable ammonite in- https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. Letulle et al.: Early Jurassic extreme polar warmth T. Letulle et al.: Early Jurassic extreme polar warmth 442 442 T. Letulle et al.: Early Jurassic extreme polar warmth Figure 5. Comparison of the new (bold outline) reconstructed Early Jurassic SST and δ18Osw with published Jurassic–Eocene proxy-based reconstructions (thin outline) and Earth system simulations. Proxy–model comparisons of SST and δ18Osw are shown for the Jurassic (a, b), the Cretaceous (c, d), and the early Paleogene (e, f). Proxy data are divided between eight time slices based on their deﬁnition in the Interna- tional Chronostratigraphic Chart v2020/03 (Cohen et al., 2013; updated); early Late Cretaceous: Cenomanian to Santonian; latest Cretaceous: Campanian–Maastrichtian. Marker colour shows sample age accordingly (see key). New data are displayed as the reported value and its as- sociated uncertainties for each samples. Datasets from the literature, from the same proxy and location, were regrouped within each time slice. The marker displays the mean of available data and the error bar the extent of the data (minimal to maximal value). Both 147 and TEXH 86 temperatures are published temperatures. δ18Ow values were recomputed using published 147 temperatures and δ18Oc with the fol- lowing fractionation equations – belemnite calcite: Coplen, (2007), aragonite: Grossman and Ku(1986), bivalve calcite: Epstein et al. (1953), foraminifera: Erez and Luz (1983), turtle bones: Barrick et al. (1999), updated by Pouech et al. (2014). Results of Earth system simulations from the literature are shown as annual averages (bold lines) and summer and winter seasonal averages (colour shading). Modern ranges of SST and δ18Osw are also shown (grey shading) for comparison. T. Letulle et al.: Early Jurassic extreme polar warmth (1953), foraminifera: Erez and Luz (1983), turtle bones: Barrick et al. (1999), updated by Pouech et al. (2014). Results of Earth system simulations from the literature are shown as annual averages (bold lines) and summer and winter seasonal averages (colour shading). Modern ranges of SST and δ18Osw are also shown (grey shading) for comparison. 147 data are from the following: Keating-Bitonti et al., 2011; Douglas et al., 2014; Petersen et al., 2016b, a; Evans et al., 2018; Wierzbowski et al., 2018; Meyer et al., 2018; Vickers et al., 2019, 2020, 2021; Price et al., 2020; Brigaud et al., 2020; Fernandez et al., 2021; de Winter et al., 2021. TEXH 86 paleothermometry data are from the following: Jenkyns et al., 2012; Lunt et al., 2012; Douglas et al., 2014; Frieling et al., 2014; O’Brien et al., 2017; Robinson et al., 2017; Cramwinckel et al., 2018; O’Connor et al., 2019; Ruebsam et al., 2020; Cavalheiro et al., 2021 . Marine turtle phosphate δ18O data are from Billon- Bruyat et al. (2005), Coulson et al. (2011), and van Baal et al. (2013). The FOAM 6× simulation is from Dera and Donnadieu (2012), the Genesis-MOM 8× simulation is from Zhou et al. (2008), IPSL-CM5A2 4× is from Laugié et al. (2020), CESMA.2 CAM5 6× is from Zhu et al. (2020), HadCM3 6× is from Tindall et al. (2010), and CCSM3 16× is from Huber and Caballero (2011), with Nx indicating CO2 levels used in the simulations as a multiple of pre-industrial level (i.e. 280 ppm). More detailed information on the building of this ﬁgure and the data compilation is available in the Supplement. ternal moulds (Suan et al., 2011). Interestingly, protobranch bivalves, to which Dacryomya belongs, are not well adapted to salinities lower than 20 ‰ (Zardus, 2002). Assuming a similar lower salinity limit for Polovinnaya River bivalves, a global mean ocean with a salinity of 34.5 ‰, and a δ18Ow of −1 ‰ VSMOW, mass balance considerations (see the Sup- plement) imply an upper limit of ∼−8 ‰ VSMOW for lo- cal δ18O of precipitation and runoff (δ18Op). This value is high relative to modern Arctic δ18Op but in agreement with the prediction that higher polar temperatures should have produced higher δ18Op than those prevailing today (Rozan- ski et al., 1992). 5.3 Early Jurassic latitudinal temperature and δ18Ow gradients The mid-paleolatitude SSTs reconstructed by our new clumped isotope data (∼25 ◦C) are in good agreement with recent Sinemurian–Pliensbachian and Toarcian TEXH 86 data pointing to summer SST ∼20–30 ◦C at slightly lower pale- olatitudes (Robinson et al., 2017; Ruebsam et al., 2020). It should be noted that the mid-latitude samples presented here are from the Davoei zone and predate the T-OAE interval recorded by the Siberian data by ∼6 million years. Neverthe- less, climate proxies from the Davoei zone indicate that the corresponding time interval, although likely slightly cooler than the T-OAE, corresponds to one of the warmest periods of the Early Jurassic (Dera et al., 2011; Bougeault et al., 2017). The new clumped isotope data from the two sites, even if they are not strictly contemporaneous, can therefore be reasonably used to tentatively estimate latitudinal gradi- ent during the warmest episodes of the Early Jurassic. The 147 data presented herein suggest a decrease in mean SST of 0.26 ± 0.05 ◦C per degree of latitude between middle and high latitudes, i.e. a reduction of the latitudinal SST gradi- ent of 32 ± 10 % relative to present, consistent with the most conservative early Eocene estimates (Evans et al., 2018). Comparing our Siberian T-OAE 147 temperatures with con- temporaneous TEXH 86 temperatures estimated for low lati- tudes (Ruebsam et al., 2020) results in an even shallower gradient of 0.17 ± 0.05 ◦C per degree of latitude between low and high latitudes. The reconstruction of δ18Ow values using proxy data pro- vides a complementary aspect to assess model capabilities, as this indicator is sensitive to both climate parameters (mois- ture, humidity, and temperatures) and paleogeography. Our mid-latitude δ18Ow values are broadly similar to those recon- structed using marine turtle bone δ18OPO4 and 147 data from Jurassic to Eocene bivalves, ammonites, and foraminifera (Fig. 5, Billon-Bruyat et al., 2005; Coulson et al., 2011; van Baal et al., 2013; Evans et al., 2018; Wierzbowski et al., 2018; Vickers et al., 2021; de Winter et al., 2021). The re- constructed δ18Ow values are, to some extent, also broadly Considering the scarcity of other Early Jurassic temper- ature proxy data, model-based SST, and δ18Ow estimates, we extend the comparison to SST and δ18Ow estimates based on various proxy data and published Earth system simulations for other Jurassic to Eocene intervals (Fig. 5; Supplement). T. Letulle et al.: Early Jurassic extreme polar warmth Such values are also comparable to the salinity of 28 ‰ estimated using a fully coupled ocean–atmosphere model for the Toarcian (Dera and Donnadieu, 2012), al- though the Arctic temperatures obtained by the same model are in strong disagreement with our data (Fig. 5). Such ob- servations should be replicated around the Arctic realm to test whether the brackish environment evidenced here was of local or more regional nature. ( g ) Our 147 SST for the Lower Jurassic can be compared to published results from Earth system models that simu- late these intervals of global warmth (see previous para- graph) to discuss model–data discrepancies, especially ap- parent at high latitudes. Proxy data indicate an atmospheric pCO2 of 1000 ± 500 ppmv during the Early Jurassic, with maximum values of 1750 ± 500 ppmv, i.e. 6× pre-industrial levels (PILs), during the T-OAE (McElwain et al., 2005; Li et al., 2020). Earth system models run at 6× PIL for the Early Jurassic (Dera and Donnadieu, 2012) or Cretaceous–Eocene paleogeography almost invariably produce lower SSTs than those inferred from our 147 data, with a maximum model– data discrepancy of > 15 ◦C at high latitudes (Fig. 5). To achieve such polar warmth, the Eocene CCSM3 simulations require 16× PIL, which is more than twice that indicated by Lower Jurassic and Eocene proxy data (Huber and Ca- ballero, 2011). Reconstructed SSTs of 14.4 ± 2.8 ◦C near the North Pole during the T-OAE, however, correspond to the maximum monthly temperatures simulated by the Tur- onian IPSL-CM5A2 model near the North Pole at 4× PIL (Laugié et al., 2020). The hypothesis of shell growth re- stricted to the warmest month in the analysed Toarcian Arc- tic bivalves, however, remains questionable given the evi- dence for summertime-only growth cessation in Eocene bi- valves from Antarctica (Buick and Ivany, 2004). Finally, Arc- tic SSTs as high as 15–20 ◦C are successfully achieved in the Eocene CESM1.2 CAM5 at 6 to 9× PIL (Zhu et al., 2020), in which climate sensitivity increases with rising CO2 due to low-altitude cloud albedo feedbacks and improved radiative parameterization. As this model produces an increase in cli- mate sensitivity with CO2 in both Eocene and modern condi- tions, our results thus support the growing body of evidence that the amplitude of future anthropogenic warming may be underestimated by conventional state-of-the-art models. 18 T. Letulle et al.: Early Jurassic extreme polar warmth Similarly, terrestrial plant n-alkane hydro- gen isotopes and paleosol siderite 147 data indicate slightly lower Arctic δ18Op of −10 ‰ to −15 ‰ VSMOW during the early Eocene (Pagani et al., 2006; van Dijk et al., 2020), another well-established warm period with evidence of polar warmth (Markwick, 1998; Sluijs et al., 2006, 2020; Douglas et al., 2014; Suan et al., 2017; van Dijk et al., 2020). As- https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 443 T. Letulle et al.: Early Jurassic extreme polar warmth terval (20–30 ◦C), with values > 5 ◦C higher than present- day SST. Mid-latitude Cenomanian–Turonian TEXH 86 SSTs are signiﬁcantly higher (> 30 ◦C) and form the highest mid- latitude temperature of the compilation. High-latitude data are much more scarce. Still, Russian Arctic Toarcian SSTs are very close to early Eocene polar SST derived from Arc- tic (Sluijs et al., 2006, 2020; Suan et al., 2010) and Antarctic (Douglas et al., 2014) sites, with polar SST > 15 ◦C warmer than present during these two distinct greenhouse periods (Fig. 5). suming a similar range of δ18Op values in the Early Jurassic Arctic and the assumption listed above for the Early Jurassic oceans, mass balance calculations indicate mean salinity of 23.9 ± 2.9 ‰ (1σ, n = 8) and 27.7 ± 1.8 ‰ (1σ, n = 8) with δ18Op of −10 ‰ and −15 ‰ VSMOW, respectively (see the Supplement), again consistent with paleontological evidence suggesting brackish waters at the Polovinnaya River during the Toarcian. Such values are also comparable to the salinity of 28 ‰ estimated using a fully coupled ocean–atmosphere model for the Toarcian (Dera and Donnadieu, 2012), al- though the Arctic temperatures obtained by the same model are in strong disagreement with our data (Fig. 5). Such ob- servations should be replicated around the Arctic realm to test whether the brackish environment evidenced here was of local or more regional nature. suming a similar range of δ18Op values in the Early Jurassic Arctic and the assumption listed above for the Early Jurassic oceans, mass balance calculations indicate mean salinity of 23.9 ± 2.9 ‰ (1σ, n = 8) and 27.7 ± 1.8 ‰ (1σ, n = 8) with δ18Op of −10 ‰ and −15 ‰ VSMOW, respectively (see the Supplement), again consistent with paleontological evidence suggesting brackish waters at the Polovinnaya River during the Toarcian. 5.3 Early Jurassic latitudinal temperature and δ18Ow gradients First, the new 147 temperatures can be com- pared with other well-established warm intervals, such as the Cenomanian–Turonian and the Eocene. The compilation shows that 147 SSTs from NE France agree with most pre- vious mid-latitude TEXH 86 and 147 SSTs for the Eocene in- https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. Letulle et al.: Early Jurassic extreme polar warmth 444 comparable with those inferred from belemnite calcite 147 data (Wierzbowski et al., 2018; Vickers et al., 2019, 2020, 2021; Price et al., 2020), although such data should be inter- preted with caution owing to the likely unique oxygen iso- tope fractionation of belemnite calcite (Price et al., 2020; Vickers et al., 2021). In line with evidence for substantial 18O depletion of Toarcian Arctic waters (relative to VS- MOW) suggested by our data, previous studies suggested low δ18Ow values in interior seas bordered by large continen- tal areas, such as in the Western Interior Seaway during the Campanian–Maastrichtian (Coulson et al., 2011; Petersen et al., 2016b; Meyer et al., 2018) and the Middle Russian Sea during the Middle to Late Jurassic interval (Wierzbowski et al., 2018). Interestingly, the 147 temperatures reported in these basins also differ markedly from those reported in more open-ocean sites of similar age and paleolatitude, suggesting the possible inﬂuence of colder Arctic water masses through southward ocean currents. Indeed, the Middle to Late Juras- sic 147 temperatures reported in the Middle Russian Sea (Wierzbowski et al., 2018) are ∼10 ◦C lower than coeval data from the Hebrides basin, Scotland (Vickers et al., 2020), and from the high mid-latitude Falkland Plateau in the South- ern Hemisphere (Vickers et al., 2019). Such anomalies in δ18Ow values and temperatures demonstrate the importance of regional patterns such as river runoff and basin connec- tions on the environmental parameters of a restricted basin (Petersen et al., 2016b). More generally, local anomalies in δ18Ow values evidenced by the new and earlier clumped iso- tope data highlight the ability of this proxy to decipher the inﬂuence of the temperature and δ18Ow values in otherwise similar δ18Oc datasets. latitudes during the early Pliensbachian and SST > 10 ◦C at polar paleolatitudes during the T-OAE. The reconstructed δ18Ow values point to a higher freshwater contribution to- ward Arctic regions, illustrating the dangers of assuming a ﬁxed global δ18Ow value for δ18O-derived temperature re- constructions. 5.3 Early Jurassic latitudinal temperature and δ18Ow gradients Although further work should clarify the in- ﬂuence of seasonal changes in the recorded SST values at polar sites, these results strengthen a growing body of evi- dence for higher climate sensitivity under high atmospheric CO2 conditions and suggest that this higher sensitivity has been a general feature of greenhouse climates since at least 180 Ma. Data availability. Detailed data supporting this study are available in the Supplement. Raw data are available on request to the author. Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/cp-18-435-2022-supplement. Author contributions. TL and GS designed the study and led the writing in close cooperation with CL, MR, and MD. MR and GS participated in the ﬁeldwork and collected the samples. TL prepared and sampled the shell material for geochemistry and performed the SEM observations. MR, JS, and OL identiﬁed the fossils. MD and TL performed the clumped isotope analyses and data processing. AVL and TL performed the stable isotopes analyses and data pro- cessing. BR, GM, and TL gathered and interpreted the Raman spec- tra. TL and GS compiled the paleotemperature proxy database. All authors were involved in the interpretation of the results. We are aware of only three Earth system δ18Ow simula- tions for the broad time interval considered here (Zhou et al., 2008; Tindall et al., 2010; Zhu et al., 2020), hence limiting model–data comparisons. The higher freshwater contribution near high-latitude land masses of the Northern Hemisphere in all these models produced lower δ18Ow values that are broadly consistent with previous data and our proxy data (Fig. 5). This good agreement, however, might be partly fortuitous, as proxy data suggest SSTs much higher than those produced by these models (Fig. 5). As mentioned above (section 5.2), such higher-than-predicted polar warmth would have substantially increased high-latitude δ18Op so that higher runoff would be required to reproduce the mag- nitude of the poleward drop in δ18Ow indicated by proxy data. This highlights the usefulness, in future models of past greenhouse climates, of systematically providing δ18Ow pre- dictions so that δ18Ow estimates derived from 147 data may serve as a constraint on Earth system models. Competing interests. The contact author has declared that nei- ther they nor their co-authors have any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements. 5.3 Early Jurassic latitudinal temperature and δ18Ow gradients We thank Ghislaine Broillet for her help with SEM analyses and Ophélie Lodyga for her help with Raman analyses. We thank two anonymous reviewers for their constructive suggestions and comments that substantially improved the paper. Financial support. This research has been supported by the Agence Nationale de la Recherche through ANR OXYMORE (grant no. ANR-18-CE31-0020) as well as joint CNRS/RFBR In- ternational Emerging Action grants from the Centre National de la Recherche Scientiﬁque (CNRS; grant no. 205700), Russian Foun- dation for Basic Research (RFBR; grant no. 21-55-15015), and Slo- vak Research and Development Agency (grant no. APVV 17-0555 to Jan Schlögl). References W., Hyland, E., In- galls, M., Jaggi, M., John, C. M., Jost, A. B., Katz, S., Kel- son, J., Kluge, T., Kocken, I. J., Laskar, A., Leutert, T. J., Liang, D., Lucarelli, J., Mackey, T. J., Mangenot, X., Meinicke, N., Modestou, S. E., Müller, I. A., Murray, S., Neary, A., Packard, N., Passey, B. H., Pelletier, E., Petersen, S., Pi- asecki, A., Schauer, A., Snell, K. E., Swart, P. K., Tripati, A., Upadhyay, D., Vennemann, T., Winkelstern, I., Yarian, D., Yoshida, N., Zhang, N., and Ziegler, M.: InterCarb: A Com- munity Effort to Improve Interlaboratory Standardization of the Carbonate Clumped Isotope Thermometer Using Carbon- ate Standards, Geochem. Geophy. Geosy., 22, e2020GC009588, https://doi.org/10.1029/2020GC009588, 2021. Coplen, T. B.: Calibration of the calcite–water oxygen- isotope geothermometer at Devils Hole, Nevada, a natural laboratory, Geochim. Cosmochim. Ac., 71, 3948–3957, https://doi.org/10.1016/j.gca.2007.05.028, 2007. Coulson, A. B., Kohn, M. J., and Barrick, R. E.: Iso- topic evaluation of ocean circulation in the Late Creta- ceous North American seaway, Nat. Geosci., 4, 852–855, https://doi.org/10.1038/ngeo1312, 2011. Craig, N.: Growth of the bivalve Nucula annulata in nutrient- enriched environments, Mar. Ecol. Prog. Ser., 104, 77–90, https://doi.org/10.3354/meps104077, 1994. Cramwinckel, M. J., Huber, M., Kocken, I. J., Agnini, C., Bijl, P. K., Bohaty, S. M., Frieling, J., Goldner, A., Hilgen, F. J., Kip, E. L., Peterse, F., van der Ploeg, R., Röhl, U., Schouten, S., and Sluijs, A.: Synchronous tropical and polar temperature evolution in the Eocene, Nature, 559, 382–386, https://doi.org/10.1038/s41586- 018-0272-2, 2018. Billon-Bruyat, J.-P., Lécuyer, C., Martineau, F., and Mazin, J.-M.: Oxygen isotope compositions of Late Jurassic ver- tebrate remains from lithographic limestones of west- ern Europe: implications for the ecology of ﬁsh, turtles, and crocodilians, Palaeogeogr. Palaeocl., 216, 359–375, https://doi.org/10.1016/j.palaeo.2004.11.011, 2005. Daëron, M.: Full Propagation of Analytical Uncertainties in 147 Measurements, Geochem. Geophy. Geosy., 22, https://doi.org/10.1029/2020GC009592, 2021. Daëron, M., Blamart, D., Peral, M., and Affek, H. P.: Absolute isotopic abundance ratios and the accu- racy of 147 measurements, Chem. Geol., 442, 83–96, https://doi.org/10.1016/j.chemgeo.2016.08.014, 2016. Blaise, T., Barbarand, J., Kars, M., Ploquin, F., Aubourg, C., Brigaud, B., Cathelineau, M., El Albani, A., Gautheron, C., Izart, A., Janots, D., Michels, R., Pagel, M., Pozzi, J.-P., Boiron, M.-C., and Landrein, P.: Reconstruction of low temperature (< 100 ◦C) burial in sedimentary basins: A comparison of geothermometer in the intracontinental Paris Basin, Mar. Petrol. Geol., 53, 71–87, https://doi.org/10.1016/j.marpetgeo.2013.08.019, 2014. de Winter, N. J., Müller, I. A., Kocken, I. J., Thibault, N., Ull- mann, C. References Brigaud, B., Bonifacie, M., Pagel, M., Blaise, T., Calmels, D., Hau- rine, F., and Landrein, P.: Past hot ﬂuid ﬂows in limestones de- tected by 147–(U-Pb) and not recorded by other geothermome- ters, Geology, 48, 851–856, https://doi.org/10.1130/G47358.1, 2020. Anderson, N. T., Kelson, J. R., Kele, S., Daëron, M., Bonifa- cie, M., Horita, J., Mackey, T. J., John, C. M., Kluge, T., Petschnig, P., Jost, A. B., Huntington, K. W., Bernasconi, S. M., and Bergmann, K. D.: A uniﬁed clumped isotope thermometer calibration (0.5–1100 ◦C) using carbonate-based standardization, Geophys. Res. Lett., 38, e2020GL092069, https://doi.org/10.1029/2020GL092069, 2021. Buick, D. P. and Ivany, L. C.: 100 years in the dark: Extreme longevity of Eocene bivalves from Antarctica, Geology, 32, 921– 924, https://doi.org/10.1130/G20796.1, 2004. Cavalheiro, L., Wagner, T., Steinig, S., Bottini, C., Dummann, W., Esegbue, O., Gambacorta, G., Giraldo-Gómez, V., Farnsworth, A., Flögel, S., Hofmann, P., Lunt, D. J., Rethemeyer, J., Tor- ricelli, S., and Erba, E.: Impact of global cooling on Early Cretaceous high pCO2 world during the Weissert Event, Nat. Commun., 12, 5411, https://doi.org/10.1038/s41467-021-25706- 0, 2021. Baghli, H., Mattioli, E., Spangenberg, J. E., Bensalah, M., Arnaud- Godet, F., Pittet, B., and Suan, G.: Early Jurassic climatic trends in the south-Tethyan margin, Gondwana Res., 77, 67–81, https://doi.org/10.1016/j.gr.2019.06.016, 2020. Barrick, R. E., Fischer, A. G., and Showers, W. J.: Oxygen iso- topes from turtle bone; applications for terrestrial paleoclimates?, PALAIOS, 14, 186–191, https://doi.org/10.2307/3515374, 1999. Chen, S., Ryb, U., Piasecki, A. M., Lloyd, M. K., Baker, M. B., and Eiler, J. M.: Mechanism of solid-state clumped iso- tope reordering in carbonate minerals from aragonite heat- ing experiments, Geochim. Cosmochim. Ac., 258, 156–173, https://doi.org/10.1016/j.gca.2019.05.018, 2019. Bernasconi, S. M., Müller, I. A., Bergmann, K. D., Breitenbach, S. F. M., Fernandez, A., Hodell, D. A., Jaggi, M., Meckler, A. N., Millan, I., and Ziegler, M.: Reducing Uncertainties in Carbon- ate Clumped Isotope Analysis Through Consistent Carbonate- Based Standardization, Geochem. Geophy. Geosy., 19, 2895– 2914, https://doi.org/10.1029/2017GC007385, 2018. Cohen, K. M., Finney, S. C., Gibbard, P. L., and Fan, J.-X.: The ICS International Chronostratigraphic Chart, Episodes, 36, 199–204, https://doi.org/10.18814/epiiugs/2013/v36i3/002, 2013. Bernasconi, S. M., Daëron, M., Bergmann, K. D., Bonifacie, M., Meckler, A. N., Affek, H. P., Anderson, N., Bajnai, D., Barkan, E., Beverly, E., Blamart, D., Burgener, L., Calmels, D., Chaduteau, C., Clog, M., Davidheiser-Kroll, B., Davies, A., Dux, F., Eiler, J., Elliott, B., Fetrow, A. C., Fiebig, J., Gold- berg, S., Hermoso, M., Huntington, K. T. Letulle et al.: Early Jurassic extreme polar warmth Review statement. This paper was edited by Luc Beaufort and reviewed by two anonymous referees. Review statement. This paper was edited by Luc Beaufort and reviewed by two anonymous referees. R., Huret, E., and Thibault, N.: Climatic and palaeoceano- graphic changes during the Pliensbachian (Early Jurassic) in- ferred from clay mineralogy and stable isotope (C-O) geo- chemistry (NW Europe), Glob. Planet. Change, 149, 139–152, https://doi.org/10.1016/j.gloplacha.2017.01.005, 2017. 6 Conclusions The clumped isotope compositions of pristine, minimally buried, marine mollusc shells yield SST > 25 ◦C at mid- Clim. Past, 18, 435–448, 2022 https://doi.org/10.5194/cp-18-435-2022 445 References V., Farnsworth, A., Lunt, D. J., Claeys, P., and Ziegler, M.: Absolute seasonal temperature estimates from clumped isotopes in bivalve shells suggest warm and vari- able greenhouse climate, Commun. Earth Environ., 2, 121, https://doi.org/10.1038/s43247-021-00193-9, 2021. Bougeault, C., Pellenard, P., Deconinck, J.-F., Hesselbo, S. P., Dommergues, J.-L., Bruneau, L., Cocquerez, T., Laffont, Clim. Past, 18, 435–448, 2022 https://doi.org/10.5194/cp-18-435-2022 T. Letulle et al.: Early Jurassic extreme polar warmth 446 leotemperatures, Geochim. Cosmochim. Ac., 139, 362–382, https://doi.org/10.1016/j.gca.2014.04.040, 2014. leotemperatures, Geochim. Cosmochim. Ac., 139, 362–382, https://doi.org/10.1016/j.gca.2014.04.040, 2014. Dera, G. and Donnadieu, Y.: Modeling evidences for global warm- ing, Arctic seawater freshening, and sluggish oceanic circulation during the Early Toarcian anoxic event, Paleoceanography, 27, PA2211, https://doi.org/10.1029/2012PA002283, 2012. Hu Huber, M. and Caballero, R.: The early Eocene equable climate problem revisited, Clim. Past, 7, 603–633, https://doi.org/10.5194/cp-7-603-2011, 2011. Dera, G., Pucéat, E., Pellenard, P., Neige, P., Delsate, D., Joachim- ski, M. M., Reisberg, L., and Martinez, M.: Water mass exchange and variations in seawater temperature in the NW Tethys during the Early Jurassic: Evidence from neodymium and oxygen iso- topes of ﬁsh teeth and belemnites, Earth Planet. Sc. Lett., 286, 198–207, https://doi.org/10.1016/j.epsl.2009.06.027, 2009. Jenkyns, H. C. and Clayton, C. J.: Black shales and carbon iso- topes in pelagic sediments from the Tethyan Lower Juras- sic, Sedimentology, 33, 87–106, https://doi.org/10.1111/j.1365- 3091.1986.tb00746.x, 1986. Jenkyns, H. C., Schouten-Huibers, L., Schouten, S., and Sin- ninghe Damsté, J. S.: Warm Middle Jurassic–Early Cretaceous high-latitude sea-surface temperatures from the Southern Ocean, Clim. Past, 8, 215–226, https://doi.org/10.5194/cp-8-215-2012, 2012. Dera, G., Brigaud, B., Monna, F., Laffont, R., Pucéat, E., Decon- inck, J.-F., Pellenard, P., Joachimski, M. M., and Durlet, C.: Cli- matic ups and downs in a disturbed Jurassic world, Geology, 39, 215–218, https://doi.org/10.1130/G31579.1, 2011. Keating-Bitonti, C. R., Ivany, L. C., Affek, H. P., Douglas, P., and Samson, S. D.: Warm, not super-hot, tempera- tures in the early Eocene subtropics, Geology, 39, 771–774, https://doi.org/10.1130/G32054.1, 2011. Disnar, J. R., Le Strat, P., Farjanel, G., and Fikri, A.: Sédimen- tation de la matière organique dans le nord-est du Bassin de Paris: conséquences sur le dépôt des argilites carbonées du Toarcien inférieur (Organic matter sedimentation in the north- east of the Paris Basin: consequences on the deposition of the lower toarcian black shales), Chem. Geol., 131, 15–35, https://doi.org/10.1016/0009-2541(96)00021-6, 1996. Kerimov, V. Yu., Shcherbina, Yu. V., and Ivanov, A. References A.: For- mation conditions and evolution of oil and gas source strata of the Laptev sea shelf ore and gas province, Izvestiya Vysshikh Uchebnykh Zavedeniy. Geologiya i Razvedka, 3, 46– 59, https://doi.org/10.32454/0016-7762-2020-63-3-46-59, 2020. Douglas, P. M. J., Affek, H. P., Ivany, L. C., Houben, A. J. P., Sijp, W. P., Sluijs, A., Schouten, S., and Pagani, M.: Pronounced zonal heterogeneity in Eocene southern high-latitude sea sur- face temperatures, P. Natl. Acad. Sci. USA, 111, 6582–6587, https://doi.org/10.1073/pnas.1321441111, 2014. Killam, D. E. and Clapham, M. E.: Identifying the ticks of bivalve shell clocks: seasonal growth in relation to temperature and food supply, PALAIOS, 33, 228–236, https://doi.org/10.2110/palo.2017.072, 2018. Epstein, S., Buchsbaum, R., Lowenstam, H. A., and Urey, H. C.: Revised carbonate-water isotopic temperature scale, Geol. Soc. Am. Bull., 64, 1315, https://doi.org/10.1130/0016- 7606(1953)64[1315:RCITS]2.0.CO;2, 1953. Kim, S.-T. and O’Neil, J. R.: Equilibrium and nonequilibrium oxygen isotope effects in synthetic carbonates, Geochim. Cos- mochim. Ac., 61, 3461–3475, https://doi.org/10.1016/S0016- 7037(97)00169-5, 1997. Erez, J. and Luz, B.: Experimental paleotemperature equation for planktonic foraminifera, Geochim. Cosmochim. Ac., 47, 1025– 1031, https://doi.org/10.1016/0016-7037(83)90232-6, 1983. Kim, S.-T., Mucci, A., and Taylor, B. E.: Phosphoric acid fractionation factors for calcite and aragonite between 25 and 75 ◦C: Revisited, Chem. Geol., 246, 135–146, https://doi.org/10.1016/j.chemgeo.2007.08.005, 2007. Evans, D., Sagoo, N., Renema, W., Cotton, L. J., Müller, W., Todd, J. A., Saraswati, P. K., Stassen, P., Ziegler, M., Pear- son, P. N., Valdes, P. J., and Affek, H. P.: Eocene green- house climate revealed by coupled clumped isotope-Mg/Ca thermometry, P. Natl. Acad. Sci. USA, 115, 1174–1179, https://doi.org/10.1073/pnas.1714744115, 2018. Krencker, F. N., Bodin, S., Hoffmann, R., Suan, G., Matti- oli, E., Kabiri, L., Föllmi, K. B., and Immenhauser, A.: The middle Toarcian cold snap: Trigger of mass extinction and carbonate factory demise, Glob. Planet. Change, 15, 64–78, https://doi.org/10.1016/j.gloplacha.2014.03.008, 2014. Fernandez, A., Korte, C., Ullmann, C. V., Looser, N., Wohlwend, S., and Bernasconi, S. M.: Reconstructing the magnitude of Early Toarcian (Jurassic) warming using the reordered clumped iso- tope compositions of belemnites, Geochim. Cosmochim. Ac., 293, 308–327, https://doi.org/10.1016/j.gca.2020.10.005, 2021. Laugié, M., Donnadieu, Y., Ladant, J.-B., Green, J. A. M., Bopp, L., and Raisson, F.: Stripping back the modern to reveal the Cenomanian–Turonian climate and temperature gradient under- neath, Clim. Past, 16, 953–971, https://doi.org/10.5194/cp-16- 953-2020, 2020. Frieling, J., Iakovleva, A. I., Reichart, G.-J., Aleksandrova, G. N., Gnibidenko, Z. 137, 205–271, https://doi.org/10.1016/S0031-0182(97)00108-9, 1998. McElwain, J. C., Wade-Murphy, J., and Hesselbo, S. P.: Changes in carbon dioxide during an oceanic anoxic event linked to intrusion into Gondwana coals, Nature, 435, 479–482, https://doi.org/10.1038/nature03618, 2005. Pouech, J., Amiot, R., Lécuyer, C., Mazin, J.-M., Martineau, F., and Fourel, F.: Oxygen isotope composition of vertebrate phos- phates from Cherves-de-Cognac (Berriasian, France): Environ- mental and ecological signiﬁcance, Palaeogeogr. Palaeocl., 410, 290–299, https://doi.org/10.1016/j.palaeo.2014.05.036, 2014. Meyer, K. W., Petersen, S. V., Lohmann, K. C., and Winkel- stern, I. Z.: Climate of the Late Cretaceous North Ameri- can Gulf and Atlantic Coasts, Cretaceous Res., 89, 160–173, https://doi.org/10.1016/j.cretres.2018.03.017, 2018. Price, G. D., Bajnai, D., and Fiebig, J.: Carbonate clumped isotope evidence for latitudinal seawater temperature gradients and the oxygen isotope composition of Early Cretaceous seas, Palaeogeogr. Palaeocl., 552, 109777, https://doi.org/10.1016/j.palaeo.2020.109777, 2020. Nikitenko, B. L. and Mickey, M. B.: Foraminifera and ostra- codes across the Pliensbachian-Toarcian boundary in the Arc- tic Realm (stratigraphy, palaeobiogeography and biofacies), Ge- ological Society, London, Special Publications, 230, 137–174, https://doi.org/10.1144/GSL.SP.2004.230.01.08, 2004. Robinson, S. A., Ruhl, M., Astley, D. L., Naafs, B. D. A., Farnsworth, A. J., Bown, P. R., Jenkyns, H. C., Lunt, D. J., O’Brien, C., Pancost, R. D., and Markwick, P. J.: Early Jurassic North Atlantic sea-surface temperatures from TEX86 palaeothermometry, Sedimentology, 64, 215–230, https://doi.org/10.1111/sed.12321, 2017. Nikitenko, B. L., Shurygin, B. N., Knyazev, V. G., Meled- ina, S. V., Dzyuba, O. S., Lebedeva, N. K., Peshchevit- skaya, E. B., Glinskikh, L. A., Goryacheva, A. A., and Khafaeva, S. N.: Jurassic and Cretaceous stratigraphy of the Anabar area (Arctic Siberia, Laptev Sea coast) and the Bo- real zonal standard, Russ. Geol. Geophys.+, 54, 808–837, https://doi.org/10.1016/j.rgg.2013.07.005, 2013. Roche, D. M., Donnadieu, Y., Pucéat, E., and Paillard, D.: Effect of changes in δ18O content of the surface ocean on estimated sea surface temperatures in past warm climate, Paleoceanography, 21, PA2023, https://doi.org/10.1029/2005PA001220, 2006. Nooitgedacht, C. W., van der Lubbe, H. J. L., Ziegler, M., and Staudigel, P. T.: Internal water facilitates ther- mal resetting of clumped isotopes in biogenic arago- nite, Geochem. Geophy. Geosy., 22, e2021GC009730, https://doi.org/10.1029/2021GC009730, 2021. Rogov, M. A., Zverkov, N. G., Zakharov, V. A., and Arkhangel- sky, M. S.: Marine Reptiles and Climates of the Jurassic and Cretaceous of Siberia, Stratigr. Geol. Correl., 27, 398–423, https://doi.org/10.1134/S0869593819040051, 2019. Rozanski, K., Araguas-Araguas, L., and Gonﬁantini, R.: Rela- tion Between Long-Term Trends of Oxygen-18 Isotope Com- position of Precipitation and Climate, Science, 258, 981–985, https://doi.org/10.1126/science.258.5084.981, 1992. O’Brien, C. L., Robinson, S. 137, 205–271, https://doi.org/10.1016/S0031-0182(97)00108-9, 1998. A., Pancost, R. D., Sinninghe Damsté, J. S., Schouten, S., Lunt, D. J., Alsenz, H., Bornemann, A., Bottini, C., Brassell, S. C., Farnsworth, A., Forster, A., Huber, B. T., Inglis, G. N., Jenkyns, H. C., Linnert, C., Littler, K., Markwick, P., McAnena, A., Mutterlose, J., Naafs, B. D. A., Püttmann, W., Sluijs, A., van Helmond, N. A. G. M., Vellekoop, J., Wagner, T., and Wrobel, N. E.: Cretaceous sea-surface tem- perature evolution: Constraints from TEX86 and planktonic foraminiferal oxygen isotopes, Earth-Sci. Rev., 172, 224–247, https://doi.org/10.1016/j.earscirev.2017.07.012, 2017. Ruebsam, W., Reolid, M., Sabatino, N., Masetti, D., and Schwark, L.: Molecular paleothermometry of the early Toar- cian climate perturbation, Glob. Planet. Change, 195, 103351, https://doi.org/10.1016/j.gloplacha.2020.103351, 2020. Schöne, B. R.: The curse of physiology—challenges and opportuni- ties in the interpretation of geochemical data from mollusk shells, Geo-Mar. Lett., 28, 269–285, https://doi.org/10.1007/s00367- 008-0114-6, 2008. O’Connor, L. K., Robinson, S. A., Naafs, B. D. A., Jenkyns, H. C., Henson, S., Clarke, M., and Pancost, R. D.: Late Creta- ceous Temperature Evolution of the Southern High Latitudes: A TEX86 Perspective, Paleoceanography and Paleoclimatology, 34, 436–454, https://doi.org/10.1029/2018PA003546, 2019. Shackleton, N. J. and Kennett, J. P.: Paleotemperature history of the Cenozoic and the initiation of Antarctic glaciation: oxygen and carbon isotope analyses in DSDP Sites 277,279, and 281, Initial Reports of Deep Sea Drilling, 29, 743–756, 1975. p p g Sluijs, A., Schouten, S., Pagani, M., Woltering, M., Brinkhuis, H., Damsté, J. S. S., Dickens, G. R., Huber, M., Reichart, G.-J., Stein, R., Matthiessen, J., Lourens, L. J., Pedentchouk, N., Backman, J., Moran, K., and the Expedition 302 Sci- entists: Subtropical Arctic Ocean temperatures during the Palaeocene/Eocene thermal maximum, Nature, 441, 610–613, https://doi.org/10.1038/nature04668, 2006. Pagani, M., Pedentchouk, N., Huber, M., Sluijs, A., Schouten, S., Brinkhuis, H., Damste, J. S. S., and Dickens, G. R.: Arctic hydrology during global warming at the Palaeocene/Eocene thermal maximum, Nature, 442, 671– 675, https://doi.org/10.1038/nature05043, 2006. Peck, L. S., Colman, J. G., and Murray, A. W. A.: Growth and tissue mass cycles in the infaunal bivalve Yoldia eightsi at Signy Island, Antarctica, Polar Biol., 23, 420–428, https://doi.org/10.1007/s003000050463, 2000. Sluijs, A., Frieling, J., Inglis, G. N., Nierop, K. G. J., Peterse, F., Sangiorgi, F., and Schouten, S.: Late Paleocene–early Eocene Arctic Ocean sea surface temperatures: reassessing biomarker paleothermometry at Lomonosov Ridge, Clim. Past, 16, 2381– 2400, https://doi.org/10.5194/cp-16-2381-2020, 2020. Petersen, S. V., Dutton, A., and Lohmann, K. References N., Schouten, S., and Sluijs, A.: Paleocene–Eocene warming and biotic response in the epicontinental West Siberian Sea, Geology, 42, 767–770, https://doi.org/10.1130/G35724.1, 2014. Li, X., Wang, J., Rasbury, T., Zhou, M., Wei, Z., and Zhang, C.: Early Jurassic climate and atmospheric CO2 concentration in the Sichuan paleobasin, southwestern China, Clim. Past, 16, 2055– 2074, https://doi.org/10.5194/cp-16-2055-2020, 2020. Grossman, E. L. and Ku, T.-L.: Oxygen and carbon isotope fraction- ation in biogenic aragonite: temperature effects, Chem. Geol., 59, 59–74, https://doi.org/10.1016/0168-9622(86)90057-6, 1986. Lunt, D. J., Dunkley Jones, T., Heinemann, M., Huber, M., LeGrande, A., Winguth, A., Loptson, C., Marotzke, J., Roberts, C. D., Tindall, J., Valdes, P., and Winguth, C.: A model– data comparison for a multi-model ensemble of early Eocene atmosphere–ocean simulations: EoMIP, Clim. Past, 8, 1717– 1736, https://doi.org/10.5194/cp-8-1717-2012, 2012. Hemingway, J. D. and Henkes, G. A.: A disordered ki- netic model for clumped isotope bond reordering in carbonates, Earth Planet. Sc. Lett., 566, 116962, https://doi.org/10.1016/j.epsl.2021.116962, 2021. Markwick, P. J.: Fossil crocodilians as indicators of Late Cretaceous and Cenozoic climates: implications for using palaeontologi- cal data in reconstructing palaeoclimate, Palaeogeogr. Palaeocl., Henkes, G. A., Passey, B. H., Grossman, E. L., Shenton, B. J., Pérez-Huerta, A., and Yancey, T. E.: Temperature lim- its for preservation of primary calcite clumped isotope pa- https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. Letulle et al.: Early Jurassic extreme polar warmth 447 137, 205–271, https://doi.org/10.1016/S0031-0182(97)00108-9, 1998. 137, 205–271, https://doi.org/10.1016/S0031-0182(97)00108-9, 1998. Late Cretaceous Western Interior Seaway, Geology, 44, 903–906, https://doi.org/10.1130/G38311.1, 2016b. Late Cretaceous Western Interior Seaway, Geology, 44, 903–906, https://doi.org/10.1130/G38311.1, 2016b. T. Letulle et al.: Early Jurassic extreme polar warmth Vihtakari, M., Renaud, P. E., Clarke, L. J., Whitehouse, M. J., Hop, H., Carroll, M. L., and Ambrose, W. G.: De- coding the oxygen isotope signal for seasonal growth pat- terns in Arctic bivalves, Palaeogeogr. Palaeocl., 446, 263–283, https://doi.org/10.1016/j.palaeo.2016.01.008, 2016. Thuy, B., Gale, A. S., and Reich, M.: A new echino- derm Lagerstätte from the Pliensbachian (Early Jurassic) of the French Ardennes, Swiss J. Palaeontol., 130, 173–185, https://doi.org/10.1007/s13358-010-0015-y, 2011. Tindall, J., Flecker, R., Valdes, P., Schmidt, D. N., Markwick, P., and Harris, J.: Modelling the oxygen isotope distribution of ancient seawater using a coupled ocean–atmosphere GCM: Implications for reconstructing early Eocene climate, Earth Planet. Sc. Lett., 292, 265–273, https://doi.org/10.1016/j.epsl.2009.12.049, 2010. Waterlot, G., Bonte, A., and Destombes, J.-P.: Carte géologique de la France à 1 : 50000, [68], Renwez, France, 1960. Wierzbowski, H., Bajnai, D., Wacker, U., Rogov, M. A., Fiebig, J., and Tesakova, E. M.: Clumped isotope record of salin- ity variations in the Subboreal Province at the Middle– Late Jurassic transition, Glob. Planet. Change, 167, 172–189, https://doi.org/10.1016/j.gloplacha.2018.05.014, 2018. Torsvik, T. H., Van der Voo, R., Preeden, U., Mac Niocaill, C., Steinberger, B., Doubrovine, P. V., van Hinsbergen, D. J. J., Domeier, M., Gaina, C., Tohver, E., Meert, J. G., McCaus- land, P. J. A., and Cocks, L. R. M.: Phanerozoic polar wander, palaeogeography and dynamics, Earth-Sci. Rev., 114, 325–368, https://doi.org/10.1016/j.earscirev.2012.06.007, 2012. Zakharov, V. A. and Shurygin, B. N.: Biogeography, facies and stratigraphy of the Middle Jurassic of Soviet Arctic (by bivalve molluscs), Transactions of the Institute of Geology and Geo- physics, Siberian Branch of the Academy of Science of USSR, 352, 1–206, 1978. Ullmann, C. V., Boyle, R., Duarte, L. V., Hesselbo, S. P., Kase- mann, S. A., Klein, T., Lenton, T. M., Piazza, V., and Aberhan, M.: Warm afterglow from the Toarcian Oceanic Anoxic Event drives the success of deep-adapted brachiopods, Sci. Rep.-UK, 10, 6549, https://doi.org/10.1038/s41598-020-63487-6, 2020. Zardus, J. D.: Protobranch bivalves, Ad. Mar. Biol., 42, 1–65, https://doi.org/10.1016/S0065-2881(02)42012-3, 2002. Zhou, J., Poulsen, C. J., Pollard, D., and White, T. S.: Simu- lation of modern and middle Cretaceous marine δ18O with an ocean-atmosphere general circulation model: Modern, mid- Cretaceous seawater δ18O, Paleoceanography, 23, PA3223, https://doi.org/10.1029/2008PA001596, 2008. van Baal, R. R., Janssen, R., van der Lubbe, H. J. L., Schulp, A. S., Jagt, J. W. M., and Vonhof, H. T. Letulle et al.: Early Jurassic extreme polar warmth 448 Suan, G., Mattioli, E., Pittet, B., Lécuyer, C., Suchéras-Marx, B., Duarte, L. V., Philippe, M., Reggiani, L., and Martineau, F.: Sec- ular environmental precursors to Early Toarcian (Jurassic) ex- treme climate changes, Earth Planet. Sc. Lett., 290, 448–458, https://doi.org/10.1016/j.epsl.2009.12.047, 2010. van Hinsbergen, D. J. J., de Groot, L. V., van Schaik, S. J., Spakman, W., Bijl, P. K., Sluijs, A., Langereis, C. G., and Brinkhuis, H.: A Paleolatitude Calculator for Paleoclimate Studies, PLoS ONE, 10, e0126946, https://doi.org/10.1371/journal.pone.0126946, 2015. Vickers, M. L., Bajnai, D., Price, G. D., Linckens, J., and Fiebig, J.: Southern high-latitude warmth during the Jurassic–Cretaceous: New evidence from clumped isotope thermometry, Geology, 47, 724–728, https://doi.org/10.1130/G46263.1, 2019. Suan, G., Nikitenko, B. L., Rogov, M. A., Baudin, F., Spangen- berg, J. E., Knyazev, V. G., Glinskikh, L. A., Goryacheva, A. A., Adatte, T., Riding, J. B., Föllmi, K. B., Pittet, B., Matti- oli, E., and Lécuyer, C.: Polar record of Early Jurassic mas- sive carbon injection, Earth Planet. Sc. Lett., 312, 102–113, https://doi.org/10.1016/j.epsl.2011.09.050, 2011. p g Vickers, M. L., Fernandez, A., Hesselbo, S. P., Price, G. D., Bernasconi, S. M., Lode, S., Ullmann, C. V., Thibault, N., Hougaard, I. W., and Korte, C.: Unravelling Middle to Late Jurassic palaeoceanographic and palaeoclimatic sig- nals in the Hebrides Basin using belemnite clumped iso- tope thermometry, Earth Planet. Sc. Lett., 546, 116401, https://doi.org/10.1016/j.epsl.2020.116401, 2020. Vickers, M. L., Fernandez, A., Hesselbo, S. P., Price, G. D., Bernasconi, S. M., Lode, S., Ullmann, C. V., Thibault, Suan, G., Popescu, S.-M., Suc, J.-P., Schnyder, J., Fauquette, S., Baudin, F., Yoon, D., Piepjohn, K., Sobolev, N. N., and Labrousse, L.: Subtropical climate conditions and mangrove growth in Arctic Siberia during the early Eocene, Geology, 45, 539–542, https://doi.org/10.1130/G38547.1, 2017. N., Hougaard, I. W., and Korte, C.: Unravelling Middle to Late Jurassic palaeoceanographic and palaeoclimatic sig- nals in the Hebrides Basin using belemnite clumped iso- tope thermometry, Earth Planet. Sc. Lett., 546, 116401, https://doi.org/10.1016/j.epsl.2020.116401, 2020. Thierry, J.: Middle Toarcian map. 8, in: Altlas Peri-Tethys Palaeogeographical Maps, edited by: Dercourt, J., Gaetani, M., Vrielynck, B., Barrier, E., Biju-Duval, B., Brunet, M. F., Cadet, J. P, Crasquin, S., and Sandulescu, M., CCGM/CGMW, Paris, 61–70, 2000. Vickers, M. L., Bernasconi, S. M., Ullmann, C. V., Lode, S., Looser, N., Morales, L. G., Price, G. D., Wilby, P. R., Hougård, I. W., Hesselbo, S. P., and Korte, C.: Marine temperatures underesti- mated for past greenhouse climate, Sci. Rep.-UK, 11, 19109, https://doi.org/10.1038/s41598-021-98528-1, 2021. 137, 205–271, https://doi.org/10.1016/S0031-0182(97)00108-9, 1998. C.: End-Cretaceous extinction in Antarctica linked to both Deccan volcanism and meteorite impact via climate change, Nat. Commun., 7, 12079, https://doi.org/10.1038/ncomms12079, 2016a. p g p Stolper, D. A. and Eiler, J. M.: The kinetics of solid-state isotope- exchange reactions for clumped isotopes: A study of inorganic calcites and apatites from natural and experimental samples, Am. J. Sci., 315, 363–411, https://doi.org/10.2475/05.2015.01, 2015. p g Petersen, S. V., Tabor, C. R., Lohmann, K. C., Poulsen, C. J., Meyer, K. W., Carpenter, S. J., Erickson, J. M., Matsunaga, K. K. S., Smith, S. Y., and Sheldon, N. D.: Temperature and salinity of the https://doi.org/10.5194/cp-18-435-2022 Clim. Past, 18, 435–448, 2022 T. Letulle et al.: Early Jurassic extreme polar warmth T. Letulle et al.: Early Jurassic extreme polar warmth B.: Oxygen and carbon stable isotope records of marine vertebrates from the type Maastrichtian, The Netherlands and northeast Bel- gium (Late Cretaceous), Palaeogeogr. Palaeocl., 392, 71–78, https://doi.org/10.1016/j.palaeo.2013.08.020, 2013. Zhu, J., Poulsen, C. J., Otto-Bliesner, B. L., Liu, Z., Brady, E. C., and Noone, D. C.: Simulation of early Eocene water iso- topes using an Earth system model and its implication for past climate reconstruction, Earth Planet. Sc. Lett., 537, 116164, https://doi.org/10.1016/j.epsl.2020.116164, 2020. van Dijk, J., Fernandez, A., Bernasconi, S. M., Caves Rugenstein, J. K., Passey, S. R., and White, T.: Spatial pattern of super- greenhouse warmth controlled by elevated speciﬁc humidity, Nat. Geosci., 13, 739–744, https://doi.org/10.1038/s41561-020- 00648-2, 2020. Clim. Past, 18, 435–448, 2022 https://doi.org/10.5194/cp-18-435-2022
https://openalex.org/W2887834225	https://infoscience.epfl.ch/record/258892/files/PhysRevAccelBeams.21.084401.pdf	English	null	Experimental stabilization of transverse collective instabilities in the LHC with second order chromaticity	Physical review. Accelerators and beams	2,018	cc-by	9,498	I. INTRODUCTION transverse actions. This approach provides potentially a more effective means for stabilization of the beams of future, high-energy hadron colliders. The reason is that Landau octupoles will be less effective for smaller transverse emittance beams because of the reduced transverse action spreads [10]. The longitudinal action spread, on the other hand, remains of the same order of magnitude, and hence the betatron detuning with an rf quadrupole can be very effective [8,10]. For example, the high luminosity upgrade of the LHC will operate with beams with a ratio of 10−4 between the one-sigma transverse and longitudinal emittances at 7 TeV [11]. For the Future Circular Collider, this number becomes another 10–100 times smaller at the top energy (50 TeV), reducing further the effect of Landau octupoles [12]. It is thus advisable to explore alternative methods that could provide Landau damping in future machines. Landau damping is a powerful stabilizing mechanism and is commonly employed to mitigate transverse collective instabilities in particle colliders [1,2]. The mechanism is present when there is an incoherent spread in the betatron tunes Qx;y of the particles in the beam. The spread is a result of machine nonlinearities often introduced by design through dedicated elements. In the Large Hadron Collider (LHC), magnetic octupoles are installed to produce an incoherent tune spread that depends on the transverse actions of the particles [3–5]. These Landau octupoles are exten- sively used for beam stabilization during LHC operation, in particular, to suppress impedance-driven head-tail instabil- ities before the beams are brought into collision [6,7]. Recently, the design and working principle of a radio frequency (rf) quadrupole has been published, including a numerical analysis of its stabilizing effect [8–10]. Unlike Landau octupoles, an rf quadrupole generates a betatron tune spread depending on the longitudinal rather than the The second order chromaticity Q00x;y ¼ ∂2Qx;y=∂δ2jδ¼0, where δ ¼ dp=p is the relative momentum error, generates an incoherent betatron tune spread as a function of longitudinal action in the same manner as the rf quadrupole as will be derived in this paper. Indeed, it can be shown that the detuning introduced by Q00 mimics the effect of the rf quadrupole in a first approximation. (Received 8 March 2018; published 17 August 2018) (Received 8 March 2018; published 17 August 2018) This paper discusses measurements on the stabilization of single bunches with second order chromaticity (Q00) in the Large Hadron Collider (LHC) at CERN. Q00 introduces an incoherent betatron tune spread which can produce Landau damping of transverse instabilities. Although the resulting stabilizing effect is similar to that provided by Landau octupoles, the underlying beam dynamics are different. Since the tune spread from Q00 is based on the longitudinal rather than the transverse action of the particles, it will not be affected by the smaller transverse emittance beams of future machines, such as the High Luminosity LHC or the Future Circular Collider, and may hence provide more efficient Landau damping than magnetic octupoles. This study serves as a proof-of-principle experiment to demonstrate Landau damping from detuning with longitudinal action by means of Q00 in a carefully prepared and well-understood accelerator environment. The agreement between measurements and PyHEADTAIL tracking simulations shows that Q00 indeed contributes to the beam stability, that the numerical model of the LHC is accurate, and that the involved beam dynamics mechanisms are understood from both the single- and multiparticle effects points of view. The results also serve as a first experimental validation of the recently proposed radio frequency quadrupole for Landau damping. michael.schenk@cern.ch DOI: 10.1103/PhysRevAccelBeams.21.084401 Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Experimental stabilization of transverse collective instabilities in the LHC with second order chromaticity M. Schenk,1,2, X. Buffat,1 L. R. Carver,1 R. De Maria,1 K. Li,1 and E. M´etral1 1European Organization for Nuclear Research (CERN), CH-1211 Geneva, Switzerland 2École Polytechnique F´ed´erale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland *michael.schenk@cern.ch Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. PHYSICAL REVIEW ACCELERATORS AND BEAMS 21, 084401 (2018) PHYSICAL REVIEW ACCELERATORS AND BEAMS 21, 084401 (2018) I. INTRODUCTION The advantage of second order chromaticity is that it can be introduced in an existing machine like the LHC without the need for installing new hardware, for example, by powering the different families of the main sextupoles in a specific Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. 084401-1 Published by the American Physical Society 2469-9888=18=21(8)=084401(12) PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) M. SCHENK et al. configuration. Despite the optics constraints which can limit the achievable dynamic range of Q00, second order chromaticity still provides a practical and cost-effective way for a first experimental verification of the stabilizing effect expected from an rf quadrupole cavity. interval ½−ˆδi; ˆδi, where ˆδi is the maximum momentum deviation amplitude of the synchrotron oscillation. By consequence, ΔQix;y has an implicit time dependence, and the effective incoherent detuning of the particle is given by the time average hΔQix;y½δiðtÞit→∞, in analogy to detuning with the transverse amplitude from magnetic octupoles [5]. Assuming linear synchrotron motion e ect e pected o a quad upo e cav ty. The main objective of this paper is to present a proof-of- principle experiment that confirms the stabilizing effect of transverse detuning with longitudinal amplitude and to validate the beam dynamics models. The observations made in LHC experiments on single-bunch stability with Q00 will be used to demonstrate that second order chromaticity has a stabilizing effect and that the main beam dynamics effects are understood and correctly modeled in numerical simu- lation codes such as MAD-X and PyHEADTAIL, from both the single- and the multiparticle dynamics points of view [13,14]. Section II briefly summarizes the detuning mech- anisms for Q00 and the rf quadrupole and shows the equivalence of the two approaches in a first approximation. Section III describes the production scheme for Q00 and the definition of two dedicated knobs to power the LHC main sextupole magnets in a specific configuration. This includes numerical calculations and an analysis of undesired side effects. Section IV discusses the characteristics of the single- bunch head-tail mode in the LHC at the top energy (6.5 TeV) and how this instability is routinely mitigated by means of the Landau octupoles in the absence of the Q00 knobs. where αx;y z ¼ ∂hΔQix;yi=∂Jiz ¼ Q00x;yQs=2ηR ð4Þ ð4Þ is the longitudinal detuning coefficient, defined in analogy to detuning with the transverse amplitude. II. ANALYTICAL CONSIDERATIONS The purpose of this section is twofold: first, to show that the incoherent betatron detuning produced by Q00 depends on the longitudinal action of the particles and, second, to briefly review the tune spread introduced by an rf quadru- pole and to prove that it is equivalent to Q00 in a first approximation. An approximate dispersion relation specifically for detun- ing with longitudinal amplitude has been derived by Berg and Ruggiero [15]. Their work shows that the incoherent tune spread leads to an increase of the stable region in the complex tune space ReðΔQcohÞ vs −ImðΔQcohÞ in equiv- alence to Landau damping from magnetic octupoles. Here ΔQcoh denotes the complex coherent tune shift that char- acterizes a particular head-tail instability. The real part is given by the tune shift of the bunch centroid oscillation with respect to the unperturbed tune of the unstable synchrotron sideband. The imaginary part corresponds to the exponential growth rate of the amplitude of the bunch centroid motion. Two incoherent tune distributions and their corresponding stability diagrams are shown in the upper and lower plot in Fig. 1, respectively, one for positive (blue) and one for negative (red) Q00, respectively. An azimuthal mode zero head-tail instability was assumed for computing the stability I. INTRODUCTION The analysis is done using both experimental data and results from the macroparticle tracking code PyHEADTAIL. It dem- onstrates that the simulations successfully reproduce the beam dynamics in the machine. Finally, Sec. V details the observations made during dedicated measurements with the Q00 knobs defined in Sec. III. Again by means of PyHEADTAIL simulations, the involved beam dynamics mechanisms are clearly identified and understood. Among others, the differences between stabilizing the single bunch with Landau octupoles or with Q00 become evident. δiðtÞ ¼ ˆδi cos ðQsω0t þ ϕδ;iÞ; where Qs is the synchrotron tune, ω0 the angular revolution frequency, and ϕδ;i the constant synchrotron phase offset of the particle, the calculation of the effective detuning becomes straightforward. It is obtained by averaging Eq. (1) over one synchrotron period Ts ¼ 2π=Qsω0: hΔQix;yi ¼ Q00x;yˆδ2 i 2 1 Ts Z Ts 0 cos2ðQsω0t þ ϕδ;iÞdt ¼ Q00x;yˆδ2 i 4 : ð2Þ ð2Þ This is equivalent to averaging for t →∞as described in Ref. [5], given that Qs is not a rational number. ˆδ2 i can be rewritten in terms of the longitudinal action Jiz of the particle, ˆδ2 i ¼ 2JizQs=ηR, with η the slip factor and R the physical radius of the accelerator ring. This leads to the final expression This is equivalent to averaging for t →∞as described in Ref. [5], given that Qs is not a rational number. ˆδ2 i can be rewritten in terms of the longitudinal action Jiz of the particle, ˆδ2 i ¼ 2JizQs=ηR, with η the slip factor and R the physical radius of the accelerator ring. This leads to the final expression hΔQix;yi ¼ Q00x;y 2 Qs ηR Jiz ≐αx;y z Jiz; ð3Þ ð3Þ A. Incoherent tune spread from Q00 Given a machine lattice with first and second order chromaticities Q0x;y and Q00x;y, a particle i with a relative momentum deviation of δi experiences a betatron tune shift: ΔQix;yðδiÞ ¼ Q0x;yδi þ Q00x;y 2 δ2 i : ð1Þ ð1Þ Since the particle undergoes synchrotron motion, its momen- tum deviation varies over time and takes on values in the 084401-2 084401-2 EXPERIMENTAL STABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. B ABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. BEAMS 21, 084401 (2018 FIG. 1. Incoherent tune distributions (top) and normalized stability diagrams (bottom) for detuning with the longitudinal amplitude from Q00 < 0 (red, solid line) or Q00 > 0 (blue, dashed line), respectively, computed employing the formalism described in Ref. [15]. ΔQix;y ¼ βx;y bð2Þ 4πB0ρ cos ωzi βc þ ϕ0 : ð5Þ ð5Þ βx;y are the transverse beta functions at the location of the device, bð2Þ is the rf quadrupolar integrated gradient in units of (Tm/m), B0ρ denotes the magnetic rigidity of the beam, ω is the angular frequency of the rf quadrupole field, zi is the longitudinal position of the particle measured with respect to the zero crossing of the main rf voltage, β and c denote the relativistic beta and the speed of light, respec- tively, and ϕ0 is a constant phase offset that determines the mode of operation of the rf quadrupole. In Ref. [10], the authors show that if the wavelength of the rf wave is much larger than the bunch length σz, i.e., ωσz=βc ≪1, the effective tune spread provided by an rf quadrupole cavity (for ϕ0 ¼ 0) reads hΔQix;yi≈∓βx;y bð2Þ 8πB0ρ ω βc 2 ηR Qs Jiz ≐˜αx;y z Jiz; ð6Þ ð6Þ where ˜αx;y z has been defined as the longitudinal detuning coefficient for an rf quadrupole FIG. 1. Incoherent tune distributions (top) and normalized stability diagrams (bottom) for detuning with the longitudinal amplitude from Q00 < 0 (red, solid line) or Q00 > 0 (blue, dashed line), respectively, computed employing the formalism described in Ref. [15]. ˜αx;y z ¼∓βx;y bð2Þ 8πB0ρ ω βc 2 ηR Qs ; ð7Þ ð7Þ in analogy to αx;y z for detuning from Q00x;y in Eq. (3). Equations (3) and (6) manifestly show the equivalence of Q00 and the rf quadrupole given the approximation ωσz=βc ≪1. A. Incoherent tune spread from Q00 Therefore, the betatron detuning introduced, respectively, by Q00 and an rf quadrupole is generated according to the same mechanism, and the effects on the beam dynamics are directly comparable. This makes Q00 a suitable tool for a first, cost-effective, experimental vali- dation of the stabilizing effect from an rf quadrupole and also allows us to thoroughly benchmark the numerical models with beam measurements. diagrams. The way to read the plots is that all the head-tail instabilities with an unperturbed coherent tune shift, i.e., measured in the absence of any tune spread, situated below the line traced out by the stability diagram will be Landau damped. The asymmetry of the stable regions for the two signs of Q00 is a result of the strictly one-sided detuning [see Eq. (3)]. It is worth noting that the average detuning in Eq. (2) contains only the term with Q00x;y. The first order chroma- ticity does not contribute to the effective tune spread and, hence, does not introduce Landau damping. It does, however, change the effective impedance, i.e., the overlap sum between the beam spectrum and the impedance, and can hence modify the head-tail instability mechanism. As such, it can, for example, be used to raise the transverse mode coupling instability threshold [2]. A similar change of the effective impedance is also observed for Q00x;y, in the theory, simulations, and experiments (see also Sec. V). This effect often plays an important role in addition to the Landau damping. III. Q00 IN THE LHC This section is split into three parts and contains information about how to generate and measure Q00 in the LHC. First, the sextupole powering scheme used to generate Q00 is analyzed by means of the LHC MAD-X model. Second, the detuning with the transverse amplitude introduced by the sextupoles is discussed and compared to the LHC Landau octupoles. The latter is a side effect that needs to be included in the study to decouple the different stabilizing mechanisms that are involved. Finally, the higher order chromaticity measurement method is explained and illustrated with example data. A. Q00 production scheme SF∶Δku 2;F1 ¼ þbu FΔF; Δku 2;F2 ¼ −bu FΔF; SD∶Δku 2;D1 ¼ þbu DΔD; Δku 2;D2 ¼ −bu DΔD: ð8Þ SF∶Δku 2;F1 ¼ þbu FΔF; Δku 2;F2 ¼ −bu FΔF; SD∶Δku 2;D1 ¼ þbu DΔD; Δku 2;D2 ¼ −bu DΔD: ð8Þ ð8Þ FIG. 2. Q00 predicted by MAD-X for the two beams (B1 and B2) for the horizontal (H) and the vertical (V) planes as a function of the QPPF (top) and QPPD (bottom) sextupole knobs, respectively. The absolute change of the sextupole strength ΔFðDÞ is the same for all the SF (SD) in the lattice, but the families in each sector are powered with a different sign denoted by bu FðDÞ. With the definitions in Eq. (8), the absolute value of Q00x;y depends quadratically on the sextupolar strength, i.e., jQ00x;yj ∝Δ2 F;D [16,17]. By choosing the signs bu F;D carefully for each sector, one can define a set of almost orthogonal knobs to change Q00 towards positive or negative values in the two beams and for both planes independently. For the experiment in the LHC, the goal is to define a sextupole scheme that produces a large negative second order chromaticity in both transverse planes. The choice is based on the fact that the real part of the coherent tune shifts for the most unstable head-tail modes in the LHC is negative [10,18]. This is a result of the inductive nature of the collimator impedances which are the main contributors to the effective impedance at the top energy [18]. The stability diagrams displayed in the lower part in Fig. 1 demonstrate that in this case negative provides more effective Landau damping than positive Q00. The best powering scheme to generate a significant amount of negative second order chromaticity for the LHC optics configuration used at the top energy has been determined by means of MAD-X/PTC [3,14,19]. The final results are listed in Table I. The two sextupole knobs are called QPPF and QPPD for the focusing and the defocusing sextupole families, respectively. The maximum possible variation in the sextupole strengths is given by Δmax F;D together with the corresponding amounts of Q00x and Q00y expected from MAD-X calculations for both beam 1 and FIG. 2. Q00 predicted by MAD-X for the two beams (B1 and B2) for the horizontal (H) and the vertical (V) planes as a function of the QPPF (top) and QPPD (bottom) sextupole knobs, respectively. A. Q00 production scheme A particle i that is subject to the transverse kicks from an rf quadrupole experiences a change of the betatron tunes [8,10]: The second order chromaticity is an energy-dependent aberration that is defined primarily by the integral of the 084401-3 PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) M. SCHENK et al. terms β0x;yðδÞk1, β0x;yðδÞDxk2, and D2xk3 around the machine lattice [16,17]. k1, k2, and k3 are the quadrupolar, sextu- polar, and octupolar strengths, respectively, β0x;y ¼ ∂βx;y=∂δ is the first derivative of the beta function with respect to the relative momentum deviation, and Dx denotes the horizontal dispersion function. In the LHC, the main sextupoles (MS) are grouped into focusing (SF) and defocusing (SD) families, installed, respectively, at focus- ing and defocusing quadrupoles in the lattice. SF and SD are further divided into two interleaved subfamilies sepa- rated by a phase advance of approximately π. They are called ðF1; F2Þ and ðD1; D2Þ, respectively. Each of the four families can be individually powered for each of the eight sectors of the LHC. With this sextupole scheme, β0x;yðδÞ can be enhanced to correct, or introduce, Q00x;y without affecting the first order chromaticity Q0x;y. This is achieved by varying the strengths of the two subfamilies with opposite signs for both SF and SD separately in all the sectors u ∈ f1; 2; …; 7; 8g of the machine: TABLE I. Definition of the sextupole powering schemes used to introduce Q00x;y < 0 in the LHC at the top energy. For each of the two knobs QPPF and QPPD, the maximum strengths are listed together with the corresponding amounts of Q00 predicted by MAD-X in the two beams (B) and planes. Knob B bð1;2;…;7;8Þ F;D Δmax F;D (m−3) 10−4Q00x 10−4Q00y QPPF 1 ++−+−+−− 0.30 −15.0 −0.2 2 −++−++++ 0.30 −14.1 0.1 QPPD 1 −+++−+++ 0.28 −0.1 −8.9 2 −−−++−−+ 0.28 −0.1 −4.3 beam 2. The values illustrate that the QPPF and QPPD knobs mainly produce Q00 in the horizontal and the vertical planes, respectively. The quadratic dependence of the second order chromaticity on the strength of the knobs is further illustrated by Fig. 2 for both beams and the two transverse planes. The plots also clearly demonstrate that the Q00 in the horizontal and the vertical planes can be controlled independently. C. Q00 measurement method To measure the amount of Q00 present in the LHC, a sinusoidal frequency modulation is applied to the main rf system of the machine. This results in a variation of the momentum deviation dp=p of the beam, as illustrated by the top plot in Fig. 4. Because of chromaticity, such a modu- lation translates into time-varying betatron tunes Qx;yðtÞ which can be determined from the base-band tune meter measurement (BBQ) [21]. The middle plot in Fig. 4 shows an example of horizontal BBQ spectral data. The fractional tune values qx are obtained from the BBQ turn-by-turn measurement with a sliding window frequency analysis using SUSSIX [22]. Several synchrotron sidebands as well as some noise lines can be seen (yellow). To eliminate systematic errors in the measurement of Q00, one synchrotron sideband must be selected consistently throughout the modulation period as illustrated by the overlaid red markers. To extract the first and second order chromaticities, the selected tune values are then binned with respect to dp=p, and a weighted second order polynomial fit of Q vs dp=p is applied. The result is shown in the bottom plot in Fig. 4. The error bars correspond to the standard deviation of the mean calculated during the binning process. ΔQxðJx; JyÞ ¼ αxx2Jx þ αxy2Jy; ΔQyðJx; JyÞ ¼ αyy2Jy þ αyx2Jx; ð9Þ ð9Þ where the cross-detuning coefficients are identical, i.e., αxy ¼ αyx. For the studies presented here, the additional detuning with the transverse amplitude is an undesired side effect. After all, the goal is to show that the beam stabilization happens as a result predominantly of detuning with the longitudinal amplitude. The additional transverse detuning coefficients complicate the process of determining the actual origin of the Landau damping, i.e., whether it is mostly a result of detuning with the transverse or longi- tudinal amplitude. However, as long as the amount of transverse amplitude detuning from the sextupoles is small enough, the two effects can be clearly disentangled by means of tracking simulations (see Sec. V B). Figure 3 illustrates the amount of detuning with the transverse amplitude introduced by the two sextupole knobs QPPF (left) and QPPD (middle), respectively. The results have been obtained with MAD-X and PTC calcula- tions. Clearly, QPPF introduces mainly αxx and αxy, while QPPD gives rise mostly to αyy. The results are shown only for beam 1, but they are similar for beam 2. A. Q00 production scheme 084401-4 EXPERIMENTAL STABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. B LIZATION OF TRANSVERSE … PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) B. Detuning with transverse amplitude absolute current but with opposite signs. For the plot presented here, positive focusing and negative defocusing currents are used. The symbol Ioct corresponds to the current in the focusing family throughout the paper. Powering the main sextupoles with the scheme described in the previous section not only introduces Q00 but also creates non-negligible transverse detuning coefficients αmn ¼ ∂Qm=∂ð2JnÞ, with m; n ∈fx; yg [20]. They give rise to detuning with transverse amplitude and hence Landau damping in the same manner as from magnetic octupoles [4]: B. Instability mitigation with Landau octupoles During routine machine operation, this instability is mitigated by means of the Landau octupoles. During the measurement campaign, the minimum Landau octupole current required to suppress the instability was determined to Ioct ¼ 96þ29 −10 A for the beam and machine parameters FIG. 4. Top: Variation of the momentum deviation of the beam as a result of the rf frequency modulation. Middle: Horizontal base band tune meter (BBQ) spectral data acquired during momentum modulation, overlaid with the selected SUSSIX tune peaks (red markers). Bottom: Weighted second order polynomial fit (blue line) to the data (red). FIG. 5. Head-tail mode patterns from LHC measurements (top) and simulations (bottom) for a single bunch at the top energy (6.5 TeV). higher orders of chromaticity is relatively low. Hence, in particular, for values of Q00 close to zero, the relative error on the second order chromaticity measurement becomes signifi- cant. Furthermore, the widths of the peaks in the frequency spectra,whichareparticularlylargeinthepresence ofthetune spread created by a strong powering of the Landau octupoles, contribute to the uncertainty of the fits as do the noise lines. The Landau octupoles were required during some of the measurements to guarantee beam stability and to determine their contribution to Q00. The noise is a result mostly of the high bunch intensity used during the measurement. A. Instability characteristics In 2016, a campaign was launched in the LHC to measure the single-bunch stability thresholds at different chromaticities [6,23]. At an energy of 6.5 TeV, with design bunch parameters, Q0x;y between 11 and 14 units, and in the presence of the transverse feedback system with a damping time of τfb ≈100 turns, the most prominent transverse instability was found to be a horizontal head-tail mode with azimuthal and radial numbers l ¼ 0 and m ¼ 2, respectively. The upper plot in Fig. 5 shows the corre- sponding head-tail pattern acquired with the LHC head-tail monitor during the measurement [24]. Using a detailed impedance model of the LHC and a simplified model of the transverse feedback system, macroparticle tracking simu- lations with PyHEADTAIL predict the same instability as observed in the machine with the correct azimuthal and radial mode numbers. The corresponding head-tail mode pattern is displayed in the lower plot in Fig. 5 and is in good agreement with the pattern observed during the measure- ment. The main beam, machine, and simulation parameters used for the study are summarized in Table II. C. Q00 measurement method For compari- son, also the detuning coefficients introduced by the LHC Landau octupoles are shown in the figure (right). The latter are subdivided into a focusing and a defocusing family, depending on whether they are installed near the focusing or defocusing quadrupoles, respectively. During machine operation, both families are powered with the same The main reason for choosing this method is that it provides a fast measurement of the nonlinear chromaticity. Modulation windows of about 60 s contain enough data to obtain a satisfying result. While usually optics measure- ments at the LHC are performed with a bunch of only 1010 p, here the measurement had to be done with bunches of 10 times larger intensities because of time constraints. As a result of the larger bunch charge, the reach in dp=p is limited to about 3 × 10−4 (restricted dynamic aperture), implying a reduced accuracy of the measurement. At these values of dp=p, the sensitivity of the betatron tunes to the FIG. 3. MAD-X calculation of the transverse amplitude detuning coefficients introduced by the two sextupole knobs QPPF (left) and QPPD (middle) and, for comparison, by the LHC Landau octupoles (right). FIG. 3. MAD-X calculation of the transverse amplitude detuning coefficients introduced by the two sextupole knobs QPPF (left) and QPPD (middle) and, for comparison, by the LHC Landau octupoles (right). 084401-5 M. SCHENK et al. PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) FIG. 4. Top: Variation of the momentum deviation of the beam as a result of the rf frequency modulation. Middle: Horizontal base band tune meter (BBQ) spectral data acquired during momentum modulation, overlaid with the selected SUSSIX tune peaks (red markers). Bottom: Weighted second order polynomial fit (blue line) to the data (red). mode is characterized and its mitigation with the Landau octupoles is explained using both experimental data and PyHEADTAIL simulations. IV. LHC SINGLE-BUNCH STABILITY AT TOP ENERGY FIG. 5. Head-tail mode patterns from LHC measurements (top) and simulations (bottom) for a single bunch at the top energy (6.5 TeV). This section introduces the single-bunch instability in the LHC observed at 6.5 TeV. The most unstable head-tail 084401-6 LIZATION OF TRANSVERSE … PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) EXPERIMENTAL STABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. B PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) TABLE II. Main parameters used in PyHEADTAIL to reproduce the LHC experimental machine setup at 6.5 TeV. Parameter Symbol Value Beam energy E 6.5 TeV Bunch intensity Nb 1.2 × 1011 p Transverse normalized emittance ϵx;y 2.0 μm rad Bunch length 4σt 1.20 ns Chromaticity Q0x;y 13 Transverse feedback system τfb 100 turns Number of macroparticles Nmp 106 Number of turns Nt 5 × 105 turns TABLE IV. MAD-X calculations vs measurements of second order chromaticity at 6.5 TeV for the two LHC beams (B) and at zero Landau octupole current for operational machine settings. 10−4Q00x 10−4Q00y B Simulated Measured Simulated Measured 1 0.0 0.0 0.2 0.0 0.0 0.2 2 0.0 0.0 0.2 0.0 0.0 0.2 Table II. The contribution from the Landau octupoles to the Q00 has also been included according to the values quoted in Table III. The final results are summarized in Fig. 6. The plot shows the instability growth rates in the horizontal and the vertical planes as a function of the Landau octupole current. They have been extracted from an exponential fit to the bunch centroid motion. Clearly, with an increasing current, i.e., more tune spread and hence more Landau damping, the growth rates are reduced until the instability is completely suppressed. The gray area marks the region where the beam is stable over the entire simulation period of Nt ¼ 5 × 105 turns. This is further illustrated by Fig. 7, where the bunch centroid motion is shown over the full simulation period for four different currents in the Landau octupoles. The stabilizing current lies at Ioct ¼ 107.5 2.5 A. At this value, the Landau octupoles con- tribute to second order chromaticity with ΔQ00x ≈5000 and ΔQ00y ≈−2400, respectively. listed in Table II [6]. The upper error corresponds to the step size used for the scan in the Landau octupole current which was performed from high to low. The lower error originates from the uncertainty in the transverse emittance measurement. IV. LHC SINGLE-BUNCH STABILITY AT TOP ENERGY The Landau damping from the octupole magnets in the LHC is mainly due to the incoherent tune spread from detuning with the transverse amplitude. However, there is also an indirect contribution of detuning with the longi- tudinal amplitude from these magnets. As explained in Sec. III A, Q00 has a contribution among others from the integral of D2xk3 around the accelerator ring. By conse- quence, the LHC Landau octupoles located in dispersive regions will introduce second order chromaticity with an amount that depends linearly on the octupolar strength k3. For this reason, the Q00 contribution from the Landau octupoles was both measured and simulated for the given optics configuration. For the measurement, the change of Q00 was determined when increasing the Landau octupole current from Ioct ¼ 40 A to Ioct ¼ 320 A. The measured amounts of Q00 introduced per current are summarized in Table III alongside MAD-X predictions. The values obtained from the model are consistent with the measurements, although the latter have rather large uncertainties due to the limited sensitivity of the measurement method (see Sec. III C). It is also worth noting that, in the absence of the Landau octupoles, the Q00x;y in the LHC is practically zero, both in measurements and in MAD-X calculations. A comparison is shown in Table IV. The model predicts a threshold current that is consistent with the measurement in the machine, which demonstrates that the main beam dynamics are well modeled in PyHEADTAIL. In particular, the reliability of the LHC impedance model for single-bunch instabilities is con- firmed for the operational machine configuration at FIG. 6. PyHEADTAIL simulations showing the stabilization of a single bunch with Landau octupoles in the LHC at 6.5 TeV. Centroid growth rates vs Landau octupole current are shown, with the gray area indicating the cases that were stable over Nt ¼ 5 × 105 turns (compare Fig. 7). To compare the measured stabilizing Landau octupole current with model predictions, a scan in Ioct has been performed in PyHEADTAIL, again using the parameters in TABLE III. MAD-X calculations vs measurements of second order chromaticity introduced by the Landau octupoles per current for the two LHC beams (B). ΔQ00x=ΔIoctð1=AÞ ΔQ00y=ΔIoctð1=AÞ B Simulated Measured Simulated Measured 1 49 57 20 −23 2 27 2 48 43 22 −21 −7 28 FIG. 6. IV. LHC SINGLE-BUNCH STABILITY AT TOP ENERGY PyHEADTAIL simulations showing the stabilization of a single bunch with Landau octupoles in the LHC at 6.5 TeV. Centroid growth rates vs Landau octupole current are shown, with the gray area indicating the cases that were stable over Nt ¼ 5 × 105 turns (compare Fig. 7). 084401-7 PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) M. SCHENK et al. FIG. 7. Horizontal bunch centroid signals over the full PyHEAD- TAIL simulation period for a selection of four different Landau octupole currents for the single-bunch head-tail instability ob- served in the LHC at 6.5 TeV. were powered with a current of Ioct ¼ 320 A to guarantee beam stability. To test stabilization with Q00, the aim was to set QPPF and QPPD such that Q00x;y ≈−4 × 104 in both beams once the current in the Landau octupoles would be reduced to zero. Because of the strong current, the addi- tional contribution to the second order chromaticity from the Landau octupoles in dispersive regions was significant and, hence, had to be taken into account during the Q00 adjustment process. At the initial current of Ioct ¼ 320 A, for example, they were ΔQ00x ≈1.5×104 and ΔQ00y ≈−0.7× 104 according to Table III. To introduce the desired amount of Q00, several iterations were made by varying the sextu- pole knobs QPPF and QPPD, remeasuring Q00, and com- paring the results to MAD-X calculations. FIG. 7. Horizontal bunch centroid signals over the full PyHEAD- TAIL simulation period for a selection of four different Landau octupole currents for the single-bunch head-tail instability ob- served in the LHC at 6.5 TeV. p g Once the targeted sextupole settings were reached, the current in the Landau octupoles was reduced in steps of 40 A from 320 down to 0 A. At Ioct ¼ 40 A, all four bunches were still stable. This already indicated a success- ful stabilizing effect from Q00x;y, recalling that Ioct ¼96þ29 −10 A was necessary to control the instability without second order chromaticity. At this point, a chromaticity measure- ment was performed, since the effect from the Landau octupoles was small enough to allow for an accurate assessment of Q00. The values are summarized in Table VI and compared to MAD-X calculations after the subtraction of the remaining contribution from the Landau octupoles (ΔQ00x ≈1.9 × 103 and ΔQ00y ≈−0.9 × 103). IV. LHC SINGLE-BUNCH STABILITY AT TOP ENERGY They demonstrate good agreement and a successful adjust- ment procedure for Q00 in the machine. The amount of second order chromaticity was consistent with the targeted −4 × 104 units in three out of four planes, while it was, in absolute terms, slightly lower than expected in the hori- zontal plane of beam 2. 6.5 TeV, which will also be used in the following for the stability studies with second order chromaticity. V. LHC SINGLE-BUNCH STABILITY STUDIES WITH Q00 This section discusses the experiments and simulations carried out with Q00 in the LHC. The experimental procedure and the observations made during the machine development session are described and explained using PyHEADTAIL simulations. The aim of the section is to demonstrate stabilization of single bunches through Landau damping predominantly from detuning with the longitudinal rather than the transverse amplitude in both experiments and simulations. A. Experimental procedure and observations When the Landau octupole current was reduced further, from 40 to 0 A, a horizontal instability occurred in beam 1 for the bunch residing in bucket 700, while the other three bunches remained stable. The reasons will be discussed in Sec. V B. For the unstable bunch, the observed head-tail instability was an azimuthal mode l ¼ −1. The top right plot in Fig. 8 displays the turn-by-turn traces acquired with the LHC head-tail monitor for the unstable bunch. It shows a pattern with three nodes, indicating a radial mode m ¼ 3. The characteristics of this instability are clearly different The experiment was performed with two bunches in each of the two LHC beams at an energy of 6.5 TeV. The main parameters of the four individual bunches, i.e., the bucket number (Bkt), bunch intensity (Nb), four-sigma bunch length (4σt), and the transverse normalized emittances (ϵx;y), are listed in Table V. Because of the presence of the transverse bunch-by-bunch feedback system as well as the large longitudinal separation of the two bunches in each beam, coupled-bunch wakefield effects can be neglected. At the beginning of the experiment, the Landau octupoles TABLE VI. Q00 MAD-X calculations vs measurements for the two LHC beams (B) with powering of the main sextupoles (MS) at zero Landau octupole current after the Q00 adjustment pro- cedure targeting an amount of Q00x;y ¼ −4 × 104 units in both beams. TABLE V. Initial bunch parameters of the four individual bunches in the two beams (B) residing in different buckets (Bkt) while performing the experiments with Q00. TABLE V. Initial bunch parameters of the four individual bunches in the two beams (B) residing in different buckets (Bkt) while performing the experiments with Q00. B. PyHEADTAIL simulations and interpretation There are three main goals for the simulation studies: (i) whether the stabilization of three out of four bunches is indeed a result of mostly detuning with the longitudinal amplitude, or whether it could have been provided by the transverse detuning coefficients αmn, with m; n ∈fx; yg, introduced parasitically by the QPPF and QPPD knobs, (ii) to understand why one of the four bunches became unstable, and (iii) why the observed head-tail mode with nonzero Q00 knobs is now an azimuthal mode l ¼ −1 rather than an l ¼ 0 as originally observed in Sec. IVA. The second set of simulations is shown in the two lower plots in Fig. 9. This simulation now also includes the effects from detuning with the longitudinal amplitude as intro- duced by the second order chromaticity. Two main obser- vations can be made. First, large regions of stability (blue) are created in the QPPF vs QPPD plane. The two main stable areas are separated by an unstable band in both the horizontal and the vertical planes which shows a different head-tail mode with an azimuthal number l ¼ −1 (red dots). The reason for that is that second order chromaticity changes the effective impedance, similarly to a first order chromaticity. This effect is described by the Vlasov formalism [2] and is currently under more detailed ana- lytical study. It affects the complex coherent tune shift and can also change the most unstable mode. The bottom plots in Fig. 9 are hence a combination of a change in effective impedance and Landau damping, both introduced by Q00. At the experimental working point QPPF ¼ 0.15 m−3 and QPPD ¼ 0.19 m−3 (beam 1), the transverse detuning coefficients generated by the sextupoles are comparable to LHC Landau octupole currents of Ioct ≈50 A (αxx), Ioct ≈20 A, (αyy) and Ioct ≈30 A (αxy), respectively (com- pare Fig. 3). They are all well below the above-mentioned single-bunch stability threshold of Ioct ¼ 96þ29 −10 A, which indicates that an important contribution to beam stability must have been provided by Q00. To understand the horizontal instability observed in one of the bunches, however, simulation studies were required. Optics results from MAD-X, among them the dependencies of Q00x;y and the transverse detuning coefficients on the QPPF and QPPD knobs, displayed in Figs. A. Experimental procedure and observations B Bkt Nb (1011 p) 4σt (ns) ϵx (μm rad) ϵy (μm rad) 1 0 0.91 0.01 1.04 0.02 1.5 0.2 1.8 0.2 700 1.08 0.01 1.06 0.02 1.9 0.1 1.7 0.1 2 30 0.92 0.01 1.04 0.02 1.2 0.1 1.5 0.1 740 1.14 0.01 1.06 0.02 1.2 0.1 1.3 0.1 B Bkt Nb (1011 p) 4σt (ns) ϵx (μm rad) ϵy (μm rad) 1 0 0.91 0.01 1.04 0.02 1.5 0.2 1.8 0.2 700 1.08 0.01 1.06 0.02 1.9 0.1 1.7 0.1 2 30 0.92 0.01 1.04 0.02 1.2 0.1 1.5 0.1 740 1.14 0.01 1.06 0.02 1.2 0.1 1.3 0.1 MS (m−3) 10−4Q00x 10−4Q00y B QPPF QPPD Simulated Measured Simulated Measured 1 0.15 0.19 −3.8 −4.4 0.6 −3.9 −4.0 0.6 2 0.15 0.26 −3.7 −3.1 0.5 −3.7 −3.7 0.6 084401-8 EXPERIMENTAL STABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. B LIZATION OF TRANSVERSE … PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) FIG. 8. Comparison of the two modes observed in the machine (top) and in PyHEADTAIL simulations (bottom) without (left) and with (right) the effects from Q00. They correspond to the working points labeled (a) and (b), respectively (compare Fig. 9). are modeled. In all the studies, the Landau octupoles are switched off to reproduce the situation of the experiment at Ioct ¼ 0 A where the bunch became unstable. The bunch parameters are taken from the measurements in Table V for the bunch in beam 1, bucket 700. Nmp ¼ 4 × 105 macro- particles are tracked over Nt ¼ 1.8 × 106 turns using again the same impedance model of the LHC. p In a first set of simulations, the tune spread from Q00 is deliberately excluded. This is to assess whether the spread from the transverse detuning coefficients alone, which is introduced parasitically by the QPPF and QPPD knobs, would be enough to provide stability at the working point (QPPF ¼ 0.15 m−3, QPPD ¼ 0.19 m−3). The results are summarized in the upper plots in Fig. 9 for the horizontal (left) and the vertical (right) planes. A. Experimental procedure and observations The color code shows the relative emittance growth over the simulation period (in percent), where blue means stable and white means unstable. The dots represent the azimuthal mode number of the instability predicted for each setting of (QPPF, QPPD) in case the bunch was unstable in the simulation. The two working points labeled (a) and (b) show the situation with and without the Q00 knobs and correspond to the exper- imental machine configuration described in Secs. IVA and VA, respectively. For the latter, the error bars correspond to the uncertainty in the Q00x;y listed in Table VI. Most of the area is unstable, in particular, the working point (b) where the Q00 measurements of Sec. VA were carried out. This demon- strates that the transverse detuning coefficients from the sextupole knobs indeed do not provide sufficient Landau damping. The experiment showed that three out of four bunches remained stable, while the model predicts instability with a large level of significance. Furthermore, the instability predicted by the model is an azimuthal mode l ¼ 0 (green dots) with two nodes in the head-tail pattern, which is not consistent with the experimental observations made in the machine at the working point (b). Rather, an azimuthal mode l ¼ −1 with three nodes is what was observed experimen- tally (Fig. 8, right). FIG. 8. Comparison of the two modes observed in the machine (top) and in PyHEADTAIL simulations (bottom) without (left) and with (right) the effects from Q00. They correspond to the working points labeled (a) and (b), respectively (compare Fig. 9). from those described in Sec. IVA, where an azimuthal mode l ¼ 0 with two nodes in the head-tail pattern was recorded (Fig. 8, top left). The main difference with respect to the situation in the previous section is the large amount of Q00x;y that was now present in the machine. The reasons for the change of the head-tail mode are explained with PyHEADTAIL simulations in the following Sec. V B. B. PyHEADTAIL simulations and interpretation An impedance-driven instability is more difficult to be stabilized for a bunch of higher intensity, since its complex coherent tune shift is larger and more likely to lie outside the stability diagram. FIG. 9. Two PyHEADTAIL studies showing the predicted head- tail instabilities as a function of the sextupole knobs QPPF and QPPD. Labels (a) and (b) mark the two working points used during the experiments. Top: Results obtained without Q00x;y effects but including the transverse amplitude detuning coeffi- cients from the sextupole knobs. Bottom: Results obtained including also Q00x;y effects. The first unstable band observed at low values of QPPF (QPPD) is the azimuthal mode zero (green dots), consistent with the experimental measurements described in Sec. IVA. The stable region between the two unstable bands l ¼ 0 and l ¼ −1 arises from sufficient Landau damping of both modes. The further increase of QPPF (or QPPD), however, leads to a change of the effective impedance, in a way that Landau damping is lost for the l ¼ −1 mode. For even larger amounts of Q00, however, all the instabilities can be suppressed. The second observation is that the working point (b), although essentially stable, lies very close to the unstable band of the l ¼ −1 mode. Indeed, the experimental data clearly revealed the observed horizontal instability to be of mode l ¼ −1. The LHC head- tail monitor signal is in good agreement with the predic- tions from simulations. Both of them feature a three-node coherent oscillation pattern along the bunch as shown in Fig. 8 on the right. Thus, the experiment and simulation agree both in the azimuthal as well as the radial mode numbers of the excited instability. In summary, the experiments can be reproduced in simulations only when including all the effects of second order chromaticity in PyHEADTAIL. This concerns both the Landau damping effect as well as the change of the effective impedance, which manifests as a change of the azimuthal and radial mode numbers. When including the effects of the second order chromaticity in the model, the observed head-tail modes are consistent with simula- tions at both working points (a) and (b). Moreover, the second order chromaticity is the main stabilizing effect, while the contribution from detuning with the transverse amplitude introduced by the sextupole knobs plays a minor role here. B. PyHEADTAIL simulations and interpretation 2 and 3, are also included in PyHEADTAIL such that both the stabilizing effects from detuning with the transverse and longitudinal amplitudes 084401-9 PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) M. SCHENK et al. FIG. 9. Two PyHEADTAIL studies showing the predicted head- tail instabilities as a function of the sextupole knobs QPPF and QPPD. Labels (a) and (b) mark the two working points used during the experiments. Top: Results obtained without Q00x;y effects but including the transverse amplitude detuning coeffi- cients from the sextupole knobs. Bottom: Results obtained including also Q00x;y effects. accuracy of the impedance model and, therefore, the uncertainty on the beam stability predictions from PyHEADTAIL. A number of considerations can be made, however, to better understand and explain the experimental observations. First, the bunches in beam 1 and beam 2 do not experience the same amount of Landau damping due to different settings in Q00x;y for the two beams (Table VI). While this may explain why beam 1 and beam 2 behave differently, it does not answer the question why the two bunches in beam 1 exhibit a different behavior. After all, they are both subject to the same amount of second order chromaticity. The only remaining obvious differences between the two bunches are their transverse emittances and intensities listed in Table V. The unstable bunch residing in bucket 700 had a slightly larger horizontal emittance compared to the stable one located in bucket 0 (1.9 0.1 vs 1.5 0.2 μm rad). The vertical emittances, on the other hand, were identical within the errors (1.7 0.1 vs 1.8 0.2 μm rad). As a result of the larger horizontal emittance, the Landau damping introduced by the parasitic transverse detuning coefficient αxx in the horizontal plane is actually larger for the unstable bunch compared to the stable one. However, the difference between the emittances is minor, and, more importantly, the trans- verse detuning coefficient is very small. It is clear from the upper plots in Fig. 9 that the Landau damping from detuning with the transverse amplitude is insignificant for the insta- bility under consideration. The bunch intensities, on the other hand, are the most likely explanation for the differences observed in terms of bunch stability. The stable bunch had a lower intensity of ð0.91 0.01Þ × 1011 p compared to ð1.08 0.01Þ × 1011 p for the unstable one. B. PyHEADTAIL simulations and interpretation The reason why one of the four bunches has gone unstable during the experiment is explained by the differences in bunch intensities as well as in the second order chromaticities present in the two beams. The simulation model cannot make a definite statement about why only one of the four bunches became unstable during the experiment. This is mostly due to the measure- ment uncertainties in the bunch length, intensity, and emittance and to some extent also due to the limited ACKNOWLEDGMENTS The authors thank the BE-OP-LHC teams and Machine Development coordinators and participants at CERN for helping with the experiments. Furthermore, they acknowledge St´ephane Fartoukh, Antoine Maillard, and Giovanni Rumolo for fruitful discussions and inputs to these studies. [1] L. D. Landau, On the vibrations of the electronic plasma, Zh. Eksp. Teor. Fiz. 16, 574 (1946) [J. Phys. USSR 10, 25 (1946)]. The single-bunch stability in the LHC at 6.5 TeV has been recapped, and the stabilizing Landau octupole cur- rents for the most unstable head-tail mode were shown to be consistent between experiments and PyHEADTAIL simula- tions. Based on the same machine configuration, experi- ments have been performed to assess the stabilization of single bunches with second order chromaticity. Beam dynamics simulations clarify that detuning from the trans- verse amplitude alone, introduced parasitically by the sextupole knobs, cannot explain the observations made in the machine. Clearly, Q00 makes a strong contribution to the beam stability as demonstrated by the combined analysis of the data and PyHEADTAIL simulations. Furthermore, the second order chromaticity leads to a change of the head-tail mode, which is expected from the Vlasov theory and consistently observed in simulations and experiments conducted here. This is due to a change of the effective impedance introduced by chromaticity, and a detailed analysis will be reported in a forthcoming paper. With the present LHC beam parameters, the magnetic octupoles provide enough Landau damping such that no additional means are required. Nevertheless, the work on beam stabilization from detuning with the longitudinal amplitude will be continued. Future studies will, among others, assess the effect of Q00 on the dynamic aperture (beam lifetime), its potential for stabilization of, e.g., electron cloud-driven instabilities, and the evaluation of various Q00 production schemes. A step in that direction has been made recently with a machine development session in the LHC to address the aforementioned questions [25]. However, the scheme that was employed created a large off-momentum beta beating around the ring and hence was not machine-safe for multibunch operation. The offline The single-bunch stability in the LHC at 6.5 TeV has been recapped, and the stabilizing Landau octupole cur- rents for the most unstable head-tail mode were shown to be consistent between experiments and PyHEADTAIL simula- tions. Based on the same machine configuration, experi- ments have been performed to assess the stabilization of single bunches with second order chromaticity. ACKNOWLEDGMENTS Beam dynamics simulations clarify that detuning from the trans- verse amplitude alone, introduced parasitically by the sextupole knobs, cannot explain the observations made in the machine. Clearly, Q00 makes a strong contribution to the beam stability as demonstrated by the combined analysis of the data and PyHEADTAIL simulations. Furthermore, the second order chromaticity leads to a change of the head-tail mode, which is expected from the Vlasov theory and consistently observed in simulations and experiments conducted here. This is due to a change of the effective impedance introduced by chromaticity, and a detailed analysis will be reported in a forthcoming paper. [2] A. Chao, Physics of Collective Beam Instabilities in High Energy Accelerators, Wiley Series in Beam Physics and Accelerator Technology (Wiley, New York, 1993). [3] O. S. Brüning, P. Collier, P. Lebrun, S. Myers, R. Ostojic, J. Poole, and P. Proudlock, LHC Design Report, CERN Yellow Reports: Monographs (CERN, Geneva, 2004). [4] J. Gareyte, J.-P. Koutchouk, and F. Ruggiero, Technical Report Nos. LHC-Project-Report-91, CERN-LHC-Project- Report-91, CERN, 1997. [5] S. Peggs and T. Satogata, Introduction to Accelerator Dynamics (Cambridge University Press, Cambridge, England, 2017), Chap. 10, pp. 107–109. [6] L. R. Carver et al., in Proceedings of the 7th International Particle Accelerator Conference (IPAC’16), Busan, Korea, 2016 (JACoW, Geneva, 2016), pp. 1434–1437. [7] E. M´etral et al., in Proceedings of the 57th ICFA Advanced Beam Dynamics Workshop on High-Intensity and High- Brightness Hadron Beams (HB’16), Malmö, Sweden, 2016 (JACoW, Geneva, Switzerland, 2016), pp. 254–259. With the present LHC beam parameters, the magnetic octupoles provide enough Landau damping such that no additional means are required. Nevertheless, the work on beam stabilization from detuning with the longitudinal amplitude will be continued. Future studies will, among others, assess the effect of Q00 on the dynamic aperture (beam lifetime), its potential for stabilization of, e.g., electron cloud-driven instabilities, and the evaluation of various Q00 production schemes. A step in that direction has been made recently with a machine development session in the LHC to address the aforementioned questions [25]. However, the scheme that was employed created a large off-momentum beta beating around the ring and hence was not machine-safe for multibunch operation. The offline [8] A. Grudiev, Radio frequency quadrupole for Landau damping in accelerators, Phys. Rev. Accel. Beams 17, 011001 (2014). [9] K. Papke and A. Grudiev, Design of an rf quadrupole for Landau damping, Phys. Rev. Accel. Beams 20, 082001 (2017). VI. CONCLUSIONS AND OUTLOOK Detuning with the longitudinal amplitude is potentially a usable technique to introduce Landau damping in the 084401-10 ABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. BEAMS 21, 084401 (2018 PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) EXPERIMENTAL STABILIZATION OF TRANSVERSE … PHYS. REV. ACCEL. B transverse planes, particularly for the high-brightness, low transverse emittance beams of future hadron colliders where conventional approaches, such as magnetic octu- poles, may not provide a sufficient tune spread. Second order chromaticity or an rf quadrupole cavity are two possible ways to introduce a betatron tune spread as a function of the longitudinal action, and it was shown analytically that the two mechanisms are equivalent in a first approximation. The second order chromaticity has the advantage that it can be introduced in a machine like the LHC by using the existing hardware and changing the optics of the accelerator. In that way, it can be used to experimentally study the stabilization from transverse detuning with the longitudinal amplitude in a cost-effective manner. The LHC represents an ideal accelerator environ- ment for such a proof-of-principle experiment, as it is a well-studied machine, in terms of both optics and collective effects. Here, a sextupole powering scheme was employed to control Q00 in both beams and the two transverse planes independently. Beam measurements and MAD-X calcula- tions show good agreement and demonstrate a thorough control of the second order chromaticity in the LHC. analysis showed, however, that this may be overcome by a change of the off-momentum collimation scheme. On the other hand, it may also be an indication of why an rf quadrupole could be a better choice for Landau damping from detuning with the longitudinal amplitude. Other than Q00, it does not depend on optics constraints but works instead as an independent device. The advantages and disadvantages of both methods, however, need to be evaluated in more detail, especially in terms of single- particle effects (e.g., dynamic aperture and resonances). ACKNOWLEDGMENTS [10] M. Schenk, A. Grudiev, K. Li, and K. Papke, Analysis of transverse beam stabilization with radio frequency quadru- poles, Phys. Rev. Accel. Beams 20, 104402 (2017). [11] G. Apollinari, I. B´ejar Alonso, O. Brüning, M. Lamont, and L. Rossi, High-Luminosity Large Hadron Collider (HL-LHC): Preliminary Design Report, CERN Yellow Reports: Monographs (CERN, Geneva, 2015). [12] M. Benedikt and F. Zimmermann, Technical Report No. CERN-ACC-2015-0164, CERN, 2015. 084401-11 PHYS. REV. ACCEL. BEAMS 21, 084401 (2018) M. SCHENK et al. [13] E. M´etral et al., Beam instabilities in hadron synchrotrons, IEEE Trans. Nucl. Sci. 63, 1001 (2016). [22] R. Bartolini and F. Schmidt, Technical Report No. SL- Note-98-017-AP, CERN, 1998. [22] R. Bartolini and F. Schmidt, Technical Report No. SL- Note-98-017-AP, CERN, 1998. [14] Methodical Accelerator Design (MAD), http://mad.web .cern.ch/mad/. [23] L. R. Carver, D. Amorim, N. Biancacci, X. Buffat, G. Iadarola, K. Łasocha, K. Li, T. Levens, E. M´etral, B. Salvant, and C. Tambasco, MD1228: Validation of single bunch stability threshold & MD1751: Instability studies with a single beam, Technical Report No. CERN-ACC- NOTE-2017-0013, CERN, 2017. [15] J. S. Berg and F. Ruggiero, in Proceedings of the 1997 Particle Accelerator Conference (Cat. No.97CH36167) (IEEE, New York, 1997), Vol. 2, pp. 1712–1714. ( ) pp [16] F. C. Iselin, Technical Report No. SL-92-199, CERN, 1994. F. C. Iselin, Technical Report No. SL-92-199, CERN, [24] T. Levens, K. Łasocha, and T. Lef`evre, in Proc. of International Beam Instrumentation Conference (IBIC'16), Barcelona, Spain, Sept. 13-18, 2016, International Beam Instrumentation Conference No. 5 (JACoW, Geneva, Switzerland, 2017), p. THAL02, . [17] S. D. Fartoukh, Technical Report No. CERN-LHC-Project- Report-308, CERN, 1999. [18] N. Mounet (unpublished). [19] F. Schmidt, E. Forest, and E. McIntosh, Technical Report Nos. CERN-SL-2002-044-AP, KEK-REPORT-2002-3, CERN, 2002. [25] M. Schenk, S. D. Fartoukh, K. Li, L. Malina, E. M´etral, and R. Tomás Garcia, MD2190: Q" Stabilization during injection, Technical Report No. CERN-ACC-NOTE-2018- 0003, CERN, 2018. [20] S. Y. Lee, Accelerator Physics, 3rd ed. (World Scientific, Singapore, 2012), Chap. 2, pp. 197–198. [21] M. Gasior and R. Jones, Technical Report No. LHC- Project-Report-853, CERN, 2005. 084401-12
https://openalex.org/W2810420630	https://ris.utwente.nl/ws/files/120013909/fphar_09_00790.pdf	English	null	Current Trends and Challenges in the Clinical Translation of Nanoparticulate Nanomedicines: Pathways for Translational Development and Commercialization	Frontiers in pharmacology	2,018	cc-by	13,242	REVIEW published: 17 July 2018 doi: 10.3389/fphar.2018.00790 REVIEW REVIEW published: 17 July 2018 doi: 10.3389/fphar.2018.00790 Current Trends and Challenges in the Clinical Translation of Nanoparticulate Nanomedicines: Pathways for Translational Development and Commercialization S H 1 2* M i B C d M t 3 J b t M M t l 4 5 d G t St 3 4 6 Susan Hua 1,2, Maria B. C. de Matos 3, Josbert M. Metselaar 4,5 and Gert Storm 3,4,6 1 Therapeutic Targeting Research Group, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW, Australia, 2 Hunter Medical Research Institute, New Lambton Heights, Newcastle, NSW, Australia, 3 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands, 4 Department of Biomaterials Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, Netherlands, 5 Department of Experimental Molecular Imaging, RWTH University Clinic Aachen, Aachen, Germany, 6 Imaging Division, University Medical Centre Utrecht, Utrecht, Netherlands Citation: Hua S, de Matos MBC, Metselaar JM and Storm G (2018) Current Trends and Challenges in the Clinical Translation of Nanoparticulate Nanomedicines: Pathways for Translational Development and Commercialization. Front. Pharmacol. 9:790. doi: 10.3389/fphar.2018.00790 The use of nanotechnology in medicine has the potential to have a major impact on human health for the prevention, diagnosis, and treatment of diseases. One particular aspect of the nanomedicine ﬁeld which has received a great deal of attention is the design and development of nanoparticulate nanomedicines (NNMs) for drug delivery (i.e., drug-containing nanoparticles). NNMs are intended to deliver drugs via various mechanisms: solubilization, passive targeting, active targeting, and triggered release. The NNM approach aims to increase therapeutic efﬁcacy, decrease the therapeutically effective dose, and/or reduce the risk of systemic side effects. In order to move a NNM from the bench to the bedside, several experimental challenges need to be addressed. This review will discuss the current trends and challenges in the clinical translation of NNMs as well as the potential pathways for translational development and commercialization. Key issues related to the clinical development of NNMs will be covered, including biological challenges, large-scale manufacturing, biocompatibility and safety, intellectual property (IP), government regulations, and overall cost-effectiveness in comparison to current therapies. These factors can impose signiﬁcant hurdles limiting the appearance of NNMs on the market, irrelevant of whether they are therapeutically beneﬁcial or not. Edited by: Xinhua Qu, Shanghai Ninth People’s Hospital, Shanghai Jiao-Tong University School of Medicine, China Reviewed by: Christoph Eugen Hagemeyer, Monash University, Australia Nazareno Paolocci, Johns Hopkins University, United States Correspondence: Susan Hua Susan.Hua@newcastle.edu.au Edited by: Xinhua Qu, Shanghai Ninth People’s Hospital, Shanghai Jiao-Tong University School of Medicine, China Edited by: Xinhua Qu, Shanghai Ninth People’s Hospital, Shanghai Jiao-Tong University School of Medicine, China Reviewed by: Christoph Eugen Hagemeyer, Monash University, Australia Nazareno Paolocci, Johns Hopkins University, United States Correspondence: Susan Hua Susan.Hua@newcastle.edu.au Correspondence: Susan Hua Susan.Hua@newcastle.edu.au *Correspondence: Susan Hua Susan.Hua@newcastle.edu.au Specialty section: This article was submitted to Translational Pharmacology, a section of the journal Frontiers in Pharmacology Received: 31 May 2018 Accepted: 28 June 2018 Published: 17 July 2018 Keywords: nanomedicine, nanoparticles, drug delivery systems, clinical translation, challenges, commercialization, biological, regulations Keywords: nanomedicine, nanoparticles, drug delivery systems, clinical translation, challenges, commercialization, biological, regulations Enhanced Permeability and Retention (EPR) Effect and Passive Accumulation of NNMs The EPR eﬀect refers to the preferential localization of NNMs in pathological tissues due to the enhanced permeability of the vasculature that supplies such tissues (e.g., tumors and inﬂammatory conditions). Deregulations in angiogenesis and/or the increased expression and activation of vascular permeability factors predominates at these sites, which can lead to fenestrations allowing passage of NNMs (Hashizume et al., 2000; Nehoﬀet al., 2014). In addition to the enhanced leakiness of tumorous and inﬂamed blood vessels, the EPR eﬀect also relates to the observation that solid tumors tend to lack functional lymphatic drainage, which limits the removal of extravasated NNMs from the target site (Maeda et al., 2013; Danhier, 2016). These pathological properties allow NNMs to accumulate at pathological sites and is referred to as passive targeting. To achieve this, it is important that NNMs with drug cargo circulate long enough in the bloodstream (i.e., show prolonged circulation kinetics). This can be achieved by conjugating polyethylene glycol (PEG) to the surface of NNMs (Figure 1). Thus, the EPR eﬀect is expected to increase the therapeutic eﬃcacy of NNMs in comparison to small molecules, which often show inferior pharmacokinetic properties (Matsumura and Maeda, 1986; Hobbs et al., 1998; van der Meel et al., 2013). The EPR eﬀect was ﬁrst observed in 1986 (Matsumura and Maeda, 1986) and has since been exploited particularly for the development of NNMs for passive tumor targeting, leading to NNMs with adequate physicochemical properties and prolonged circulation half-life that accumulate in tumors over time (Maeda et al., 2013; Nakamura et al., 2015; Danhier, 2016). The EPR eﬀect and thus extent of passive targeting is highly dependent on the tumor pathophysiology. Currently, it is recognized that EPR is a very heterogeneous phenomenon as it depends on the type of tumor and can vary signiﬁcantly within the same tumor type (Lammers et al., 2012; Ojha et al., 2017). The degree of tumor vascularization and passive targeting of NNMs has been observed to be positively correlated (Theek et al., 2014). For example, Doxil R⃝(pegylated liposomal doxorubicin) is the ﬁrst FDA-approved NNM and has demonstrated superior eﬃcacy in ovarian cancer and AIDS-related Kaposi’s sarcoma compared to standard conventional therapies (Nichols and Bae, 2014). When doxorubicin is encapsulated within PEGylated liposomes, it delays and minimizes uptake and clearance by the reticuloendothelial system (RES), thereby prolonging circulation half-life. INTRODUCTION Nanomedicine applies nanotechnology to highly speciﬁc medical interventions for the prevention, diagnosis, and treatment of diseases (Teli et al., 2010). In the last several decades, the application of nanotechnology for medical purposes has received signiﬁcant attention from researchers, academia, funding agencies, government, and regulatory bodies (Allen and Cullis, 2004; Sercombe et al., 2015; Hare et al., 2017). One particular aspect of the nanomedicine ﬁeld which has July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 1 Clinical Translation of Nanomedicines Hua et al. received a great deal of attention is the design and development of nanoparticulate nanomedicines (NNMs) for drug delivery (i.e., drug-containing nanoparticles), which are most often given by parenteral (particularly intravenous) administration. NNMs are intended to increase the therapeutic index of drugs (i.e., increase eﬃcacy and/or reduce toxicity) by delivering them via various mechanisms: solubilization, passive targeting, active targeting, and triggered release (Figure 1). Nanoencapsulation gives the opportunity to protect fragile compounds that degrade easily in biological environments and to provide solubilization, i.e., to deliver compounds which have physicochemical properties that strongly limit their aqueous solubility and therefore systemic bioavailability (Talekar et al., 2015; Kim et al., 2016; Larsson et al., 2017; Mishra et al., 2017; Shajari et al., 2017). Targeted drug delivery and triggered release of NNMs have been shown to be beneﬁcial for increasing the therapeutic index of compounds, by improving the in vivo fate of drug molecules such that more eﬃcient delivery to the target site is achieved (to yield improved therapeutic eﬀects) with less accumulation in many healthy body sites (to reduce toxicity). Also NNMs have been studied for their ability to stimulate target cell uptake and improve intracellular traﬃcking, processes sometimes required when they have localized in target tissues (Mastrobattista et al., 1999; Hua, 2013; Hua et al., 2015). exploiting the underlying biology of these conditions (Milane and Amiji, 2017). Non-cancerous inﬂammatory diseases that have been explored with NNM therapy include rheumatoid arthritis, inﬂammatory bowel disease, asthma, multiple sclerosis, diabetes, and neurodegenerative diseases (Milane and Amiji, 2017). Enhanced Permeability and Retention (EPR) Effect and Passive Accumulation of NNMs This allows the NNM to accumulate in the tumor tissue by exploiting the locally increased permeability of the tumor blood vessels, rather than in non-target healthy tissues which do not have such leaky vessels (Rahman et al., 2007). Furthermore, the use of pegylated liposomal doxorubicin avoids high plasma Although NNMs have demonstrated signiﬁcant therapeutic advantages for a multitude of biomedical applications, their clinical translation has not progressed as rapidly as the plethora of positive preclinical results would have suggested (Luxenhofer et al., 2014). In order to move a NNM from the bench to the bedside, several experimental challenges need to be addressed. From a biological perspective, these include studies focused on understanding the in vivo fate and interactions of NNMs with the blood, tissue, cellular, and intracellular compartments in the host in healthy and diseased states (Nehoﬀet al., 2014; Sercombe et al., 2015; Hare et al., 2017). For NNMs to have clinical translation potential, the complexity in their design and development also needs to be minimized as much as possible to create systems that are able to be reproducibly prepared and characterized (Lammers, 2013; Barz et al., 2015). This review will address the current trends and challenges in the clinical translation of NNMs as well as the potential pathways for translational development and commercialization. CURRENT TRENDS IN THE CLINICAL TRANSLATION OF NANOMEDICINES NNMs are often studied to improve drug targeting to speciﬁc sites of disease (i.e., site-speciﬁc drug delivery) and/or attenuate localization in healthy non-target tissues (i.e., site-avoidance drug delivery; Rizzo et al., 2013). The vast majority of NNMs in preclinical and clinical development as well in clinical use are for targeting a wide variety of cancers and tumors (Hare et al., 2017). The application of NNM-based therapies for drug targeting to non-cancer conditions has increased in recent years. In particular, NNMs have been developed to address the clinical challenge of eﬀectively managing inﬂammatory diseases by Frontiers in Pharmacology \| www.frontiersin.org July 2018 \| Volume 9 \| Article 790 2 Hua et al. Clinical Translation of Nanomedicines FIGURE 1 \| Schematic representation of different strategic designs for nanoparticulate nanomedicines (NNMs). (A) Conventional NNM—These NNMs can be modiﬁed with charged lipids/polymers, thermosensitive lipids/polymers and/or components for triggered release (e.g., pH-sensitive coating). (B) PEGylated NNM—Nanoparticle characteristics and behavior in vivo can be modiﬁed by the addition of a hydrophilic polymer coating, polyethylene glycol (PEG), to the NNM surface to confer steric stabilization. (C) Ligand-targeted NNM—Nanoparticles can be used for active targeting by attaching ligands (e.g., antibodies, peptides and carbohydrates) to its surface or to the terminal end of the attached PEG chains. (D) Theranostic NNM – These NNM systems consist of an imaging component and a therapeutic component, and may include a targeting element. peak levels of free drug (Lyass et al., 2000) and signiﬁcantly reduces the risk of cardiotoxicity by preventing doxorubicin release through the heart vasculature (Rahman et al., 2007). macrophages, ﬁbroblasts) that can acquire a tumor survival- promoting phenotype in response to cytokines in the tumor microenvironment (Coimbra et al., 2010; Danhier et al., 2010; Kuijpers et al., 2010; Danhier, 2016). There is still much debate about whether ligand-targeted NNMs are capable of signiﬁcantly enhancing NNM accumulation at target sites over non-targeted NNMs (passive-targeting), with conﬂicting results reported in the literature (Ferrari, 2005; Puri et al., 2009; Riehemann et al., 2009; van der Meel et al., 2013). Enhanced therapeutic eﬀects have been demonstrated with ligand-targeted NNMs, despite showing no diﬀerences in accumulation in target tissues compared to non-targeted NNMs. For example, similar high levels of tumor tissue accumulation were achieved with both non-targeted liposomes and liposomes conjugated with HER2 monoclonal antibody fragments (7–8% injected dose/g tumor tissue) in HER2-overexpressing breast cancer xenografts models (Kirpotin et al., 1997, 2006). However, signiﬁcantly superior therapeutic results was demonstrated with the doxorubicin-loaded anti- HER2 immunoliposomes in comparison to all other control groups, including recombinant anti-HER2 Mab trastuzumab, non-targeted liposomal doxorubicin, and free doxorubicin (Park et al., 2002). Diﬀerences in pharmacodynamics of the targeted NNM formulation in vivo was suggested as the reason for the Frontiers in Pharmacology \| www.frontiersin.org NNMs for Triggered Release gg A third targeting strategy based on stimuli-responsive NNMs, referred to as triggered drug release, is currently receiving much attention from academia and industry. This class of NNMs is designed with the goal of enhancing drug release in tumors by means of endogenous or exogenous stimuli. Endogenous stimuli-responsive NNMs exploit factors associated with the local environment at the site of disease (Figure 1). For example, low pH, presence of redox gradients or certain enzymes in the tumor microenvironment. Exogenous-responsive NNMs respond to external stimuli to trigger drug release, such as temperature, light, magnetic ﬁeld or ultrasound. Of these strategies, the use of an external hyperthermic trigger to release therapeutic compounds from NNMs (e.g., thermosensitive liposomal doxorubicin, ThermoDox R⃝) appears to be the most promising to date (Needham et al., 2000). ThermoDox R⃝was shown to be superior to its counterpart Doxil R⃝in an in vivo model of non-resectable hepatocellular carcinoma (Torchilin, 2006; Sawant and Torchilin, 2012; Oude Blenke et al., 2013; Bertrand et al., 2014; Min et al., 2015; Jang et al., 2016; Shi et al., 2017). Thermosensitive liposomes are typically modiﬁed with temperature-sensitive lipids (e.g., distearoyl phosphocholine, DSPC) and/or polymers [e.g., poly(N-isopropylacrylamide)]. This composition allows the NNM to remain stable and retain their contents at physiologic temperatures, and undergo a phase change that makes them more permeable upon heating, thereby triggering the release of the cargo (Kono, 2001). The advantages of these NNMs can be further extended with the incorporation of imaging moieties (Figure 1) to enable monitoring of biodistribution, target accumulation and eﬃcacy. NNMs and Active Targeting g g Active targeting, also termed ligand-targeting or receptor- mediated targeting, involves the use of ligands (e.g., antibodies, peptides or sugar moieties) which are physically or chemically conjugated onto the surface of NNMs to facilitate localization to and/or uptake by target cells (van der Meel et al., 2013; Danhier, 2016; Figure 1). Ligand-targeted NNMs have enormous potential for site-speciﬁc delivery of therapeutic compounds to designated cell types in vivo, which selectively express or over- express speciﬁc receptors (e.g., cellular receptors or cell adhesion molecules) at the site of disease (Willis and Forssen, 1998; Hua, 2013). For example, three sets of cellular targets are generally considered for active targeting in cancer—(i) targeting of cancer cells, which present overexpression of receptors for transferrin, folate, epidermal growth factor or glycoproteins; (ii) targeting of the tumor endothelium overexpressing vascular endothelial growth factors, integrins, vascular cell adhesion molecule-1 or matrix metalloproteinases; and (iii) targeting of stroma cells (e.g., July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 3 Clinical Translation of Nanomedicines Hua et al. improved anti-tumor eﬀect, by enhancing intracellular drug delivery to HER2-overexpressing cancer cells (Kirpotin et al., 2006). every year (Table 2), and again liposomal formulations represent the biggest share of the NNMs under clinical evaluation. The most frequently observed clinical beneﬁt so far has been a reduction in toxicity with little evidence of improved eﬃcacy. However, recently approved liposomal NNM, Vyxeos R⃝ (daunorubicin/cytarabine liposomal formulation), demonstrated improved survival and response rates, with tolerable toxicity in phase III clinical trials in older patients with therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (AML-MRC; Kim and Williams, 2018). CHALLENGES IN THE CLINICAL TRANSLATION OF NANOMEDICINES The clinical translation of NNMs is an expensive and time- consuming process. NNM technology is usually far more complex in comparison to conventional formulation technology containing free drug dispersed in a base (e.g., tablets, capsules and injections; Teli et al., 2010; Tinkle et al., 2014; Sainz et al., 2015). Key issues related to the clinical development of NNMs are listed in Table 3, and include biological challenges, large-scale manufacturing, biocompatibility and safety, intellectual property (IP), government regulations, and overall cost-eﬀectiveness in comparison to current therapies (Allen and Cullis, 2004, 2013; Zhang et al., 2008; Sawant and Torchilin, 2012; Narang et al., 2013). These factors can impose signiﬁcant hurdles limiting the appearance of NNMs on the market, irrelevant of whether they are therapeutically eﬃcacious or not. Biological Challenges Traditionally, NNM development has been based on a formulation-driven approach, whereby novel delivery systems are ﬁrstly engineered and characterized from a physicochemical perspective. It is only when attempting to align the NNM with a pathological application that limitations in the clinical translation of the system have been identiﬁed. Understanding the relationship between biology and technology, including understanding the inﬂuence of disease pathophysiology on nanomedicine accumulation, distribution, retention and eﬃcacy, as well as the biopharmaceutical correlation between delivery system properties and in vivo behavior in animals versus humans are important determinants for the successful translation of NNMs. Therefore, applying a disease-driven approach by designing and developing NNMs that are able to exploit pathophysiological changes in disease biology has been suggested to improve clinical translation (Hare et al., 2017). July 2018 \| Volume 9 \| Article 790 NNMs Approved and in Clinical Trials pp A number of NNM products are on the market with more in clinical development. The majority of NNMs in clinical development incorporate already approved drugs and are based on a variety of drug delivery platforms, including polymeric micelles, liposomes, dendrimers, and inorganic nanoparticles (Torchilin, 2006; Wagner et al., 2006; Sercombe et al., 2015). Despite the arsenal of nanoparticulate targeted systems currently under preclinical development or in clinical trials, it is indisputable that liposomes are dominant on the NNM market (Table 1) and were the ﬁrst FDA-approved NNM (Caster et al., 2017; Shi et al., 2017). In fact, liposomes have all the necessary features to allow formulation of highly toxic and/or poorly soluble drugs, such as paclitaxel and amphotericin B (Min et al., 2015; Caster et al., 2017). Soon after their discovery in 1965 (Sessa and Weissmann, 1968; Deamer, 2010), liposomes were proposed as drug delivery vehicles for both small molecules as well as macromolecular drugs (Gregoriadis and Ryman, 1971; Gregoriadis et al., 1971). Years of research led to the development of the ﬁrst FDA-approved NNM (Doxil R⃝/Caelyx R⃝) as well as additional therapeutics (Allen and Cullis, 2013). Expectedly, many more NNMs are progressing to clinical investigation From the outset in NNM development, it is essential to consider the relationship between disease pathophysiology and the heterogeneity of the disease in humans, and the importance of physicochemical characteristics of diﬀerent NNMs to overcoming biological barriers to enable improved targeting to diseased tissue and/or reduced accumulation in non-target organs. Considerably less research eﬀort has been dedicated to comprehensively understanding the correlations between NNM behavior and patient biology in speciﬁc clinical applications as well as disease heterogeneity in patients—which are likely Frontiers in Pharmacology \| www.frontiersin.org July 2018 \| Volume 9 \| Article 790 4 Clinical Translation of Nanomedicines Hua et al. TABLE 1 \| NNM formulations currently approved for marketing. NNMs Approved and in Clinical Trials Type Name Drug Indication Liposomal NNMs Doxil/Caelyx Doxorubicin HIV-related Kaposi’s Sarcoma, metastatic breast cancer, advanced ovarian cancer, multiple myeloma AmBisome Amphotericin B Fungal infections DaunoXome Daunorubicin HIV-related Kaposi’s Sarcoma Myocet Doxorubicin Metastatic breast cancer Abelcet Amphotericin B Fungal infections Lipo-Dox Doxorubicin HIV-related Kaposi’s Sarcoma, ovarian cancer, multiple myeloma Marqibo (Onco-TCS) Vincristine Adult AML Onivyde Irinotecan Pancreatic cancer Vyxeos (CPX-351) Cytarabine and daunorubicin AML Visudyne Verteporﬁn Wet AMD, myopia, ocular histoplasmosis DepoDur Morphine Postoperative analgesia DepoCyt Cytarabine Lymphomatous meningitis Micellar NNMs Genexol PM Paclitaxel Metastatic breast cancer, advanced lung cancer Nanoxel M Paclitaxel Advanced NSCLC, breast cancer, pancreatic cancer, ovarian cancer Protein NNMs Abraxane Paclitaxel Breast cancer, NSCLC, pancreatic cancer (Ref: ema.europa.eu; drugs.com; fda.gov). TABLE 1 \| NNM formulations currently approved for marketing. (Ref: ema.europa.eu; drugs.com; fda.gov). the major reasons for the failure seen in the translation of promising NNMs in clinical trials (Hare et al., 2017). These biological challenges can be a signiﬁcant deterrent for pharmaceutical industry investment into nanomedicines. In order to reduce investment risk for NNMs, the preclinical data sets need to comprehensively evaluate therapeutic eﬃcacy, safety, biodistribution, and pharmacokinetics in appropriate animal models of the disease that are relevant to human disease. Evaluation of NNMs in multiple preclinical animal models that represent aspects of the clinical disease is preferred to achieve reproducibility of results for the speciﬁc disease and not for a speciﬁc animal model. In addition, animal models that reﬂect only a narrow spectrum of the clinical disease may provide useful data that can predict their suitability for treating a speciﬁc patient sub-group (Hare et al., 2017). Diﬀerences in the anatomy and/or physiology of the animal species compared to humans should be taken into account based on diﬀerent routes of administration. Preclinical studies of NNMs should also be conducted under appropriate randomization and blinding to reduce bias, as well be evaluated against proper controls, including the gold standard treatment and not just free drug solution. These factors are currently lacking in many published studies, which makes it diﬃcult to assess clinical applicability and translatability. Other considerations include designing preclinical studies to optimize NNM performance in vivo, dosing schedules, and treatment combinations based on the speciﬁc clinical disease, as well as understanding the inﬂuence of disease progression and severity on nanomedicine performance. This will determine whether speciﬁc patient sub-groups may respond more favorably to NNM-based treatment. in the last two decades alone (Park, 2017). Frontiers in Pharmacology \| www.frontiersin.org NNMs Approved and in Clinical Trials Despite the large number of publications, the translation of the published studies to clinical applications has been disappointing. Cancer targeting of NNMs has generally been universally based on the EPR eﬀect, despite the fact that EPR-mediated accumulation has only been reported for some tumor types (Maeda, 2015). Tumors, like other clinical diseases, can be highly heterogeneous and can show inter-patient and intra-patient variability as the disease progresses. Hence a one-size-ﬁts-all approach when designing NNM-based treatment is unlikely to translate to clinically beneﬁcial outcomes. The EPR eﬀect has increasingly been exploited for NNM targeting in other non-cancer conditions, especially those involving an inﬂammatory component that causes leakiness of inﬂamed blood vessels (e.g., rheumatoid arthritis, atherosclerosis, and inﬂammatory bowel disease; Metselaar et al., 2003, 2004; Maiseyeu et al., 2009; Crielaard et al., 2012; Hua and Cabot, 2013; Hua et al., 2015; Milane and Amiji, 2017). It should be appreciated that not all diseases can be accessed with NNMs due to biological barriers and that the EPR eﬀect is unlikely to be present in all clinical diseases. EPR is also not the only determinant of NNM eﬃcacy. NNM activity is also inﬂuenced by the extent of cellular uptake and kinetics of drug release within target tissues (Hare et al., 2017). the major reasons for the failure seen in the translation of promising NNMs in clinical trials (Hare et al., 2017). These biological challenges can be a signiﬁcant deterrent for pharmaceutical industry investment into nanomedicines. In order to reduce investment risk for NNMs, the preclinical data sets need to comprehensively evaluate therapeutic eﬃcacy, safety, biodistribution, and pharmacokinetics in appropriate animal models of the disease that are relevant to human disease. Evaluation of NNMs in multiple preclinical animal models that represent aspects of the clinical disease is preferred to achieve reproducibility of results for the speciﬁc disease and not for a speciﬁc animal model. In addition, animal models that reﬂect only a narrow spectrum of the clinical disease may provide useful data that can predict their suitability for treating a speciﬁc patient sub-group (Hare et al., 2017). Diﬀerences in the anatomy and/or physiology of the animal species compared to humans should be taken into account based on diﬀerent routes of administration. Preclinical studies of NNMs should also be conducted under appropriate randomization and blinding to reduce bias, as well be evaluated against proper controls, including the gold standard treatment and not just free drug solution. NNMs Approved and in Clinical Trials These factors are currently lacking in many published studies, which makes it diﬃcult to assess clinical applicability and translatability. Other considerations include designing preclinical studies to optimize NNM performance in vivo, dosing schedules, and treatment combinations based on the speciﬁc clinical disease, as well as understanding the inﬂuence of disease progression and severity on nanomedicine performance. This will determine whether speciﬁc patient sub-groups may respond more favorably to NNM-based treatment. Furthermore, the advantages of ligand-targeted NNMs in the clinical research phase have so far been negligible, despite the enhanced accumulation in target sites and therapeutic outcomes in a number of preclinical studies (Sercombe et al., 2015). Potential reasons for this discrepancy have previously been reviewed (Sawant and Torchilin, 2012; Allen and Cullis, 2013), and include factors such as target accessibility and expression, disease-dependent anatomical and physiological barriers, and formulation stability. In addition, the optimal targeting ligand density on the surface of each NNM has yet Interestingly, the majority of the NNM formulations in development and clinical trials are focused on cancer targeting, including more than 80% of the publications on nanomedicine July 2018 \| Volume 9 \| Article 790 5 Clinical Translation of Nanomedicines Hua et al. TABLE 2 \| NNM formulations in clinical trials. NNMs Approved and in Clinical Trials Type Name Drug Indication Status Lipid NNMs LiPlaCis Cisplatin Advanced or refractory solid tumors, metastatic breast cancer and skin cancer Phase I/II ThermoDox Doxorubicin Hepatocellular carcinoma, breast cancer Phase I/IIIII 9NC-LP 9-Nitro-20 (S)-Camptothecin Ewing’s sarcoma and other solid tumors with lung involvement, endometrial cancer Phase I/II completed SPI-077 Cisplatin Ovarian cancer, relapsed/progressive osteosarcoma metastatic to the lung Phase I/ II/ III Lipoxal Oxaliplatin Colorectal cancer, glioma Phase II EndoTAG-1 Paclitaxel Pancreatic cancer, liver metastases, HER2 and triple negative breast cancer Phase II completed OSI-211 Lurtotecan SCLC Phase I/II completed LE-DT Docetaxel Solid tumors, pancreatic cancer Phase I/II completed LEP-ETU Paclitaxel Breast cancer, neoplasm, gastric carcinoma Phase I/II/IV TKM-080301 siRNA against PLK1 Advanced hepatocellular carcinoma, solid tumors or lymphomas that are refractory to conventional therapies; colorectal, gastric, breast and ovarian cancers with hepatic metastases Phase I/II completed Atu027 siRNA against PKN3 Advanced solid tumors, pancreatic cancer Phase I/II completed 2B3-101 Doxorubicin Advanced solid tumors, brain metastases, lung and breast cancers, melanoma, malignant glioma Phase I/II completed MTL-CEBPA saRNA Liver cancer Phase I TLI Topotecan SCLC, ovarian cancer, solid tumors Phase I MM-398 Onivyde Irinotecan Solid tumors, ER/PR positive and triple negative breast cancer, metastatic breast cancer with active brain metastasis, SCLC, metastatic pancreatic cancer Phase I/II/III MM-302 Doxorubicin Breast cancer Phase I ATI-1123 Docetaxel Advanced solid tumors Phase I completed SGT-53 p53 pDNA Solid tumors, recurrent glioblastoma Phase I/II SGT-94 RB94 pDNA Solid tumors, recurrent glioblastoma Phase I, Phase II Anti-EGFR-IL-DOX Doxorubicin Solid tumors Phase II RNL Rhenium-186 Glioblastoma and astrocytoma (treatment and imaging) Phase I/II Patisiran siRNA TTR-mediated amyloidosis Phase I/II/III Polymeric NNMs Paclical Paclitaxel Ovarian cancer Phase III completed NK105 Paclitaxel Gastric cancer Phase III completed BIND-014 Docetaxel NSCLC, solid tumors Phase II completed CALAA-01 RRM2 siRNA Solid tumors Phase II terminated CRLX101 Camptothecin NSCLC Phase II completed (R f li i lt i l ) TABLE 2 \| NNM formulations in clinical trials. Large-Scale Manufacturing Frontiers in Pharmacology \| www.frontiersin.org Large-Scale Manufacturing to be determined and will likely depend on characteristics of the molecular target (e.g., expression, location, internalization rate and immunogenicity; Puri et al., 2009; Hua and Wu, 2013; Kraft et al., 2014). Detailed analysis of the degree of NNM accumulation, cellular internalization, intracellular functionality and intracellular degradation will also be important considerations for clinical validation and translation (Puri et al., 2009). Through extensive experimentation, we are gaining a better understanding of the more appropriate clinical applications for ligand-targeted NNMs. Therefore, by taking a disease-driven approach to NNM development, it will be possible to build comprehensive preclinical data sets that best predict eﬃcacy for patient sub-groups and support translatable clinical development. to be determined and will likely depend on characteristics of the molecular target (e.g., expression, location, internalization rate and immunogenicity; Puri et al., 2009; Hua and Wu, 2013; Kraft et al., 2014). Detailed analysis of the degree of NNM accumulation, cellular internalization, intracellular functionality and intracellular degradation will also be important considerations for clinical validation and translation (Puri et al., 2009). Through extensive experimentation, we are gaining a better understanding of the more appropriate clinical applications for ligand-targeted NNMs. Therefore, by taking a disease-driven approach to NNM development, it will be possible to build comprehensive preclinical data sets that best predict eﬃcacy for patient sub-groups and support translatable clinical development. One of the important factors contributing to the slow pace in the clinical translation of NNMs is the structural and physicochemical complexity of the formulation itself. Platforms that require complex and/or laborious synthesis procedures generally have limited clinical translation potential, as they can be quite problematic to pharmaceutically manufacture on a large-scale (Teli et al., 2010; Tinkle et al., 2014; Barz et al., 2015; Sainz et al., 2015). Pharmaceutical manufacturing development is centered on quality and cost. Quality includes the manufacturing process and stability of the formulation, with NNM manufacturing being challenged by potential issues related to: (i) poor quality control; (ii) scalability complexities; (iii) incomplete puriﬁcation from contaminants (e.g., by-products July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 6 Clinical Translation of Nanomedicines Hua et al. TABLE 3 \| Considerations for the translational development of nanomedicines. without the need for numerous manufacturing steps or the use of organic solvents (Jaafar-Maalej et al., 2012; Kraft et al., 2014). The challenges arise when the NNM system becomes more complex. Key Considerations Simpliﬁcation of development pathways from invention to commercialization to minimize time and expense p Evaluation of safety/toxicity in humans (acute and chronic) Evaluation of therapeutic efﬁcacy in patients Key Considerations Need for validated and standardized assays for early detection of toxicity Evaluation in appropriate animal models of disease Evaluation in appropriate animal models of disease Adequate understanding of in vivo behavior, incl. cellular and molecular interactions ◦Pharmacokinetics (absorption, distribution, metabolism and excretion) ◦Pharmacodynamics (intracellular trafﬁcking, functionality, toxicity and degradation) Current Obstacles Lack of clear regulatory guidelines speciﬁc for NNMs Complexity of NNM patents and IP Limited understanding of the biological interaction of NNM with the biological environment (incl. target site) in the body of patients Limited understanding of the biological interaction of NNM with the biological environment (incl. target site) in the body of patients and starting materials); (iv) high material and/or manufacturing costs; (v) low production yield; (vi) insuﬃcient batch-to-batch reproducibility, consistency and storage stability of the ﬁnal product (e.g., regarding size distribution, porosity, charge and mass); (vii) lack of infrastructure and/or in-house expertise; (viii) chemical instability or denaturation of the encapsulated compound during the manufacturing process; and (ix) scarcity of venture funds and pharmaceutical industry investment (Teli et al., 2010; Narang et al., 2013; Hafner et al., 2014; Tinkle et al., 2014). Large-Scale Manufacturing For example, with the addition of surface modiﬁcation with coatings and/or ligands, inclusion of multiple targeting components, or by encapsulating more than one therapeutic agent. Integration of multiple components into a single nanosized carrier requires multiple steps in the production process, which inevitably poses problems for large- scale good manufacturing (cGMP) production, increases the cost of production, and makes the quality assurance and quality control (QA and QC) evaluation of such products more diﬃcult (Teli et al., 2010; Svenson, 2012; Tinkle et al., 2014). TABLE 3 \| Considerations for the translational development of nanomedicines. NANOPHARMACEUTICAL DESIGN Key Considerations Route of administration Reduce complexity in formulation design Final dosage form for human use Biocompatibility and biodegradability Pharmaceutical stability (physical and chemical) Current Obstacles Large-scale production according to GMP standards ◦E.g., Reproducibility, infrastructure, techniques, expertise and cost Quality control assays for characterization ◦E.g., Size and polydispersity, morphology, charge, encapsulation, surface modiﬁcations, purity and stability PRECLINICAL EVALUATION Key Considerations Need for validated and standardized assays for early detection of toxicity Evaluation in appropriate animal models of disease Adequate understanding of in vivo behavior, incl. cellular and molecular interactions ◦Pharmacokinetics (absorption, distribution, metabolism and excretion) ◦Pharmacodynamics (intracellular trafﬁcking, functionality, toxicity and degradation) Current Obstacles Development of more specialized toxicology studies for nanomedicines Adequate understanding of the interaction of NNM with tissues and cells Adequate structural stability of NNM following in vivo administration Limited degree of accumulation of nanomedicines in target organs/tissues/cells CLINICAL EVALUATION FOR COMMERCIALIZATION Key Considerations Simpliﬁcation of development pathways from invention to commercialization to minimize time and expense Evaluation of safety/toxicity in humans (acute and chronic) Evaluation of therapeutic efﬁcacy in patients Optimal clinical trial design Current Obstacles Lack of clear regulatory guidelines speciﬁc for NNMs Complexity of NNM patents and IP Limited understanding of the biological interaction of NNM with the biological environment (incl. target site) in the body of patients and starting materials); (iv) high material and/or manufacturing costs; (v) low production yield; (vi) insuﬃcient batch-to-batch reproducibility, consistency and storage stability of the ﬁnal product (e.g., regarding size distribution, porosity, charge and mass); (vii) lack of infrastructure and/or in-house expertise; Frontiers in Pharmacology \| www.frontiersin.org Current Obstacles Large-scale production according to GMP standards ◦E.g., Size and polydispersity, morphology, charge, encapsulation, surface modiﬁcations, purity and stability Characteristics of the manufactured NNM need to be well- deﬁned and reproducibly generated to allow initiation of clinical translation. Chemistry, Manufacturing, and Controls (CMC) information is required for investigational new drugs (IND) at each phase of investigation to ensure proper identity, strength or potency, quality, and purity of the drug substance and drug product (FDA, 2003). The type of information submitted will depend on the phase of the investigation, the extent of the human study, the duration of the investigation, the nature and source of the drug substance, and the drug product dosage form (FDA, 2003). The characterization and validation of more complex NNMs can be particularly challenging due to the sheer number of parameters to address (e.g., size distribution, morphology, charge, purity, drug encapsulation eﬃciency, coating eﬃciency, and density of conjugated ligand/s; Teli et al., 2010). Batch-to-batch variation of NNMs can potentially lead to signiﬁcant changes to their physicochemical properties (e.g., polarity and size), pharmacokinetic parameters (i.e., absorption, distribution, metabolism and excretion), and/or pharmacodynamic interactions (e.g., cellular interaction and activity; Teli et al., 2010; Tinkle et al., 2014; Barz et al., 2015). In addition, NNMs need to be stable after the manufacturing process, during long-term storage, and upon clinical administration (i.e., to avoid massive drug release or aggregation in the bloodstream en route to the site of action). Current Obstacles q y g Limited degree of accumulation of nanomedicines in target organs/tissues/cells Biocompatibility and Safety p y y Detailed toxicology is essential for the clinical translation of NNMs to determine the overall safety for human use (Nystrom and Fadeel, 2012). Pharmaceutical regulatory authorities generally recommend that the sponsor carefully assess for any changes in the drug substance and drug product manufacturing process or drug product formulation at any phase of clinical development, in order to determine if the changes can directly or indirectly aﬀect the safety of the product. CMC modiﬁcations throughout the IND process that can aﬀect safety include: (i) changes in the synthetic pathway or reagents used to manufacture the drug substance, product or formulation; (ii) changes resulting in a diﬀerent impurity proﬁle; (iii) changes in the actual manufacturing method (e.g., chemical synthesis, fermentation, or derivation from a natural source); (iv) changes in the source material; (v) changes in the method of sterilization of the drug substance or drug product; (vi) changes in the An essential requirement for clinical translation is to have access to a preparation method that allows the production of large scalable quantities of NNMs, which is also consistently manufactured at the same high level of quality and batch- to-batch reproducibility to set speciﬁcations (Grainger, 2013; Lammers, 2013; Barz et al., 2015). Suitable methods for the industrial scale production of several basic nanomedicine platforms, such as liposomes, have been successfully developed Frontiers in Pharmacology \| www.frontiersin.org July 2018 \| Volume 9 \| Article 790 7 Clinical Translation of Nanomedicines Hua et al. testing are essential to screen for potential hazards prior to preclinical evaluation in animal models (Gaspar, 2007). For example, standardized in vitro protocols using diﬀerent cell culture models (i.e., blood, liver, lung, brain, placenta, gastrointestinal system) to assess potential risk of cytotoxicity, immunotoxicity, and genotoxicity of NNMs (Accomasso et al., 2018). This is particularly important with the development of NNMs incorporating many new materials with the goal for use in the clinical setting. In order to do this eﬀectively across the board, standardized reference materials would need to be established and the testing would also need to be relevant for the intended route of administration (Tinkle et al., 2014). Although current testing approaches are limited and insuﬃcient for nanotoxicology evaluations for clinical translation, a number of techniques that are more speciﬁc for nanomedicines are under development. Biocompatibility and Safety This includes alternative test strategies, high-throughput screening techniques, high-content screening, and computational modeling (Nel et al., 2013; Oomen et al., 2014; Dusinska et al., 2015; Accomasso et al., 2018). These techniques have the potential to analyze in a comparative way many NNMs simultaneously. route of administration; (vii) changes in the composition and/or dosage form of the drug product; (viii) changes in the drug product manufacturing process that can aﬀect product quality; and (ix) changes in the drug product container closure system that can aﬀect product quality (e.g., dose delivery; FDA, 2003). If any changes are identiﬁed, stringent procedures are in place to ensure appropriate comparison testing of the drug substance and/or drug product produced from the previous manufacturing process with the changed manufacturing process to evaluate product equivalency, quality, and safety (FDA, 2003). When analytical data demonstrate that the materials manufactured before and after are not comparable, sponsors should perform additional qualiﬁcation and/or bridging studies to support the safety and bioavailability of the material to be used in the proposed trials (FDA, 2003). Knowledge of the activity and toxicities of the free drug, the behavior of diﬀerent NNM delivery systems and their interaction with biological components, and the inﬂuence of drug release rate on target and oﬀ-target concentrations of bioavailable drug allow the ability to predict potential side eﬀects or toxicities in vivo (Hare et al., 2017). In particular, the rational design of NNMs from the early phase of material selection, production method optimization, and product puriﬁcation is of fundamental importance to increase their clinical translation potential (Accomasso et al., 2018). Although the safety of some common materials such as phospholipids and biodegradable polymers have been studied previously (Storm et al., 1993), increasing the complexity of NNMs, such as the use of diﬀerent synthetic compositions, coatings and ligands, can have a signiﬁcant eﬀect on the biocompatibility, biodistribution and toxicology proﬁle of nanomedicines following in vivo administration (Allen and Cullis, 2004, 2013; Zhang et al., 2008; Sawant and Torchilin, 2012; Narang et al., 2013; Tinkle et al., 2014). For example, complement activation-related pseudoallergy (CARPA) is an acute adverse immune reaction caused by many NNMs (Szebeni, 2005; Sercombe et al., 2015; Szebeni and Storm, 2015; Jackman et al., 2016). The complement system is part of the innate immune response and is involved in a range of inﬂammatory and immunological processes (Moghimi and Hunter, 2001). Biocompatibility and Safety CARPA is an immediate, non-IgE-mediated hypersensitivity reaction that can cause symptoms, including anaphylaxis, facial swelling, facial ﬂushing, chills, headache, and cardiopulmonary distress (Szebeni, 2005). This adverse reaction is generally managed by slowing the infusion rate or ceasing therapy, as well as the use of standard allergy medications (e.g., antihistamines, corticosteroids and epinephrine; Sercombe et al., 2015; Szebeni and Storm, 2015). The development of immunogenic reactions to NNM-based therapies may lead to altered pharmacokinetics, loss of eﬃcacy, and the rise of potentially serious toxicities (e.g., anaphylaxis; Szebeni and Moghimi, 2009; Szebeni and Storm, 2015). There is also a need to perform specialized toxicology studies in animal models to assess both short-term and long- term toxicity, as circulation half-lives and drug retention times are generally signiﬁcantly increased with nanoencapsulation. A thorough understanding of the absorption, distribution, metabolism, and excretion of emerging nanomaterials in vivo is important to predict the toxicological responses to NNMs (Dobrovolskaia and McNeil, 2013; Tinkle et al., 2014). Adequate assessment protocols are needed to monitor various aspects of the NNM drug delivery process, including pharmacokinetics, biodistribution, target site accumulation, local distribution at the target site, localization in healthy tissues, kinetics of drug release, and therapeutic eﬃcacy (Kunjachan et al., 2015). Incorporation of real-time imaging techniques have enabled better understanding of the interaction of NNMs with biological organs and tissues following in vivo administration (Gaspar, 2007; Nystrom and Fadeel, 2012; Dobrovolskaia and McNeil, 2013; Kunjachan et al., 2015). In addition, biocompatibility, immunotoxicological, and inﬂammatory potential should be assessed, with functional outcomes correlated with mechanisms of tissue uptake and clearance (Gaspar, 2007). These parameters need to be well- investigated based on dose, dosage form and route of administration to establish safe limits prior to clinical trials (Gaspar, 2007; Nystrom and Fadeel, 2012). This is of particular importance for NNMs composed of materials that have never been used before in clinical applications. Even in the clinical trial phase, regulatory protocols should be in place to detect any toxicity caused not only by the encapsulated therapeutic compounds, but also novel mechanisms unique to nanotechnology (Gaspar, 2007; Nystrom and Fadeel, 2012). For example, short- and long-term eﬀects of NNM accumulation in RES organs (esp. Frontiers in Pharmacology \| www.frontiersin.org Intellectual Property (IP) Given the complexities of incorporating nanotechnology into biomedical and clinical applications, there needs to be more precise deﬁnitions on what constitutes novel IP of a nanomedicine (Satalkar et al., 2015). Nanomedicines are complex as they have a number of variable components, and bridge between the ﬁeld of medicine and medical device (Paradise et al., 2009). Generally, the control of a NNM product requires an IP position on: (i) the encapsulated cargo; (ii) the carrier technology; and (iii) the characteristics of the drug and carrier together. Although this deﬁnition is straightforward, it does open up a number of problems with the issuing of patents to date (Bawa, 2007; Bawa et al., 2008). For example, NNMs that incorporate existing drugs with novel carrier technology, or those that incorporate existing drugs with existing carrier technology for a new biomedical or disease application. The IP situation becomes even more confusing with more complex drug delivery systems, such as those which incorporate commercially available targeting ligands (e.g., antibodies) or coatings (e.g., Eudragit R⃝) that are owned by other companies. IP strategies may likely involve multiple patents associated with any given technology and the need for cross-licensing arrangements (Murday et al., 2009). Therefore, new IP practices and protocols are required to simplify the pathway from invention to commercialization to reduce the time and expense required for negotiating collaboration and licensing agreements (Murday et al., 2009). NNMs are currently regulated within the conventional framework governed by the key regulatory authority of each country (e.g., FDA, TGA, and EMA). Although NNMs have been on the market for nearly two decades, the ﬁrst generation of NNM products passed regulatory approval by only having to meet general standards, applicable to medicinal compounds. These regulations are no longer appropriate to conﬁrm the quality, safety, and eﬃcacy of NNMs for clinical use (Gaspar, 2007; Tinkle et al., 2014; Sainz et al., 2015). Reasons for this are based on the complex structure of NNMs, their unclear interaction with cells and tissues within the human body, increased complexity of clinical use, and the multifunctional nature of some formulations (e.g., integration of therapeutics with imaging diagnostics; Gaspar, 2007; Tinkle et al., 2014; Sainz et al., 2015). Regulatory standards and protocols validated speciﬁcally for nanoparticles are needed that bridge both medicine and medical devices regulations. Government Regulations g Nanomedicines have signiﬁcant potential to increase the growth of the pharmaceutical market and improve health beneﬁts, however the current scientiﬁc and regulatory gap for nanomedicines is large and challenging. Commercialization of nanomedicines is highly dependent on a number of regulatory factors based on government policies in the area of manufacturing practice, quality control, safety, and patent protection (Gaspar, 2007; Tinkle et al., 2014; Sainz et al., 2015). The lack of clear regulatory and safety guidelines has aﬀected the development of NNM products toward timely and eﬀective clinical translation (Gaspar, 2007; Tinkle et al., 2014; Sainz et al., 2015). For example, polymers have been widely investigated as an eﬀective platform for NNM strategies; however, their safety and eﬃcacy is highly dependent on the polymer molecular weight, polydispersity, molecular structure, and conjugation chemistry (Gaspar and Duncan, 2009; Diab et al., 2012). Due to the increased number of novel polymeric materials and complex polymeric-based NNM formulations, there is an urgent need for an appropriate regulatory framework to assist in evaluation (Gaspar and Duncan, 2009). As each polymer-based NNM is diﬀerent, it is important to consider each individually based on doses, administration routes, dosing frequency, and proposed clinical use. This would be the same for most other NNM platforms. Biocompatibility and Safety liver, kidneys, spleen, lungs, lymph nodes, and bone marrow; Senior, 1987; Szebeni and Barenholz, 2009; Szebeni and Moghimi, 2009), which are the main sites for NNM accumulation following systemic administration (Poste et al., There is a regulatory need for validated, sensitive and standardizable assays incorporating in vitro, ex vivo and in vivo protocols to appropriately assess the nanotoxicology of NNMs during the early stages of clinical development (Dobrovolskaia and McNeil, 2013; Jackman et al., 2016; Accomasso et al., 2018). Comprehensive in vitro or ex vivo assays for nanosafety Frontiers in Pharmacology \| www.frontiersin.org July 2018 \| Volume 9 \| Article 790 8 Clinical Translation of Nanomedicines Hua et al. 1976; Senior, 1987). The cells of the RES are also part of the innate immune system, which has raised concerns regarding whether macrophage saturation by NNMs can cause immunosuppression and increase the risk of infections (Sercombe et al., 2015; Liu et al., 2017). There have been no reports of clinically signiﬁcant immunosuppression at therapeutic doses of non-cytotoxic NNMs, despite suggestions that excessive NNM deposition in macrophages may impair their phagocytic capacity or modulate other cellular functions (Szebeni and Barenholz, 2009; Szebeni and Moghimi, 2009). However, NNMs that contain cytotoxic compounds are capable of inducing macrophage destruction following uptake (Szebeni and Barenholz, 2009; Szebeni and Moghimi, 2009), with indirect signs that suggest the possibility of some immune suppression (Storm et al., 1998; Szebeni and Barenholz, 2009; Szebeni and Moghimi, 2009). For example, administration of Doxil R⃝in mice was reported to interfere with the clearance of bacteria from the blood due to macrophage suppression (Storm et al., 1998; Szebeni and Barenholz, 2009). Addressing these issues are necessary to safeguard the application of emerging NNMs in the clinical setting. nanotech-related prior art that resided in scientiﬁc publications world-wide, including earlier publications that preceded the emergence of online publication databases (Tinkle et al., 2014). Patent examiners also require expertise and training with respect to the emerging ﬁelds of nanotechnology and nanomedicine. The complexities with nanotechnology have led to the so called “patent thickets”, which can lead to costly litigation and halt commercialization eﬀorts (Tinkle et al., 2014). Therefore, improved clarity on IP and patenting surrounding nanotechnology in health and medicine is required, and will need to involve implementation of universal regulations and policies that are tailored toward this niche commercialization ﬁeld. Frontiers in Pharmacology \| www.frontiersin.org Intellectual Property (IP) This is of particular importance due to the limited availability of contract manufacturing organizations world-wide that specialize in producing NNM products in accordance with the requirements for good manufacturing practice (GMP; Hafner et al., 2014). It should be noted that this limited number of manufacturing organizations may be further divided based on their infrastructure capabilities of producing speciﬁc NNM platforms (e.g., liposomes, polymeric nanoparticles, dendrimers and drug-polymer conjugates). Therefore, NNMs produced in these manufacturing organizations will likely be marketed in multiple countries and thus should be governed under the same regulatory standards (Hafner et al., 2014). There will need to be complete evaluation and documentation of production processes for NNMs, incorporating appropriate industrial standards for both quality control and prevention of environmental issues (Gaspar, 2007). Manufactured NNMs will still need to meet general pharmaceutical standards such as purity, sterility, stability, manufacturing operations, and related industrial control standards (Gaspar, 2007). In addition, new analytical tools and standardized methods will need to be implemented to evaluate key physical characteristics of NNMs that can aﬀect in vivo performance such as particle size and size distribution, surface chemistry, morphology, surface area, surface coating, hydrophilicity, porosity, and surface charge density (Gaspar, 2007; Tinkle et al., 2014; Sainz et al., 2015). These methods will vary for diﬀerent nanomaterials and nanostructures. Thus, regulatory authorities should work together to develop the testing methods and appropriate standardized protocols for toxicity studies and regulatory requirements, which will be needed to ensure the eﬃcacy and safety of current and emerging NNMs Complexity in dosage form design is a key factor in the ability for a NNM formulation to be translated to the clinic, irrelevant of its therapeutic eﬃcacy. Simpliﬁcation in formulation design is required to allow eﬃcient and reproducible large- scale manufacturing (Grainger, 2013; Lammers, 2013; Barz et al., 2015). Any added complexities to the basic NNM platform would need to show signiﬁcantly improved beneﬁts that is reliable and reproducible in animal models and patients, due to the added costs and complexity in the manufacturing process. For example, further studies are required to examine the beneﬁts of ligand-targeted delivery systems over basic NNM platforms, in particular the reliability and consistency of the expression of the target across disease severity and in diﬀerent patients (Hua et al., 2015; Sercombe et al., 2015; Hare et al., 2017). Intellectual Property (IP) This should take into account a NNM’s complexity, route of administration, pharmacokinetics, pharmacodynamics and safety proﬁle, as well With the signiﬁcant increase in the number of nanotechnology patent applications over the last few decades, other key issues that need to be addressed include patent review delays, patent thickets, and issuance of invalid patents (Bawa, 2005, 2007; Bawa et al., 2005). There needs to be a universal nano-nomenclature on identical or similar nanostructures or nanomaterials, and more reﬁned search tools and commercial databases to avoid the issuing of multiple nanopatents on the same invention (Bawa et al., 2005; Bawa, 2007). Databases used by the Patent and Trademark Oﬃce (PTO) need to be able to search through Frontiers in Pharmacology \| www.frontiersin.org July 2018 \| Volume 9 \| Article 790 9 Clinical Translation of Nanomedicines Hua et al. results to warrant progression in the R&D process (Hua et al., 2015). Eﬃcacy in an animal model also does not necessarily equate to eﬃcacy in humans, as drug delivery within the human body is complex and can be highly variable, especially when associated with disease (Hare et al., 2017). Therefore, this concept of designing nanomedicines that act like a “magic bullet,” which refers to the exclusive delivery of active compounds to speciﬁc organs, tissues or cells, is just not realistic when taking into account the pharmacokinetic and pharmacodynamic processes that occur following administration into the body (Barz et al., 2015). This term should refer to the development of realistic therapeutic platforms, in which therapeutic eﬀects are maximized, doses are minimized, and complexity in dosage form design is reduced (Barz et al., 2015). as provide information on the most appropriate clinical trial design and patient selection (Tinkle et al., 2014). There needs to be a ﬁne balance to ensure the safety and quality of NNMs without over-regulation, which can negatively aﬀect the progress of innovative products to the market, by escalating costs for achieving regulatory approval and/or consuming a signiﬁcant portion of the life of a patent. Development of global regulatory standards for NNMs should be established alongside key countries with invested interest. Although major steps have been taken in the last 5 years, a closer collaboration between regulatory agencies, academia, research and industry is needed (Gaspar, 2007; Murday et al., 2009; Hafner et al., 2014). Intellectual Property (IP) In addition, when translating ﬁndings from animal models to humans, we need to determine how to modify these formulations so that they are appropriate for human administration (Hua et al., 2015). In vivo studies are typically conducted in animal models of experimental diseases, especially in mice and rats, which can place limitations on the size and consistency of the dosage form that can be administered—for example, via oral, topical or intraperitoneal delivery (Hua et al., 2015; Sercombe et al., 2015). The practicability of designing dosage forms that are both acceptable to humans and eﬃcacious should be further explored for clinical studies. Thus, there needs to be a balance between complexity, therapeutic eﬃcacy, and clinical translation. To transition NNMs to the clinic, attention should be given to nanosized carriers that are stable following in vivo administration, easily able to be up-scaled for manufacturing with high control over their physicochemical properties (e.g., size and polydispersity, morphology, drug encapsulation eﬃciency, and charge), as well as being composed of materials that are biocompatible, biodegradable, and non-toxic. As nanoparticles are able to enter cells and interfere with molecular pathways, synthetic polymers and lipids should be carefully evaluated for potential short-term and long-term toxicity for clinical application (Gaspar and Duncan, 2009). For example, potentially toxic in vitro and in vivo eﬀects have been identiﬁed with the use of cationic polymers and lipids, including reduced number of mitoses, cell shrinking, detrimental eﬀects on key cellular proteins (e.g., protein kinase C), and vacuolization of the cytoplasm (Lv et al., 2006). PATHWAY TO TRANSLATION AND COMMERCIALIZATION Nanomedicines generally face a number of regulatory approval hurdles. The control of materials in the nanosize range often presents greater scientiﬁc and technical challenges compared to conventional formulations (Gaspar, 2007; Teli et al., 2010; Hafner et al., 2014; Tinkle et al., 2014; Sainz et al., 2015). NNMs encompass a number of diﬀerent types of nanomaterials and nanostructures, which make it even more challenging to establish appropriate regulatory protocols and tools to ensure standardized GMP manufacturing and characterization, safety and toxicology evaluation, and clinical trial design. These procedures are paramount to conﬁrming therapeutic eﬃcacy and safety prior to marketing approval for use in patients on a larger scale. Eﬀective clinical translation will require an interdisciplinary approach to develop novel protocols, assays and infrastructure for the manufacturing and characterization of NNMs (Gaspar, 2007; Teli et al., 2010; Hafner et al., 2014; Tinkle et al., 2014; Sainz et al., 2015). This will need to involve experts from academia and industry with specialty in pharmaceutics, engineering, biology, medicine, and toxicology. Potential approaches to fast-track promising novel NNMs to clinical trials include the establishment or coordination of laboratories and centers that have expertise in (i) characterizing NNM platforms, (ii) conducting preclinical studies on NNMs for submission to regulatory agencies, (iii) scale up laboratory preparation of nanomaterials according to regulatory and industry standards for early clinical trials, and (iv) designing and conducting clinical trials of NNM platforms (Hafner et al., 2014). The experimental development of NNMs is progressing at a fast pace, however signiﬁcant challenges still exist in promoting these platforms into clinically feasible therapies (Table 3). The majority of NNMs in the clinic are for the treatment of cancer, predominantly by the parenteral route of administration. They are structurally based on simple nanomedicine platforms, in particular basic nanoparticles, surface charge-modiﬁed nanoparticles, and PEGylated nanoparticles (Hafner et al., 2014; Sainz et al., 2015). Although clinical applications of nanotechnology for non-cancer diseases are increasing based on promising experimental results, there are several barriers that have slowed progress in the preclinical and, especially, clinical stages of development. This includes issues surrounding complexity in manufacturing and characterization, lack of understanding of in vivo pharmacokinetics and pharmacodynamics, acute and chronic toxicity, and cost- eﬀectiveness (Gaspar, 2007; Teli et al., 2010; Hafner et al., 2014; Tinkle et al., 2014; Sainz et al., 2015). These challenges are even greater with increasing complexity of the NNM design. PERSPECTIVES ON THE TRANSLATIONAL DEVELOPMENT OF NANOMEDICINES From a therapeutic perspective, increasing drug accumulation at target tissues and minimizing systemic adverse eﬀects are still the biggest design challenges to meet when developing new drug delivery systems. Even though promising NNMs may demonstrate signiﬁcant eﬃcacy in in vitro or ex vivo studies, it is important to evaluate the platforms in vivo using appropriate animal models of the disease. It is here where many of the current NNM platforms have shown limited speciﬁcity, accumulation and/or stability, therefore providing unsatisfactory July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 10 Clinical Translation of Nanomedicines Hua et al. PATHWAY TO TRANSLATION AND COMMERCIALIZATION g g p y g The pace for the clinical translation of NNMs has been relatively slow as the development trajectory is very costly, complex and time-consuming, which has aﬀected the attitudes of the pharmaceutical industry and capital investors. There has to be a clear positive beneﬁt-to-risk ratio that will accompany the clinical implementation of products and procedures based on nanotechnology. In particular, the cost-beneﬁt analysis may be a limitation to the clinical translation of some NNMs when compared to an approved counterpart or existing therapies. This analysis has to be clear before starting the development process. Emerging NNM products, which are more complex in structure and more expensive than conventional therapies, need to provide an overall reduction in health care costs and provide a worthwhile opportunity for the pharmaceutical industry to invest its R&D budgets (Hafner et al., 2014). This reduction in health care costs is likely to be obtained by increasing therapeutic eﬃcacy, improving quality of life, reducing adverse eﬀects or toxicities in non-target organs, and/or reducing the need for surgical or other high-risk interventions (Gandjour and Chernyak, 2011). Nanopharmaceuticals can oﬀer the ability to extend the economic life of proprietary drugs and create additional revenue streams (Tinkle et al., 2014). In addition, market analysis, investment risk, potential proﬁt margins, and value proposition of novel NNMs are important factors for the pharmaceutical industry and investors. Typically, pharmaceutical products that are developed to address larger disease populations with treatment expected in a primary or secondary care setting are preferred by the pharmaceutical industry. From a business perspective, the necessary infrastructure, understanding of NNMs, and skill set required for the commercial development of NNMs are not currently well represented at most pharmaceutical companies. These factors should be taken into account when assessing the overall cost-eﬀectiveness of NNMs in comparison to existing therapies. Frontiers in Pharmacology \| www.frontiersin.org REFERENCES nanomedicine: lessons from NanoTEST. Nanotoxicology 9 (Suppl. 1), 118–132. doi: 10.3109/17435390.2014.991431 nanomedicine: lessons from NanoTEST. Nanotoxicology 9 (Suppl. 1), 118–132. doi: 10.3109/17435390.2014.991431 Accomasso, L., Cristallini, C., and Giachino, C. (2018). Risk Assessment and Risk Minimization in Nanomedicine: A Need for Predictive, Alternative, and 3Rs Strategies. Frontiers in Pharmacology. 9:228. doi: 10.3389/fphar.2018.00228 FDA (2003).Guidance for Industry - INDs for Phase 2 and Phase 3 Studies: Chemistry, Manufacturing, and Control (CMC) Information Ferrari, M. (2005). Nanovector therapeutics. Curr. Opin. Chem. Biol. 9, 343–346. doi: 10.1016/j.cbpa.2005.06.001 Allen, T. M., and Cullis, P. R. (2004). Drug delivery systems: entering the mainstream. Science 303, 1818–1822. doi: 10.1126/science.1095833 Gandjour, A., and Chernyak, N. (2011). A new prize system for drug innovation. Health Policy. 102, 170–177. doi: 10.1016/j.healthpol.2011.06.001 Allen, T. M., and Cullis, P. R. (2013). Liposomal drug delivery systems: from concept to clinical applications. Adv. Drug Del. Rev. 65, 36–48. doi: 10.1016/j.addr.2012.09.037 Gaspar, R. (2007). Regulatory issues surrounding nanomedicines: setting the scene for the next generation of nanopharmaceuticals. Nanomedicine 2, 143–147. doi: 10.2217/17435889.2.2.143 Barz, M., Luxenhofer, R., and Schillmeier, M. (2015). Quo vadis nanomedicine? Nanomedicine 10, 3089–3091. doi: 10.2217/nnm.15.156 Gaspar, R., and Duncan, R. (2009). Polymeric carriers: preclinical safety and the regulatory implications for design and development of polymer therapeutics. Adv. Drug Deliv. Rev. 61, 1220–1231. doi: 10.1016/j.addr.2009.06.003 Bawa, R. (2005). Will the nanomedicine “patent land grab” thwart commercialization? Nanomedicine 1, 346–350. doi: 10.1016/j.nano.2005.10.009 Bawa, R. (2007). Patents and nanomedicine. Nanomedicine 2, 351–374. doi: 10.2217/17435889.2.3.351 Grainger, D. W. (2013). Connecting drug delivery reality to smart materials design. Int. J. Pharm. 454, 521–524. doi: 10.1016/j.ijpharm.2013.0 4.061 Bawa, R., Bawa, S. R., Maebius, S. B., Flynn, T., and Wei, C. (2005). Protecting new ideas and inventions in nanomedicine with patents. Nanomedicine 1, 150–158. doi: 10.1016/j.nano.2005.03.009 Gregoriadis, G., Leathwood, P. D., and Ryman, B. E. (1971). Enzyme entrapment in liposomes. FEBS Lett. 14, 95–99. doi: 10.1016/0014-5793(71)80 109-6 Bawa, R., Melethil, S., Simmons, W. J., and Harris, D. (2008). Nanopharmaceuticals - patenting issues and FDA regulatory challenges. Am. Bar Assoc. SciTech Lawyer. 5, 10–15. Gregoriadis, G., and Ryman, B. E. (1971). Liposomes as carriers of enzymes or drugs: a new approach to the treatment of storage diseases. Biochem. J. 124:58P. doi: 10.1042/bj1240058P Bertrand, N., Wu, J., Xu, X., Kamaly, N., and Farokhzad, O. C. (2014). Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv. Drug Deliv. Rev. 66, 2–25. REFERENCES doi: 10.1016/j.addr.2013.11.009 Hafner, A., Lovric, J., Lakos, G. P., and Pepic, I. (2014). Nanotherapeutics in the EU: an overview on current state and future directions. Int. J. Nanomedicine. 9, 1005–1023. doi: 10.2147/IJN.S55359 Calcagno, C., Lobatto, M. E., Dyvorne, H., Robson, P. M., Millon, A., Senders, M. L., et al. (2015). Three-dimensional dynamic contrast-enhanced MRI for the accurate, extensive quantiﬁcation of microvascular permeability in atherosclerotic plaques. NMR Biomed. 28, 1304–1314. doi: 10.1002/nbm.3369 Hare, J. I., Lammers, T., Ashford, M. B., Puri, S., Storm, G., and Barry, S. T. (2017). Challenges and strategies in anti-cancer nanomedicine development: an industry perspective. Adv. Drug. Deliv. Rev. 108, 25–38. doi: 10.1016/j.addr.2016.04.025 Caster, J. M., Patel, A. N., Zhang, T., and Wang, A. (2017). Investigational nanomedicines in 2016: a review of nanotherapeutics currently undergoing clinical trials. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 9:e1416. doi: 10.1002/wnan.1416 Hashizume, H., Baluk, P., Morikawa, S., McLean, J. W., Thurston, G., Roberge, S., et al. (2000). Openings between defective endothelial cells explain tumor vessel leakiness. Am. J. Pathol. 156, 1363–1380. doi: 10.1016/S0002-9440(10)65006-7 Coimbra, M., Banciu, M., Fens, M. H., de Smet, L., Cabaj, M., Metselaar, J. M., et al. (2010). Liposomal pravastatin inhibits tumor growth by targeting cancer-related inﬂammation. J. Control Release. 148, 303–310. doi: 10.1016/j.jconrel.2010.09.011 Hobbs, S. K., Monsky, W. L., Yuan, F., Roberts, W. G., Griﬃth, L., Torchilin, V. P., et al. (1998). Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc. Natl. Acad. Sci. U.S.A. 95, 4607–4612. doi: 10.1073/pnas.95.8.4607 Crielaard, B. J., Lammers, T., Schiﬀelers, R. M., and Storm, G. (2012). Drug targeting systems for inﬂammatory disease: one for all, all for one. J. Control Release. 161, 225–234. doi: 10.1016/j.jconrel.2011.12.014 Hua, S. (2013). Targeting sites of inﬂammation: intercellular adhesion molecule- 1 as a target for novel inﬂammatory therapies. Front. Pharmacol. 4:127. doi: 10.3389/fphar.2013.00127 Danhier, F. (2016). To exploit the tumor microenvironment: since the EPR eﬀect fails in the clinic, what is the future of nanomedicine? J. Control. Release. 244 (Pt A), 108–121. doi: 10.1016/j.jconrel.2016.11.015 Hua, S., and Cabot, P. J. (2013). Targeted nanoparticles that mimic immune cells in pain control inducing analgesic and anti-inﬂammatory actions: a potential novel treatment of acute and chronic pain condition. Pain Physician. 16, E199–E216. Danhier, F., Feron, O., and Preat, V. (2010). To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Control Release. 148, 135–146. CONCLUSION Overall, the use of nanotechnology in medicine has the potential to have a major impact on human health. It has been suggested to facilitate the development of personalized medicine for speciﬁc patient sub-groups, in which therapy is tailored by the patient’s individual genetic and disease proﬁle (Teli et al., 2010; Mura and Couvreur, 2012; Laroui et al., 2013). For example, disease- speciﬁc characteristics such as capillary permeability (Calcagno et al., 2015), cellular receptor expression and molecular pathway activation could be analyzed and used to design personalized nanomedicines (Teli et al., 2010; Mura and Couvreur, 2012; Laroui et al., 2013). The physicochemical properties (e.g., size and structure) of the delivery system can also be modiﬁed according to the severity of the disease for optimal therapeutic beneﬁts (Hua et al., 2015). This concept would signiﬁcantly advance the way in which we treat patients. However, for this to occur, there are still a number of issues that need to be addressed as detailed in this review—from our basic understanding of the biology of speciﬁc diseases and the biological interaction of NNMs in patients, to commercialization hurdles related to manufacturing, costs, and regulatory standards. Finally, researchers need to consider minimizing the complexity of NNMs and take into account the ﬁnal dosage form for human use, in order for a formulation to have the potential to be translated into a clinically applicable therapeutic. Reducing complexity to the minimum required for pathophysiological or medical need is paramount in nanoparticle July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 11 Clinical Translation of Nanomedicines Hua et al. ACKNOWLEDGMENTS design and synthesis to generate clinically translatable nanosized therapeutics. The authors wish to thank The Pharmacy Research Trust of New South Wales, The Rebecca L. Cooper Medical Research Foundation, Gladys M Brawn Fellowship, ausEE Research Foundation, and The University of Newcastle for providing ﬁnancial support for our research. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. REFERENCES doi: 10.1016/j.jconrel.2010.08.027 Hua, S., Marks, E., Schneider, J. J., and Keely, S. (2015). Advances in oral nano- delivery systems for colon targeted drug delivery in inﬂammatory bowel disease: Selective targeting to diseased versus healthy tissue. Nanomedicine 11, 1117–1132. doi: 10.1016/j.nano.2015.02.018 Deamer, D. W. (2010). From “banghasomes” to liposomes: a memoir of Alec Bangham, 1921-2010. FASEB J. 24, 1308–1310. doi: 10.1096/fj.10-0503 1117–1132. doi: 10.1016/j.nano.2015.02.018 Hua, S., and Wu, S. Y. (2013). The use of lipid-based nanocarriers for targeted pain therapies. Front. Pharmacol. 4:143. doi: 10.3389/fphar.2013.00143 Diab, R., Jaafar-Maalej, C., Fessi, H., and Maincent, P. (2012). Engineered nanoparticulate drug delivery systems: the next frontier for oral administration? AAPS J. 14, 688–702. doi: 10.1208/s12248-012-9377-y Jaafar-Maalej, C., Elaissari, A., and Fessi, H. (2012). Lipid-based carriers: manufacturing and applications for pulmonary route. Expert Opin. Drug Deliv. 9, 1111–1127. doi: 10.1517/17425247.2012.702751 Dobrovolskaia, M. A., and McNeil, S. E. (2013). Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines. J Control Release. 172, 456–466. doi: 10.1016/j.jconrel.2013.05.025 Jackman, J. A., Meszaros, T., Fulop, T., Urbanics, R., Szebeni, J., and Cho, N. J. (2016). Comparison of complement activation-related pseudoallergy in miniature and domestic pigs: foundation of a validatable immune toxicity model. Nanomedicine 12, 933–943. doi: 10.1016/j.nano.2015.12.377 Dusinska, M., Boland, S., Saunders, M., Juillerat-Jeanneret, L., Tran, L., Pojana, G., et al. (2015). Towards an alternative testing strategy for nanomaterials used in July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 12 Hua et al. Clinical Translation of Nanomedicines Jang, B., Kwon, H., Katila, P., Lee, S. J., and Lee, H. (2016). Dual delivery of biological therapeutics for multimodal and synergistic cancer therapies. Adv. Drug Deliv. Rev. 98, 113–133. doi: 10.1016/j.addr.2015.10.023 Matsumura, Y., and Maeda, H. (1986). A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 46 (12 Pt 1), 6387–6392. Kim, H. J., Kim, A., Miyata, K., and Kataoka, K. (2016). Recent progress in development of siRNA delivery vehicles for cancer therapy. Adv. Drug. Deliv. Rev. 104, 61–77. doi: 10.1016/j.addr.2016.06.011 Metselaar, J. M., Van den Berg, W. B., Holthuysen, A. E., Wauben, M. H., Storm, G., and Van Lent, P. L. (2004). Liposomal targeting of glucocorticoids to synovial lining cells strongly increases therapeutic beneﬁt in collagen type II arthritis. Ann. Rheum. Dis. 63, 348–353. doi: 10.1136/ard.2003.009944 Kim, M., and Williams, S. (2018). REFERENCES Development of next-generation macromolecular drugs based on the EPR eﬀect: challenges and pitfalls. Expert Opin Drug Deliv. 12, 53–64. doi: 10.1517/17425247.2014.955011 Lammers, T. (2013). Smart drug delivery systems: back to the future vs. clinical reality. Int. J. Pharm. 454, 527–529. doi: 10.1016/j.ijpharm.2013.02.046 Narang, A. S., Chang, R. K., and Hussain, M. A. (2013). Pharmaceutical development and regulatory considerations for nanoparticles and nanoparticulate drug delivery systems. J. Pharm. Sci. 102, 3867–3882. doi: 10.1002/jps.23691 Lammers, T., Kiessling, F., Hennink, W. E., and Storm, G. (2012). Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress. J. Control Release. 161, 175–187. doi: 10.1016/j.jconrel.2011.09.063 Laroui, H., Rakhya, P., Xiao, B., Viennois, E., and Merlin, D. (2013). Nanotechnology in diagnostics and therapeutics for gastrointestinal disorders. Dig. Liver Dis. 45, 995–1002. doi: 10.1016/j.dld.2013.03.019 Needham, D., Anyarambhatla, G., Kong, G., and Dewhirst, M. W. (2000). A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model. Cancer Res. 60, 1197–1201. Larsson, M., Huang, W. T., Liu, D. M., and Losic, D. (2017). Local co- administration of gene-silencing RNA and drugs in cancer therapy: State- of-the art and therapeutic potential. Cancer Treat. Rev. 55, 128–135. doi: 10.1016/j.ctrv.2017.03.004 Nehoﬀ, H., Parayath, N. N., Domanovitch, L., Taurin, S., and Greish, K. (2014). Nanomedicine for drug targeting: strategies beyond the enhanced permeability and retention eﬀect. Int. J. Nanomedicine 9, 2539–2555. doi: 10.2147/IJN.S47129 Liu, Y., Hardie, J., Zhang, X., and Rotello, V. M. (2017). Eﬀects of engineered nanoparticles on the innate immune system. Semin. Immunol. 34, 25–32. doi: 10.1016/j.smim.2017.09.011 Nel, A., Xia, T., Meng, H., Wang, X., Lin, S., Ji, Z., et al. (2013). Nanomaterial toxicity testing in the 21st century: use of a predictive toxicological approach and high-throughput screening. Acc. Chem. Res. 46, 607–621. doi: 10.1021/ar300022h Luxenhofer, R., Barz, M., and Schillmeier, M. (2014). Quo vadis nanomedicine? Nanomedicine 9, 2083–2086. doi: 10.2217/nnm.14.140 Lv, H., Zhang, S., Wang, B., Cui, S., and Yan, J. (2006). Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control Release. 114, 100–109. doi: 10.1016/j.jconrel.2006.04.014 Nichols, J. W., and Bae, Y. H. (2014). EPR: evidence and fallacy. J. Control Release. 190, 451–464. doi: 10.1016/j.jconrel.2014.03.057 Lyass, O., Uziely, B., Ben-Yosef, R., Tzemach, D., Heshing, N. I., Lotem, M., et al. (2000). Correlation of toxicity with pharmacokinetics of pegylated liposomal doxorubicin (Doxil) in metastatic breast carcinoma. Cancer 89, 1037–1047. doi: 10.1002/1097-0142(20000901)89:5<1037::AID-CNCR13>3.0.CO;2-Z Nystrom, A. M., and Fadeel, B. (2012). REFERENCES Daunorubicin and cytarabine liposome in newly diagnosed therapy-related Acute Myeloid Leukemia (AML) or AML with myelodysplasia-related changes. Ann. Pharmacother. 1:1060028018764923. doi: 10.1177/1060028018764923 Ann. Rheum. Dis. 63, 348–353. doi: 10.1136/ard.2003.009944 Metselaar, J. M., Wauben, M. H., Wagenaar-Hilbers, J. P., Boerman, O. C., and Storm, G. (2003). Complete remission of experimental arthritis by joint targeting of glucocorticoids with long-circulating liposomes. Arthritis Rheum. 48, 2059–66. doi: 10.1002/art.11140 Kirpotin, D. B., Drummond, D. C., Shao, Y., Shalaby, M. R., Hong, K., Nielsen, U. B., et al. (2006). Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models. Cancer Res. 66, 6732–6740. doi: 10.1158/0008-5472.CAN-05-4199 Milane, L. S., and Amiji, M. (2017). Nanomedicine for Inﬂammatory Diseases. New York, NY: CRC Press Kirpotin, D., Park, J. W., Hong, K., Zalipsky, S., Li, W. L., Carter, P., et al. (1997). Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro. Biochemistry 36, 66–75. doi: 10.1021/bi962148u Min, Y., Caster, J. M., Eblan, M. J., and Wang, A. Z. (2015). Clinical translation of nanomedicine. Chem. Rev. 115, 11147–11190. doi: 10.1021/acs.chemrev.5b00116 Mishra, D. K., Balekar, N., and Mishra, P. K. (2017). Nanoengineered strategies for siRNA delivery: from target assessment to cancer therapeutic eﬃcacy. Drug. Deliv. Transl. Res. 7, 346–358. doi: 10.1007/s13346-016-0352-5 Kono, K. (2001). Thermosensitive polymer-modiﬁed liposomes. Adv. Drug Deliv. Rev. 53, 307–319. doi: 10.1016/S0169-409X(01)00204-6 Moghimi, S. M., and Hunter, A. C. (2001). Capture of stealth nanoparticles by the body’s defences. Crit. Rev. Ther. Drug Carrier Syst. 18, 527–550. doi: 10.1615/CritRevTherDrugCarrierSyst.v18.i6.30 Kraft, J. C., Freeling, J. P., Wang, Z., and Ho, R. J. (2014). Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems. J. Pharm. Sci. 103, 29–52. doi: 10.1002/jps.23773 and Couvreur, P. (2012). Nanotheranostics for personalized m Mura, S., and Couvreur, P. (2012). Nanotheranostics for personalized medic Adv. Drug Deliv. Rev. 64, 1394–1416. doi: 10.1016/j.addr.2012.06.006 Kuijpers, S. A., Coimbra, M. J., Storm, G., and Schiﬀelers, R. M. (2010). Liposomes targeting tumour stromal cells. Mol. Membr. Biol. 27, 328–340. doi: 10.3109/09687688.2010.522204 Murday, J. S., Siegel, R. W., Stein, J., and Wright, J. F. (2009). Translational nanomedicine: status assessment and opportunities. Nanomedicine 5, 251–273. doi: 10.1016/j.nano.2009.06.001 Kunjachan, S., Ehling, J., Storm, G., Kiessling, F., and Lammers, T. (2015). Noninvasive imaging of nanomedicines and nanotheranostics: principles, progress, and prospects. Chem. Rev. 115, 10907–10937. doi: 10.1021/cr500314d Nakamura, H., Fang, J., and Maeda, H. (2015). REFERENCES Safety assessment of nanomaterials: implications for nanomedicine. J. Control Release. 161, 403–408. doi: 10.1016/j.jconrel.2012.01.027 Ojha, T., Pathak, V., Shi, Y., Hennink, W. E., Moonen, C. T. W., Storm, G., et al. (2017). Pharmacological and physical vessel modulation strategies to improve EPR-mediated drug targeting to tumors. Adv. Drug Deliv. Rev. 119, 44–60. doi: 10.1016/j.addr.2017.07.007 Maeda, H. (2015). Toward a full understanding of the EPR eﬀect in primary and metastatic tumors as well as issues related to its heterogeneity. Adv. Drug Deliv. Rev. 91, 3–6. doi: 10.1016/j.addr.2015.01.002 Maeda, H., Nakamura, H., and Fang, J. (2013). The EPR eﬀect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv. Drug. Deliv. Rev. 65, 71–79. doi: 10.1016/j.addr.2012.10.002 Oomen, A. G., Bos, P. M., Fernandes, T. F., Hund-Rinke, K., Boraschi, D., Byrne, H. J., et al. (2014). Concern-driven integrated approaches to nanomaterial testing and assessment–report of the NanoSafety Cluster Working Group 10. Nanotoxicology 8, 334–348. doi: 10.3109/17435390.2013.802387 Maiseyeu, A., Mihai, G., Kampfrath, T., Simonetti, O. P., Sen, C. K., Roy, S., et al. (2009). Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis. J. Lipid. Res. 50, 2157–2163. doi: 10.1194/jlr.M800405-JLR200 Oude Blenke, E., Mastrobattista, E., and Schiﬀelers, R. M. (2013). Strategies for triggered drug release from tumor targeted liposomes. Expert. Opin. Drug Deliv. 10, 1399–1410. doi: 10.1517/17425247.2013.805742 Paradise, J., Wolf, S. M., Kuzma, J., Kuzhabekova, A., Tisdale, A. W., Kokkoli, E., et al. (2009). Developing, U.S. oversight strategies for nanobiotechnology: learning from past oversight experiences. J. Law Med. Ethics. 37, 688–705. doi: 10.1111/j.1748-720X.2009.00441.x Mastrobattista, E., Koning, G. A., and Storm, G. (1999). Immunoliposomes for the targeted delivery of antitumor drugs. Adv. Drug. Deliv. Rev. 40, 103–127. doi: 10.1016/S0169-409X(99)00043-5 July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 13 Hua et al. Clinical Translation of Nanomedicines Park, J. W., Hong, K., Kirpotin, D. B., Colbern, G., Shalaby, R., Baselga, J., et al. (2002). Anti-HER2 immunoliposomes: enhanced eﬃcacy attributable to targeted delivery. Clin. Cancer Res. 8, 1172–1181. Szebeni, J., and Barenholz, Y. (2009). “Adverse immune eﬀects of liposomes: complement activation, immunogenicity and immune suppression,” in Harnessing biomaterials for nanomedicine: preparation, toxicity and applications, ed P. S. Publishing (Singapore: Pan Stanford Publishing), 1–19. Park, K. (2017). The drug delivery ﬁeld at the inﬂection point: Time to ﬁght its way out of the egg. J. Control Release. 267, 2–14. REFERENCES doi: 10.1016/j.jconrel.2017.07.030 Szebeni, J., and Moghimi, S. M. (2009). Liposome triggering of innate immune responses: a perspective on beneﬁts and adverse reactions. J. Liposome Res. 19, 85–90. doi: 10.1080/089821009027 92855 Poste, G., Papahadjopoulos, D., and Vail, W. J. (1976). Lipid vesicles as carriers for introducing biologically active materials into cells. Methods Cell Biol. 14, 33–71. doi: 10.1016/S0091-679X(08)60468-9 Puri, A., Loomis, K., Smith, B., Lee, J. H., Yavlovich, A., Heldman, E., et al. (2009). Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit. Rev. Ther. Drug Carrier Syst. 26, 523–580. doi: 10.1615/CritRevTherDrugCarrierSyst.v26.i6.10 Szebeni, J., and Storm, G. (2015). Complement activation as a bioequivalence issue relevant to the development of generic liposomes and other nanoparticulate drugs. Biochem. Biophys. Res. Commun. 468, 490–497. doi: 10.1016/j.bbrc.2015.06.177 Rahman, A. M., Yusuf, S. W., and Ewer, M. S. (2007). Anthracycline-induced cardiotoxicity and the cardiac-sparing eﬀect of liposomal formulation. Int. J. Nanomedicine 2, 567–583. Talekar, M., Tran, T. H., and Amiji, M. (2015). Translational nano-medicines: targeted therapeutic delivery for cancer and inﬂammatory diseases. AAPS J. 17, 813–827. doi: 10.1208/s12248-015-9772-2 Riehemann, K., Schneider, S. W., Luger, T. A., Godin, B., Ferrari, M., and Fuchs, H. (2009). Nanomedicine–challenge and perspectives. Angew. Chem. Int. Ed. Engl. 48, 872–897. doi: 10.1002/anie.200802585 Teli, M. K., Mutalik, S., and Rajanikant, G. K. (2010). Nanotechnology and nanomedicine: going small means aiming big. Curr. Pharm. Des. 16, 1882–1892. doi: 10.2174/138161210791208992 Rizzo, L. Y., Theek, B., Storm, G., Kiessling, F., and Lammers, T. (2013). Recent progress in nanomedicine: therapeutic, diagnostic and theranostic applications. Curr. Opin. Biotechnol. 24, 1159–1166. doi: 10.1016/j.copbio.2013.02.020 Theek, B., Gremse, F., Kunjachan, S., Fokong, S., Pola, R., Pechar, M., et al. (2014). Characterizing EPR-mediated passive drug targeting using contrast- enhanced functional ultrasound imaging. J. Control Release. 182, 83–89. doi: 10.1016/j.jconrel.2014.03.007 Sainz, V., Conniot, J., Matos, A. I., Peres, C., Zupancic, E., Moura, L., et al. (2015). Regulatory aspects on nanomedicines. Biochem. Biophys. Res. Commun. 468, 504–510, doi: 10.1016/j.bbrc.2015.08.023 Tinkle, S., McNeil, S. E., Muhlebach, S., Bawa, R., Borchard, G., Barenholz, Y. C., et al. (2014). Nanomedicines: addressing the scientiﬁc and regulatory gap. Ann. N. Y. Acad. Sci. 1313, 35–56. doi: 10.1111/nyas. 12403 Satalkar, P., Elger, B. S., and Shaw, D. M. (2015). Deﬁning Nano, Nanotechnology and Nanomedicine: Why Should It Matter? Sci. Eng. Ethics 22, 1255–1276. doi: 10.1007/s11948-015-9705-6 Sawant, R. R., and Torchilin, V. P. (2012). Challenges in development of targeted liposomal therapeutics. AAPS J. 14, 303–315. doi: 10.1208/s12248-012-9330-0 Torchilin, V. REFERENCES P. (2006). Multifunctional nanocarriers. Adv. Drug Deliv. Rev. 58, 1532–1555. doi: 10.1016/j.addr.2006.09.009 Senior, J. H. (1987). Fate and behavior of liposomes in vivo: a review of controlling factors. Crit. Rev. Ther. Drug Carrier Syst. 3, 123–193. van der Meel, R., Vehmeijer, L. J., Kok, R. J., Storm, G., and van Gaal, E. V. (2013). Ligand-targeted particulate nanomedicines undergoing clinical evaluation: current status. Adv. Drug Deliv. Rev. 65, 1284–1298. doi: 10.1016/j.addr.2013.08.012 factors. Crit. Rev. Ther. Drug Carrier Syst. 3, 123–193. Sercombe, L., Veerati, T., Moheimani, F., Wu, S. Y., Sood, A. K., and Hua, S. (2015). Sercombe, L., Veerati, T., Moheimani, F., Wu, S. Y., Sood, A. K., and Hua, S. (2015). Advances and challenges of liposome assisted drug delivery. Front. Pharmacol. 6:286. doi: 10.3389/fphar.2015.00286 ( ) Advances and challenges of liposome assisted drug delivery. Front. Pharmacol. 6:286. doi: 10.3389/fphar.2015.00286 Wagner, V., Dullaart, A., Bock, A. K., and Zweck, A. (2006). The emerging nanomedicine landscape. Nat. Biotechnol. 24, 1211–1217. doi: 10.1038/nbt1006-1211 Sessa, G., and Weissmann, G. (1968). Phospholipid spherules (liposomes) as a model for biological membranes. J. Lipid. Res. 9, 310–318. Willis, M., and Forssen, E. (1998). Ligand-targeted liposomes. Adv. Drug Deliv. Rev. 29, 249–271. doi: 10.1016/S0169-409X(97)00083-5 Shajari, N., Mansoori, B., Davudian, S., Mohammadi, A., and Baradaran, B. (2017). Overcoming the challenges of siRNA delivery: nanoparticle strategies. Curr. Drug. Deliv. 14, 36–46. doi: 10.2174/1567201813666160816105408 Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S., and Farokhzad, O. C. (2008). Nanoparticles in medicine: therapeutic applications and developments. Clin. Pharmacol. Ther. 83, 761–769. doi: 10.1038/sj.clpt.61 00400 Shi, J., Kantoﬀ, P. W., Wooster, R., and Farokhzad, O. C. (2017). Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer 17, 20–37. doi: 10.1038/nrc.2016.108 Storm, G., Oussoren, C., Peeters, P., and Barenholz, C. (1993). “Tolerability of liposomes in vivo,” in Liposome Technology. 3, ed G. Gregoriadis (Florida: CRC Press), 345–83. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Storm, G., ten Kate, M. T., Working, P. K., and Bakker-Woudenberg, I. A. (1998). Doxorubicin entrapped in sterically stabilized liposomes: eﬀects on bacterial blood clearance capacity of the mononuclear phagocyte system. Clin. Cancer Res. 4, 111–115. Copyright © 2018 Hua, de Matos, Metselaar and Storm. July 2018 \| Volume 9 \| Article 790 REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Svenson, S. (2012). Clinical translation of nanomedicines. Curr. Opin. Solid. State Mater. Sci. 16, 287–294. doi: 10.1016/j.cossms.2012.10.001 Szebeni, J. (2005). Complement activation-related pseudoallergy: a new class of drug-induced acute immune toxicity. Toxicology 216, 106–121. doi: 10.1016/j.tox.2005.07.023 July 2018 \| Volume 9 \| Article 790 Frontiers in Pharmacology \| www.frontiersin.org 14
https://openalex.org/W4250103104	https://www.biodiversitylibrary.org/itempdf/116680	English	null	Care and training of trotters	null	1,914	public-domain	34,204	F TROTTERS _@ QFS4\ WS Cornell University Library OF THE Tew Dork State College of Agriculture The date shows when this tolume (wad taken. To renew this book copy the call No, and giveto > the librarian hsié 2 + MAY 1 4/1978 &® MAY 01973 L 3 HOME USE RULES, All Books subject to Recal All books must be r turned at end of colleg year for inspection an repairs. Students must r turn all books befor leaving town. Office should ‘arrange for t return of books wante during their absenc from town. Books needed: b more than one perso are held on the reserv list. ‘Volumes of period cals and of pamphlet are held in the libra ay much as possibl Special putpose are given out f ited time. » Baowen shoul not use their libra privileges for the bene fit of other persons.. Books of. speci en and gift books when the giver wish it, are not allowed The date shows when this tolume (wad taken. To renew this book copy the call No, and giveto > The date shows when this tolume (wad taken. To renew this book copy the call No, and giveto > the librarian hsié 2 + MAY 1 4/1978 &® MAY 01973 L HOME USE RULES, All Books subject to Recall All books must be re- turned at end of college year for inspection and repairs. Students must re- Students must re- turn all books before leaving town. Officers should ‘arrange for the return of books wanted during their absence from town. Books needed: by Books needed: by more than one person are held on the reserve list. ‘Volumes of periodi- 3 ‘Volumes of periodi- cals and of pamphlets are held in the library ay much as possible. Special putposes are given out for ited time. » Baowen should » Baowen should not use their library privileges for the bene- fit of other persons.. Books of. special Books of. special en and gift books, when the giver wishes it, are not allowed to circulate. Readers are asked to Readers are asked to report all cases of books marked or mutilated. Do not deface books by marks and writing, Do not deface books by marks and writing, SF 341.H wii Hee" University Library raining of trott wi mann DATE DUE i? Vt A Cray), ES Rms, GAYLORD PRINTEOINU.S.A, DATE DUE CARE AND TRAINING OF TROTTERS VOLUME I OF “THE\ HORSEMAN LIBRARY" PREPARED BY THE EDITORIAL STAFF OF “THE HORSEMAN and SPIRIT OF THE TIMES” FROM INFORMATION .FURNISHED BY THE LEADING TRAINERS AND DRIVERS OF THE DAY BS PRICE $1.00, CLOTH, POSTPAID.- PUBLISHED AND COPYRIGHTED 1914 BY* CHICAGO HORSEMAN NEWSPAPER CO. 538 SO. DEARBORN STREET., CHICAGO, ILL. VOLUME I OF “THE\ HORSEMAN LIBRARY" PRICE $1.00, CLOTH, POSTPAID.- PUBLISHED AND COPYRIGHTED 1914 BY* CHICAGO HORSEMAN NEWSPAPER CO. 538 SO. DEARBORN STREET., CHICAGO, ILL. Introduction. There has been a great demand in recent years for an authoritative treatise on the “Care and Training of Trotters.” The only books on the subject are out of date. We have endeavored to fill this demand. This book does not represent the ideas of one This book does not represent the ideas of one man, as in past treatises, nor of a few men, but of many. Practically all the leading authorities have kindly collaborated with four members of the editorial staff of “The Horseman,” during a period of about six months, in the preparation of this work. The authorities listed herein have assisted either by letter, personal interview, or in an editorial capacity, to the contents of this book. We desire to extend our thanks to all. Quotations have been made, in a few instances, from the works of Charles: Marvin and John Bradburn, but credit is given each case. THE PUBLISHERS. THE PUBLISHERS. CONTRIBUTORS AND COLLABORATORS. Roland C. Drake Charles H. Gelo Frank -E. Alley Warren Bacon Dr. W. A. Barber L. E. Brown Asa Danforth John L. Dodge Frank G. Jones S. J. Fleming L. C, Kinney STOCK FARM Harry Burgoyne John Dagler John H. Dickerson Budd Doble : James M. Hazleton Joe Heather Jos. M. McGraw Ed, Allen oscar Ames W. J. Andrews ied. Benyon Jas. Benyon Ed. Bither Geo. Bowerman Mike Bowerman James Carpenter J. B. Chandler Zach Chandler Harold Childs Walter R. Cox Dick Curtis Wick Curry Crit Davis Charley Dean Chas. De Ryder Budd Doble Billy Dunham Will G. Durfee Fred Egan John Fleming’ Will Fleming W. 0. Foote J. Y. Gateomb Ed. F. Geers Jd. O. Gerrity Geo. T, Haag W. 4H. Harrison Geo. B. Hayes H. H. Helman James Hogan H. H. James Fred Jamison H. M. Jones Chet Kelly Ren Kenney ‘George W. Leavitt Ned McCarr W. H. McCarthy Scott McCoy Dan McEwen 1 . §. Alexander (Arranged Alphabetically.) EDITORIAL STAFF. Wn. H. Shields Arthur C.. Thomas OWNERS eCoy Walter Palmer Ed. L. Peckham A. C. Pennock O. H. Sholes W. Hz. Smollinger Amos Whiteley. SUPERINTENDENTS. John Ruppert A. B. Scott A. L. Thomas Ben White iid. Willis John Young TRAINERS AND DRIVERS. Mike McDevitt Joe McLaughlin Alonzo McDonald Dick McMahon Jobn McQuaig Guss Macey TReamey Macey ~ Roy Miller Hunter C. Moudy Thomas W. Murphy \| ‘rom Nolan Vance Nuckols kupert Parker Robt. Introduction. Proctor Harry Putnam Nat Ray “Joe Rea Ben Rennick A. S. Rodney Millard Sanders Geo. W. Saunders Jos. L. Serrill Bert Shank Bt Shively W. W. Shuitt Ray Snedeker Wm. L. Snow W. L. Spears John Splan George Starr Harry C. Stinson Charles Tanner J. L, Tarlton Henry H. Thomas Douglas ‘Thomas Henry Titer e Charley Valentine Al Whitney Diek Wilson Paul C. Wilson Henry Williams Matt Williams v BN ABTANS. . S. R. Howard v BN ABTANS. . S. R. Howard Dr. _L. M. Oldham Dr. Jack Seiter CARE AND TRAINING OF TROTTERS.- Chapter I—The Suckling Colt. Chapter I—The Suckling Colt. 1 HE mare about to foal.should be - placed in a warm box §stall, especially if the colt comes early and in a cold climate. The colt might contract pneu- monia from the shock to its delicate constitution by ex- posure to cold air. The first attention to the colt should be to the ETM — Dae Sait iif 1 H ETM — Dae Sait iif The first attention to the colt should be to the umbilical cord (navel string). There are two methods of treatment, each has its adherents. The older horsemen, and some younger ones, pre- fer to tie up the cord about a half inch from the body, and then cut it off, just below where it has been tied. The younger horsemen and many vet- erinarians advise against the tying up of the um- bilical cord, since it has been found in some cases to imprison the germs of navel disease which had gained entrance to the navel before being disin- fected. James Hazelton of Boice Stock Farm, Frankfort, Ind., who has raised hundreds of colts. successfully, is one we recall who does not tie up the umbilical cord. As soon as possible after the colt is born squeeze out the gelatinous contents of the umbil- ical cord, tie it up if you will, but in any event paint it with an antiseptic solution, or, better still, dip it into such a solution. Dr. A. S, Alexander THE SUCKLING COLT. 5 prefers an antiseptic solution containing 2 drahms of powdered corrosive sublimate to a pint of boiling water, to which when cold has been added, 3 drahms of tincture or solution of chloride of iron (Label bottle “Poison”). Use this solution twice a day until the cord drops off. Dr. W. A. Barber of Springfield, O., uses “a solution of 9 parts of carbolic acid, dissolved in 1 part of alcohol, with 25 parts of camphor added, giving a clear oily ‘so- lution that may be applied without fear of cauter- izing and many times more efficacious, I think, than corrosive sublimate.”’ A different method of accomplishing the same A different method of accomplishing the same end is advised by the Percheron Society of Amer- ica in its pamphlet, “Facts About Percherons,” and excellent results are reported by those breed- ers who have tried it. Chapter I—The Suckling Colt. This method of treatment was recommended to the Percheron society by Dr. R. R.. Dykstra. Tincture of iodine is first ap- -plied to the cord and the area immediately sur- rounding it. After this apply a drying powder every half-hour for a period of three or four hours, or until the cord is thoroughly dried up. This drying powder is composed of equal parts. of powdered gum camphor, starch and alum. After the navel is attended to the next thing on After the navel is attended to the next thing on the program is to inject some warm water into the colt’s rectum so as to start the bowels working reg- ularly. Some men use an injection of one ounce of sweet oil in a quart of warm water. Others use CARE AND TRAINING OF TROTTERS. 6 castile soap suds in warm water, but this is not widely recommended as the soap is thought to be too irritating. Users of the soap suds injection claim it is not irritating, especially if glycerine is added. Olive oil makes a satisfactory in- jection. In extreme cases give an ounce of castor oil. A horseman writes: “For enemas would sug- gest the use of a few tablespoonfuls of liquid soap instead of castile soap-suds. It is non- irritating, and ‘being of an oily nature serves a double purpose. Great care should be exercised in giving injections. Serious harm may result from roughness as the tissues that you are working upon are.in a very delicate condition at this time. Gentleness should be observed in all things per- taining to the handling of.a youngster. He is bet- ter off without your medicine if you have to use force in administering it. Nature will do a lot for the colt if you let it alone in almost every case.” Dr. S. R. Howard says: “The best rectal in- jections, in my opinion, are: emulsion of slippery elm bark or warm cow’s milk. No harm can be done by any amount used, as they are natural in their action.” First Milk Important. the colt arrives it will, First Milk Important. the colt arrives it will, Soon after the colt arrives it will, if strong and healthy, struggle to its feet (more or less clumsily at first, of course), and suck.. If it is too weak to rise it should be assisted, for the first nour- ishment will strengthen the colt at a time when THE SUCKLING COLT. 7 strength is absolutely necessary. The first milk also contains a natural laxative which is beneficial to the colt. If the colt is weak and will not suck even when assisted, the mare should be milked and the first milk, while still warm, given the colt from a bottle with nipple. Tf the colt will not suck nipple use a dessert spoon and pour the warm milk down its throat. Do not be in too great a hurry to get the colt up to suck. Any time within the first hour will do. We will say nothing of the care of the mare We will say nothing of the care of the mare after foaling (such as the removal and sanitary destruction of the after-birth) for we are dealing only with the colt. As soon as possible after foaling, which we will As soon as possible after foaling, which we will assume takes place indoors, the stall should be thoroughly disinfected; the bedding should be burned, the floor sprinkled with lime, and fresh bedding laid down. For several weeks the bedding’ should be changed often. Cleanliness will usually prevent navel disease. In Kentucky and farther South. most colts are foaled out doors. Harry Burgoyne of Walnut Hall Farm, Donerail, Ky., and Ed. Willis of Patchen Wilkes Farm, Lexing- ton, Ky., prefer to have the colts foaled out doors, if the weather is not too cold. Treatment for Diarrhea. e colt is from seven to ten Treatment for Diarrhea. e colt is from seven to ten When the colt is from seven to ten days old it may be troubled with diarrhea, due to the mare coming in heat or for other causes, in which event CARE AND TRAINING OF TROTTERS. 8 keep both mare and colt in a quiet place’ Milk the mare dry and doctor the colt. John Bradburn strongly advised giving colts troubled with diarrhea a blackberry cordial, full directions for the making of which are described in his book “Breeding and Developing the Trotter.” A horseman writes: “John Bradburn’s black- A horseman writes: “John Bradburn’s black- berry cordial has not proved sufficient in my ex- perience, especially in obstinate cases. I have se- cured the best results from paregoric and aro- matic syrup of rhubarb, equal parts.” A favorite prescription of Al Thomas, and one with which he once saved the life of Alta Ax-: worthy, 3, 2:1014, when she was suffering from a severe case of diarrhea, is: -Take colt out of sun (if turned out) and give it the yolk of an egg to which has: been added 10 to 20 drops of tincture of opium. Three or four doses a day should be given until relief is obtained. Another prescrip- tion is an egg in a pint of milk given three times a day until cured. Another good prescription is a teaspoonful of lime water in several of milk, given. every three hours. At the Allen Farm, Pittsfield, Mass., the remedy for diarrhea is limewater. Roy Miller writes: “As to diarrhea in colts, let me give you a suggestion which I received from Major Daingerfield, to whom every breeder in this world has to ‘take off-his hat.’ He told me that his colts were never bothered with diarrhea, to speak of, and especially soon after foal- THE SUCKLING COLT. 9 ing, as he made it a point to put the. mare on a diet that tended. to make milk about four months before foaling time. About two to three weeks before foaling time, he fed the mare the same amount of feed she would receive after foaling, and never increased the feed of the mare for several weeks after she had foaled. He claimed that, in doing this, the blood of the foal was of the same richness as the blood of the mare. Treatment for Diarrhea. e colt is from seven to ten He claimed that, in nearly all cases, diarrhea came from increasing the feed of the mare too soon after her colt was foaled. Care must be taken to keep the colt’s navel Care must be taken to keep the colt’s navel clean, so as to prevent “navel disease,” -but if trouble develops call a veterinarian at once and in- sist on a serum treatment. If no veterinarian is accessible (and in that emergency only) insert into the navel, with a bulb-syringe catheter, any good antispetic solution. For instance a one-quarter ounce of creolin to two ounces of boiled water. Insert the catheter as far as an opening can be made without forcing. The injection should be continued from time to time till the navel is healed and closed. The symptoms of navel trouble are: Colt acts dull, there is a leakage from navel; colt is stiff in knees, hocks, or hips, with sometimes a formation of pus around knees, hocks or abdomen. Some people advise lancing to relieve the swelling, others prefer letting Nature remove the foreign matter, CARE AND TRAINING OF TROTTERS. 1¢ We will state here that it is not the intention to make this in any sense a veterinary treatise. Where thought best, simple home treatment for common ailments will be made mention of in simple terms. In most cases a veterinarian should be called. In Pasture. As soon as the colt is strong enough and when weather is good, mare and colt should be turned into a paddock an.hour or two, morning and after- noon. The length of time the colt is turned out should be gradually increased each day until finally colt is out all day if weather permits, but taken in _at night. When warm weather comes (it is as- sumed we have a spring foal), the mare and colt should be turned out in pasture. If large pas- tures are used too many mares should not be turned out together. No matter how large or small the pasture, turn the mares and colts into the pas- ture at intervals until the desired number are in but never turn out the whole drove together, as the mares may run about and kick each other, and: injure either themselves or the colts. In the South mates stay in pasture practically the year around. Large open sheds are built for them, with southern exposure, for such occasional protection from weather as they will need. The system of feeding brood mares with suck- ling foals, and young horses, at Palo Alto. Farm was somewhat different from that usually prac- THE SUCKLING COLT. ne ticed. They ran to grass, were fed hay, and night and morning were fed steamed or cooked food—sometimes oats and sometimes barley; they were also fed carrots. Long before the colt was weaned it learned to poach on its mother’s meal, and when at four months of age it was weaned, it was fed the same as a yearling or two- year-old. ‘If the mare comes in season at thirty days, the colt may again be troubled with diarrhea.. By this time the colt will ordinarily be strong enough to go through such trouble without ill effects. If the diarrhea continues, however, take the mare from pasture and keep her in a cool, quiet place, take her off of grain feed entirely, and feed her hay or grass for from five to eight days till colt’s condi- tion is normal. When the colt is two months old its feet may When the colt is two months old its feet may be leveled for the first time with a rasp; do not use pinchers. The feet should be leveled once’ a month, otherwise the wall of the foot will grow down and break off unevenly. In Pasture. Some claim it is not necessary to trim the feet till weaning time. This subject is treated in detail in a later chapter on “Care of Colt’s Feet,” by Dr. Jack Seiter. If the mare does not thrive on pasture at first, If the mare does not thrive on pasture at first, she should be taken up each morning and fed some crushed oats and bran equally mixed, say four quarts each morning. She may also be given green corn, sorghum, or alfalfa. She should stay in- CARE AND TRAINING OF TROTTERS. iS doors until the middle of the afternoon, when she should be fed again, omitting the green feed, and turned back into pasture. Feeding Paddock. About the middle of summer colts will require additional nourishment. Feeding paddocks should be built in the pastures, as illustrated, which will be built in the pastures, as illustrated, which will allow only the colts access to the troughs. On most farms the troughs are placed about 2% feet from the ground. It is well to line them with zinc so that the attendant can‘keep them clean. Some have troughs only four inches from the ground. These low troughs are not widely used because colts are liable to jump over them and. knock their allow only the colts access to the troughs. On most farms the troughs are placed about 2% feet from the ground. It is well to line them with zinc so that the attendant can‘keep them clean. Some have troughs only four inches from the ground. These low troughs are not widely used because colts are liable to jump over them and. knock their allow only the colts access to the troughs. On most farms the troughs are placed about 2% feet from the ground. It is well to line them with zinc so that the attendant can‘keep them clean. Some have troughs only four inches from the ground. These low troughs are not widely used because colts are liable to jump over them and. knock their THE SUCKLING COLT. 13 ankles and skin their legs. Musty food should not be allowed to accumulate and troughs should. be cleaned out after each meal. Colts may be fed once a day, or twice, according to judgment, de- pending on amount of extra nourishment neces- sary. Feed as much as colts will eat up clean. Some horsemen use a thoroughly mixed feed in proportions of two bushels of oats, one of wheat, one-half of cracked corn, fifty pounds of bran and twenty pounds of oil meal (not oil cake but ground flaxseed meal). Colts, as well as mares, should be salted once or twice a week, or leave rock salt where it can be licked as desired. Every stock farm owner should know the per- centage of limestone in his soil. Fast trotters never came from lime-deficient regions. Horses raised in such localities are apt to be week-boned, therefore unsound. Feeding Paddock. If your soil is deficient in lime take a piece of fresh-burned lime the size of a hen’s egg and drop it into the water troughs once or twice a week. Speaking of water troughs they should be cleaned out regularly. “As the twig is bent the tree inclines,” so par- “As the twig is bent the tree inclines,” so par- ticular attention should be given to a colt in the first few months of.its life. As Jahn Splan once wrote: “Anyone who can not control his temper should never be allowed to have anything to do with a colt. * * * You should begin to impress the colt from its earliest life that man is his friend, and the foundation of his education is laid.” CARE AND TRAINING OF TROTTERS. 14 Mannering the Colt. Many good horsemen put ‘an easy-fitting halter on the colt when it is ten days old, which makes the colt easier to manage in the stall. On several large farms, such as Allen Farm, Pittsfield, Mass., and White River Stock Farm, Muncie Ind., colts are thoroughly halter broken at this time. The halter should be taken off when the colt is turned out for the summer. The colt’s feet should be picked up and handled from time to time. Amos Whiteley of White River Stock Farm, says: “We commence mannering the colts almost as soon as they are foaled, and soon accus- tom theni to being handled. Our colts are haltered when they are two or three weeks old. We use an ordinary halter, and get a piece of half-inch hemp rope, about two and a half feet long, thor- oughly wrapped at one end, or near the end, and with a snap hook attached to the other end, and snap the rope into the halter, letting the colt carry or drag it so as to become accustomed to carrying something, and in a way,-to be guided by it. Our brood mares are all nicely mannered and compan- ionable; in fact; when I go into the brood mare pasture, the mqres always expect some sugar, car- rots, apples, or something that they like, and while they are enjoying their little treat, the colts are becoming accustomed to being handled, and soon begin to. look for something for themselves. By treating the colts in this way, they are almost THE SUCKLING COLT. Feeding Paddock. 15 broken before they are weaned, and in many cases our colts wean themselves,:or substantially so, as they are put on ground feed, oats with a smal! portion of rye and bran. We try to have them learn to eat good before they are weaned.” The main things to observe in the care of suck- The main things to observe in the care of suck- ling colts are common-sense and kind treatment. Nature should be allowed to do as much of the work as possible. , Ai:dale, 2:15% ‘(in 1912), World's Champion Yearling Trotter, , Ai:dale, 2:15% ‘(in 1912), World's Champion Yearling Trotter, CARE AND TRAINING OF TROTTERS. 16 Chapter 2—The Weanling Colt. . HEN fall comes, the suckling colt should be weaned. Some brood mare handlers take the colt away from the dam and milk the mare dry each day. This Ne UUTOONLEe is done on the Savage Farm “and McKennan farm. Others allow the colt to suck twice a day for sev- eral days, then once a day for a while, till the mare dries up of her own accord. One prominent colt handler says: “The first method is the right one and best when you have experi- enced help or can look after mare and colt your- self, otherwise the second. method should be used.” Another says: ‘Don’t tantalize the colt by keep- ing its mother around while wens is like cutting off a dog’s tail a little at atime.” Ne UUTOONLEe “ Ne UUTOONLEe “ _A horseman writes: “I wean my colts by tak- ing them off the mares at once and taking the mares far enough away so that they will not hear each other. Of late years IT have been weaning by the signs of the Zodiac, This may sound a lit- tle ancient, at least to’ some people, but it does not cost anything, and Ihave found that my mares and colts both do a great deal better. Before I tried this I had more or less trouble with mares’ bags caking and colts worrying a good deal for THE WEANLING COLT. 1? a short time, but under this system I have had no trouble either way.” Joseph McGraw writes: “We wean a colt by a short time, but under this system I have had no trouble either way.” Joseph McGraw writes: “We wean a colt by taking it away from dam and milk mare for four days, twice a day, then once a day till dried up. I use equal parts spirits of camphor, tincture of belladonna and lard (no salt) on mare’s bag.” The colt should be placed in a box stall and The colt should be placed in a box stall and haltered. Use a good, strong five-ring leather. halter, one that fits right and is not too tight nor too loose. One horseman then proceeds to break the colt to stand tied. Chapter 2—The Weanling Colt. . His advice is: “Takea 4- inch rope, maKe a small noose in one end, pass the rope around the colt’s girth, slip the plain end through the noose, and draw the rope tight around the girth, pass the loose end of the rope between the colts’ legs and up through the halter ring. Tie end of rope to a ring in the stall and leave colt stand for an hour or so each day. This will break the colt to stand hitched, which is an im- portant part of its education.” Other horsemen do not tie colts in the stall until after they are thor- oughly halter broken. Joe Heather of Hopper farm writes: “I put Joe Heather of Hopper farm writes: “I put a, good fitting halter on the colts the first thing. I attach a lead and just let it drag. The next thing is to get a good quiet man that likes colts and put him in with them, brushing them and fussing with them. I find that when a colt has run loose in the stall a few days with the halter CARE AND TRAINING OF TROTTERS. 18 lead dragging it is no trouble at all for it to learn to lead. I like this way better than putting ropes around their girths and hind quarters, and such things as that, as I have known a good many cases where colts were injured more or less.” Feeding. After weaning, the colt should bé well fed. Oats is the usual food, together with timothy or prairie-hay. Bran may be mixed equally with the oats, although this will not be necessary if clover or alfalfa are available. Some authorities con- sider oats too hard for young colts to masticate and give ground feed and clover hay. Roy Miller writes: “I take it we are all trying Roy Miller writes: “I take it we are all trying to raise colts, fit in constitution, size and éndur- ance, to start in the futurities; therefore the feed- ing of the little fellow, just at this age, is a very important consideration indeed. I note you men- tion oats and timothy hay. I suggest two quarts rolled oats, one quart bran, one pint cracked’ screened corn, and a handful of rolled barley, to a feed, three times a day, with all the straight clover hay that a colt will eat, twice daily. If a breeder is forced to eliminate any of the bill of fare I have mentioned, I suggest he leave out anything he sees fit except the.clover hay.” One horseman writes: “As to feeding them, I give them clover hay, with a small allowance of corn, and all the good oats they will clean up. I THE WEANLING COLT. 19 also like sowed cane, which I think makes an ex- cellent feed for young colts in winter.” J. L. Dodge writes: “Regarding feeding, I be- lieve that too much is worse than too little. You seldom see a sick hungry man. If your colt doesn’t thrive and you increase his feed and he improves you know the reason. If he gets sick and you in- crease his feed and he gets worse, what do you do? Over-feeding causes nearly all the sickness. One big strong colt eats no moré than some runts. It’s what they digest, not what they eat, that does them good. Too much rich food makes too much expensive manure and heavy doctor bills. Feed the colts all the good oats and timothy they will eat up clean, and see that they get exercise enough to warrant such feeding. Reduce the feed when sick or not exercising. Don’t feed rich food at any time. During the time of strenuous work, feed crushed oats, bttt don’t practice this. Feeding. Fletcheriz- ing would leave us no stomachs at all in a few generations and concentrated foods do only for emergencies.” . Ben White writes: “I think the most important thing of all is the feeding of the weanling, and no man could improve on what Roy Miller says in regard to feeding youngsters. I like a few car- rots three times a week to feed to colts. They will drive worms from a colt and keep their bowels in good shape.” CARE AND TRAINING OF TROTTERS. 20 20 The attendants should handle the colts carefully so as to inspire confidence and friendship. The colt should be groomed daily in order to improve its coat and to break it to the use of the currycomb and brush. : Halter-Breaking. After the colt has become accustomed to a stall, the next thing is to halter-break it. The horse- man who advised tying the colt in stall, with a rope around girth, makes this suggestion: “The first day the colt is weaned and after it has stood in the stall for awhile, the rope should be taken off and a plain leather strap substituted. Bring the dam out. Let the colt go up to her, then have an assistant lead the mare away. Then lead the colt-after her, until the colt gets used to being led about. Then take the mare away, without let- ting the colt see you do so, and continue the les- son in leading.” : ‘ Dr, W. A. Barber writes: “My idea as to the best time to halter-break a colt is the next day after it is foaled. Slip a good fiting halter on the colt and handle it at every opportunity. If you have a boy’ that loves a horse he will soon have the colt broken to halter as well as to lead at will. From that time’ ne cole will grow up to know what restraint is.’ Ned McCarr, colt man at the Savage Farm, writes: “We halter-break a colt by putting a piece of three-eighths-inch bell cord around it, THE WEANLING COLT. 21 the same as a breeching, and then a short piece to run over the back, directly over the flanks,‘ con- necting both sides, to keep it from falling down over the heels; then the two long pieces are run through the halter ring. The colt breaker takes the halter shank in one hand, and the two ends of the cord in the other, then gently pulls on the halter shank and gives the cord a sharp jerk. The colt will generally make.a jump forward, and in some cases attempt to kick, but it takes, as a rule, only one lesson for the colt to grasp the idea that with a pull of the halter shank it will also re- ceive a jerk on the cord, so that after a few les- sons it will obey and lead on the first pull of the shank.” Frank E. Halter-Breaking. Alley of Roseburg, Ore., uses -this same method to break his colts to halter ex- cept he is careful to give ‘an equal pull on the halter rope and the rope which goes around the colt. Give a steady pull and the colt will always step forward to get away from the pressure be- hind.” “T have always considered that I knew some- thing about breaking colts, but my superintend- ent, Mr. McDonald, showed me a new wrinkle in bitting.a colt, which is the finest thing I have ever seen. He uses the ordinary bitting rig, consist- ing of a surcingle, back band (with a ring on either side), crupper attachment, plain, open bridle without check, and a soft leather bit with a leather guard at either side of the mouth. The CARE AND TRAINING OF TROTTERS. Lo lo colt is led out into the yard or paddock.and a short. strap attached to the bit on one side and tied through one ring on the back band, tight enough to draw the colt’s head around to one side. The colt is then turned loose and allowed to wander where it will. As the head is turned to one side, ‘the colt will continue to go in a circle and cannot run. After a few minutes the strap is’ changed to the other side, and in a very few lessons the colt is perfectly bridle-wise. An important advan- tage of this method is that you can go up to the colt at any time, for it is impossible for the colt to get away from you, and the colt will learn the purpose of the bit without inflicting any dam- age whatever.” This method of breaking is cer- tainly a success with Mr. Alley, for he furnishes us a picture of four weanlings in motion, hitched four-in-hand to a light cart, and all well-behaved. Leading Beside Pony. You will now have to decide whether you are going to break your colt to lead beside a pony, or not. Authorities are at variance on the subject. One man will say it “makes speed,” another “‘it is harmful,” while still another will take the middle ground and say “it does not make speed, it is not harmful, and it will add to a colt’s value if it passes through a sale ring, or if you wish to show it to advantage to a possible purchaser.” Even among those who break their colts to lead beside THE WEANLING COLT. 23 a pony, there is some difference of opinion as to when this should be done. One prominent colt man advises breaking the colt in this manner with- in a day or two after weaning. He says the advantage of breaking beside a pony the next day after weaning is: the colt will be lonesome and will follow the pony naturally. Start the lesson in a yard with an assistant to go behind the colt till it is used to leading. The leading should be done every day till the colt is well broken. If the colt handler is not an expert in leading colts beside a pony, give only a few lessons in this direction, as the colts will get to side pulling and will learn other vices. If the handler is expert in this direc- tion, the colt’s leading may be increased, with an occasional brush at nearly the limit of its speed, but do not overdo it. Let the colt have his head and trot as naturally as possible. O. H. Sholes is one who does not believe in O. H. Sholes is one who does not believe in breaking a colt to lead beside a pony. He says: “Tt is time thrown away. It is effort in the wrong direction. We drive horses in races, we do not lead them. It does not add to their value, but diminishes their worth. It costs money to make speed beside a pony and you have to do it all over again when the time comes for driving. Leading Beside Pony. I don’t think a colt should be taught to follow.” Roy Miller writes: “I am not in favor of lead- Roy Miller writes: “I am not in favor of lead- ing colts, never was, and I don’t believe I ever will be; however, a great many of our very best colt CARE AND TRAINING OF TROTTERS. 24 trainers are advocates of this method, and as you say, if carefully done, and within reason, by some one that understands his business, good results can be expected.” Booting. Now comes the disputed question of when to boot a colt. One colt man writes: “Before being led at any speed the colt should be fitted with a few boots for protection against injury. Put on shin boots in front and behind, quarter boots in front and scalpers behind. If the colt has not been shod, then in place of scalpers use a small rubber bell-boot behind. In booting remember that an ounce of prevention is worth a pound of cure. If the little fellow once hits himself he is apt.to lose confidence. It is, better to put on too many boots than too few. When the colt is turned in paddock, put on front shin and quarter boots.” Another man writes: “Don’t get too anxious Another man writes: “Don’t get too anxious to put on boots. When a colt gets sick because it has scalped a little, then it is time to inject a little new blood into your great stable. When you see marks.on your colt’s feet, call them to the atten- tion of your blacksmith, or write the shoeing editor of The Horseman. The chances are the angle of the foot is wrong.” Ned McCarr says: “I never put boots on colts Ned McCarr says: “I never put boots on colts or shoe them until they are perfectly broken and ready to be trained for speed. This idea of boot- ing and shoeing a colt, that is not perfectly broken, THE WEANLING COLT. 25 is, to my mind, a bad one, as they cannot hurt themselves if they are not shod, and the boots are a decided hindrance to freedom of action, and are apt to give a wrong.idea as to How the colt is gaited. I prefer to train them in the afternoon, turning them out in the morning and then handling them after they are brought in. In this way they are not apt to be so frisky and consequently be- have far better, and there is less-danger of their being injured.” While the colt is receiving its lesson (which While the colt is receiving its lesson (which is usually in the morning), have its stall cleaned and bedded and a little hay thrown in for it to nibble at on its return. Booting. Let the colt stay in the stall about an hour, or until it is entirely cooled off, and then turn it out in a paddock for a féw hours. The colt’s feet will need attention. One horse- The colt’s feet will need attention. One horse- man’s advice is: ,“Keep: hoofs rasped to proper angle and level once a month.” Some authorities insist the colt’s feet should Some authorities insist the colt’s feet should be cleaned with a foot pick every morning, others object to using a pick, but agree that the feet should be handled. The preponderance of. opin- ion is with the former method. Nature requires a certain amount of moisture in the foot, and if colts stand on dry ground, some horsemen advise packing the feet, at least three times a week, with some kind of hoof dressing. Some use clay, while others object to it, because it draws out the nat- CARE AND TRAINING OF TROTTEHRS. 26 ural oils. One horseman says: “Cut out the oil meal packing, as it was discarded with the 1492 methods. I prefer clay.” A packing recommended by a well-known horseman is old-process oil meal and water mixed to the consistency of bread dough, with any good indisfectant added to pre- vent souring. This packing supplies moisture and oil and prevents thrush. Other horsemen object strenuously to packing of any kind on colts. One horseman writes: “Don’t pack the colts’ feet. If you can’t think of anything else to do, sit down and smoke. If you don’t smoke, play with the dog.” Another horseman writes: “Outside of the feet being kept’ properly trimmed and kept clean, we do not use any packing or hoof dressing until after they are shod. Nature provides for this and a foot will keep in a good, healthy and soft condition, without any artificial methods, until the hoof is shod. Then it is time to provide the necessary moisture, that the shoeing takes away.” Bitting. A prominent colt man writes: “I bit all my colts with a halter, using a double snap, with one snap in the halter ring and the other in the bit. This can be used for half an hour in the morning and evening. After the colt has become used to the bit, then by all means use a bitting harness, first for twenty minutes to half an hour morning and evening, and, after a few days a little longer, THE WEANLING COLT. 2 and it isn’t a bad idea to turn a colt out in a pad- dock for a half an hour with it on.” , Joseph L. Serrill writes: “I think looking after the colt’s teeth a very important thing. If a colt fights the bit much, look at his mouth, and you will probably find a sharp tooth has cut his cheek, which is very sore.’ Ground Breaking. w ready to be grou The colt is now ready to be ground-broken, that is, broken to harness. Get a harness that will fit it properly and put it on carefully and slowly. Let the colt stand.in the stable with the harness on a short time each day till accustomed to it. Then the colt should be driven in the barn. Have two men at first, one to lead the colt, the other to hold the reins. One horseman objects to the preceding sentence. “Don’t have two men to handle a poor little colt. A fifteen-year-old boy can give a colt its first lessons, or one man can, if he is fearless and not too heavy in the arms and-doesn’t make a sled of his feet,” Teach the purpose of reins, that is, to turn in either direction, or to back. It is im- portant to teach the colt the command “whoa,” and to stand still, especially when tied to a hitch- ing post. Be careful in harnessing not to check your colt too high. Many colts are unbalanced by checking too ‘high. Ned McCarr describes his method of ground- Ned McCarr describes his method of ground- breaking: “After our colts are broken to lead, a harness is applied. Care is taken to see that it CARE AND TRAINING OF TROTTERS. 28 fits snug, especially the bit, which is a leather cov- ered one. One attendant leads the colt and an- other takes up the lines and drives it. We do not have to repeat this performance very often, as the colts can be driven alone, after a. few «ays, depending naturally on the headway made.’’ Joe Heather writes: “After colts are thorough- Joe Heather writes: “After colts are thorough- ly gentle I slip a harness on them, using a com- mon slip-noose halter, with a long lead, and com- mence to break them to drive, using the halter to control them. This avoids bruising and hurting their mouths which are very tender at this time. I do not need any attendant to help me drive them the first time or two as some others have suggest- ed, as they don’t work together all the time, mak- ing more or less confusion with the colts, irritat- ing them unnecessarily and so on. Ground Breaking. w ready to be grou I have never tried to drive a colt under this plan that within thirty minutes I could not drive him almost any- where I wanted to go. Of course this depends on his having been properly handled by the man at the barn. After he is well ground-broken and thor- oughly bridle-wise, there are very, few that will give any trouble when hitched to a light cart.” One colt man writes: “TI hitch my colts to the lightest kind of a low-wheel rubber-tire cart. With an attendant to lead them a short distance; and the driver up, they are started off, and, as a rule, they go on and pay no attention to the rig at.all.” THE WEANLING COLT. 29 Before hitching the colt to cart, some horsemen run two light poles through the shaft. holders and let the colt become used to feeling them along his sides. If.this is done, they claim it is not neces- sary to hitch the colt to a heavy breaking cart. This advice is objected to by one colt man, who says: “Don’t run poles through the shaft holders. Don’t play dog with the colt.” Another expert writes’: ‘Cut out the poles, as we are, at this day and date, breaking intelligent horses, and the broncho methods don’t go. ‘Any colt that is prop- erly ground-broken, and has been driven for three weeks, at the age of a weanling, will be ready to hitch to the lightest bike cart that is made.” Many good colt breakers object to the use of a very heavy cart, preferring a lighter one from which the driver can descend quickly and easily. On the other hand, one man says: “Don’t think about a cart you can get out of quickly, but one you can stay in easily. If you are a coward and too good to die, give up breaking colts. They know when you are afraid and will show you a fast time.” Charles Marvin, in his book, wrote against the use of carts, and advised a skeleton wagon, but later in life he told Al Thomas he had changed his mind and given up the use of the skeleton wagon. Hitching to Cart. The colt is now ready to be hitched up and Hitching to Cart. The colt is now ready to be hitched up and driven. Ground Breaking. w ready to be grou Take an assistant along, as his help will be required in cases of accident. An objection is CARE AND. TRAINING OF TROTTERS. 30 raised to this by a horseman, who writes: “Don’t take an assistant along. It is no time for visiting. If you are afraid of the colt, let your wife drive it the first few times.” Some colt breakers prefer to hitch the colt alongside an older and well broken horse for its first few lessons, but this method is somewhat antiquated and little used’ Some advise carrying a whip from the first, so that the colt will become used to one, but do not be in too great a hurry to use it. An objector to this advice says: “Don’t carry a whip the first few times. The colt doesn’t need whipping.” Be careful about pulling on the lines so as not to make the colt a “puller.” One prominent trainer, in the-early lessons, always uses a rope halter under the bridle, with the rope extending back to the seat of the cart and always pulls the colt to a stop with this, and even guides to certain extent with it. Another trainer says: “Don’t be afraid of the bridle making pullers. It’s the driver who does that.” Do not break a colt before a crowd of specta- Do not break a colt before a crowd of specta- tors. Do not lose your temper. Do not pull the colt over backwards. ; Dr. J. C. McCoy says: “The way to begin breaking a colt is to always have the same man harness it and the colt won't be scared. Let the one who hitches the colt drive it around with the harness on for akout a week and be sure not to hurt its mouth. After the week is up, hitch the colt to a cart and walk it for another week. Never THE WEANLING COLT. 31 going faster than a walk, if possible. When this has been done, the colt is ready for anything, and he will soon show whether,-he is of any account. or not. Above éverything, in breaking the colt, don’t use any check for about.a month.” With the exception of leading beside a pony With the exception of leading beside a pony nothing has been said in this chapter about “mak- ing speed,” as that will be treated in the next installment. Ground Breaking. w ready to be grou — . Shoeing. Shoeing. y reserved We have purposely reserved for the closing par- agraphs of this chapter the subject of shoeing, as authorities differ as to just when the colt should be shod. Some have gone so far as to suggest light front shoes on colts in pasture late in the summer, but this is a theory and seldom, if ever, practiced. Others shoe shortly after weaning, or just before leading beside a pony,.especially when the soil is sandy. Others do not shoe until the spring of a colt’s yearling form. This subject will be treated in a separate chapter by Dr. Jack Seiter. The natural gaited colts will need only light shoes The natural gaited colts will need only light shoes for protection. Double gaited colts require heavier shoes at first, but the weight should be gradually reduced, if possible. In applying weight somé horsemen object ‘to: In applying weight somé horsemen object ‘to: the use of toe weights, unless sure a colt needs. them, and prefer heavy shoes. Toe weights have their uses, they claim, but sometimes get a colt to hitting his elbows. On the other hand, Roy Miller CARE AND TRAINING OF TROTTERS. 82 writes: “TI consider toe weights one of the most important inventions made to assist trainers in the training of horses ofall ages, and especially colts. It is safe to estimate, I should think, that fully go per cent of thé progressive trainers of to- day use toe weights. A heel weight (or loaded heel boot), is just as essential for some colts, and has been fised with a great deal of success here at Lexington, for the past half-dozen years.” In cases of defective gait, a colt handler must use his own judgment or consult an experienced farrier. It is just as necessary to have the advice of an experienced farrier in cases of defective gait, as it is to call a veterinarian when a colt is sick. The colt’s feet should be trimmed regularly. Finally, remember, in the words of Charles Finally, remember, in the words of Charles Marvin: “There is nothing more senseless and injurious than punishing ‘a horse or a colt for not doing what he does not understand you want him to do.” MAKING SPEED. 33 Chapter 3—“Making Speed.” S)RAINERS are not agreed on when to commence with a colt to “make speed.” Some train- ers do considerable speed work in the fall, right after breaking, but the majority wait till the spring of a colt’s yearling form. In these articles we. are assuming that a colt is being trained for the futurities. In the last chapter our colt had been broken to S) S) In the last chapter our colt had been broken to drive. After breaking the colt should be jogged. until it is accustomed to all strange sights and ex- periences. This jogging does not call for any speed, But sooner or later the time comes for speed work, or “making speed.” The old way was to drive a colt for a certain distance, say half a mile, at a slow gait, and on each succeeding occasion drive him a little faster. This system is still in use but has been superseded to a great degree by the “brush” system as introduced by Gov. Leland Stanford and made popular by the success of the colts trained at Palo Alto Farm by Charles Mar- vin. Marvin is often credited with having intro- duced: the brush system of training but in his book he tells us that it was in use when he be- came connected with the famous California estab- lishment. CARE AND TRAINING OF TROTTERS. 34 Before proceeding with an explanation of the brush system, the opportunity here -presents itself to mention a few hints about the hitching and driving of a colt for speed work. Pulling. “No. foot, no horse” is an old and true axiom, but “no mouth, no horse” is just as true. Great care should be exercised not to spoil a colt’s mouth. Do not teach a colt to “pull,” by “taking too strong a hold” on the reins. Drive with a light hand. Hold the reins just tight enough to “steady” the colt in his gait and to prevent stum- bling or swerving. (It will be understood that these remarks refer to colts just being broken. An old-time confirmed puller cannot be driven with.a loose line, unless one is lucky enough “to break him” of the habit—Ed.) If you take too strong a hold the colt will “fight the bit,” or “lug,” or become unbalanced in gait or acquire a bad temper. The habit of pulling is sometimes caused by checking the colt too high. Many successful trainers do not use a check until the colt has had several weeks of speed work. When the check is put on let it hang a trifle loose at first, adjustment can be made later as experience demands. If the colt starts to pull when you jog him, bring him to a walk. Then start him up again slowly. Re- peat this till he learns to jog without pulling. © MAKING SPEED. 35 It is important to teach the colt to trot at uni- form ‘speed on a lightly held line, when started at a certain gait. An intelligent colt will soon learn this habit. A colt should be taught to eer to the voice, A colt should be taught to eer to the voice, so that when he shows an inclination to “take the bit in his teeth,” the driver can “talk him back.” As a colt’s speed increases a-firmer grip on thé reins will be necessary to steady him in his gait, and keep him in his stride—but don’t pull. It is important for the driver to learn how to It is important for the driver to learn how to “catch” a colt when it makes a break. Marvin wrote: “My plan is to give him a square pull back, and swing him very slightly to one side, giv- ing him a chance to catch in the cross stride.” “Catching” is an accomplishment which can be acquired only by practice, certainly not through written advice. Pulling. Don’t lose your temper when a colt leaves its Don’t lose your temper when a colt leaves its feet, don’t jerk nor snatch nor see-saw. It is hardly necessary to advise against teaching a colt to be a “handy breaker.” This was an old time idea’ that happily has. been practically discarded. Do not allow the colt to learn the side-pulling Do not allow the colt to learn the side-pulling habit, which may be caused by an uneven hold on. the lines, or by poor teeth, or by too large a bit. If a colt starts to hitch, scalp, or forge, correct If a colt starts to hitch, scalp, or forge, correct the evil before you go on with its speed lessons. As to a whip, don’t carry one unless you know As to a whip, don’t carry one unless you know how and when to use it. CARE AND TRAINING OF TROTTERS. 36 With these preliminary remarks we will pro- ceed to take the yearling to the track for a speed lesson on the brush system; but first let us warn the driver not to start in speed work until the colt is properly hardened and “legged up” for the coming ordeal. Inasmuch as Marvin was the great exponent of this system we will quote his own words: “Colts need practically no jogging, yearlings cer- tainly none whatever. Of course no colt or horse should be worked soon-after a meal. You will find the yearling (althougha trifle nervous) ready to work as soon as you get him on the track. Start him up at a good, fast jog for about 150 to 200 yards. Then turn slowly, giving him time to get his breath, and let him brush back a little faster. After going about the same distance, stop again, turning slowly, and send him back again, this time carrying him right up to his clip at.some point of the brush, preferably near the end of it. In all his work, especially when the brushes are sharp, be careful to let him get his wind at each turn, and after this sharp brush that I have just described give him a little longer to breathe than you did before. Pulling. Now straighten him out and brush him up the stretch again about the same distance, going up to his clip about the last of it, and that will be enough work for that day. “Take him in where no cold draft can blow on him, “Take him in where no cold draft can blow on him, and take off the harness and boots. Give him a swal- low or two of water, rub him off lightly, and let the boy walk him a little, then put him in his box and leave him undisturbed, so that he can lay down, as a colt youngster will, and rest. “As I have said, young colts require little jogging “As I have said, young colts require little jogging aud no sweating or scraping. Young animals do not take on fat internally like matured ones, and there is in fact no Superfluous flesh in this rapidly growing period. The colt requires not to be reduced, but rather to be. made stouter and stronger. Physicing, sweating and scraping are just the things no colt can MAKING SPEED. 37 take and thrive. It stops his growth and muscular development to strip him of his flesh, for the growing body, the maturing muscle and bone, need that nour- ishment which is only afforded in a condition of marked thriftiness. Only in this condition will the colt be in good fettle and spirit, and capable of taking his work with relish and being benefited by it. it therefore behooves the trainer to watch constantly that the colt does not go back in condition) for this loss of condition may be at first almost impercept- ible. It is all the better if the colt carries a fair de- gree of flesh, which will not be of the soft kind with the work here prescribed. Keep him in good, vig- orous condition, so that he will perspire freely with work, but leave heavy blankets, hoods, sweating and scraping alone. “The first day’s training in harness should be ad- hered to without any increase for the first ten days or so. From four to six brushes will be sufficient at first, but in say, two weeks, it can be increased a lit- tle. Pulling. Don’t increase the length of the brushes, but the number and speed of them, but this increase must be slow and gradual, according to the size and capacity of the colt, and the relish he shows for the work. “Tt is a good plan to let the colt up for two or three “Tt is a good plan to let the colt up for two or three days, every three or four weeks, for a run out and a rest. This will freshen him up, and these breaks in the monotony will, if he is not overdone or harshly worked be an effective preventive of track sickness and staleness. After each little let up he will go to work again with more keenness and vim. Barring these rests, the colt’s work will go on every day— Sundays excepted—presuming that he has been kept well and right. When he is two years old he will take more work, but not a greatly increased distance. I am not prepared to say that the length of the brush should ever be increased to over a quarter of a mile. We are now, mark you, working our colt for speed. You will, no doubt, inquire how a horse can trot a race without being worked mile heats. You cannot cut much of a figure in a race without speed, and, after you have developed speed sufficient to go away from home with, it will be time enough to condition him to carry it. You must have the speed before you CARE AND TRAINING OF TROTTERS. 38 can win races. It is of no use to condition your horse to go mile heats, if you haven’t first got the speed to beat somebody else. You will see, then, that the Palo Alto system proceeds on the logic of the author of the recipe already quoted for cooking the hare: First catch your hare. We aim to first de- velop the speed, and after that to condition the horse to carry it. The merit of this system of training in short, sharp brushes lies in the fact that it is the quickest and most effective way of at once toning up and hardening the muscles, and bringing out a high rate of speed—of teaching the colt to trot fast.” Marvin’s book was published in 1890. Pulling. We have always considered it unfortunate that he did not publish a revised edition before his death, be- cause he changed a number of his methods before his death. However, he never discarded the brush system explained above. Marvin’s book was widely read—especially by owners, who tried ty pass along their absorbed knowledge to their trainers, with sometimes laugh- able results, so that the term ‘Marvin-book- trained-owners” became quite common. As time flew by Marvin’s book and other contemporaneous works became out-of-date. There was a call for a modern treatise on the subject of training trot- ters, which we are endeavoring to fill by the pub- lication of this book. By combining the ideas of practically all of the various authorities on the subject, we hope to escape the criticism which has been heaped on past effort. Special care should be taken at this period of a colt’s life to see that it has plenty of water, MAKING SPEED. 39 Fix a hoop in the corner of his stall so that the water bucket may be kept at all times in reach -of the colt. Naturally, you will not allow him to drink much after a work-out until he is cooled off, While it is not wise to load a colt up with boots, he shoitld have enough for protection. You will doubtless have discovered, during the first lessons to cart, what -boots he will need. Be especially careful the colt doesn’t speedy-cut otherwise he will get to going “sideways” and in an otherwise bad-gaited manner. Do not put the boots on too tight, but as snug as possible, without interfering with circulation. Of course, the boots should be cleaned each time after they are used. If, in breaking, the colt has not been taught If, in breaking, the colt has not been taught ‘to stand still, while being hitched or unhitched, you had better complete that neglected part of his education before doing much speed work. In unhitching be sure everything is loose before backing the cart away. Horsemen disagree about when to start ban- Horsemen disagree about when to start ban- daging’a colt. One collaborator writes: “After speeding or jogging a colt I always apply leg wash, then wrap the legs in cotton and put on a set of bandages. Pulling. After the colt is thoroughly cooled out, the bandages should be removed and the legs brushed out and given a good hand rubbing of about fifteen minutes to each leg. CARE AND TRAINING OF TROTTERS. 40 Brush the colt off and then put him away in cotton. Be sure and pack his feet, for they are growing and developing and need plenty of moisture. The bandages should be changed again in the evening and legs given a good, hard rubbing.” ie “As to blanketing,” one horseman writes, “the guy at the wheel is supposed to know when to blanket. I always ‘put on a blanket and hood on cold, windy days. Never allow a direct draft to hit the colt when he is in a heated con- dition.” We have riow given the essential features of speed work. The subject might be extended in- definitely, but further details would make our re- marks too cumbersome. _ As Charles Valentine very expressively set forth: “This thing of be- ing able to find out in a book how to break, shoe, train and feed a colt is all a frost. As you know, you can’t handle all colts alike. The first thing owners should do with a colt ready for speed de- velopment is. to send it to a first class trainer.” The man who wants to win a futurity, and who can afford it, should either hire a good trainer, or send his colt to one. But if an owner cannot afford this, or if he is one who owns colts for the pleasure of training them himself, he must carefully study what others have done. and apply the knowledge gained to his own colts to the best MAKING SPEED. 41 of his ability. He must never do anything with a colt unless he knows why he is doing it. One of our collaborators suggests that this One of our collaborators suggests that this chapter include a short story of the methods’ of training some of the famous yearlings of both the present and past. We are glad to accept this suggestion, and find our work lessened by refer- ence to Roland Drake’s article in the 1912 Christ- mas number of “The Horseman.” Airdale, 1, 2:1534, the world’s champion year- Airdale, 1, 2:1534, the world’s champion year- ling trotter, was foaled in April, 1911. Pulling. Before he was weaned he had learned to eat grain and was halter-broken. In October, 1911, he was broken to harness, and after twelve hitchings or less was turned out for the winter. He. was in training. (at Lexington, Ky.) where this could be done. Incidentally, without any effort to, “search” the colt; he stepped an eighth in 25 seconds. About March 1, 1912, Airdale was taken up and shod and jogged on the road for a month; then sent to the track for training. He was brushed every other good day, for a short distance, to make speed. On June 25 he was driven a half in 1:1234 and a quarter in :35, and turned out for. a month. After his short vacation he was taken up, and after another month’s training was driven his first full mile in 2:50, about Aug. 25. In the next two weeks he was given three miles better than 2:40, the fastest in 2:2814. In the mean- time, he had been a half in 1:0734 and a quarter CARE AND TRAINING OF TROTTERS. 42 in 33 seconds. On September 18 he was started for a record, and trotted in 2:20, A few days later he trotted in 2:41, then 2:27. On Sept. 30 he was driven a mile in 2:2134, last half in 1 :0634, last quarter in :3214. The next day he was given slow jog work, and the following day (Oct. 2) started publicly to beat 2:20, and trotted a mile in 2:1534, breaking all yearling trotting records. This was the last of his fast work for che year. He was kept up and jogged for a while and let down gradually. Airdale was broken as a wean- ling and trained and driven as a yearling by Hunter C. Moody, who uses the system explained above on almost all of the colts he trains. He does not favor leading colts beside of, or ahead of, a pony. Peter Volo, 1, 2:19, that was the champion yearling trotter for about six weeks, and second only to Airdale, was foaled April 25, 1911. He “was weaned about October 1, and halter broken and led beside a pony. Pulling. The usual custom at the farm where he was foaled is for colts to be grotind broken shortly after January 1, but not hitched in shafts until about ‘March 1, when they are driven through the fields barefooted.. But-in the case of Peter Volo it was expected to sell him at auction in May, so he was not ground broken. but trained beside a pony in the spring. He went to the sale, but was bid in and returned to the farm, He was hitched to a cart for the first time MAKING SPEED. 43 about May 15. He was practically broken the first time he was hitched. The third time in har- ness he was hooked to a bike cart and trotted a quarter in 40 seconds. Before long he trotted a quarter in 36 seconds. Note that he was broken to drive about seven months after Airdale, but soon stepped a faster quarter than Airdale had trotted at the same time: About July 1 Peter Volo was driven a mile in 2:33. In two weeks he trotted in 2:26%, and a few days later in 2:23%. On August 16 he was started to beat 2:3014, and trotted in 2:19, lowering the world’s yearling record, held by Miss Stokes, by one-quar- ter of a second. Later he was driven a half in 1:06, with the last quarter in 3114 seconds. Peter Volo was‘trained and driven by Ed Willis, who was also responsible for Miss Stokes. Hester C., 2:21Y%, was foaled in the spring of 1g1t. She was weaned and halter broken in the fall, then harnessed and driven ahead of a pony, not to make speed, but to teach her how to behave in harness; then she was turned out, without being hitched, shod or booted. About April 1, 1912, she was hitched to a cart and jogged without shoes until ready for speed work. She was given full miles in training, not driven any extremely fast quarters or halves, as her trainer (Henry Williams) believed they take too much out of a colt. She took her record at Lexington on Octo- ber II, 1912. CARE AND TRAINING OF TROTTERS. 44 Wickliffe Curry, who has given records to more yearlings than anyone except Moody, halter breaks as soon as colts are weaned, then has them shod and leads them beside a pony. He ‘boots his colts for protection. Pulling. After being led a few times, he ground-breaks them, and later on, dur- ing the winter months, they are hitched and broken to drive. The speed making comes in the spring. Stewart Chandler handles his colts in much the same way as Curry. Previous to Peter Volo, the champion yearling Previous to Peter Volo, the champion yearling trotter was Miss Stokes, 1, 2:1914%4, that was trained by Ed Willis in his usual manner, as pre- viously outlined. — Previous to Peter Volo, the champion yearling trotting stallion, was Wilbur Lou 2:19%, devel- oped by the late Frank H. Holloway, of Hemet Stock Farm, Hemet, Calif. Wilbur Lou was weaned and halter-broken in the month of De- cember, 1909. When he was nicely halter-broken, he was bitted and ground broken for about a month. He was then hitched and driven a few times, and then turned out. On the 2oth of April he was taken up again and driven for several days before being shod with 6 oz. half round shoes in front, and 4 oz. plain shoes behind. The next day he stepped an eighth in :30%4, two days later in :28%4, and two weeks later in :2214' sec- onds. Up to this time he had not been asked to go further than an eighth of a mile, and in MAKING SPEED. 45 working was not turned around, but kept on going the same way of the track. For the next two weeks, he was repeated quarters every other day. He made speed so fast that through the month of June he was worked only once a week. The 25th of June he trotted a quarter in 39%4 seconds, with an eighth in 18 seconds. He was not worked again until the 6th of July, and on through that month was brushed quarters’ twice a week. July 29th he trotted a quarter in 36%, an eighth in 174%. August Ist he was worked his first mile in 2:55, last quarter in 39 seconds. August 5th, 2:45; August oth, 2:421%4, last quarter in, 3634; August 12th, 2:37%, last quarter in 36. On August 16th he was brushed quarters, one in 3514, and one-eighth in 1744; August 2oth, mile in 2:50; August 25th, mile in 2:40%4; Sept. Ist, mile in 2:30%; Sept. 8th, mile in 2:281%4; Sept. 12th, 2:45; Sept. 16th, mile 2:33%; Sept. 19th, mile 2:2734, last quarter in :36. Pulling. All this work was over the farm half mile track. The last mile was 634 seconds faster than the world’s half-mile-track yearling trotting record made the same year by Benear. This concluded-his work at home, as he was shipped to Phoenix, Arizona, where he got his record. His first workout on a mile track was in 2:24%4. A few days later, on Nov. 8th, he trotted a public mile in 2:23, equaling Adbell’s record. Three days later he trotted in 2:19%4, with the quarters in :3434, working was not turned around, but kept on going the same way of the track. For the next two weeks, he was repeated quarters every other day. He made speed so fast that through the month of June he was worked only once a week. The 25th of June he trotted a quarter in 39%4 seconds, with an eighth in 18 seconds. He was not worked again until the 6th of July, and on through that month was brushed quarters’ twice a week. July 29th he trotted a quarter in 36%, an eighth in 174%. August Ist he was worked his first mile in 2:55, last quarter in 39 seconds. August 5th, 2:45; August oth, 2:421%4, last quarter in, 3634; August 12th, 2:37%, last quarter in 36. On August 16th he was brushed quarters, one in 3514, and one-eighth in 1744; August 2oth, mile in 2:50; August 25th, mile in 2:40%4; Sept. Ist, mile in 2:30%; Sept. 8th, mile in 2:281%4; Sept. 12th, 2:45; Sept. 16th, mile 2:33%; Sept. 19th, mile 2:2734, last quarter in :36. All this work was over the farm half mile track. The last mile was 634 seconds faster than the world’s half-mile-track yearling trotting record made the same year by Benear. This concluded-his work at home, as he was shipped to Phoenix, Arizona, where he got his record. His first workout on a mile track was in 2:24%4. A few days later, on Nov. 8th, he trotted a public mile in 2:23, equaling Adbell’s record. Three days later he trotted in 2:19%4, with the quarters in :3434, CARE AND TRAINING OF TROTTERS. 46 13434, :36 and :35. The same system was used on Harry R. (1) 2:24% and Hemet, p., (3) 2:08. 13434, :36 and :35. The same system was used on Harry R. (1) 2:24% and Hemet, p., (3) 2:08. Pulling. Previous to Miss Stokes, the world’s champion yearling was Adbell, 2:23, that took his record at San Jose, Calif., Sept. 27, 1894, driven by Walter Maben, although his speed was developed by John S. Phippen. We know little about the method in which his speed was made. His first start was on Aug. 17, when he won a dash on a bad day and on a slow track in 2:28, a new record for a. yearling colt in a race. On Aug. 27, in another dash, he won in 2:26, further reducing the. -year- ling race record, and also the yearling stallion record of Athadon, 2:27. On Sept. 27 he trotted a mile against time in 2:23, quarters in :36, :36. 3554, :3574. Previous to Adbell, the champion yearling trot- ting stallions were Athadon, 2:27, driven by Matt Dwyer at Stockton, Calif., Nov. 28, 1891, and Freedom, 2:2934, driven by John A. Goldsmith. at Napa, Calif., Oct. 18, 1890. Previous to Adbell, the champion yearling trot- ters, without regard to sex, were: Pansy Mc- Gregor, 2:2334, driven by O. M. Keets at Hor- ton, Kas., Nov. 18, 1893; Frou Frou, 2:25%, driven by Millard Sanders, at Stockton, Calif., Nov. 28, 1891; Bell Bird, 2 :26%4, driven by Charles Marvin, at Stockton, Calif., Oct. 21, MAKING SPEED. 47 1891; then Freedom, 2:2934, already mentioned, the first yearling to trot in 2:30. Charles Marvin, the great colt trainer of his Charles Marvin, the great colt trainer of his day, drove three yearling champions to their rec- ords: Hinda Rose, 2:36% (1881), Norlaine, 2:31%4 (1887) and Bell Bird, 2:264 (1891). ' We quote from Marvin’s book, which describes We quote from Marvin’s book, which describes the training of Norlaine. With less than .a month’s preparation, she reduced the world’s rec- ord for her age 4% seconds: “From the day that Hinda Rose made her record of 2:36%4 in 1881 there was no yearling produced in America to threaten that record until the season of 1887, and as long as it was not menaced we made no effort to improve it. But a surprise caine from Kentucky in the year last mentioned, when the deeds of Sudie D. made her famous. * * * ° George Bowerman started her at Lexington, October 15th; and she went the mile in 2:3534. Pulling. When the news arrived that the Palo Alto yearling record had been eclipsed we at once set to work to bring the honor back. The time was short, and we had to pick a good one of our youngsters and push development at high pressure. The most forward of our yearlings ‘was -the filly Norlaine, by Norval’ (present ‘record 2:17%), out of Elaine, -2:20—the fast mare by Mes- senger Duroc, out of Green Mountain Maid. whose history I have already given. She was a rather dull brown in color, a trifle pony-built in some respects. hut with a long, low-set body, short sloping hip of the pacing formation, and low at the withers. Her legs and feet were of the best quality, and she had a level head. Norlaine was not impressive in ap- pearance until you saw her go. She was always fast from her first lesson on the miniature track, and I began working her in April, but gave her only the easiest of work, as the intention was not to start her until she was: two years old. But Sudie D.’s brilliant performance in October changed all - this, “From the day that Hinda Rose made her record of 2:36%4 in 1881 there was no yearling produced in America to threaten that record until the season of 1887, and as long as it was not menaced we made no effort to improve it. But a surprise caine from Kentucky in the year last mentioned, when the deeds of Sudie D. made her famous. * * * ° George Bowerman started her at Lexington, October 15th; CARE AND TRAINING OF TROTTERS. 48 and I then began training the filly in earnest, work- ing her twice a day. In doing this, of cotirse, I took chances of injuring her, and, indeed, of breaking her down. Had we begun earlier she could have beer given more work, and could have been developed to a higher point, with little or no risk, but we never allow such considerations to stand in the way when the suprematy of Palo Alto in colt records is at stake. . The filly took her hard work with relish, and improved under it until November 12th, when we felt that she was equa] to the task of plucking the fresh laurels from Sudie D.’s brow. MAKING SPEED. 49 (Note.—The reader will note a reference to the training paddock system used at Palo Alto Farm. This was an improvement over ‘the ordinary paddock. Corners were rounded off, so that when colts were chased around they would not trot up into a corner and stop. Later two covered tracks were constructed, one 313 feet in circumference, the other 506 feet around. After the colts were thoroughly halter broken they were turned into these tracks and chased about to develop their speed. ‘The Palo Alto miniature track system had quite a vogue, as did the other old-time plan of hitching a colt by the side of a runner, but both methods have practically gone out of use.) Edna the Great, 2:2914, former champion year- Edna the Great, 2:2914, former champion year- ling trotter on a half-mile track, was trained and driven by Dr. W. A. Barber, a‘dentist of Spring- field, Ohio, who finds \| ‘recreation in horses. The following is his own story: “Edna proved to be a bear-cat to subdue, be- ing unbroken when I bought her early in her yearling form. We found it necessary to pad her stall with baled ‘straw, in order that she would not do injury to herself in her attempts to get away from her tor- mentors. Halter breaking and bitting required all of April and a portion of May before we hitched her. She was a broncho when we hitched her. The brush system was not used, as she had all the speed on tap that was‘necessary. She was low in flesh and gentle exercise was all that we aimed to give her for the next few weeks. She was hitched daily for a couple of weeks at a time, then a run in the paddock -for a few days and she began to take on flesh. With an abundance of feed, plenty of grass and a tonic to tone up her system, we started in to give her a mile every morning, very slow at first, with a brush home, gradually increasing the distance that she was stepped at speed until we were going a pretty fair quarter in almost every workout, keeping always in mind not to ask her for more than she could do well within herself, never at any time did I carry her to the extreme limit of her effort, or to the point of exhaustion. Pulling. The trial was made at the Bay Distgict track, San Francisco, and she trotted the mile in 2:311%, a yearling record that has a good chance to last as long as Hinda Rose’s. The time by quarters was :39, :36, :38, :3814.” We will quote also from Marvin’s description of Hinda Rose’s training: “Hinda Rosa was our first youngster that earned fame at_the early period of yearling form. She was foaled February 22, 1880,:and is a brown mare, by Electioneer, out of Beautiful Bells, 2:2934. ° She was well broken early, and in her yearling fem I began working her. -Her serious training began July 5, 1881; I had now gotten well into the Palo Alto system of training, and could work the new fangled ideas pretty skillfully. She was worked on the method described in chapters’ further ‘on, until November 5th, the date of her first public perform- ance. The yearling record was then 2:5634, and at the Bay District Track a set of harness was offered to yearlings to trot against this record. The first trial was made by the filly Pride, by Buccaneer, owned by Count Valensin, and driven by John Gold- smith, who -has since handled Guy Wilkes, Sable Wilkes and other horses so successfully “for Mr. Corbitt. Pride made the mile in 2:44%4.: I then drove Hinda Rose and she went from wire to wire in 2:43%. On the 24th we gave her another trail, when she went in 2:36%, and this stood as the yearling record until 1888, when it was lowered successfully by the Kentucky filly, Sudie D., and our lost. Palo Alto star, Norlaine.” MAKING SPEED. MAKING SPEED. The chief thing in the training of Edna the Great was to be able to say ‘whoa’ often enough, as she had ambition enough to try to beat any horse on the track. It was not very long before she began to go miles. A mile around three minutes seemed a romp for her, and she was given a mile CARE AND TRAINING OF TROTTERS. CARE AND TRAINING OF TROTTERS. 50 every morning that we had favorable weather con- ditions, with a brush at the end of the mile. A little later in her work we began to take her down to the eighth pole at a good, stiff clip, then ease her up to within a short distance of the wire and let her step a short distance at the end of the mile. “She was worked very early in the morning in “She was worked very early in the morning in order that she would have a light rub, and then walked through the dewy grass and allowed to have a good lunch of grass; and made an effort to have her legs well bathed in the cool dew each morning when it was at all possible. She never had a bandage on, and her legs or her general physical condition would not indicate that she had ever worn harness. “She was gradually dropped down in her work to the 2:40 mark and beat that notch upon two oc- casions prior to her record mile. One mile was in 2:33, with the last half in 1:10. On the 29th day of August, at the Columbus, O., State Fair Grounds, upon a track that was exceedingly slow due to rainy weather, she was sent against the record of 2:34% made by Benear at Goshen, N.-Y., with the result well known to all that love the American trotter. “Wer shoeing and rigging was of the simplest kind; in front ‘she wore a 4%4-oz. shoe, no toe weight at any time, with a short toe, ‘and behind she wore a shoe as light as could be made to afford sufficient protection to her feet. “Her harness was plain, breast collar, blind bridle with a nose band attached to a standing martingale, and carried her head levél with her body. Her boots were the lightest that I could procure and she never showed any marks on them.” The present champion yearling trotter over a half-mile track is U. Forbes. Despite a sticky track and high wind he trotted a mile in 2:21%, driven by Hunter C. Moody, at Louisville, Ky., September 17, 1913. The colt was sent away slow, first eighth in :1934. The next eighth was in 17 seconds, making the quarter in :3634. The MAKING SPEED. CARE AND TRAINING OF TROTTERS. 51 next quarter was in :33)4 (a 2:13 gait), making the half in 1:10. The next quarter was in :35%4 and home in :36. Peter Volo, 2:04% (in 1913). World’s Champion Two-year-old Trotter. Peter Volo, 2:04% (in 1913). World’s Champion Two-year-old Trotter. CARE AND TRAINING OF TROTTERS. 52 Chapter 1V—Shoeing Colts. By Dr. Jack SEITER. HEN shoeing a colt I have found that a study of the gait of its parents, when possible, is of great assistance, for, in correcting a fault, it is well to know whether it is individ- ual or hereditary. And before going into the subject of this chapter I wish to register a note of warning as regards heredity of gait. How often have I seen a breeder attempt to produce a colt of good conformation, by crossing a horse of excellent structure (one with which the most exacting judge of horse flesh could find no fault) with a spindle-legged, knee-knocking mare, simply because she was well bred, or had ‘considerable speed. Naturally he figured that the stallion would predominate in this union, and the colt would be of the desired conformation. I have seen this mistake made year after year. The influence of heredity (for bad as well as good) can not be better illustrated. The result is usually a leaning toward the bad; the colt is almost always an animal of faulty conformation in one or more points. Naturally this condition will also exist if we reverse the order of things, and cross an ill structured stallion with a perfectly developed ETT EN met: CA Tintin ETT EN met: CA Tint SHOEING COLTS. 53 mare. The bad will almost always crop out in preference to the good. If more attention were paid to the conformation of both the sire and the dam, we would not be obliged to cope with the large number of misfit animals that are raced to- day. It is not uncommon to hear some of our best horsemen make the remark, that “such a colt has license to be very fast, but he hits his knees, he toes out with one foot,” or some other malforma- tion handicaps him from being a world beater. After several years of training, during which time the horse shoers and the boot makers derive enough money out of him to buy a good animal, the colt is given up as a bad racing prospect. If a filly, she is retired to the broodmare ranks, to produce more of the same type; if a stallion, to do stud service, to fill the country with more trouble makers of the sort that drive prospective owners out of the game. Chapter 1V—Shoeing Colts. By Dr. Jack SEITER. But this thing has been going on for ages, and the chances are that it will continue as long as the breeders insist on breeding their “pets,”’ regardless of conformation, expecting to get perfectly developed animals, that -will do to race and to fix a type of race horse. Many promi- nent stallions, standing at high fees, have been handicapped because wealthy horsemen would in- sist on breeding their worn-out favorite road mares to the stallion then in the lime-light-—Axtell and Bingen are recalled as two examples and there are many more. CARE AND TRAINING OF TROTTERS. 54 First Trip to the Blacksmith. When the colt arrives at the age of two months its feet should be examined carefully. If dressing is needed feet should be attended to at this time. Just as a human baby becomes bow-legged, the colt is liable to be foaled with, or acquire, a faulty con- formation. If the toes are excessively long, they must be shortened; if: the heels are abnormally high, they must be cut down; and if the foot shows more growth on one side than the other, the high side should be trimmed down far enough so that the lower side will also receive its share of the weight and bearing. If the colt shows the slightest inclination of being deformed, knock- kneed, or toes otit, we must dress down the out- side-of the hoof, especially the otitside toe, and it is also advisable, in cases of this kind, to rasp off the edges of the wall at the outer toe, enough to reduce it to the same thickness as the inner one. By following these instructions, atleast once a month, one-can work wonders with a foot of this type. Under no consideration should one apply a knife. to the sole, bars or frog of the foot. Ex- cessive cleaning out of the feet is not advisable, either; naturally we must look after the cleanli- ness of the feet, but, unless there are positive signs of thrush, one must not go to extremes, such as the free use of the foot pick, which is often the cause of forcing filth into the cleft of the frog and the bars, whereas, if the parts are left SHOEING COLTS. Chapter 1V—Shoeing Colts. By Dr. Jack SEITER. 55 intact and filled up with the natural growth of horn that nature provided, it will become almost impossible for the seat of the trouble to become infected. In case of thrush one must not go to extremes in an endeavor to cure it, and cut away the bars and frog. One must try and save all of the frog that is not'infected,.consequently only the ragged edges should be removed, for, by carving out the healthy portions of the frog or bars, we only invite future trouble, in the form of contrac- tion. In the majority of cases the knife is entirely unnecessary, but a good washing out with warm water, to which a good antiseptic solution has been added, will remove the trouble. After this, the foot must be thoroughly dried, generally it will dry out naturally in a few minutes. Then the parts involved, the cleft of the frog and sur- rounding bars, must' be packed with some good antiseptic powder; it is a good plan to force some cotton or oakum into the crevices to hold the powder in place. Several'treatments of this kind generally suffice to cure the most stubborn case of thrush. But, as in all other afflictions that horse flesh falls heir to, an ounce of prevention is worth a pound of cure. Care in Dressing Hind Feet. In dressing the hind feet, it is, as a rule, advis- able to keep the toes short and well rounded off, but the conformation must never be lost sight of. If there is the slightest sign of curby hocks, we CARE AND TRAINING OF TROTTERS. 56 can not cut the toes too short, nor keep the heels too high, and where there is a strong predisposi- tion to this unsoundness, early shoeing is strongly recommended, the shoe to be square toed and set back from the toe, the heels of the shoe to be of a good length and a heel calk turned up on them. Now, the above are simple rules, so simple, in fact, that they are known alike by the humble stable boy and the prosperous owner, but we often overlook small details; consequently, the oftener we are reminded of them, the more apt we will be to remember them. Chapter 1V—Shoeing Colts. By Dr. Jack SEITER. If we overlook the most minute detail, which goes to build up the animal, we will have a:-weakness somewhere, and the chain is as strong as its weakest link only. With the above precautions and attentions ever before us, we will have the proper sort of a foot to work on, when the time arrives for the first shoeing. The first shoes should be applied for protection only, consequently they must be as light and thin as possible, and the nail holes as few as possible, and punched toward the toe, to allow for the natural expansion of the foot. The foot must be leveled with the rasp only, no knife should be allowed to mutilate the sole, the bars, or the frog; if we leave these structures intact, and apply a thin shoe, we do not rob the frog of its function, that of acting as a cushion, not only to the foot, but to the limb as well. The frog is SHOEING COLTS. 57 the one thing that we can depend upon to, keep the foot in its natural elastic state, the sole and bars depend upon the frog to furnish them with moisture, and they in turn, when pliable, protect the structures that are above them. If the frog and bars are left intact, as nature intended they should be, we will not be troubled with contrac- tion, and its sequels, such as corns and quarter cracks. The frog takes care of the entire foot, there is no substitute, that man has discovered as yet, that will take the place of the good, healthy, unmutilated frog as a moisture secreting organ, and never under any conditions, should it be cut into. It is permissible to trim off the ragged edges, and rightly, too, but there are few, indeed, who can resist the temptation to cut off just a little more than is necessary—the idea being to give the frog a symmetrical appearance—to make it take on the appearance of some of the pictures we occasionally see labeled—a natural foot. Chapter 1V—Shoeing Colts. By Dr. Jack SEITER. The fact of the matter is, that a natural foot, un- touched by the hand of man, or his misery pro- ducing tools, is about as unsymmetrical a piece of handiwork as the Creator ever endowed an ani- mal with, yet we attempt to make a model shaped organ out of this crude appearing mass of sensi- tive and insensitive tissue. It is not desirable to interfere with the growth of the foot at all, outside of shortening the wall sufficiently to enable us to get a good level bearing for the shoe. The frog, bars CARE AND TRAINING OF TROTTERS. 58 and sole should not be touched; the more sole we leave, the less danger of bruises and corns we will ‘have to contend with; the more frog bearing we can obtain, the less jar and concussion the foot and limb are subject to, and the less corns and quarter cracks we are liable to have and we pre- serve the natural moisture. This evaporates the moment we apply a knife and open the cells or pores. Hard Frog Unnatural. An animal will go lame if it steps on a pebble or a rock, especially if the sole, bars and frog have been excessively pared out. A frog that has been trimmed to the extent of robbing it of its natural function and trimmed so it is forever kept off of the ground, will dry up.and become as hard as a ‘piece of stone. Most horsemen will admit that a stone will bruise a foot, but it is difficult for some to realize that a dried-up frog is just as hard as a stone. The fact of the matter is, they both do the same damage to the foot, with this slight dif- ‘ference, the stone acts on the sole only, whereas. the hard frog acts on the sensitive structures that underly it, the fatty frog, the preforans tendon, where it runs over the navicular bone to find its attachment on the semi-lunar ridge of the coffin bone and above this the nacivular bone. Can the frog protect those parts when it is robbed of the power to do so? Well hardly. SHOEING COLTS. 59 3s GE. “No foot, no horse,’ “no frog, no foot,’ are two true sayings, consequently we must consider the frog to be a link in the chain in order to have a perfect working animal, all parts must work in unison; if only one and the most insig- nificant structure is out of order, we are in trouble. The chain has a weak link, consequently it matters not how powerful, speedy or game an animal is, when the crucial test arrives, the en- tire structure will be found to be no stronger than its weakest organ. We hate to be told the truth. We do not like We hate to be told the truth. We do not like to have the little things that go to build up the large ones drilled into us, and the majority of horsemen, upon reading’ the above, will say: We know that much ourselves.. Certainly you do— but it’s the things that we know the most about, that we grow careless of; we are too anxious to learn something new, consequently forget the old and fundamental principles of our work. Hard Frog Unnatural. For instance, if a horse becomes lame, it: matters not where, we look for something to cure the lame- ness, a hot iron, or a liniment that may be still hotter. We do not understand the action of them, but they are the things we invariably go after; instead of looking after the little things, things we understand, things that are the direct cause of our troubles, and if any one should en- deavor to explain them to us, we would exclaim, “Why, I know that much myself,” Certainly you CARE AND TRAINING OF TROTTERS. 60 do, but why don’t you use your knowledge before you are in trouble? Changes in Colt’s Gait. The changes that take place in a Changes in Colt’s Gait. The changes that take place in a colt’s gait, after being shod, are due, to a great: extent, to the abnormal changes that the structure of the foot often has to undergo, changes that are not due to the ignorance of the persons in charge so much as to the carelessless. In a natural foot, the sole is perfectly flat, the frog, the bars and the sole all have an equal bearing upon the ground. If we take off just enough of the wall to get a level bearing surface for the shoe, and then apply a thin strip of steel, the thickness not to exceed the amount of wall we have taken off, we will shoe according to nature, or as near as possible to nature; of course it is to be understood that the bars, sole and frog are left intact. But here is the general procedure: the sole is carved out, the bars are also cut out, and the frog is cut away and shaped up, then a shoe is applied that is usually from a quarter to a half inch thick. The moment this shoe is applied, the sole, frog and bars are robbed of their functions as weight car- riers and concussion destroyers, they dry out and become atrophied, and as hard as a stone. The colt is worked, and goes well for the time being, but after a few weeks he shows signs of going rather short gaited, does not extend himself as he should, or as he did when first shod. Again he is SHOEING COLTS. Hard Frog Unnatural. 61 taken to the shop; we all know what the orders will be, do not take a thing off his feet, and apply a still heavier—and naturally thicker—shoe in an effort to improve the action. In this manner the frog and sole are still further elevated from the ground which nature intended it should come in contact with at every step. After this change, we have, following in rapid succession, the dropping in of the quarters, contraction of the feet, fol- lowed by corns and quarter cracks, and, also, the foundation is laid for that dreaded of all foot troubles, navicular disease. When the hard, atro- phied frog comes in contact with a stone or a, rock, and the sensitive structures that it is supposed to protect, with its rubber-like elasticity, are bruised, then there will loom up in the near future, a bloodshot sole, a bruised tendon or navicular disease. REPLYING TO AN INQUIRY. Dear Sir: In a recent issue of The Horseman your advice on colt shoeing was to let the frog, sole and bars have ground bearing, so they would perform their natural duties and retain a healthy con- dition. Now we find cases where such would be impossible’and what I wish to know is this, if the wall, sole, frog, and bars were rasped perfectly flat and a perfectly flat disk of steel, shoe shape, was nailed on, would bad results follow from concussion on sole, bars, or frog. or all three? In case of open heeled shoeing should pressure be allowed between sole and shoe inside of lamni? I ask this because our shoer forbids the least sole pressure and another says without it, the wall will be split loose from the foot.—L. E., Calif. the wall will be split loose from the foot.—L. E., Calif. The horse in its natural state has an equal amount of frog, bar and sole bearing along with the wall bearing, and in nine cases out of ten the average race horse, after wearing his shoes for several weeks, will be found to have the same bearing distributed over the entire surface of the hoof, especially is this the case when the shoe is made of very thin material and devoid of calks. Cases are rare, {ndeed, where the sole does not grow down after a few weeks of shoeing, so that it is perfectly even with the wall, and shoe, it matters not how much of an effort we may make to prevent or avoid this condition by excessive paring out the sole, the bars and the frog. It is not good policy to attempt to rasp the wall, sole and frog perfectly flat; but it is good practice to rasp the sole, wall and bars level, but in all cases we must strive to preserve the frog, every particle of it. It matters not how much froz pressure we obtain, the more the better. A Natural Dressed Foot. foot is dressed in the pro A Natural Dressed Foot. foot is dressed in the pro When the foot is dressed in the proper manner, and, after it is shod, receives the proper attention and care—it is just as essential, or more so, to keep the feet of a colt soft and pliable, as it is to be- stow that care on a race horse—it will be found that not one-half of the weight usually applied is necessary to balance a colt. Now this may seem a broad statement to make, but it is a fact, as I have discovered during twenty years’ work with light harness horses, and considerable of that time was spent shoeing colts and taking care of their feet, CARE AND TRAINING OF TROTTERS. 62 on some of the most prominent stock farms in the country, and farms are the ideal school for any one interested in this work. No, what I say is not theory, but fact, as the average horseman or horseshoer is well aware. They all know better, but they often overlook the small details, looking for the larger ones. Instead of getting at the seat of the trouble, the cause as it were, and removing it, they entirely overlook it in their endeavor to correct gait with new-fangled shoes, toe weights, pads, bits, straps, and the like. REPLYING TO AN INQUIRY. This idea, or rather notion, that we save the sole and frog from concussion by cutting them away so that they cannot The horse in its natural state has an equal amount of frog, bar and sole bearing along with the wall bearing, and in nine cases out of ten the average race horse, after wearing his shoes for several weeks, will be found to have the same bearing distributed over the entire surface of the hoof, especially is this the case when the shoe is made of very thin material and devoid of calks. Cases are rare, {ndeed, where the sole does not grow down after a few weeks of shoeing, so that it is perfectly even with the wall, and shoe, it matters not how much of an effort we may make to prevent or avoid this condition by excessive paring out the sole, the bars and the frog. It is not good policy to attempt to rasp the wall, sole It is not good policy to attempt to rasp the wall, sole and frog perfectly flat; but it is good practice to rasp the sole, wall and bars level, but in all cases we must strive to preserve the frog, every particle of it. It matters not how much froz pressure we obtain, the more the better. This idea, or rather notion, that we save the sole and frog from concussion by cutting them away so that they cannot SHOEING COLTS. 63 come in contact with the ground is all nonsense, pure and simple. ) If an animal were not supposed to have frog and sole pressure, why is it almost the universal rule of all of our foremost drivers and horsemen to invariably shoe with a leather pad under the shoe and then pack the space be- tween the sole of the foot and the pad with hoof ointment and then lay several layers of oakum.upon it? This is gen- erally packed in as snug as possible. Do they not do this to avoid concussion by distributing the blow of the foot, as it hits the ground, over the entire sole surface? Cer- tainly they do, otherwise what would be the use of the pad and packing? Would it not be just as well to carve out the frog, sole and bars and then not use the leather and packing? CARE AND TRAINING OF TROTTERS. When the job was completed the sole, frog and bars were left intact, in identically the same manner as though the animal were bare-footed, the function of the shoe being merely to prevent breakage of the wall. Years ago, especially in the old countries, the Charlier shoe (named, no doubt, after its inventor), or more often a “tip” after the same pattern, was widely used. It was made out of very narrow material and instead of being hailed upon the wall of the foot, as is the custom now in vogue, there was a groove cut out of the wall extending in and up for about three-eighths of an inch, depending upon the size and strength of the wall, and into this groove was fitted the shoe, or plate. When the job was completed the sole, frog and bars were left intact, in identically the same manner as though the animal were bare-footed, the function of the shoe being merely to prevent breakage of the wall. This shoe was and is successfully used on hunters and jumpers. Our trotters have only their own weight to earry and the concussion naturally depends greatly upon the action of the animal. But when we take a hunter and put a hundred and fifty pounds, or more, upon: his back and then ask him to take a four or five-foot fence, it can readily be seen that the possibilities for concussion are greatly magnified, yet we hardly ever hear of one of this kind being laid up on account of bruised feet. This shoe was and is successfully used on hunters and jumpers. Our trotters have only their own weight to earry and the concussion naturally depends greatly upon the action of the animal. But when we take a hunter and put a hundred and fifty pounds, or more, upon: his back and then ask him to take a four or five-foot fence, it can readily be seen that the possibilities for concussion are greatly magnified, yet we hardly ever hear of one of this kind being laid up on account of bruised feet. In England they have races for ponies that stand fourteen hands high. In order not to exceed this heighth it is often necessary. CARE AND TRAINING OF TROTTERS. CARE AND TRAINING OF TROTTERS. 64 taught to dress the foot with a rasp only. In our own army the rules say, you must not, under any consideration, touch the sole, the frog, or the bars of the feet with a knife. In the veterinary colleges (very few, 1 am sorry to state, make any pretense of giving this all important structure its due share of study) they also teach us that by applying a knife to the sole, bars and frog of the foot we rob it of its natural function and elasticity and its natural moisture secreting qualities. taught to dress the foot with a rasp only. In our own army the rules say, you must not, under any consideration, touch the sole, the frog, or the bars of the feet with a knife. In the veterinary colleges (very few, 1 am sorry to state, make any pretense of giving this all important structure its due share of study) they also teach us that by applying a knife to the sole, bars and frog of the foot we rob it of its natural function and elasticity and its natural moisture secreting qualities. If a foot is dressed perfectly’ flat and the frog and bars are left in their natural state, the chances for concussion are remote indeed. The foot lands upon the heels. The frog. if in its natural state, breaks the concussion. There is no further chance for concussion after tne foot comes in contact with the ground, for after it strikes the ground on the heels and frog the rest of the movement is simply a continuation of a roll until the foot again leaves the ground. . Years ago, especially in the old countries, the Charlier shoe (named, no doubt, after its inventor), or more often a “tip” after the same pattern, was widely used. It was made out of very narrow material and instead of being hailed upon the wall of the foot, as is the custom now in vogue, there was a groove cut out of the wall extending in and up for about three-eighths of an inch, depending upon the size and strength of the wall, and into this groove was fitted the shoe, or plate. REPLYING TO AN INQUIRY. It may do, to the theory of some, but if there were any merit in this procedure, why do not the leading horsemen shoe in this manner? : The old-time trotting horse men, and shoers, too, can vouch for the fact that the old-timers were more in favor ef paring out the soles of their horses feet, in fact a far- rier’s reputation depended to quite an extent upon his ability to do a nice job of paring. By this is meant to see hew much of the frog, sole and bars he really could cut out without drawing blood.. But history tells us that the eld-time racer invariably had foot trouble of some sort or other, generally corns, quarter cracks and toe cracks were common, and contraction and its subsequent sequel, the dreaded navicular disease. There is no denying the fact that excessive paring of the sole ultimately hastens the above named foot diseases. If our colts had their feet dressed in the proper manner from colthood up and were shod according to nature, the entire structure would be pliable, as nature intended it~ should be, without artificial hoof packings and oils, and as it is when untouched by the hand of man. No one ever saw a hard frog or sole in the foot of an No one ever saw a hard frog or sole in the foot of an animal that had plenty of frog and sole pressure. The action of the frog supplies the needed expansion to the hoof at every step and this action necessarily keeps the bars and sole in constant activity and consequently pliable and resistant, to concussion. ‘ In all the schools for farriers, especially those of the foreign countries (and there is no denying the fact that they are far advanced and pay more attention to the foot of the horse than we do, as a general rule) students are CARE AND TRAINING OF TROTTERS. to go to extremes in dressing the feet, the wall, at times, being rasped considerably lower than the sole, and then the thinnest possible piece of steel is SHOEING COLTS. 65 applied, often the Charlier shoe or ‘‘tip’’ being used. I have it from reliable information that invariahly the feet of these ponies that undergo this shortening process are the best and are free from corns, bruises and quarter “cracks. I know of a large teaming stable in Chicago, the owner of which is a graduate veterinarian, and he tells me that the best feet on many of his horses are those that occa- sionally lose a shoe while on the road, his orders to the drivers being, drive to the nearest shop and have an old flat Shoe tacked on to the foot, and do not allow the smith to dress the foot in any way. Especially is the use of the Knife forbidden. Those horses travel over the rock paved streets on their frogs, bars and soles, they do not go lame or sore, simply because those parts are left intact and able to perform their duty as nature intended they should. Of what use is a pad under a light shoe? Some say a pad covering the foot and packed with oakum only adds to the concussion of the sole. Others say it breaks concus-~ sion, so there you are. I believe that outside of holding moisture to keep the sole of the foot soft that pads are, in most cases, unnecessary. If the foot were dressed as it ought to be from the beginning, and the sole, frog and bars left intact, the use of pads; especially the full pad, could be dispensed with. The structures named would secrete sufficient moisture naturally, that is if they ‘are permitted to fulfill their function as weight carriers and concussion destroyers. I believe that where a pad is indi- cated, on account of the track being extremely hard, the rim pad of very thin leather will answer the purpose very well. The animal would get along better yet if we left just a trifle more sole and wall. There is just about as much elasticity in the natural sole and wall as we could expect to find in the average leather pad. CARE AND TRAINING OF TROTTERS. To sum up; I believe, in fact I know from experience, that if we dress the foot of the colt in this manner and keep the wall dressed down at regular intervals, and then follow the same principle when we shoe him, we will have considerably less of the common ailments that the foot falls heir to. On the other hand, I would not advise taking an animal, On the other hand, I would not advise taking an animal, especially an aged one, that has had its frog, bars and sole trimmed out until they were thin enough to give under the pressure of the thumb, and where the frog is dried up and shrunk out of all semblance of a natural frog, and the foot CARE AND TRAINING OF TROTTERS. CARE AND TRAINING OF TROTTERS. 66 is contracted and hard, and attempt to drop this sort of a sole and frog upon the ground, certainly it would not be advisable unless the feet were well softened first, and horse then turned out into some placé where the footing was soft. When a condition as this presents itself, it is best to dress the foot down in gradual stages. I never heard of a case where sole pressure lamed an animal, nor have I ever heard of a wall splitting loose from the foot for the want of sole pressure. Of course there are exceptions to all cases, but generally when we find rare cases of this sort there may have been a hundred ‘and one different causes that have brought them on, causes that probably seem too insignificant for the average horse- man, or horseshoer, to notice in their incipiency. Colorado E,, 2:04% (in 1910),, World’s Champion Three-year-old Trotter, Colorado E,, 2:04% (in 1910),, World’s Champion Three-year-old Trotter, SHOEING COLTS. 67 Chapter V—Preparing for Two-year-old Futurities. E HAVE written of the care and training of. the colt from the day it is foaled till the time: when it is desired to “make speed.” We assumed that the speed-making was to be done in the colt’s yearling ‘form—we have even shown how colts are worked for yearling records. If it is not desired to make speed in a yearling, this part of the colt’s education may be postponed a year or two, at the owner’s option, but even if colt is not to be raced until its aged form it is desirable to “make speed” while it is young and impftessionable, for the colt will be easier to train later.. This chapter is to be devoted to preparing two-year-olds for the futuri- ties. By this we do not mean to advise that all two-year-olds be prepared with that purpose in view, but in case it is desired to train a two-year- old this chapter will be found to contain valuable hints from noted trainers. In many cases the two-year-old that is to be SATE = ie ret SATE = ie ret SATE = ie ret In many cases the two-year-old that is to be trained for the futurities. will have been running out during the winter, especially in southern clim- ates. Some trainers, of whom J. B. Chandler’ is one, do not believe that colts that are to be trained for-the two-year-old futurities should be turned CARE AND TRAINING OF TROTTERS. 68 out at all, but should be kept up all winter, and carefully fed, perhaps jogged a little, and occasion- ally turned out in a paddock. Even if turned out in the fall, it is advisable to take the colt up early (some advise January 1) so that it will be- come well-muscled and hardened in flesh before the spring speed-work commences. Almost every trainer has a different way of working colts, some give no jog work at all (only brushing), some jog a great deal, and others com- bine or alternate jogging and brushing. We will illustrate the various methods by citing specific cases. ‘One prominent horseman, who usually trains in the South and who is too modest to allow the use of his name, writes: “We begin as early as possi- ble and jog and brush otrr two-year-olds, begin~ ning with two mile jogs which include two or three brushes of % mile. Chapter V—Preparing for Two-year-old Futurities. These jogs are gradually in- creased in length and speed until we are jogging four miles and brushing quarters. Then we begin working miles around 3:30 three times a week, dropping down two seconds a week until we are going miles in 2:40, when we begin repeating. Now we work miles in 3:00 and another one in the same time. We gradually reduce the time of both miles according to how the colts progress. We do-not work three heat repeats until about two weeks before we expect to race, and not at all if we do not expect to start.” SHOKING COLTS. 69 J. B. Chandler writes: “I.do not believe in jog work for two-year-olds. I only brush my colts. When the colt gets so-he can brush a quarter in 32 seconds then I commence working miles. I be- lieve in working colts in training a little every good day. I start in miles about as fast as the colt can go without tiring and let the colt drop himself down at successive workouts as he learns to trot. When you have speed enough (a man will have to judge for himself) you can commence working two-heat repeats. I do not believe in three-heat repeats for two-year-olds.” Amos Whiteley writes: “It is my opinion that Amos Whiteley writes: “It is my opinion that two-year-olds should never be prepared for futuri- ties of that age. We bring our two-year-olds along, commencing with them abotit the Ist of April of their ‘two-year-old form, and go right along mannering and jogging them for the ‘first thirty days ; then we commence making speed with them for short distances, say..a sixteerith to an eighth-of a mile, and keep brushing them for about sixty days. We never give them any full miles where they can step, but we do brush them quar- ters, give them slow miles, stepping them the last quarter. What we want is goed three-year-olds, four-year-olds, and five-year-olds, and we do not approve of over-developing them as two-year- olds.” Dr. W. A. Barber writes: “I believe in starting Dr. W. A. Barber writes: “I believe in starting early with a two-year-old, say January 1, so as to CARE AND TRAINING OF TROTTERS. 70 harden. it.up:for the brush work’ later. I believe:in jogging every good day, but as to how’ much.of it, that depends.on the colt. Many. Chapter V—Preparing for Two-year-old Futurities. colts take as much work as an aged horse and thrive on ‘it. .I-begin working. heats as soon-as weather and ‘trackget’good inthe spring. I work every other iday; at first, if colt is strong, starting in with miles from 3:30 to 3:5o:and drop down very gradually. T:.begin’giving slow repeats within 40 days after working a-full mile, the first one very slow; the second: ane the. sarhe to past the: three-quarters, with a brush home. I néver-give three-heat re- ‘peats-until within a few weeks: sels first enanee ment.” : \| ae “James Benyon writes: “An the colts T have been wenbectsd with were worked a little in the fall as a yearling and jogged all winter.. In my opinion, a colt should be taken up in its two-year-old form as soon as possible. A colt has everything 'to learn ‘and the more chance you get to’ school him or- her, tye more‘it 15 bound to learn. The colt should be jogged: every day that is favorable. I never jog a two-year-old over three miles and the next day after working one I usually jog only two miles. We start working them one-heat, every other day, from the middle of March, when the weather per- mits. How fast the colt should be worked at first depends entirely on how fast the colt is. “We usually go very slow miles-and step the last eighth ‘or quarter near its limit, say. miles around 3:00 SHOEING COLTS. 71 to 3:30 to start on.. I would drop a colt from 3:00 in the latter part of March to 2:30 by the first of May. Commence to repeat colt when you-cut down to 2:30, that is by fore part of May.. When we start repeating we usually go the first heat in 2:40 to 2:45 and second heat in 2:30 to 2:35. About dropping one down, I can give you my idea from Sweet Alice’s work as a two-year-old. She worked in 2:30 about May roth, went from there to 2:25 by June 5th. Was working her three heat repeats by May: 20th. Two heats the forepart of week .and three the latter part, altogether five heats a week. She went in 2:22 by June.zoth, going seven heats a week then, three first of: week and four latter part. During Grand Rapids meet she worked in 2:19, at Kalamazoo two. heats. Chapter V—Preparing for Two-year-old Futurities. the same day in 2:19%4 and 2:19, at Detroit,two miles in 2:17% and 2:17, at Cleveland two. miles in 2:18 and 2:16%. Then went to the post the next’ week at Pittsburgh. This is about all I can tell about working colts. You can seldom, work any two colts alike because you seldom see two of the same kind, some want more work and some don’t need as much. Some want to be brushed. a lot and others need very little brushing.” Ed F. Geers writes: ‘Two-year-old colts shoula to 3:30 to start on.. I would drop a colt from 3:00 in the latter part of March to 2:30 by the first of May. Commence to repeat colt when you-cut down to 2:30, that is by fore part of May.. When we start repeating we usually go the first heat in 2:40 to 2:45 and second heat in 2:30 to 2:35. About dropping one down, I can give you my idea from Sweet Alice’s work as a two-year-old. She worked in 2:30 about May roth, went from there to 2:25 by June 5th. Was working her three heat repeats by May: 20th. Two heats the forepart of week .and three the latter part, altogether five heats a week. She went in 2:22 by June.zoth, going seven heats a week then, three first of: week and four latter part. During Grand Rapids meet she worked in 2:19, at Kalamazoo two. heats. the same day in 2:19%4 and 2:19, at Detroit,two miles in 2:17% and 2:17, at Cleveland two. miles in 2:18 and 2:16%. Then went to the post the next’ week at Pittsburgh. This is about all I can tell about working colts. You can seldom, work any two colts alike because you seldom see two of the same kind, some want more work and some don’t need as much. Some want to be brushed. a lot and others need very little brushing.” Ed F. Geers writes: ‘Two-year-old colts shoula Ed F. Geers writes: ‘Two-year-old colts shoula be taken up in February, if possible, and jogged two or three miles every day, except Sunday. After the colt is seasoned he should be brushed a little every other day at three-quarter speed. CARE AND TRAINING OF TROTTERS. Chapter V—Preparing for Two-year-old Futurities. 4 te After working him a month or six weeks that way, he could go an easy mile, twice a week, let- ting him move a little strong at the finish. Along in June, after having several-of these easy miles, he might be repeated, well: within himself, finish- ing a little strong through the stretch. Drop him down a couple of seconds every week. Three or four weeks before his race I would give him three heat repeats to key him up for his race. Care should be taken not to tire the colt ; the main thing is to keep him cheerful. Use an easy bit, handle his mouth gently, and have him drive good and straight on the bit.” Roy Miller writes: ‘“A two-year-old should be jogged every clear day, except. Sunday, from three to six miles, or enough to keep him quiet. After four or six weeks commence giving him slow miles every .other day, with a skip, depending on a colt’s spirits and his ability to take.work. I would train .him from then on just as I did Justice Brooke. Enclosed find a summary of his work.” (This will be presented. later. ) Charles A. Valentine writes: “There are no two colts that can be trained alike. It would: be impossible for any man to tell. you. how to train colts. There are a few general rules—colts should be broken when they are eight months old’ and have nice big paddocks with plenty of grass and plenty of good oats and then have a competent trainer, who will train them according to what SHOEING COLTS. 73 they can stand. My way of handling colts, after I break them, is to keep them going from that time on, according to their condition. No two can be trained exactly alike.” Budd Doble writes: “I have had but very little experience in handling colts, having devoted most of my time to aged horses. However, in my judg- ment, you should commence with the two-year-old as early as possible. As to how far and often to jog, and when to speed, etc., depends very much on the colt, and has to be done entirely on judg- ment. Hardly any two will need the same training.” Jos. L. Serrill writes: “I start in November of Jos. L. Chapter V—Preparing for Two-year-old Futurities. Serrill writes: “I start in November of a colt’s yearling form to prepare him for the two- year-old futurities and jog him every clear week day from three to five miles. I start working heats, twice a week, as soon as the weather per- mits. I work quite a lot of miles at first in 3:30, then drop down two to three seconds. Very soon after I get the colt down two or three seconds a week. After I get him down to 3:15 I work re- peats and after 2:50, three heat repeats.” O. H. Sholes writes: “Nowadays we expect a ten-year-old finished race horse at two years of age, so time is the most essential thing; therefore commence as soon as the colt is born and keep busy, teach it something every day. Ask yourself every day what you have taught the colt that day. The most necessary things are speed, manners, and CARE AND TRAINING OF TROTTERS. 74 condition, Teach it manners first and then. speed, then. more manners, and‘ then more speed. :: The condition -is edsy, it will usually come: itself. To make manners, be gentle and kind, and ‘not‘always too firm. Treat the colt as you would your'son, if he cracks a ‘joke laugh at it. It will be. your turn:to ‘track a‘ jokenext. To-make speed, never tet a colt: know how fast he can go. Drive hiri his best-diten,-butidon’t let him know it. The way to do:'this is by' letting it step fast for an eighth ora sikteenth ; speak to it, tap it with the whip,:and let-it go''for fifty to one huthdred feet,:and take it right back to the clip it was going before it makes aibreak. I don’t believe in making speed by. forc- ing ‘toa break, as many do. Don’t let the colt make a hop or a skip. If ‘it does, and continues, také him!to the blacksmith. . Have perfect bal- ance anda perfect gait. Boot him for protection only and if he should hit himself, don’t wait for him.to wear his boots out (thinking there are more where yours came from) but take colt to the smith. When you think he can step:4n eighth in'the spring in sixteen seconds, take out your ‘watch on him arid ifthe steps an eighth in twenty or. twenty- threezseconds he is a goodicolt. Chapter V—Preparing for Two-year-old Futurities. When you hear of a:colt stepping an eighth iti sixteen seconds the fourth time it was: hitchéd it ‘is usually a lie or they lost.track, f° his workouts. Such talk is misléading to ‘the’ new ‘teamer’and to the’ owner. ‘Along in June I- would work colt two repeats, SHOEING COLTS. 15 about twice a week for a few weeks, say at 2:40 to 3:00; and then go back to short-brushes through July and get more brush. In- August: the work would be quite severe, say 2:40.down to about 2:20. The week before. his race I would'work him to step one mile in 2:14 and ‘then'I would’ be ready to beat Lord Allen-in 2:11. If the.colt came out of his first. race sound, I. would expect him: to: race well the next week,.and'then.I would notbe great- ly disappointed. if he trained off. Mariy of ‘them do, and you must expect it. Possibly I would get another good race out: of him later. Condition is like an ax, once you lose the edge it is-hard to’ get back. Manners in shipping is a big help. Many race horses work good at home, ‘but ‘as soon as loaded on the cars, the stuff is off. Anna ‘Axme 2:081%4,.the futurity winner of 1912,:would.lay down on the cars and snore’ while ‘they -were: running. She was at home ‘wheteéver:.I was. >The futurities. should be won by’ men with. only' one colt, as ‘they have more tire to educate it than we fellows do who have a great many. 1 think all colts should be worked. and raced in bandages. I do not believe in working a two-year-old three “repeats.” ’ Harold.M. Childs writes: “A colt to be trained about twice a week for a few weeks, say at 2:40 to 3:00; and then go back to short-brushes through July and get more brush. In- August: the work would be quite severe, say 2:40.down to about 2:20. The week before. his race I would'work him to step one mile in 2:14 and ‘then'I would’ be ready to beat Lord Allen-in 2:11. If the.colt came out of his first. race sound, I. would expect him: to: race well the next week,.and'then.I would notbe great- ly disappointed. if he trained off. Mariy of ‘them do, and you must expect it. Possibly I would get another good race out: of him later. Chapter V—Preparing for Two-year-old Futurities. Condition is like an ax, once you lose the edge it is-hard to’ get back. Manners in shipping is a big help. Many race horses work good at home, ‘but ‘as soon as loaded on the cars, the stuff is off. Anna ‘Axme 2:081%4,.the futurity winner of 1912,:would.lay down on the cars and snore’ while ‘they -were: running. She was at home ‘wheteéver:.I was. >The futurities. should be won by’ men with. only' one colt, as ‘they have more tire to educate it than we fellows do who have a great many. 1 think all colts should be worked. and raced in bandages. I do not believe in working a two-year-old three “repeats.” ’ Harold.M. Childs writes: “A colt to be trained ’ Harold.M. Childs writes: “A colt to be trained with a view. of-starting in the two-year-old fu- turities. should, in- the first place, have natural speed and-be good headed and good gaited.... I think also that they.should have enough. work as CARE AND TRAINING OF TROTTERS. 16 yearlings to. thoroughly manner them and de- velop and grow them. They should commence jogging about February 1 and, when the footing is good, should be jogged fast, right up to their gait. I do not jog over two miles at first, and never over three miles later. There is nothing so harmful to a colt as slow jogging over a long distance. They get thoroughly tired and sick of the game and learn all the bad habits in the cata- logue. As soon as there ‘is a track in the spring they should be worked the Gov. Stanford (Palo Alto) brush system, working them a little every day, except Sunday, and being very careful to not do too much with them any one day. After a month of this kind of work they can be worked two heats of the brush work, every other day, jogging two to three miles the day between or, what is still better, be turned in a nice grass pad- dock the day between the repeats. I will say here, that. the failures I have seen in the use of the brush system have been because trainers make too much:use of their colts. Chapter V—Preparing for Two-year-old Futurities. They think because they are using the brush system, that they must keep their colts right up on their toes all the time, and they go too far with them, not stop- ping to consider the distance they have been. If they would stop and figure the quarters they have been, it would often be from a mile and a half to two miles at speed. This would soon make a colt’ stale and tired of the game. After three or ' SHOEING COLTS. G7 four weeks of the repeat work at the brush sys- tem they can.then be given two repeats every other day. Start them at 2:50 and drop them down three or four seconds a week, letting them step the last eighth within themselves, but up close to their speed limit. Gradually increase the brush:at the finish of the miles until they can step the last quarter fast, then, later on, increase the fast brush to a half mile and so on, in the same manner as you would prepare an old horse for a race. I think colts should be worked some every other day, or three times a week, but after they can brush a fast quarter or half, the fast work should be limited to about once a week, going the other two workouts of that week say in 2:40 and 2:35 each day, letting them step the last- part of the last heat up near the limit. When you get your colt'to within a month of a race he should have three heats, once each week, letting’ him step the last one within five seconds of: when you think he will have to go, provided he can do it well within himself.” W. H. Smollinger writes: “It seems to me that the trainers should be able to give more practical information than those who, like myself, can only speak from the experience gained by observation. And yet, taking: into consideration that you can count, almost on the fingers of one hand, the trainers that have been successful with colts, and that. their methods are as many and varied as the CARE AND TRAINING OF TROTTERS. 78, 78, religious denominations, it is doubtful if even they can lay down any hard'and fast rules which will be of value. Chapter V—Preparing for Two-year-old Futurities. I apprehend that it all depends on the hand and temperament of the driver and on the colt to be trained. Some years ago I was very much interested in colt training, and took advantage of every opportunity to observe and to question the successful colt trainers. Only one, a Kentuckian, who had a large measure of suc- cess with colts, gave anything like an answer to my question ‘What is the best way to train colts?’ ‘Train ’em like aged horses, sir,’ he said. In answer to my inquiry if such a method did not produce lameness he replied ‘Yes, sir, but it don’t hurt ’em.’ As there seems to be so’ many good ways to train colts, any method adopted, if it happens to be suited to the colt, is like the old lady’s opinion of the doctrine of total depravity— ‘a good thing if well lived*up to.” J appreherid it is after all a question of training the colt to make what he lacks. If it is true, as I believe, that, ow- ing to our advance in breeding, ‘speed is born with the foal’ more frequently every year, it may be that the mile on mile system, with a good stiff brush at the.end, will now produce more useful colts than the brush system. Every farm seems to have an abundance of speed. We seem to have found or stumbled onto a way to produce speed. Useful speed is now what we want. For that reason I think the colt should be in harness all SHOBEING COLTS. 79 winter every day to develop manners and muscle, if he is to be trained for the futurities. Manners and individuality, I believe, will mean more every year. In conclusion I am forced to admit that I know nothing practical about the matter.” The present champion two-year-old trotter is The present champion two-year-old trotter is Peter Volo, 2:0434, that trotted faster than any three-year-old trotter or pacer: or any four-year- old trotter up to his time. After Peter Volo had made his yearling record, he was let down for the winter and sent back to Patchen Wilkes Farm. On January 9 of his two-year-old form he was shipped to Thomas W. Murphy at Poughkeepsie, N. Y: He was carried along slowly. Chapter V—Preparing for Two-year-old Futurities. At Grand Rapids, the week before his start in The Horse- man Futurity, Murphy worked him a mile in 2:1234, last half in 1:05, last quarter in :3134, and back in 2:133%4, last half in 1:04%4, last quarter in :3134. The following Tuesday saw his first futurity. Adbella Watts led to the three-quarters in the phenomenal time of 1:3534. Peter Volo then moved up from trailing and carried her to a break and won by five lengths, pulled up, in 2:09, a new world’s trotting record for his age and sex. In the second heat Murphy began to drive from the word “go,” doubtless hoping to: reduce the world’s record of 2:0734, held by Native Belle. He was at the three-quarters ‘in 1:3534. At the head of the stretch, when about 15 lengths in the lead, he went to a break on a CARE AND TRAINING OF TROTTERS. 80 wet spot where the sprinkler had turned in the track. He soon caught and won by ten lengths in 2:101%. Peter Volo’s next start was in the Horse Breeder Futurity at Salem on August 20. On account of heavy track Murphy made no effort to step a fast mile. In the first heat, while Peter ‘Volo was leading in the stretch, Airdale came up very fast. Murphy sat still, thinking he had the heat won, but Airdale was coming faster than Murphy judged and, before he realized it, he had lost the heat in 2:151%%. Peter Volo won the next two heats easily in 2:1234 and 2:14%4, although he made a break in the first turn in the second heat. The next start was in The Horse Review Futurity at Columbus, O., on September 25. Peter Volo showed slightly lame in warming up. (some hidden trouble in front). He won both heats off in front’ in 2:06%4 and 2:07, although Alma Forbes or Lady Wanetka was after him all the way. The last start of the year was in the Kentucky Futurity. In the first heat Lucile Spier led to the three-eights but made a break, and from there on Peter Volo was never headed, win- ning in 2:09%. Lady Wanetka made him trot the last quarter in 3134 seconds and the two went under the wire, a half length apart, in better than a two minute gait. Chapter V—Preparing for Two-year-old Futurities. In the second heat Peter Volo went out for a record and was never headed in 2:0414, although Lady Wanetka moved up along- side him at the three-quarters in 1:33. Peter: SHOEING COLTS. 81 Volo was given very little fast work between races, The champion two-year-old filly is Native Belle 2:0734 that took her record in the second: heat of the Kentucky Futurity on Oct. 6, 1909, at Lexington, Ky., driven by Thomas W. Murphy. She was perhaps the most wonderful two-year-. old that had then shown. She reduced by three full seconds the mark of Arion, against time, that had stood for 18 years. She was born great because she was not broken till April, only six months previous to her great performance. She had shown fast beside a pony the previous winter, however. Murphy went slow in her training at first, so as not to spoil her, yet by the last of July he worked her a mile in 2:23. About Sept. 15 she was worked in 2:1434, last half in 1:04. The next week she was worked in 2:14144. The following. week she started in her first futurity and won in 2:1334 and 2:1214, equaling, in the last heat, the two-year-old trotting race record of her day. She stepped the final quarter of this heat, against a wind, in 3134 seconds. In the Kentucky Futurity she won the first heat in 2:1234 and was then sent for a world’s record in the second heat. She made the quarters in :33, 31, :31 (middle half in 1:02) and :323%4. The previous two-year-old record, as has been The previous two-year-old record, as has been mentioned, was held by Arion. His mile was in 2:1034, made to high-wheels (no ball-bearings) CARE AND TRAINING OF TROTTERS. $2 against time, on Nov. 10, 1891, over the Stockton, Calif., kite-shaped track, driven by Charles Mar- ‘vin. Samuel Gamble once wrote that Arion fin- ‘ished this mile strong, while Palo Alto and Stami- boul finished theirs, over the same track, “like drunken sailors.” Gamble timed Arion in a race an eighth in 1434 seconds and three-eighths in:47. Arion wore a peculiar six-ounce shoe in front, when he made his two-year-old record. On the inside from the middle of the toe to half way down the side, the shoe was wider and heavier than elsewhere. Chapter V—Preparing for Two-year-old Futurities. Marvin was of the opinion that this side-weight shoe kept Arion from brushing his knees and arms. Incidentally Arion wore al- most all the boots in the catalogue in this record performance, except elbow boots. The quarter time of the record mile as reported in “The Horseman,” was :33!4, 31 (the fastest quarter on the track), .:33%4, 73234 (this quarter was slightly up hill). The first two-year-old trotting colt to lower Arion’s record, was Justice Brooke, 2:091%, that took his record October 5, 1910, as did Native Belle, in the second heat of a winning Kentucky Futurity. He was a late foal (June 2).’He was broken in the fall of his yearling form by Dromore Farm Su- perintendent, A. B. Scott. On March 1 of -his two-year-old form he was turned over to Roy Miller, who then began his duties as farm trainer. SHOBING COLTS. 83 He was jogged on the road till March 25. Then he was brushed on the covered speedway at the farm, in connection with road work until April 10. The next day he was given his first mile over the farm half mile track. The diary of his work- outs (and a diary is a good thing to kéep on all colts) and races is kindly furnished. us by Mr. Miller. May Ie 2:52, an :4214, quarter in :38. ? ? May gi—Brashed in’ nesaway {rain}. June 4—2:30, 2: June 9—2:56, 2: 26, 2: 40%, quarter in :37%. June 13—2:52, 2 BP 2:42, June 13—2:52, 2 BP 2:42, June 15—2: 59, 2:514. June 17—2:46, 2:3534, 2:35%4, quarter in :37. June a ae to mile track at Detroit. June a ae to mile track at Detroit. June 28—2:5: 2:3414, quarter in :35, June a ae to mile track at Detroit. June 28—2:5: 2:3414, quarter in :35, June 28—2:5: 2:3414, quarter in :35, June 30—2:46%, 2 38%, 2:28%, half 1:1334, qua June 30—2:46%, 2 38%, 2:28%, half 1:1334, quarter :36. duly 6—2:49. me pov quarter in 135%. duly 6—2:49. me pov quarter in 135%. : ae : ae July 26—2:49, 2 4: 3444, quarter in :3 sul 28—2:45, 2:80, 2:21, 2: 18% pale 1 208 July 26—2:49, 2 4: 3444, quarter in :3 sul 28—2:45, 2:80, 2:21, 2: 18% pale 1 208%, sul 28—2:45, 2:80, 2:21, 2: 18% pale 1 208%, quarter :33. r in :3 sul 28—2:45, 2:80, 2:21, 2: 18% pale 1 208%, quarter :33. CARI AND TRAINING OF TROTTERS. CARI AND TRAINING OF TROTTERS. $4 October 5—Fourth start, won in 2:11%, 2:09%4. October 10—2:414%, 2:27%. October 5—Fourth start, wo October 10—2:414%, 2:27%. October 18—2:45, 2:40%4. October 10—2:414%, 2:27%. October 18—2:45, 2:40%4. October 17—Shipped home. October 17—Shipped home. . October 19—Shoes off and October 17—Shipped home. : : . October 19—Shoes off and retired for season. Shoes weighed 5% ounces forward, 3 ounces behind. S October 17—Shipped home. : . October 19—Shoes off and retired for season. Shoes weighed 5% ounces forward, 3 ounces behind. S The two-year-old training of Axtell, a cham- pion two-year-old trotting stallion, on both mile and half-mile tracks, was rather peculiar, and will doubtless be of interest. He was driven to a record of 2:23 at Lexington, Ky., Oct. 8, 1888, by C. W. Williams. The following-is an excerpt from Mr. Williams’ own story from the Christ- mas number of “The Horseman” in 1889: “Some time between March 1st and 15th he was: taken up and jogged from four to six miles a day. It will be! hard for any one to believe that he could be made to eat the amount of feed given him during March and April; as, think of a two-year-old being fed five quarts of oats, two of bran, two of carrots, and three’ or four ears of corn, three times a day, and all the coarse feed he would eat. This Is not exaggerated in the least. As the weather became warmer he was fed less, but could not, at this time, trot a quarter in: less than a minute, while I am informed Sunol could, at ‘that time, go the same distance in thirty-five seconds. Up to this time I had driven Axtell but a few times, but as the man that had been: jogging him was sent to Michigan with some mares, I took him‘to work. A first I was not pleased with the way he drove, as he was stubborn, and wanted to have his own way a little too much to suit me. “After driving him two or three times I became disgusted, and and wanted to have his own way a little too much to suit me. “After driving him two or three times I became disgusted, and one day struck him rather sharply with the whip. He squared away and went straight. Chapter V—Preparing for Two-year-old Futurities. r in :3 August. 4—2: pe "2: 34," a (half A 08, ait, :83), 2:24. August hipped De To) August hipped De To) i 29-—2:4714, 2: a? o :28 August hipped De To) 4 i 29-—2:4714, 2: a? o :28Y,, quarter fa 13 August hipped De To) 4 i 29-—2:4714, 2: a? o :28Y,, quarter fa 1344, September 1—8:45%5, 2:33, 2:20, 2:23, half 1 07%, “qu September 1—8:45%5, 2:33, 2:20, 2:23, half 1 07%, September Iga hetween)—2: 343, 2: 30, 2:20, 2:15%4, % in :33%. tember 10—2: tember 10—2: So emer ner £40, 2 2: Ea 2:16% (4%, 1:04%; %, :81%), 2:15%. tember 17-— 2%. t 1 So emer ner £40, 2 2: Ea 2:16% (4%, 1:04%; %, :81%), 2:15%. tember 17-— So emer tember 17-— oe miber i eeoad eect won in 2:27%, 2:22%. oe miber i eeoad eect won in 2:27%, 2:22%. September 21—Shipped to Columbus. Ohio. oe m September 21—Shipped to Columbus. Ohio. September 28—2:42, 2:29, 2:21, 2: 19%, quarter in :3 September 21—Shipped to Columbus. Ohio. September 28—2:42, 2:29, 2:21, 2: 19%, quarter in September 21—Shi September 28—2:42, 2:29, 2:21, 2: 19%, quarter in :30%. Septeniber 24—Third start, 1-2-2 in 2:1494, 2:09%, 2:11% September 28—2:4 Septeniber 24—Third start, 1-2-2 in 2:1494, 2:09%, 2:11%4. September ee ae to ‘Lexington (delayed in shipping). September Septeniber 24—Third start, 1-2-2 in 2:1494, 2:09%, 2:11%4. September ee ae to ‘Lexington (delayed in shipping). Septeniber 24—Th September ee ae to ‘Lexington (delayed in shipping). 3—2:428, 2:28%, quarter in :35%%. ptember ee ae to ‘Lexington (delayed in shipping tober 3—2:428, 2:28%, quarter in :35%%. CARI AND TRAINING OF TROTTERS. far enough and fast enough to convince me he would make a trotter if I developed him’ as-I should. Up to this time I had never worked a-colt that could trot in 3:00, did not know how others worked their colts, and the only thing for me to’ do was to use what little common sense nature had given me. bi “I had for years been quite a pedestrian, practicing a great deal “I had for years been quite a pedestrian, practicing a great deal for pastime, and the exercise. I knew by experience how long it took to get the muscles in condition for hard work, and how sore and lame. it made me after any great effort, be the distance ever so short, and this. after I had supposed I was in condition for this kind: of work. I. also knew to have great speed for a short distance it was necessary to cultivate the muscles for such efforts, and the only way this could be done was to make these great efforts every two or three days, but not too often. I also knew by experience that it was necessary for me to consume plenty of muscle-making food. In fact, I had learned how to condition myself for this kind of work and: how to take care of myself after a great effort. “After considerable thought I decided to work Axtell as I work and: how to take care of myself after a great effort. “After considerable thought I decided to work Axtell as I developed myself and see what the result would be. His road-work was continued, with an occasional brush where the footing was good, and every time I started him up he could go faster than he ever had before. About the 20th of May he was hitched to the sulky for the first time and taken‘ to the track. Up to this time I had no oppor- tunity of knowing how fast he could go, but the first time he was 85 CARE AND TRAINING OF TROTTERS. CARE AND TRAINING OF TROTTERS. 86 SHOEING COLTS. moved to harness I found he could go an eighth in less than 0:20. That was not very fast, still it showed a big improvement over the speed he had shown in the fall. “Being pleased with the colt, I was determined to do the best I “Being pleased with the colt, I was determined to do the best I could with him. He only saw the track about two days in a week; the other days (he was never harnessed on Sunday) being set aside for Jogging on the road for eight or ten miles in an hour. He was driven without a check and always in an open bridle. The days he was given track-work I jogged him about three miles the wrong way of the track, then turned and went the right way about two miles, and started him, up from two to four times in that distance. I would drive him about thirty or forty rods at speed, then jog him a short distance before asking him for another burst of speed. After I thought him in condition I drove him in these brushes about as fast_as he could go. : “During all this time he was fed large quantities of grain and “During all this time he was fed large quantities of grain and all the hay and grass he would eat. About the middle of July he was asked to go his first half-mile and did it handily in 1:15. Ten days from that time he covered the same distance in -1:12, About August Ist he was driven a easy mile in 2:38%4, the first one he had ever gone. In this mile he was brushed four or five times, and the rest of the time only moved along at about a 3:00 gait. This mile was about as fast as he was driven in his work in his two-year-old form, and on August 9th he started at Keokuk, Iowa, in his first race. All of the other starters were three-year-olds. In the third heat, over a poor half-mile track, he distanced the field in 2:31%, and the next morning Axtell’s name appeared in the daily papers for the first time. Since that time none has appeared as often.’’ Chapter VI—Three-year-olds. HE training of three-year-olds does not differ greatly from that of the younger colts except, with EE MTT eS = Ch : added age, most youngsters re- = i il { = . ioe alt “th Ble quire more work, . It has. been Aiud \| thought advisable to devote a separate chapter to the three- "year-olds, as such a division admits of a more care- ful study of several prominent colts of that age, whose training will be of interest to the reader. If a colt has been trained as a yearling, or as a EE MTT eS = Ch : = i il { = alt “th Ble Aiud \| EE MTT eS = Ch : = i il { = alt “th Ble Aiud \| If a colt has been trained as a yearling, or as a two-year-old, its training as a three-year-old will be along the lines previously written of, but ex- tended as, in the opinion of the trainer, best suits the individual case. If a colt has not been previously trained; and the owner desires to race the colt as a three-year- old, it will be necessary to proceed with early training, as previously set forth for colts of a younger age, except that the education and train- ing will have to be rushed and crowded into a shorter space of time. To save repetition of advice we will assume that the colt has been previously trained—raced as a two-year-old if you wish. THREE-YEAR-OLDS. 87 In the late fall of its two-year-old form, the trainer must decide whether the colt is to be turned out. Some horsemen turn two-year-olds out from November 1 to February 1 and then commence jogging, while others prefer to jog the colt all winter—each owner or trainer must de- ee this point for himself. ' The jogging will: consist of from re to six bi ' The jogging will: consist of from re to six sles a.day (trainers’ opinions differ) except Sun- day. As the jogging progresses a little brushing may be indulged in, at the end of the jog, if de- sired. About April 1st the trainer should begin to work colts for speed. Some drivers start with a full mile in about. three minutes, others start with half-mile heats and gradually increase the distance till a mile is reached. Chapter VI—Three-year-olds. Each succeeding work day the colt is asked to go a little faster mile, but the drop must be gradual, and, if the colt gets to going rough or bad-gaited, the fault should be corrected, if possible, before much more is done, unless the colt be one that improves in gait as he improves in speed. The dropping down has to be done as the trainer thinks best. W. O. Foote says: “The more speed a colt shows at this time the less fast work I give him.” This is good ad- vice, for many colts are made speed crazy by dropping them too fast. In some families the. colts come to their speed more quickly than oth- ers, and,.as a consequence, careless trainers have militated against the success of such families, by CARE AND TRAINING OF TROTTERS. 88 ruining colts. In addition to working miles, it is assumed that those who believe in the brush sys- tem will make speed with it as described before. After the colt has been worked mile heats, and After the colt has been worked mile heats, and from two weeks to two months later (according to individual judgment, location, or weather), the colt should be worked two heat repeats. Later, say from two to four weeks (by this time it will be May or June) three heat repeats are in order. Still later most trainers work four heats, includ- ing the “opener,” but very few go beyond that, as most futurities are decided under the 2 in 3, or three-heat system. In midsummer, if it is ex- pected to start in the Kentucky Futurity (a 3 in 5 event) it may be thought advisable to work five heats. It is important that a colt be worked in com- pany as much as possible and taught to trail, also to come out from behind and race beside another horse without trying to rush past. Experience Not Always a Criterion. It would be useless to go into the minor de- tails of training and working colts. No set rules can be laid down. If the colt shows considerable speed he will doubtless be placed in the hands of an experienced trainer. Experience even is not always a teacher. Take, for instance, the case of Don Chenault and Etawah in 1913. Don Chenault won the Review futurity at Columbus, THREE-YBHAR-OLDS. 89 defeating Etawah. Two weeks later Etawah turned tables on him. There are many features of interest in both races, and about what took place between and afterwards. At Columbus Don Chenault defeated Etawah by an eyelash the first heat. The second and final heat was easily won by Don Chenault, after an early break had put Etawah out of the contest. After this race Don Chenault was worked very little. Two weeks later he started in the Kentucky Futurity and won the first heat, off in front all the way. In the second heat he seemed very rank, made two breaks and finished seventh. In the third heat he became practically unmanageable and was dis- tanced for running. Don Chenault’s driver at- tributed his defeat in the Kentucky Futurity to the fact that the colt had not been worked enough between the two races. He points to the fact that after the Kentucky Futurity the colt was given a stiff workout and, six days after his de- feat, won the Championship stake in two straight heats. As Etawah was not a starter in this last race it cannot be brought into comparison with the other’two. Etawah, that had been defeated at Columbus on September 24, participated in a race against aged horses on October 4 and trotted five hard heats, finishing 5, 3, I, I, 2 in 2:08%, 2:10%, 2:10, 2:09%4, 2:13, and then was drawn, as he was becoming exhausted, and the Kentucky classic was in mind. In the Kentucky Futurity CARE AND TRAINING OF TROTTERS. 90 Etawah was a sorry looking spectacle. After Don Chenault had won a heat in 2:0534, and }Peter Johnston one in 2:0834, Etawah was the freshest horse in the race and won the next three heats handily in 2:0834, 2:10, and 2:12. Experience Not Always a Criterion. The driver of Etawah, who had been criticised for giving his, colt so much work between futurities (the same people criticised the driver of Don Chenault for not doing the same thing!) is of the opinion that the hard work between the two futurities put Etawah “on edge,” as the saying goes, for the big race. Others believe’ that Etawah won on’ his gameness and, would. have won without such harsh treatment. And so we say, even experi- ence is not an indisputed criterion for training colts. a Henry .M. Jones writes: ‘“Three-year-olds should’ be taken up not later than February 1 and jogged five to six miles a day, except Sun- day. One should start to work heats not later. than April 1, starting with miles every other day in three minutes and dropping down. If colt has shown speed in its two-year-old form it can be dropped down to 2:45 in two weeks. I brush an eighth away from the wire and an eighth home. In two weeks from the time of working heats the colt should be given two heat repeats, and from two to four weeks later three heat repeats, first mile in 2:40, last two in 2:35 or 2:30, with first and last quarters in 35 seconds. When the THREE- YEAR-OLDS. 91 colt has been dropped down to 2:20, brush it to the half in one heat and then home from the half in the other fast heat, but rating the slow half so both miles will be practically the same. Finally the colt may be worked four heats, the first an opener, the second mediunily fast, and the last two the fastest but in about the same notch. Never allow the colt to slow up immediately after passing the wire. I trained Waverly, p., 2:041%4 (which I drove a quarter in 28 seconds as a three- year-old), Maggie Winder (3, p.), 2:06%4, and Fleeta Americus (3), p., 2:09%4, in this manner.” W. O. Foote, who trains in Texas, writes: W. O. Foote, who trains in Texas, writes: “The colt should be taken up the fall or winter before he. is three years old and jogged three to five miles a day, Sunday excepted. In March start working half mile heats and gradually in- crease to a mile. The time of these heats depends on speed shown. Experience Not Always a Criterion. The more speed a colt shows, the less fast work it should have. ‘No two colts are alike, so cannot give any advice about drop- ping them down. Two heat repeats may com- mence in April in a warm climate, then three heats in May. Four heats are plenty. A very important thing in educating colts is their shoeing and balancing. Unless this is done carefully and correctly it will be almost impossible:to win a futurity. Colts should be shod as lightly as pos- sible, and, as a- rule, with as short feet as possible. The balancing of a colt depends CARE AND TRAINING OF TROTTERS. 92 largely on the proper angle of his feet. He should also be taught to go on as light a line as possible, but, of course, all will not do this. Colts should be taught to trail other horses and work beside them without wanting to rush by as fast as to cause them to break or trot themselves out before the end of a mile. Colts do better with an occasional let up of a week or ten days.and jogged every day or turned in a paddock to exercise them- selves. J never work my colts miles as fast as they will go. Governor Francis’ fastest workout as a .three-year-old was in 2:14%, yet he trotted in 2:11, 2:1114, 2:121%4—the fastest three heats trotted by a three-year-old stallion until 1913. The Climax’s fastest workout as a three-year-old was in 2:12)4, yet he paced in 2:07 ina race.” Sam J. Fleming writes: “Futurity prospects should be jogged three to six miles daily all win- ter after two-year-old training or campaigning. Start to work mile in three minutes and drop down slowly to 2:40. Then 30 to 60 days later start repeating. The trainer will have to use his own judgment from here on. Baroness Virginia (3), 2:0814, was worked in 2:1714 as a two-year- old and could have trotted in 2:15 or better. As a three-year-old she was not worked faster than 2:26), and was raced into condition over the half- mile tracks, beating the half-mile track record for her age. At Indianapolis, in the Western Horseman Futurity, she trotted four heats around THREE-YEAR-OLDS. 93 2:15. At Columbus the next week Murphy worked her in 2:14 and 2:10% and won the Stock Farm Futurity around 2:10 three times. At Lex-. Experience Not Always a Criterion. ington he worked her two heats around 2:15 and then won the Kentucky Futurity. All together she had less than 20 heats better than 2:20, in- cluding all her work and races. In my opinion colts need to be kept fresh. They should not have too many miles, but plenty of speed making and conditioning. They will race with this work if game, and if not game no amount of staying- up will get them to Futurity form.” Shoeing of 1911, 1912 and 1913 Colts. Speaking of shoeing, we made a comp Speaking of shoeing, we made a comparison of the shoes worn by the fastest three-year-olds of IQII, I912, and 1913, as illustrated in the Christmas numbers of “The Horseman” of the respective years. The following tabulation of av- erages. tells its own story: No. of Wt. Angle Length Wt. Angle Length colts Front Front Front Hind Hind Hind Year. observed. Shoes. Feet. Feet. Shoes. Feet. Feet. MOLL, ccineeinaiierisicewmas 16 7 48 35% 4 53 3% QTD soiarssveieieecarcinvedueds 12 7 49 35% 4 54 35% TOG: jo: iisieserndace: Raver ersten 35 ™% 48, 35% 4 52 3% Av. for 3 years.... 7% 48% 35% 4 52%, 3% Av. for pacers..... va 48 8% 4 81% 3 The table contains the average for both gaits. The table contains the average for both gaits. A line is added at the bottom to show the aver- age for pacers. As there were only Io pacers considered out of a total of 63 colts, their influ- ence in the main table is not considerable. CARE AND TRAINING OF TROTTERS. 94 Let us see what style of shoes are being worn by colts. The following table gives the different styles, with the names and total number of colts that wore each. All aged colts appear in this table, from yearlings to three-year-olds: FRONT SHOES. Trotters. Pacers. Plain ShOC 21... cece ee cece cere cee e cent eee e ence eee enee 11 (Lady Wanetka, Peter Johnston, ‘Airdale, Anna Ax Me,. Colonel Cochran Jr., Ima Jay, Pine Knot, Royal Hall, Ruby Watts, Mediumore, Sweet Alice and Mightellion.) Plain shoe with heel calks 21... cc cuee cscs e reas ences 4 (Bonnie Setzer, Don Chenault (2), Onset and King Clansman.) Plain shoe with creased toe ........- ssdssaacest askesars(@ienohets# et (Etawah, Homer Baughman, Judge. Experience Not Always a Criterion. Jones, Todd Forbes and Mamie Guy.) Plain square toe shoe ......s...60: sasiauseandesasatduslsiegss seat oints 2 (U. Forbes, Atlantic Express.) Plain square toe shoe with heel calks ...............- aise (Don Chenault (3), The Right.) Plain shoe with creased toe and heel calks .... ...... 1 . (Willow Mack.) Plain shoe with grab ...... ation gs eras SURG SEES 1 (Grace O. C.) Plain shoe with grab and heel calks .............se.00- 1 (Roberta Bingen and Herman Wenger.) Plain shoe with four calks .......c scence eee eees bY (Mahomet Watts.) : Bar shoe, plain .......... cyaiiavaceyaih Sve fig nuateeevaecrevandeasevapatoteed, .3 ' (Miss Stokes, Rhythmell, Binville, and Miss De Forest.) Bar shoe with heel calks ........... eee cee cece ence eens 5 (Peter Volo, Manrico, Nowaday Girl, Barbara Overton and Sweet Spirit.) Bar shoe with creased toe ..........0ccseceeeeeeeeeeaes 3 (Adlon, Burt Axworthy and Minna Ward.) Bar shoe with heel calks and creased toe .............. <4 (Princess Nelda.) te Bar shoe with grab and heel calks ...... tea eMart E.S Nek So vol (William, Box.) ; Bar shoe with four calks ............. ataliveSuavare atavaenteiasi rea (Fay Kirk—1 forward calk missing.) Swedged shoe ..........-eeeeee eigen sera Mere DRM 5 1 THREE-YEAR-OLDS, 95 (Bonington, Little Bernice, Tilly Tipton, Brighton B., Lord Brussels, Margaret Parrish, Hydrad, King Daphne and The Climax.) Swedged shoe with heel calks .............cccecaeeeees 1 (Hall Mark.) Swedged bar shoe with square toe .............2.0000% 1 . (Justice Brooke.) Swedge bar shoe with heel calks .......... pamecxgadna 0 (Direct Gentry.) Swedged ars SHO, cscsensieeseavencnenrcahyeamets ame atta kahmeunis 2 (Lucile Spier and Dillon Axworthy.) Rim shoe ............ lke Sats eed ona use eau aNaasate, pairs ave "els 0 (Impetuous Palmer and The Conclusion.) Memphis bar shoe ........ aesveretaparersse shenciteriereete a wishes 1 (Baldy McGregor—only one bar used.) Memphis nub shoe ...........ccceeeccneseseecenseeenee 1 (Sure Mike.) Plain shoe ...... gas abe a cau ere vente wadenn PCr nde Wal eek oa TOIR eso ZeHAN 6 (Lady Wanetka, Peter Johnston, Anna Ax Me, Todd Forbes, Mediumore, Fay Kirk and Grace 0’C.) Plain shoe with heel calks .........seseeeeees widebiseavsandens 12 (Atlantic Express, Binville, Bonnie Setzer, Di- rect Gentry, George Rex, Ima Jay (1 calk only), Nowaday Girl, Onset (1 calk only), Roberta Bin- gen, Royal Hall, King Clansman, Hydryad and Col. Cochran Jr. (1 calk only.) Plain shoe with grab and heel calks .................- 2 (William, Box and Mamie Guy.) Plain shoe with creased toe and heel calks......... Experience Not Always a Criterion. 1 (Etawah.) Plain shoe with heel calks and outside grab ........... 1 (King Daphne (oblique toe) and The Conclusion.) Plain shoe with square toe and heel calks ............. 2 (Mahomet Watts and The Right.) Bar shoe ‘with grab: 2 sis2 ccs ee oie es ee een cect acanvere (Princess Nelda.) Rar shoe with grab and heel calkS ........cce ccc ee ees 0 (Herman Wenger.) = Swedged shoe ....6- 0 -seeeeeee eee e cece eee eeeenaee oe D (Peter Volo, Don Chenault, Miss Stokes, Adlon, Minoa Ward, Brighton B., Justice Brooke, Mar- garet Parrish, Mightellion: and The Climax.) Swedged shoe with heel calks .........sseeecceeececees 6 (Don Chenault, Baldy McGregor, Bonington, Pine ‘Knot, Lord Brussels and Sweet Alice.) Swedged shoe with square toe ........ ays rayne ce aaa NS 3 vente wadenn PCr nde Wal e CARE AND TRAINING OF TROTTERS, 96 (Airdale, U. Forbes and Judge Jones.) Swedged shoe with square toe and heel calks .......... 3 1 (Dillon Axworthy, Homer Baughmar, Mahomet Watts and Burt Axwortby.) Swedged bar shoe with heel calks ......cccccceeeveeees 1 mt) (Manrico.) Half swedged shoe 22... c ccc e cece teen e ae ee ee eetncenee 1 o (Ruby Watis.) Half swedged shoe with heel calks .......-.:eeeccerees 4 2. (Hall Mark, Lucile Spier, Sure Mike, Tilly Tip- ton, Willow Mack, Miss De Forest.) Rim shoe with heel calks ........ccccseeeeeeeeeeeeeee Ci) 1 (Little Bernice.) Flalf rim Shoe ..ccccnessencces cence canes scscecseeenne 0 1 (Impetuous Palmer.) It will be noticed that the most popular front shoe for trotters is a plain one. Next in popu- larity comes the bar shoe with heel calks, and the swedged shoe. The most popular pacing shoe is the swedged. . ‘The most popular hind shoe for trotters is the plain shoe with heel calks, followed- by the swedged shoe, then the plain shoe. In speaking of calks on a front shoe we mean the long narrow calks welded on the shoe par- allel to the direction of travel. sometimes called jar calks. Heel calks on the hind shoe are those formed from the body of the shoe itself. The classification above is not elastic enough to accurately indicate the wide difference between shoes of the same class. Take the plain front shoes used by 11 trotters and 1 pacer. According, to our classification all are alike, yet each one dif- fers from the other in some particular, either as THREE-YEAR-OLDS. 97 to weight, shape-or size.’ The same is. true of any other kind of shoe. To describe each colt’s shoes accurately a description and cut would have to be printed separately. ‘This would make tire- some reading and would defeat. the purpose of our table, in which it is impossible to show such minor differences as trailing heels, side clips, heavier toes, etc. It will be noticed that certain trainers seem to It will be noticed that certain trainers seem to prefer certain shoes. Thus Walter R. Cox used plain shoes in front on Lady Wanetka and; Peter Johnston. Thomas W. Murphy used a bar shoe with heel calks on Peter Volo, Barbara Overton and Sweet Spirit. Ed. F. Geers used a_ plain shoe with creased toe. vente wadenn PCr nde Wal e on Etawah, and Homer Baughman, although the former, was a. trotter and the latter a pacer. Many other similar com- parisons might be drawn. Champion Three-Year-Olds. ‘Let us conclude our chapter with a consideration of the champion threé-year-olds. The present champion is Colorado E,_ 2:0434, trained and driven by Guss Macey. The previous champion was General Watts, 2:0634, trained and driven by Mike Bowerman. Previous to that was Fantasy, 2:0834, trained and driven by Ed. F. Geers. Pre- vious to that was Sunol, 2:10%, trained and driven by the late Charles Marion. Previous to that was Axtell, 2:12, bred, owned, trained, and CARE AND TRAINING OF TROTTERS. $8 driven’ by C. W. Williams, practically an ama- teur at the time. This takes us back to 1889, beyond. which there is little to be learned that would be of interest or benefit to present-day horsemen. . : Colorado E., 2:0434. Colorado E., 2:0434. Colorado E was sensational as a yearling and was driven a quarter at that age in :33%4 by W. W. Evans. This resulted in the colt’s sale to Geo. H. Estabrook for $5,000. ‘Colt was then turned over to Guss Macey. As a two-year- old the colt sprung a curb and could not be worked properly, yet in the Kentucky Futurity he was second to the great Native Belle, 2:0734. After the Futurity, Colorado E. was turned out at Lexington. He was taken up December 1 and shipped to Denver. His jogging began on his arrival in Colorado. He was jogged about four miles a day, except Sunday, until March 1, when he was double-headed miles in from 3:15 to 3:00 ‘every other day through March., By April 1 he had worked a mile in 2:35 and was dropped down about 3 seconds a week. In April Macey began giving him two-heat workouts, twice a week. By May 1 he had been in about 2:20. During this month he was given two heats the first of the week and three heats the last of the week. By June 1 he had been in 2:15. On June 14 at Detroit he worked in 2:11. At Grand Rapids, on July 22, he won easily in 2:1234 and 2:1234. THREE-YEAR-OLDS. 99 At .Kalamazoo, on July 29, driven by Reamey Macey, young .son of Guss, he worked a mile in 2:06% or a half second faster than the world’s _record, last half in 1:01%4, third quarter in 30 seconds. At Cleveland he worked in 2:0734 and 2:0714%. At Empire City, in his second start of the year, the, Matron Stake, he won easily in 2:0714, 2:0734, the fastest two heats trotted by a three-year-old up to that time. At Boston he won the Horse Breeder Futurity pulled up in 2:06%4 and 2:0734. At Syracuse he won the Horse World futurity in 2:1334, 2:0814. At Co- lumbus he won the Stock Farm futurity in 2:0834 and 2:0534. In the Kentucky Futurity every- body was. out to beat him, and here he suffered his only defeat. In his final start, the Kentucky stake, he won in 2:11%4 and 2:0434, a new world’s record. His winnings for the year were $109,- 790.25. Colorado E., 2:0434. Colorado E was a large rangy colt, with a sweeping yet frictionless stride, which enabled him to cover ground in a deceiving manner. At .Kalamazoo, on July 29, driven by Reamey Macey, young .son of Guss, he worked a mile in 2:06% or a half second faster than the world’s _record, last half in 1:01%4, third quarter in 30 seconds. At Cleveland he worked in 2:0734 and 2:0714%. At Empire City, in his second start of the year, the, Matron Stake, he won easily in 2:0714, 2:0734, the fastest two heats trotted by a three-year-old up to that time. At Boston he won the Horse Breeder Futurity pulled up in 2:06%4 and 2:0734. At Syracuse he won the Horse World futurity in 2:1334, 2:0814. At Co- lumbus he won the Stock Farm futurity in 2:0834 and 2:0534. In the Kentucky Futurity every- body was. out to beat him, and here he suffered his only defeat. In his final start, the Kentucky stake, he won in 2:11%4 and 2:0434, a new world’s record. His winnings for the year were $109,- 790.25. Colorado E was a large rangy colt, with a sweeping yet frictionless stride, which enabled him to cover ground in a deceiving manner. Colorado E. wore four ounce square toe plain Colorado E. wore four ounce square toe plain shoes all around, very light close-fitting quarter boots in front, ankle and speedy-cutting boots be- hind, two-minute harness, blind bridle, snaffle bit, and Crabb overcheck bit. General Watts, 2:0634. ; General Watts was trained lightly as a two- year-old by Harold Childs and given-a time rec- ord of 2:27%4. He was then the property of CARE AND TRAINING OF TROTTERS. 100 Senator J. W. Bailey and Gen. C. C. Watts. Shortly thereafter. he became the exclusive prop- erty of the latter gentleman, who placed him in the hands of Mike Bowerman on March 18, 1907. The colt was thin and weak, following a severe illness, and was naturally small in size, so his training was done carefully, yet he made speed fast. He was jogged until May 1, then was given slow repeats from-2:40 to 2:30, twice a week. Around July 1 he showed a mile in 2:1514. At Cleveland; Aug. 1, in his first start, he was in- terfered with: in the first heat and was 5-4 in 2:13%4 and 2:1114. At Readville, on Aug. Colorado E., 2:0434. 20, in the Horse Breeder futurity, he won in 2:0914 (middle half: in 1:0134) and 2:09%, lowering the three-year-old stallion record of 2:10%, held by Arion. In the Stock Farm futurity at Co- Iumbus he led to the half in 1:021%4, but was beaten by Kentucky -Todd in 2:0834, which equaled the world’s record held by: Fantasy. Gen. Watts seemed -to tire and was only fifth the next heat in 2:11. Mr. Bowerman attributed his de- feat to lack of work. ‘The race seemed to leg hin up and ‘the next week, at Columbus, he won the Review futurity in 2:11, 2:09%4' and 2:09%, losing the third heat. At Lexington he won the’ Kentucky futurity. over a/slow track in 2:12%, 2:1134 and 2:11.' The second week at Lexington he trotted the first heat ‘in 226634 (last half-in t :0234—loweritig thé: world’s fecotd by two sec- THREE-YEAR-OLDS. 101 onds) and 2:0934. His year’s winnings were over $20,000 and he retired sound. The follow- ing is quoted from Mike Bowerman’s account of the training of Gen.- Watts, which appeared. in the Horse Review of December 17, 1907: As to any system of training colts, I have none. I train horses differently from_any other man that I ever saw train. After I thought I had Gen, Watts’ thoroughly seasoned, I did less jogging than any one generally does. I went ‘to the track less than al- most any one else goes.. If I can get out on the road I care little about the track. only for speeding purposes. After getting him into condition I would work out Gen. Watts twice a week; good fast miles, rated all _the way, not driving him any fast quarters or eighths, After I drove him a mile in 2:15 ,» Which, my recol- lection is, was some time the first part of uly, I began to teach him to get’ away from the wire fast; after going about an eighth taking him back and stepping the last eighth at the end of 4 mile. I have seen trainers go out to work their horses, it being what they called ‘‘work out day’’ for certain ones. Possibly the horse, for some unknown reason, would not, work as well as‘ he had at other times. Then the trainer would whip him and run him and say, ‘‘Well. Colorado E., 2:0434. I will work-him another heat; I will make him worse or better.’’ J never do that. When I start to work a horse and he does not act right, I take him to the stable, for it is a sure thing that there is something wrong. with him, and he not being able to talk, I will wait and see what the trouble is or was. I use less bandages than any one, I use no body wash at all, nothing in the way it is mostly used. In short I trained General Watts as I do all other horses in my charge. Trained him when he felt good, if it was his time to work. As to what shoes he wore and the angle of his feet, he wore a As to what shoes he wore and the angle of his feet, he wore a bar shoe in front, weighting 614 ounces, with 8-ounce quarter boot, the angle of his foot being 47 degree, with a 3-inch toe. His hind shoe weighed less than three ounces, 38-inch toe, angle 51. He, wore shin boots behind with a very light coronet boot, a very: light knee boot for protection only, for I had driven him without any boots a little ways as fast as he could go, he never touching a hair. I wore an open bridle on him with his head checked a lit- tle above the level of back. No martingale. I fed him twelve quarts of grain a day. with all the hay that he would eat. His stomach and digestive organs were always in perfect order. After I said he was ready to trot he should never have lost a_ heat, and the only time that he did I myself was to blame for it, not him. While he has done what no other three-year-old ever ap- ‘proached, the world does not know what his real ability was, for I am just as sure in my own’ mind, had I hit the right day and track, he would have trotted a mile in 2:05 or better, as I was sure that he would trot a mile in 2:07% or better, which I did not hesitate to say the day he trotted in 2:06%. ; Fantasy, 2:0834. broken and wor Fantasy was broken and worked as a two- year-old and showed speed at once. As a three- year-old she started seven times and was never CARE AND TRAINING OF TROTTERS. 102 defeated, except in one heat. At Nashville after winning the fast heat. in 2:1634, distance was waived and a runner sent along to prompt Fan- tasy and she trotted the second heat in 2:0834. She was a nervous, high-strung mare, but very steady. Sunol, 2:0874. Sunol, 2:0874. Sunol (the last of the high wheel three-year- old champions) was a mare of somewhat whim- sical form. She was 15.2 hands high at the withers and 16 hands behind. She was fast in the paddock but gave trouble in. breaking, being high strung and cranky and had to be handled gently. She was broken to harness at a year old, worked a little beside a steady going horse, then tried to single harness while she was very. anruly. At first. it took about an hour to hitch her up. She was worked carefully during the winter of her yearling and two-year-old form, but brushed at speed a short distance (say a quar- ter in 35 seconds) every other day or two. On Aug. 1 of her two-year-old form, four davs be- fore her first start, she worked her first full mile at Los Angeles in 2:4014 and repeated in 2:38. The next day she was. merély exercised and on the following was repeated in 2:36 and 2:3314. She won her race in 2:341%4 and 2:25. She was taken home and brushed quarters and_halves. She. was shipped to Petaluma and on Aug. 20 worked in. 2:38 and 2:33. Two days later. she THREE-YEAR-OLDS. 103 won in 2:28% and 2:2634. and was returned to Palo Alto until the second week in October. She was then shipped to San Francisco, where she was given two repeats, the first in 2:32 and 2:35, the second in 2:28) and 2:23, the latter on Oct. 18. Two days later she started against the world’s two-year-old record of 2:21 held by Wildflower, and although suffering from sexual excitement reduced the record half a second. A week later, with only brush work in the mean- time, she trotted in 2:18. She was then taken home and jogged all winter to skeleton wagon. She was turned out in a paddock and one day, in playing, strained a tendon in her right hind leg. The resultant swelling was kept down by cooling lotions and cold showers. Jogging was commenced on May 19, but she was not brushed for a month, as her hind ankles-looked suspicious. She was then given the brush system till she could step a quarter in 30 seconds, then mile and repeat work. On being shipped to San Francisco she. contracted a very severe case of distemper. Sunol, 2:0874. At Napa the weather was hot and this, with her nervous temperament, kept her much reduced in strength. At Petaluma she was defeated by Lil- lian Wilkes after she had won the fast heat in 2:21%. A week later, at Oakland, she turned the tables and won in straight heats, best time 2.:20. At Sacramento, on Sept. 12, she walked over. in 2:161%4%. Five days later she won, best CARE AND TRAINING OF TROTTERS. 104 tithe’ 2:18:At' Fresno, on Oct. 2, she went against time in 2:1334. At San Francisco, Oct. 12, she walked. over in 2:1534, and again on November g in 2:1034. -She closed the season at Napa, on Nov. 16, when she trotted against time in 2:15. Axtell, 2:12. Axtell’s training as a’ two-year-old, as re- counted by C. W.. Williams, appeared in the last chapter. From November 1 of his two-year-old form till the first of March following he was not jogged, but turned out every day and fed all the oats, bran and carrots he could eat, with corn- stalks for coarse feed. On March 1 his jogging commenced with one mile. This was increased a mile a day till he was taking twelve miles of road work a day. This was kept up till May 1, when he was worked on the track twice a week, but jogged twelve miles every other day. His track work was on the brush system. In addition to all this work he was bred to 22 mares between March 1 and July 4, but was not given much work the days he served mares. Up. to the last of June he had not been a mile faster than 2 :40, but had been speeded fast quarters, and halves. The last week in June, at Cedar Rapids, Ia., he reduced the three- -year-old half mile track record from-2:26% to 2:2134. On July 2, at Minneapolis, Minn., he started against the three- -year-old stallion - record of’ 2:18, held by Sable Wilkes, and, although it THREE-YEAR-OLDS, 103 was a cold raw day, with a light rain falling, he trotted in 2:1534. On July 4, at Independence, Ta., he reduced the half-mile track. record to’ 2:20%. For the next few days he was jogged eight to ten miles, then shipped to St.Paul, Minn., and started to beat 2:1534, and equalled it, which constituted a losing performance; the track was dead and cuppy and thought to be about three sec- onds slow. Axtell was then shipped to Independ- ence for a few days’ rest, then to Cleveland, O., where he further reduced his record to, 2:1434. Then on to Chicago,. where he went an exhibition mile in 2:1514 and, two days later, won a race, best time 2:14. The next week at Independence he worked in 2:22, the following week at Des Moines in the same notch. He was’-then’ sent home and jogged three weeks. At St. Louis he worked in 2:19, then was shipped to Terre Haute. Early in the week he was driven an exhibition in 2:141%4. Axtell, 2:12. The next day he was not harnessed, the following day he was jogged six miles to cart on a country road. The next day,,Oct. 11, he reduced the three-year-old record, and also the all-aged stallion, record to 2:12. That night he was sold for $105,000. : In Conclusion. In Conclusion. followed the colt We have now followed the colt from the day of its birth to the fall of its three-year-old: form. By this time the reader. if he has absorbed and di- gested all that has been written, by the famous CARE AND TRAINING OF TROTTERS, 106 horsemen who have contributed to this work, and: even if he is an amateur, will have formulated ideas of his own on the proper way to train and race.. When a man reaches that stage, further ad- vice is superfluous. Native Belle, 2:073%4 (in 1909), _World’s Champion Two-year-old -Trotting Filly. ' Native Belle, 2:073%4 (in 1909), _World’s Champion Two-year-old -Trotting Filly. ' APPENDIX. 107 Appendix—Chronic Indigestion, By Dr. A. S. ALEXANDER. \| There is on almost every stock farm, where horses are kept, at least one horse that does not “do well.” His coat is coarse and stands on end; his urine at times is plentiful, but at other times scant and like honey. When he stands in the barn for one day without work or exercise, his hind legs stock .up and when he is put to work he sweats easily and without hard labor or sweats profuse- ly after he is placed in the barn at noon or night. He is a ravenous eater and gets all he wants to eat, but is not satisfied and so proceeds to eat his bedding. If he is watched closely, it will be seen that he has the habit of raising his upper lip as if yawning; his manure differs in composition from time to time. Sometimes it is composed of small, hard, dry balls; at other times the balls are larger and covered with slime; again it comes in masses, undigested and foul smelling. _ The horse is troubled with chronic indigestion _ The horse is troubled with chronic indigestion and such animals usually bolt their food without proper mastication. Examination will sometimes disclose the fact that the molar teeth are in such condition that proper mastication is impossible. It should be understood by all owners of horses that the molar teeth in the horse do not com- pletely pass each other when chewing. The outer CARE AND TRAINING OF TROTTERS. 108 edge of the upper molars and the inner edge of the lower molars in time becoming long and sharp and interfere with proper mastication. This neces- sitates having the ‘teeth “floated” (filed down) once a vear after the horse is seven or eight years of age. ‘The first step, ore in Sue to cure the chronic indigestion alluded to is to have the teeth put in good order by a veterinary dentist. To go back a little, it should be mentioned that as a rule in practice we find most of these cases in barns that are close from poor ventilation, dirty from lack of cleaning and damp from improper drain- age of the building site or from inadequate meas- ures adopted’ for removal or absorption of liquid excreta. Appendix—Chronic Indigestion, By Dr. A. S. ALEXANDER. \| We also find the same condition existing where horses are given very hard water to drink, where sewage impregnates the drinking water, and where drinking water is habitually given after instead of before feeding. ; ‘How to Feed and Water. In the feeding of horses suffering f In the feeding of horses suffering from chronic indigestion, ‘it should be remembered that the horse digests oats in the stomach and takes ‘care of water and hay in the large intestines. For this reason the feeding and watering should be con- ducted upon the following theoretical plan: Water first, then hay, then oats. Mastication of the food is absolutely necessary in horses that are to do well and escape indigestion of any kind. Where APPENDIX. 109 ground food is given it is taken into the stomach without much: mastication, hence it is not properly mixed with saliva which is necessary for its sub- sequent digestion. When oats are in process of mastication, four times its bulk of saliva is se- creted in the mouth and this is needed to digest it. Where corn meal is fed, but a quarter of the proper amount of saliva is secreted and so on with the other ground foods. The teeth having been attended to, the next The teeth having been attended to, the next thing is to stop bolting of the food, and for this purpose ‘practical horsemen place the feed in an extra large, shallow feed box or put a few cobble stones in the feed box or mix a handful of old, dry, hard shelled corn or field peas among the oats or use one of the patent boxes. To stimulate secretion of saliva, rock salt should To stimulate secretion of saliva, rock salt should be kept before the horse at all times, and when this is done he will drink more water and should always have it befote but not after feeding. How to Water a Horse. How to Water a Horse. The stomach of the horse holds but 314 gallons. The stomach of the horse holds but 314 gallons. If he eats his oats, which remain in the stomach for digestion, and then drinks four or five gallons of water, the latter, as explained above, passes through the stomach and lodges in the large. Appendix—Chronic Indigestion, By Dr. A. S. ALEXANDER. \| in- testines, and in doing so must of necessity wash out undigested oats into the intestines, where they are not digested, but decompose and set up irrita- tion or give forth gas, which causes colic. CARE AND TRAINING OF TROTTERS. CARE AND TRAINING OF TROTTERS. 110 How to Feed a Horse.’ Feed the horse hay after drinking water the first thing in the morning and his oats after the small amount of hay has been eaten; at noon, water, then oats; at night, water, then oats, and two hours afterwards all the hay he will clean up before morning. More than twenty pounds of hay should never be offered to any horse, no mat- ter how large and heavy he may be. Hay should not be kept in front of a horse all of the time, as it is on many farms; so used it is a positive detri- ment, rather than a ‘benefit to the animal. INDEX. 111 INDEX Adbell, 1, 2:28...........- joe “AGE \| FROGS, W:. Oh ea: ised soeasezarorecocere 91 Adbella Watts.. “ Freedom, 1, 2:29%. 47 Airdale, 2:15%. Frou Frou, 2:254...........-- 46 Gamble, Samuel........ aes, 82 Geers, Ed. F............ 97 General Watts. 3, ae 06542. 99 Goldsmith. John A....... 46, 48 Governor Francis..........-+ 92 Halter-Breaking ........... 20 . ' Hazelton, James.. ce RBandseing xx sees ess yea ts 89 \| Heather, Joe..... -17, 28 Barber, Dr, W. A..5, 20, 49, 69 Peel Boots eases yee: Be Baroness Virginia, 3, 2: 08 yy 92 \| Hest Cir ts es oye «eee 48 Bell Bird, 2:264%......... 46, 47 \| High- oWheel Sulky........ 81, 102 Renear ........ .... 45 \| Hinda Rose. 1, 2:36%....47, 48 Benyon, James.. 70 \| Holloway, Frank H 44 Blanketing ..............005 40 \| Hoofs. Packing...... 25 Bowerman, Mike. -100, 101 \| Howard, Dr. S. R 6 Bradburn. John............. Brush Svstea .33, 36 Burgoyne, Harry............ Jones, Henry M............ 90 E. Justice Brooke, 2, 2:09%... 82 Chandler, J. B............ Chandler, Stewart. an EY Keets, O. Mu... ...csecsvaee 46 Childs, Harold M..... Kentucky Todd, 3, 2:08%.... 100 Chronic Indigestion.... -. 107 Colorado E., 3, 2:04%....... 98 Lady Wanetka ............. 79 Com. Walter Re scccscscacesinletecosers 97 Curry. Wickliffe............ 44 \| Marvi in, qc Ripert, cas bua arbre alee age , 88, 36, 38, 46, 47, 82 Ite 46 Diarrhea .. 7 : Macey, Ream Does, Budd: aa Maggie Winder, 3, p., 2:06%4 Don Chenault, 88 MCCaIT) NOG iii cicero cc Dwyer, Matt..........0..... 46 BCom DEE ak 17 Dykstra.. Dr. 3 \| Miller. Roy o8. 18, 33, 81,72, 82 Moody, Hunter C........: 42, 50 Edna the Great, 1, 2:29%.. 49 Murphy, Thomas ‘W... CARE AND TRAINING OF TROTTERS. 79, 97 itaw: pee 88 : Bstabrody, ‘go, Bo. 8 \| Native, Bette, " Navel Disease. . 9 Fantasy, 3, 2 Norlaine, 1, 2:31% 47 Eocding Paddbck.. 2 ‘ee addock.. Feet, Dressing 52 \| Palo Alto Training ‘Paddock 49 Fleeta Americus: 3. D., 2 209% 91 \| Pansy McGregor, I, 2:23%.. 46 Fleming, Sam J............- 92 \| Peter Volo, 1. 2:19.......+.+ 42 CARE AND TRAINING OF TROTTERS. CARE AND TRAINING OF TROTTERS. 112 Peter Volo, .2, 2:04%........ 79 Phippen, John §....... ~. 46 Pony, Leading Beside Pride: sscessierese2esa Pulling ......... Serrill, Joseph L. Sh hoeing see 6. BT Smollinger, W. H Stanford, Gov. Leland.. Suckling Colt.......... 4 Sudie D........-. 47 Sunol, 3, 2: 10%. : +» 102 Sweet Alice..........000000, , 71 The ‘Climax, Bis czxjeowas sane eve 92 Thomas, A. L.............4- 7 Three-yr. olds; How to Train 86 \| Toe Weights ............... 32 Training Paddock poet agepaisieicgeitts 49 Two-year-olds, How to Train 67 U. Forbes, 1,. 2:21%........ ). Valensin, Count.'........-... Valentine, Chas. A....... 40, Yearlings, How to Train.... Illustrations. Airdate, 2:15%4,. Champion Yearling Peter Volo, 2:04%4, “World's Champion. Two - year - old (, HEQOLLER: .. sccarh scauedant eases Colorado E., 2:04%, World’s Champion “Three-year - old FRPOBLOR iciassjeusiacovacsteteaen Greets Native Belle, 2:07%, World’s repels ion Two - year - old ‘World's: 51 66
https://openalex.org/W2119777793	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0000535&type=printable	English	null	The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System	PLoS neglected tropical diseases	2,009	cc-by	8,063	Abstract nding: This work was supported by National Institutes of Health Grants AI 077033 and AI 060005. The funders had no role in study desi alysis, decision to publish, or preparation of the manuscript. supported by National Institutes of Health Grants AI 077033 and AI 060005. The funders had no role in study design, data collection and ish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: douglas.hooper@jefferson.edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: douglas.hooper@jefferson.edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: douglas.hooper@jefferson.edu ¤ Current address: Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH, 44195 USA ¤ Current address: Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH, 44195 USA ¤ Current address: Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH, 44195 USA mechanism to compromise the barrier function of the neurovas- culature, circulating rabies virus-specific immune effectors, whether raised by the infection or by active or passive immunization, would be unable to mediate an antiviral response in CNS tissues. This may explain why conventional post-exposure treatment of human rabies, consisting of active and passive immunization, is unsuccessful if begun after the appearance of signs of the disease [2–4]. At this stage of the infection the virus has likely begun to replicate in the CNS. Thus, the primary function of current post-exposure regimens may be limited to preventing the virus from reaching CNS tissues. The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System D. Craig Hooper1,2, Timothy W. Phares1¤, Marzena J. Fabis1, Anirban Roy1 D. Craig Hooper1,2, Timothy W. Phares1¤, Marzena J. Fabis1, Anirban Roy1 1 Center for Neurovirology, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, Un D. Craig Hooper1,2, Timothy W. Phares1¤, Marzena J. Fabis1, Anirban Roy1 1 Center for Neurovirology, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America, 2 Department of Neurological Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America 1 Center for Neurovirology, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United Neurological Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America www.plosntds.org www.plosntds.org Abstract Background: The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus. Methodology/Principal Findings: Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus– neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus- specific antibodies. Conclusions/Significance: The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues. Citation: Hooper DC, Phares TW, Fabis MJ, Roy A (2009) The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System. PLoS Negl Trop Dis 3(10): e535. doi:10.1371/journal.pntd.0000535 itor: Charles E. Rupprecht, Centers for Disease Control and Prevention, United States of America Received May 6, 2009; Accepted September 24, 2009; Published October 6, 2009 oper et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. pyright: 2009 Hooper et al. This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Real-time quantitative RT-PCR Total RNA was isolated from CNS tissue samples and mRNA expression levels of rabies virus nucleoprotein, CD4, CD8, IFN-c and L13 in CNS tissues were measured by quantitative reverse- transcriptase (RT)-PCR as previously described [10]. Real-time quantitative RT-PCR was carried out on cDNA using specific primer and probe sets and a Bio-Rad iCycler iQ Real Time Detection System (Hercules, CA). The number of copies of specific mRNAs in each sample was determined as previously described [10] and normalized to the mRNA copy number of the housekeeping gene L13 in that sample. Data are expressed as Immunohistochemistry For immunohistochemical analysis, brains from perfused mice were snap frozen in Tissue-Tek O.C.T. Compound (Sakura Finetex, Torrance, CA), sectioned using a Thermo Shandon cryostat (Pittsburgh, PA), and fixed in either 80% acetone or 95% ethanol. Immunoglobulin (Ig) was detected using either biotiny- lated monoclonal rat anti-mouse kappa light chain (1 hour at 1:50) (BD Pharmingen, San Jose, CA) followed by Alexa Fluor 568 streptavidin (1 hour at 1:1000) (Invitrogen, Eugene, OR) or the VECTASTAIN ABC-AP KIT with polyclonal rabbit anti-mouse biotinylated IgG (1:200) developed using the peroxidase antiper- oxidase method and 393-diaminobenzidine as substrate (Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. For the additional staining shown in one of the figure panels (#3D), a 1 hour incubation with 1 mg/ml 1112 was performed prior to detection of Ig. To assess virus infection sections were stained for 1 hour with FITC-conjugated anti-rabies virus nucleoprotein monoclonal antibody (1:50) (Centocor, Mal- vern, PA). Photographs were taken with a Nikon digital camera on an Olympus BX-60 microscope. Assessment of blood-brain barrier integrity In this study, we show that the functional changes in the BBB required to deliver immune effectors to the CNS tissues can be induced in mice infected with a lethal rabies virus strain by immunization with a live-attenuated virus vaccine strain but not by administration of killed virus vaccine. Furthermore, our data suggests that clearance of rabies virus from CNS tissues is dependent upon the production of virus-specific antibodies by infiltrating B cells. Antibody Delivery to the CNS in Rabies Clearance from the in-house breeding colony at Thomas Jefferson University. RAG-22/2 mice on a 129/SvEv background were obtained from Taconic (Germantown, NY). Mice were infected or immu- nized intranasally (i.n.) with 105 focus forming units of CVS-F3, CVS-N2c or UV-inactivated CVS-F3 in PBS as previously described [10]. In some experiments, mice were infected and immunized with a combination of the viruses. Where indicated, CVS-F3-infected JHD2/2 mice were treated intraperitoneally with 1 mg of the monoclonal, rabies virus glycoprotein-specific, virus-neutralizing antibody 1112 in 500 ml of saline or with the vehicle alone at the time points noted in the figure legends. All procedures were carried out according to the protocols approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University. www.plosntds.org Assessment of blood-brain barrier integrity g y BBB integrity was assessed by quantifying the leakage of a low molecular weight fluorescent marker (Na-fluorescein, 376 kDa) from the circulation into CNS tissues as previously described [10]. Briefly, 100 ml of 10% solution of Na-fluorescein was injected intraperitoneally and after 10 minutes mice were anesthetized and cardiac blood was collected followed by transcardial perfusion. Serum samples as well as supernatants of homogenized and centrifuged tissues were clarified by precipitating proteins with 15% TCA and the level of fluorescence measured with a CytoFluor TMII fluorimeter. The amount of Na-fluorescein in the CNS tissue is normalized to its level in serum by (mg of Na-fluorescein in CNS tissue/mg of tissue)/(mg of Na-fluorescein in serum/ml of serum) and is expressed as a fold increase in fluorescence uptake by comparison with the results obtained from naı¨ve controls. the CNS, most of the attenuated rabies virus variants that we have tested spread to and replicate in the CNS but are cleared by immune effectors that cross the BBB and infiltrate neural tissues [7]. In contrast, BBB integrity is maintained and immune effectors do not accumulate in the CNS tissues during infection of mice with common pathogenic rabies virus strains, despite the development of virus-specific immunity in peripheral lymphoid organs and innate immunity in the infected CNS tissues [7,8]. These observations have led us to speculate that the lethal outcome of infection with wildlife and pathological strains of rabies virus is at least in part due to the evasion of immune clearance as a consequence of the maintenance of BBB integrity [8]. Perhaps the best evidence that this may be the case is that disruption of the BBB in mice infected with a highly lethal silver-haired bat- associated rabies virus (SHBRV), by triggering autoimmune CNS inflammation, promotes the clearance of the virus from the CNS tissues and survival [9]. Due to the associated pathology, the approach of using an autoimmune response to induce elevated BBB permeability and permit rabies virus-specific immune effectors to infiltrate CNS tissues is clearly inappropriate for use in human rabies. On the other hand, the neuroimmune response induced by infection with attenuated rabies virus, which also has the appropriate specificity, is not associated with significant pathology. Author Summary Every year over 50,000 people die from rabies worldwide, primarily due to the poor availability of rabies vaccine in developing countries. However, even when vaccines are available, human deaths from rabies occur if exposure to the causative virus is not recognized and vaccination is not sought in time. This is because rabies virus immunity induced by the natural infection or current vaccines is generally not effective at removing disease-causing rabies virus from brain tissues. Our studies provide insight into why this is the case and how vaccination can be changed so that the immune response can clear the virus from brain tissues. We show that the type of immune response induced by a live-attenuated rabies virus vaccine may be the key. In animal models, live-attenuated rabies virus vaccines are effective at delivering the immune cells capable of clearing the virus into CNS tissues and promote recovery from a rabies virus infection that has spread to the brain while conventional vaccines based on killed rabies virus do not. The production of rabies-specific antibody by B cells that invade the CNS tissues is important for complete elimination of the virus. We hypothesize that similar mechanisms may promote rabies virus clearance from individuals who are diagnosed after the virus has reached, but not extensively spread, through the CNS. Introduction Rabies viruses spread from peripheral sites of entry to the central nervous system (CNS) tissues via axonal transport thereby bypassing the specialized features of the neurovasculature known as the blood-brain barrier (BBB). Once the virus reaches CNS tissues three alternative outcomes are likely: (1) the BBB remains intact and the infection is lethal due to the absence of an antiviral CNS immune response (2) immune effectors cross the BBB and mediate a CNS antiviral immune response with extensive immunopathology that contributes to the disease, or (3) immune effectors cross the BBB and clear the virus from the CNS without significant pathological consequences. It is well known that in humans naturally infected with rabies virus the latter outcome is exceedingly rare. In addition, CNS inflammation is generally limited in individuals who succumb to rabies [1]. Consequently, it is probable that the BBB remains intact through much of the course of rabies infection in humans. In the absence of a Unlike humans where rabies viruses may take weeks to reach the CNS from the site of exposure [5], the spread of most rabies virus strains to the CNS in mice is rapid with virus generally being detectable in CNS tissues within 48 hours of infection [6]. Nevertheless, normal mice survive infection with laboratory- attenuated strains of rabies virus [7]. While certain of these viruses may be deficient in the capacity to spread from the periphery to October 2009 \| Volume 3 \| Issue 10 \| e535 1 October 2009 \| Volume 3 \| Issue 10 \| e535 Antibody Delivery to the CNS in Rabies Clearance Antibody Delivery to the CNS in Rabies Clearance Antibody Delivery to the CNS in Rabies Clearance the number of copies of mRNA for a particular gene in a sample per copy of mRNA for the housekeeping gene L13 in that sample. virus. CVS-F3 was administered first because the virus spreads to, and replicates in the CNS more slowly than CVS-N2c (data not shown). The delays were limited to 3 and 5 days so that the CVS-N2c infection would have the 48 hours required to spread to the CNS before the appearance of serum rabies virus-specific antibodies approximately 8 days following CVS-F3 infection [10]. To control for unanticipated effects caused by the administration of either immunogen, groups of mice were also given both live and inactivated CVS-F3. As shown in Table 1, administration of an inactivated CVS-F3 vaccine preparation that is effective when given several weeks before a CVS-N2c challenge does not protect when given 3 or 5 days prior to challenge. On the other hand, the majority of mice infected with CVS-F3 as recently as 3 days previously survive CVS-N2c infection regardless of whether or not inactivated virus is also administered. These results suggest that the processes required to clear pathogenic rabies virus from CNS tissues are induced by infection but not immunization with CVS-F3. Isolation of mononuclear cells, flow cytometry, and ELISPOT analysis y Mononuclear cells were prepared from peripheral blood collected by retro-orbital bleeding in heparinized capillary tubes by centrifugation at 300 g for 20 minutes. The white cell layer was washed in PBS twice before analysis. Mononuclear cells were isolated from CNS tissues as described elsewhere by isolation at the interface of a 30/70 Percoll (Sigma) step gradient centrifuged at 800 g for 25 minutes [11]. For flow cytometry, mononuclear cells were suspended in staining buffer (PBS with 2% FBS and 0.1% NaN2) and incubated with anti-CD16/32 (1 ug/106 cells) (2.4G2 BD Pharmingen, San Jose, CA) antibody to prevent non- specific binding. Cells were washed in PBS and incubated with anti-mouse CD19 (1:1000) (1D3, BD Pharmingen, San Jose, CA) and MHC class-II (1:1000) (120.1, BD Pharmingen, San Jose, CA) antibodies. Phenotypic characterization of antibody-labeled cells was performed on a BD-FacsCaliber Flowcytometer. CD19-MHC class II double-positive cells were defined as B cells. Numbers of rabies virus-antigen specific antibody secreting B cells were assessed using Millipore Multiscreen HAH ELISPOT plates coated with 5 ug/mL of UV-inactivated whole rabies virus. Peripheral blood or brain derived mononuclear cells were suspended in RPMI media supplemented with 25 mM HEPES and 10% FBS and 200,000 cells were incubated in each well for 18 hours. Plates were washed and bound rabies virus-specific antibodies were detected by addition of alkaline-phosphatase conjugated anti- mouse IgG antibody (1:500) (Sigma, St. Louis, MO) followed by BCIP/NBT substrate. Spots were counted using a dissecting microscope. T cell activities promote long-term survival in CVS-F3- infected mice but cannot clear the virus / JHD2/2 mice lack B cells but have functional T cells and, unlike RAG-22/2 mice, which lack both T and B cells, are capable of elevating fluid-phase BBB permeability in response to the infection (Fig. 1A). These mice are therefore suitable for analyzing the effects of antibody administration on CVS-F3 infection. As a preface to such studies we compared the course of CVS-F3 infection in JHD2/2 and RAG-22/2 mice. As is the case for wild-type mice infected with CVS-F3 [10], both JHD2/2 and RAG-22/2 mice lose weight as the infection progresses (Fig. 1A). However, while RAG-22/2 mice continue to lose weight and die approximately 20 days following infection, up to 70% of JHD2/2 mice survive past this time-point, most showing a modest weight gain (Fig. 1B). At the same time virus replication, which continues to increase in RAG-22/2 mice, becomes reduced in the JHD2/2 mice (Fig. 1C). These JHD2/2 mice exhibit signs of rabies infection including ataxia and partial paralysis but survive the infection for extended periods of time ( .40 days). This raises the possibility that T cell activities may be able to partly control the virus infection independently of antibody. To gain insight into the contributing T cell subsets, we compared the levels of CD4, CD8 and IFN-c mRNAs in CNS tissues from wild-type conventional mice 24 days after CVS-F3 infection when there is little virus replication remaining (see below) and JHD2/2 mice 40 days after Statistical analyses Results are expressed as the mean6standard error mean (S.E.M.). Statistical significance of the differences between groups was tested using the Mann-Whitney test and the symbol indicates a p value,0.05. www.plosntds.org Methods Animals, viruses, and antibody treatment Eight to 12-week old wild-type control 129/SvEv and C57BL6 mice and JHD2/2 mice on a C57BL6 background were obtained Animals, viruses, and antibody treatment October 2009 \| Volume 3 \| Issue 10 \| e535 2 www.plosntds.org October 2009 \| Volume 3 \| Issue 10 \| e535 Lethal rabies virus infection can be prevented by immunization with live-attenuated, but not killed rabies virus Mouse models are not considered to be particularly suited to studies of post-exposure prophylaxis (PEP) with rabies due to the rapid spread of the viruses to the CNS. However, our prior studies suggest that the lethal outcome of rabies in mice is more a consequence of the inability to deliver immune effectors into CNS tissues than its spread [8]. In our view, the key feature is that BBB integrity is maintained during infection with lethal rabies viruses, while infection with attenuated rabies virus variants causes enhanced BBB permeability and a virus-clearing CNS immune response [7,10]. The reason for this difference could be that infection with highly pathological rabies virus strains causes the inhibition of immune mechanisms that mediate the changes in BBB function necessary for rabies-specific immune effectors to cross. Alternatively, these may not be triggered due to a subtle difference in rabies virus immune mechanisms induced by pathogenic and attenuated viruses. To distinguish between these two possibilities, mice were infected with the attenuated CVS-F3 variant or immunized with killed CVS-F3 and then 3 or 5 days later were super-infected with the pathogenic CVS-N2c rabies Table 1. Survival of mice immunized or infected with CVS-F3 prior to infection with CVS-N2c. Table 1. Survival of mice immunized or infected with CVS-F3 prior to infection with CVS-N2c. Table 1. Survival of mice immunized or infected with CVS-F3 prior to infection with CVS-N2c. Table 1. Survival of mice immunized or infected with CVS-F3 prior to infection with CVS-N2c. Initial Infection or Immunization Secondary Infection (Delay in Days) Survival (Percent) None CVS-N2c 0/5 (0) CVS-F3, live none 5/5 (100) CVS-F3, live CVS-N2 c (3) 4/5 (80) CVS-F3, live CVS-N2c (5) 5/5 (100) CVS-F3, inactivated CVS-N2c (3) 0/5 (0) CVS-F3, inactivated CVS-N2c (5) 0/5 (0) CVS-F3, inactivated CVS-N2c (21) 10/10 (100) CVS-F3, live and inactivated CVS-N2c (3) 3/5 (60) CVS-F3, live and inactivated CVS-N2c (5) 5/5 (100) doi:10.1371/journal.pntd.0000535.t001 3 Antibody Delivery to the CNS in Rabies Clearance Figure 1. The T cell response to CVS-F3 infection in the absence of B cells mediates enhanced BBB permeability and long-term survival. The extent of BBB permeability in the cerebellum of wild-type, JHD2/2 and RAG-22/2 mice, infected i.n. with CVS-F3 10 days previously, is shown in panel A. Lethal rabies virus infection can be prevented by immunization with live-attenuated, but not killed rabies virus Weight loss over the course of infection in JHD2/2 and RAG-22/2 mice is shown in panel B, while the number of copies of rabies virus nucleoprotein mRNA at different times of infection in these animals is presented in panel C. BBB permeability changes are shown as the mean6S.E.M. fold increase in Na-Fluorescein uptake in the tissues with the levels from uninfected mice taken as 1. Weight is expressed as the mean6S.E.M. percent body weight with the weight on day 0 being taken as 100% and copies of nucleoprotein mRNA in the tissues are expressed per copy of L13 mRNA in the same sample. doi:10.1371/journal.pntd.0000535.g001 Figure 1. The T cell response to CVS-F3 infection in the absence of B cells mediates enhanced BBB permeability and long-term survival. The extent of BBB permeability in the cerebellum of wild-type, JHD2/2 and RAG-22/2 mice, infected i.n. with CVS-F3 10 days previously, is shown in panel A. Weight loss over the course of infection in JHD2/2 and RAG-22/2 mice is shown in panel B, while the number of copies of rabies virus nucleoprotein mRNA at different times of infection in these animals is presented in panel C. BBB permeability changes are shown as the mean6S.E.M. fold increase in Na-Fluorescein uptake in the tissues with the levels from uninfected mice taken as 1. Weight is expressed as the mean6S.E.M. percent body weight with the weight on day 0 being taken as 100% and copies of nucleoprotein mRNA in the tissues are expressed per copy of L13 mRNA in the same sample. doi:10.1371/journal.pntd.0000535.g001 infection. As can be seen in Fig. 2, the levels of CD4 and CD8 mRNA are somewhat lower in the JHD2/2 mice but IFN-c mRNA levels have remained relatively high and may therefore be contributing to the control of virus replication. detectable [12]. Nevertheless, it may be expected that some antibody would cross the BBB in conjunction with infiltrating immune cells and, over time, sufficient levels may accumulate to impact virus replication. To examine this possibility, JHD2/2 mice were treated with 1 mg of the mouse IgG1 monoclonal rabies virus-neutralizing, glycoprotein-specific antibody 1112, which is highly effective in post-exposure treatment models [13], on each of days 7 and 9 post-infection when BBB permeability is at a peak. Lethal rabies virus infection can be prevented by immunization with live-attenuated, but not killed rabies virus Several hours later, CNS tissues were obtained and stained with antibodies specific for rabies nucleoprotein and for mouse IgG to determine if there was any antibody associated with infected cells. While extensive infection of Purkinje cells can be readily detected with nucleoprotein-specific antibodies in sections from the cerebellum of JHD2/2 mice (Fig. 3A), as expected, there is no evidence of IgG in sections from animals that had not received antibody (Fig. 3B). IgG-specific staining of Purkinje cells in sections from mice receiving 1112 antibody could be detected (Fig. 3C) but the treatment of these sections with additional 1112 antibody in vitro prior to IgG detection resulted in more extensive staining (Fig. 3D). When cells stained for both nucleoprotein (Fig. 3E,G green) and antibody (Fig. 3F,G red) were examined more closely, distinct inclusions of nucleoprotein and antibody/glycoprotein can be seen. These findings suggest that low levels of 1112 antibody can leak from the circulation to interact with rabies virus-infected cells in the CNS tissues provided that their application coincides with elevated BBB permeability. Leakage of antibody across the BBB may not be sufficient to clear CVS-F3 from CNS tissues Rabies virus specific antibodies administered to CVS-F3-infected JHD2/2 mice have limited access to infected CNS tissues. At 7 and 9 days post-infection, CVS-F3-infected JHD 2/2 mice were treated with 1 mg of the rabies glycoprotein-specific, virus-neutralizing mouse monoclonal antibody 1112 (A, C, D, E, F, G) or saline vehicle alone (B). Several hours after the second treatment mice were transcardially perfused, CNS tissues removed and sections from the cerebellum were stained for rabies virus infection with FITC-anti-nucleoprotein monoclonal antibody (panels A and E; green), polyclonal rabbit anti-mouse biotinylated IgG (panels B and C; brown), additional 1112 antibody followed by the polyclonal rabbit anti- mouse biotinylated IgG (panel D; brown), or a combination of FITC-anti-nucleoprotein monoclonal antibody and rhodamine-conjugated anti-mouse Ig. Arrows in panels A-D identify Purkinje cells, a cell population that is extensively infected. Panels E-G show close-ups of a single cell stained with FITC-anti-nucleoprotein monoclonal antibody and rhodamine-conjugated anti-mouse Ig photographed using filters for FITC (panel E), rhodamine (panel F) and a combination red/green filter that shows both stains simultaneously (panel G). doi:10.1371/journal.pntd.0000535.g003 Figure 3. Rabies virus specific antibodies administered to CVS-F3-infected JHD2/2 mice have limited access to infected CNS tissues. At 7 and 9 days post-infection, CVS-F3-infected JHD 2/2 mice were treated with 1 mg of the rabies glycoprotein-specific, virus-neutralizing mouse monoclonal antibody 1112 (A, C, D, E, F, G) or saline vehicle alone (B). Several hours after the second treatment mice were transcardially perfused, CNS tissues removed and sections from the cerebellum were stained for rabies virus infection with FITC-anti-nucleoprotein monoclonal antibody (panels A and E; green), polyclonal rabbit anti-mouse biotinylated IgG (panels B and C; brown), additional 1112 antibody followed by the polyclonal rabbit anti- mouse biotinylated IgG (panel D; brown), or a combination of FITC-anti-nucleoprotein monoclonal antibody and rhodamine-conjugated anti-mouse Ig. Arrows in panels A-D identify Purkinje cells, a cell population that is extensively infected. Panels E-G show close-ups of a single cell stained with FITC-anti-nucleoprotein monoclonal antibody and rhodamine-conjugated anti-mouse Ig photographed using filters for FITC (panel E), rhodamine (panel F) and a combination red/green filter that shows both stains simultaneously (panel G). doi:10.1371/journal.pntd.0000535.g003 peaked [10]. However, B cells that have infiltrated the CNS tissues express high levels of k-light chain mRNA during this time period indicating that there is likely to be substantial antibody production in the CNS tissues [10]. Leakage of antibody across the BBB may not be sufficient to clear CVS-F3 from CNS tissues The inability of JHD2/2 mice to clear CVS-F3 from the CNS reaffirms the importance of rabies virus-specific antibodies in this process. However, little is known with respect to how these antibodies may be delivered to infected CNS tissues. Our studies of mice clearing CVS-F3 suggest that the leakage of naturally developing antibodies from the circulation into the CNS tissues may be minimal since elevated BBB permeability occurs before serum antibody titers peak [10]. In addition, over the short term, extensive fluid phase exchange across the BBB is seen but little accumulation of markers of the molecular mass of antibody is Figure 2. Prolonged IFN-c mRNA expression in the CNS tissues of CVS-F3-infected JHD2/2 mice. Wild-type and JHD2/2 mice were infected i.n., with CVS-F3 and euthanized at 24 or 40 days following infection to assess CD4, CD8 and IFN-c mRNA levels using quantitative RT-PCR. Data is expressed as the mean6S.E.M. fold increase with the levels in tissues from uninfected mice taken as 1. doi:10.1371/journal.pntd.0000535.g002 To determine whether 1112 antibody administration to CVS- F3-infected JHD2/2 mice leads to the clearance of the virus from CNS tissues, we administered saline or 1 mg of the antibody 5 times at two day intervals between days 7 and 15 post-infection. This antibody dose regimen achieved a half-maximal serum rabies-specific antibody titer of approximately 1/240, which is roughly equivalent to the serum titer found in normal mice 8 days post-infection with CVS-F3, during the period of time when BBB permeability is maximal. Viral nucleoprotein mRNA levels in the CNS tissues of surviving animals, both saline and antibody treated, were substantial several weeks later (Fig. 4) at a time when they are virtually undetectable in wild-type mice [10]. Moreover, no impact on the health or survival of the mice was noted. Figure 2. Prolonged IFN-c mRNA expression in the CNS tissues of CVS-F3-infected JHD2/2 mice. Wild-type and JHD2/2 mice were infected i.n., with CVS-F3 and euthanized at 24 or 40 days following infection to assess CD4, CD8 and IFN-c mRNA levels using quantitative RT-PCR. Data is expressed as the mean6S.E.M. fold increase with the levels in tissues from uninfected mice taken as 1. doi:10.1371/journal.pntd.0000535.g002 October 2009 \| Volume 3 \| Issue 10 \| e535 October 2009 \| Volume 3 \| Issue 10 \| e535 4 www.plosntds.org Antibody Delivery to the CNS in Rabies Clearance Figure 3. Clearance of rabies virus from CNS tissues is dependent upon antibody production by infiltrating B cells Clearance of rabies virus from CNS tissues is dependent upon antibody production by infiltrating B cells CVS-F3 clearance from the CNS tissues of wild-type mice occurs prior to the development of high titers of circulating virus- neutralizing antibodies (VNA) and after BBB permeability has Figure 4. The administration of rabies virus neutralizing antibodies fails to clear CVS-F3 from the CNS of JHD2/2 mice. JHD 2/2 mice were infected i.n. with CVS-F3 and treated at 7, 9, 11, 13, and 15 days post-infection with saline vehicle alone or the rabies- specific antibody 1112. Three weeks after the final treatment mice were transcardially perfused, cerebellar tissues were removed, and virus replication was estimated by quantifying nucleoprotein mRNA levels. Virus replication was similarly estimated in cerebellar tissues from wild- type mice infected with CVS-F3 24 days previously. Virus replication is expressed as the mean6S.E.M. copies of rabies virus nucleoprotein mRNA per copy of the housekeeping gene L13 mRNA in the tissue sample. doi:10.1371/journal.pntd.0000535.g004 Leakage of antibody across the BBB may not be sufficient to clear CVS-F3 from CNS tissues To assess this possibility more directly, we used antibodies specific for mouse IgG to stain CNS tissues from wild-type mice infected 12 days previously with CVS-F3. Extensive foci of antibody are seen throughout the cerebellum (Fig. 5). At higher magnification the antibodies appear to be diffusing in stellate patterns from the foci (Fig. 5). To determine if B cells may be the source of these antibodies and whether or not they are likely to be rabies virus-specific, we assessed rabies virus- specific antibody production by B cells from the peripheral blood and CNS tissues of CVS-F3-infected mice. While the proportion of CD19+ B cells in mononuclear cells recovered from the CNS tissues of CVS-F3-infected mice is lower than in peripheral blood from the same animals, the fraction of the cells that produce rabies virus-specific antibodies is considerably higher (Fig. 6). This suggests that B cells producing rabies virus-specific antibodies either selectively invade or expand in the CNS tissues in response to CVS-F3 infection. Antibody Delivery to the CNS in Rabies Clearance www.plosntds.org www.plosntds.org Discussion Prompt administration of PEP is the recommended course for an individual who has come in contact with a rabid animal. Since this prevents the development of clinical rabies it is impossible to be certain how many of the tens of thousands of people who receive PEP on an annual basis have actually been infected with the virus. It is also impossible to know how far the rabies virus may have spread before being cleared by the immune effectors provided or induced by PEP and the infection. The commonly held view that pathogenic wildlife rabies virus that has spread to the CNS cannot be cleared by immune mechanisms is supported by the absence of significant immune cell infiltration into the CNS tissues of individuals who die from rabies [1] and the failure of Figure 4. The administration of rabies virus neutralizing antibodies fails to clear CVS-F3 from the CNS of JHD2/2 mice. JHD 2/2 mice were infected i.n. with CVS-F3 and treated at 7, 9, 11, 13, and 15 days post-infection with saline vehicle alone or the rabies- specific antibody 1112. Three weeks after the final treatment mice were transcardially perfused, cerebellar tissues were removed, and virus replication was estimated by quantifying nucleoprotein mRNA levels. Virus replication was similarly estimated in cerebellar tissues from wild- type mice infected with CVS-F3 24 days previously. Virus replication is expressed as the mean6S.E.M. copies of rabies virus nucleoprotein mRNA per copy of the housekeeping gene L13 mRNA in the tissue sample. p doi:10.1371/journal.pntd.0000535.g004 October 2009 \| Volume 3 \| Issue 10 \| e535 5 Antibody Delivery to the CNS in Rabies Clearance Figure 5. Pattern of antibody appearance in the CNS tissues of CVS-F3-infected mice. Sections from the cerebella of wild-type mice either uninfected (A and C) or infected with CVS-F3 12 days previously (B and D) were stained for IgG (brown). Photomicrographs taken at low magnification are shown in panels A and B and at higher magnification in C and D. doi:10.1371/journal.pntd.0000535.g005 Figure 5. Pattern of antibody appearance in the CNS tissues of CVS-F3-infected mice. Sections from the cerebella of wild-type mice either uninfected (A and C) or infected with CVS-F3 12 days previously (B and D) were stained for IgG (brown). Photomicrographs taken at low magnification are shown in panels A and B and at higher magnification in C and D. doi:10.1371/journal.pntd.0000535.g005 PEP in individuals that have developed signs of rabies [2,4]. Discussion Our studies in animal models of rabies suggest that this is a consequence of the inability of virus-specific immune effectors to cross the BBB and enter CNS tissues infected with pathogenic rabies viruses [7,8]. The rabies virus-specific immune effectors that are raised in lethally infected mice are able to clear rabies virus from the CNS if provided access across the BBB. For instance, when the BBB is compromised by the induction of autoimmune CNS inflammation, rabies-specific immune effectors infiltrate CNS tissues and can clear the highly pathogenic SHRBV [9]. In addition, the adoptive transfer of immune effectors recovered from mice lethally infected with SHBRV results in clearance of the attenuated CVS-F3 virus from the CNS tissues of mice lacking T and B lymphocytes [8]. In contrast, the transfer of cells from mice clearing CVS-F3 has no impact on the outcome of SHBRV infection [8]. Regardless of the infecting virus strain, elements of the innate immune response that are important for the early control of virus replication and for attracting immune cells into infected tissues are induced [7,8,10]. These findings led us to speculate that functional changes at the BBB required to provide immune effectors access to the CNS tissues are induced during infection with attenuated rabies virus strains but not during pathological rabies virus infection [7–9]. A key issue examined in this study is whether or not this is due to an inhibitory process triggered by infection with pathogenic rabies virus. If so, it may be PEP in individuals that have developed signs of rabies [2,4]. Our studies in animal models of rabies suggest that this is a consequence of the inability of virus-specific immune effectors to cross the BBB and enter CNS tissues infected with pathogenic rabies viruses [7,8]. The rabies virus-specific immune effectors that are raised in lethally infected mice are able to clear rabies virus from the CNS if provided access across the BBB. For instance, when the BBB is compromised by the induction of autoimmune CNS inflammation, rabies-specific immune effectors infiltrate CNS tissues and can clear the highly pathogenic SHRBV [9]. In addition, the adoptive transfer of immune effectors recovered from mice lethally infected with SHBRV results in clearance of the attenuated CVS-F3 virus from the CNS tissues of mice lacking T and B lymphocytes [8]. In contrast, the transfer of cells from mice clearing CVS-F3 has no impact on the outcome of SHBRV infection [8]. www.plosntds.org Antibody Delivery to the CNS in Rabies Clearance This leads us to conclude that elements of the T cell response, likely including IFN-c production by CD4 and CD8 T cells, can control certain features of the infection that make significant contributions to its lethality but that antibody is required for virus clearance. To examine the contribution of circulating antibody to virus clearance from CNS tissues, we administered high levels of the rabies virus neutralizing mouse monoclonal 1112 antibody to CVS-F3-infected JHD2/2 mice during the stage of infection when BBB permeability is maximal. While leakage of a 150 kDa molecular weight marker from the circulation into the CNS tissues of CVS-F3-infected mice is minimal over a 4-hour period [12], antibodies present in the circulation over a more extensive period of time can evidently leak into the CNS tissues of the infected mice. 1112 antibody was found associated with the Purkinje cells in the cerebellum that express high levels of rabies virus antigen. The antibody was primarily localized in inclusion bodies which is consistent with previous in vitro studies showing that 1112 antibody is rapidly internalized by rabies virus-infected neuroblastoma cells where it accumulates in intracellular vesicles [13]. Of note in our studies is that the intracellular inclusions of glycoprotein-specific 1112 are generally distinct from inclusions of the virus nucleoprotein. The amounts of antibody reaching rabies virus-infected cells in vivo appears to be relatively low as considerably greater amounts of the antibody can bind to the cells when applied to tissue sections in vitro. While it is possible that even low levels of virus-neutralizing antibody may impact the replication and spread of the virus while BBB permeability is enhanced, treatment of CVS-F3-infected JHD2/2 mice with 1112 antibody failed to clear the virus. y q As certain of the aspects of BBB function required for immune cell infiltration are unchanged by CNS infection with pathogenic rabies viruses [8], once the virus has reached the CNS a PEP protocol capable of altering the BBB, so that virus-specific immune effectors can reach the infected tissues, is required. Inactivated CVS-F3 can induce rabies virus-specific T and B cells, but fails to promote recovery from CVS-N2c infection over a time frame during which the administration of live CVS-F3 is therapeutic. We consider that this is a consequence of the inability of the inactivated virus to induce the functional changes in the BBB that are required for antiviral immune effectors to enter CNS tissues. Antibody Delivery to the CNS in Rabies Clearance expected that BBB integrity would be maintained during infection with both pathogenic and attenuated rabies viruses and the outcome would be lethal, but it is not. Infection with an attenuated rabies virus induces BBB integrity changes and immune effector entry into CNS tissues regardless of whether or not there is also an ongoing infection with pathogenic rabies virus. However, protection is not provided by immunization with killed virus. We therefore conclude that the generation of a rabies virus-specific immune response in the periphery is not sufficient to clear pathogenic rabies viruses from the CNS tissues. A mechanism selectively induced by infection with attenuated rabies virus, likely manifested at the BBB, is necessary to provide immune effectors access to CNS tissues. examine the possibility that rabies virus-specific antibodies are produced by infiltrating B cells. The current findings indicate that this is the case. Focal concentrations of antibodies can be readily detected in the CNS tissues of mice clearing CVS-F3 and a high proportion of B cells recovered from the tissues produce rabies virus-specific antibodies in vitro. This leads us to conclude that the high levels of antibodies required for rabies virus clearance from the CNS tissues are produced at the site of infection rather than diffusing in from the circulation. In this case, passively adminis- tered antibody during PEP would primarily impact virus in the periphery and an active immune response leading to elevated BBB permeability and immune effector delivery to the CNS tissues would likely be required to clear virus from the CNS. To gain further insight into the mechanism of rabies virus clearance from the CNS tissues, we have used gene-deleted mice to study the role(s) of different antiviral immune effectors in the CNS tissues of mice clearing the attenuated rabies virus CVS-F3. Mice lacking T and B cells cannot clear this virus and die from the infection [8,14]. CD8 T cells contribute to, but are not required for the clearance of CVS-F3 as clearance is merely delayed in mice without this cell population [14,15]. On the other hand, JHD2/2 mice, which lack B cells but have functional CD4 and CD8 T cells, often survive CVS-F3 infection over extended periods despite being unable to clear virus from CNS tissues and exhibiting neurological symptoms. 1. Murphy FA (1977) Rabies pathogenesis. Arch Virol 54: 279–297. 2. Centers for Disease Control and Prevention. Human Rabies - New York, 1993 (1993) MMWR Morb Mortal Wkly Rep 42: 799, 805–806. 3. World Health Organization Expert Committee on Rabies (1992) 8th report. WHO Tech Rep Ser 824: 1–84. 4. Wilde H (2007) Failures of post exposure prophylaxis. Vaccine 25: 7605–7609. Acknowledgments We thank Rhonda B. Kean and Fatu Badiane for valuable contributions to this work. It should be noted with respect to the origin of the antibodies that participate in rabies virus clearance that serum rabies virus- specific antibody titers peak some time after BBB integrity has been restored [10]. The presence of cells expressing the B cell phenotypic marker CD19 and mRNAs specific for k- light chain in the CNS tissues of mice clearing CVS-F3 [8,10] led us to Antibody Delivery to the CNS in Rabies Clearance In our view, administration of a live-attenuated rabies virus vaccine is the most reasonable, currently available, approach to providing the appropriate immune effectors access to the CNS tissues. The results of our experiments with a new, highly attenuated recombinant rabies virus vaccine which expresses three copies of a mutated glycoprotein gene, strongly support this hypothesis [16]. In these studies, the triple G vaccine was shown to promote immune effector delivery into CNS tissues and normal mice were found to survive the intracranial injection of a mixture of the vaccine virus and a highly pathogenic dog strain which was nearly 100% lethal when administered alone [16]. The triple G vaccine also proved effective in the post-exposure treatment of mice infected with a highly pathogenic dog rabies virus several days previously [16]. However, when UV-inactivated and given peripherally to mimic conventional post-exposure vaccination, there was little protective effect [16]. In addition to boosting the antiviral response, attenuated rabies virus vaccines spread to the CNS where they trigger the mechanisms required for T cells and B cells to enter the tissues and clear, not only the attenuated, but also pathogenic rabies viruses. It is clear from the commonly lethal outcome of rabies that these mechanisms are not induced in a timely fashion in the context of the spread of a wildlife rabies virus to the human CNS. Author Contributions Conceived and designed the experiments: DCH TWP AR. Performed the experiments: DCH TWP MJF AR. Analyzed the data: DCH TWP MJF AR. Contributed reagents/materials/analysis tools: DCH. Wrote the paper: DCH TWP AR. Discussion Regardless of the infecting virus strain, elements of the innate immune response that are important for the early control of virus replication and for attracting immune cells into infected tissues are induced [7,8,10]. These findings led us to speculate that functional changes at the BBB required to provide immune effectors access to the CNS tissues are induced during infection with attenuated rabies virus strains but not during pathological rabies virus infection [7–9]. A key issue examined in this study is whether or not this is due to an inhibitory process triggered by infection with pathogenic rabies virus. If so, it may be Figure 6. Rabies virus-specific antibody secreting B cells accumulate in the CVS-F3-infected CNS. Mononuclear cells isolated from peripheral blood lymphocytes (PBL) and brain (CNS) were assessed for surface phenotype using flow cytometry and for the numbers of rabies virus-specific antibody secreting cells by ELISPOT. The percentage of B cells, identified as positive for both CD19 and MHC class II, among total mononuclear cells is presented on the left side of the figure while the proportion of the total mononuclear cells producing rabies virus-specific antibodies is presented on the right side of the figure. doi:10.1371/journal.pntd.0000535.g006 Figure 6. Rabies virus-specific antibody secreting B cells accumulate in the CVS-F3-infected CNS. Mononuclear cells isolated from peripheral blood lymphocytes (PBL) and brain (CNS) were assessed for surface phenotype using flow cytometry and for the numbers of rabies virus-specific antibody secreting cells by ELISPOT. The percentage of B cells, identified as positive for both CD19 and MHC class II, among total mononuclear cells is presented on the left side of the figure while the proportion of the total mononuclear cells producing rabies virus-specific antibodies is presented on the right side of the figure. g doi:10.1371/journal.pntd.0000535.g006 October 2009 \| Volume 3 \| Issue 10 \| e535 October 2009 \| Volume 3 \| Issue 10 \| e535 6 Antibody Delivery to the CNS in Rabies Clearance 3. World Health Organization Expert Committee on Rabies (1992) 8th report. WHO Tech Rep Ser 824: 1–84. y 11. Tschen SI, Bergman CC, Ramakrishna C, Morales S, Atkinson R, et al. (2002) Recruitment kinetics and composition of antibody secreting cells within the central nervous system following viral encephalomyelitis. J Immunol 168: 2922–2929. 10. Phares TW, Kean RB, Mikheeva T, Hooper DC (2006) Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J Immunol 176: 7666–75. 5. Tsiang H, Ceccaldi PE, Lycke E (1991) Rabies virus infection and transport in human sensory dorsal root ganglia neurons. J Gen Virol 72: 1191–1194. J 9. Roy A, Hooper DC (2007) Lethal silver-haired bat rabies virus infection can be prevented by opening the blood-brain barrier. J Virol 81: 7993–7998. 6. Hooper DC (2005) The role of immune response in the pathogenesis of rabies. J Neurovirol 11: 88–92. J 7. Roy A, Hooper DC (2008) Immune evasion by rabies viruses through the maintenance of blood-brain barrier integrity. J Neurovirol 14: 401–411. 8. Roy A, Phares TW, Koprowski H, Hooper DC (2007) Failure to open the blood- brain barrier and deliver immune effectors to the CNS tissues leads to the lethal outcome of silver-haired bat rabies virus infection. J Virol 81: 1110–1118. 5. Tsiang H, Ceccaldi PE, Lycke E (1991) Rabies virus infection and transport in human sensory dorsal root ganglia neurons. J Gen Virol 72: 1191–1194. 6. Hooper DC (2005) The role of immune response in the pathogenesis of rabies. J Neurovirol 11: 88–92. 7. Roy A, Hooper DC (2008) Immune evasion by rabies viruses through the maintenance of blood-brain barrier integrity. J Neurovirol 14: 401–411. 8. Roy A, Phares TW, Koprowski H, Hooper DC (2007) Failure to open the blood- brain barrier and deliver immune effectors to the CNS tissues leads to the lethal outcome of silver-haired bat rabies virus infection. J Virol 81: 1110–1118. 9. Roy A, Hooper DC (2007) Lethal silver-haired bat rabies virus infection can be prevented by opening the blood-brain barrier. J Virol 81: 7993–7998. 10. Phares TW, Kean RB, Mikheeva T, Hooper DC (2006) Regional differences in blood-brain barrier permeability changes and inflammation in the apathogenic clearance of virus from the central nervous system. J Immunol 176: 7666–75. 11. Tschen SI, Bergman CC, Ramakrishna C, Morales S, Atkinson R, et al. (2002) Recruitment kinetics and composition of antibody secreting cells within the central nervous system following viral encephalomyelitis. J Immunol 168: 2922–2929. References 3. World Health Organization Expert Committee on Rabies (1992) 8th report. WHO Tech Rep Ser 824: 1–84. 4. Wilde H (2007) Failures of post exposure prophylaxis. Vaccine 25: 7605–7609. 3. World Health Organization Expert Committee on Rabies (1992) 8th report. WHO Tech Rep Ser 824: 1–84. www.plosntds.org October 2009 \| Volume 3 \| Issue 10 \| e535 www.plosntds.org 7 Antibody Delivery to the CNS in Rabies Clearance Antibody Delivery to the CNS in Rabies Clearance Antibody Delivery to the CNS in Rabies Clearance 12. Fabis MJ, Phares TW, Kean RB, Koprowski H, Hooper DC (2008) Blood–brain barrier changes and cell invasion differ between therapeutic immune clearance of neurotrophic virus and CNS autoimmunity. Proc Natl Acad Sci U S A 105: 15511–15516. 13. Dietzschold B, Kao M, Zheng Y, Chen Z, Maul G, et al. (1992) Delineation of putative mechanisms involved in antibody mediated clearance of rabies virus from the central nervous system. Proc Natl Acad Sci U S A 89: 7252–7256. 14. Phares TW, Fabis MJ, Brimer CM, Kean RB, Hooper DC (2007) A peroxynitrite-dependent pathway is responsible for blood-brain barrier perme- ability changes during a CNS inflammatory response; TNF-a is neither necessary nor sufficient. J Immunol 178: 7334–7343. 15. Hooper DC, Morimoto K, Bette M, Weihe M, Koprowski H, et al. (1998) Collaboration of antibody and inflammation in the clearance of rabies virus from the CNS. J Virol 72: 3711–3719. 16. Faber M, Li J, Kean RB, Hooper DC, Alugupalli K, Dietzschold B (2009) Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus. Proc Natl Acad Sci U S A 106: 11300–11305. www.plosntds.org October 2009 \| Volume 3 \| Issue 10 \| e535 www.plosntds.org 8 www.plosntds.org
https://openalex.org/W3038866735	https://durham-repository.worktribe.com/preview/1263199/30987.pdf	English	null	Policy, institutional fragility, and Chinese outward foreign direct investment: An empirical examination of the Belt and Road Initiative	Journal of international business policy	2,020	cc-by	14,778	Abstract The Belt and Road Initiative (BRI) is an important policy agenda undertaken by the Chinese government. We explore how the BRI – as well as an associated policy, the creation of Chinese overseas special economic zones – influences Chinese outward foreign direct investment (FDI). We find that host country institutional fragility positively influences Chinese FDI volumes and that the impact of institutional fragility on Chinese inward FDI to the host is amplified in the presence of the BRI. Specifically, BRI policy facilitates FDI to countries with weaker rule of law and less government accountability. We argue that while the BRI may actively facilitate economic growth (i.e. via infrastructure development) and in turn aspects of human development, particularly in less developed economies, its likely impacts on political rights may not be so promising. Keywords: Bilateral relationships; BRI, investment policy; political risk; governance, Chinese OFDI Bilateral relationships; BRI, investment policy; political risk; governance, Chinese 1 1 INTRODUCTION Until quite recently MNEs from developed market economies were largely responsible for driving global FDI flows. The general tenor of policy advice given to less developed countries seeking to receive greater FDI volumes, predominantly from developed market economies, was to move their economies towards market friendly, rules based, well-governed and highly accountable institutional frameworks (World Bank, 1997). This school of thought is perhaps best captured and most closely associated with the now well-known “Washington consensus” view of development policy (Williamson, 1993; World Bank, 1993). Under this set of broad guidelines and overarching philosophy for policy implementation, host countries have been encouraged to foster sound judicial institutions, promote transparency, provide accountability, and safeguard property rights, while maintaining macroeconomic stability under small, lean professional bureaucracies. With the growth of MNEs from emerging markets, particularly those from China, the international investment environment and related policy advice for attracting inward FDI has changed considerably (Alon et al., 2018). China has become an essential source of FDI for many developing countries, in particular in African and Southeast Asian nations. Chinese MNEs, moreover, are sometimes considered different from developed market MNEs (Sutherland et al., 2018). They are “latecomers”, for example, looking to catch-up (via technology seeking) with their developed market counterparts (Sutherland et al., 2020; Anderson et al., 2015; Anderson & Sutherland, 2015a); they may exhibit high levels of state-ownership or involvement (Anderson & Sutherland, 2015b), and associated mercantilist investment strategies (Clegg et al., 2018); and, importantly, they may have so-called “special” ownership advantages – which allows them to more easily do business in markets that are imperfect – i.e. characterized by weak and “fragile” institutions (Buckley et al., 2007; Shi et al., 2017). As such, the types of policies required to attract EMNE FDI could well be different to 2 those prescribed by the Washington Consensus. As China has started to emerge as a global economic power, an alternative set of policies to the Washington consensus began to emerge – provocatively but accurately referred to as the “Beijing consensus” (Huang, 2017; Hlover & Shaibu, 2019). Founded upon China’s successful economic development experience over the past four decades, the Beijing consensus offers ‘an alternative to the policy toolkit offered to developing countries by the International Monetary Fund (IMF) and the World Bank, the so called “Washington Consensus”’ (Yagci, 2016: 2). INTRODUCTION This policy toolkit is typified by things such as incremental reform, innovation and experimentation, export-led growth, and state capitalism (Hlover & Shaibu, 2019; Williamson, 2012). The Beijing consensus ‘has begun to remake the whole landscape of international development, economics, society and, by extension, politics’ (Yao, 2015: 3). According to Williamson (2012), ‘the Beijing consensus basically refers to the Chinese way of doing things’ (p. While some scholars have questioned the efficacy of the Beijing consensus as a sustainable economic model (Huang, 2011; Williamson, 2012), host countries are not required to follow a prescriptive set of pro-market or political (i.e. democratic) ideologies as a precondition for attracting FDI and other associated financial and trade related support measures. The advancement of social institutions linked to democratic political values, such as greater transparency, accountability, strong and independent judicial systems, functioning independent media, are, therefore, not part of Beijing’s economic engagement plan. Rather, Beijing is seemingly content, and indeed at times may prefer, to work with what may be considered by Western standards institutionally fragile states. Sometimes these countries are controlled by unelected authoritarian leaders. Such leaders, faced with economic development needs, have a choice between free market, democratic institutions imposed by Western world order on the one hand, and the Beijing model of a quasi-liberal market economy coupled with 3 authoritarianism on the other hand. As a result, despite theoretical arguments that institutional voids and political risk will negatively impact FDI, much of the research on Chinese outward FDI to date supports the opposite view: Chinese (C)MNE investment significantly increases as institutions weaken and political risk levels increase (Buckley et al., 2007; Kolstad & Wiig, 2012). The BRI was initiated in 2013 (Duan et al., 2018) and is ‘designed to stimulate economic development by dramatically enhancing regional interconnectivity’ (Rolland, 2017: 127). More specifically, BRI was ‘devised to reconfigure China’s external sector in order to continue its strong growth’ (Huang, 2016: 314). This is done in several ways, such as infrastructure development and “connectivity” investments (Swaine, 2015). Past studies found that host country engagement in the BRI significantly increases FDI volumes into those countries. More specifically, infrastructure-based projects were found to be primarily driven by state-owned firms, while non-infrastructure projects are primarily driven by private Chinese firms (Du & Zhang, 2018). In the case of private investment in BRI host countries, the development of overseas special economic zones (SEZs) play a particularly germane role. INTRODUCTION Overseas SEZs are Chinese government funded areas which seek to promote Chinese FDI into host countries with a focus on, for example, logistics, export processing, and manufacturing (Bräutigam & Tang, 2012). In many cases, Chinese overseas SEZs are created for Chinese MNEs exclusively (Bräutigam & Tang, 2014). While these SEZs are not created only in BRI countries, they have become an important policy pillar within the BRI structure and strategic approach. As a policy initiative, BRI – and it subcomponents, such as SEZs – is intricately linked to the internationalization of Chinese firms. Accordingly, this raises a question: does the BRI increase the attractiveness of host- countries associated with institutional weakness (which we refer to hereafter as fragility) for CMNE related FDI? 4 We contribute to the literature on FDI from China by proposing and testing the impact of Chinese policy initiatives on the counter-intuitive relationship found in past articles between institutional fragility and Chinese investment. We confirm that Chinese FDI is more strongly attracted by weak institutional environments. In addition, we find the BRI policy amplifies (i.e. positively moderates) the impact of host country institutional fragility on Chinese FDI volumes. We also find evidence that Chinese FDI is sensitive to weaknesses in specific institutional domains in the presence of the BRI: it is higher in countries with weaker rule of law and less accountability under the BRI policy. We consider why this might be so, focusing on the difficulties of implementing large-scale infrastructure projects, an important component of the BRI, in nations with strong legal institutions and greater accountability. In addition, we consider how the depth of host country engagement with the BRI (proxied by SEZ creation), may amplify (i.e. moderate) the impacts of institutional fragility on FDI volume. We do so by exploring the simultaneous engagement of host countries in the BRI as well as China’s strategic initiative to create overseas SEZs in FDI recipient host countries. As mentioned, Chinese overseas SEZs are an additional component of the BRI and thus creation of an SEZ indicates fuller engagement with the BRI by host countries. In such cases we find the moderation effects of BRI engagement on institutional fragility to be even greater. Our discussion notes that from a host country perspective, approaches for attracting Chinese FDI appear to stand in direct contrast to those advocated in Washington consensus style policy toolkits (i.e. promoting sound institutions). INTRODUCTION This is because Chinese FDI is more strongly associated with BRI countries when there is less transparency, accountability and weaker rule of law. The paper proceeds as follows: we first develop three hypotheses, related to the BRI’s impacts The paper proceeds as follows: we first develop three hypotheses, related to the BRI’s impacts on Chinese outward (O)FDI volumes, incorporating the moderating impacts on institutional fragility as well as the development of overseas SEZs as representative policies. We then outline our 5 methodological approach using a panel data set on Chinese greenfield and acquisition equity investments in host countries world-wide, accounting for potential endogeneity concerns. Finally, we discuss our results, highlighting the positive moderation impacts on institutional fragility of the BRI initiative and the further amplifying impacts of the accompanying creation of overseas SEZs. LITERATURE REVIEW AND HYPOTHESES DEVELOPMENT Host country political and diplomatic ties with China and Chinese outward FDI volumes That state agencies intervene in the FDI decision of CMNEs, especially state-owned ones, has been established by previous research and is well documented (Quer et al., 2012, 2018, 2019, 2020; Li & Alon, 2019). The state engages its multinationals both directly (e.g., ownership and control), indirectly (e.g., regulation and incentives) and in relation to specific markets (e.g., idiosyncratic bilateral and multilateral institutional relationships). A number of prior studies have investigated the link between bilateral international relationships and inward FDI volumes from China. According to Zhang, Jiang and Zhou (2014), bilateral diplomatic activities, such as senior government official visits, increase Chinese FDI, especially in resource-rich countries. Quer et al. (2019) report similar findings specifically in visits to Latin American countries. Further, it has been found that ties between political actors in different countries, through UN voter similarity, for example, increases the likelihood that a Chinese firm will establish a foreign subsidiary in that host country (Li et al., 2018). Duanmu (2014) also reports somewhat similar findings, but clarifies that the relationship depends on ownership. These papers provide a foundation for understanding the relationship between creating and strengthening bilateral relationships and increasing levels of Chinese FDI. However, to date this research has largely tested this relationship through implicit means (e.g. senior official visits, UN voter similarity). 6 6 Our purpose here is to expand this research by testing the influence of more explicit bilateral investment policies on Chinese OFDI, specifically the BRI. This policy initiative, created by the Chinese government and consisting of two components (the Silk Road Economic Belt and a 21st Century Maritime Silk Road (Fei, 2017: 838)), involves ‘the funding and construction of a system of roads, railways, oil and natural gas pipelines, fiber-optic and communication systems, ports, and airports’ (Lairson, 2018: 40) and deepening economic integration and engagement with China in order to create new economic opportunities and assert greater international influence (Huang, 2016). It has been referred to as an ‘ambitious $1tn project, stretching from the South Pacific to the fringes of Europe and Latin America’ (Weinland, 2019: 18). LITERATURE REVIEW AND HYPOTHESES DEVELOPMENT Along with being a massive construction project to fund and construct infrastructure of all kinds, it aims to further integrate Chinese and participant host countries, which covers ‘cooperation in all aspects, from policy dialog to trade, from financial cooperation to people-to-people exchange’ (Zhang, et al., 2018: 2). The BRI is intended to be a multilateral system of deep interdependence where all participants gain from China’s ‘efforts in supplying capital, defining and implementing a system designed to provide mutual growth and potentially providing the rules, norms, and institutions to facilitate the operation and management of such a system’ (Lairson, 2018: 38). It has set up, for example, specific international dispute resolution mechanisms for BRI related investments (Tao & Zhong, 2018). Finally, it is important to note that policy orientation has also ‘shifted from direct financial assistance to the output of development experience….as a sharing of China’s expertise and development success’ (Brautignam & Tang: 812) (emphasis added). To date, surprisingly, there are comparatively few studies that have explored the economic impacts of the BRI. Perhaps, as Zhai (2018) notes, this is because of some of the challenges in pinning it down: ‘the BRI is still a flexible conceptual initiative and far from a well-defined action plan with 7 top-down design. The vagueness of the BRI program leads to difficulties in quantitatively evaluating its economic impact’ (Zhai, 2018: 85). Or, as Hillman (2018) puts it: ‘the BRI label evades classification. There is no agreed-upon definition for what qualifies as a BRI project’ (p. 3). Extant studies have focused primarily on its potential impacts in facilitating trade flows, particularly between Europe and China (i.e. via land routes) and in doing so spurring economic growth (Zhai, 2018; Maliszewska & Van Der Mensbrugghe, 2019; Herrero & Xu, 2016; Villafuerte et al., 2016). However, as well as improved trading relationships, increased Chinese FDI to host countries would also appear an inevitable outcome of the BRI. There is anecdotal evidence, for example, that those host countries involved with the BRI have increased their levels of overall Chinese OFDI. Hillman (2018), for example, notes how ‘Chinese outbound capital restrictions appear to be more relaxed for BRI-related transactions. Deals that might be classified as advancing the BRI are more likely to be approved, and to be approved faster. In contrast, China has been reining in outbound deals for foreign real estate, entertainment, and sports teams’ (p. 3). LITERATURE REVIEW AND HYPOTHESES DEVELOPMENT Our purpose, as noted, is to further explore the impacts of the BRI on Chinese FDI. What types of FDI are BRI countries likely to receive? What types of FDI are BRI countries likely to receive? China has begun many sizable infrastructure projects throughout the BRI countries, examples include $1.73 billion for transport, energy, and communication projects in Central Asia (Indeo, 2018), China General Nuclear Power Corporation’s $7.8 billion investment in a Romanian nuclear power plant and another $11.1 billion for a special investment fund under the BRI for other Eastern European ventures (Vangeli, 2017). Other current infrastructure projects include $1.6 billion Batang Toru dam being built on the island of Sumatra, Indonesia, and in Kenya the construction of a $2 billion coal powered power station (Wilson, 2019). While construction projects may involve the 8 establishment of foreign subsidiaries, often related to very large state-owned construction firms (China now has eight of ten largest construction firms in the world (Hillman, 2018)), it is important to note such projects are often associated with a range of accompanying private sector initiatives (Parente et al., 2019). Initial construction projects, underwritten at first by soft loans, in time also provide opportunities for all Chinese firms to learn more about foreign markets, develop networks (with local firms and politicians) and thus lead to new market opportunities. Infrastructure, therefore, has been found to act as an important initial catalyst for further subsequent FDI of different types (Parente et al., 2019). As such, we would expect countries that engage with the BRI to, in general, receive higher FDI inflows. Hypothesis 1: Host country engagement with the BRI is associated with greater volumes of FDI from China. 1 The concept of institutional fragility Shi et al. (2017) develop is somewhat different from ours. They argue that rather than focus on a more static view of institutional change, different dimensions of institutions progress (or regress) at different rates, which creates internal friction and conflict during institutional reform. Do formal policy initiatives moderate the impact of institutional fragility? Recently, scholars have found that Chinese FDI appears to counter conventional wisdom on the relationship between institutional fragility and FDI (Buckley et al., 2018). Institutional effectiveness stems from complementarity across three set of political institutions: the state, rule of law, and political accountability (Fukuyama, 2014). When these are absent or weak, institutional fragility occurs (Shi et al., 2017).1 Countries with high levels of internal institutional friction have, at least for developed market MNEs, long been viewed as highly risky for FDI and to be avoided (Kobrin, 1979; Brewer, 1981; Alesina & Tabellini, 1989; Howell & Chaddick, 1994; Alon & Martin, 1998). Yet, a range of cross-sectional and panel studies have found Chinese FDI volumes appear to 9 correlate positively with a country’s level of institutional weaknesses (which we also refer to here as “institutional fragility”). Buckley et al. (2007), for example, find a positive and significant impact of institutional fragility on Chinese FDI. Using political stability, Ramasamy et al. (2012) finds similar results. Kolstad and Wiig (2012) find an interaction between institutions and natural resources is negative and usually significant, suggesting Chinese natural resource seeking firms invest in weak institutional climates. Blomkvist and Drogendijk (2013) concluded that the larger the differences in the political systems between China and a potential target market, the less Chinese firms invest in that market. They suggest Chinese FDI is attracted to countries that disregard human rights and have relatively high political risk profiles. Kang and Jiang (2012) argue Chinese firms prefer to locate in risky locations as those locations are similar to their domestic business environment. The heft of the Chinese government in backing investments of CMNEs through explicit policies, such as the BRI, might create institutional fragility attractiveness for CMNEs. The Chinese state, for example, has the ability to critically engage host countries via its policies, which subsequently may redefine conventional risks commonly associated with institutional fragility. This potentially creates opportunities for CMNEs, as well as a new path for host countries to generate FDI from China. One aim of the BRI is to provide an institutional system to facilitate investment and other economic activity between BRI countries and China. This structural system of cooperation and interdependence through the BRI signals to CMNEs that they have the ability to leverage their government’s external policies. Do formal policy initiatives moderate the impact of institutional fragility? This may reduce actual as well as perceptions of risk for Chinese MNEs investing in participant countries and thus increase investment, despite the persistence of institutional fragility within them. Indeed, what may be considered a politically risky unattractive investment location by a Western developed market MNE, owing to lack of legal structures, accountability, corruption, and weak regulatory systems (and no domestic governmental support – 10 rather hindrances – i.e. laws outlawing corrupt practices like bribery), may be perceived as the exact opposite by Chinese MNEs. Infrastructure related development projects are a significant component of the FDI projects undertaken in BRI countries (Hillman, 2018). There are also reasons for thinking that undertaking such projects may be easier in institutionally weak environments. The construction of dams, roads, ports, power plants and the like may entail complex political consultation processes when undertaken in institutionally strong environments. Interest groups, represented by NGOs and civil society organizations, must be consulted in institutionally developed settings. Legal restrictions (for example, governing environmental standards, safeguarding of biodiversity etc.), moreover, may potentially cause long delays or even scupper projects in institutionally developed countries. Legal interventions can certainly add uncertainty as to whether some infrastructure projects can be completed. By contrast, such infrastructure projects might be more easily negotiated and implemented in weaker institutional environments. While the financing channels available to BRI countries are significant (involving, among other things, soft loans from Chinese development banks), these cannot be accessed if projects cannot be approved. The legal complexity of many such projects means they may be more easily initiated and completed in authoritarian regimes where rule of law is mandated, often by dictatorships. To illustrate how the BRI may positively moderate the impact of institutional fragility on FDI, the case of Chinese investments made by the Queensway Group in Angola (a BRI member) and subsequently other developing countries, is instructive. After a 27year civil war (ending in 2002) Western donors (such as the IMF) were unwilling to support Angola. China, by contrast, quickly developed political ties with Angola, in part owing to it large oil reserves (which China lacks). To date, Chinese engagement with Angola has provided more than 60 billion US dollars in loans for infrastructure projects, including power plants, bridges, 2,800 kilometers of railways, 20,000 11 kilometers of roads, 100 schools, 50 hospitals and 100,000 houses (He, 2018). Do formal policy initiatives moderate the impact of institutional fragility? Much of this financing has been paid for in kind, by Angolan oil exports (now known as the “Angolan model”, where infrastructure packages are paid for by resource deals). Detailed award-winning journalism published in the Financial Times documents how China’s Queensway Group skillfully nurtured its links with leading Angolan political and business figures, cutting deals via backhanders to high-level insiders, to obtain a significant share of Angola’s oil reserves (Burgis, 2014). The Angolan model which was to emerge from Queensway’s early interventions was facilitated by an unaccountable authoritarian regime and involved questionable business practices which allowed insider political elites, in both China and Angola, to enrich themselves via secretive deals. In open, transparent and rule-based societies, these oil for infrastructure deals, would likely never have been completed. Subsequently, the Queensway Group has been active in a range of other poorly governed African countries (Guinea, Mozambique, Zimababwe), using identical tactics (developing and exploiting close relationships with unaccountable political elites) to those employed in Angola (Burgis, 2014). The BRI has provided the necessary financial support and legitimacy to Chinese investors like Queensway (for example, the group invited Angola’s leaders to meetings in Beijing with China’s vice president, Zeng Peiyan), required to grease the wheels of corrupt officials in unaccountable regimes, subject to judicial voids. Political legitimacy and close state to state relations have been important to groups like Queensway, now considered a specialist in working in these types of business environments. Queensway is privately controlled but has received high-level government support in China. By contrast, western developed market MNEs hoping to engage in foreign corrupt business cannot expect their home governments to support them. For example, Swedish network equipment giant Ericsson recently 12 agreed to pay over $1 billion in fines to US regulators after pleading guilty to bribing government officials in multiple countries over a 17-year period.2 We argue the BRI may moderate the impact of institutional weakness on Chinese FDI. Firstly, it supports investments in particular areas – construction and infrastructure development – which may be easier to complete in institutionally weak environments. It provides ample financing mechanisms through Chinese development banks to support these projects. It is prepared, moreover, to work with authoritarian political systems. Secondly, via stronger interstate relationships, investments risks (and perceptions of those risks) in FDI projects are reduced. Chinese FDI has historically been attracted to institutionally weak countries - owing to “special” ownership advantages. Do formal policy initiatives moderate the impact of institutional fragility? BRI positively moderates this tendency owing to the stronger state to state relations it may foster (enhancing CMNE bargaining positions), increased financial resources and lower perceived and actual risk it creates for investing businesses. Hypothesis 2: Host country formal engagement in the BRI positively moderates the impact of institutional fragility on Chinese FDI inflows. 2 International law, however, is starting to catch-up with Queensway, whose founder is now being pursued by US authorities. law, however, is starting to catch-up with Queensway, whose founder is now being pursued by US What are the impacts of the BRI when accompanied by explicit outward FDI promotion? The further moderating impact of China’s overseas Special Economic Zones The BRI is one of the most ambitious foreign policies ever launched. It consists, however, of a number of associated policy measures although owing to the opaque nature of BRI it is not exactly clear which measures are associated with the BRI and which ones are not. As noted, ‘the vagueness of the BRI program leads to difficulties in quantitatively evaluating its economic impact’ (Zhai, 2018: 85). Hillman (2018), notes, for example, ‘BRI is more a brand than a master plan with specific criteria 13 for project inclusion’ (p. 5). For example, it is allegedly integrated with the establishment of multilateral development banks (particularly the Asian Infrastructure Investment Bank, AIIB) and cross-regional economic cooperation programs (Clarke, 2018). From the point of view of understanding CMNE OFDI, however, a key plank of the BRI initiative, and one specifically related to promoting FDI (as opposed to infrastructure development), has been the addition of overseas SEZs. It has been noted, for example, that ‘SEZ development has been adopted energetically at the heart of the BRI…to provide Chinese companies (and executives) with a controlled channel for building familiarity with and exposure to international markets and the global economy’ (Fei, 2017: 840). According to MOFCOM (2015), the BRI is not only ‘taking advantage of international transport routes, relying on core cities along the Belt and Road’ (p. 839) it is also ‘using key economic industrial parks as cooperation platforms’ (State Council, 2015, highlights added by the author, quoted in Fei, 2017: 839). As of 2015, 77 of 118 Chinese overseas SEZs were to be built in BRI partner countries (MOFCOM, 2015). The creation of overseas SEZs have the expressed purpose of improving the institutional environment for CMNE foreign investments, primarily in manufacturing, through ‘filling information gaps and reducing risks and high transaction costs’ (Brautigam & Tang, 2012: 800). They are also meant to be mutually beneficial; in exchange for furthering Chinese strategic objectives, China is willing to transfer some of its own economic success to other developing countries (Brautigam & Tang, 2014). For example, the SEZ created by China jointly with the Egypt government in Suez reduces red-tape and other institutional costs through a “one-stop-shop” administrative services building housed within the complex (El-Gohari & Sutherland, 2010). In several other African and Cambodian SEZs, the Chinese government intervened in disputes between host governments and Chinese firms (Brautigam & Tang, 2012). What are the impacts of the BRI when accompanied by explicit outward FDI promotion? The further moderating impact of China’s overseas Special Economic Zones Chinese MNEs are also eligible for financial assistance 14 from major national banks such as the China Development Bank, which offers subsidies of up to $61 million for resource extraction industries and $46 million for others, concessional loans and other incentives to eligible companies operating in SEZs within BRI countries (CDB, 2016; cited in Fei, 2017). The zones are intended to help offset the considerable liability of foreignness that CMNEs may face, particularly in their initial stages of internationalization. Overseas SEZs provide agglomeration benefits to small and medium-sized Chinese MNEs that have limited experience working in foreign environments (Brautigam & Tang, 2014). Many of the countries CMNEs are attracted to, moreover, may often be poor countries with low labor costs but, in addition, very weak institutional environments (i.e. in sub-Saharan Africa). Some of China’s private sector manufacturing firms, for example, are looking to exploit lower cost labor (i.e. in Laos, Cambodia, Democratic Republic of Congo, etc.) owing to shrinking size (and increasing wages) of China’s working age population (i.e. its “demographic dividend” is being lost) (Lin, 2012). Hence, the Chinese government has been keen to promote zone development as private sector initiatives. An important component of the SEZ strategy relates to its exclusivity in restricting investors to Chinese companies alone. By specifically concentrating Chinese businesses together in zones their strength in numbers, as a group, can be leveraged. Their group influence, moreover, is ultimately underpinned by the Chinese state, which may give host countries food for thought in any attempt to take advantage of zone based CMNEs. Host countries, instead of picking-off Chinese companies one by one (as China has been able to do to its foreign investors in China – forcing them into unfavorable joint ventures, for example, in which technologies can be assimilated), are in a far weaker position to negotiate with the economic mass of an entire zone with many affiliated companies supported by “China Inc.”. The reasoning is somewhat similar to Duanmu’s (2014) finding that stronger trading 15 relationships weaken the impacts of expropriation risk on CMNEs – because host countries have potentially far more to lose when dependency levels are higher. Host countries clearly have a great deal to lose by alienating CMNEs based in zones. Zones may therefore provide focal points for state to state negotiations (regarding preferential policies, for example). What are the impacts of the BRI when accompanied by explicit outward FDI promotion? The further moderating impact of China’s overseas Special Economic Zones Why then might zones also moderate the impact of institutional fragility on Chinese investments? It is likely that Chinese zones enhances the bargaining position of the Chinese state (as representative of its investors) and, furthermore, that there is greater scope for bargaining in institutionally weak environments, where more can be negotiated for (as there are fewer legal constraints on what can and cannot be done). There is, moreover, comparatively less concern about being held accountable by other non-governmental and civil society interest groups. The avoidance of institutionally fragile countries by Western MNEs due to the perceived risk, moreover, provides a “blue ocean” for Chinese OFDI. By engaging relatively untapped markets through policy initiatives such as the BRI, which provides multilateral infrastructure and cooperation, combined with overseas SEZs, which provides additional bilateral support, the Chinese government decreases the investment risks associated with these countries and increases their attractiveness for Chinese MNEs. Hypothesis 3: Host country formal engagement in the BRI combined with the associated SEZ policy strengthens the positive moderation effects on institutional fragility on Chinese FDI inflows. METHODS Data and model Data and model Data and model Our model specification is along similar lines to Buckley et al. (2007) albeit we estimate models using the annual equity value of FDI projects drawn from commercially sourced FDI flow 16 data. 3 Officially collected, nationally aggregated FDI data from all countries is increasingly recognized for its biases (Sutherland et al., 2019). This is driven to a large extent by the bilateral way in which FDI is collected. As MNEs often transit FDI via offshore tax havens and financial centers these destinations are greatly overstated in conventional FDI data (OECD, 2015). China’s MOFCOM data is no exception, with heavy biases towards Hong Kong, the Cayman Islands, BVI and, in developed markets countries like the Netherlands and Luxembourg (Sutherland & Anderson, 2015). Value data are estimated using random effects (following the results of a Hausman test) generalized least squares (GLS) models and utilize panel data of FDI flows from China to the rest of the world (173 countries) during the time period of 2003-2017. Estimations are found to be unbiased due to the use of both within and between group variation. This lengthy time period helps capture pre and post BRI impacts on FDI volumes and moderating effects. Our primary model is defined as: FDIti = f (β1BRIti, β2Institutional fragilityti, β3Country risk premiumti, β4Cultural proximityti, β5Geographic distanceti, β6GDPti, β7GDP growthti, β8Natural resource exportsti, β9Exchange rateti, β10Inflationti, β11Open to FDIti, β12AIIB memberti, β13Bilateral trade agreementti, β14Pct agree UN voteti) Where t is time and i is host country. Where t is time and i is host country. Where t is time and i is host country. Count data were also estimated as a robustness check. Results were quantitatively similar to valu Dependent variable As noted, Chinese outward FDI data is drawn from the commercial databases Thomson ONE Banker and the Financial Times fDi Markets. This consists of 2,031 acquisitions and 4,402 greenfield investments (totaling 6,433 investments). Of these, 782 were positively identified as SOEs. The average investment values for greenfield and acquisition investments are $111 and $161 million, 17 17 respectively. We focus on all projects in which Chinese ownership exceeds 10%, following standard FDI definitions. Note that our approximation of Chinese FDI does not include intra-company loans or reinvested earnings. It can be thought of as an approximation of first entry equity FDI. In reality, given the difficulties of using officially recorded FDI data, using commercially available data in this way is one of the few realistic ways of gauging CMNE activity (and approximates to the methods used by influential think-tanks like the Heritage Foundation’s measurement of Chinese MNE activity). Independent variables Our main independent variables are based around the widely used Political Risk Services Group (PRS) institutional stability measures. Disaggregated this includes: control of corruption; political stability; rule of law; regulatory quality; and government accountability. A downside of using these disaggregated measures is the relatively high level of collinearity between them. We also, therefore, create an index based around the average of the five measures (both are used to test hypothesis 2, regarding moderating impacts of BRI). We invert our aggregated and decomposed measures. Higher scores for institutional risk variables therefore represent higher levels of institutional fragility. Two dummy variables are used to capture affiliation to BRI (hypothesis 1) and the overseas SEZ policy (hypothesis 3), as reported by MOFCOM. To test hypothesis 1 we run a model with the BRI variable. In addition, we incorporate a number of additional policy initiatives, including AIIB relationship, UN voting similarity and bilateral trading relationships, to explore the impacts of other measures on Chinese FDI volumes. Some of these initiatives, such as the AIIB, have been associated with the BRI initiative, but are in fact only loosely tied to it. The AIIB, for example, was initiated and is led by the Chinese government and is self-described as ‘a multilateral development bank with a mission to improve social and economic outcomes in Asia’ (AIIB, 2019: 1). The main motivation 18 behind the development of AIIB is for it to be used as ‘an important financial tool for China in increasing its geopolitical influence in the region and increasing the international momentum of the [BRI] strategy’ (Yu, 2017: 359). But it is not officially part of the BRI. Similarly, trade relationships are not included. They thus provide interesting contrasts with the BRI itself. Other comparators included, are non-explicit policies such as UN voting similarity with China, expressed as a percentage. To test hypothesis 2 we run models that incorporate the PRS index in our base model combined with an interaction term (PRSBRI). We also test this hypothesis by looking at the outcomes for individually decomposed elements of PRS indicators (Table 4). Finally, to test hypothesis 3 we incorporate a three-way interaction (BRISEZPRS) (Table 5). Control variables Control variables Control variables are similar to those used in other Chinese location choice studies (Buckley et al. 2007). They include: country risk premium (country risk premiums matched to averaged credit default swaps spreads and bond ratings – Moody’s and Bloomberg); GDP (World Bank); GDP growth (World Bank); natural resource endowment (Fuels, ores, and metals exports as share of GDP – World Bank); inflation (inflation rate – World Bank); geographic distance (Distance between capital of host country and China – CEPII/World Bank); exchange rate (Host country annual average exchange rate against RMB (fixed to dollar) – World Bank); Chinese diaspora (Ohio University); and openness to FDI (inward FDI stock as a share of GDP – World Bank). State ownership is defined as a greater than 50% government ownership (Orbis). See Table 1 for a summary of variables and data sources. Insert Table 1 about here 19 RESULTS Table 2 presents the descriptive statistics and a correlation matrix. It is noteworthy that many of the PRS elements (control of corruption, political stability, accountability, law, regulatory quality) are correlated with each other. Multicollinearity, however, does not bias our results, though it can lead to complications in hypothesis testing by inflating standard errors. Beyond the PRS variables, multicollinearity is not an issue with regards to other variables in our model (Table 2). Insert Tables 2-3 about here ------------------------------ Table 3 reports the aggregated institutional fragility measure plus BRI as a dummy, and a combined interaction term (BRIPRS). Model 1 (without the interaction) suggests FDI volumes to BRI countries are higher than for non BRI countries (supporting hypothesis 1). However, when introducing the interaction term this variable becomes insignificant. This may be because the interaction introduces multicollinearity into the model, an issue common in interaction models. As regards hypothesis 2, the interaction term itself is positive and significant (5% level), supporting the idea that the BRI positively moderates the impact of institutional fragility on inward Chinese FDI. The other policy measures we include in our models (AIIB and bilateral trade agreements) do not exhibit this same moderating impact. Based on the aggregated results, Table 4 further decomposes the PRS institutional fragility measure into its five different components. Table 4 again (corroborating Table 3) shows that the BRI dummy variable is significant, both in the fully specified model (last column) and most of the models reporting PRS components individually. This again supports hypothesis 1. 20 Hypothesis 2 predicts that the BRI policy positively moderates the impact of host country institutional fragility on FDI volume. The interaction term in Table 3, as noted, is significant and positive, suggesting the coefficient on PRS (aggregated) is larger for BRI countries than non-BRI countries. In Table 3 the PRS coefficient on its own is also significant, suggesting that even when not explicitly a recipient of the BRI policy, Chinese MNEs are more strongly attracted to institutionally fragile countries. Table 4 further decomposes the PRS institutional fragility measure into its five main components. Table 4 shows that legal institutional fragility stands out as a positive moderator. Accountability is also a significant and positive moderator, albeit the evidence is not quite as clear cut. Only in one of the models (last column) is the interaction term significant. Hypothesis 3 predicts the BRI policy initiative when combined with the creation of overseas SEZs is a fuller and more complete set of BRI policy measures and thus strengthens the moderation impacts originally hypothesized. Table 5 reports estimations for our full sample. Table 5 shows that the three way interaction of BRI, SEZ and PRS is significant and positive. This implies that a BRI partner hosting an SEZ further positively moderates the impact of institutional fragility. Insert Tables 4-5 about here Insert Tables 2-3 about here Model 3 in Table 5 supports our earlier hypothesis 2, albeit significance is lost in model 4 (possibly owing to multicollinearity). 4 We do lag our variables (by one year). Such lags, however, are an insufficient solution on their own to correct for endogeneity and potential biases in coefficient estimates. 4 We do lag our variables (by one year). Such lags, however, are an insufficient solution on their d i d i l bi i ffi i i We do lag our variables (by one year). Such lags, however, are an insufficient solution on their endogeneity and potential biases in coefficient estimates. ur variables (by one year). Such lags, however, are an insufficient solution on their own to correct fo ( y y ) g , , nd potential biases in coefficient estimates. Supplementary tests: endogeneity and the BRI policy Supplementary tests: endogeneity and the BRI policy Do host countries engage in the BRI policy because they have already received significant levels of Chinese FDI, rather than the other way around, as we hypothesize? Do governments that are prone to entering the BRI also have a stronger likelihood/propensity to attract Chinese FDI? Is there 21 endogeneity or selection problem, in other words? 4 Conceptually, we argue there are strong arguments in favor of our hypothesized direction in causality, namely from policy to inward FDI. This is because the BRI is intimately connected to China’s foreign policy and its underlying geopolitical strategic intentions. Strong political drivers motivate the BRI, which is concerned with enhancing China’s foreign influence, via both hard and soft power. In doing so, China hopes to create a sphere of political influence around China via engagement in economic development programs subsumed under the BRI (and SEZs). Promoting economic interlinkages is certainly one way of achieving this target. Put simply, if current bilateral economic ties are weak, there would appear to be stronger political incentives to implement the BRI than in the case of already strong economic ties. We believe, therefore, that China’s promotion and endorsement of the BRI in a host country is unlikely to be driven by the strength of prior economic linkages or FDI. This reasoning is reflected in many of the countries that have entered the BRI, which includes many less developed, low income countries (i.e. Kenya, Djibouti, Ethiopia, DRC, Egypt, etc.) with comparatively weak economic ties to China – albeit with the potential for considerable expansion. Nonetheless, to further empirically explore the possibility of endogeneity, we used an instrumental variable (two-stage least square) estimation to explore whether reverse causality is a potential issue in our modelling (Baum, 2006). By establishing a suitable instrument, a variable correlated with the endogenous variable in question (BRI) but not with the error term, we can run endogeneity tests. One potential instrument is the measure the quality of diplomatic relations between China and the host country as measured by the affinity of the two countries’ votes in the United Nations (UN) General Assembly. In previous studies, Li et al. Supplementary tests: endogeneity and the BRI policy We cannot reject the null hypothesis based upon our results. This implies that the BRI is exogenous of prior Chinese inward FDI.5 hypothesis for these tests is that the variables are exogenous. We cannot reject the null hypothesis based upon our results. This implies that the BRI is exogenous of prior Chinese inward FDI.5 5 In addition, we use the instrument to test for omitted variable bias. Sargan and Basmann tests, with a null hypothesis that the instrument set is valid and the model is correctly specified, cannot be rejected, implying our model is correctly specified. Supplementary tests: endogeneity and the BRI policy (2018) and Duanmu (2014) have used the affinity measure of UN votes (based on Strezhnev & Voeten, 2013) in their investigation of the 22 impact of political ties on Chinese FDI (as a main explanatory variable, however, not as an instrument). Using this measure as an instrument, however, may be suitable. It is correlated with our BRI explanatory variable as countries joining the BRI are, in general, likely to share closer political ties and likeness to China. According to Li et al. (2018), for example, UN voting similarity: “demonstrates the public stance on a large number of issues, including military, security, social, political, and economic concerns (Voeten, 2000). Voting at the general assembly does not bind countries and thus countries are relatively free to express their sincere opinions (Gartzke, 1998). Countries voting similarly are expected to have a good relationship and act cooperatively because they share similar views and understanding on world issues (Gartzke, 1998). Thus affinity of UN votes has been frequently used to capture interstate political relations in political science and international strategy” (Li et al. 2018: 668). They note that diverse issues, moreover, are discussed at the UN general assembly and that voting decisions are not likely ‘to be shaped by corporate interests in a foreign country’ (Li et al. 2018: 668). Using this measure in their models, they argue, alleviates potential reverse causality to some extent. This measure uses two categories of voting data (1 = “yes” or approval for an issue; 2 = “no” or disapproval for an issue) and ranges from -1 (least similar interests) to 1 (most similar interests). We use a similar approach. To establish whether UN General Assembly voting similarity is a viable instrument and can therefore be used to test for endogeneity we undertake several tests. First, we test whether UN voting similarity satisfies the requirements defining an independent variable. From our first stage regressions we find it does have a statistically significant and positive effect on our main measure of BRI. We find the F-value of the first stage regression achieves a value of 49.55 (p=0.00) exceeding the critical value of the Wald test (5% level) which has a maximum level of 16.38. We then perform the Durbin (=1.293, p=0.255) and Wu-Hausman (=1.280, p=0.258) tests of endogeneity. The null 23 hypothesis for these tests is that the variables are exogenous. DISCUSSION Why does the BRI policy positively moderate the impacts of institutional fragility on Chinese FDI? We have found, like a number of other studies (Buckley et al. 2007; Kolstad & Wiig, 2012), that countries with fragile institutions attract greater volumes of FDI from China. Under the BRI, moreover, this affect was found to be even stronger (i.e. that the BRI positively moderates this already counter intuitive result). How might the BRI policy amplify the impact of institutional fragility on Chinese FDI? Arguably, one key competitive advantage Chinese businesses exploit is their special relationships to governmental and quasi-governmental actors (Yiu, 2011). The Chinese developmental state, in other words, has close relationships to both private (and of course) state sector businesses (Brautigam & Tang, 2014). This relationship extends from the domestic to international arena, where the government has been keen to promote the internationalization of Chinese businesses (particularly large groups, i.e. the “national team” business groups) (Sutherland, 2009). Previous research, as noted, has established how different measures of bilateral political ties (such as, for example, official visits and trade agreements) are leveraged to ease Chinese FDI in foreign markets (Duanmu, 2014, Li et al., 2018; Quer et al., 2018). Such linkages reduce expropriation risks, afford Chinese firms relevant information and help overcome a range of liabilities of foreignness (Quer, 2018). Using a similar line of reasoning, we hypothesized that the Chinese state, via its BRI policy, may make it easier for CMNEs to do business in weaker institutional environments (i.e. those with 24 greater fragility). How specifically does the state intervene and what are the mechanisms underlying this positive moderation effect in the case of the BRI? To further explore this question, we can examine in more detail our results pertaining to institutional fragility and decompose them by their various sub-components. Are there any specific institutional factors where the moderation effects can be identified and might these results help us better understand how BRI policy interacts and facilitates Chinese FDI? Interestingly, our results on the decomposed institutional measures showed that the impacts of weaker rule of law and less government accountability (more authoritarian regimes) were positively moderated by the BRI policy. Why would the BRI make investments to countries with weak accountability and legal systems more attractive or viable? One plausible explanation, relates to the aforementioned industrial composition of many BRI related FDI projects. p p g p was a let-down and failed, however, owing to the challenges of finding viable ‘ready to go’ projects 6 President Obama came into office on the promise of a massive infrastructure programme. This programme ultimately p p g p g y failed, however, owing to the challenges of finding viable ‘ready to go’ projects. DISCUSSION Such projects are associated with large-scale infrastructure developments (dams, railways, ports, bridges, roads, etc.) (Parente et al., 2019). These giant construction projects, by their nature, are often politically sensitive and typically have long gestation periods prior to commencement. Project planning may involve many consultation procedures, for example, as such projects typically displace local residents and often entire communities.6 In most instances, such projects cannot therefore be easily and quickly rolled out. This is likely to be particularly the case in countries with stronger legal and political institutions, where planning procedures are more rigorous. The erection of the BRI’s Batang Toru dam in Indonesia, for example, has led to fierce local resistance from local NGOs, as it endangers a critically rare species of orangutan threatened with extinction (Wilson, 2019). Similarly, Indonesia’s flagship BRI project, a $6 billion high-speed rail project linking Jakarta with Bandung (140km away), has run into chronic delays and controversies partly over land acquisition disputes (Wilson, 2019). In Kenya, the construction of a $2 billion coal-fired 25 power station in close proximity to the World Heritage site of Lamu, envisages creation of the largest power plant in east Africa. According to the Chinese backer it will ‘solve the power shortage for millions in the region’ (Wilson, 2019: 1). However, recent court rulings have halted the development on environmental grounds, fearing the massive coal-powered station will destroy the pristine natural environment of the region (Wilson, 2019). These infrastructure projects, moreover, require very large, long-term investments and thus commitments by governments to take on significant long-term debt. In some instances, owing partly to poor project appraisal it has been suggested, these have turned out to be unserviceable. Examples include multi-billion dollar deep sea ports in Malaysia, Myanmar and Sri Lanka (Crabtree, 2019). Negotiating these very large infrastructure deals, however, may be more easily undertaken in the context of regimes that are less accountable to democratic processes and therefore sudden removal. Parente et al. (2019), for example, outline in detail the important impacts of regime stability on the commitment to FDI projects of Chinese MNEs in the Democratic Republic of Congo. These factors may also lead to the positive moderation impact of institutional fragility that we observe, specifically with regards to accountability. DISCUSSION Many Chinese infrastructure-based foreign investments involve projects in which domestic governments have attempted to push through infrastructure developments and related FDI projects with the help of Chinese financial largesse combined with construction expertise. However, many have been, to a greater or lesser degree, frustrated by legal and other political pressures, largely driven by NGOs and other civil society groups. These examples illustrate why BRI countries with weaker legal systems and less accountability may actually suit Chinese BRI related FDI projects. Such projects can be more easily initiated and completed in countries with less governmental accountability (in part related to democratic institutions, freedom of press, media and internet) and weak legal 26 systems (which otherwise would block heavy handed governmental interference, as in the case of the coal-fired power station in Lamu, Kenya). Powerful institutional forces, therefore, may lead countries with weak legal redress and limited accountability to be first in line to receive Chinese BRI related projects. Ample financing exists, of course, for Chinese projects in BRI partner countries. The challenge many Chinese construction groups face is in finding feasible projects. Arguments related to speed of infrastructure development initiation are also supported by consideration of the BRI’s multifarious objectives. One of these is the creation of new markets for export of Chinese surplus capacity. Implementation of infrastructure development overseas creates immediate markets for Chinese products, such as iron and steel, cement, chemicals and a host of other building materials (glass, tiles, porcelain, gypsum, etc.) as well as manufactured products like machinery and equipment (cranes, bulldozers, tunnel boring equipment, etc.). Clearly, BRI related policy-making, and those tasked in undertaking BRI projects, may opt for and emphasize projects that can be implemented quickly with a relatively fast turn-around. Again, speed of project initiation may be faster in less democratic countries where rule of law and accountability is weaker. Chinese FDI projects related to BRI are often tied to Chinese soft loans and aid giving. The very significant sums of money invested creates ample opportunities for rent seeking and corruption – and in turn for insiders to privately benefit from these development projects (i.e. Queensway in Angola) (Burgis, 2014). Such windfall opportunities (for the executives involved) may again be more easily exploited in less accountable societies where rule of law is weaker and there is less public scrutiny (via, for example, an independent media). 7 Albeit we have little way of evaluating what kind of value for money the BRI offers DISCUSSION Executives, in other words, may be attracted to these types of environments when appraising projects. Recently the central inspection team of the Chinese Communist Party has assigned members to work with firms undertaking FDI in BRI countries, aware of the growth in large-scale corruption that foreign markets provide for Chinese 27 executives - who face much tougher scrutiny at home (Weinland, 2019). The aforementioned case of Queensway Group in Angola very much supports the argument that insiders, including senior Chinese executives and politicians embedded in SOEs (Queensway’s investments were linked to Sinopec, whose chief executive was later charged with corruption), have much to gain personally (in terms of private wealth) by working in such environments (Burgis, 2014). In short, these factors, when combined, may potentially explain why the BRI policy amplifies the impact of institutional fragility on Chinese FDI. These findings, of course, can be interpreted in both positive and negative lights. On the one hand, for example, they can be interpreted to suggest China’s BRI policy is supporting, intentionally or otherwise, unaccountable and less democratic regimes where legal systems are weak – so undermining political rights of citizens in the BRI countries. From another, more positive perspective, they can be interpreted to show that the BRI policy may help in lowering or mitigating the potential liabilities of foreignness and challenges associated with working in what may be institutionally fragile and difficult business environments, often low income less developed countries. In this sense, the BRI policy can be interpreted as having more positive impacts on the potential economic development of the BRI countries it targets.7 SEZs and the impact of a fuller package of BRI supports As noted, SEZs are also considered an integral element and lie ‘at the heart of the BRI’ (Fei, 2017: 840). BRI and SEZs are being jointly leveraged as investment platforms for CMNEs (Fei, 2017). Firstly, our findings show that SEZs are significantly associated with increased Chinese FDI. This suggests SEZs may provide islands of stability in fragile institutional environments. They may, for 28 example, potentially lower the liabilities of foreignness that CMNEs face – one of their intended purposes. We hypothesized that engagement with the SEZ policy would signal stronger commitment to the BRI policy, and in turn may foster stronger state to state relationships between China and the host country. SEZs, moreover, may also directly leverage the bargaining power of Chinese businesses vis- à-vis the host country, providing additional support to them in negotiating favorable policies – thus attracting greater investment. Within institutionally weak regimes scope for preferential policy negotiation may also be greater (i.e. normal rules may be bent) – thus further amplifying the impact of institutional fragility on FDI. In countries like Egypt, for example, which created the Suez special economic zone, highly preferential policies have been negotiated for members of the Chinese zone (approved via a presidential decree). These policies included: no tariffs or taxes of any kind or permits and other restrictions on imports of raw materials and capital equipment (allowing the Suez SEZ to act as a Chinese import/export processing trade hub); granting of its own customs and taxation administration system; prohibition of the nationalization of zone assets or asset sequestration; simplified labor and employment laws; and minimal taxes on goods which are exported (El-Gohari & Sutherland, 2010; El-Rashidy, 2016). These approved policies were ‘greatly inspired’ by regulations developed in China’s domestic Shenzhen SEZ (El-Rashidy, 2016: 85). SEZs may amplify the potential for preferential policy negotiation in institutionally weak settings. More generally, SEZs when complemented with other favorable BRI policies (particularly financial supports) may potentially lower the liabilities of foreignness and other challenges faced by CMNEs in institutionally weak foreign settings. In line with our original hypothesis, we interpret this to suggest that adoption of SEZs and BRI in tandem implies a stronger overall commitment to the BRI leading to stronger overall moderation impacts 29 One unexpected modeling result of interest was the finding that the SEZPRS interaction was negative and significant, albeit only weakly significant (at the 10% level) (Table 5). SEZs and the impact of a fuller package of BRI supports In the SEZs there are more private sector focused initiatives and Chinese FDI in these cases may be associated with better institutional quality. This also points to a possible duality underlying China’s BRI engagement which requires further investigation. Perhaps as the private sector grows in importance these impacts will intensify and the role of SEZs as a catalyst for greater private sector market oriented FDI activities will intensify, overturning the currently observed relationships. SEZs and the impact of a fuller package of BRI supports This suggests that investors in SEZs alone (i.e. not in conjunction with the BRI) behave in a more similar way to MNE investors from developed markets. Namely, they do care about institutional fragility. To further explore this interesting result, we further decomposed our sample by ownership to explore whether the role of China’s private sector may be playing a stronger role in this outcome. Recall that SEZs were originally developed in China to attract private investments and businesses more strongly driven by market forces. This is also the case for overseas SEZs: while some zones were developed with state-owned businesses in mind (the Chambishi zone in Zambia, for example), even these zones were designed to include strong private sector participation. In contrast to infrastructure development, therefore, SEZs have a stronger focus on private sector investments. The private sector is arguably more affected by institutional fragility and the economic and political risks this engenders (because it does not have the immediate and direct backing of government). In China, private entrepreneurs can be disposed of when they no longer serve a political purpose or serve the “wrong” purpose (Zhang et al., 2014). Thus, private entrepreneurs are arguably not as “protected” as state enterprises and are may be more exposed to adverse political and social actions in the host market. Decomposing our sample by state and private ownership in additional supplementary analysis (see appendices), our results did suggest that it was privately owned firms driving this observed, albeit weak, negatively significant interaction. The BRI appears to be exporting components of the Chinese development model – in line with the general ideas associated with the widely debated Beijing consensus. While some aspects of this model may be of concern when looked at from a Western, democratic liberal free-market perspectives (i.e. such as the Washington Consensus), not all aspects of the development model 30 associated with overseas SEZs are necessarily antithetical to them. In the SEZs there are more private sector focused initiatives and Chinese FDI in these cases may be associated with better institutional quality. This also points to a possible duality underlying China’s BRI engagement which requires further investigation. Perhaps as the private sector grows in importance these impacts will intensify and the role of SEZs as a catalyst for greater private sector market oriented FDI activities will intensify, overturning the currently observed relationships. associated with overseas SEZs are necessarily antithetical to them. CONCLUSION Using the BRI and associated policies China is arguably diffusing aspects of its own development model and experience to other countries, particularly developing economies. The Chinese approach to development, however, may favor certain institutional configurations, including weak legal institutions and limited accountability. Whether by design or otherwise (i.e. simply by the play of market forces), our study shows the influence of BRI appears to have amplified the impacts of institutional fragility on promoting Chinese inward FDI to host countries. Looked at from a different angle, the BRI policy appears to be supporting the growth, and possible subsequent integration with, countries more similar to China itself – namely those that lack fully functioning legal systems and high levels of accountability. Viewed from the perspective of liberal, developed market democracies, this may appear as a worrisome trend. However, it is important to note that many of the countries that have received support from the BRI are exactly those that the West has often been unwilling to meaningfully engage with. Through Chinese-led policies, such as the BRI, the economic development of these very same countries may be promoted. At one level, this may not bode well for the future of a liberal democratic world order (as envisioned, for example, in the original post second world war Marshall Plan). The geopolitical vision Chinese leaders have in mind today, 31 arguably, is one in which Western legal, political and economic institutions might be supplanted by alternatives more akin to those found in China itself. Our findings show that Beijing, deliberately or otherwise, appears to be promoting less accountable and more autocratic governments not subject to inconvenient legal rulings. Policy Implications Policy Implications The Chinese development model the BRI currently promotes involves the downplaying of individual human rights and emphasizing, above all else, the paramount gains of economic development. Further, it seems the BRI has enhanced the potential for CMNEs to engage in corruption. The Chinese government will do well to extend their hard line on domestic corruption to the international arena. While there may be downsides in terms of political freedoms, there may, as mentioned, also be considerable upsides in terms of economic development, including poverty reduction. Without Chinese growth, for example, the world would never have gotten anywhere near meeting many of the United Nation’s eight Millennium Development Goals. Chinese (domestic) policies have lifted many millions of people out of poverty at home. Externally-focused policies, such as the BRI, may have the same poverty-alleviating impact abroad. Further, the BRI’s goal of enhancing the volume of Chinese OFDI is working. It is possible the innovative developmental policies China has created is relevant to other countries – both developing and emerging. Better understanding and acknowledging the impacts of China’s growing global influence, whatever one’s moral or ethical standpoint on it, is vitally important if Western policy-makers are to successfully engage in shaping the future international geopolitical landscape. International political relations between the US and China, for example, are nearing an all-time low as the US embarks upon an inward-looking phase. The European Union is struggling to successfully counter Chinese BRI 32 related influence in its own backyard. Better understanding the nature of the BRI policy, including its impacts on Chinese FDI and growing economic integration between China and other BRI member countries, may help inform high level policy making in the major developed market economies as we grow into a post-Washington consensus era of international political and economic engagement. The OECD led developed market economies, in particular, must further reflect on how best to engage with lower income emerging market economies in the face of an ever more assertive China. Looked at from a host country perspective, our findings suggest countries that do engage with Chinese BRI policy can potentially attract higher levels of FDI and that this, in turn, could benefit their economies. Special economic zones, as a Chinese development tool, may also hold out considerable longer-term potential for the attraction of a wide variety of Chinese MNEs, particularly from the private sector. Policy Implications Future research would do well to explore the impacts of these zones specifically and the BRI more generally. While we have shown they do impact on FDI, better understanding other impacts, such as on employment, exports, domestic linkages between Chinese and host market firms, as well as economic growth and poverty reduction more generally, is required. . 33 REFERENCES AIIB. (2019). Who we are. Accessed at: https://www.aiib.org/en/about-aiib/index.html Alesina, A., & Tabellini, G. (1989). External debt, capital flight and political risk. Journal of International Economics, 27(3), 199-220. Alon, I., Anderson, J., Munim, ZH. & Ho, A. (2018). A review of the internationalization of Chinese enterprises. Asia Pacific Journal of Management, 35(3), 573-605. Alon, I., & Martin, M. (1998). A normative model of macro political risk Assessment. Multinational Business Review, 6(2), 10-19. Anderson, J. & Sutherland, D. (2015). Developed economy investment promotion agencies and emerging market foreign direct investment: The case of Chinese FDI in Canada. Journal of World Business, 50(4), 815-825. Anderson, J. & Sutherland, D. (2015). Entry mode and emerging market MNEs: An analysis of Chinese greenfield and acquisition FDI in the United States. Research in International Business and Finance, 35, 88- 103. Anderson, J., Sutherland, D. & Severe, S. (2015). An event study of home and host country patent generation in Chinese MNEs undertaking strategic asset acquisitions in developed markets. International Business Review, 24(5), 758-771. Baum, C. (2006) (Eds). An Introduction to Modern Econometrics Using Stata. Stata Pre 6) (Eds). An Introduction to Modern Econometrics Using Stata. Stata Press. Blomkvist, K. & Drogendijk R. (2013). The Impact of Psychic Distance on Chinese Outward Foreign Direct Investments. Management International Review, 53, 659-686. Bräutigam, D. & Tang, X. (2012). Economic statecraft in China's new overseas special economic zones: soft power, business or resource security?. International Affairs, 88(4), 799-816. Bräutigam, D., & Tang, X. (2014). “Going Global in Groups”: Structural Transformation and China’s Special Economic Zones Overseas. World Development, 63, 78-91. Brewer, T. (1981). Political risk assessment for foreign direct investment decisions: better methods for better results. Columbia Journal of World Business, 16(1), 5-12. Buckley, P., Clegg, L., Cross, A., Liu, X., Voss, H., & Zheng, P. (2018). A retrospective and agenda for future research on Chinese outward foreign direct investment. Journal of International Business Studies, 49(1), 4-23. Buckley, P., Clegg, L., Cross, A., Liu, X., Voss, H., & Zheng, P. (2007). The determinants of Chinese outward foreign direct investment. Journal of International Business Studies, 38(4), 499-518. Burgis, T. (2014). China in Africa: how Sam Pa became the middleman. Financial Times, 8 August 2014. Burgis, T. (2014). China in Africa: how Sam Pa became the middleman. Financial Time CDB (China Development Bank). (2016). Plans for Supporting Overseas Trade and Economic Zones. Retrieved June 2, 2016, from http://www.cdb.com.cn/cpfw/gjyw/zxfw_440/201602/ t20160222_2678.html Clarke, M. (2018). Clarke, M. (2018). The Belt and Road Initiative: Exploring Beijing’s Motivations and Challenges for its New Silk Road. Strategic Analysis, 42(2), 84-102. REFERENCES The Belt and Road Initiative: Exploring Beijing’s Motivations and Challenges for its New Silk Road. Strategic Analysis, 42(2), 84-102. 34 Clegg, L. J., Voss, H., & Tardios, J. A. (2018). The autocratic advantage: Internationalization of state-owned multinationals. Journal of World Business, 53(5), 668-681. Crabtree, J. (2019) China needs to make BRI more transparent and predictable. Global infrastructure project must move beyond mish-mash of opaque bilateral deals. 25 April 2019. Du, J. & Zhang, Y. (2018). Does One Belt One Road initiative promote Chinese overseas direct investment? China Economic Review, 47, 189-205. Duan, F., Ji, Q., Liu, B. & Fan, Y. (2018). Energy investment risk assessment for nations along China’s Belt & Road Initiative. Journal of Cleaner Production, 170(1), 535-547. Duanmu, J. L. (2014). State-owned MNCs and host country expropriation risk: The role of home state soft power and economic gunboat diplomacy. Journal of International Business Studies, 45(8), 1044-1060. El-Gohari, A., & Sutherland, D. (2010). China's Special Economic Zones in Africa: The Egyptian Case. In 21st CEA Annual Conference, University of Oxford, UK, July (pp. 12-13). El-Rashidy, S. (2016). The Role of China’s Overseas Special Economic Zones in Economic Development. The American University in Cairo School of Humanities and Social Sciences. A Thesis Submitted to Department of Political Science by Salah El-Rashidy under the supervision of Dr. Javed Mswood. August 2016. Fei, D. (2017). Worlding developmentalism: China's economic zones within and beyond its border. Journal of International Development, 29(6), 825-850. Fukuyama, F. 2014. Political order and political decay: From the industrial revolution to the globalization of democracy. New York: Farrar, Straus and Giroux. Gartzke, E. 1998. Kant we all just get along? Opportunity, willingness, and the origins of the democratic peace. American Journal of Political Science, 42(1): 1–27. Hillman, J. (2018). “China’s Belt and Road Initiative: Five Years Later”. Statement Before the U.S.-China Economic and Security Review Commission, 25 January 2018. He, W. (2018). BRI injects new energy into China-Angola cooperation. CGTN, 9 October 2 Herrero, A. G., & Xu, J. (2016). China’s belt and road initiative: Can Europe expect trade gains? Bruegel working paper series. Issue 5. Hlovor, I & Shaibu. Y. (2019). Searching for a development paradigm in Ghana: Can the ‘Beijing Consensus’ provide and Anchor? Journal of Economics and Political Economy, 6(1), 39-57. Howell, L. & Chaddick, B. (1994). Models of political risk for foreign investment and trade: an assessment of three approaches. REFERENCES The Columbia Journal of World Business, 29(3), 70-91. Huang, Y. (2011). Rethinking the Beijing consensus. Asia Policy, (11), 1-26. Huang, Y. (2016). Understanding China’s Belt & Road Initiative: Motivation, framework, and assessment. China Economic Review, 40, 314-321. Huang, Y. (2017). Debating China’s Economic Growth: The Beijing Consensus or the Washington Consensus. Academy of Management Perspectives, 24(2), 31-47. Indeo, F. (2018). The impact of the Belt and Road Initiative on Central Asia: Building new relations in a reshaped geopolitical scenario. In Zhang, W., Alon, I., & Lattemann, C. (Eds.). (2018). China's Belt and Road Initiative: Changing the Rules of Globalization. Springer. 35 Kang, Y. & Jiang, F. (2012). FDI location choice of Chinese multinationals in East and Southeast Asia: Traditional economic factors and institutional perspective. Journal of World Business, 47(1), 45-53. Kobrin, S. (1979). Political risk: A review and reconsideration. Journal of International Business studies, 10(1), 67-80. Kolstad, I. & Wiig, A. (2012). What determines Chinese outward FDI? Journal of World Business, 47(1), 26-34. Lairson, T. (2018). The global strategic environment of the BRI: Deep interdependence and structural power. In Zhang, W., Alon, I., & Lattemann, C. (Eds.). (2018). China's Belt and Road Initiative: Changing the Rules of Globalization. Springer. Li, S., & Alon, I. (2019). China’s intellectual property rights provocation: A political economy view. Journal of International Business Policy, 1-13. Li, J., Meyer, K. E., Zhang, H., & Ding, Y. (2018). Diplomatic and corporate networks: Bridges to foreign locations. Journal of International Business Studies, 49(6), 659-683. Lin, J. (2012). Youth Bulge: A Demographic Dividend or a Demographic Bomb in Developing Countries? World Bank Blogs, January 5, 2012. Accessed at: https://blogs.worldbank.org/developmenttalk/youth-bulge- a-demographic-dividend-or-a-demographic-bomb-in-developing-countries. Maliszewska, M., Van Der Mensbrugghe, D. (2019). The Belt and Road Initiative: Economic, Poverty and Environmental Impacts; Policy Research Working Paper Series 8814, The World Bank. Milne, R. (2018). Maersk makes waves with pure focus on shipping and logistics. Financia 8). Maersk makes waves with pure focus on shipping and logistics. Financial Times, 2. MOFCOM (Ministry of Commerce). (2015). Nation-Level Economic Development Zones. Retrieved May 29, 2016, from http://english.mofcom.gov.cn/article/zt_invest/lanmub/201302/ 20130200031461.shtml. OECD. (2015). Implementing the latest international standards for compiling foreign direct investment statistics: FDI statistics by the ultimate investing country. March. Parente, R., Rong, K., Geleilate, J. M. G., & Misati, E. (2019). Adapting and sustaining operations in weak institutional environments: A business ecosystem assessment of a Chinese MNE in Central Africa. Journal of International Business Studies, 50(2), 275-291. REFERENCES Quer, D., Claver, E., & Rienda, L. (2012). Political risk, cultural distance, and outward foreign direct investment: Empirical evidence from large Chinese firms. Asia Pacific Journal of Management, 29(4), 1089- 1104. Quer, D., Claver, E., & Rienda, L. (2018). The influence of political risk, inertia and imitative behavior on the location choice of Chinese multinational enterprises: Does state ownership matter?. International Journal of Emerging Markets, 13(3), 518-535. Quer, D., Rienda, L., Andreu, R., & Miao, S. (2019). Host country experience, institutional distance and location choice of Chinese MNEs: the moderating effect of government official visits. Cross Cultural & Strategic Management, 26(1), 24-45. Quer, D., Rienda, L., & Andreu, R. (2020). FDI drivers and establishment mode choice of emerging-market MNEs: the role of state ownership. European Journal of International Management, 14(1), 144-172. Ramasamy, B., Yeung, M., & Laforet, S. (2012). China's outward foreign direct investment: Location choice and firm ownership. Journal of World Business, 47, 17-25. 36 Rolland, N. (2017). China’s “Belt and Road Initiative”: Underwhelming or Game-Changer? The Washington Quarterly, 40(1), 127-142. Shi, W. S., Sun, S. L., Yan, D., & Zhu, Z. (2017). Institutional fragility and outward foreign direct investment from China. Journal of International Business Studies, 48(4), 452-476. Strezhnev, A., & Voeten, E. (2013). United Nations general assembly voting data. http://thedata.Harvard.edu/dvn/dv/Voeten/faces/study/StudyPage.Xhtml?studyId=38311&studyListingInd ex=0_dee53f12c760141b21c251525332. Accessed January 8, 2015. Sutherland, D. (2009). Do China's “national team” business groups undertake strategic‐asset‐seeking OFDI? Chinese Management Studies 3(1): 11-24. Sutherland, D. & Anderson, J. (2015). The pitfalls of using foreign direct investment data to measure Chinese multinational enterprise activity. The China Quarterly, 221, 21-48. Sutherland, D., Anderson, J. & Hertenstein, P. (2018). Is the strategic asset seeking investment proclivity of Chinese MNEs different to that of developed market MNEs? A comparative analysis of location choice and orientation. Management International Review, 58(6), 911-933. Sutherland, D., Anderson, J. & Hu, Z. (2020). A comparative analysis of location and non-location-bounded strategic asset seeking in emerging and developed market MNEs: An application of new internalization theory. International Business Review, 29(2), article 101635. Sutherland, D., Hennart, J.F. & Anderson, J. (2019). How does the routing of FDI to and via tax havens confound our understanding of Chinese MNE activity? A critical review of the empirical literature on Chinese MNEs. Asian Business and Management, 18(5), 337-359. Swaine, M. (2015). Chinese views and commentary on the ‘One Belt, One Road’ initiative. China Leadership Monitor, 47, 1-24. Tao, J. & Zhong, M. REFERENCES (2018). The changing rules of international dispute resolution in China’s Belt and Road Initiative. In Zhang, W., Alon, I., & Lattemann, C. (Eds.). (2018). China's Belt and Road Initiative: Changing the Rules of Globalization. Springer. Vangeli, A. (2017). China's engagement with the sixteen countries of central, east and southeast Europe under the belt and road initiative. China & World Economy, 25(5), 101-124. Villafuerte, J., Corong, E., & Zhuang, J. (2016). The one belt, one road initiative: Impact on trade and growth. Paper presented at the 19th annual conference on global economic analysis. Voeten, E. (2000). Clashes in the assembly. International Organization, 54(2): 185–215. Weinland. D. (2019). China to tackle corruption in Belt and Road projects: Beijing plans to embed anti-graft officers in BRI ventures abroad. Financial Times, 18 July 2019. Williamson, J. (1993). Democracy and the “Washington consensus”. World Development, 21(8), 1329-1336. Williamson J (2012) Is the “Beijing Consensus” now dominant? Asia Policy (13) 1 16 Williamson, J. (1993). Democracy and the “Washington consensus”. World Development, 21(8), 1329-1336. Williamson, J. (2012). Is the “Beijing Consensus” now dominant?. Asia Policy, (13), 1-16. Wilson, T. (2019). Kenyan court blocks China-backed power plant on environment grounds. Blow for Belt and Road initiative as judges revoke licence for project near Unesco site. 27 June 2019. World Bank. (1997). World Development Report. Available at: http://documents.worldbank.org/curated/ en/518341468315316376/World-development-report-1997-the-state-in-a-changing-world 37 World Bank. (1993). World Development Report. Available at: http://documents.worldbank.org/curated/en/468831468340807129/World-development-report-1993- investing-in-health Yao, Y (2015). Beijing Consensus. In The Wiley Blackwell Encyclopedia of Race, Ethnicity and Nationalism. Wiley. Yiu, D. (2011). Multinational Advantages of Chinese Business Groups: A Theoretical Exploration. Management and Organization Review, 7(2), 249-277. Yagci, M. (2016). A Beijing Consensus in the Making: The Rise of Chinese Initiatives in the International Political Economy and Implications for Developing Countries. Perceptions: Journal of International Affairs, 21(2), 29-56. Yu, H. (2017). Motivation behind China’s ‘One Belt, One Road’ initiatives and establishment of the Asian infrastructure investment bank. Journal of Contemporary China, 26(105), 353-368. Zhai, F. (2018). China’s belt and road initiative: A preliminary quantitative assessment. Journal of Asian Economics, 55, 84-92. Zhang, J., Jiang, J. & Zhou, C. (2014). Diplomacy and investment – the case of China. International Journal of Emerging Markets, 9(2), 216-235. 38 Table 1: Variables, descriptions, and data sources. Table 1: Variables, descriptions, and data sources. shows significance at the 0.01 level REFERENCES Variable Proxy Data source Chinese outward FDI Value of Chinese FDI projects in host country Financial Times fDi Markets; Thomson ONE Country risk premium Mature equity market premium plus additional country risk premium Moody’s; Bloomberg Cultural proximity Dummy variable where percentage of host country population is Chinese is 1% or greater = 1; zero otherwise Ohio University Geographic distance Geographic distance from Beijing to the capital of the host country CEPII/World Bank GDP Host country gross domestic product World Bank GDP growth Gross domestic product growth World Bank Natural resource exports Fuels, ores, and metals exports as a share of GDP World Bank Exchange rate Host country annual average exchange rate against RMB (fixed to dollar) World Bank Inflation Inflation rate World Bank Open to FDI Inward FDI stock as a share of GDP World Bank Institutional fragility Index based around the average of five institutional fragility factors: control of corruption; political stability; rule of law; regulatory quality; and government accountability Political Risk Services Group SEZ Dummy variable where the host country has at least one nationally approved Chinese overseas special economic zone = 1; zero otherwise China Ministry of Commerce BRI Dummy variable where host country has committed to at least one BRI project = 1; zero otherwise China Ministry of Commerce AIIB member Dummy variable where AIIB member country = 1; zero otherwise Asian Infrastructure Investment Bank Bilateral trade agreement Dummy variable where having a bilateral trade agreement between the host country and China = 1; zero otherwise China Ministry of Commerce Pct agree UN vote Percentage of United Nations votes the same for the host country and China United Nations , p , Variable Proxy Data source Chinese outward FDI Value of Chinese FDI projects in host country Financial Times fDi Markets; Thomson ONE Country risk premium Mature equity market premium plus additional country risk premium Moody’s; Bloomberg Cultural proximity Dummy variable where percentage of host country population is Chinese is 1% or greater = 1; zero otherwise Ohio University Geographic distance Geographic distance from Beijing to the capital of the host country CEPII/World Bank GDP Host country gross domestic product World Bank GDP growth Gross domestic product growth World Bank Natural resource exports Fuels, ores, and metals exports as a share of GDP World Bank Exchange rate Host country annual average exchange rate against RMB (fixed to dollar) World Bank Inflation Inflation rate World Bank Open to FDI Inward FDI stock as a share of GDP World Bank Institutional fragility Index based around the average of five institutional fragility factors: control of corruption; political stability; rule of law; regulatory quality; and government accountability Political Risk Services Group SEZ Dummy variable where the host country has at least one nationally approved Chinese overseas special economic zone = 1; zero otherwise China Ministry of Commerce BRI Dummy variable where host country has committed to at least one BRI project = 1; zero otherwise China Ministry of Commerce AIIB member Dummy variable where AIIB member country = 1; zero otherwise Asian Infrastructure Investment Bank Bilateral trade agreement Dummy variable where having a bilateral trade agreement between the host country and China = 1; zero otherwise China Ministry of Commerce Pct agree UN vote Percentage of United Nations votes the same for the host country and China United Nations 39 Policy, Institutional fragility and Chinese FDI Table 2: Pairwise correlations Standard errors are in parenthesis (* p<0.01, p<0.05, * p<0.1) Table 2: Pairwise correlations es (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) DI 1.00 orruption -0.12* 1.00 olitical instability 0.04 0.02 1.00 gal fragility -0.06 0.70* 0.11* 1.00 eg. fragility -0.13* 0.79* -0.08* 0.64* 1.00 naccountability -0.04 0.53* -0.30* 0.29* 0.57* 1.00 sk premium -0.12* 0.65* 0.11* 0.63* 0.61* 0.38* 1.00 ult. proximity 0.15* -0.05 -0.04 0.01 -0.15* -0.00 -0.08* 1.00 eo. distance -0.06* 0.11* 0.07* 0.40* 0.17* -0.10* 0.21* -0.11* 1.00 GDP 0.44* -0.25* 0.04 -0.19* -0.31* -0.22* -0.25* 0.17* -0.03 1.00 GDP growth -0.02 0.16* -0.18* 0.10* 0.19* 0.15* 0.13* 0.04 -0.07* -0.07* 1.00 Natural resources 0.00 0.04 0.09* 0.07 0.13* -0.02 0.07* 0.00 0.16* -0.07* 0.07* 1.00 Exchng. rate -0.00 0.11* 0.04 0.01 0.02 0.04 0.15* -0.01 0.01 -0.01 -0.08* 0.02 1.00 nflation -0.01 0.13* 0.05 0.14* 0.13* 0.09* 0.17* 0.05 0.05 -0.06* -0.05 0.05 0.12* 1.00 Open to FDI -0.01 -0.19* 0.01 -0.16* -0.21* -0.14* -0.09* -0.03 0.09* -0.04 -0.03 -0.02 -0.00 -0.04 1.00 AIIB member 0.19* -0.01 0.01 -0.02 -0.01 0.12* -0.01 0.13* -0.20* 0.01 -0.01 -0.00 -0.00 -0.02 -0.01 1.00 Bilat. trade_agree 0.12* -0.15* 0.08* -0.08* -0.16* 0.04 -0.14* 0.31* -0.21* -0.02 0.04 0.09* -0.01 -0.03 -0.02 0.25* 1.00 CT UN_vote -0.15* 0.58* -0.27* 0.49* 0.60* 0.61* 0.43* -0.00 0.16* -0.37* 0.20* 0.11* 0.02 0.05 -0.13* -0.06* 0.04 1.00 BRI 0.11* 0.07* 0.08* 0.02 0.08* 0.11* 0.05 0.03 -0.24* -0.02 -0.04 -0.04 -0.01 -0.03 -0.02 0.56* 0.13* -0.13* 1.00 EZ 0.13* 0.12* 0.15* 0.17* 0.14* 0.10* 0.07* 0.13* -0.09* 0.03 -0.01 -0.04 -0.00 0.13* -0.02 0.16* 0.18* 0.04 0.12* 1.00 nstitutional frag. -0.10* 0.89* 0.06 0.79* 0.89* 0.70* 0.71* -0.06 0.20* -0.29* 0.15* 0.08* 0.04 0.16* -0.19* 0.02 -0.09* 0.65* 0.09* 0.17* 1.00 315.76 0.45 0.66 0.63 0.59 0.7 0.03 0.16 8931.9 4 33168 6.9 0.04 0.09 28400 00 0.06 3.04 0.03 0.07 0.73 0.07 0.03 0.61 1362.7 6 0.2 0.13 0.21 0.24 0.26 0.03 0.37 3851.5 2 13300 00 0.06 0.15 1.38E +08 0.26 32.72 0.17 0.25 0.16 0.25 0.18 0.16 s significance at the 0.01 level 40 Policy, Institutional fragility and Chinese FDI Table 3: Aggregated institutional fragility interacted with BRI and other policy measures. (1) (2) (3) (4) (5) (6) (7) (8) (9) FDI value FDI value FDI value FDI value FDI value FDI value FDI value FDI value FDI value Country risk premium -2705.26 -2773.37 -2108.73 -2124.86 -1615.76 -1625.48 -1349.12 -1082.94 -1525.86 (3420.42) (3414.01) (3384.93) (3385.09) (3389.01) (3397.21) (3237.80) (3241.31) (3063.11) Cultural proximity 253.89 259.72 216.11 213.06 207.00 204.85 360.69 380.33 226.65 (189.68) (189.20) (186.24) (186.76) (186.56) (187.38) (168.72) (168.93) (158.77) Geographic distance -0.01 -0.00 -0.01 -0.01 -0.02 -0.02 -0.01 -0.01 0.01 (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) GDP 0.00* 0.00* 0.00* 0.00* 0.00* 0.00* 0.00* 0.00* 0.00*** (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) GDP growth -116.15 165.93 -267.45 -284.93 -887.50 -898.90 -465.43 -445.42 475.74 (1285.18) (1290.55) (1277.34) (1280.13) (1280.59) (1281.58) (1281.92) (1281.57) (1263.09) Natural resource exports 914.74* 888.71* 804.62* 803.97* 756.10 748.83 791.09* 743.72* 542.43 (485.41) (484.37) (477.30) (477.07) (474.65) (476.84) (439.28) (439.85) (406.32) Exchange rate 0.05 0.05 0.04 0.04 0.03 0.04 0.05* 0.05* 0.04 (0.03) (0.03) (0.03) (0.03) (0.03) (0.03) (0.03) (0.03) (0.03) Inflation 1315.40 1180.54 1232.83 1238.73 1091.77 1082.91 844.63 926.37 1294.95 (985.22) (985.83) (978.96) (979.53) (985.09) (986.18) (956.99) (958.69) (934.24) Open to FDI 282.97* 287.46* 276.55* 276.20* 278.72* 277.59* 121.23 114.83 69.55 (75.63) (75.50) (74.77) (74.78) (74.71) (74.97) (78.53) (78.57) (74.52) Institutional fragility 1123.45* 1019.65* 1137.39* 1148.49* 1329.73 1382.90 985.03 3250.34 469.03 (610.19) (610.84) (600.32) (602.45) (593.08) (604.57) (618.53) (2009.48) (1922.76) BRI 952.34* -922.46 -1591.82 (195.84) (925.44) (985.42) BRI#Ins.fragility 4297.36 5482.65 (2073.34) (2311.50) AIIB member 1441.59* 1619.67* 2468.74** (275.84) (862.04) (967.75) AIIB member#Ins.frag -428.33 -3970.06* (1966.20) (2320.31) Bilateral trade agreement 483.27 701.06 149.63 (202.37) (500.83) (497.97) Bilat.trade agree#Ins.frag -645.70 358.44 (1351.06) (1293.25) Pct agree UN vote -54.77 1264.01 792.10 (492.86) (1215.82) (1141.76) Pct agree UN vote#Ins.fr. -3528.23 -584.46 (2975.71) (2820.98) cons -529.88 -508.88* -469.46* -470.25* -440.32* -450.67* -277.08 -1084.69 -777.73 (263.07) (262.58) (257.44) (257.36) (253.71) (255.37) (336.43) (760.39) (709.60) Obs. 1204 1204 1204 1204 1204 1204 1191 1191 1191 Pseudo R2 .235 .238 .239 .244 .227 .227 .222 .2214 .z Standard errors are in parenthesis (* p<0.01, p<0.05, * p<0.1) 41 Policy, Institutional fragility and Chinese FDI Table 4: Institutional fragility decomposed and individually interacted with BRI. p * p<0.01, p<0.05, * p<0.1 Standard errors are in parenthesis Policy, Institutional fragility and Chinese FDI (1) (2) (3) (4) (5) (6) (7) fdi_all_val fdi_all_val fdi_all_val fdi_all_val fdi_all_val fdi_all_val fdi_all_val Country risk premium -2093.17 -2491.95 -2039.45 -1536.23 -2034.89 -2029.16 -795.70 (3465.87) (3463.23) (3476.30) (3293.07) (3424.32) (3473.58) (3562.75) Cultural proximity 229.46 235.83 232.23 235.44 226.62 223.31 189.35 (245.89) (243.30) (247.65) (243.83) (243.61) (248.18) (197.59) Geographic distance -0.02 -0.02 -0.02 -0.01 -0.02 -0.02 -0.01 (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) (0.02) GDP 0.00* 0.00* 0.00* 0.00* 0.00* 0.00* 0.00*** (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) GDP growth 333.45 509.06 243.94 184.16 289.28 494.27 221.25 (961.45) (968.97) (967.75) (930.20) (962.33) (1039.99) (1351.95) Natural resource exports 957.31* 915.63* 964.47* 943.47* 957.65* 946.21* 890.90* (515.84) (515.38) (517.59) (513.94) (517.18) (516.78) (501.46) Exchange rate 0.04 0.04 0.04 0.03 0.04 0.04 0.03 (0.04) (0.04) (0.04) (0.03) (0.04) (0.04) (0.03) Inflation 1566.61 1382.61 1562.38 1512.55 1588.98 1541.84 1520.44 (1534.07) (1438.88) (1550.18) (1466.44) (1524.45) (1486.60) (1052.84) Open to FDI 261.94 268.52 266.07 272.70 260.64 259.52 262.21* (182.96) (184.91) (183.50) (185.70) (183.43) (184.05) (77.07) BRI 868.64 1691.65 2180.07 3129.52 769.57 1191.82 4917.92*** (419.71) (849.68) (2081.70) (1481.32) (560.43) (785.10) (1901.72) Corruption -170.21* -184.76** -174.28* -171.97* -170.41* -172.52* -199.01** (91.47) (91.31) (90.79) (91.03) (90.98) (92.49) (98.60) Political instability 58.05* 57.88* 55.01* 59.26* 57.91* 56.71* 50.89 (32.76) (32.81) (31.74) (32.51) (32.53) (33.13) (38.75) Legal fragility 181.16 185.75 256.45 146.13* 179.82 184.20 140.20 (85.29) (85.80) (85.51) (81.06) (85.88) (85.54) (90.27) Regulatory fragility 37.89 41.35 38.98 44.81 40.96 44.01 105.43 (137.15) (135.32) (137.48) (134.18) (134.79) (135.48) (141.94) Unaccountability 112.81 115.90 119.90 123.84 112.54** 101.11* 95.88 (52.05) (51.12) (52.00) (49.83) (51.87) (55.53) (62.64) BRI#corruption 341.02 438.63 (369.21) (458.76) BRI#political Instability 256.45 155.27 (308.36) (261.17) BRI#legal fragility 990.42 938.64* (467.74) (307.12) BRI#regulatory fragil. -45.79 -946.24 (459.25) (392.71) BRI#unaccountability 221.19 341.99 (185.73) (167.30) cons 1023.75* 1021.38* 1009.18* 933.44* 1023.37* 989.05* 799.28 (557.50) (559.76) (554.60) (559.23) (556.49) (577.05) (504.11) Obs. 1181 1181 1181 1181 1181 1181 1181 R2 .241 .244 .242 .249 .241 .242 .254 Standard errors are in parenthesis * p<0.01, p<0.05, * p<0.1 utional fragility decomposed and individually interacted with BRI. Table 4: Institutional fragility decomposed and individually interacted with BRI 42 Policy, Institutional fragility and Chinese FDI p * p<0.01, p<0.05, * p<0.1 Standard errors are in parenthesis Policy, Institutional fragility and Chinese FDI Table 5: Total FDI regressed on SEZs and BRI combined, including moderation. (1) (2) (3) (4) FDI value FDI value FDI value FDI value Country risk premium -2950.22 -2969.02 -2912.78 -2507.80 (3382.22) (3384.62) (3391.18) (3305.28) Cultural proximity 240.00 245.10 244.97 262.17 (185.65) (186.15) (187.18) (180.36) Geographic distance -0.00 -0.00 -0.00 -0.01 (0.02) (0.02) (0.02) (0.02) GDP 0.00* 0.00* 0.00* 0.00* (0.00) (0.00) (0.00) (0.00) GDP growth 112.48 313.73 194.04 -572.14 (1281.93) (1287.37) (1288.78) (1270.66) Natural resource exports 862.36* 847.69* 821.15* 900.00* (476.41) (477.46) (479.94) (464.04) Exchange rate 0.03 0.03 0.02 0.01 (0.03) (0.03) (0.03) (0.03) Inflation 1332.25 1224.33 1369.37 1499.19 (979.00) (981.17) (985.04) (965.44) Open to FDI 283.00* 286.56* 288.46* 289.91* (74.60) (74.71) (74.92) (72.84) SEZ 829.21* 754.58* 3876.72** 3251.08* (273.05) (276.79) (1865.32) (1832.60) BRI 862.55*** -597.41 -659.41 1345.01 (197.52) (930.86) (930.93) (964.93) Institutional fragility 913.35 852.51 933.94 1135.06* (603.76) (605.95) (609.84) (592.05) BRI#Institutional fragility 3365.16 (2096.67) 3501.24* (2096.70) -2452.27 (2252.83) SEZ# Institutional fragility -5998.08* (3541.70) -6343.53* (3475.59) BRI#SEZ#Institutional fragility 7353.63*** (1123.04) cons -487.11* -475.80* -505.40* -487.21* (257.87) (258.58) (260.37) (251.21) Obs. 1204 1204 1204 1204 Bet/within R2 .241 .245 .248 .275 Standard errors are in parenthesis Table 5: Total FDI regressed on SEZs and BRI combined, including moderation. 43 Policy, Institutional fragility and Chinese FDI Appendix Appendix A: SOE FDI, impacts of SEZs and BRI combined, including moderation. (1) (2) (3) (4) fdi_soe_val fdi_soe_val fdi_soe_val fdi_soe_val Country risk premium -2172.60 -2173.47 -2173.40 -2106.84 (908.95) (910.79) (913.07) (905.15) Cultural proximity 141.92* 142.97* 142.92* 144.63* (46.72) (46.92) (47.14) (46.60) Geographic distance 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0.00) GDP 0.00 0.00 0.00 0.00 (0.00) (0.00) (0.00) (0.00) GDP growth -598.40* -562.65 -568.85 -697.43* (361.92) (363.76) (364.60) (363.57) Natural resource exports 158.33 156.15 155.28 171.04 (122.05) (122.51) (123.11) (121.86) Exchange rate 0.02 0.02 0.02 0.02 (0.01) (0.01) (0.01) (0.01) Inflation 129.76 108.54 116.11 145.18 (272.50) (273.50) (275.14) (273.23) Open to FDI 15.92 16.53 16.62 16.80 (19.82) (19.87) (19.93) (19.75) SEZ 75.25 62.03 212.23 105.61 (76.43) (77.58) (525.56) (522.62) BRI 220.27*** -42.51 -45.48 307.90 (56.40) (264.64) (264.99) (277.60) Institutional fragility 271.43* 260.35 263.95* 301.57* (157.48) (158.33) (159.31) (158.01) BRI# Ins.fragility 606.00 612.49 -437.24 (596.25) (597.01) (648.20) SEZ#Ins.fragility -288.27 -363.78 (997.83) (991.12) BRI#SEZ#Ins fragility 1305.32* (324.88) cons -37.74 -35.55 -36.82 -35.48 (65.41) (65.70) (66.11) (65.38) Obs. 1204 1204 1204 1204 Pseudo R2 .063 .0639 .064 .077 Standard errors are in parenthesis * p<0 01 ** p<0 05 * p<0 1 Appendix A: SOE FDI, impacts of SEZs and BRI combined, inclu pendix A: SOE FDI, impacts of SEZs and BRI combined, including moderation. 44 Policy, Institutional fragility and Chinese FDI Appendix B: Private FDI value, impacts of SEZs and BRI combined, including moderation. (1) (2) (3) (4) fdi_private_val fdi_private_val fdi_private_val fdi_private_val Country risk premium 85.58 116.31 37.10 125.29 (1271.06) (1267.70) (1266.10) (1222.21) Cultural proximity 27.92 33.12 31.97 35.26 (58.23) (58.11) (58.01) (56.00) Geographic distance 0.00 0.00 0.00 -0.00 (0.01) (0.01) (0.01) (0.01) GDP 0.00* 0.00* 0.00* 0.00* (0.00) (0.00) (0.00) (0.00) GDP growth 468.95 622.48 547.36 146.62 (571.41) (572.66) (572.70) (554.49) Natural resource exports 24.11 12.84 -6.42 61.21 (157.75) (157.38) (157.36) (152.07) Exchange rate 0.01 0.01 0.00 -0.00 (0.01) (0.01) (0.01) (0.01) Inflation 1014.55 904.64 1020.24 1182.88* (415.37) (416.22) (418.75) (404.60) Open to FDI 17.87 20.76 22.26 21.39 (27.54) (27.48) (27.45) (26.49) SEZ 741.92* 685.35* 2486.33* 2112.39* (117.71) (119.23) (816.76) (789.44) BRI 471.80* -650.58 -690.47 571.84 (92.01) (422.59) (422.27) (429.26) Institutional fragility -200.79 -249.66 -213.05 -76.55 (210.42) (210.62) (210.91) (204.12) BRI#Institutional fragility 2593.66* 2679.91* -1082.59 (953.23) (952.43) (1003.09) SEZ#Institutional fragility -3456.21 -3821.02 (1550.70) (1497.41) BRI#SEZ#Institutional fragility 4815.40* (513.36) cons -24.91 -15.28 -27.99 -24.74 (82.88) (82.73) (82.79) (79.92) Obs. Appendix 1204 1204 1204 1204 Pseudo R2 .118 .0123 .1267 .187 Standard errors are in parenthesis 0 01 0 0 0 1 te FDI value, impacts of SEZs and BRI combined, including moderation. (1) (2) (3) (4) Private FDI value, impacts of SEZs and BRI combined, including moderation. 45
https://openalex.org/W4281781787	https://zenodo.org/records/6620381/files/Does%20gender%20have%20an%20impact%20on%20developing%20entrepreneurial%20skills.pdf	English	null	Does gender have an impact on developing entrepreneurial skills?	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	3,400	JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION │ │ │ Volume 1 Issue Number 7 February – May 2022 February – May 2022 Volume 1 Issue Number 7 JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 Volume 1 Issue Number 7 February – May 2022 February – May 2022 Abstract This study focused on determining the relation between gender and entrepreneurship. Hence, the researchers conducted an interview via virtual focus group discussion on female and male participants who were classified with the criteria of: participants who graduated or currently taking any business-related courses, and those who have an existing business in the National Capital Region of the Philippines. Thus, the researchers found out that gender does not have an impact on one's development of entrepreneurial skills and in handling a business. Secondly, the study was able to determine those skills that are perceived as entrepreneurial skills. And lastly, the study discovered the factors that influence the development of entrepreneurial skills. Keywords: entrepreneurship; business skills; innovation. Keywords: entrepreneurship; business skills; innovation. 1. Introduction Entrepreneurship is the process of creating a new enterprise and bringing new value to the market. It is one of the most critical aspects of economic development and social growth as it creates jobs, opportunities, and innovation. Hence, entrepreneurship is a significant career choice that teaches individual life skills, creativity, problem-solving, adversity, and critical thinking. However, in the discussion of Ward et al., (2019), regarding gender in developing entrepreneurial potential and intention, it has been argued that currently, the majority of the population who pursue entrepreneurship are men, thus indicating that entrepreneurship is male-dominated field. Furthermore, in research by Marín et al. (2019), the probability of men being involved in entrepreneurship is higher than women, and this gap rises as a country's development progresses. This is because men are more interested in achieving and gaining financial gains, whereas women are more focused on obtaining and achieving social value goals. Hence, it is acclaimed that males are more likely to have a positive view of entrepreneurship than females because compared to women, men are viewed as the most successful in the field as they hold a bigger share of firms in society (European Institute for Gender Equality, 2017). Thus, males are considered the majority of business role models. However, European Institute for Gender Equality (2017) also stated that women are most likely to pursue self-employment to balance work and life. Hence, women entrepreneurs’ entrepreneurial activity is to create jobs for themselves and others as they support diversification, improve women’s equality and promote women empowerment. Therefore, women entrepreneurs’ objective is to raise the employment rate by providing practical solutions to business problems. Furthermore, according to Jaiswal (2020), when comparing the success of female and male new ventures, it is critical to know what factors influence these two groups differently. According to the human capital theory of Jaiswal (2020), increasing an employee's abilities should result in an increase in overall cognitive capacity, which in turn 2 2 BME 1102│ENM 1207│MGT 1101│MKTG 1106 JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION Volume 1 Issue Number 7 Volume 1 Issue Number 7 February – May 2022 February – May 2022 should result in an increase in production and efficiency. A greater level of human capital means that people will have an easier time finding and exploiting valuable economic opportunities. In research from Camelo-Ordaz et al. (2016), self-efficacy in entrepreneurship refers to a person's confidence in their capacity to perform or succeed in various entrepreneurial tasks and roles. It also influences one’s choices, effort, and perseverance, and it even plays a significant role in developing an individual’s entrepreneurial intentions. Therefore, gender stereotypes and preconceived roles have led to differences in how people run their businesses. Women begin considering becoming entrepreneurs for social rather than economic reasons, as it is more appropriate for their emotional goals and roles than commercial enterprises, which are the primary motives for men. Moreover, the purpose of this study is to have ample knowledge regarding the impact of gender in entrepreneurship. Therefore, the researcher will analyze the effect of gender on developing entrepreneurial skills. Thus, the study will specifically answer the particular questions: how gender and entrepreneurial skills are related; what skills are perceived as entrepreneurial skills; what are the factors that determine the development of entrepreneurial skills. 2. Research Method This case study is mainly about determining the impact and role of gender in entrepreneurship. Thus, the study will disclose the entrepreneurial skills that each gender possesses. Moreover, this case study will be qualitative research which according to McLeod (2019), is a process of acquiring, evaluating, and analyzing non-numerical data that can be utilized to discover how a person objectively sees and interprets their social structure. As a result, data will be collected via in-depth focus group discussion and analyzed using a theoretical framework or case study. 3 BME 1102│ENM 1207│MGT 1101│MKTG 1106 3 JOURNAL OF UNDERGRADUATE RESEARCH IN Volume 1 Issue Number 7 February – May 2022 February – May 2022 3. Results and Discussion Table 1. Presentation of data analysis for research question number 1 using codes and corresponding descriptive themes. Table 1. Presentation of data analysis for research question number 1 using codes and corresponding descriptive themes. Research Question 1 Codes Themes How gender and entrepreneurial skills are related? Set of Codes 1: ● Traits of both genders ● Representation of genders ● Men are for manufacturing industries ● Women are more task-oriented ● Man’s job skill ● Experience of the man ● Females are risk takers. ● Gender Skills. ● Males are more qualified. ● Gender Differentiation Theme 1: Gender Roles 5 JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 Volume 1 Issue Number 7 February – May 2022 February – May 2022 For this study, the researchers will use snowballing or word of mouth techniques to gather data from different participants. According to DeCarlo (n.d), the researcher uses snowball sampling to find a small number of potential participants for their study before seeking the participation of these primary participants in the search for further individuals. Thus, as the study progresses, the researcher's sample becomes bigger, like a snowball rolling through the snow. Tongco (n.d.), agreed that the inherent bias of the method contributes to the efficiency, and the method stays right even when tested against non- probability sampling. According to Vasileiou et al. (2018), the sample size in qualitative research is a crucial guideline to justify and evaluate the quality of the study - samples in research are intended to aid the ground of discussion. Thus, sample sizes are subject to demonstrating qualitative studies’ greater efficiency. Therefore, this research paper will have 16 participants: eight female and eight male business people (Ritchie et al., 2003). And these participants have to meet the criteria set by the researchers, which are: 1) the participants will have an existing business in the National Capital Region of the Philippines; 2) the participants have graduated or are currently taking up any business-related courses. In this study, it is essential to clearly define the research instrument. According to Paradis et al. (2016), a research instrument is chosen to assist the researcher in collecting data. Also, the most common qualitative research instruments used in qualitative research are observation, interview, and document analysis. Thus, the data for this study is gathered through an interview through virtual focus group discussion to collect the data by the researchers. Our interview methods are one by one through the virtual room. And after the interview, we will take a screenshot photo with the interviewer and interviewee to serve as proof. 4 4 BME 1102│ENM 1207│MGT 1101│MKTG 1106 BUSINESS ADMINISTRATION ss │Semi-annually │Course final output Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 February – May 2022 Set of Codes 2: ● Gender does not define the skills ● Everyone can be an entrepreneur ● Gender should not be an indicator for development of entrepreneurial skills ● It is based on confidence, personal interest, knowledge and self-efficacy ● Equal View ● Cultural diversity ● Gender and skills ● Successful Entrepreneur Set of Codes 3: ● Acquiring capabilities and skills ● Confidence ● Marketing Capability ● Skills needed to entrepreneurs work ● Job related skills Theme 2: Gender is not just the one influencing one’s entrepreneurial skills Theme 3: Entrepreneurial traits that must be acquired 6 │ │ │ Set of Codes 2: ● Gender does not define the skills ● Everyone can be an entrepreneur ● Gender should not be an indicator for development of entrepreneurial skills ● It is based on confidence, personal interest, knowledge and self-efficacy ● Equal View ● Cultural diversity ● Gender and skills ● Successful Entrepreneur Set of Codes 3: ● Acquiring capabilities and skills ● Confidence ● Marketing Capability ● Skills needed to entrepreneurs work ● Job related skills Theme 2: Gender is not just the one influencing one’s entrepreneurial skills Theme 3: Entrepreneurial traits that must be acquired Theme 2: Gender is not just the one influencing one’s entrepreneurial skills ● Gender does not define the skills ● Everyone can be an entrepreneur ● Gender should not be an indicator for development of entrepreneurial skills ● It is based on confidence, personal interest, knowledge and self-efficacy 6 BME 1102│ENM 1207│MGT 1101│MKTG 1106 JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 ● Learning through skills. JOURNAL OF UNDERGRADUATE RESEARCH IN Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 As for the result of the virtual group discussion, participants categorized how gender and entrepreneurial skill are related into three themes: gender roles, gender is not just the one influencing one’s entrepreneurial skills and entrepreneurial traits. Gender roles, it is consist of people’s beliefs and expectations about how gender should play a role in the entrepreneurial field (Li et al., 2020). Meanwhile, gender is not just the one influencing entrepreneurial skills, it is defined that self-efficacy is generally acknowledged to develop one’s entrepreneurial skills, which self-efficacy refers to the capability of gender to perform their roles in the entrepreneurial field (Newman et al., 2019, p. 404). And lastly, entrepreneurial traits, it is referred to as the instinctive characteristic of an entrepreneur that defines to become one (Indeed Editorial Team, 2021). Table 2. Presentation of data analysis for research question number 2 using codes and corresponding descriptive themes. Research Question 2 Codes Themes What skills are perceived as entrepreneurial skills? Set of Codes 1: ● Marketing ● Organization ● Management ● Leadership ● Communication Skills ● Technical Abilities Theme 1: Soft Skills 7 JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION Volume 1 Issue Number 7 February – May 2022 ● Problem-Solving Skills ● Listening Skills Set of Codes 2: ● Job Related Skills ● Appreciation & recognition of skills ● Resiliency ● Risk taking Set of Codes 3: ● Patience ● Strong Mindset ● Stress Management Ability ● Accepting Criticisms ● Broad Mindset ● Decision-making ● Critical and creative thinking Theme 2: Capabilities of the Entrepreneurs that are important in the field of expertise. Theme 3: Cognitive Skills As a result of the conducted virtual group discussion, the skills perceived as entrepreneurial skills by the majority are soft skills, capabilities, and cognitive skills. Soft skills are the traits needed to shape someone’s way of working and interacting with others (Indeed Editorial Team, 2021). At the same time, capabilities are the set of skills that drive business success (Hamilton, 2021). Furthermore, lastly is cognitive skills, which are skills that utilize the brain to learn, think, pay attention, and remember (Mind Matters, n.d). Thus, 8 8 BME 1102│ENM 1207│MGT 1101│MKTG 1106 JOURNAL OF UNDERGRADUATE RESEARCH IN Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 ● Communication ● Negotiation Set of Codes 3: ● Focus ● Opportunities ● Risks ● Planning ● Good in Budgeting ● Handling Situations ● Acceptance of challenges ● Managerial Perspective ● Responsiveness ● Aspiration ● Upbringing Codes: ● Educational Background ● Joy of learning ● Learn ● Engaging ● Willingness ● Career Choice ● Own perspective Theme 3: Strategic analysis of the business surroundings. Theme 4: Surroundings that influence the development of entrepreneurial skills. ● Communication ● Negotiation Set of Codes 3: ● Focus ● Opportunities ● Risks ● Planning ● Good in Budgeting ● Handling Situations ● Acceptance of challenges ● Managerial Perspective ● Responsiveness ● Aspiration ● Upbringing Codes: ● Educational Background ● Joy of learning ● Learn ● Engaging ● Willingness ● Career Choice ● Own perspective Theme 3: Strategic analysis of the business surroundings. Theme 4: Surroundings that influence the development of entrepreneurial skills. 10 JOURNAL OF UNDERGRADUATE RESEARCH IN Volume 1 Issue Number 7 February – May 2022 February – May 2022 these are perceived as entrepreneurial skills as, according to Gibb (1993), entrepreneurial skills include the traits, can be in the form of attributes, and are business management skills. Table 3. Presentation of data analysis for research question number 3 using codes and corresponding descriptive themes. Research Question 3 Codes Themes What are the factors that determine the development of entrepreneurial skills? Set of Codes 1: ● Environmental experiences ● Acknowledgement of experiences ● Work related experience ● Environmental awareness ● Environment ● Quality of life ● Social Factor Set of Codes 2: ● Attitude ● Mindset ● Interest ● Ability ● Creativity ● Leadership Theme 1: Experiences affecting people’s way of living. Theme 2: Innate characteristics of an Entrepreneur. 9 BME 1102│ENM 1207│MGT 1101│MKTG 1106 JOURNAL OF UNDERGRADUATE RESEARCH IN Volume 1 Issue Number 7 February – May 2022 As for the result of the virtual group discussion, the participants were able to answers the factors that determine the development of entrepreneurial skills by the participants perspective experiences affecting people’s way of living, innate characteristics of an entrepreneur, strategic analysis of the business surroundings and surroundings that influence the development of entrepreneurial skills. According to Staff (2021), it's apparent that entrepreneurial growth is influenced by a wide range of elements, and it's possible that some of these factors may impact their decision to become an entrepreneur. However, it's also obvious that in the last two decades, the community, society, business, and technology have developed dramatically. Today, entrepreneurship is much easier for a wider range of people to get involved in, including those from different socioeconomic backgrounds, educational levels, and ages, from young people to older people (Factors Affecting Entrepreneurship Growth - Harappa, 2021). BME 1102│ENM 1207│MGT 1101│MKTG 1106 JOURNAL OF UNDERGRADUATE RESEARCH IN BUSINESS ADMINISTRATION Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 JOURNAL OF UNDERGRADUATE RESEARCH IN Volume 1 Issue Number 7 Volume 1 Issue Number 7 February – May 2022 February – May 2022 skills, marketing, and technical abilities. Thus, these stated skills are presumed by the participants to be learned through universities, on-hand experience, and environment. Moreover, the researcher came to the conclusion of the importance of education and upbringing to one’s development of entrepreneurial skills. And this eliminates the idea of relationship and impact of gender on the development of entrepreneurial skills. Nonetheless, the researchers recommend this research paper to future studies that tackle and address the topic of gender and entrepreneurship to have in guide doing research. skills, marketing, and technical abilities. Thus, these stated skills are presumed by the participants to be learned through universities, on-hand experience, and environment. Moreover, the researcher came to the conclusion of the importance of education and upbringing to one’s development of entrepreneurial skills. And this eliminates the idea of relationship and impact of gender on the development of entrepreneurial skills. Nonetheless, the researchers recommend this research paper to future studies that tackle and address the topic of gender and entrepreneurship to have in guide doing research. References: 1. Ward, A., Hernández-Sánchez, B. R., & Sánchez-García, J. C. (2019). Entrepreneurial potential and gender effects: The role of personality traits in University students’ entrepreneurial intentions. Frontiers in Psychology, 10. https://doi.org/10.3389/fpsyg.2019.02700 2. Marín, L., Nicolás, C., & Rubio, A. (2019). How Gender, Age and Education Influence the Entrepreneur’s Social Orientation: The Moderating Effect of Economic Development. Sustainability, 11(17), 4514. https://doi.org/10.3390/su11174514 12 4. Conclusion The main purpose of this study is to determine the impact and relationship of gender on the development of entrepreneurial skills. Hence, this study allows the researchers to give enlightenment regarding the discrimination among genders in the entrepreneurial field. Thus, the study identified how gender and entrepreneurial skills are related according to the majority of the participants, gender does not reflect one’s entrepreneurial skills. Additionally, the participants remark that gender has nothing to do with and is not an indicator of one’s development of entrepreneurial skills. Moreover, as for the perceived entrepreneurial skills, the participants stated that the most perceived entrepreneurial skills are leadership, critical thinking, creative thinking, risk-taking, interpersonal skills, analytical 11 BME 1102│ENM 1207│MGT 1101│MKTG 1106 5. Acknowledgement The researchers would like to pay special thankfulness, warmth and appreciation to the persons below who made the research possible: The participants, for their support, patience, consideration, and coordination while they are conducting this Focus Group Discussion (FGD). Their participation helps us to accomplish this research. Finally, to our family, for their moral support while conducting this research and understanding that the researchers needed to spare time for their research. 0594-7 10. Legard, R., Keegan, J., & Ward, K. (2003). In-depth interviews. In J. Ritchie & J. Lewis (Eds.), Qualitative research practice: A guide for social science students and researchers (pp. 138-169). 11. Paradis, E., O'Brien, B., Nimmon, L., Bandiera, G., & Martimianakis, M. A. (2016). Design: Selection of Data Collection Methods. Journal of graduate medical education, 8(2), 263–264. https://doi.org/10.4300/JGME-D-16-00098.1 12. Li, C., Bilimoria, D., Wang, Y., & Guo, X. (2020, December 17). Gender role characteristics and entrepreneurial self-efficacy: A comparative study of female and male entrepreneurs in China. Frontiers. https://www.frontiersin.org/articles/10.3389/fpsyg.2020.585803/full 13. Newman, A., Obschonka, M., Schwarz, S., Cohen, M., & Nielsen, I. (2019). Entrepreneurial self- efficacy: A systematic review of the literature on its theoretical foundations, measurement, antecedents, and outcomes, and an agenda for future research. Journal of Vocational Behavior, 110, 403-419. https://doi.org/10.1016/j.jvb.2018.05.012 14. Indeed Editorial Team. (2021, August 31). What are the characteristics of successful entrepreneurs? Indeed Career Guide. https://uk.indeed.com/career-advice/career-development/characteristics-of- entrepreneurs 13 13 JOURNAL OF UNDERGRADUATE RESEARCH IN s │Semi-annually │Course final output Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 February – May 2022 3. European Institute for Gender Equality. (2017). Gender in entrepreneurship. EIGE’s publications. 978-92-9493-610-3 3. European Institute for Gender Equality. (2017). Gender in entrepreneurship. EIGE’s publications. 978-92-9493-610-3 4. Jaiswal, M. (2020), "Gender differences and new venture performance", New England Journal of Entrepreneurship, Vol. 23 No. 1, pp. 41-61. https://doi.org/10.1108/NEJE-08-2019-0038 5. Camelo-Ordaz, C., Diánez-González, J. P., & Ruiz-Navarro, J. (2016). The influence of gender on entrepreneurial intention: The mediating role of perceptual factors. BRQ Business Research Quarterly, 19(4), 261–277. https://doi.org/10.1016/j.brq.2016.03.001 6. Mcleod, S. (2019, July 30). What’s the difference between qualitative and quantitative research? Simply Psychology. https://www.simplypsychology.org/qualitative-quantitative.html 7. DeCarlo, M. (n.d.). 10.2 Sampling in qualitative research – Scientific Inquiry in Social Work. Pressbooks.com. Retrieved from https://scientificinquiryinsocialwork.pressbooks.com/chapter/10-2- sampling-in-qualitative-research/ 8. Tongco, D. C. (n.d.). Purposive Sampling as a Tool for Informant Selection. Retrieved from https://core.ac.uk/download/pdf/5093226.pdf 9. Vasileiou, K., Barnett, J., Thorpe, S., & Young, T. (2018). Characterising and justifying sample size sufficiency in interview-based studies: systematic analysis of qualitative health research over a 15- year period. BMC medical research methodology, 18(1), 148. https://doi.org/10.1186/s12874-018- 0594-7 JOURNAL OF UNDERGRADUATE RESEARCH IN ess │Semi-annually │Course final output Preprint │Open access │Semi-annually │Course final output Volume 1 Issue Number 7 February – May 2022 February – May 2022 15. Indeed Editorial Team. (n.d.). Soft Skills: Definitions and Examples. Indeed Career Guide. https://www.indeed.com/career-advice/resumes-cover-letters/soft-skills 16. Hamilton, J. (2022, March 30). How to Describe the Difference Between a Capability and a Skill. Medium. https://medium.com/the-future-of-workplace-learning/how-to-describe-the-difference- between-a-capability-and-a-skill-13f1ac42434f 17. Mind Matters. (n.d.). What Are Cognitive Skills? https://www.mindmattersjo.com/what-are- cognitive-skills.html 18. Gibb, A. A. (1993). Enterprise Culture and Education. International Small Business Journal: Researching Entrepreneurship, 11(3), 11–34. https://doi.org/10.1177/026624269301100301 19. Staff, B. B. (2021, January 11). Factors Influencing Entrepreneurship Development. beta-bowl.com. https://beta-bowl.com/factors-influencing-entrepreneurship-development/ 20. Factors Affecting Entrepreneurship Growth - Harappa. (2021, October 28). Harappa; harappa.education. https://harappa.education/harappa-diaries/factors-influencing-entrepreneurship- growth-and-development/ 14
https://openalex.org/W4380078438	https://www.mdpi.com/2077-0383/12/12/3944/pdf?version=1686297816	English	null	A Novel Irrigation System to Reduce Heat Generation during Guided Implantology: An In Vitro Study	Journal of clinical medicine	2,023	cc-by	8,308	Citation: Parvizi, S.; Cameron, A.; Tadakamadla, S.; Figueredo, C.M.S.; Reher, P. A Novel Irrigation System to Reduce Heat Generation during Guided Implantology: An In Vitro Study. J. Clin. Med. 2023, 12, 3944. https://doi.org/10.3390/ jcm12123944 Keywords: dental implants; surgical guide; irrigation channels; thermocouples; 3D printing; CAD/CAM Somayeh Parvizi 1, Andrew Cameron 1 , Santosh Tadakamadla 2, Carlos Marcelo S. Figueredo 1,3,* and Peter Reher 1 1 School of Medicine & Dentistry, Grifﬁth University, Brisbane, QLD 4111, Australia; somayeh.parvizi@grifﬁthuni.edu.au (S.P.); a.cameron@grifﬁth.edu.au (A.C.); p.reher@grifﬁth.edu.au (P.R.) 2 y p g g p g 2 Dentistry and Oral Health, Department of Rural Clinical Sciences, La Trobe Rural Health Sc Bendigo, VIC 3552, Australia; s.tadakamadla@latrobe.edu.au g 3 Division of Oral Diseases, Department of Dental Medicine, Karolinska Institutet, OF Odontologi, OF Orala sjukdomar, 171 77 Stockholm, Sweden g j * Correspondence: c.dasilvaﬁgueredo@grifﬁth.edu.au Abstract: The purpose of this in vitro study is to evaluate the effectiveness of incorporating a new irrigation system into a surgical guide and monitor its effect on heat generation during implant bed preparation. A total of 48 surgically guided osteotomies were performed on 12 bovine ribs divided into 4 groups, using different irrigation techniques: Group A (test) had entry and exit channels incorporated into the guide; Group B had a similar design with an entry channel only; Group C had conventional external irrigation; and Group D (control) had no irrigation. Heat generation during the osteotomies was measured using thermocouples placed at a depth of 2 mm and 6 mm. The lowest mean temperature was observed in Group A (22.1 ◦C at 2 mm and 21.4 ◦C at 6 mm), which was statistically signiﬁcant when compared with Groups C and D (p < 0.001). Group A showed a lower mean temperature compared with Group B as well; however, it was statistically signiﬁcant only at 6 mm depth (p < 0.05). In conclusion, the proposed surgical guide has signiﬁcantly reduced heat generation during implant osteotomy compared to conventional external irrigation. The integration of an exit cooling channel can resolve limitations found in previously designed surgical guides such as debris blockage and can be easily incorporated into computer designing and 3D printing software. Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Article A Novel Irrigation System to Reduce Heat Generation during Guided Implantology: An In Vitro Study Parvizi 1, Andrew Cameron 1 , Santosh Tadakamadla 2, Carlos Marcelo S. Figueredo 1,3,* R h 1 Somayeh Parvizi 1, Andrew Cameron 1 , Santosh Tadakamadla 2, Carlos Marcelo S. Figue and Peter Reher 1 1. Introduction Academic Editors: Francisco Mesa and A. Noelle Larson Surgical guides are important tools to aid in the insertion of dental implants in the prosthetically driven, digitally planned ideal position with a high level of accuracy. More- over, regional anatomy is visualized on the implant designing software before surgery and the possibility of iatrogenic injury to vital structures can be minimized [1]. However, 3D- printed guides act as a barrier surrounding implant bed preparation and consequently will minimize or block irrigation ﬂuid from reaching the osteotomy site adequately. Inadequate irrigation during implant drilling can excessively increase the temperature and damage those vital cells crucial for bone remodeling and osteointegration [2–4]. Received: 25 April 2023 Revised: 19 May 2023 Accepted: 5 June 2023 Published: 9 June 2023 Received: 25 April 2023 Revised: 19 May 2023 Accepted: 5 June 2023 Published: 9 June 2023 Histological evaluation of implant failures stated bone overheating as the most plausi- ble cause of implant failure [5]. Although there is no clear threshold value for heat-induced osteonecrosis available in the literature, most related articles have suggested that during bone drilling temperature should not exceed 47 ◦C for up to 1 min [6]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Due to the importance of using proper irrigation on heat control during guided implan- tology, several studies have investigated the efﬁciency of different irrigation systems during guided osteotomies. Using chilled ﬂuid and combined irrigation techniques, external and internal cooling systems have been evaluated in numerous in vitro studies [7,8] and animal https://www.mdpi.com/journal/jcm J. Clin. Med. 2023, 12, 3944. https://doi.org/10.3390/jcm12123944 J. Clin. Med. 2023, 12, 3944 2 of 10 studies [9]. The results of these studies indicated that the highest temperatures were noted when a conventional handpiece irrigator was used with a surgical guide, conﬁrming that guided surgery interferes with proper irrigation during implant drilling. Subsequently, the possibility of thermal osteonecrosis could be higher in cases using conventional external irrigators when compared to internal ones [8,10,11]. Up to now, external irrigation is the most acceptable technique to avoid thermal injury during free hand implantology; however, the beneﬁts of using this technique to minimize the heat in deeper osteotomy sites are controversial, leading researchers to ﬁnd a solution to facilitate irrigation on entire osteotomy surface, including any drilling tissues covered with surgical guides. The incorporation of a cooling channel into a surgical guide has been suggested as an efﬁcient technique to lead the ﬂuid under the guide barrier during implant bed preparation [11,12]. Conversely, a recent study showed that adding a supplementary irrigation system to the surgical guide did not signiﬁcantly reduce the temperature compared to conventional irrigation [13]. In this experiment, blockage of irrigation channels by bone debris was found to be a possible reason for reducing the efﬁciency of the proposed cooling system. The beneﬁts of using capillary drills as an internal irrigation system to lead the ﬂuid directly on the drilled bone surface are also controversial [14]. Considering all the proposed strategies to facilitate irrigation during guided implan- tology, the aim of this in vitro study is to improve the current design of surgical guides for implantology by incorporating a new irrigation system with an entry and exit channel into a 3D-printed surgical guide and evaluate its effect on heat generation during osteotomies. 2. Materials and Methods Ethics approval was not required for this study; however, an animal ethics exemption was received from the animal ethics committee of Grifﬁth University, as bovine ribs were used. This was an in vitro study using fresh bovine ribs, as they have similar thermal conductivity and density as human bones [15]. After a power analysis study (85–90% power, signiﬁcance at 0.05, detecting 2 ◦C difference with StDev of 1.5 ◦C), a sample size of 12 was considered ideal. All bone samples were sourced from a local butcher and all the residual soft tissues were cleaned off. Samples were marked by creating identiﬁcation numbers carved into the bones using a rotary grinder (Ozito 170, Sydney, Australia) to facilitate identifying them after scanning. 2.1. Data Acquisition for 3D Planning A pre-planning CBCT scan was obtained for all samples (Carestream CS 9300C, New York, NY, USA), set at 69 kV, 10 mA, and exposed for 11.30 s. The resolution was set to 180 voxels/mm3, allowing for 0.1 mm thick scans, and the ﬁeld of view was 10 × 5 cm. A digital surface impression was also taken for all samples using an intraoral scanner (Trios 3, 3Shape A/S, Copenhagen, Denmark). The surface scan ﬁles were exported as standard tessellation language (STL) ﬁles. Then, the CBCT and the STL ﬁles were imported into the 3Shape Implant Studio software (3Shape A/S, Copenhagen Denmark) version 2018 1.2, a conventional commercial implant planning software for use in both clinical and laboratory environments. 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on The osteotomy sites for each group are on the top of the guide, the fixation at the edges of the guide, and the thermocouple sites perpendicular to the osteoto mm depths. Figure 1. Surgical guide created for each sample, with groups A, B, C and D incorporated into the design. The osteotomy sites for each group are on the top of the guide, the ﬁxation screw sites are at the edges of the guide, and the thermocouple sites perpendicular to the osteotomies, at 2 and 6 mm depths. design. The osteotomy sites for each group are on the top of the guide, the fixation at the edges of the guide, and the thermocouple sites perpendicular to the osteoto mm depths. Four drilling sites were planned per guide (one for each group) with an Four drilling sites were planned per guide (one for each group), with an distance of 3 mm, all parallel to each other utilizing the software’s autom (Figure 2). Titanium sleeves (Steco System Technik, Hamburg, Germany) w were added to guide the implant drill during the procedure. Each surgical gu to the bone sample with two fixation screws and the holes for these were Four drilling sites were planned per guide (one for each group), with an inter-implant distance of 3 mm, all parallel to each other utilizing the software’s automated function (Figure 2). Titanium sleeves (Steco System Technik, Hamburg, Germany) with Ø 2.2 mm were added to guide the implant drill during the procedure. Each surgical guide was ﬁxed to the bone sample with two ﬁxation screws, and the holes for these were also incorporated into the guide design. distance of 3 mm, all parallel to each other utilizing the software’s autom (Figure 2). Titanium sleeves (Steco System Technik, Hamburg, Germany) w were added to guide the implant drill during the procedure. Each surgical gu to the bone sample with two fixation screws, and the holes for these were rated into the guide design. rated into the guide design. Figure 2. The digital planning of the guide was done using implant software (Im Shape A/S, Denmark). The osteotomy sites for the 4 groups, the fixation screws site mocouples insertion guides were made mimicking implants, and surgical sleeves w Figure 2. The digital planning of the guide was done using implant software (Im Shape A/S, Denmark). 2.2. Study Groups To evaluate the changes in temperature observed with the new surgical guide designs, four groups were created: Group A: irrigation built-in guide + exit channel (new design); oup B: irrigation built-in guide (no exit channel); Group B: irrigation built-in guide (no exit channel); G C l h d i i i i ( i l) Group C: external handpiece irrigation (conventional); Group D: no irrigation (control group). J. Clin. Med. 2023, 12, 3944 3 of 10 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gu same surgical guide mimicking on To ensure consistency at room temperature and allow for similar experimental condi- tions, we created one surgical guide for each sample, with the four groups built into the same surgical guide, mimicking one implant insertion per group (Figure 1). The digital planning for the guides was conducted using the 3Shape Implant Studio software (3Shape A/S, Copenhagen Denmark). This software allows the alignment of the two scans so accurate planning of implant or drilling site can take place. y p p ditions, we created one surgical guide for each sample, with the four group same surgical guide, mimicking one implant insertion per group (Figure planning for the guides was conducted using the 3Shape Implant Studio sof A/S, Copenhagen Denmark). This software allows the alignment of the tw curate planning of implant or drilling site can take place. same surgical guide, mimicking one implant insertion per group (Figure planning for the guides was conducted using the 3Shape Implant Studio sof A/S, Copenhagen Denmark). This software allows the alignment of the tw curate planning of implant or drilling site can take place. Figure 1. Surgical guide created for each sample, with groups A, B, C and D incorp design. The osteotomy sites for each group are on the top of the guide, the fixation at the edges of the guide, and the thermocouple sites perpendicular to the osteoto mm depths. Figure 1. Surgical guide created for each sample, with groups A, B, C and D incorporated into the design. The osteotomy sites for each group are on the top of the guide, the ﬁxation screw sites are at the edges of the guide, and the thermocouple sites perpendicular to the osteotomies, at 2 and 6 mm depths. Figure 1. Surgical guide created for each sample, with groups A, B, C and D incorp design. The osteotomy sites for each group are on the top of the guide, the fixation at the edges of the guide, and the thermocouple sites perpendicular to the osteoto mm depths. Four drilling sites were planned per guide (one for each group) with an de created for each sa i f h Figure 1. Surgical guide created for each sample, with groups A, B, C and D incorp design. 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on The osteotomy sites for the 4 groups, the fixation screws sit mocouples insertion guides were made mimicking implants, and surgical sleeves w porated into the guide. The varying osteotomy sites are indicated from A–D. Figure 2. The digital planning of the guide was done using implant software (Implant Studio, 3 Shape A/S, Denmark). The osteotomy sites for the 4 groups, the ﬁxation screws sites and the thermocouples insertion guides were made mimicking implants, and surgical sleeves were then incorporated into the guide. The varying osteotomy sites are indicated from A–D. rated into the guide design. Figure 2. The digital planning of the guide was done using implant software (Im Shape A/S, Denmark). The osteotomy sites for the 4 groups, the fixation screws sit l i i id d i i ki i l d i l l Figure 2. The digital planning of the guide was done using implant software (Im Shape A/S, Denmark). The osteotomy sites for the 4 groups, the fixation screws sit mocouples insertion guides were made mimicking implants, and surgical sleeves w porated into the guide. The varying osteotomy sites are indicated from A–D. Figure 2. The digital planning of the guide was done using implant software (Implant Studio, 3 Shape A/S, Denmark). The osteotomy sites for the 4 groups, the ﬁxation screws sites and the thermocouples insertion guides were made mimicking implants, and surgical sleeves were then incorporated into the guide. The varying osteotomy sites are indicated from A–D. J. Clin. Med. 2023, 12, 3944 4 of 10 iamet Two temperature measurement points were created into the guide, set perpendicular to the implant perforation at 2 mm depth (TC1) and 6 mm depth (TC2) by placing additional ‘phantom’ implants in this location to create a drilling hole (Figure 2). To precisely position these, insertion guides for the thermocouples were planned to reach a precise distance of 1 mm from the drilling surface. Sleeves of 1.3 mm diameter (Steco system technik, Hamburg, Germany) were incorporated into these insertion guides for the thermocouples. A surgical guide was then designed with a thickness of 2.2 mm with additional support structures added to allow the incorporation of irrigation channels. The surgical guide was then exported as a standard tessellation (STL) ﬁle (Figure 1). couples. 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on A surgical guide was then designed with a thickness of 2.2 support structures added to allow the incorporation of irrigation c guide was then exported as a standard tessellation (STL) file (Figure To design the irrigation channels, the STL file of the surgical g ported into another computer software: Meshmixer (Autodesk, San For group A (new design): two irrigation channels were incorpora guide, so they emerged under the metal sleeve (Figure 3). The entry p g To design the irrigation channels, the STL ﬁle of the surgical guide designs was imported into another computer software: Meshmixer (Autodesk, San Francisco, CA, USA). For group A (new design): two irrigation channels were incorporated into the surgical guide, so they emerged under the metal sleeve (Figure 3). The entry channel was used to insert the irrigation tubing, and the exit channel was used to vent the ﬂuid and any debris to create a greater ﬂow of coolant across the bur. For group B: only one entry irrigation channel was incorporated for the placement of the irrigation tubing. The channels were designed with a 3 mm diameter in a funnel shape, using the ‘add tube’ function, to place a 2.5 mm diameter irrigation tubing (Figure 3). The funnel shape allowed the tight and secure placement of the irrigation tubing. For groups C and D no irrigation channels were incorporated into the surgical guide. g y g g y insert the irrigation tubing, and the exit channel was used to vent the to create a greater flow of coolant across the bur. For group B: only channel was incorporated for the placement of the irrigation tubin designed with a 3 mm diameter in a funnel shape, using the ‘add tu a 2.5 mm diameter irrigation tubing (Figure 3). The funnel shape a secure placement of the irrigation tubing. For groups C and D no irri incorporated into the surgical guide. Figure 3. See-through illustration of the irrigation channel design showing designed in Meshmixer (Autodesk, San Francisco, CA, USA). Groups A an h l ( th ) d G A h d l it li h l ( Figure 3. See-through illustration of the irrigation channel design showing all groups. These were designed in Meshmixer (Autodesk, San Francisco, CA, USA). Groups A and B had entry irrigation channels (near the arrows), and Group A had also an exit cooling channel (marked in green). 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on Group C was used with a standard handpiece irrigation while group D had no irrigation. Figure 3. See-through illustration of the irrigation channel design showing designed in Meshmixer (Autodesk, San Francisco, CA, USA). Groups A an Figure 3. See-through illustration of the irrigation channel design showing all groups. These were designed in Meshmixer (Autodesk, San Francisco, CA, USA). Groups A and B had entry irrigation channels (near the arrows), and Group A had also an exit cooling channel (marked in green). Group C was used with a standard handpiece irrigation while group D had no irrigation. ( ), p g ( C was used with a standard handpiece irrigation while group D had no irri The surgical guides were then additively manufactured in a su (NextDent 5100, 3D Systems Inc., Rock Hill, SC, USA) and post-proc ufacturer’s instructions with the metal sleeves being incorporated p On the day of the experiment, the specimens were allowed to ture, and all experiments were performed on the same day to ensure line for temperature measurements. The surgical guides were fixed t with two screws (Zenith, Mumbau, India) so they would not move t The surgical guides were then additively manufactured in a surgical guide material (NextDent 5100, 3D Systems Inc., Rock Hill, SC, USA) and post-processed as per the manufacturer’s instructions with the metal sleeves being incorporated prior to post curing. On the day of the experiment, the specimens were allowed to reach room temperature, and all experiments were performed on the same day to ensure a standardized baseline for temperature measurements. The surgical guides were ﬁxed to the bony specimens with two screws (Zenith, Mumbau, India) so they would not move throughout the experiment. The thermocouple insertion channels were drilled using ParaPost X Drills with a 4.5 size and a 1.14 mm diameter (Coltene, Cuyahoga Falls, OH, USA). The channel depth was measured individually in the software for each site, so it was at 1 mm of the implant drill. After cleaning the channels from any debris, the thermocouples (1 mm diameter) were inserted, and the holes were sealed and secured in place with an orthodontic wax (Figure 4). J. Clin. Med. 2023, 12, 3944 J. Clin. Med. 2023, 12, x FOR 5 of 10 5 of 10 Figure 4. Preparation of the thermocouple channels using the ParaPost® burs (Coltene, Cuyahoga Falls, OH, USA)with 1.14 mm diameter. 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on A drill was inserted into the other side to show where the exit channel is located. The temperature was recorded during each osteotomy using K-type thermocouples with a 1 mm diameter (TC Instruments, 5SRTC-TT-KI-36-0.5M, OMEGA, Knoxville, TN, USA) Th b l d t 8 h l d t l (OM HL EH TC K CAL The temperature was recorded during each osteotomy using K-type ther with a 1 mm diameter (TC Instruments, 5SRTC-TT-KI-36-0.5M, OMEGA, Kno USA) Th b l d t 8 h l d t l (OM HL EH T For Groups A and B the irrigation tubing was connected to the surgical guide, while for Group C it was connected directly to the handpiece (external irrigation), and for Group D the osteotomy was performed without irrigation (control). USA). These probes were coupled to an 8-channel data logger (OM-HL-EH-TC-K-CAL, OMEGA, Knoxville, USA), as seen in Figure 6. The accuracy parameters were: ±0.8 °C ± 0.2%, and the reading time was for a minimum of 1 s sampling and 2 s logging intervals. All recorded data was transferred and stored on a personal computer using the OM-HL Logpro Software (OMEGA, Knoxville, TN, USA). The data was later downloaded in Excel format for statistical analysis. After each group was measured, the specimen was cleaned, dried, and the thermocouples were repositioned for the next group measurement. USA). These probes were coupled to an 8-channel data logger (OM-HL-EH-T OMEGA, Knoxville, USA), as seen in Figure 6. The accuracy parameters were 0.2%, and the reading time was for a minimum of 1 s sampling and 2 s logging All recorded data was transferred and stored on a personal computer using th Logpro Software (OMEGA, Knoxville, TN, USA). The data was later download format for statistical analysis. After each group was measured, the specimen w dried, and the thermocouples were repositioned for the next group measurem The temperature was recorded during each osteotomy using K-type thermocou- ples with a 1 mm diameter (TC Instruments, 5SRTC-TT-KI-36-0.5M, OMEGA, Knoxville, TN, USA). These probes were coupled to an 8-channel data logger (OM-HL-EH-TC-K- CAL, OMEGA, Knoxville, USA), as seen in Figure 6. The accuracy parameters were: ±0.8 ◦C ± 0.2%, and the reading time was for a minimum of 1 s sampling and 2 s logging intervals. All recorded data was transferred and stored on a personal computer using the OM-HL Logpro Software (OMEGA, Knoxville, TN, USA). 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on The thermocouples had 1 mm in diameter and depth was measured individually in the software for each site, so it was at 1 mm of the osteotomy site. Figure 4. Preparation of the thermocouple channels using the ParaPost® burs (Coltene, Cuyahoga Falls, OH, USA)with 1.14 mm diameter. The thermocouples had 1 mm in diameter and depth was measured individually in the software for each site, so it was at 1 mm of the osteotomy site. Figure 4. Preparation of the thermocouple channels using the ParaPost® burs (Coltene Falls, OH, USA)with 1.14 mm diameter. The thermocouples had 1 mm in diameter and measured individually in the software for each site, so it was at 1 mm of the osteotomy The osteotomies for each group were then performed by using a 20:1 reduc angle (W&H, Bürmoos, Austria) with a pilot drill (Straumann Ø 2.2 mm, short) to an implant electric motor (Satelec, Viry-Châtillon, France), using the follow 800 t t 45 N i i ti fl id t t 35 L/ i (Fi 5) F Figure 4. Preparation of the thermocouple channels using the ParaPost® burs (Coltene, Cuyahoga Falls, OH, USA)with 1.14 mm diameter. The thermocouples had 1 mm in diameter and depth was measured individually in the software for each site, so it was at 1 mm of the osteotomy site. Figure 4. Preparation of the thermocouple channels using the ParaPost® burs (Coltene, Cuyahoga Falls, OH, USA)with 1.14 mm diameter. The thermocouples had 1 mm in diameter and depth was measured individually in the software for each site, so it was at 1 mm of the osteotomy site. The osteotomies for each group were then performed by using a 20:1 reduc angle (W&H, Bürmoos, Austria) with a pilot drill (Straumann Ø 2.2 mm, short) to an implant electric motor (Satelec, Viry-Châtillon, France), using the follow The osteotomies for each group were then performed by using a 20:1 reduction contra angle (W&H, Bürmoos, Austria) with a pilot drill (Straumann Ø 2.2 mm, short) connected to an implant electric motor (Satelec, Viry-Châtillon, France), using the following setup: 800 rpm, torque at 45 Nm, irrigation fluid rate at 35 mL/min (Figure 5). For consistency, all osteotomies were performed by one calibrated operator (SP), to a depth of 8 mm. The drills were changed regularly as per the manufacturer’s instructions. 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on F G A d B h i i i bi d h i l id hil The osteotomies for each group were then performed by using a 20:1 reduction contra angle (W&H, Bürmoos, Austria) with a pilot drill (Straumann Ø 2.2 mm, short) connected to an implant electric motor (Satelec, Viry-Châtillon, France), using the following setup: 800 rpm, torque at 45 Nm, irrigation ﬂuid rate at 35 mL/min (Figure 5). For consistency, all osteotomies were performed by one calibrated operator (SP), to a depth of 8 mm. The drills were changed regularly as per the manufacturer’s instructions. 800 rpm, torque at 45 Nm, irrigation fluid rate at 35 mL/min (Figure 5). For co all osteotomies were performed by one calibrated operator (SP), to a depth of drills were changed regularly as per the manufacturer’s instructions. For Groups A and B the irrigation tubing was connected to the surgical gu for Group C it was connected directly to the handpiece (external irrigation), and D the osteotomy was performed without irrigation (control). p g g g g , for Group C it was connected directly to the handpiece (external irrigation), and for Group D the osteotomy was performed without irrigation (control). Figure 5. Surgical guide fixed in place with two fixation screws, showing the irrigation tube inserted into the channel (Group A). A drill was inserted into the other side to show where the exit channel is located. Figure 5. Surgical guide fixed in place with two fixation screws, showing the irrigation t into the channel (Group A). A drill was inserted into the other side to show where the is located. Figure 5. Surgical guide ﬁxed in place with two ﬁxation screws, showing the irrigation tube inserted into the channel (Group A). A drill was inserted into the other side to show where the exit channel is located. Figure 5. Surgical guide fixed in place with two fixation screws, showing the irrigation tube inserted into the channel (Group A). A drill was inserted into the other side to show where the exit channel is located Figure 5. Surgical guide fixed in place with two fixation screws, showing the irrigation t into the channel (Group A). A drill was inserted into the other side to show where the is located. Figure 5. Surgical guide ﬁxed in place with two ﬁxation screws, showing the irrigation tube inserted into the channel (Group A). 2.3. Surgical Guide Design and Manufacture To ensure consistency at room ditions, we created one surgical gui same surgical guide mimicking on The data was later downloaded in Excel format for statistical analysis. After each group was measured, the specimen was cleaned, dried, and the thermocouples were repositioned for the next group measurement. J. Clin. Med. 2023, 12, 3944 J. Clin. Med. 2023, 12, x FOR 6 of 10 6 of 10 Figure 6. Implant osteotomy being performed with the irrigation entry and exit channel (Group A), measuring the temperature with thermocouples inserted into the thermocouple channels TC1 (2 mm) and TC2 (6 mm). These probes were coupled to an 8-channel data logger (OM-HL-EH-TC-K- CAL, OMEGA, Knoxville, TN, USA). Figure 6. Implant osteotomy being performed with the irrigation entry and exit channel (Group A), measuring the temperature with thermocouples inserted into the thermocouple channels TC1 (2 mm) and TC2 (6 mm). These probes were coupled to an 8-channel data logger (OM-HL-EH-TC-K-CAL, OMEGA, Knoxville, TN, USA). Figure 6. Implant osteotomy being performed with the irrigation entry and exit channel (Group A), measuring the temperature with thermocouples inserted into the thermocouple channels TC1 (2 mm) and TC2 (6 mm). These probes were coupled to an 8-channel data logger (OM-HL-EH-TC-K- CAL, OMEGA, Knoxville, TN, USA). Figure 6. Implant osteotomy being performed with the irrigation entry and exit channel (Group A), measuring the temperature with thermocouples inserted into the thermocouple channels TC1 (2 mm) and TC2 (6 mm). These probes were coupled to an 8-channel data logger (OM-HL-EH-TC-K-CAL, OMEGA, Knoxville, TN, USA). considered 3. Results 3. Results The highest temperatures measured among the three test groups (A, B and C) at 2 mm depth (TC1) and 6 mm depth (TC2) were 47.2 °C and 36.9 °C, respectively. Both were recorded in Group C (external irrigation), which was still less than the maximum temper- ature in Group D (control), with no irrigation (TC1: 51.4 °C, TC2: 52.6 °C), as shown in Table 1. There was a clear increase in the mean temperature from Group A to Group D (A The highest temperatures measured among the three test groups (A, B and C) at 2 mm depth (TC1) and 6 mm depth (TC2) were 47.2 ◦C and 36.9 ◦C, respectively. Both were recorded in Group C (external irrigation), which was still less than the maximum temperature in Group D (control), with no irrigation (TC1: 51.4 ◦C, TC2: 52.6 ◦C), as shown in Table 1. There was a clear increase in the mean temperature from Group A to Group D (A < B < C < D), at both measurement points. < B < C < D), at both measurement points. There were significant differences in recorded temperatures between all groups, both at 2 mm depth (Table 2 and Figure 7) and 6 mm depth (Table 2 and Figure 8). Group A (new guide) had highly significant lower temperatures (p < 0.001) compared to the control and external irrigation groups (mean temperature at TC1; 22.1 °C, TC2; 21.4 °C). Similar observations were noted in Group B, with significantly lower temperatures than Groups C and D (p < 0.01). When comparing Groups A and B, the new guide design showed better results, but these only reached significance in the TC2 measurement point. Table 1. Mean, standard deviation and maximum temperature recorded in all groups, at 2 mm depth (TC1) and 6 mm depth (TC2). A (Guide with Entry + Exit) B (Guide with Entry Only) C (External Handpiece) D (Control) TC1 Mean 22.1 ◦C 22.3 ◦C 24.9 ◦C 28.2 ◦C (2 mm depth) StDev 1.5 ◦C 1.7 ◦C 2.1 ◦C 3.8 ◦C Maximum 33.9 ◦C 45.9 ◦C 47.2 ◦C 51.4 ◦C TC2 Mean 21.4 ◦C 22.1 ◦C 23.7 ◦C 27.2 ◦C (6 mm depth) StDev 0.8 ◦C 0.9 ◦C 1.3 ◦C 2.8 ◦C Maximum 28.9 ◦C 27.5 ◦C 36.9 ◦C 52.6 ◦C < B < C < D), at both measurement points. 2.4. Statistical Analysis 2.4. Statistical Analysis SPSS (version 27.0) was used for statistical analysis. Normality of the outcome varia- bles, TC1 (2 mm depth temperature) and TC2 (6 mm depth temperature) were assessed using Shapiro–Wilk test. TC1 was not distributed normally. Median and interquartile ranges for presenting the data that were normally distributed, while mean and standard deviation were used for normally distributed data. Therefore, a non-parametric, Kruskal– Wallis test, was used to compare the TC1 temperatures between the four groups. Mann– Whitney U test was used to conduct pair-wise comparisons between the groups for TC1. One-way ANOVA was used to compare TC2 temperature between the groups and Games–Howell post hoc test was used to pair-wise comparison. A p value of <0.05 was id d i ifi SPSS (version 27.0) was used for statistical analysis. Normality of the outcome vari- ables, TC1 (2 mm depth temperature) and TC2 (6 mm depth temperature) were assessed us- ing Shapiro–Wilk test. TC1 was not distributed normally. Median and interquartile ranges for presenting the data that were normally distributed, while mean and standard deviation were used for normally distributed data. Therefore, a non-parametric, Kruskal–Wallis test, was used to compare the TC1 temperatures between the four groups. Mann–Whitney U test was used to conduct pair-wise comparisons between the groups for TC1. One-way ANOVA was used to compare TC2 temperature between the groups and Games–Howell post hoc test was used to pair-wise comparison. A p value of <0.05 was considered signiﬁcant. considered 3. Results There were significant differences in recorded temperatures between all groups, both at 2 mm depth (Table 2 and Figure 7) and 6 mm depth (Table 2 and Figure 8). Group A Table 1. Mean, standard deviation and maximum temperature recorded in all groups, at 2 mm depth (TC1) and 6 mm depth (TC2). J. Clin. Med. 2023, 12, 3944 7 of 10 C C 7 of 10 C C There were signiﬁcant differences in recorded temperatures between all groups, both at 2 mm depth (Table 2 and Figure 7) and 6 mm depth (Table 2 and Figure 8). Group A (new guide) had highly signiﬁcant lower temperatures (p < 0.001) compared to the control and external irrigation groups (mean temperature at TC1; 22.1 ◦C, TC2; 21.4 ◦C). Similar observations were noted in Group B, with signiﬁcantly lower temperatures than Groups C and D (p < 0.01). When comparing Groups A and B, the new guide design showed better results, but these only reached signiﬁcance in the TC2 measurement point. Table 2. Pair-wise comparison of groups (the significance level is 0.05). Groups in Comparison TC1 (2 mm Depth) TC2 (6 mm Depth) A-B 0.3897 0.0352 A-C 0.0005 0.0000 A-D 0.0000 0.0000 B-C 0.0013 0.0005 Table 2. Pair-wise comparison of groups (the signiﬁcance level is 0.05). Groups in Comparison TC1 (2 mm Depth) TC2 (6 mm Depth) A-B 0.3897 0.0352 A-C 0.0005 0.0000 A-D 0.0000 0.0000 B-C 0.0013 0.0005 B-D 0.0000 0.0000 C-D 0.0073 0.0004 B-D 0.0000 0.0000 C-D 0.0073 0.0004 The maximum temperature measured by TC1 was higher than TC2 for all groups; however, this difference did not reach significance (p > 0.05). The mean deviation from baseline temperature in Group A with the new design never exceeds more than 2 °C. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signifi- cant, * p < 0.001. Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signiﬁcant, * p < 0.001. REVIEW 8 of 10 Table 2. Pair-wise comparison of groups (the signiﬁcance level is 0.05). 4. Discussion In the present study, surgical guides for implant placement were modiﬁed to incorpo- rate a single cooling and a double cooling channel. The cooling channels were theorized to reduce heat generation and debris buildup on the bur. The results showed that a surgical guide with cooling channels incorporated improved irrigation ﬂuid ﬂow on the bur and into the osteotomy site. Furthermore, the addition of an exit channel has addressed the risk of obstruction with bone debris, resulting in even better temperature reduction. This ﬁnding supports the hypothesis that the addition of an irrigation channel into the surgical guide would minimize heat during the drilling of bone [11]. In the current experiment, the incorporation of one (Group A) or two (Group B) irrigation channels into the surgical guide have been shown to signiﬁcantly reduce the temperature compared to external irrigation (Group C) or no irrigation (Group D). In the recent study by Stocchero et al. [13], the incorporation of the cooling channel did not make a statistically signiﬁcant difference compared to the group with no irrigation or with a free hand technique without a surgical guide. In their study, a CBCT was taken of the surgical guides after the experiment with the obstruction of cooling channels being observed. A high-density drilling product was suggested as a possible cause for the insufﬁciency of coolant irrigation for this proposed surgical guide design. The cooling channels, in the current study, direct the cooling ﬂuid directly onto the bur and around the metal sleeve, which without being cooled have signiﬁcant overheating of bone [16]. This may also aid in reducing debris accumulated on the bur during an osteotomy [13]. Consequently, the integration of irrigation with this design has the potential to overcome one of the concerns related to guided osteotomy: heat-induced osteonecrosis of the bone [10,17]. The integration of an entry and exit irrigation channel may solve this problem as it would allow any debris to be vented from the bur during the osteotomy process. y g y p In our study, the mean deviation from baseline temperature in Group A with the new design never exceed more than 2 ◦C, indicating that the surgical guide design would have the potential to keep the temperature within the safety zone in a clinical context during the cortectomy procedure. considered 3. Results Groups in Comparison TC1 (2 mm Depth) TC2 (6 mm Depth) A-B 0.3897 0.0352 A-C 0.0005 0.0000 A-D 0.0000 0.0000 B-C 0.0013 0.0005 B-D 0.0000 0.0000 C-D 0.0073 0.0004 B D 0.0000 0.0000 C-D 0.0073 0.0004 The maximum temperature measured by TC1 was higher than TC2 for all groups; however, this difference did not reach significance (p > 0.05). The mean deviation from baseline temperature in Group A with the new design never exceeds more than 2 °C. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. Table 2. Pair-wise comparison of groups (the signiﬁcance level is 0.05). B D 0.0000 C-D 0.0073 Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signifi- cant, * p < 0.001. Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signiﬁcant, * p < 0.001. REVIEW 8 of 10 Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signifi- cant, * p < 0.001. Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signiﬁcant, * p < 0.001. REVIEW 8 of 10 Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signifi- cant, * p < 0.001. Figure 7. Average temperature measurements at 2 mm depth (TC1) in all groups (NS, non-signiﬁcant, * p < 0.001. EVIEW 8 of 10 Figure 8. Average temperature measurements at 6 mm depth (TC2) in all groups (NS, non-signifi- cant, * p < 0.05, Figure 8. Average temperature measurements at 6 mm depth (TC2) in all groups (NS, non-signiﬁcant, * p < 0.05, *** p < 0.001. Figure 8. Average temperature measurements at 6 mm depth (TC2) in all groups (NS, non-signifi- cant, * p < 0.05, Figure 8. Average temperature measurements at 6 mm depth (TC2) in all groups (NS, non-signiﬁcant, * p < 0.05, *** p < 0.001. J. Clin. Med. 2023, 12, 3944 8 of 10 The maximum temperature measured by TC1 was higher than TC2 for all groups; however, this difference did not reach signiﬁcance (p > 0.05). The mean deviation from baseline temperature in Group A with the new design never exceeds more than 2 ◦C. considered 3. Results It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. 4. Discussion In most related articles, which investigated heat generation during osteotomies, the safety of the proposed irrigation systems was assessed based on the threshold value of 47 ◦C, which was ﬁrst stated in 1984 by Eriksson, Albrektsson and Albrektsson [6]. Subsequently, if the temperature did not exceed 47 ◦C, the proposed system was considered safe for bone drilling, but it should be taken into consideration that in those studies, drilling performed on nonvital animal bone models and baseline temperature was room temperature (20–24 ◦C) at the time of the experiment, which was on average 15 ◦C lower than the baseline temperature on a human bone (37 ◦C) at the time of drilling [8,9,18]. Therefore, the amount of deviation from baseline temperature should be considered as the safety of any proposed irrigation system. In the present study, the temperature value increased from Group A to Group D, with a limited increase from TC2 to TC1. This conﬁrms that if the design of the irrigation system leads and maintains the ﬂuid close to the osteotomy site and under the guide barrier, it is more likely to reduce the bone temperature during osteotomy. This ﬁnding is in accordance with the ﬁnding by Liu et al. (2016), who concluded that guided osteotomy using a drill with the internal hole was 3.6 times, and when using a cooling channel in the surgical guide was 1.95 times, more effective than conventional handpiece irrigation in heat control during osteotomy [10]. However, another study did not ﬁnd any differences between internal and external irrigation systems in terms of heat generation during bone drilling [14]. In our J. Clin. Med. 2023, 12, 3944 9 of 10 9 of 10 study, the maximum temperature measured by TC1 was higher than TC2 for all groups, indicating that drilling of dense cortical bone with or without irrigation will generate more heat and highlights the need for effective irrigation to minimize thermal injury in dense cortical bone. This reinforces previous observations that the density of bone plays a more important role than the osteotomy depth in terms of heat generation during the drilling of the bone [7,13]. This in vitro study has limitations to a complete connection to a clinical context. The bones utilized were not living or had circulating blood which may have affected the outcome. Different structures in each bone may have also inﬂuenced the temperature readings. 4. Discussion Future directions for this research could see this method conducted on a cadaver or live animal model to better simulate clinical conditions. Informed Consent Statement: Not applicable. Data Availability Statement: Data generated in this research project is available by contacting the last author of this paper via email. It is stored electronically as Excel worksheets. Data Availability Statement: Data generated in this research project is available by contacting the last author of this paper via email. It is stored electronically as Excel worksheets. Acknowledgments: The authors would like to acknowledge the technical staff members at the Anatomy Laboratory, School of Medical Sciences, Grifﬁth University, for their support during the execution of the project and for the use of its facilities. Acknowledgments: The authors would like to acknowledge the technical staff members at the Anatomy Laboratory, School of Medical Sciences, Grifﬁth University, for their support during the execution of the project and for the use of its facilities. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions The proposed surgical guide with built-in entry and exit irrigation channels has signiﬁcantly reduced heat generation during implant osteotomy compared to conventional external irrigation. The integration of an exit cooling channel has reduced heat and can resolve limitations related to the constant obstruction of irrigation channels found in previously designed surgical guides. This has an immediate practical application and can be incorporated into computer designing and 3D printing software. Author Contributions: Conceptualization, P.R. and A.C.; methodology, S.P., A.C. and P.R.; software, S.P. and A.C.; validation, P.R., S.T. and C.M.S.F.; formal analysis, P.R. and S.T.; investigation, S.P.; resources, S.P. and P.R.; data curation, P.R.; writing—original draft preparation, S.P.; writing—review and editing, P.R. and C.M.S.F.; visualization, A.C.; supervision, P.R.; project administration, S.P., P.R. and C.M.S.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Ethics approval was not required for this study; however, an animal ethics exemption was received from the animal ethics committee of Grifﬁth University as bovine ribs were used. Informed Consent Statement: Not applicable. p [ ] [ ] 7. Sener, B.C.; Dergin, G.; Gursoy, B.; Kelesoglu, E.; Slih, I. Effects of irrigation temperature on heat co drilling depths. Clin. Oral Implants Res. 2009, 20, 294–298. [CrossRef] [PubMed] 6. Eriksson, A.R.; Albrektsson, T.; Albrektsson, B. Heat caused by drilling cortical bone: Temperature and animals. Acta Orthopaedica Scandinavica 1984, 55, 629–631. [CrossRef] [PubMed] References 1. Vermeulen, J. The Accuracy of Implant Placement by Experienced Surgeons: Guided vs Freehand Approach in a Simulated Plastic Model. Int. J. Oral Maxillofac. Implants 2017, 32, 617–624. [CrossRef] 1. Vermeulen, J. The Accuracy of Implant Placement by Experienced Surgeons: Guided vs Freehand Approach in a Simulated Plastic Model. Int. J. Oral Maxillofac. Implants 2017, 32, 617–624. [CrossRef] f p 2. Misir, A.F.; Sumer, M.; Yenisey, M.; Egrioglu, E. Effect of surgical drill guide on heat generated from implant drilling. J. Oral Maxillofac. Surg. 2009, 67, 2663–2668. [CrossRef] 3. Migliorati, M.; Amorﬁni, L.; Signori, A.; Barberis, F.; Silvestrini, B.A.; Benedicenti, S. Internal bone temperature change during guided surgery preparations for dental implants: An in vitro study. Int. J. Oral Maxillofac. Implants 2013, 28, 1464–1469. [CrossRef] 4. dos Santos, P.L.; Pereira Queiroz, T.; Margonar, R.; Gomes de Souza Carvalho, A.C.; Betoni Jr, W.; Rodrigues Rezende, R.R.; dos Santos, P.H.; Garcia, R., Jr. Evaluation of bone heating, drill deformation, and drill doughiness after implant osteotomy: Guided surgery and classic drilling procedure. Int. J. Oral Maxillofac. Implants 2014, 29, 51–58. [CrossRef] [PubMed] J. Clin. Med. 2023, 12, 3944 10 of 10 8. Strbac, G.D.; Unger, E.; Donner, R.; Bijak, M.; Watzek, G.; Zechner, W. Thermal effects of a combined irrigation method during implant site drilling. A standardized in vitro study using a bovine rib model. Clin. Oral Implants Res. 2012, 25, 665–674. [CrossRef] [PubMed] [ ] 9. Stocchero, M.; Jinno, Y.; Toia, M.; Ahmad, M.; Papia, E.; Yamaguchi, S.; Becktor, J.P. Intraosseous temperature change during installation of dental implants with two different surfaces and different drilling protocols: An in vivo study in sheep. J. Clin. Medicine 2019, 8, 1198. [CrossRef] [PubMed] 10. Liu, Y.; Wu, J.; Zhang, J.; Peng, W.; Liao, W. Numerical and Experimental Analyses on the Temperature Distribution in the Dental Implant Preparation Area when Using a Surgical Guide. J. Prosthodont. 2016, 27, 42–51. [CrossRef] [PubMed] p p g g 11. Teich, S.; Bocklet, M.; Evans, Z.; Gutmacher, Z.; Renne, W. 3D printed implant surgical guides with inter temperature reduction during osteotomy preparation: A pilot study. J. Esthet. Restor. Dent 2022, 34, 796–8 11. Teich, S.; Bocklet, M.; Evans, Z.; Gutmacher, Z.; Renne, W. 3D printed implant surgical guides with internally routed irrigation for temperature reduction during osteotomy preparation: A pilot study. J. Esthet. Restor. Dent 2022, 34, 796–803. [CrossRef] [PubMed] 12. Alevizakos, V.; Mitov, G.; Von See, C. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References Guided implant placement using an internally cooling surgical template: A technical note. J Oral Implantol 2020 46 533–535 [CrossRef] [PubMed] 11. Teich, S.; Bocklet, M.; Evans, Z.; Gutmacher, Z.; Renne, W. 3D printed implant surgical guides with internally routed irrigation for temperature reduction during osteotomy preparation: A pilot study. J. Esthet. Restor. Dent 2022, 34, 796–803. [CrossRef] [PubMed] 12. Alevizakos, V.; Mitov, G.; Von See, C. Guided implant placement using an internally cooling surgical te J. Oral Implantol. 2020, 46, 533–535. [CrossRef] [PubMed] p 13. Stocchero, M.; Sivolella, S.; Brunello, G.; Zoppello, A.; Cavallin, F.; Biasetto, L. Bone temperature variation using a 3D-Printed surgical guide with internal irrigation. Appl. Sci. 2021, 11, 2588. [CrossRef] g g g 14. Benington, I.C.; Biagioni, P.A.; Briggs, J.; Sheridan, S.; Lamey, P.J. Thermal changes observed at implant sites during internal and external irrigation. Clin. Oral Implants Res. 2002, 13, 293–297. [CrossRef] [PubMed] 15. Davidson, S.R.H.; James, D.F. Measurement of thermal conductivity of bovine cortical bone. Medical Engin. Physics 2000, 22, 741–747. [CrossRef] [PubMed] 16. Barrak, I.; Joób-Fancsaly, Á.; Braunitzer, G.; Varga, E.; Boa, K.; Piffkó, J. Intraosseous heat generation during osteotomy was performed freehand and through a template with an integrated metal guide sleeve. Implant. Dent. 2018, 27, 342–350. [CrossRef] [PubMed] 17. Frösch, L.; Mukaddam, K.; Filippi, A.; Zitzmann, N.U.; Kühl, S. Comparison of heat generation between guided and conventional implant surgery for single and sequential drilling protocols-an in vitro study. Clin. Oral Implants Res. 2019, 30, 121–130. [CrossRef] [PubMed] 18. Boa, K.; Barrak, I.; Varga, E.; Joob-Fancsaly, A.; Varga, E.; Piffko, J. Intraosseous generation of heat during guided surgical drilling: An ex vivo study of the effect of the temperature of the irrigating ﬂuid. Br. J. Oral Maxillofac. Surg. 2016, 54, 904–908. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W3172502655	https://ora.ox.ac.uk/objects/uuid:9d6ee798-3be1-4b96-883b-b13b1d317b64/files/r7h149q320	English	null	Support needs and barriers to accessing support: Baseline results of a mixed-methods national survey of people bereaved during the COVID-19 pandemic	Palliative medicine	2,021	cc-by	9,416	43372 PMJ0010.1177/02692163211043372Palliative MedicineHarrop et al. 43372 PMJ0010.1177/02692163211043372Palliative MedicineHarrop et al. Original Article Palliative Medicine 2021, Vol. 35(10) 1985–1997 © The Author(s) 2021 1Marie Curie Palliative Care Research Centre, Division of Population Medicine, Cardiff University, Cardiff, UK 2School of Dentistry, Cardiff University, Cardiff, UK 3PRIME Centre, Division of Population Medicine, Cardiff University, Cardiff, UK 4Keele University, Keele, UK 5Palliative and End of Life Care Research Group, Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK 6Queen’s University, Belfast, Northern Ireland, UK 7Clinical Psychology, School of Health in Social Science, University of Edinburgh, Edinburgh, UK Abstract Background: The COVID-19 pandemic is a mass bereavement event which has profoundly disrupted grief experiences. Understanding support needs and access to support among people bereaved at this time is crucial to ensuring appropriate bereavement support infrastructure. Aim: To investigate grief experiences, support needs and use of formal and informal bereavement support among people bereaved during the pandemic. Aim: To investigate grief experiences, support needs and use of formal and informal bereavement support among people bereaved during the pandemic. Design: Baseline results from a longitudinal survey. Support needs and experiences of accessing support are reported using descriptive s from a longitudinal survey. Support needs and experiences of accessing support are reported using descriptive analysis of free-text data. Design: Baseline results from a longitudinal survey. Support needs and experiences of accessing support are rep tatistics and thematic analysis of free-text data. Setting/participants: 711 adults bereaved in the UK between March and December 2020, recruited via media, social media, national associations and community/charitable organisations. Results: High-level needs for emotional support were identified. Most participants had not sought support from bereavement services (59%, n = 422) or their General-Practitioner (60%, n = 428). Of participants who had sought such support, over half experienced difficulties accessing bereavement services (56%, n = 149)/General-Practitioner support (52%, n = 135). About 51% reported high/ severe vulnerability in grief; among these, 74% were not accessing bereavement or mental-health services. Barriers included limited availability, lack of appropriate support, discomfort asking for help and not knowing how to access services. About 39% (n = 279) experienced difficulties getting support from family/friends, including relational challenges, little face-to-face contact and disrupted collective mourning. The perceived uniqueness of pandemic bereavement and wider societal strains exacerbated their isolation. Conclusions: People bereaved during the pandemic have high levels of support needs alongside difficulties accessing support. We recommend increased provision and tailoring of bereavement services, improved information on support options and social/ educational initiatives to bolster informal support and ameliorate isolation. Conclusions: People bereaved during the pandemic have high levels of support needs alongside difficulties accessing support. We recommend increased provision and tailoring of bereavement services, improved information on support options and social/ educational initiatives to bolster informal support and ameliorate isolation. Support needs and barriers to accessing support: Baseline results of a mixed-methods national survey of people bereaved during the COVID-19 pandemic https://doi.org/10.1177/026921632110433 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/02692163211043372 journals.sagepub.com/home/pmj Emily Harrop1 , Silvia Goss1 , Damian Farnell2, Mirella Longo1, Anthony Byrne1 , Kali Barawi1, Anna Torrens-Burton3 , Annmarie Nelson1 , Kathy Seddon1, Linda Machin4 , Eileen Sutton5, Audrey Roulston6 , Anne Finucane7 , Alison Penny8, Kirsten V Smith9 , Stephanie Sivell1 and Lucy E Selman5 8National Bereavement Alliance/Childhood Bereavement Network, London, UK 9Centre for Anxiety Disorders and Trauma, Department of Experimental Psychology, University of Oxford, Oxford, UK Corresponding author: Emily Harrop, Marie Curie Palliative Care Research Centre, Cardiff University, 8th Floor Neuadd Meirionydd, Heath Park Way, Cardiff, CF14 4YS, UK. Email: harrope@cardiff.ac.uk Keywords Bereavement, grief, pandemics, coronavirus infections, social support, bereavement services Palliative Medicine 35(10) 1986 Implications for policy and practice •• Further investment in the provision of tailored bereavement support is needed to meet the diverse needs and back- grounds of bereaved people, including support that is culturally and crisis/context competent, and group-based support for those with shared experiences and characteristics. •• Further investment in the provision of tailored bereavement support is needed to meet the diverse needs and back- grounds of bereaved people, including support that is culturally and crisis/context competent, and group-based support for those with shared experiences and characteristics. •• To raise awareness of support options, information on grief and bereavement services should be provided proactively following a death and made available in online and community settings, with General Practitioners (GPs) and other primary care providers better resourced to signpost to appropriate support. •• To raise awareness of support options, information on grief and bereavement services should be provided proactively following a death and made available in online and community settings, with General Practitioners (GPs) and other primary care providers better resourced to signpost to appropriate support. •• Following compassionate communities approaches, expanded provision of informal community-based support and activities could help with isolation, whilst longer-term educational and societal initiatives are needed to bolster com- munity support for people experiencing death, dying and bereavement. •• Following compassionate communities approaches, expanded provision of informal community-based support and activities could help with isolation, whilst longer-term educational and societal initiatives are needed to bolster com- munity support for people experiencing death, dying and bereavement. structured, reflective support, beneficial for those with moderate needs, estimated at c.30% of the bereaved pop- ulation.15,17–19 Third-tier support, including specialist grief, mental health and psychological interventions, should be targeted at the small minority (c.10%) of people at high risk of prolonged grief disorder.15,17,18 A review of bereave- ment interventions following mass bereavement events confirmed the value of social networks, psycho-educa- tional approaches, group-based support and specialist psychological support for those with complex needs, alongside early, proactive outreach to bereaved families.16 What this paper adds •• Participants had high level needs for emotional support, especially dealing with/expressing feelings, with 51% experi- encing high or severe vulnerability in grief; however, 74% of this group were not accessing formal bereavement service or mental health support. •• Participants had high level needs for emotional support, especially dealing with/expressing feelings, with 51% experi- encing high or severe vulnerability in grief; however, 74% of this group were not accessing formal bereavement service or mental health support. •• Most participants had not tried to access bereavement services, for reasons such as lack of appropriate support, dis- comfort in asking for help and uncertainty of how to access services; of the 41% who tried, 56% experienced difficulties such as long waiting lists or ineligibility. •• Most participants had not tried to access bereavement services, for reasons such as lack of appropriate support, dis- comfort in asking for help and uncertainty of how to access services; of the 41% who tried, 56% experienced difficulties such as long waiting lists or ineligibility. •• A substantial proportion of people (39%) reported difficulties accessing support from friends and family; reduced in- person contact affected the perceived quality of support and disrupted collective mourning practices, whilst the wider social difficulties of the pandemic compounded feelings of isolation. •• A substantial proportion of people (39%) reported difficulties accessing support from friends and family; reduced in- person contact affected the perceived quality of support and disrupted collective mourning practices, whilst the wider social difficulties of the pandemic compounded feelings of isolation. What is already known about the topic? s of pandemic bereavement, such as traumatic death experiences, exacerbate family distress and add to th ity of grief. •• In pre-pandemic times most people mainly relied on the informal support of friends and family to cope with their bereavement, but an estimated 40% required more formal therapeutic support from bereavement or mental health services. •• In pre-pandemic times most people mainly relied on the informal support of friends and family to cope with their bereavement, but an estimated 40% required more formal therapeutic support from bereavement or mental health services. •• Bereaved people experience difficulties getting the support that they need from bereavement services and their social networks. •• Bereaved people experience difficulties getting the support that they need from bereavement services and their social networks. Background The COVID-19 pandemic has resulted in widespread bereavement on an unprecedented global scale. Lack of access to, and physical contact with, loved ones at the time of death, restrictions surrounding funerals and the sudden nature of most Covid-19 deaths have caused high levels of distress to those bereaved during the pan- demic.1–4 Traumatic end-of-life and death experiences add to the complexity of grief,5–11 whilst limited access to usual support networks and severe societal disruption are also likely to increase risks of poor bereavement outcomes.11–14 However, there is evidence that bereaved people expe- rience problems getting the right support. These include lack of understanding and compassion amongst family and friends, and difficulties expressing their feelings and needs.20–24 The limited evidence on pandemic bereave- ment suggests these experiences are intensified by the physical isolation brought about by lockdown and social distancing restrictions, as well as a sense of feeling forgot- ten.25,26 Disparities between the amount of formal sup- port available and the level of demand have been Bereavement support is a core part of health and social care provision, and is of heightened importance at times of mass bereavement.12,15,16 Public health approaches to bereavement care recognise the differing needs of bereaved people and recommend a tiered approach to support.15,17,18 The first-tier includes universal access to information on grief and available support, recognising that (in pre-pandemic times) c.60% of bereaved people cope without formal intervention, supported by existing social networks.15,17,18 The second-tier includes 1987 Harrop et al. organisations, including those working with ethnic minor- ity communities. Organisations helped disseminate the voluntary (non-incentivised) survey by sharing on social media, web-pages, newsletters, on-line forums and via direct invitations to potential participants (Supplemental File 2, example advertisement). For ease of access, the survey was posted onto a bespoke study-specific website with a memorable URL (covidbereavement.com). Two participants completed the survey in paper format. Summaries of interim survey results (released November 2020) were posted on the website and provided to inter- ested participants. Results The survey was administered via JISC (https://www. onlinesurveys.ac.uk/) and was open from 28th August 2020 to 5th January 2021. It was disseminated to a con- venience sample on social and mainstream media and via voluntary sector associations and bereavement support Study design and aim Baseline results from a longitudinal survey which aims to investigate the grief experiences, support needs and use of bereavement support by people bereaved during the pandemic. The Checklist for Reporting Results of Internet E-Surveys32 was followed. Data analysis Descriptive statistics and frequency tables were used for demographic and categorical response data. Statistical calculations were carried out using SPSS V26. No statisti- cal correction was conducted. Free-text survey responses were analysed using inductive thematic analysis, involving line-by-line coding in NVivo V12 and identification of descriptive and analytical themes.35 A preliminary coding framework was developed based on a sample of survey responses (ES). The framework was revised and applied in an iterative process (EH, ES, SG, KB), moving between the data and the analytical concepts to develop codes and themes grounded in the data. This involving independent double coding of 10% of the dataset (70 responses), regu- lar discussion and cross-checking within the study team and review of final themes by the qualitative team. About 85% of participants (n = 606) provided comments related to experiences of accessing support. Quantitative and qualitative data are integrated under two thematic head- ings which describe experiences of accessing (a) formal and (b) informal support, with the qualitative results used to expand upon and further explain quantitative observations. Background identified before and during the pandemic.27–29 Barriers to support include lack of information and knowledge of how to get support, and discomfort or reluctance to seek help from services.16,27 Limited awareness of available support and a lack of culturally competent services are particular barriers for people from minority ethnic communities.30,31 This mixed-methods longitudinal study is the first to investigate bereavement support needs and experiences in the UK during COVID-19, adding to the emerging evi- dence-base on pandemic bereavement.1–3,11 This paper reports baseline survey results. Using quantitative and qualitative free-text data it describes support needs and experiences of accessing formal and informal support, to inform support provision during and beyond the current crisis. Inclusion criteria: aged 18+; family or close friend bereaved since social-distancing requirements were intro- duced in the UK (16/03/2020); death occurred in the UK; ability to consent. The initial section of the survey requested informed consent and details data protection. Via contact and demographic information we identified 12 surveys completed in duplicate; the first completed survey was retained for these participants. Two surveys were excluded as only the consent question had been answered. Survey development An open web survey (Supplemental File 1) was designed by the research team, which includes a public representa- tive (KS), with input from the study advisory group. It was piloted, refined with 16 public representatives with expe- rience of bereavement and tested by the advisory group and colleagues. Survey design and questions were informed by study aims and previous research.19,24,27,33 Non-randomised open and closed questions covered end- of-life and grief experiences, and perceived needs for, access to and experiences of formal and informal bereave- ment support. Grief was measured using the Adult Attitude to Grief (AAG) Scale,34 which gives an overall index of vulnerability (0–20 = low vulnerability, 21– 23 = high vulnerability and 24–36 = extreme vulnerabil- ity). Most free-text data reported here was from question C4: ‘If relevant, please briefly describe any difficulties you faced getting support from friends, family or bereave- ment services’. Sample characteristics 711 bereaved participants completed the survey (Table 1). Participants represented diverse geographical areas, deprivation indexes and levels of education. About 88.6% Palliative Medicine 35(10) 1988 Table 1. Characteristics of the bereaved person. Age Mean [Median] SD Min–Max Age (years) 49.5 [50.0] 12.9 18–90 n Percentage Gender identity Male 74 10.4 Female 628 88.6 Other 7 1.0 Ethnicity Non-BAME (total) 676 95.3 White British 438 64.8 White English 111 16.4 White Welsh 41 6.1 Northern Irish 22 3.3 White Scottish 40 5.9 Any other white 17 2.5 White Irish 7 1.0 BAME (total) 33 4.7 White and Black Caribbean 12 36.4 White and Asian 5 15.2 Indian 4 12.1 Black Caribbean 4 12.1 Any other mixed background 3 9.1 Pakistani 1 3.0 Bangladeshi 1 3.0 Arab 1 3.0 White and Black African 1 3.0 Any other Asian 1 3.0 Religious beliefs Buddhism 8 1.2 Christian 251 36.7 Hinduism 3 0.4 Islam 5 0.7 Judaism 6 0.9 Sikhism 2 0.3 Other or agnostic 107 15.7 No 301 44.1 Highest qualification None or GCSEs* 108 15.3 A-level or apprenticeship or ONC 132 18.6 HND or university degree* 468 66.1 Region England 517 78.5 Wales 63 9.6 Scotland 53 8.0 Northern Ireland 26 3.9 Unemployed during the pandemic? Yes 55 7.9 No 645 92.1 Bereavements in previous year? Yes 158 22.5 No 543 77.5 GCSE = general certificate of secondary education for 15 and 16 year olds in the UK. A Levels = Advanced Level subject-based qualification for students i h UK d 16 d b ONC O di N i l C ifi Table 1. Characteristics of the bereaved person. of participants were female (n = 628); the mean age of the bereaved person was 49.5 years old (SD = 12.9; range 18–90). The most common relationship of the deceased to the bereaved was parent (n = 395,55.6%), followed by partner/spouse (n = 152,21.4%). Seventy-two people (10.1%) had experienced more than one bereavement since 16th March 2020. Thirty-three people (4.7%) self- identified as from a minority ethnic background. The mean age of the deceased person was 72.2 years old (SD = 16.1; range 4 months gestation to 102 years) (Table 2). About 43.8% (n = 311) died of confirmed/sus- pected COVID-19, 21.9% (n = 156) from cancer and 16.7% (n = 119) from another life-limiting condition. Most died in hospital (n = 410; 57.8%). Sample characteristics Questionnaires were com- pleted a median of 152 days (5 months) after the death (range 1–279 days). Qualitative findings We identified six major themes, with subthemes, pre- sented in Table 3. Three major themes related to access- ing formal support and three to accessing informal support. Support needs and access to formal support Support needs were quantitively assessed in 13 domains (Table 4). In six emotional-support domains, between 50% and 60% of participants reported high/fairly high lev- els of need. The most common were: ‘dealing with my feelings about the way my loved one died’ (60%), ‘express- ing my feelings and feeling understood by others’ (53%) and ‘feelings of anxiety and depression (53%). Over half of participants also demonstrated high or severe levels of overall vulnerability in grief, assessed via the AAG Scale (Severe = 28%, high = 23%, low = 48%, Table 5). Despite this, 72% (n = 230) of people with high/severe vulnerabil- ity and who were more than 6 weeks bereaved (n = 318), were not using formal tier 2/3 support (Figure 1). Most participants had not tried to access support from a bereavement service (59%, n = 422, %) or their GP (60%, n = 428); just under a half of whom had high or severe vul- nerability (45/44% respectively; n = 190). Amongst the 267 participants who sought support from bereavement services, 56% (n = 149) experienced access difficulties, with similar proportions observed for General Practitioner (GP) services (52%, n = 135/159). Reasons for not using bereavement services included: not needing the support due to adequate support from friends and family (29%, n = 207); feeling uncomfortable asking for support (27%, n = 195); a perception that the support will not help (18%, n = 130); unavailability (15%, n = 103), and not knowing how to get support (14%, n = 96). 1989 Harrop et al. Table 2. Characteristics of the deceased. Age Mean [Median] SD Min–Max Age (years) 72.2 [74.0] 16.1 Pregnancy-102 n Percentage Relationship of the deceased person to the bereaved Partner (Male/Female) 152 (129/23) 21.4 (18.1/3.2) Parent (Father/Mother) 395 (218/197) 55.6 (30.7/27.7) Grandparent 54 7.6 Sibling (Brother/Sister) 23 (15/10) 3.2 (2.1/1.4) Child (Son/Daughter) 15 (12/4) 2.1 (1.7/0.6) Other family member 46 6.5 Colleague or friend 26 3.7 Cause of death COVID 273 38.5 Suspected COVID 38 5.4 Non-COVID (total) 399 56.2 Cancer 156 21.9 Other LLC 118 16.7 Non LLC* 112 15.8 Don’t know 12 3.0 Not specified 1 0.2 Was the death expected? A small minority paid for private counselling to access more timely help. A small minority paid for private counselling to access more timely help. Free-text comments expanded upon these reasons, with two main themes identified: availability of (appropri- ate) support, and knowledge and attitudes towards sup- port use. These themes were represented across gender and ethnic groups; group-specific findings were also iden- tified, described below. The services are so overloaded that there [are] huge waits to get help. I phoned a local bereavement charity that the hospital recommended when I called them to say that I wasn’t coping well. However, the charity informed me that they would add me to their waiting list, but probably wouldn’t be able to get back to me for 7 months. It made my grief and anxiety even worse knowing that I couldn’t get access to support. (Bereaved mother, RID696) Support needs and access to formal support Yes 113 16.0 No 552 78.0 Don’t know 43 6.1 Place of death In hospital 410 57.7 In their home 158 22.2 In a hospice 37 5.2 In a care home 91 12.8 Other/Don’t know 13 1.8 Multiple bereavements recorded by participants explain discrepancies between overall totals in sibling, child and parent groups and their sub- categories. LLC = Life-limiting condition, for example, heart disease, COPD, dementia. *NLLC = Non-life-limiting condition, for example, stroke, heart attack, accident, suicide. Multiple bereavements recorded by participants explain discrepancies between overall totals in sibling, child and pare categories. Multiple bereavements recorded by participants explain discrepancies between overall totals in sibling, child and parent groups and their sub- categories. ements recorded by participants explain discrepancies between overall totals in sibling, child and parent groups and their s LLC Life limiting condition, for example, heart disease, COPD, dementia. **NLLC = Non-life-limiting condition, for example, stroke, heart attack, accident, suicide. Availability of (appropriate) support Several problems relating to the availability of support were described. Assumptions of over-stretched services dissuaded some participants from trying to get help, whilst others described experiences of unreturned calls, needing to wait a minimum period after the death (6 weeks to 6 months), long waiting lists and post-code- based ineligibility. Some people experienced difficulty getting through to helplines or struggled with discontinu- ity between call handlers or the limited sessions available. Although many participants who received telephone or web-based support described positive experiences, some felt uncomfortable discussing sensitive and personal mat- ters remotely. People with children at home or who were working in shared offices reported problems with privacy and having the time and space to access remote support. Some perceived a need for COVID-19 loss-specific Palliative Medicine 35(10) 1990 Table 3. Overview of qualitative themes. Too long a wait for counselling; don’t feel I have the strength to retell the same story to different people on a help line. (Bereaved daughter, RID315) I can’t bear the idea of having a bereavement service talk to me about loss in the traditional ways, because losing someone to Covid and in the middle of a lockdown isn’t like other types of loss. What possible advice could they give? There just isn’t any way of observing the sorts of traditions or rituals that would be healing. And how can I move past this when the pandemic is still all around me? (Bereaved daughter, RID336) I am reluctant to reach out to bereavement services because I feel uncomfortable about the idea of making myself vulnerable to a complete stranger. (Bereaved grandson, RID071) I’ve found because there is no continuity of care at my own GP surgery that I have to make my case and talk about difficult things on the phone is so hard, especially when you have to make the case to a receptionist to start with, just to get to talk to a doctor. . .. All those things, like asking for help and finding the right words of what you’re going through, are hard. It’s easier not to do anything and to stick to your friends and family. (Bereaved wife, RID458) It’s been difficult for myself and my husband to get back into work and the pandemic related social isolation made it difficult for us to grieve openly with friends and family. Availability of (appropriate) support This in itself put an emotional barrier up between us and them. As they hadn’t been through the unique trauma we’d experienced, it was difficult to set up a line of communication and build relationships again. (Bereaved mother, RID267) I’ve had to contend with managing my own grief with also supporting my children through theirs and dealing with a heavy workload, home- schooling and being unable to meet with my own friendship group or family for the support I would normally have looked for from them. (Bereaved granddaughter, RID348) Disrupted grieving • Disruption to mourning rituals/ collective support • Harder accepting and coming to terms with death I think working through the anger and sadness I feel about how mum died and what we have consequently suffered in terms of loss of normal grief ‘rights’. (For example I have still not hugged my Dad or sister) is something that I need more help with now and I think the government has failed to take account of the damage done to bereaved families by not making allowances for them to have bereavement contact during lockdowns, crying on Zoom is just not the same. All I wanted when Mum first died was to get out of this house and go and have a cup of tea with a friend. I have been stuck in the house most of this year and for a long time with my husband and kids, home-schooling. I had no escape and nowhere to go. (Bereaved daughter, RID734) Lack of understanding and empathy • Difficult/unsupportive conversations • Time limited support • Exceptional bereavements • Insensitive behaviours and attitudes towards pandemic We have been unable to assist our relatives, especially us from Black Minority Ethnic (BME), in our background culture staying [close] with friends, visiting them frequently in time of bereavement is the most important thing we do. But right now, no one was [able] to do that which make the challenges even harder. (Bereaved niece, RID680) Other people don’t want to keep hearing it and some people who believe Covid is a hoax or conspiracy, it’s heart-breaking to have to listen to that crap continuously. People look at you like you are lying if you say Mum died if Covid. The ignorance out there is stifling sometimes. Availability of (appropriate) support (Bereaved daughter, RID318) We have been unable to assist our relatives, especially us from Black Minority Ethnic (BME), in our background culture staying [close] with friends, visiting them frequently in time of bereavement is the most important thing we do. But right now, no one was [able] to do that which make the challenges even harder. (Bereaved niece, RID680) Other people don’t want to keep hearing it and some people who believe Covid is a hoax or conspiracy, it’s heart-breaking to have to listen to that crap continuously. People look at you like you are lying if you say Mum died if Covid. The ignorance out there is stifling sometimes. (Bereaved daughter, RID318) support, rather than ‘generic’ support, or reflected on a lack of contextual understanding among support provid- ers. Needs for culturally-relevant support and group-spe- cific support for those with similar experiences were also described; for example widows, young widows, parents, same-sex couples and following particular types of death (COVID-19, childbirth-related, pregnancy-loss). support, rather than ‘generic’ support, or reflected on a lack of contextual understanding among support provid- ers. Needs for culturally-relevant support and group-spe- cific support for those with similar experiences were also described; for example widows, young widows, parents, same-sex couples and following particular types of death (COVID-19, childbirth-related, pregnancy-loss). couldn’t make the time they offered me. I am still waiting for a place in a group at a time I can attend. As I write I wonder if they have forgotten about me. I would also want to meet with others who have lost a same sex partner (not necessarily the same group). Again I can’t find a group that’s meeting. (Bereaved wife/partner, RID487) Knowledge and attitudes towards support use I wrote to the hospice asking for group support. I definitely NEED to connect with people who have been bereaved during lockdown not particularly because of covid. This hasn’t been possible. The hospice closed their groups and when they were able to offer a zoom group I had returned to work and Other barriers related to a lack of information about how to get support, feeling too uncomfortable or upset to seek formal support, feeling unable to open up to ‘strangers’ or 1991 Harrop et al. Table 4. Support needs ranked by mean level of need. High or fairly high level of support needed (%) Moderate level of support needed (%) Little or no support needed (%) Mean (95% CI) Median Dealing with my feelings about the way my loved one died 59.8 21.5 18.7 3.71 (3.62–3.80) 4 Dealing with my feelings about being without my loved one 49.9 29.3 20.8 3.48 (3.39–3.57) 3 Expressing my feelings and feeling understood by others 53 23.9 23 3.48 (3.38–3.57) 4 Feeling comforted and reassured 51.8 26.7 21.6 3.46 (3.37–3.55) 4 Feelings of anxiety and depression 52.8 21.1 26.1 3.45 (3.35–3.55) 4 Loneliness and social isolation 52.0 19.1 29 3.36 (3.26–3.46) 4 Finding balance between grieving and other areas of life 45.0 27.9 27 3.29 (3.20–3.39) 3 Regaining sense of purpose and meaning in life 46.7 21.6 31.7 3.26 (3.15–3.36) 3 Managing and maintaining my relationships with friends and family 36.2 26.4 37.4 2.98 (2.88–3.08) 3 Participating in work, leisure or other regular activities (e.g. shopping, housework) 33.8 23.9 42.1 2.87 (2.76–2.97) 3 Getting relevant information and advice for example, legal, financial, available support 24.3 22.3 53.3 2.51 (2.41–2.61) 2 Practical tasks for example, managing the funeral, registering the death, other paperwork, etc. 23.5 21.7 54.7 2.48 (2.38–2.58) 2 Looking after myself/family, for example, getting food, medication, childcare 15.2 22.8 62 2.25 (2.16–2.34) 2 Note that to interpret means and medians: no support = 1; little support = 2; moderate support = 3; fairly high support = 4; and high support = 5). Table 5. Descriptive statistics for the AAG questionnaire.34. Table 4. Support needs ranked by mean level of need. and medians: no support = 1; little support = 2; moderate support = 3; fairly high support = 4; and high support = 5). Table 5. Descriptive statistics for the AAG questionnaire.34. Accessing GP support In relation to GPs, people described difficulties getting appointments or feeling inadequately supported during telephone appointments. GP support commonly involved providing medication, helpline information and sick-notes for time-off work, with a lack of information about, or referral to, bereavement and mental health services also noted. Knowledge and attitudes towards support use Highest level of support accessed by IOV group using three tiers of the Public Health Model.17 Tier 1 a: Friends and family only; Tier 1 b: Informal and information-based support: GP, helpline, online community support, informal support group, other for example, websites, podcasts, self-help material; Tier 2/3: Formal bereavement service and mental health support: One to one support/ counselling, bereavement support group/group counselling, mental health support. >6 weeks; participants who completed questionnaire at least 42 days post-death I am having difficulties coming to terms with my loss. GP can only offer medication, or helpline advice which has not helped really. I need company really. (Bereaved husband, RID671) unsure how bereavement support could help. People who lost elderly parents to long-term illnesses (and in some cases COVID-19), felt less entitled or worthy of support, whilst a woman grieving her female partner explained ‘Because of our relationship I do not know where to turn to for help’ (RID667). Some participants who answered the survey soon after the death considered it too early to be thinking about accessing formal support, but others perceived a need for early intervention. Accessing support from friends and family A substantial proportion of participants (n = 279, 39%) reported difficulties getting support from friends and fam- ily. Across the sample, 25% (n = 175) reported that their friends or family were unable to support them in the way that they wanted, whilst 19% (n = 195) reported feeling uncomfortable asking for help. I feel awkward making a phone call to say that I am struggling, especially as a couple of months has passed since she died. I had hoped that time would settle things down (it has to an extent) and now it seems too late to seek help. No one offered/directed me to any support either so I wondered if not actually available? (Bereaved daughter, RID024) Three main themes were identified in free-text data: difficulties connecting and communicating with friends and family, disrupted grieving and lack of understanding and empathy. These themes were again evident across gender and ethnic groups with other group-specific find- ings described. Knowledge and attitudes towards support use Subscales/Total score n % Missing Mean (95% CI) [Median] SD AAG Overwhelmed 705 0.8 8.53 (8.31–8.72) [9.00] 2.79 Controlled 700 1.5 6.61 (6.41–6.82) [7.00] 2.71 Reversed resilience 701 1.4 5.28 (5.07–5.49) [5.00] 2.82 IOV 698 1.8 20.41 (20.06–20.77) [21.00] 4.77 IOV grouped into categories Low n (%) High n (%) Severe n (%) 338 (48.4) 163 (23.4) 197 (28.2) The AAG has three subscales (overwhelmed, controlled and reversed resilience). Subscale scores are calculated by summing the scores for each item for each subject, giving a range from 0 to 12 for each subscale, with increasing scores indicating higher levels of feeling overwhelmed, controlled and reversed resilience (i.e. vulnerability). The overall index of vulnerability (IOV) is calculated by adding the scores for each subscale together, where IOV score 0–20 = low vulnerability, 21–23 = high vulnerability and 24–36 = extreme vulnerability. Table 5. Descriptive statistics for the AAG questionnaire.34. The AAG has three subscales (overwhelmed, controlled and reversed resilience). Subscale scores are calculated by summing the scores for each item for each subject, giving a range from 0 to 12 for each subscale, with increasing scores indicating higher levels of feeling overwhelmed, controlled and reversed resilience (i.e. vulnerability). The overall index of vulnerability (IOV) is calculated by adding the scores for each subscale together, where IOV score 0–20 = low vulnerability, 21–23 = high vulnerability and 24–36 = extreme vulnerability. Palliative Medicine 35(10) 1992 Figure 1. Highest level of support accessed by IOV group using three tiers of the Public Health Model.17 Tier 1 a: Friends and family only; Tier 1 b: Informal and information-based support: GP, helpline, online community support, informal support group, other for example, websites, podcasts, self-help material; Tier 2/3: Formal bereavement service and mental health support: One to one support/ counselling, bereavement support group/group counselling, mental health support. >6 weeks; participants who completed questionnaire at least 42 days post-death. Figure 1. Highest level of support accessed by IOV group using three tiers of the Public Health Model.17 Tier 1 a: Friends and family only; Tier 1 b: Informal and information-based support: GP, helpline, online community support, informal support group, other for example, websites, podcasts, self-help material; Tier 2/3: Formal bereavement service and mental health support: One to one support/ counselling, bereavement support group/group counselling, mental health support. >6 weeks; participants who completed questionnaire at least 42 days post-death. Figure 1. Difficulties connecting and communicating with friends and family Opportunities for in-person contact were minimised due to social distancing measures and geography. Needing physical comfort and ‘hugs’ was widely reported, with 1993 Harrop et al. many describing difficulties talking openly about their feelings with friends and family, especially over the phone or internet. Isolation during lockdown and early bereave- ment made it harder to reconnect with friends over time. In some cases, isolation and disconnectedness were wors- ened by pre-existing strained relationships, or conflict sur- rounding end-of-life or early bereavement experiences. Only my cousin’s wife [was] allowed to say good bye to her husband. His two children or any other relatives were not allowed. She returned home with her children. There was no other relatives there to support her (due to isolation and Covid regulations) [even] her mother was not there to [console] her. This was like pouring salt on wounds. Sharing ones grief reduces pain and help overcome the pain. Normally hundreds of relative would have been visiting her and sharing her grief which would have helped her and all of us to accept the death. But not being to visit her personally made it very difficult to overcome. (Bereaved male cousin, RID653) I have not really sought support from family as they are affected too. We talk about Dad in a positive way, and joke about him as well. This helps. I would not ‘seek support’ from any friends - what would I ask them? No idea. I suppose what could happen would be getting a bit drunk together and getting a few things off my chest, but this isn’t likely to happen in times of Covid. (Bereaved son, RID340) Lack of understanding and empathy A lack of understanding and empathy within social net- works was commonly described. Participants perceived friends and family members as feeling awkward and uncomfortable talking about grief or the deceased person, changing the subject or implying that they should have ‘moved on’. Many described receiving frequent calls in the first weeks of bereavement, but noted the decline as the months went on. One younger participant reflected that since parental loss was unusual in her peer group, her friends were unable to provide the support that she needed. People worried about being a burden, and felt unable to trou- ble others also grieving. The perceived need to keep grief to oneself and stay strong was particularly acute for parents, especially those home-schooling children during lockdown. Owing to the widespread stress caused by the pandemic, many feared adding to the emotional and mental health bur- den of others. People who lost elderly parents to non-COVID illnesses described further inhibitions about asking for help. There is no doubt that the COVID restrictions have made this period even worse than they would have been in ‘normal’ times. It has sometimes made it hard to ask for help when I am aware that everyone is having a difficult time due to COVID and equally there are things that I might have been able to do to help myself - e.g. volunteering activities, which have not been possible. Two terrible life experiences happening at exactly the same time has been very hard and continues to be. (Bereaved wife, RID469) [Not] there in person, feel lonely while others get on with their lives where one doesn’t want to intrude. Also nobody who’s not lost a partner, can really understand. . ..It’s about time you pulled yourself together and got on with your life. But my life is gone, from language, to food, to walks in woods, to friendship and sharing. . . (Bereaved wife/partner, RID111) People also described how the exceptional nature of COVID-19 bereavement (including the anger associated with it) made it impossible for friends to understand, compounding their sense of loneliness and isolation. Experiences of social contacts disregarding regulations, questioning the seriousness of the pandemic or sharing conspiracy theories on social media were also distressing, further contributing to their alienation. Strengths and weaknesses This mixed-methods study will be the first to longitudi- nally investigate peoples’ experiences of bereavement support during the Covid-19 pandemic in the UK. The baseline quantitative data demonstrates the extent of the difficulties experienced by the bereaved, whilst explana- tory qualitative data provide rich insights into participant experiences. However, lack of random sampling means that the survey is not representative and by recruiting mostly online we were less likely to reach the very old or other digitally marginalised groups, who are likely to experience enhanced vulnerability in the current context. By promoting the survey via bereavement organisations we were also more likely to hear from those seeking help, thus potentially underestimating the proportions of those bereaved not engaging with any services. Despite signifi- cant efforts and targeted recruitment, people from minor- ity ethnic backgrounds and men are underrepresented in the data. However, group sizes were sufficient to enable comparisons (although not to the level of specific ethnic groups), with group-specific observations reported where relevant in our qualitative findings. Quantitative analysis of the effects of demographic and clinical characteristics on support use is forthcoming. Main findings This study quantifies and describes the support needs and difficulties accessing support experienced by bereaved people during the COVID-19 pandemic. We identified high level needs for emotional and therapeutic support, along- side difficulties accessing both formal and informal sup- port. Barriers included feeling uncomfortable asking for help and not knowing how to get help, as well as a lack of availability of support from bereavement services and GPs. Pandemic-specific challenges included high propor- tions of people perceiving social support to be inade- quate, reduced in-person contact affecting the perceived quality and functionality of support and disruption to col- lective mourning and grieving. The unique nature of pan- demic bereavement and wider societal strains compounded the difficulties and isolation experienced by people bereaved during these exceptional times. Disrupted grieving Being unable to meet close family/friends for support dis- rupted the grieving process, making the death seem ‘sur- real’ and harder to accept. A bereaved daughter described this as ‘a constant prolonging of a goodbye’ (RID112). Some felt that grieving families should have been permit- ted to meet during lockdown. The covid bereavement group on Facebook have been a great source of comfort, as have immediate family. But whilst friends try to be helpful and kind - they don’t understand the anger which is also part of this grief. My Mother passed away 9 years ago from a severe stroke and whilst this was as big a shock as my father’s death - there was more of an acceptance about it. Friends offered tea and sympathy and a shoulder to cry on but then you picked yourself up and got on with it. With Covid it’s very different, the social isolation obviously doesn’t help but there is this underlying anger that not enough infection control procedures were put in place within our hospitals and therefore this death was avoidable! (Bereaved daughter, RID635) Just not being able to hug and be in the same room. After funeral I would have liked to have been in a room with my mother’s friends, my friends and family, sharing memories and stories, crying and laughing, etc. For a while I thought I would still organise a wake after COVID but now I think the moment has passed and that ritual will be missing too. (Bereaved son, RID723). Disruption to mourning rituals and collective support appeared especially salient for people from minority eth- nic backgrounds, affecting extended family and commu- nity as well as immediate family. Palliative Medicine 35(10) 1994 questioned the efficacy or appropriateness of the support on offer. Lack of face-to-face support and to a lesser extent COVID-specific support dissuaded people from tak- ing up formal support. We identified preferences for sup- port groups based on shared experiences or characteristics, the benefits of which have been described previously.16,19 For the 40% of people who did try to access bereavement service support, just over half experienced difficulties such as long waiting lists or ineligibility, supporting the findings of other studies.27–29 The main problems affecting the accessibility and quality of GP support were difficul- ties getting appointments or unsatisfactory telephone appointments,3,37 with inadequate signposting and refer- ral to bereavement and mental health services also noted. What this paper adds About 50% to 60% of bereaved people reported high or fairly high needs for help with processing feelings sur- rounding the death and loss, anxiety and depression and communicating and connecting with friends and family, suggesting considerable needs for social/emotional sup- port as well as reflective grief-focussed support. Only a third perceived no need for bereavement service support due to adequate support from friends and family. This is significantly less than the 60% estimation in the pre-pan- demic public health model,17,18 but validates recent pan- demic-based predictions.13 As in pre-pandemic studies, people commonly reported lack of understanding and compassion amongst friends and family, alongside difficulties expressing their feelings and asking for help.20–24 We found that these experiences have been exacerbated by the physical isolation and diminished opportunities for in-person support,25,26 the disruption to collective mourning caused by pandemic restrictions, as well as additional concerns over burdening others also experiencing hardship. Loneliness was com- pounded by the perceived uniqueness and anger associ- ated with pandemic grief and COVID-19 deaths,36 alongside the distressing effects of people questioning or disregarding the pandemic. Implications for research This study includes follow-up surveys at c.7 and 13 months post-death, longitudinal qualitative interviews and research exploring the impact of the pandemic on volun- tary sector bereavement services and their response. Although our interviews target underrepresented partici- pants such as those from minority ethnic backgrounds, same-sex couples and men, further research with these groups and older bereaved people is needed. Research exploring the needs of bereaved children and young peo- ple, longer-term bereavement experiences and experi- ences of statutory sector bereavement support providers is also recommended. However, whilst most participants felt that they needed additional support, most had not tried to access help from bereavement services or their GP. Strikingly, around three quarters of people with high/severe vulnerability were not accessing tier 2/3 support. As in pre-pandemic stud- ies, reasons included lack of knowledge/information on how to get support as well as psycho-social barriers such as feeling uncomfortable asking for help.16,27 Some people felt less entitled to support at this time of crisis; others 1995 Harrop et al. Declaration of conflicting interests The author(s) declared the following potential conflicts of inter- est with respect to the research, authorship, and/or publication of this article: All authors except AP declared no potential con- flicts of interest with respect to the research, authorship and/or publication of this article. AP declared a potential financial inter- est relating to lobbying by the Childhood Bereavement Network and National Bereavement Alliance for additional financial sup- port for the bereavement sector. 1. Increased provision and tailoring of services, including greater resourcing and expansion of national support as well as regional services in areas with long waiting lists. Safe ways to access face-to-face individual and group support as well as online and telephone support should be identi- fied, with specific support available for groups with shared experiences and characteristics. This should include support which is culturally competent16,30,31 and crisis/context competent.3,16 Training in core competencies specific to COVID-19 and identifying and sharing best practice amongst bereavement and palliative care providers would facilitate this. Author contributions E.H. and L.E.S. designed the study, led the application for fund- ing and are co-principal investigators; E.H. drafted the paper; M.L., A.B., D.F., A.N., A.T.B., L.M., A.F., K.S., K.V.S., A.R., A.P., S.S. are members of the research team or the study advisory group and contributed to the design of the study and survey. D.F. con- ducted the statistical analyses with data management assis- tance from M.L. and S.G., E.H., K.B., E.S. and S.G. conducted the thematic analysis of qualitative data. All authors contributed to drafting the paper and read and approved the final manuscript. These results demonstrate high levels of need for emo- tional and therapeutic support, and the significant diffi- culties bereaved people face getting these needs met. Our results elaborate upon pre-pandemic inadequacies in formal and informal support, while demonstrating new pandemic-specific challenges including more complex, crisis-specific needs, diminished opportunities for face-to- face and group support, acute social isolation and disrup- tion to collective grieving and the wider societal consequences of the pandemic. Based on study findings we make three recommendations for improving the sup- port available for bereaved people: ORCID iDs Emily Harrop https://orcid.org/0000-0003-2820-0023 Silvia Goss https://orcid.org/0000-0002-9728-5756 Anthony Byrne https://orcid.org/0000-0002-1413-1496 Anna Torrens-Burton https://orcid.org/0000-0002-2162-3739 Annmarie Nelson https://orcid.org/0000-0002-6075-8425 Linda Machin https://orcid.org/0000-0001-6956-7214 Audrey Roulston https://orcid.org/0000-0002-7446-6116 Anne Finucane https://orcid.org/0000-0002-3056-059X Kirsten V Smith https://orcid.org/0000-0002-6973-2846 Stephanie Sivell https://orcid.org/0000-0003-0253-1860 Lucy E Selman https://orcid.org/0000-0001-5747-2699 Funding The author(s) disclosed receipt of the following financial sup- port for the research, authorship, and/or publication of this article: This study was funded by the UKRI/ESRC (Grant No. ES/ V012053/1). The project was also supported by the Marie Curie core grant funding to the Marie Curie Research Centre, Cardiff University (grant no. MCCC-FCO-11-C). E.H., A.N., A.B., S.S. and M.L. posts are supported by the Marie Curie core grant funding (grant no. MCCC-FCO-11-C). ATB is funded by Welsh Government through Health and Care Research Wales. K.V.S. is funded by the Medical Research Council (MR/V001841/1).The funder was not involved in the study design, implementation, analysis or interpretation of results and has not contributed to this manuscript. 2. 2. Strategies to improve awareness of bereavement support options, including providing information on grief and bereavement services proactively fol- lowing a death and ensuring accessible public information is available online and in community settings. GPs and other primary care providers should be better resourced to signpost bereaved patients to appropriate support.38 Ethical approval The study protocol and supporting documentation was approved by Cardiff University School of Medicine Research Ethics Committee (SMREC 20/59). The study was conducted in accord- ance with the Declaration of Helsinki and all respondents pro- vided informed consent. 3. More help with loneliness and isolation, including flexible support bubble arrangements for the recently bereaved when restrictions are in place.26 Following compassionate communities approaches,39,40 informal community-based inter- ventions should be expanded, whilst educational and society level initiatives are needed to improve how, as a society, we communicate and support peo- ple experiencing death, dying and bereavement.40–42 Conclusions and implications for policy and practice Author contributions References 1. Hanna JR, Rapa E, Dalton LJ, et al. A qualitative study of bereaved relatives’ end of life experiences during the COVID-19 pandemic. Palliat Med 2021; 35(5): 843–851. 1. Hanna JR, Rapa E, Dalton LJ, et al. A qualitative study of bereaved relatives’ end of life experiences during the COVID-19 pandemic. Palliat Med 2021; 35(5): 843–851. 16. Harrop E, Mann M, Semedo L, et al. What elements of a systems’ approach to bereavement are most effective in times of mass bereavement? A narrative systematic review with lessons for COVID-19. Palliat Med 2020; 34(9): 1165– 1181. 2. Mayland CR, Hughes R, Lane S, et al. Are public health measures and individualised care compatible in the face of a pandemic? A national observational study of bereaved relatives’ experiences during the COVID-19 pandemic. Palliat Med. Epub ahead of print 31 May 2021. DOI: 10.1177/02692163211019885. 17. Aoun SM, Breen LJ, O’Connor M, et al. A public health approach to bereavement support services in palliative care. Aust N Z J Public Health 2012; 36: 14–16. 3. Pearce C, Honey JR, Lovick R, et al. ‘A silent epidemic of grief’: a survey of bereavement care provision in the UK and Ireland during the COVID-19 pandemic. BMJ Open 2021; 11(3): e046872. 3. Pearce C, Honey JR, Lovick R, et al. ‘A silent epidemic of grief’: a survey of bereavement care provision in the UK and Ireland during the COVID-19 pandemic. BMJ Open 2021; 11(3): e046872. 18. Aoun SM, Breen LJ, Howting DA, et al. Who needs bereave- ment support? A population based survey of bereavement risk and support need. PLoS One 2015; 10(3): e0121101. 19. Harrop E, Morgan F, Longo M, et al. The impacts and effec- tiveness of support for people bereaved through advanced illness: a systematic review and thematic synthesis. Palliat Med 2020; 34(7): 871–888. 4. Yardley S and Rolph M. Death and dying during the pan- demic. BMJ 2020; 369: m1472. 5. Harrop E, Morgan F, Byrne A, et al. “It still haunts me whether we did the right thing”: a qualitative analysis of free text survey data on the bereavement experiences and support needs of family caregivers. BMC Palliat Care 2016; 15(1): 92. 20. Aoun SM, Keegan O, Roberts A, et al. The impact of bereavement support on wellbeing: a comparative study between Australia and Ireland. Palliat Care Soc Pract 2020; 14: 2632352420935132. 6. Sanderson C, Lobb EA, Mowll J, et al. References Signs of post-trau- matic stress disorder in caregivers following an expected death: a qualitative study. Palliat Med 2013; 27(7): 625– 631. 21. Aoun SM, Breen LJ, White I, et al. What sources of bereave- ment support are perceived helpful by bereaved people and why? Empirical evidence for the compassionate com- munities approach. Palliat Med 2018; 32: 1378–1388. 7. Dumont I, Dumont S and Mongeau S. End-of-life care and the grieving process: family caregivers who have experi- enced the loss of a terminal-phase cancer patient. Qual Health Res 2008; 18(8): 1049–1061. 22. Breen LJ and O’Connor M. Family and social networks after bereavement: experiences of support, change, and isola- tion. J Fam Ther 2011; 33: 98–120. 23. Breen LJ, Aoun SM, Rumbold B, et al. Building commu- nity capacity in bereavement support: lessons learnt from bereaved former caregivers. Am J Hosp Palliat Med 2017; 34: 275–281. 8. Kentish-Barnes N, Chaize M, Seegers V, et al. Complicated grief after death of a relative in the intensive care unit. Eur Respir J 2015; 45(5): 1341–1352. 9. Kun P, Han S, Chen X, et al. Prevalence and risk factors for posttraumatic stress disorder: a cross-sectional study among survivors of the Wenchuan 2008 earthquake in China. Depress Anxiety 2009; 26(12): 1134–1140. 24. Harrop E, Scott H, Sivell S, et al. Coping and wellbeing in bereavement: two core outcomes for evaluating bereave- ment support in palliative care. BMC Palliat Care 2020; 19(1): 29–35. 10. Kristensen P, Weisæth L and Heir T. Bereavement and mental health after sudden and violent losses: a review. Psychiatr Interpers Biol Process 2012; 75(1): 76–97. 25. National Bereavement Alliance. Covid 19: the response of voluntary sector bereavement services, https:// nationalbereavementalliance.org.uk/ourpublications/ covid-19-the-response-of-voluntary-sector-bereavement- services (2020, accessed 8 June 2021). 11. Eisma MC, Tamminga A, Smid GE, et al. Acute grief after deaths due to COVID-19, natural causes and unnatural causes: an empirical comparison. J Affect Disord 2021; 278: 54–56. 26. Sue Ryder. Bereaved people claim lockdown deaths became just a statistic, https://www.sueryder.org/news/ bereaved-people-claim-lockdown-deaths-became-just-a- statistic (2020, accessed 8 June 2021). 12. Eisma MC, Boelen PA and Lenferink LIM. Prolonged grief disorder following the Coronavirus (COVID-19) pandemic. Psychiatry Res 2020; 288: 113031. 27. Sue Ryder. A better grief report. London: Sue Ryder. https://www.sueryder.org/sites/default/files/2019-03/a- better-grief-report-sue-ryder.pdf (2019, accessed 8 June 2021). 13. Aoun S. Supplemental material Supplemental material for this article is available online. 15. National Institute for Clinical Excellence (NICE). Guidance on cancer services: improving supportive and palliative care for adults with cancer. The manual. London: National Institute for Clinical Excellence, 2004. Acknowledgements Our thanks to everyone who completed the survey for sharing their experiences, and to all the individuals and organisations that helped disseminate the survey. We would also like to thank the project assistants, collaborators and advisory group mem- bers who are not co-authors on this publication: Dr. Emma Carduff, Dr Daniella Holland-Hart, Dr. Catriona Mayland, Prof. Bridget Johnston, Dr. Donna Wakefield. Palliative Medicine 35(10) 1996 Data management and sharing palliative care: key note presentation at towards solutions for global challenges, 17-18 November 2020. Published in Palliative Care and COVID-19: grief, bereavement and mental health, https://palliativecare.org.au/palliative- care-and-covid-19-grief-bereavement-and-mental-health (2020, accessed 8 June 2021) palliative care: key note presentation at towards solutions for global challenges, 17-18 November 2020. Published in Palliative Care and COVID-19: grief, bereavement and mental health, https://palliativecare.org.au/palliative- care-and-covid-19-grief-bereavement-and-mental-health (2020, accessed 8 June 2021) Full study data sets will be made available following study clo- sure in February 2022. Data sharing requests will be considered prior to this and should be directed to Dr. Emily Harrop, har- rope@cardiff.ac.uk. 14. Smith KV, Wild J and Ehlers A. The masking of mourning: social disconnection after bereavement and its role in psy- chological distress. Clin Psychol Sci 2020; 8(3): 464–476. References Public health approaches to bereavement care – through the lens of the pandemic, at the second inter- national research seminar on public health research in Harrop et al. 1997 28. Ochieng C, Harrop E, Nelson A, et al. A scoping survey of bereavement services in Wales, https://gov.wales/sites/ default/files/publications/2019-12/scoping-survey-of- bereavement-services-in-wales-report.pdf (2019, accessed 24 August 2021). 35. Braun V and Clarke V. Using thematic analysis in psychol- ogy. Qual Res Psychol 2006; 3(2): 77–101. 36. Selman LE, Chamberlain C, Sowden R, et al. Sadness, despair and anger when a patient dies alone from COVID- 19: a thematic content analysis of Twitter data from bereaved family members and friends. Palliat Med 2021; 35: 1267–1276. 29. Wakefield D, Fleming E, Howorth K, et al. Inequalities in awareness and availability of bereavement services in north-east England. BMJ Support Palliat Care. Epub ahead of print 23 September 2020. DOI: 10.1136/bmjsp- care-2020-00242. 37. Mughal F, Hossain MZ, Brady A, et al. Mental health sup- port through primary care during and after covid-19. BMJ 2021; 373: n1064. 30. Mayland CR, Powell RA, Clarke GC, et al. Bereavement care for ethnic minority communities: a systematic review of access to, models of, outcomes from, and satisfaction with, service provision. PLoS One 2021; 16: e0252188. 38. National Bereavement Alliance. Life after death, https:// nationalbereavementalliance.org.uk/ourpublications/life- after-death (2014, accessed 8 June 2021). 39. CC-UK. www.compassionate-communitiesuk.co.uk/ (accessed 8 June 2021). 31. Murray K. National-Mapping-of-BAME-Mental-Health-Services. pdf, bamestream.org.uk (2020, accessed 8 June 2020). 40. Breen LJ, Kawashima D, Joy K, et al. Grief literacy: a call to action for compassionate communities. Death Stud 2020; 18: 1–9. 32. Eysenbach G. Improving the quality of web surveys: the checklist for reporting results of internet E-surveys (CHERRIES). J Med Internet Res 2004; 6(3): e34. 41. Breen LJ. Harnessing social support for bereavement now and beyond the COVID-19 pandemic. Palliat Care Soc Pract 2021; 15: 2632352420988009. 33. Claessen SJ, Francke AL, Sixma HJ, et al. Measuring rela- tives’ perspectives on the quality of palliative care: the con- sumer quality index palliative care. J Pain Symptom Manag 2013; 45(5): 875–884. 42. Islam I, Nelson A, Longo M, et al. Before the 2020 pan- demic: an observational study exploring public knowl- edge, attitudes, plans, and preferences towards death and end of life care in Wales. BMC Palliat Care 2021; 20: 116. 34. Sim J, Machin L and Bartlam B. Identifying vulnerability in grief: psychometric properties of the adult attitude to grief scale. References Qual Life Res 2014; 23(4): 1211–1220.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.